Peter J. Barnes - Tobacco Documents

In Account With
SHOOK, HARDY''AND BACON

Attorneys At Law
One Kansas City Place
1200 Main
Kansas City, Missouri 64105
September 30, 1991
Philip Morris , Inc .

Invoice : 602816~
IRS ID No . 44-058'5497
(8I16) 474-6550
SLEP
Philip Morris
SLEP .24065 REN
Special Liti g ation Ex p enses

(Shared by 1 Company)
REVISED 10/16/91
EXPENSES
Reproduction Systems Incorporated 14212

08/15/91 Consultox, Ltd .-Consultation fee, (Dr . Parent) -491660
July 1991 (International)
08/26/91 George L . Barnes-Investigation fees (J . Wilson) 40191
08/26/91 OnrLine :Investigations-Investigation fees 1391
(Hamilton)
08/26/91 SHB Research Account-PM contribution to Clifton 33458
F . MountainiResearch Proj!ect
08/26/91 First Security Services-Expert services
520
( Hami lton)
08/26/91 David T . Westcott-Consultation fees
2!092
(Toxicology) (International)
08/26/91 Tucker, Hendryx & Gascoyne-Distributor's Local
108 :4
Counsel fees (Carlisle)
08/26/91 Burson-Marsteller-Consultation fee (General)i
263'.5
08!/26/'91 Ralph L . Keeney-Consultants fees (Risk Utility)
160.0
(General),
08/28/'91 Frantisek Palecek--Consuiltationifee
&6010
(Toxicology)(IInternational)
08/29/91 James M' . Cholokis, Ph .D .--Consultation fee
(Toxicology) (Ingredients)
08/29/'91 Southern Consulting Group, Inc .--Consultation
fees - Irving,H . LaValle (Risk Decision
Theorist) (General)
http://legacy.library.ucsf.edu/tid/geq87e00/pdf
.75
.40
.00
.00
.00
.00
.00
.80
.76
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.00
210 .00
194 .0 .11
Continued Next Page .
2015002959
Philip Morris Inc .

CLIENT'NUMBER : SLEP
INVOICE NO . : 602816
September 30, 1991 PAGE 2'
08/2'9/91 James M . Cholokis, Ph .D .--Consultation fee
367 .50 .
08/01/91 Professor Peter J . B'arnes-- consultation fee
759 .03
('Toxicology)i (International)
(Thoracic Medicine) (International)
08/01/91 Prof . Dr . Victor Feron-- consultatio n f ee

08 / 01 /'91 Dr . Diana,Anderson-- consultation fee
2196 .15
08/01/91 Professor Paolo Pani-- consultation fee
2863 .35
08/01/91 Prof . Werner Burkart-- consultation fee
90 .72
08/01/'91 Prof . Dr . H .R . Glatt-- consultation fee
1159 .71
444 .15
(Pathology) ('International)
08'/ 01i /'91 Dr . D .O . Chanter-- consultationifee

0 8 / 01 /'91 Prof . Enrico :Malizia-- consultation fee
(,Toxicology) (International)
08/0'1/91 Dr . Alain F . Pelfrene-- consultation fee
( ;Toxicology) (International)
08/01/91 Prof . S' .D . Ferrara-- consultationifee (Forensic
Toxicology) (International)
08/01/'91 MECC EOROTOX 1991-- Eurotox conference fee, Mr .

Peck and Mr . Gregg (Interrnational) .
08/01/91 Dr . Di. Prentice-- consultation fee (Toxicology)
('International) .
08/08/91 Dr . D . Appleton-- consultation fee (Toxicology)
(International)
0'.8/14!/91 Prof . Dr . Eisenbrand-- consultation .fee
08/16/91 St . James .Ciourt H!otel-- hotel expense, Dr .
Parent (International)
08/16/91 Prof . Dr . Van Biekkum-- consultation fee
(Toxicology) (International) .
08/16/91 Dr . J .J' . Kolk-- consultation fees ('Toxicology),
(International) .
08/19/91 University of Newcastle-Upon-Tyne-- Dr .
Hendrick, consultationifee (Toxicology)
(International) .
08/27/91 Prof . Dr . Strubelt-- consultation fee
(Toxicology) (International)l
08/27/91 Dr . R . Combes-- consultation'fee (International),
693 .07
2188 .622
2011 .016
7483 .516
1097' .04
78'9 .02
907 .20
1671 .20
6026 .61
1617 .01
1335 .19
24'5'7 .00
2284 .48
871 .29 .
Total Expenses Share
$'196,908 .78
SUBTOTAL FOR MATTER SLEP .24065
$'1916,90'8 .78
http://legacy.library.ucsf.edu/tid/geq87e00/pdf
3 Ys
Continued Next Page
2015002'9G0
Confidential
Attorney Work Product
Attorney-Client Privileg e
Expert / Consultant Submissions Regardin g

ETS to Regulatory Agencies on Behalf of Philip Morri s
Working Draft
August 11, 1998
(1190809.01
http://legacy.library.ucsf.edu/tid/vbh77a00/pdf
t
Confidential
Attorney Work Produc t
Attorney-Client Privileg e
Holcomb, Larry (HES)(3-1065 )
Layard, Maxwell W. (Layard Assoc .)(3-1067)
Leber, A. Philip (Chem-Tox Consultanting)(3-1085)
Lee. Peter (PNL Stats . & Comp . Ltd.)(3-932, 3-1195)
Leslie, George B. (BIOASSAY)(3-1194)
Levy, Leonard S . (Univ. Birmingham)(3-967)
Lewis, Trent R. 0(3-537)
Moore, Richard E . (AAL)(3-1085)

Reasor, Mark J . (WVU-HSC)(3-1071)
Will. James A.
Robertson, Gray (HBI)(L3-1182)
Roe. Francis J.C. 0(3-515)
Switzer, Paul (SU)(3-1066)
The Tobacco Institute (3-1086, L3-1188)
Witorsch, Philip (GWU)(3-1045)
Wu. Joseph M . (NYMC)(3-1080)

Tab B : OSHA Notice of Proposed Rulemaking - Public (NPR)(04/15/94)
Ashford, John .R(EHIS)(9-26467)(06/00/94)
Barnes. P.J. (9-105472)
Benda, George (9-47596)
Bridges, J .W. (RIIEHS)(9-40331 )
Caldwell, John (St . Mary's Hosp .)(9-101053, 9-8672, 9-59160, 9-47799)
Chanter, Dennis 0. (Bookwood Stats., Ltd.)(9-26010, 9-26026)
Chappell, Willard R. (UC-Denver)(9-27972 )
Cline, Martin J . (LICR)(9-62029, 9-27687)
Constangy, Brooks & Smith (9-2202, 9-7175, 9-24993, 9-91211, 9-22884,
9-100505, 9-84161 . 9-85545, 9-69816 )
Smith, David L.
Tyson, Patrick R.
Wasser. Neil H.
Devine, Thomas (RMT)(9-47508)
Robinson, H . Coleman
Skipper, Steven
deWolff Frederik (Univ. Amsterdam)(9-105467)

Fletcher, George (Fletcher Group)(9-27972)
Gratt, Lawrence B . (IWG Corp.)(9-27972)
Greenfield, Stanley (SAI)(9-1612 ; 9-27989)
0190809.01
http://legacy.library.ucsf.edu/tid/vbh77a00/pdf
PHILIP MORRIS
INTERNATIONAL CONSULTANTS
"EUROQUOTES"
http://legacy.library.ucsf.edu/tid/kbc90c00/pdf
TABLE OF CONTENTS
Tab
As hf ord
. 1
Barnes
. 2
Bridges
. 3
. 4
. 5
: 6
Caldwell
Chanter
*Cline
. 7
de Wolff
*Idle
. 8
James
. 9
*Lee
. 10
. 11
Litchfield
Mannaioni
. 12
Nilsson
. 13
Palacek
. 14
. 15
. 16
. 17
. 18
Roberf roid
Samanek
* Springa 1l
Strubelt
* 16 Comments that are particularly useful examples .
PROFESSOR PETER J . BARNES

SELECTED BIOGRAPHICAL INFORMATION
Professor of Thoracic Medicine, University of London
Chairman,
MA, DM, DSC, Fellow of the Royal College of Physicians
Department
of
Thoracic
Medicine
Consultant Physician, Royal Brompton Hospital,
11012906
and
Honorary
London
PROFESSOR PETER J . BARNES

SELECTED QUOTATIONS
Pulmonary Diseases and Conditions :

"I have several concerns about the interpretation of the
evidence quoted in this document . While there is naturally
concern about the effects of ETS in the workplace, many of the
studies purporting to show adverse effects on airway function
could be interpreted differently and there are several
relevant studies that have not been referred to . The OSAA
report reviews an extensive literature on the effects [of] ETS
on airway function and has reached a conclusion that appears
to extend beyond the available data ." (Written submission, p .
1)
"In the review by Tredaniel et al, 6 studies which show no
effect of ETS exposure on lung function are discussed, whereas
the OSHA document refers to only two of these studies ;
although of the positive studies cited in this review, 7/8 are
referred to in the OSHA document . This appears to indicate a
biased selection of references ." (Written submission, p . 1)
"There is no convincing evidence for a dose-response
relationship between the amount of [ETS] exposure and the risk
of airway disease and there is little quantification of the
amount of exposure in the workplace ." (Written submission, p .
2)
"There is certainly a pressing need for large longitudinal
studies to address these issues and accurately quantify any
risk of ETS in the workplace . Until such evidence is
available, it may be premature to conclude that exposure to
ETS in the workplace is significantly increasing the risks of
airway disease ." (Written submission, p . 2)
11012806
MINUTES
SRG Meeting
October 19`h-22" d 1998
Lainston House, Sparsholt, Winchester, UK
Attendees :
Richard Baker (Chairman)
Scott Appleton
Eva Sshumacher-Wittkopf
Stewart Massey
Adrian Payne
Graham Read
Derek Irwin
Anna-Lisa Fisher (Secretary)

Mike Dixon
Eian Massey
David O'Reilly
Graham Smith
Christopher Proctor
Antonio Augusto Rodrigues
Welcome and Introduction - RB welcomed all members . The objectives of this

meeting were :
To assess the progress on projects currently funded by the SRG and consider new
areas for funding.
To review the scientific content of the new position papers
To be updated on topics relevant to smoking and health issues
To hear the views of external experts in smoking and health areas in the form of a
mini-symposium
Matters Arising
In response to RB's communication, Erwin Kausch, had indicated that it would be
appropriate for updates of relevant projects funded by the Verband der
Cigarettenindustrie to be presented at future SRG meetings .
(Action ES-W)
The previous work performed by BAT on the effect of humectants on lung

retention has been re-analysed . Further work is likely to be undertaken in
Southampton and/or B&W .
(Action MD/SA)
The TMA funded Covance study on nicotine retention is due to next month . The
study is expected to take approximately two months . If the study,is completed, the
results will be presented at the next SRG meeting .
(Action MD)
Following publication of a paper on ammonia/nicotine chemistry by Pankow et al,

1997, John Lauterbach (B&W) has written a chemical critique of this paper . The
SRG recommends that this critique should be submitted for publication in an
appropriate journal .
(Action SA)
BAT Position Papers on Smoking and Health

The currently available position papers are :
Smoking and lung cancer,
321494969
Smoking and respiratory disease,

Smoking and coronary heart disease,
British American Tobacco's research and the "safer" cigarette,
Cigarette tobacco ingredients,
Smoking and "addiction",
Environmental Tobacco Smoke .
These have been updated and distributed world-wide .
Three proposed new position papers have been considered :
Smoking and reproduction (in draft form)
Mortality statistics
Smoke constituents
AP reviewed the new Reproduction Position Paper . There was general agreement with
the views expressed by Professor Jean Golding (see Mini-Symposium) . AP will check
references and send the final version to the SRG members .
(Action AP)
A request for a "Smoke Constituents" position paper from the Board has not been
minuted . This position paper will not be progressed until a firm mandate is given .
Current SRG Projects - AF presented updates of some of the projects currently
funded by the S RG. DO reviewed the project on the putative tumour suppressor gene,
DUTTI, by Sundaresan . This was considered an important area of science . Success in
this project will be defined by determining whether DUTTI has a tumour suppressor
role, particularly in lung cancer . Sundaresan has also requested extra funds (45k) to
purchase some specialised equipment (a FISH workstation) . It was agreed that the
SRG would fund this equipment but Sundaresan would need to advise the SRG on
how he wished the equipment purchase to proceed .
(Action AF/DO'R)
Due to unforeseen difficulties, the nicotine protection project by Gray was unable to
continue . It was decided that funding for this project would no longer be provided or
redeployed to Gray's second project on nicotine modulation of pre-pulse inhibition . It
was agreed that the nicotine modulation project would continue to its natural
conclusion . In view of Gray's imminent retirement, it was agreed that his future role
as a consultant to the SRG would be explored .
(Action AF)
Tovee made a request for extra consumables (4k) . MD will assess the protocol and
justification for these consumables .
(Action MD)
It was decided that AF should send out the project briefs and full detailed project
updates in advance of future SRG meetings .
(Action AF)
Ingredient Toxicity Testing
SA described B&W's intention to set up facilities to perform biological and toxicity
testing on all cigarette ingredients not otherwise covered by some other body . The
SRG recommend that there should be a co-ordinated approach across BAT .
[Following further discussion at the Smoke Science Team meeting on 22/23 October
1998, it was subsequently agreed that SA and GS would develop appropriate
protocols for testing additives] .
(Action SAGS)
2
321494970
aQ1lcg
It was recommended that SA would make a presentation of the proposed protocols to

the TSC/REC meeting in Macon in November 1998 .
(Action SA)
SRG Budget
See attachment
SRG Conference Funding
See budget document attached
SRG Issues
SM gave an overview of Federal Regulations in British Columbia regarding toxic
constituents and described his communication to ????? of information resulting from
the monitoring of a reduced toxicity cigarette .
AP reviewed the current perspectives in COPD and Asthma . AP had visited two
experts in these fields, Professors Peter Barnes and Clive Page . PB works at the
National Heart and Lung Institute and is the leading world expert on asthma who
would be willing to consult for the SRG and may submit a proposal to investigate "the
genetic influences on susceptibility to COPD" . CPg, at Kings College, London, is a
leading world expert on asthma . As well as being willing to provide consultation
services to the SRG, CPg may also submit a research . These proposals (if submitted)
will be assessed at the next SRG meeting .
Project Proposals
PJ presented his project proposals :
1.
"Developmental and Respiratory function follow-up of infants from
antenatal study" by Paul Johnson . It was agreed that this project was of
relevance to the SRG portfolio and of sufficiently high quality to fund . AF
will inform Johnson and make the necessary arrangements to commence
funding.
(Action AF)
"An Interventionist Study of nutrition supplementation during pregnancy
to see whether foetal and postnatal development can be altered in smokers"
by Paul Johnson . Due to several problems associated with this project
highlighted by an expert in this area who reviewed the project, it was
decided that this project could not be funded in its present form. A F to
inform Johnson of the SRG's decision .
(Action AF)
"Ni
cotininc-Muscarinic
interaction
in
the
modulation
of
dopamine
release
in
2.
.
This
project
has
been
favourably
peer
rat striatal preparations" by Sue Wonnacott
reviewed . Since this project was not discussed at the SRG meeting, a decision will be
made in the next two weeks by contacting SRG members by lotus notes/fax/telephone
as to whether this project will be funded by the SRG .
(Action AF)
"Intra-Uterine effects on adult non-insulin dependent diabetes in South India ;
3.
a risk factor for coronary hear disease" by Caroline Fall and David Barker .
Unfortunately this project was submitted too late for SRG review . It will be
considered at the next meeting .
(Action AF)
3
32149497 1
To :
Graham Read/Southampton/GB/BATCo@BAT, Graham R .

Smith/Southampton/GB/BATCo@BAT, David
O'ReillyfSouthampton/GB/BATCo@BAT, Eian
Massey/SouthamptonlGB/BATCo@BAT, Mike
Dixon/SouthamptonlGB/BATCo@BAT, Scott Appleton/Macon/US/BAT@BAT,
Stewart Massey/ITL@ITL @ BATCOEXTERNAL, Eva
Schumacher-Wittkopf/Bayreuth/DE/BAT@BAT, Leopoldo Caruso/Complexo
Amorim/BR/SouzaCruz@BAT, Christopher Proctor/Staines/GB/BATCo@BAT,
Derek Irwin/Southampton/GB/BATCo@BAT, Richard
Baker/Southampton/GB/BATCo@BAT, Antonio Augusto Rodrigues/Complexo
Amorim/BR/SouzaCruz@BAT
Adrian Payne/Staines/GB/BATCo@BAT, Linda Rudge/Pagewood/AU/BAT@ BAT
Anna-Lisa Fisher/SouthamptonlGB/BATCo
18/11 /98 16 :48 :02
Molecular Genetics of COPD
cc :
From :
Date :
Subject :
Dear All,
Adrian has received this review of the Molecular Genetics of COPD by Professor Peter Barnes
(that is now in press (Thorax), on the " Molecular Genetics of COPD" and will probably be
published in Feb/March 1999) and suggested that I forward it FYI
Copdgefn .doc
This review should give you all an overview of what is known about this area, associated
factors and what/why further research may be important .
Professor Barnes is considering submitting a proposal to the SRG next year on genetic markers
of susceptibility to developing COPD .
Regards
. . .Anna-Lisa
322017516
http://legacy.library.ucsf.edu/tid/pfs60a99/pdf
24 September 199 9
Department of Thoracic Medicine
National Heart and Lung Institute
Dovehouse Street
LONDON SW3 6LY
Dear Professor Barnes

As a recognised expert in this field, we would like to invite you to write a
review on mechanisms of chronic obstructive lung disease (COPD) with
special reference to smoking. The review will be your property to publish
if you wish, and it is not a requirement on our part to acknowledge the
source of funding (although some journals may of course require this) . For
our purposes, this review would be a reference document for British
American Tobacco scientists in the context of current awareness of
scientific opinion in smoking and health research, as part of our duty of
care responsibility .
Although we do not want to influence or set specific guidelines for the
content of the review, issues that we are particularly interested in include :
Concise Review of the Most Recent Literature and the impact on Current
Scientific Opinion
Current views on important Risk Factors and Possible Disease
Mechanisms
Emerging Trends in the Understanding of Possible Genetic Influences
Gaps in what is known about Disease Mechanisms and Suggested
Research that could address thes e
We will, of course, provide a mutally agreed appropriate sum to
compensate you for the time spent on this project and any reasonable
expenses that you incur . There is no particular deadline for this review to
be finished, but a guideline would be about six months .
I would be grateful if you could contact me to confirm that this
proposition in acceptable to you in principle and, if so, to discuss the
length of the review and remuneration .
32526181 5
&11c'. US DO', Philip Moms
My contact details are :
direct line 01703 79330 9

email anna-lisa-fisher@britamtob .com
Best regards
...Anna-Lisa Fisher
32526181 6
PATfl, I11 DO,] v Philir Mnmc
Professor Peter J Barnes MA, DM, DSc, FRCP

Professor and Head of Thoracic Medicin e
Honorary Consultant Physician, Royal Brampton Hospital
Direct Lime: 01713518174 Fax: 01713515675
Email : p j .barnes@iaac.u k
Imperial College School of Medicine
National Heart & Lung Institute
Dovehouse Street, London SW3 SL Y
Imperial Colleg e
6 October 1999
OF SCIENCE, TECHNOLOGY AND MEDICIN E
Anna-Lisa Fisher
R&D Centre
Regents Park Road
Millbrook
Southampton SO15 8TL
Dear Ms Fisher
Thank you for asking me to write a report on mechanisms of COPD with special reference to
smoking. I will try to let you have this within the next six months . I briefly discussed this
with Adrian Payne who suggested that an article of approximately 20 pages with references
would be ideal .
Best wishes.
Yours sincerely
e c"-,
Peter J Barnes MA DM DSc FRCP

Professor of Thoracic Medicine
t--bLo'L
Prvj . Q c~.~r-c S
t'l6se .-e .
http://legacy.library.ucsf.edu/tid/bzk23a99/pdf
32526190 6
FLAT.-' U% 0(U v Philip -
Professor Peter J Barnes MA, DM, DSc, FRCP

Professor and Head of Thoracic Medicin e
Honorary Consultant Physician, Royal Brompton Hospital
Direct Line : 01713518174 Fax: 0171 351 5675
Email : p .j .barnes@ic.ac.uk
National Heart & Lung Institute
Dovehouse Street, London SW3 6LY
22 December 1999
Imperial Colleg e
OF SCIENCE, TECHNOLOGY AND MEDICIN E
Dr E 0 Gregg
Scientific and Regulatory Studies Manager
British American Tobacc o
R & D Centre
Regents Park Road
Millbroo k
Southampton SO15 8TL
Dear Euan
Re : Application of Dr R P Youn g
I apologise for the delay in sending you my report . This is an ambitious project that is
important and Dr Young has expertise in molecular genetics . This is an area where several
other larger labs are already actively involved . I am surprised about the concern about
confidentiality since these precise studies are already underway in at least two locations and
these are very obvious studies to do !
I think that the project is worthy of funding .
Best wishes and happy New Year .
Yours sincerely
Peter J Barnes MA DM DSc FRCP

Professor of Thoracic Medicin e
Enc.
http://legacy.library.ucsf.edu/tid/znu71a99/pdf
32527993 6
BATCo US DOJ v Philip Myna
Grant Application of Dr R P Youn g

The genetic predisposition to COPD and emphysema is an important area of research
which is currently attracting a lot of interest . Only a small proportion (approx. 15%)
of smokers develop COPD, suggesting that genetic factors are important . Since
several studies have shown that certain matrix metalloproteinases and neutrophil
elastase are released in increased amounts in patients with emphysema, it is possible
that gene polymorphisms of MMPs and their tissue inhibitors (MMP) may play a role
in predisposing to this disease . The approach taken by Dr Young is therefore logical
(although several other groups are already looking at this same area). Most gene
polymorphisms in common diseases are in the promoter region and affect
transcriptional control of gene expression, so the emphasis on promoter
polymorphisms is sensible . However variations in sites on the promoter may also
affect transcription by changing the conformation of DNA .
The patient selection is sensible and it is clearly important to closely match smokin g
history in emphysema and normal subjects .

Dr Young proposes to use heteroduplex analysis, but most investigators believe that
SSCP may detect more polymorphisms.
It will be important to establish that any polymorphisms detected more frequently in
emphysema are of functional importance, using a gene expression system .
Preliminary data to show the existence of polymorphisms in the MMP/MMP

promoters would be useful before embarking on this project .
Dr Young has a background of research in molecular genetics and is familiar with the
methodology .
The funding requested is reasonable for the proposed work.
http://legacy.library.ucsf.edu/tid/znu71a99/pdf
325279937
&4TC, US COJ v Philip Mane
O. B. Cohan
cfe:- c7^t' '*-%*1 %* IVKUai.l *\st K \ ( l k m ) \ ) U | P i t i
* I * * * - . " * " f**U* l*r ti*a** raSnni- f f^jim-*.} i*M9>%ra>lz*mt*S<al lAaV.nwi**
Characterization of Beta Adrenoceptor Subtypes in Canine

Airway Smooth Muscle by Radioligand Binding and Physiological
Responses1
PETER J. BARNES/ JAY A. NADEL. BENGT-ERIC SKOOGH/ and JAMES M. ROBERTS'
Cardiovascular ResearchtaitiMnand Departments of Matiicmo and Physiology. University of CaMorma. Sail Francisco. CaUtomm
Accepted for puWcaton FaDrusry 22.1983
ABSTRACT
Bata adrenoceptor subtypes m canine tracheal smooth muscle
have been investigated by radioligand binding and by physiological responses to beta agonists and sympathetic nerve stimulation in vim. Specie binding of | ffjdihydroaiprenotol to tracheal
smooth muscle membranes was of high affinity (K * 1.0 0.08
nM). as in peripheral lung membranes from the same animals.
but the concentratnn of binding sites (95.0 4.7 fmol/mg of
protein) was much lower than m lung (532 48 imoymg of
protein). Binding was stereoselective and agonists competed
with the rank order of potency isoproterenol > epinephrine >
norepinephrine, signifying a preponderance of beta-2 receptors.
Airway* an? relaxed both in vitnt and in avn hy beta adrenergic agunit*. indicating the presence of beta adrenoceptor!*
tin airway smooth muscle. Lands el at. (1967) originally proposed Ihm beta adrenoceptors could be aubdivided into two
classes based on the varying patencies of adrenergic agonists
in different organs. Heto-l receptors present in adipose tissue
mediated lipolysis. and in the heart mediated chr inotropic and
isniropic response*, whereas brtw2 receptor* medhted relaxation nl smooth mtiM-le in airway*, blood vessels and uterus.
Thix organ-specific subclaiwiljcation wa* challenged by later
work using selective beta adrenoceptor antagonist A which
shiwed a mixed frrta-1 and frrfa-2 receptor response i n isolated
heart preparations iCarlsson ft at.. 1972: Ablad et at., IB731.
Similar mixed responses were also found in airway smooth
muscle of dog iMoissier 7 a/.. 1971), guinea pig iFurrhgott vt
H n ^ l " r | > u M H o l i . m l ^ - n J - f .'V |SJU
*Tlii * ! * * MipiH-ntil in pun In NslH>nl I f t M i l i i W n f H r s l l h I ' m i r i m
1'itwni l.Mtii H l . - i t l m and rntann
M i l I rum i b r Ceonnl lr Toliirro
ftrwnb
* 1 ' i o r n i W - M I l r I V I M - I BrB. l>piimfni.4MfdK>>.Hini7wrHiih
H n - m l n l D u c a l * H<wl. | j * H - i U I . ' I K K K V H M I I il M r d i m l H o r M r h
Citonul l t.ftv* I I H L H I I f ratrHmr l > * " * h i H
' N J | I | ' M M I h i m u m Irrnn ihr SrtliO> N m r i r I A K I * > H I H A K I I I H M H
flnrtfhM
t>murr*Jinrtlnain/in1tnwn('i)i<l1uirfflmjlii.ili*ui|j>in\ iSummtt.
VI i
*Hrc>|wni l Satmnul lriiiu<r. til Mrslik K r v i K h l r i r l Ih-trltpntrni
Using selective beta antagonists, w a detennined the ratio of

bttO'i/bBta-2 receptors in tracheal smooth muscle membranes
to be 1-4. The relaxation response of tracheal smooth rnuteie
stnps to exogenous tats agomsts was mediated by Oata-2
receptors, with a very small coninbutton from befe-1 receptors.
However, the relaxation response to electrical field stimulation
of sympathetic nerves was mediated predomtnantty by Dett-1
receptors- Our results suggest that most Data receptors * i dog
tracheal smooth muscle are of the oera-2 subtype and meoiate
responses to circulating catecholamines, but there w a small
concentration of oafa-l receptors which mediate the response
to neuraiy released norepinephrine.
of.. 1975: Omini ft a/.. 1979: lakovidiH vt <if. 1980: Jnhnnamm

and WaldccJt. 1981) and cat (LulR-h rt at., una}. suggesting the
presence of/n'.o-l receptors in addition to fx'tn-2 rereptiirs in
airway smoot h muscle.
Direct binding studies using labeled beta adrenoceptor antagonists! hove confirmed the coexistence of brto-l *n thta-'J
receptors in homogenotc* of king in se.'crat specirs iKujig rl
o/.. 1978: Minnemnn el al., 197Jln; Kngrl. 19H1I, and using
selective beta agonists it has been possible to determine the
ratio of beta-Xfbeia-'l receptors. Hut lung contains oer -ti>
different cell types and we have recently shmvn that ihi> \m\
majnriiy of beta receptor* in lung are asuodated with alvenlar
walls rather than with nirway smooth muscle (Haines r nl..
1932). As the lung has a heterogeneous cell population, it is not
known whether some cell types of erfo-l rerepinrs. whereas
other* have oc/a-2 receptiHs. it in not pnsslhlr to study' intrapulmonary airway smooth muscle by direct binding assav. as
insufficient tissue would be available to prepare membrAm*
hamogenatr.H. We have therefore invextigated trm-henl smooth
muscle of the dag /is thin can be disserted free or surrounding
tiSHues and have used ('HJDHA to study ihr charnrtprimiiv f
beta receptnrs in hnmngenates of thin tisnue. For ciimpnrixiui.
we a1*> stiidiitl m ri'fn* beta adrcnergir resjionses in the wiimtiHsup lining Imth exogenoun beta agunism nnd etn-tricai stunulmion t f vvrnpnlhrtir nerves.
AMHEVIS-TION: DHO o.nyofoaOfenoiol
4M
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then contracted with serotonin tOilaMl ami heft the contravtinn WHM
stable t.lD rain!, the retasation response to eectrical field stumiUuvn
Do tracbeatis t w h w i i f p n f i n i l n . We obtaiwd tracheae was determmed. This response was inhibited by I iiM propranolol. The
tiom Ayp- which were being used in other etpenrarm* which did net effect of seleciivr brio adrenergic agonists on the response tr> tlehl
imvhe (he useol drug* other than anesthetic*. Dogs were anesthf tired stimulation was determined by prior mcubatiun 130 nun) with either
nuh pewuhartnwl sodium t.10rng/kg t.v.t.and the trachea was rapidly prxctolul t:lp.M) or IPS :i&) 10.1 uMl.
Data analyale. Result* are espresied as mtin * S . Statistk-al
removed. The posipnor membrane portion, of ihe trachea containing
the trachealw muscle was dissected free of loou connective tissue and comparisons were made by unpaired Student** l test. Binding data
the epiihelium as stopped awry. The tracheal*! muscle was the* were analyzed by a nonlinear least.squire* cutve fitting computer
separated and finely mmred with *ct*or* in 10 vulume* of ice-cold program. For competition studies parameters were chosen to yield the
incubation buffer i50 mM Tri* HO. iH .!!. thru homogenized in a best fit ol data, as determined by the mmunal variance of experimental
Polytron tissue homogenizer (Bnhkmann Instruments. Inc, U'estbury. data about a curve generated by these parameters iMurlasrtaf.. I9B2J.
NVi at setting d tor A x IX tec period*. The resulting homogenate wa* Parameters Tor one and two affinity site interactions were tested. The
filtered through two layer* of cheesecloth 10 remove unbroken cell* and inhibitory dissociation constant l Ki I was determined from the tela! iunconnective tissue. The supernatant was centrifuged at aojOOD x for ship15 min aad the pellet worked and rccentrifuged in buffer at a concenIC
tration of OA to l.o ma of protein* per ml. Thi* particulate preparation
K.
I + |L]/K
wax either used directly in ihe binding away or stored at TOT for up
to .1 month* without change in binding characteristics. Protein wa* where U\. is the concentration of drug causing -Wi inhdiition of
determined by the method of Bradford 119781. using buviiir serum spenfir i'HIDHA bmdinK. IM is the concentration of I'HIDHA used
alrmmtn as the standard. Particulate* were aim prepired from periph- and Kit the dissociation constant of |'H|DHA determined in equiliberal lunjj of the u m r animals after dissecting sway majur atrwoy* and rium binding assay*. Saturation isotherm* were analyted arcoriiing l o
blond vessels, using the seme procedure.
a one siteor two site interaction and the best tit determined by triimmal
I'HIDHA binding assay. Membrane* lapproximately IdO vie of variance. The estimated dissociation constant I KM I for antagonist* tn
protein per aay> were incubated with | 'H]DHA in a final volume of the physiological studies wa* determined from the relationship:
025 ml. Nonspecific binding wait determined by inrubntion* in the
pretence or I *M /-propranolol. Each data point wis determined in
KM<
duplicate, and duplicate* did not vary by >ltici. Equilibrium incuba1LVL - I)
tiorjs were carried out at 2VC for IS min and termiaated by dilution
with & ml of ice-cold bull* r and rapid filtration through \Vbatman GF/ where | l | is the concentration of amagonsn. L' is the EDv.of agoniat
C filter*. toNowed by two further Aral washes. Fiher* were counted by in the presence of antagonist and L t>e EDv. of agonist alone."
Drugs and chemical*. |'H|DHA <>prcific activHy 101 ri/mmoll
Squid scintillation spectrometry- Specific binding, which was determined from the difference between total and nonspecific count* bound was obtained from New England Nuclear (Boston. MAI. Drug* ware
to the fitter*, comprised 60tnHo', ol total bindingat ligand concentra- obtained from the following sources: f-isoproterenril. /-eiwirphrihr hytion* ol leu* than 2 n\l. For equilibrium binding, concent rat HWK of drochloride, f-nurepmephnnrliydrochloride.atfopinesuluic. serinomn
I'HIDHA varying irom O.l 108 nM were used, and Inr competition and creatinine sulfate. acetylcholinrrhloride.oY.propranolol iSigmn Chemical Co.. iit. Umi*. MO): practolol. d- and f-proprahiilnl lAyerst Labokinetic studie*. a concentration hrtwern I and 2 nM wan u*ed.
J* vitro beta adrenergic responses. For physiological studies the ratories. New York. NYl: IPS.139 hydrochwrid* < AB Hassle, (ioteborx.
excited canine trachea wa* immediately immersed in Krehs-Henseleit Sweden): terbutaline sulfate (Astra Pharmaceutical Pmdurm. Inc..
solution with the billowing composition: NaCI. I ID mM: KCI. $.9 mM: Worcester. MAI: and phentolamine mesylate tCiba-leigy Corp..SumCaCI-. 3.* mM: MgSO.. 1.3 mM; NaH.PO.. 1.2 mM: NaHCO.. 2W mit. NJ). All drugs were made up freshly in distilled water immedf stely
mM: and glucose. 3.6 mM. which was gassed with 9l* O, and tf CO,. before use. Catecholammes ware made up in O.l mM ascorbic acid.
Alter separation ol the epithelium the iracheatit muscle was cut transvcrsel.v into strips - t o 3 mm wide which were mounted vertically in
Results
glass chambers filled with |A ml of Krebs-Henwleit solution, maint ^ i D H A biNdlng anturatlan atudlet. Specific binditiR or
tained at 3i*C and aerated with 94' O, and 6!7 CO,. l*oetric tension
was measured with strain gauges <0rass model FT 0.03 lorre-dlsplace* I'HIDHA t o don trachealiB membranes won saturable and o f
mem transducer) and recorded continuously (Grass model <D poly- high offinity. Saturation iaothemta were beat defined by inter*
graph*. The tissue baths were fitted with platinum elecirndes for action of | *H|DHA with a single populnlion of hindinit otic*
electrical field stimulation using bipharic pulses (supramaximal voltage and Scatchitrd analysis save an equilibrium dinsticiatiiin contt* msec duration. 12 Hr frequency! for 20 sec. Strips were allowed to Mant (Kul of 1.0 0.00 nM <n > 61. which was very simitar t o
eiaiilibrate lor 1 hr and retting tension was adjusted to 10 R. which was that determined in peripheral lung membranes from the same
optimal fur determining changes iti tension. Only strip* that developed
animals (0.98 0.0ft n M , n fil (fix. 1)., T h e maximum
a tension ol titeaier than 10 g to electrics! field stimulation at 12 H*
concentration of binding; sites ( B ^ , I to tracheal smooth muscle
far 20 sec srere wed in the sludv. ,
Muscle >tnri were ranirarted by 5 MM acetylrhutine. taeii. when membranen was 9A.6 4.7 fmol/mg of protein, whiih wan
cent radium, were stable, a cumulative dose-responne was performed tn considerably less than that determined in whole lunu memiMfproterenti) tiMii-lini Ml ut terbutaline il-.l M). using a 2-min branen {R32 4 8 fmol/mg of protein).
expmure tmte to each concentratibn. Theelfecl ol selective orio adre.
K i n e t i c s t u d i e a . Specific bindinx was rapid T , . '.S min),
nergic Wockade on these agonwt dW-Ksponse turn* was determined reachinx equilibrium al t n min. and was revemiUIrun aildition
h\ preincubation with erther thefti-o-lselective antagonist prartolnl orfpropranolol i T , u 3Jt mm). The kinetic Ki. calralatrd from
in sMl r the orfa-2 selrrthe amagoniM IPS X 0.1 M. These the ratKi o f the reverse rale curjHtnnt tn the forward rate
conrentratwn* ol antagani*! were chosen on the basis of cimpelitiDn constant wait calculated as 0.7(1 0.1.1 nM (n .*!). which was
studVk nh I "HIDHA hindmg. so that seleciivitv wiHild be retained.
in ttood ngreement with the Ki. deirrminrrl tn equilibrium
The ellert il selective Mit adrenergic antsgitaists on the beta adreneriiir telataiiim renim>e to electrical field Mimutoliim was also de- Murlir*.
C o m p e l l t i o n aludlea. Sprrinr liindinK t o f rm-hrnlis memtermined. The cholim-rgn re|HinewHlicl(edb\ 1 tM atrtipine. and
iheaîhaaJreaergn riKmMh> lt)M pheniiilsmme The muwle wa branes was stereoselective, with / pmpMnoNI opiwoHiinntely
Methods
J!L
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Basnet at at.
W.225
8max - ' ' 8 fmoi/ina crorsm
R 9 . I . BKXJmsoM'HPHAtoacatncfiaaismooinrxrtda
membfarts. UHpanatspaoiCtxtxfngi*) art nonieaolie
bmomg mine presence ot 1 *Mpropranoioi{0)are shown.
Right panti: Seaward antfyas snowing a smgia daw ol
binding s>t* with equ*bhui aseooation constant () or
1.2 nM ami maximum receptor concentration (B*) of
1112 frnoymg ot prottn. Daja bom a nog* espenment
performed m duptcate are snown and art types! ol a
such experiments.
Mg.^. tnhibiHonotspaoli[aH)DHAbindinoioaog
tracheal smooth mueeie mtrnpranes by adrenergic
drugs. Left panel: inhibition by agonists, /-aoprottfnot ( ) . /-epintpnrine (O). Mwrnpncprmna (A) and
taroutaime (A). Right p a w inhibition by /propranolol ()) andtf-propranoioip ) . Each pant 11froma
angle experiment performed m duplicate and *
typical of three) auch expenmeme.
-9
(OB (AGONIST)
-S
CM)
-8
-7
-6
log < ANTAGONIST)
TABLE 1.
DiaaartaHon oonstaats WW ot adrenergic aganta lor mhibNon of
specJflcfolQHAbhxk^ 1 0 0 ^ t r a v e l emcothrnuecte
inai by the aelective brio antagonists IPS 339 and practolol waa
beat described by interactions with' two binding sites (fig. 31.
With IPS 3.19 most specific ' H binding 179.8 &??;. n - o>
waii to a high-affinity binding site which is presumably tht
betO'2 receptor, whereas the remainder of the binding waa to a
Agonats
site of lower affinity which U presumably the btto-\ receptor.
Msoprotarafwt
0.12 0.01
With practolol. the converse pattern waa seen with most spe/Epnephnne
1.2*01
cific J H I D H A binding <78.0 2.4rc*. n . - 4) to a low-affinity
ANwapnepnhna
153*0.3
aite (the 6t/o-2 receptor). As | 'HJDHA binding has equal affinTorbutakne
37.3*209
ity to 6rta*l and frefa-2 rereptora. the ratio of tota-l/bcta-i
Antagonist*
/Propranolol
0.0026x0.0007
aitea using either antagonist waa therefore approximately 1:4.
o^Proptanoioi
019*0.03
ltt vitro response*; 6e/a aajoniatt. Isoproterenol caused a
relaxation of dog trachealis strips, which had been contracted
fmSttl
K
with acetylcholine, with an E D * , of 4.2 0.7 **M (n 8). Thtre
was no evidence for aignificant beta receptor deaensitiiation
79.8*27
0.0013 * 0.0002 0.29 0 04
during the cumulative dose-response to isoproterenol, aa in
IPS 339
0.30*0.11
16.3*2.7
22.0*2.5
Practolol
preliminary atudiea we found that the final cumulative doae
<10u * M 1 caused a similar relaxation in strips not exposed to
ICM
j j * W f M K # f C t t * thaVCOfVCeVttTV
K dHaanmo from tnt equation K ;
progressive increases in concentration. IPS 339 (O.t M> gave
t>anolsesfttceusffiQ9p%iflfttst*Qnof apaonc|*H)DHA twiOAQ fram a computer a marked parallel shift to the right in the isoproterenol dose*
ctsâring program.fc)iB^concentration ol j'H|0MAui<>d the away (1-2
response curve <ED- 78 1.5 p M ) with a K of 4.6 n M which
fiiMl and Ko it Mia oissoothon constantfromoojueonum among {l 0 oM)
for aw sweckve aniagonau K. * the Ossoeeten oj*a hish-aftrtiy ana and waa in good agreement with its K i for the high-affinity site 11.0
K, tftt lewarftnmi ite TM otta shown are meanslt $ t ot tnraa to vo n M ) determined from competition with | "HJDH/*. binding (fig.
asperats xperanantE.
4). Practolol (3 p M ) produced only a small hift in the doseMO timet more potent than 'propranolol (fig. 2: tabte 11. response curve to isoproterenol ( E D . , 9.7 2.S iM) with a KM
Among agonists the rank order of potency wae /-isoproterenol of 2.2 *iM which la intermediate between the high- and low.
> /-epinephrine > /-norepinephrine, indicating that t h t major- affinity binding sites determined by competition with J 'HIDHA
ity of beia receptor* were of the bcta-'2 subtype. The beta-2 binding. Thia may suggest interaction with both ftr/o-1 and
wleriheaapniatterrwtalinewaKapproxtrnaieiyequipoteniMiith rxfa-2 recrptors physiologicallv. With trrbutalinetED^ IrV)
rMrepinephrine which 1* similar In the finding of other* using 27 iM) IPS 339 gave a matked inhibition t E D . . :l.VH> lutu
lane tarmhranes which have predominantly brfa-2 receptor* nM) with a K H of 4 ^ n M . which was similar to that found with
isoproterenol. Practolol, however, had almoM no elfect <>n the
(Minntman el ol.. 19T91i. Inhibition ot specific ( HJDHA bind-
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Kg. 3. Wvci'jon ot soecr&e I^HJDHA taMing

to 009 tracheal muse*) membranes by setae*
far* bsfa adrenoceptor antagonists. Lett panetinhotMo by (PS 339 (MM-2 selective}. The
data pomia ar Bast wsed by computer-generated curve which, assumes two sites. The
calculsted Association constants for the high
( I U - and low K X l l M y sues and the perceoiagect total sites toreec* are snown. Right
panel: mrsbrtioo by pracioJot (oeta-1 selective}
showng a two-site interaction Each point
thamaanotdupueate'datefnunaiionsinasingle
expenmem and is type*) ot four to fcve sucn
expenmants.
It/"
so.
10
-a
-a
-7
tea tms > oio
-a
toer
.<
Fig. 4. I n vitro relaxation response ot dog tracheal

smooth muscle strips to beta adrenoceptor agonists.
Left panel: response to /-aoprcierenoi a>one (> ana in
the presenceof 3 M practotoi (O) and O.t M IPS 339
<). Right panel: response to tertxitabr alone ( ) and
in the presence ot practotoi K3 and IPS 339 (>. The
response is measured, as percentage ot maximum control response. Each pent is the mean * S.C. ol tnree
to sat separate strips.
-a
-r
-a
-
toe <an*CTOLOU u
100
so
OL
LOB llSOPnOlERENOL) ( M l
terbutaline dose-response (ED-.. 198 2 4 iM) w i t h a !!> of 12

/ i M . suggesting interaction at the low-affinity site which corresponds t o the beia-2 receptor. T a k e n together, these results
suggest t h a t the physiological response t o exogenous 6cfo agonists is mediated predominantly by beta-2 receptors with only
a minor contribution from beta-l receptors.
E l e c t r i c a l f i e l d a t l m a l a t l o n . Electrical field stimulation,
after cholinergic a n d alpha adrenergic blockade, produced a
relaxation o f serotonin-induced contraction which was inhibited by 1 iM propranolol. T h i s response waa significantly
inhibited by both 3 pM practolo! ( P < .001} and by O.I p h i I P S
339 ( P < . 0 1 ) . but t h e inhibition was significantly greater ( P <
.OODwithpractotoI ifig.5h This suggent* that t h e beta receptors
mediating t h e response to endogenously released norepinephrine are predominantly o f the or/a-1 subtype.
DlaeusBjon
Specific binding of I ' H J D H A t o canine tracheal smooth
. muscle membranes had t h e characteristics expected o f interactions w i t h bcla receptors, {finding wan o f high affinity and
similar to t h a t determined in peripheral lung membranes from
t h e same animals and also in hing membranen o f other specie*
fRugg *t at.. 1978; Barnes tt at.. 1979: Barnes c/ of., 1980).
Binding was stereoselective and the r a n k order of potency
among agonists suggested that the beta receptors in tracheal
smooth muscle were predominantly nf t h e beta-2 subtype. T h e
oero-2 selective agonist terbutaline Uad a low potency which
was simitar t o that reported in king membranes (Minnerann rt
LOO (TEflBUtALINE) <M)
at. 1979h>. Competition Tor f TOJDHA binding by the beta-2

selective antagonist I P S 339 revealed two classes o f binding
site. Most, o f t h e binding was to a high-affinity site which in
presumably beta-2 receptor and t h e remainder t o a loweraffinity aite which in presumably the btta-i receptor. T h e
binding affinities correspond well w i t h those determined for
the beta-1 a n d 6rto-2 receptors in cot and guinea-pig heart,
with the same compound rHedberg el aL, 19801. Competition
with the 6cfn-1 selective antagonist practotoi similarly gave a n
inhibitory c u r v e which was best characterized by two binding
sites but With t h e converse pattern, as most o f t h e binding waa
to a tow-affinity site <tbe beta-2 receptorl. As t h e results with
both antagonists are complementary, we estimate that t h e ratio
of beta- \/beta-2 receptors in dog tracheitis smooth musrte is
approximately 1:4.
Our physiological studies on dog trachealia muscle supported
the evidence f r o m the binding studies that bcta-2 receptors a r e
predominant, but in addition showed ttuW 6 e / a - l receptors
contribute t o t h e relaxation response, particularly when i n duced by sympathetic nerve stimulation. I P S 3 3 9 had potent
inhibitory effect on isoproterenol- a n d terbutaline-induced relaxation, w i t h a n apparent dissociation constant ( K M ) which is
in good agreement w i t h the K determined i n the binding
studies for t h e beta-2 receptor aite. B y contrast, practotoi had
atmnst no effect on terbutaline-indured relaxation, w i t h a K
which was similar to the K i . of the low-affinity bcto-2 receptor
site. Practolul was more potent against the isoproterenol responnf with a K * which waa intermediate between its high- and
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OanwaataL
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consistent with the sparse distribution of >ympeehw nerve*

in this sjiecies (Suzuki ft at.. 1976). In human irarhr.il .-mouth
muscle, which is only sparsely innervated by cympuihwic
nerveB. we would predict that tnta-I receptorsrnuzhtplay only
a
a minor role, but thin has nut yet been examined. The den*it\
of sympathetic innervation decreases in more prripnerdl miways, suggesting that beta-l receptor-mediated effects may nl*<>
be less in smaller airways. In cm peripheral lung strips, the
relaxation response to beta agonists is predominantly mediated
by bcta-Z receptors (presumably in smooth rauwle a*? small
airways), whereas in tracheal smooth muscle. brto-\ receptor*
are predominant (Lulichrr at.. 19761.
The relevance of tVfo-1 receptors in airway smooth muscle
in human asthma is not certain. In asthmaticsubjerts, elective
beta-] adrenoceptor antagonists, such as practoioi and atenolol,
CONTROL
P S 3 3 9 PRACTOLOL
may cause bronchoconstnetion. although this is lex* likely than
FJa. 5. In vitro reubtation ratporae of dog tracheal smooth muscle strips with nonselective antagonist* such as propranolol iThirinper
to electrical held stinnuiatian. Muscle strips were- pretreated with atropne andSvedmyr. 1976:firihhin*/ at.. 19791. This brunt hucn>t rii'(1 MM). phentolanune (10 M) and serotonin tO.3 *M) Electrical Held tor response to 6rta-l selective antagonists may indicate the
atamnatwn was a t i 2 H i for 20 sac. Both IPS 339(0.1 pM) and practoioi presence of tVra-l receptors in human, airway*, but is more
(3 pM) caused a significant reduction m response compared to control
strips fP < .01 and P < .001. resoactwety). Theseconcantranont of IPS likely to reflect the lack oi"selectivity of these drugs in clinically
339 and practoioi would cause approximately 8 0 * innibttion of bera-2 used doses. Thefccia-1selective agonist prenalterol is reported
and fteie-l receptors, respectively, which mhibrtog the other receptor to have no bronchodilating effect in asthmatic subject* at rest,
subtype by t%. according to m vitro Binding results. Practoioi caused which argues against an important role tor btia-l receptor* in
wgnteantty greater reduction <P < .001) than IPS 339. Means a S.E. human airways (Lofdahl and Svedmyr. 19821.
of six separate stops are shown.
We have demonstrated by direct binding assay that beta
low-affinity binding. This suggests that, although the relaxa- receptors of dog airway smooth muscle are predominantly of
' tion response to isoproterenol is mediated predominantly by the beta-2 subtype, and mediate relaxation in response to
beta-i receptor*, a small component of the response may be exogenous agonist sand presumably circulating cat echolnniine*.
due to activation of Arte-1 receptors.
In addition, there is a small population lapproximaielv 2uN
Field stimulation using the electrical parameters described of beta-l receptors which appear to mediate the relaxation
causes the release cf neurotransmitters from nerve terminals response to sympathetic nerve stimulation. It ii> proposed that
within the smooth muscle atrip (Russell. 1978). After blockade the proportion of orlo-1 receptors may lie directly relmed to
of cholinergic and alpha adrenergic receptors, electrical stimu- the density of sympathetic innervation oi airway smooth muslation produced a relaxation response which was blocked by cle. It is possible that tVra-l and bcta-'i receptors may be
propranolol, suggesting that it was due to activation of beta differentially regulated and there is some evidence to suggest
receptors by neurally released norepinephrine. Practoioi, in the thatftefn-1receptors in guinea-pig tracheal may be more resistsame concentration that had little effect on exogenous beta anttodesensitizatkmthanbpro-S receptors (Omini era/.. 1979).
agonists, caused significantly more inhibition of the relaxation Acknowtoitsmenu
reaponseiofield stimulation than IPS 339. This is an indication
W> Ikaiik Beth C M and Pailv Snfll h't Ifcrir * U I M C * in ptfpanne ihw
that tVra-l receptors predominate in the beta adrenergic re- mamnenpt.
sponse to sympathetic nerve stimulation, in contrast to the Rafarrarrt
beta-'Z receptor predominance in responsa to exogenous beta AtLMi. B.. C A S U S O N . E. AMI Ks. L.: f*ttaNnarl<wiral Mudio l t nrw
agonists. Inhibition or presynaptic bela-2 adrenoceptors could
rstduvrirrlivr adtenenir tw-m*|rto amajBiiu^n. Ltlr Sri 12: t* 114.
ISTS.
theoretically reduce norepinephrine released by nerve stimuARIENS. E- 4.; Thr ctaMifiratmn oJ hrta-adremnttlnr. Titnd- Phimvtwl Sr
lation. but under the conditions of the experiment, this effect
2i I71M7.1. ISJII.
is likely to be minimal (Langer. 1980).
BaMtt*. P. J BASSAI-M. r . B-. KAIIEL. J . A. A M I RiWKHr*. J M_ l...!tinn
I tola adrr ntxtptor* in mammalian tuna >> iMthi M nwific atHoMcliuaraOur findings support the suggestion that beta-l receptors,
hy. Natar* ItuniLl S9: 444- 44:. I9H2.
which Have a high sensitivity to norepinephrine, are related to B M N E S . C . J.. KAMUMRM.J.X. AMilHMXiJtV.r. T.: Human lunxai!mii*-r|Hr
nuaVrd
hv radiuliaand ramltaic tint. Sri SH: *X- 4*1. l:"i
sympathetic innervation, whereas 6cto-2 receptors are unre- BothES.P.KULIDI.H..I.
HAMILTON.C A . AM>Dalll.MV.' llmiimî.mm
lated to innervation and respond t o circulating epinephrine
er alphacMrrmiiTptiira in auawa piie tunic tinny lUbtmicm. Lttr >ii 35>
120T.IJ14.1m
(Ariens, I9SI I. As the pattern of sympathetic innervation varies
I F AMVIAHI>.I1.M*I->.Mihr
considerably among species, it might he predicted that the ratio Bnaiaca.J.R.AmrMKH.r..i;umrci.i.t,
naurrtlbfuM-hulhtia-adrtawYBiiii'. Kur .1 rhnrmn>l IS; IB) li< l*>"l
of brta'l/beta'2 receptors show a similar variation. Some evi- fiauirtiMi. M M : A rapid sail wimivt nirl hm? lr iMaamiini wi-J at* tnttxm
quii)l>lt.il pnHn Aflat.ttnrhrn 7 t : J l * J ^ . IHTH
dence in support ril this hypothesis in provided by the (lading
K.AHLAK, B , BHtMH>TMOH. A. AMlt*MttlV B ' !M!rr, tlL.tlrH
that in both guinra pig and cat. which have a dense adrenergic CASLSkOH.
MurkaaV <>1 Ike cSmni>1fii*K HlrrK ! %mtiu dibrnrraK Mimoli in ilw <<i
nerve supply to tracheal smooth muscle (Richardson. 1979).
httn LH* Sri. 11:9VI.9M1 IS7.'
tracheal smooth muscle shows pronounced tVta-l receptor- EM;u_ti Satirla><rti|haiadrrniirr|>tor%AiiiMntiiaiitrrînuuiinioihri.i,
and hrWi-adiriiiirratwr* m mnara | M * and hwixjo Inn* I'mijiuil \M I A?:
mediated effect* (Furchgott el erf. 1975; Omini <*f of.. 1979;
aapul 1.7T-H.1.1!*)
lakovidisrr of.. !<*8ih .Johansson and Watdeck. 1981: l.ulich cl Pt-arm.oir. K. r*. WAHAWK-T D . S i n i a r r . R A i M i M n l i k I " lÎf
rrsi*!tio'hIrlai anrll*a J -m*r' > r*" tttiirxmIKirjiiK.il-!-ilinraiIt
at.. 197B1. In contrast, the V/e-I receptor-mediated ejects, in
aarf ihe lanaiHtn al lit* but, Ivia^raDo in iMlrtrM aiiinut- i.\'ir.i>t> t.'
dog appear to be relatively minor, an observation which in
fnt 3 d 7U. I<J
JL
PUBLICATIONS
10345200
026964
19*3
fllraiaii Bala Bar ^ a M ^ i J I I M I M O
Cauauc H. R , BAI D M S . C. 4. AKnT*rrtM*m*.

A. S* QuaMrtMivr am***
K M of htaarhal hMa-odnrimtpiarblarkadtin man. Br J. CEn. Pfcarmanl.
7:A!M-VW.IKIL
H n W K . A , M I M C O U N . K: f. A M Moturor. P. B : IMtr,-rntn) rfntnbuMa
of httn-l aael hna-2 arfiantrjor imftat* la cat and guinrtpi* fcrari. J.
Phamaol. Eaa. Thtr. S I S : Mt-iHA. lantt
I A K C U M * . D , MALTA. E . StcPmauDV C . A. AND RANCH. d la viuo artinty
of R O t t l atoUi-admoctptor iwltrln* agon*!. Br. J . PhtmatoL M i Sit'
411
M'MMHAht. K. P.. HKOSTIUND. L. R. Akp M O U M W . P. B-: T h t aktwrtrmto*tea* saanflcky of M and o" * admwrf* trrrpuM* ia am (wan aai tana
viuo Mol PkaraarftL Ifc 31-tl. iron.
MI-MA*. C NAOXL. J. A. AN* R o i c m , J M^ TJ .fencarinnrmtotHi a.

Jrmiy amooUi tauatlt: Thtir chrranamitien ia n{ 4. Appl. PayHofc U I
I8M-IW). UU.
OMm. C SoNuniux, A. R. Fotro, C. C ROMDM. n AMI Fvuttf. UtflMracusa tetwrtn bii- and otUradmMrtptari in iha iatttattd I
trachea. Pharmacol. Rr*. Caataiaa. I I t ill-tU, l I T t .
u,iwa
RKMAMMOTt. J . B-: SUM of I W an. N r n t aa(y is Iha hull*. Aim. Rtv. Raipir.
JONANUOM. L". AN* WALMCK. B J BtMi-aaVmoNpton owdiaunt ratouttan of;
Ci*.lI:7A3-Mim
h point* W uarhn: EastrinwnU wrtb prrmkaraJ. a ariaratlrctivt adrany R u n . C. L.. B M W I T . D. B. ami N A K O M W . S. f_- Csniuanc* of W u , a i
actpiaraooaia.4.PhanB.PariaroL3S:3U-a*Ubi.
'
HataaVanoctotaraiaaNKalianIut^fNidmc*frMdiraetlM*diataiiidit.
LANIM. A. M . Awrout. A . M c A t U r r . 4 . P.. L C M C K A . P. P. AND BHOWX. T.
MaLPnirnacal.14tPM-IO0S.ltM.
6 , J*.- DiMrnmnatiea of ttctptor (vttnaa anivattrf by *ymptkaMitnttk
RttAnx, J. A.: Raaponn of iaolaitd ctniaa mm** w alteirinl attawiladoa
aainta. Narutt OaadlRldj M 7 - M * . INT.
ndaryJthoit.J.ApLPh-ol.48ia-*,iB7i.
LAKCKK. S. Zi Ptttymftte mndanon of ih* ttkaao f cattehoIaBHM*. FfcatSctirnt. R . MomrA. K. ANA KvmMatl. H^ Inamauan aad ptoiNltlt* of Iha
atacol. Rtv. a t : 337-ltt. JMO.
MKXh nuirla of <ba dot tMrhta. Jtfn. J. Phjmol. t i 393-310. ISM.
Lor&MtL. C..C AM> S t l u m i . Ji_- EfTnrt of nrtaakriol in atihrnauc patlanu.
TtttilNGCA. G. ANB SvKotavn. N. : tajtractie* of orallv adaHnftitrad mrtoareM.
Eur. 4. CKa. Phaimrol, M : 397-303.1M2.
LVUCH.K. M_ MncNStU H. W. AHD!>nuUB>M. M. P J Threat hint atrip a* ' mctslol and pwpianalol with ttepnnallttt M aMhmaiif*. Bur. J. Clla. Phar>
*<*). l i 1S3-IT0. I K S .
m in viitppupaiation 1 prnphtrit airway*: A <wnpaiion of otta-adftnoctpteraiamMiâMocaidiaadaaaVhyianircaaUtiiitreaitlwIuaiiimpanduacfata.
Br. J. Pharmaml. M l 7t-T. 1S7&
MINNKMANV K. P.. H n t m u N n . L, JL AM> MOUNorr. P. B.: Simuliantoiu Sand rtprtnt raautata tat Dr. Prttr J. tatnii. Orpanattnl of Mtdkino.
dn tnntnaiioa of bia.| and btta-3 a*tnnrir m*pion> in timet coniumnjt HanNnrnunith Hatptttt. Dwrant Road. LaadoR W.|3. U.K.
both ttcrptorKihtypt*. Moi. Pharmacol. ! : M H C I9tfta.
" '
PtJBLICflnONS
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026965
FAMILIAL FACTORS IN BLADDER CARCINOMA
451
study of ureelected twins. J.A..M A.. 1S8: 749.1063.

11' Fraunwni. J F , Jr. and Thomas. L. B.: Malignant bladder turscrs
.
in a man and his three sons. J A M A.. 201: SOT. 1967.
17. Chan. H. and Pratt. C. B J A new famtlial cancer syndrome? A
12. McCulIoufth. D. I, Lamm. D L, McLaughlin. A P.. III and Cities,
spectrum of malignant and benign tumors including retinoblasR. F-! Familial transit tonal cdl carcinoma of the bladder. J. Urol.
toma, carcinoma of the bladder, and other genitourinary turgors
113: 629. 1975.
thyroid adenoma, and a probable case of multifocal est eosarcorra.
J. Natl. Cancer Inst, 5: 205,1977.
13. Petfcova-Bocharova. T.. Chemozemsky. !. N_ Nikolov. L G. and
Stoyanov. L & Families with multiple caae* of urinary system i a Ahcrne. G- Retinoblastoma associated nilh other primary malir,.
tumors: brief communtcatioa. J. Natl. Cancer Inst, 59: 1419,
nanl tumours. Trans. Ophthalmol. Soc. UK. 94: 938,1974.
1877.
19. Smith, 4 L . & Histotory and spontaneous regression of retinoblas14. Chemozemsky. L N-. Stoyanov, I. S - Pctkova-Bocharoia, T. 1C
toma. Trans. Ophthalmol. S o c UK. 94: 953,1974.
Nirolov. I. C , Draganov. I. V.. Stoichev, 1. L. Tancbev. Y 20. Lynch. H. T.. Mutcahy. G. M , Karris, R. B.. Guirns, H. A. and
Naidenov. D. and Kalcheva, N. D.: Geographic correlation beLynch, J. F - Genetic and pathologic findings, in a kindred with
tween the occurrence of endemic nephropathy and urinary tract
hereditary sarcoma, breast cancer, brain tumors. leukemia, tunc,
tumours in Vratza district. Bulgaria. Intern. J. Cancer, 19: 1,
laryngeal, and adrenal cortical carcinoma. Cancer, 41:2055.1978.
1977.
21. Zincke, H.. Aguilo. J. J Farrow. G. M Utz. D. C. and Kahn. A. USigniftcance of urinary cytology in the early detection of transi15. Morganti. C Gianferrari, L., Crrsseri. A., Arngoni, G. a.id Lovati,
tional cell cancer of the upper urinary tract. J. Urol, 118: 781,
G~ Recherches clinico-statistiques et gehetiques sur lea neopla1977.
s i a dels vesste. Acta Genets fi: 306. I954J.
16. Harvald, B. and Haute. M.: Heredity o f cancer elucidated by a
PUBLICATIONS
10343202
024025
Ctr Acknowledged
D. B . Cohert + 4s-
t` -
~~ Autoradiographic Localization o~ Autonomic Receptors
C7 ti
. In Airway Smooth Muscle
Marked Differences Betweorl Large and Small AirwaysI-3
PETER J. BARNES; CAROL B . BASBAUM, and JAY A . NADEL

Introduction
Airway smooth muscle tone is regulated in pan by rhe autonomic nervous
system acting on specific receptors :
muscarinic and alpha-adrenergic receptors, which are excitatory, and betaadrenergie and possibly pepaidergic
receptors, which are inhibitory . In airway disease, particularly asthma, atuonomic control appears to be abnormal,
and an imbalance between excitatory
and inhibitory effects may contribute
to bronchial byperreactivity . /Sutonomic receptors in airway smooth muscle have previously been studied in
vitro by measuring tension in isolaled
strips of muscle exposed to autonomic
drugs, and in vivo by making direct
and indirect measurements of airway
caliber after administration of autonomic drugs systemically or by inhalation (1). However,it is not possible to
study smooth muscle of small airways
by these methods. The smallest airways
that have been isolated are small bronchi (2) . Il has been suggested thal the
bronchioles are the sites of early
changes in airway disease (3) . and they
are a major site or obstruction in experimensal asthma (4), yet these airwcyshavebeendifficult tosludybyexisting methods . One approach has been
the measurement of tension in Isolated
lung parenchymal strips (S-8) . Because
these strips contain multiple types of
contractile elements (including bronchioles, alveolar ducts, small bronchi,
blood vessels, and interstitial cells), the
interpretation or tension measurements
In these preparations is difficult .
Another approach to the study of
autonomic receptors is the use of direct
receptor binding assays. Autonomic receptors have been Identified in lung homogenates or several species by direct
receptor binding, using radiolabeled
adrenergie and cholinergic antagonists
aaMMARY Auroracltqnpruemrprod, war
.uaMtodaumnnrlrMdlatribulionolaulon0mlonle pptnA in airway fmooth muscle ol tanN rrnrn traehea 10 IamMMI brplcMain : PWldlhytlrnrF4
penaol . fuguaraala and PN/quinudidlnyl iumltata w .n used to nbal Ortwmannak . alpna
adraMple, and murerNnlc ncaptnrs, ra.paenraty- using aeperimental cprdllluns that aara
ma.rmal specific eaerptor hindlnp wsraad dlner .ncaa wnrr rnund In Iha longitudinal disldGdion
el .aCh qe.plor and en dirrelbutlun of the rarlous neaplan In .eeh pan0er alnray . aalaROaP
tots rran present in Mah density th .ouanaullM alrwaya . r.ah Iha hlpMat Wrn11y In MnneNnVar.
Alplur .e.ptnrs wan eprrau In large sinraya, but numereur In small bronehlo4s, whareat eho
einerale eAn.ptaf w .n nWrNroua b1 WOneMN .mooB, muscee, apana /n prorlnla) Wa1eMnN1,

and almost Nu.nt rrom dlual Maremotea . TNr malhod mpy be useful In atudyma anaranons a/
auloncmlc racainors dirldbutlon in aman and 4rar anwarr atter oayMmantal manlpulatlnn and
indi .eaaa .
su
Rnr
naann
on
/aax
ranrM .ra
(radioligands) (9-12) . Because lungs nized glus microscope sections . Thesv slidec
are made up of over 40 different cell were eilher used directly in the binding aslypes, assays alone are inadequate to say or stored al -70 C .
determine receptor distribution in spe- Renpro B/nding

cific intrapulmonary structures, such Incubation and washing conditiom hasr
as airway smooth muscle . We have re- pfeviousiy been shown to produce optimal
censly shown that autonomic receptors speciflc receptor binding ror each radiocan be labeled specifically in frozen )igmd (13-1!) ; I'Hldihydroalprenolol
sections of h :ng and localized by auto- (qIH)DHA)(speci/icaclivily, 101 Ci/mmol;
radiography (13-15). Using experimen- New England Nuclear Corp ., Boston. MA)
lal conditions, which proved to be opti- was used to label bel .-adrenocepsors,
mal for specific receptor binding, we 1'Hiprarosin qrHIPZllspecUic activity, 20
have now studied the distribution of Cf/mmol ; New England Nuclear) was used
alpha-adrenergic, beta-adrenergic, and to label alpha-recepton, and Irrt)qulnuclimuscarinic receptors in smooth muscle dinylbenrilale(IIH)QNR)(speciRcanivily,
of airways from trachea to terminal 33 Ci/mmop wa~ used to labe)muscarinic
.
receptors . With ) H)DHA d 11HIPZ, seebronchides
tions wele incubated at 256 C for 20 min,
Methoda
(Mnnvd 6r oNNffN/Wen Onoan rJ, rpl2 orrd

.wry a. 19a)
Tissur PnparettoA in rerurd/onn feb
IFerrets (Musrelo parorius) were anesthetized From the Cardlovascular Research Institute
Avilh pentobarbilal sodium (35 mg/kg by In- and the Depanmmis of Analomy and Medicine .
craperiloneal inJeclionh and the trachea Univershy of Californla- San Francisco, San
and thoracic contents werequickly removed . Francisco . Califomia .
Endividual lobes of Ihe lung werr separated, e Supponed /n pan by Propram Project Grant
eannulated- and inOMed wlth tissueambed- No . HL .24136 from Ihe National Inunule, of
ding fluid (OCT : Lab-Tek Produqs, Naper- Heabh and by Contran No . 1111 from rhe
vllle, IL) diluted 1 :4 with phosphale-buf- Council for Tobaao ResearcRUSA . Ine .
reprinn should be addrcaed to
fered saline . The infiated lobe and tracheal ' Requeus tor
. Barnn, Depanmenl of Medicine .
rinas 5 mm in length w~ere rapidly froten inHammerrmith
~' R J
Hmpilal, Ducane Road-Londun,
freon 22 cooled by tiquid nilrogen. Frozen w .12. UK .
secdons 61o 8 pm thick were em on a eryo- Recipient of a Tra .elElna Fello-ship bom
stat at 1S C and thaw-moumed onto aelati- the Medical Research Counca of Great Bnlnn .
Taf
http://legacy.library.ucsf.edu/tid/bke59c00/pdf
PUBLICRTrONS
10346180
NaaaY aYOOrM rNaCLr Auro1/OYIC aECEPyoaa
d and with l'H/QNB, for 60 min . Concennalionsof radloligand were used that were ap
- prorimately equal to the dissociation constanl (Ka) for binding to ferret lung sections .
as previously delermined (13-15). Nonspecific binding was,dnumined by incubation
of adjacenl secrions from the same animal
in ihe same eoncentration of radioligand
but with an exceas of unlabeled anlagonin,
so that ccptrtr binding was competitively
inhibited . For /tiHIDHA we used I PM
(-)propranolol, for pH)PZ . IOvM Dhentolamine, and for l'H/QNB, I pM atropine .
After washing for 10 min in ice-cold buffer
(S0 mht Tris Hdi pH . 7.4), sections were
rapidly dried in a stream of cold air to prevenl diffusion of tadioligand, and stored In
a dessicalor overrbight .
Arrrorodrogmphy
Autoradiogmphy was performed using the
method of Young and Kuhar (16) to retain
reversibly bound (i .e ., diffusible) mdioligands at reeeploo sites. Glass coverslips .
which had previously been eoaled in pisoto
graphic emulsion, were pieced over the sections and 6xed to one end of the slide with
cyanoacrylme adhesive . The emulsion was
held in contact whh the section by binder
clips, then stored in light-proof boxes at
4' C . Optimal exposure times were found
io be 3 months for pHJDHA, 4 months for
lrHIPZ, and S months for pHJQNB. After
exposure, the cov'erslip was partially sepa
raled from the slide so that the emulsion
could be developed and the section stained
with 20a cresyl vuolet . The sections were
mounted and viewed under brightfield and
dartfield illuminsition . Autoradiographic
grain counts were performed using a cali
brated eyepiece and a x 100 objective lens .
Grains were counted over areas of smooth
muscle in several airways from each section,
and the airway dimensions were recorded .
There is some eonfusion over the definition
or "small airways," particularly when differenl species are compared . In our ssudy,
imrapulmonary airways were categorized as
cither O) cartilaginous airways (bronchi)
measuring I to 2 min in diameter and corra
+ponding to subsegmental bronchi of
sourth or fs(Ih gene,ration, or (?) noncanila
ginous airways (bkanehiotes) . Bronchioles
aere arbitrarily defined as proximal (> 0 .3
mm) or dislal (< 0.3 mm). Distal bronehi
ales Included terminal bronchioles and re
spiratory bronchioles.
Dald Ana)ys&
Resuhs are expressed as means m SE . Speafrc grain counts were determined by
c,+unming grains/unit area . then subtracting
background counts and nonspecific counts
flom the same area in an adjacent section.
R:ceptor density was then determined by
a+rreclion for specific activity of the radiohgand and the time of exposure using the
fprmula :
It = (gd/1) x (A/5.C) x 2
7>9
TABLE I
DISTRIBUTION OF 6ETA-ADRENEROIC . ALPHA#DRENERGIC . AND CHOLINERGIC RECEPTORS
IN AIRWAY SMOOTH MUSCLE OF FERRET
Reeeptor Dansay
IOIndlny s/rerr r m')
Oumeler A'M'ar tmm) BetaAdrenerpm AMpha-Adrenerprc Chormeroc
Tbacnea
6
1D7
s4
-1
T9 :0.0 - 263 x
BronohN 1-2 BO-0 ar 6 .1
9.1 29-9 7852
Pioxlmat aronchiolas O.y-09 122 a 20 99 s 7
.t 139 a
D/slar erpnemotes e 0 .3 160 z 15 163 : 6.9 102 o
Ba
96
77
tl
- RKrptp dM41Y eNlrieiryn hym .n .el,nn eefCxMn ,n MR"npf Rtlyln e,e nyyn r eF 110m e, Nen ] 4re,e
frMn Nch yl 3 an,marr
where R is the number of binding sites/unh

area of tissue, g is the number of specific
grains in the same area, d is the number of
decays required to give one developed grain
(a 7lms section - 34.01), t is the exposure
time (days) . A is Avogadro's number (6.023
X 1020 moleculeslmmo)), S is the specific
activity of the radioligand (Ci/mmoq, and
C is a disintegration constant (3 .2 x l0'
de,eays/day/Ci) (17) . Because the concentration of each radioligand uxed was approximasely equal to its dissociation eonstanl, approximately 30% of total binding
sites should have been occupied, Therefore,
the total receptor concentration was ealcu
iated by multiplying the producl by 2.
Ratulls
There was a striking difference in the
pattern of labeling of airway smooth
muscle with the different radioligands
~~
'a1aYe'
'
(table 1). With [rH)DHA, smooth

muscle was heavily labeled in all airways (figure 1), but the density of labeling was inversely related to the size
of airway. With 1sH)PZ, there was little specific labeling of smooth muscle
in large airways, but there was a high
density of labeling in bronchioles, parlicularly distally (figure 2) . The
(sH)QNB showed the converse pattern
with the greatest densny of labeling in
smooth muscle of large airways, parlicular)y in the intrapulmonary bronchi .
There was much less labeling of proximal bronchioles and almost no labeling
of distal bronchioles. The distribuyon
of each autonomic receptor in airway
smooth muscle, therefore, depends on
the generation of the airway, and each
caliber airway shows a distinctive re-
F/g. 1 . 6oCa11raUpi o1lMtaidranoraesplON in snwar amootll muscle 01 (aMet (al Darallaia llluminer
tioqatqna the distribution or auloraalo9raphie e1rMt qralna 66 br/pht datla in an inlralnrtmpnary branehu9: PHIDNA deneely labela sm qth muaete ISM). (b) . B/lahlliate view 61 the aba shown InteL (e) OarMnatQ waw ot 7ha sune araa In an adlarant section incubated wnh PM)DNA In the preunea ar an areesa
or pFopranol4l, showinp rq speeilie IaIN1rnB . (ol Dsr4hald view Ol a ametl nranehlo4 ahowinp danN
Iabeling of sn1oolh musete . Note the rarlehn0 ot aunoundinB aN/plir wana . (ar Br/qhthete Yiew or Ina
atea ahown ln (d1-(1) Deraaatd view ot the aame anta trCm an adjacent aaction Incu Wletl wnn l'MJOMA
and Dropranolol. Sule bat . 100 rrn.
026945
10346181
10
1M/1N1a a/NaaYa . aNa NaaaL
Fig. a Lorallranon ot aipnaatlunocePlon .. arnsy rmootn muecN 01 ferral Is) Duldbrd vrww o1 an
/myprununuy broncttus . arowina /abs4na by plriprarosrn of eonlrobum (EP)oul arewsf no labeuna of
ampoth muscro taM). Baeeprouns rs conautlerably hlaner than,n Npura I ID) . ananerom wew of the er.a
Shown In (s) (e) . Darkllettl view of tM same araa nom an adlaceni seenon incubaleP with PMloraroNn
antl an etcess 01 Dflente4Mns . (e/ Dark6ela wew o/ a amao bronelnoM sMwma tlenR labeling o1 aU
watl smDDM muubtaMl n) BrrpMnvta wsw of the area snown m(tl) lD Darkl,Ntl view o11M esme aroa
/rorinanadpCeresaetlon,ncuwtetlwunpNlprarosmantlane .eeasofpnemmaro,ne.aca+eWr . r0a,.n .
ceptor pattern . Thus, for trachea and

tnt2apulmonary bronchi, the rank
irder of receptor der.sity was muscar
ini > beta-adrenergic >> alpha-adrenergir, for proximal bronchioles betaadrenergic > alpha-adrenergic > mus
carinic, whereas for distal bronchioles
beta-adrenergic = alpha-adrenergic
>y muscarinic.
olneuukn
Autonomic regulation of airway
smooth muscle has been studied mainly
in trachea and major bronchi, because
h is possible to isolate and test the effects of drugs on these airways . The
peripheral lung strip preparation was
introduced to study the effects of pharmacologic agents on smooth muscle or
airways that were too small to be iso
lated (5) . In that preparation, however,
other contractile elements are present
to a variable extent, making interpretation of contractile responses difficult.
For example, eontractile responses of
peripheral lung strips in response to
notepinephrine and other alpha-agon
ists have been taken to indicate the
presence of alpha-receptors in "peripheral airway" smooth muscle (18-20),
but contraction of vascular smooth
muscle and contractile interstitial cells
(21) could explain these responses (22)
equally well .
We have used a different approach
to studyitdg autonomic rei-eptors in airway smooth muscle using autoradiographic localization of specific radioligands . Wc have previously established
that specific binding of each radioligand had the characteristics expected
of interaction with its receptor in lung
sections, and were able to determine
the experimental conditions ner :essary
to produce maximal specific binding . It
may be difficult to precisely determine
receptor densities from autoradiographic grain counts, but it is possible
to obtain estimates of relative reoeptor
density (17). Such estimates involve
several as'sumptions, but berarrse these
apply equally to each radioligand, it Is
valid to compare both the relative distribution of different retxptors in the
same structure as well as the relative
density of each reecptor in differant
structures .
Beta-adrenoceptors were numerous
in smooth muscle of all airways, but
the density increased al airways became smaller, with the smallest bronchioles having a very high density of
beta-receptors. This is in agreement
with the finding that beta-agonists produce relaxation of lung parenchymal
strips as well as isolated tracheal and
bronchial smooth muscle (3-B) . Alphaadrenergic contractile responses have
been described in smooth muscle strips
in many species (23-23), but they may
PUBLICATIONS
10346182
only become evident after beta-adrenergic blockade or after precontraction

of the muscle strio . In large airways
allthareceptors were present in smooth
muscle but were far outnumbeved by
beta-reccptors, whieh may explain why
beia-blockade is necessary to see the
alpha-adrenergic contractik response .
The density of alplhareceptors was
hip.her in bronchi than in trachea,
wFiich is consistent with the finding
th>tt alpha-adrenergic responses are
greater in the bronchi than in the trachca or dogs (26) . There was a surpris
ingly high density of qlpha-receptors in
small airways, particularly in the distal
bronchioles where the, density of alphareceptors was equal to that of beta-receptors . This suggests that at least part
of the contractile response to lung paremchymal strips may be due to contraction of small airways .
The significance ol' the high density
of alpha-receptors in small airways is
una ;ertain . Alpha-adrenergic responses
in airway smooth muscle may only become apparent after beta-adrenergic
bltlckade, after prgcontracting the
muscle with histamine, serotonin or
potassium chloride, or in the presence
of airway disease (25,; 27) . We have reeeatly shown th3t the activation of
alpha-adrenergic responses is not due
to a change in smooth muscle alpha-re
ceptors, but is due to some postrecep
tor mechanism (28) . In the presence of
mediators or airway disease, the alphareceptors that are alr,eady present become capable of initiating a contractile
response . The high density of alpha-reeeptors in small airways may be of importance in diseases, such as asthma, in
which mediators such as histemine may
be released in the vicinity of small airways and "turn on" alpha-adrenergic
responses. The physiologic role of
these peripheral alpha-receptors ls obscure . It is possible that they are involved in matching of ventilation to
perfusion In peripheral lung units .
Adrenergic axons releasing norepinephrine may produce both constriction of pulmonary arterioles and (by
diffusion to alpha-receptors in nearby
bronchioles) bronehoconstriction, so
that ventilation is reduced in thc same
areas that have a redqsced blood flow .
Such a mechanism would be an advantage in the case of pulqtonary embolism
Muscarinic cholinerJ{ic receptors were
most numerous in large airways ; of
these. the smooth muscle of intrapulmonary bronchi had a higher density
026946
re+
.Imw sY00TM Yut:Lr aulowCsoe arerllona
eated, but ahey may be difficult ro interpret in the face of coc%ictenl abnormalities in large airway function .
Studies on peripheral lung strips snffer
.*om the problem of helerogencity in
contractile efements, as di.cussed
above . The autoradiographic method .
however, has the advantage that
smooth mttscle of Ihe smallest airwac+
may be studied . This method may be
useful in delermining the c%istencc of
abnormalities in airway autonomic receptors in disease. /n a guinea pip
model of aslhma, a decrease in pulmonary beta-receptors was accompanied
by an increase in alpha-receptors (35) .
and in chronic airway obstruction there
was an apparent increase in alpha-receptors (11).
Because these studies involved radioligand binding to homogenates of
whole lung, it was uncertain whether
the changes occurred in airway smooth
muscle, or In other lung components.
The autoradiographic method we have
described should provide answers to
these important questions . Although
we have studied adrenergic and cholinergic receptors in the airways, the technique is equally applicable to other rtceptors (e .g ., for histamine, serotonin,
prostaglandins. and regulatory peptides) . This technique offers a new strategy for the study of regulation of
small airways and other structures
within the lung . The demonstration of
specific binding sites does not necessarily indicate that tbey are functionally
signifieant, and in the future it will be
important to correlate the presence or
changes in binding sites in a tissue.
with some measurement of receptor
function in that tissue.
Fig. 3. LOCatiranbn of muiear/nie rKeptora in aineay smooth muscle ol tana,t . ia) DVkiield view pl an
mrmpidmanary siOnenus showing dense labeling by rNioNa of arnootn muaclo {arA) Di- erialltaeid
vl.o of ineatN rhown in (a) .OCI. DarMiald vifw o11M aanNarra fromanad/acpnt .action incutNUdwitn
rlqnNa and an auce9{ of atropille. (d). Damturld vlew o/ a pro}linai broncniole Showing aparsN labeling
of 691001111 musnr Bldl . (e) ariamludd view ot 1M area shown in (dl dt Darknald vUnr ot iM same area
Irom an tidReenl paction inoubalUd wtth pH1DNa and an a .oe.as of atroplna . (a). Darkfleid view of d4,ai
6ronciuete srunrinp no upeeifn : labeling by rlI2ON8 terta111 . (n) . anpntliao view of aL. snown in (at %
DarYhald viaw at {ama arE NPm adiacent sectWn inoubanld with PNiONa and an .npsa of atMqna .
Scale bar w 1os :.n
than did tracheal smooth muscle . Thus, muscarinic cholinergic recepSmooth muscle of proximal bronchi- tors should be closely related to cholinoles had a much lower density, and the ergic axoas . This is in marked contrast
distal bronchioles had almost no cho- to the distribution of adrenergic receplinergic receptors . This distribution is tors . In most species, direct sympatheconsistent with physiolegic studies in tic Bsnervation of airway smooth musanimals showing that the greatest bron- cle is rather sparse in large airways and
choconstriction after va$al stimulation absent from bronchioles (33) . Adreneror cholinergic agonists Is in large rather gic axons in ferret lung show a similar
than in small airways, both in vivo (29, distribution (determined by fluores3D) and (n vitro (31) . Similarty, in asth- cence histochemistry) (13) . Adrenergic
matie subjects, antichtilinergie drugs receptors in small airway smooth musappear to cause dilation mainly in cle would, therefore, be regulated by
bronchi, whereas beta-qgonisas dilate circulating calecholamines .
all airways (32). The pattern of distri- Although there may be marked spebution of cholinergic reorpton Is prob- des differences in the response of airably related to parasympathetic inner- way smooth muscle to autacolds and
vation, which Is dense in bronchial prostaglandins, differences in responses
smooth muscle but sparser in bronchi- to autonomic drugs are much less apolar smooth muscle and vinually ab- parrnt (34) . Our findings in ferret airsent from terminal bronchioles (33) . ways can, therefore, probably be cxThis Is not surprising because acetyl- trapolated to other species . Including
choline, when released from choliner- humans . Littte Is known about the
gic a\ons, is rapidly Inactivated by cho- function of small airways in human
lineslerase and does not function as a disease . Several physiologic tests of
circulating hormone. small airway function have been advo-
AeknowkdamrNr
The writers wish lo Ihank Leona Lauricelta
for expen photographic assistance, and
Beth Cost and Palty Snell for their assittanee in preparina the manuuript .
Rafarane.a
1 . Hahn HL, Nadel JA . MetDod& of uudy of
airway smooth muscle and Its phynoloay In:
Widdhombe JO. ed . Internalional enryclopedla
of pharmacoloay, tand IheraPeuiicn : ra.piraiory
phnmacotoay. London : i'tryawn Prnt, 19a1 :
sor-at .
2. Penson CY3A, Ekmen M . Coniractlleeffens

of hiuamine in laqe and small mrayw Aarnn
Aalons 1976; 6:3a9-92.
3 . Mosa JC, Maektem PT. Thunbeck wM. Sile

and naiure of airway, obururlion in chronic ob
uruni.r lung disene . N Enp J Mcd 1968 ; 178:
p!s-do.
4. Draren JM, Ausren KF . Eftens of Inlrave
~ rcArraus
10346183
026547
. 762
nous adminiuration of slow aainq substance
of anaphSla%dw histamine. bradykinin and pmsglandin F1 .*r,r on pulmqnar5 mechanics in the
,aurcaa pig . 1 Clin Inveu 1974; 3~:1679-83 .
S . Lulich V :\1 . Mitchell, HW, Spqrrou MP .
The car lung strip as an in sitm preparaion of
. peripheral ain.a>s : a compariwn of bcraodrenoaplor agoriists. amacoids and anaphyiactic
chaTlenge o1,the luns strip and trachea . Br 1
Pharmarol 1976; 3g :11-9.
6. Seigl PKS. Rossi V. L7ruchorski RR . 1so
Ined lung strips of guinea pigs : respunses of
bera.adrenergic agonins and amalntiists . Eur J
Pharmacol 1979 ; 34:1-7.
7. Kldnniver PM'. Eyre P. The lunl Wrenchyma strip preparation of nd and dog. Responses
to anaphylaaic mediason and sympathetic bron, cAudilators . Ra Comun Chem Pathol Phvmaco! 1980: 27 :431-67 .
B . Goldle RG. Paterson JW. Wate JL A comparatise study of betaadrr,enoceprors in human
and porcine lung parenchyma strip. Br J Pharmacol 1982 : 76:323-6.
9. Rugf EL. Bamen DR N4honti SR . Coenisrence of P,- and A:-adrenocepton in mammalian
lung : evidence from dired binding studies . Mol
Ph .rmml 1978; 14:996-1003.
10. Bames P. Rarliner J ., Hamilton CA . Dollery C- Demonsuation of plpha,-adrenoceplors
in guina pi8 lung using I'Hlpraeosin . Life Sci
1979: 25 :f207-14 .
II. Barnes PJ . Karliner 1S. Dollery CT . Human lung-adrenocepfoas sthdied by radioligand
binding . Clin Sd /980: 51:437-61 .
12. Chens JB. Tornley RG . Comparimn of
muscarinic and bera-adrenergic receptors bewxn bovine peripheral fung and tracheal
smooth muu4es: a slNking differcnee . Life Sei
1982 ; 50:2079-86 .
13 . Bames Pl, Bnbaum CB, Nadel lA. Robens
:M . Localisarion of beraadrenorepton in mammaihrn lung by li;hl microscopic autoradlo8raphy. Nmure 1982 : 299:444-7 .
eMarrw baagWY . awo Awn
14. Ba.nes PJ, Basbaum CB . Nade1 JA. Roberts

JM. Putmonaryalphaadrenocepton : amoradlographic localization using 1'Hlpraeodu . Ear J
PhannKol. in press.
IS . Bames PJ . Nade1 lA. Roberu JM. Basluum
Co . Muscarinic reeeplon in .ung and trachea:
autoradiographic localization using I'H)Quinu
elidinyI benaHate . Eur J Piurmacol 1913: 86:
103-6.
16. Young WS. Kuhar MJ . A ner method for
receplorautoradlography:sH-opioid receptorlabellin; in mounted tissue sections. Brain Res
1979; 179:233-70
.17
. Lane MA, Sasue
A. Law M . Salperer MM.
Cholinergic and adrenerNc receptors on mouu
cyrdiocyies in vitro . Des Bioi 1977; 37 :254-69 .
IS . Chand N . DeRmh L . PMrmacological
characterization of the rabbit lung strip . Res
Commun Chem Pathol Pharmarol 1979 ; 23 :
223-30.
19. Chand N . Evidence for exBtam of alpha
adrenocepton in the wt lung . Res Commun
Chem Pathol Phumaml 1979: 2S :21S-2A
20. Black J. Turner A . Shaw J . Alphavdrmo
eeptors in human peripheral lung. Eur J Pharnu
col 1981 : 72 :83fi.
21 . Kapanci Y, . Assimaropouios A . ]rlr C.
Zrahlen A . Gabbioni G . Contractile interstitial
cells in pulmonary alveolar septa : a possible regulator of semilmionMperfusion ratio? J Cell Biol
1974; 60:373-92 .
22 . Mirbahar KP. Eyre P. Bovine lung paren

chyma strip hn botn airray and sascular charao
teristln . Res Commun Chem Pmhoi Pharmacol
1980 : 29 :1 S-23 .
.
23. Fleisch JH, Maling AM, Brodie BB . Eci
dence for esistence of alphwdrenergic receiaors
in the mammalian trachea. Am 1 Physiol 1970 ;
258 :596-9.
24. Mathe AA. Astrom A . Persson N-A. Some
bronchoconwricting and bronchodilatine responses of human isolated bronchi : esidence for
the esisiace of alphaadrenottpton. J Pharm
PUBLICATIONS
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Phmmacol 1971 ; 23:9oS-10.

2S . Kneussl MP . Richardson JR . Alpha.adren.
eryie recepton in human and canine tracheal and
bronchial mmmh muscle . J Appl Phyxiol 1978 :
45 :307-/1 .
26 . Left A . Palhophysioloey of anhmauir

brrtrnchocarstdclion . Chen 1982 : 82:133-215 .
27. Ohno Y, Waranabe M . Knuri Y . Ma~:.rra
tation of Inent alpha .eciulory responst inlhe
canine tracheal smooth muscle preparation . Re
lat,ion to basal tone. Arch Im Pharmacodfn Thn
1981 : 231 :20AI6.
28 . Barnes PJ . Skoogh BBE. Brown JK. Nrdel

1R. . Anisalion of alpha-adrmergk responses in
eriMeal smooth musde : a posr-reeepmr meeha
nism . J Appl Physiol, in press.
29. Coleb.reh HJH . Olsen CR . Nadel JA. Ef
fect of histamine. serotonin and acnyieholine on
peHpheral airways. J Appl Physbl 1966: 21 :217
; Nadel JA. Cabeaas GA. Austin JHM . In."

visp roentgenographic eaamination of parasfm
paahnk innerralion of slllkll airways : Yse of
po;rdered unwlum and a fine foal spot .-ray
tube. In.esl RadWl 1971 : 6 :9-i7.
31 ; Drarm JM, Schneider MW. -Comparadve
responses of tracheal spirals and parenchymal
srrips to histamine and carbachol in vitro . J Oin
In~va 1978 ; 61 :1M1-8 .
32. McFadden ER Jr. Ingram RH Jr.'Hayaes
AL . N'ellman JJ . Predominant sile U Row gmi
rmton and mechanisms of poneaneional asthma .
J Appl Physiol 1977; 42 :746-52 .
33., Richardson 1B. Nerve supply tonhe lurqr .
Am Rev Respir Dis 1979; 119:785-802. ,
34. Fielsch JH . Pharmaeologinl aspects of airsvaq smooth musde. In : Nadel JA . ed . Physiol
ogl and phmmacotogY of t:ae airways . Ner
Ymk : Marcel Dekker, 1980:191-216
., Barnes PJ, Dollery CT, MacDerrnol
.3
J . In
ereased pulmonary dpha-adrennsic and reduced
bda,adrenergic ceptots in enperimental anh
ma : Namre 19g0; 288 :569-71 .
026948
274
Am Rev Respir Dis 127(-uppl 2) :274 , 1983
gTREPTOfACCUS PNEUMONIAP CELL LYSATE IIOfIBITS HUHAN

NEUTROPHIL ELt5TA5E . T .g . Collins and R .A . Santlhaus
Department of Medicine, aclona ev an Nospita and tbe
Unleerslty of Colorado School of Medicine . genver . CO.
StreptocoCpW pOeumnNae l5trep Pn) inducea a marked
sutrophil (PMN) influx into the lung . Coplte thie avail. illty of large amounts of pttagocyte protelnenee, the
` reumonltla aeldgm reaulta in paronchymal necrosia or
residual eephyaematoUa changes . To test the hypothesis that
this sparing or the lung parenchyma 13 due to inhlbitlon or
Inssn P!!1 elutaase cell lyeate frwm a clinical Ssolate of
Strsp Po type 11 was tested [or ira ability to Inhibit
e3aeCOlylla by porcine pancrtetic slmetaae (PE) and by hlman
P18t granular extract (CE) . Stspnylococcm aurcua ATCC 259z3
(Staph) vaa used u the anntrol . In>aluble- crltleted .
bov)ne ligamentm nuceoe elaatln eerved u LDe substrate .
Purified PE, and Ge prepared frem p/4( of normal auman volunteepa aeewed ae the elaateae sources . Hlaetaea activity v.a
caleuleted tr label x)uD(lfzed during 5 hours of Sneub .. tion . Toe bacteria vere grown at 37oC in trypticase soy
broth vontalning /f dextrose . Staph was harvested at a
hours and Strap Pn at the end of Sta erponentlal grovth
phaae . The ce11a vera uashed, spun, and ground with
alw(nus oxide in the cold . The resultant lysatea were apun
at 20,000 a a for 20 m3nutes and dialysed vs . O .1M TrSs HCl,
pH 7 .6- We round that neither lysate had endogenous
elaetase sotivlty . Nelther lysete Snhiblted P6 Dut there
vsre algnlflesnt differencen In their SnhibittOn of Pa9V
elastaee . Strep Pn reached greater than S0i inhibition
(731) at 6 to 10 mg proteta/ml, the lowest concentration
tegted, vhSle Staph reached 50% inhlbitfon at 65 to 75 mg
frotainlal . In one experlment . the lysate frOm a nCCrotiaing type 3 Strep Pn failed to inhib .t P10( elastase . Ln
suamary . lynete ot Strep Pn type 1t appears to contain one
or eore non-dialyzable oonst3tuents which inhlDit humsn PIQ(

elastase but not P8 . Staph cell ]yspte inhietta PfD+ alastase poorly at <omparable protein concentrations and also
faila to inhibit PC. This lnhlbitory phenomenon may help

explain vby Strep Pa pnemonla ael .aom result . In parmchymal
necrosis or reaidual itmg tlamage . (Supported by NIH HLo1U01 and yR-D7og5)
'kcNOLINtRCIC RESPONSES IN HUNAN LUNG - A

STkUCTUHt/FOdCTION CURNELaT1UN .
J .L . glack . N .3 .6 . Ptnney snd N . Herend,
Departreot of Ph.rwcology, and the Deparrmenc of

Nedaclna, Coocord gospltal, un3veteicy of Sytsey,
N .S .V .e anatTalla .
ln contrast to the reproducible reapooses of

busao lung paranchymi strlps (HLPS) el/cited vith
aaaeiata aucN as noradrenallne (Ilack et al . Eur J
PLarucoy 72- 83, 1951) reepooeer to cerbacnol tn
[hfa preparation were found to be highly varfable .
.latiee 10 masSaal coqtvactile
The marked vr
response to arbachol between dtffereot 11123 could
not be etplsined by dlfferences in the veight of
the preparations (c - 0 .06 P> 0 .05 0- 53) .
To datmrmine if contracttou to carbachol vas a
function of a varying proportion of a tlssue
cosponeat, a ateueture/fuoctlon correlation study
tqs performed . Pittaeo dI.PS uere aelected for
sorphosetrie snalysi> so tamt a wide range of
ccotractile responses to urDschnl Sa v[tro vaa
tapresented . Comp>oeots of the gf3S vere
u[agori.ed p etthar 1) alrray 2) blood veaeela
3) parenchyaa 4) noo-parenchysa . Proportione by
volume of these tisnue eoeponentm oere determined
by rsne of a pofnt-cuut systen (Duani)1, Thorax
17, 720, 1962) . Pifteen histological sectlone
uere taken tros esch atrip and 5,000 - 10,000
poiets aere tounted to sach strip . There eaa a
etgniftunt correlation (r - 0 .77 P < 0 .01)
between the percentage volgae of slrwy Sm a strip
and the masbum Coatractllt respcnae of that atrlp
to eaebachol . 'Ihera rraa vo slgoiElGUt
coyrelmeion betveen the percentage volume of any
other structure Category sea the mawlaum
vontxactioo . It is concluded that sirvay mmootb
~ euscle Sg the component of the at-P5 chiefly
eeponaiele for the contractile mffactm of
.arbachnl in vltto . Supported 2 . part by the
Nat3ooel Hulth and Madieel Reaaarrb Counetl of
Auetralll .
http://legacy.library.ucsf.edu/tid/idz49c00/pdf
RBrY-STRDCTURE i'M gR)L00y
gi
/
H.PPIMG 0F VASOACTIVE INTESTINAL PEPTIDE (qIP) RECEPTORS
USING. CYCLIC A38 (cAfff) O9NHOGYIOGNGMISTRY . S-U . tura_y
C . . baabam, P .J . Harnes and u .M- culd . Grdlovasc .IIar
Research Iottlcute, dCSF . Sen Fran<fsco . Gt .

VTV, flzet tsolated from porcine Intestine, alao ealsts in
postgsng11og1c autonomic axone iu mnny eisaues . 8ee,ve (abers
eontaining iugoreactive VIP have been demonstracad in the
respiratory tract of guinen plgs . rabblts, ots . doga, aag
huamns . Recenrly . VIP nev been implicated In the regulat!eo
of respiratory mucus secretioo . a pretess involving release of
macromleculee frou esocrlne celle, and transport of fons and
vater aeross mucosa . Hecauee aubaueosel gbnda ond mucosa
both cousiet of sised Cell populations, it vee raot clear uhicb
speclflo ulls poaeessed VIP receptors and eon[rlbti ;ed to Vlyeooked responses . Since VIP 1s known to accivate ac)enyl Lyclase, ve used an 1menmcytocLemical Probe for rJ4tP (0 loaotify
thoae celln acfauLted by VIP . Dog tracheal poaterlor membrane and ferret tracheal rlage vere equ111bra[ed x 60 mio in
ottyBeveted LLam' F-11 medium conC a1n!ng indosethoelq
. (10-6)1)
and becirreclo (Img/nl), ehes traesfarred to bea$enq cantelq1ag fresh medium~ VIP (3 x 10-7tn, or VIP (3 a 10-7)t) plus
proPrenolel (10-0l1), pbaocalaadna (10-6M), atrop).ne (10`6M),
and et[mdotoaln (3 a 10-yM) . After 5 .1.0, Clseuer vere
fraseo and cryCSt .t >ecriona luined fbr Saun>reetRive cA`P,
u>ittg the unlabeled antibody eneyme mathod . In fer;ec trache ;
VIP lacreased imuunoreectlve cAHP 1v both sarous arlQ mcoue
eellc tn submucossl glands, and this Sne .use veb enx inMabIted Ly the blockers tested . VIP produced 1itle f4 any
Imcrease in cAt(P In ciliated eplthellal eella of the ferretIn dog tracheal eplphellum, VIP markedly increased cAMP In
c111oted and basel cells, but not Sn goblec cella, and thia
in<reasa vse not blocked by the antagonfate Ce>ied : Yhoe . ue
have .ueed levunocytochemical localizecenn of cAMP i a marker
for VIP receptor activation In heterogeneous tia0uufn which
cellespeclfle receptors cennet be meaaurad d3rectlj, ; We
coaClude that seroua and mucVVa [ella of ferret tracheal submucodal glande and e111ated end basel cello of the dog
cracheal ep(thelium contain VIP receptors, and ate .responsible
for i+edisting physiological responses evoked by VIP ;
(Supqorted by USPNS PPC (0 .-24136 and Council for 7obatco
Resesrch-USn . Inc . Grant l327R1)
CFFC(.T OF TNIOG1YCOy .1TE ON HafL9lER 11/NG MACROPMACE .~VrfIGin

QU .WRITAT nY FI/JM Cy]y)tSfRY . ] . .7 . Godleati, M- Mortars .
N, Nacler and d .D . araln . Department of Thysinlnqy, Harvacd
School
of
Public
Health . Boston . nA
we have produc .d n nronoclonal antibody which ieAccs vit .`, a

rell xusface antiqen on bamatea lung eucrophaqea birt not vsth
other lung cells- eirc~elating eronocytea, or peritoneal vucrophag9s . Pluorescein tagqed antlbody can be used to~auanti .'y
the 8nount of antigen using flov cytometry . Nhen the asount
of tluo[eecent anubody un the cell aurtac* vas quhntitated
and plotted aa a histogram ef eell number versus amount of
fluorescence (xr cell, we found that hamater lung tsacropheges
heve a broed syetrieal Gaussian dlstriluRion . In previous
work . we noted that the length of time tllat mncrephnqes bavm
been In the lung In an important daterminant Of thb quantlty
ef antlgen . In tS,is study we ccmpare chengea in the number
and e(re of lung mecrophagea and their aonunt of antigen after
a single intiacrecheal inst111at1on of 0-15 ml of 6e thloqlyc-olate 1TG) . One day after SC, the mmOer of Pagrophages
recovered by repe$ted lavage increased fros 2 .4 a 10 in cantrols to 8 .1 a 10 r thesa levels pereiecad for 30 days . Flow
c)tometry analysls of macrophaqes frust TG animals Indicated an
inltial dacrease in mean cell eit4 ss a.easured by kl[1tl light
losa one day after instilletion from a control vm14e af 51 .0:
2 .2 to '/H .5a1 .7- By the third day and thereafter, the sise
of the 9G macrophaqes Increased to 88 .624 .0 (not aiqnificantly
different from control) . Nean fluorescence ef the mectaopnsqa
populatlon fe decteaead from the eontrol value of 51-7s1 .0
15 .0 . of the mean of 6 animals) to 19-6e) .3 In the TG treated
aaisele an day 1 . Menn fluorrcance per cell mcaa0ily
increased to 65 .9lT .1 on dey 10 at which time it surpassed
control voluea, Ostng the ratio of fluorescence to assal
light 1ose, this sane trend ras seen . On day 1, thm ratio
decreased from the control velue of ]0 .7!1 .0 to d-9ta .2 . 'rM
ratio lncreased to 42 .1 :6 .6 at day 10 . Rhese dlta suggevt
that TG sevrvits inantwa nbcrophagee which lack aucface
aneiqen into the lung, . With t Ue . theme cells nature and
incretse both in srre and Sn the number of eati}enic sitea
on the cell 9urface . Tbua this sonoclonnl antibody may be
Ih,
ly :
A1
do
War
pa
Er .
Ana
by
tht
ret
oot
the
(
ad
ove
tre
te)
Lah
aaa
prc
rel
and
asc
of
v1t
vit
tor
v1e
1e
lni
useful In studies of the mturation of alveolas macropneqea .

(SUpported rry NIH Grante Nt.-]7]ee and r5-aUnn] .)
PUBLICATIONS Q1
10333448
-45
Ctr /\c! :r-rvied
D. B . Cohea `'
Characterization of Purified Dog Mastocytoma Cells

4PAutonomic
Membrane Receptors and Pharmacologic Modulation

of Histamine Release' -`
MARTIN J . PHILLIPS,' PETER d . BARNES,' and WARREN M . GOLD

' Intlnductton
a Mast cells appear to play a cent ral role
in many innammatory and immunologic

reactions in the lung because they release
diverse chcimicals capabic of initiating
and modulating such reacuons (1)- Assem and Schild (2) showed that a variety
of beta-adrenergic agonists co W d intubil
antigen-induced release of histamine
from passively sensitized human lung tissue, presumably by their effects on lung
mast cells . Since then, the autonoroic
receptors and biochemical mechanisms
by which vanous neurol ransout ters, hor
mones, and pharmacologic agents modu~te release of mast cell mediators have
een the subject of intense investigation
but controversial conclusions . This situalion appears to be due to expelvoena
using heterogeneous lung iissue and beoerogeneous cells, and differences in behavior hetween purified cells from different smlrce-a (ng ., rodent mast cells and
mast cell tumors, human basophits and
human lung mast cells). These discrepancies might be due to differenees in mast
cells from different species ar from different locations in the same species, or they
might be due to effetts on the mast cells
caused by ttie puriGcation techniques, Alternal ively, m a heterogeaeous ceU population such as the lung, pharmacologlc
effects on mediator release may he indirect, acuag via other cells present fn
the essue.
We have studied dog mastocytoma

eaUs for several reasons-the dog has
. been used Jddely for the study of bron-
chial reactivity and is the only mammal

other than Aumans that develops a nat ui rally occurring respiratory poUeuosts .
However, charactcrizaaon of dog mast
~cells has depended on studies of heteroncous tissucs such as lurlg(3)and bronchl (J). or ceUs harvested by bronchial
lavage (5), and is Ihus subjeet to uncertainties inherent in all studies of m1Red
cell popula8ions. Dog mauocyeoma cells
http://legacy.library.ucsf.edu/tid/hez49c00/pdf
TM .q la conlllQlny atvyanra u to whlcn oulon4o~C .eeeprCr . rnaat CMM PnaMa .

anG wn.rMr nM1lNpi0lf anfJprpia orr1lGEWamrp netllaryr LMOa vI . nMeuMNY CCa maaro4y
loma celY nn'auM tMr a . . Ml4ale In 4Nf numWra In a MnMty yun tom1 . unlrar npmal yty
9UMMM.riY
maal GYRk . Maalorytoma nYCUIp rNm a YYp .wn iIaM anE aLayysQ+rwd wM . cellaypvaa to
Pnvlos a an fuapenawnot maatorytwna Ma er ~ 93M wntr . TM pw.~ ef aurunomk waP
lo/a nI/ aiN1JM ay nUnl rOleOllpp~tlbin0l.q aiYr . ..a 4r aMUimg pnYm .eObyk .nDU.NLon
d mMlator rlNau In eM rbbllparM Elndlny asrya MFamN,Mnle nleePton aMO aaMnMtad
.U ehou,.w,
p'r 1'Mp~nF'oa .penewl mnouq, nlr .e . .r..qia rseepew. W PHlwaamin a1nOn'p v
yk r . .>pmn by hH14ulnuGltllnrl Dantliab OinClnq . Nen.yepnc OuWmp wn oaurm/natl m seoa
eae' nr ineuen~enln ur pe .u .e.oltM .p .[mc anaqo .YSn Moypqlw.On.^fMamus, anC ans
plnq sapadMlr. TnaNnel of aNanemk apoMab on Imrnunebpk .ne non/mmunaioylc Mals
R11M
raNaM
wq
mmnNJ . uab.a tn1 beTeOleM,plC ayenl .e YOPAtarIql alntl Y .Wlanna, fM
alpnYWrlnarple apornet pMrqlpbnna wrthantl wnlrout p10prYW WI . arW tM enelhw .9ta aFn41
aearyrcnonna ows...pnnao cu,. .a . awrvst .A noN k . Ure eulonomk ayonbb anC rM

1+islan.usraNd .p /aent . Maulb trom t1y raElOayanG buWln) wq the pM1Finawkqic anMw
:
rw curmdanl . rMw Yey ma.foryton~ eell. MO a nqn e.elry of pey..ac.pb.a lZf .5nn
SE ML-nppMNpll . n-31 et+tneh IM p .aGOmIMm aubrrpa .pparaU m tw Eau,.
3,aGG: mean .
Mtlenoew..kunCbrtMpns.~+w W .Ipnfee ..r.a .qb e .r~ .dl .r .px

.aeayro . olnWnp ot oy tinv acvone on nlp .Nn . ..kur
No
~epow. .Nw IM dirscl
.r nrv .as.a os .rs:
closely resemble normal dog mast cells

(6) and may be obtafned in large numbers in a relatively pure form without the
need for extensive pWPcation proccduru. Study of these cells may aid in understauding the physiology of bronchial
byperrcactiviry and the role of the maet
cell in this phenomenon as well as the
general features of the phalmacologic
control of mast cell function . Moreover,
these oeLLs appear to have advantages over
rodent mast ceU tumors because they contain similar mediators to human mast
ceLls (including a large amount of hismmine rather than of serotonn), bind 1gE,
and releasc theu mediatorswith both immunologic and nonimmunologc challen ge- features that are preunt only variably in rodent mast cell tumors (7) .
Funhermo[e, prrvious studies have
tended to test for the presencc of pharmacologic receptors by evaluating the effect of specific agonistsand antagonists
on ceU function (eg-, change in cyclic
AMP concentration or modulation of
mediator release) . Converscly, other
workers have assumed that the prescnce
of receptors (by figand binding studies)

or the increase in cyclic AMP implied an
effecl on release of mast cell mediators.
Ho.vever-the presence of receptors cannot be assumed to correlate itrvariably
tReer. .N w onF .nul Jo .m Febn.ary 4 1988 wrd
.n ievsM Jo>m JWy 1, 1985)
-' From Ne OMiwaseWar Reavrm tusutuleaod
tkpanmun of Med4cine . Uw+avty of CalifornsaSan Fraoos<o. San FrannsttA CJiforaia .
Supporud in prt by C+rant Nn ffL-7A1]6 from
the Natwoel Hean . Lungaod Blood Imorutef, by
Graar No. 133]R from the fouod for lbbre<o
Ikseartb-tiSA . lo4 and by graau num ITe Srmotl
Md]fcal Resea/ea Fund (Amerinn tuny Assadauuu of Sea Fuocnce). the Um .erary of CaWornur Rzua1N Ewluaoon and Alloonoo Cammiruz, aod tne California Rcealeb and Medleal
Educauon Fund (Ameriean LueB Associadnn of
Cal3fornfa) .
' Prrseotetl m preliminsry form m W e Aunual
Metnng of Wa Amerum Thoreoc Sooery. IDS Angelea- CA. May 18. 7952 .
Aequen5 tor Rpnnu shoWd be addressed to
Warreo M-Gold, M .R, Caedo.meutar Reeeuc .
losuruu: UISM . Umwrsry of Califomia . 6an
F.anoscu sen Frano.co. CA 94143.
Rmprcol of a Thvenlna FeBmstlip frum Ihe
Medical geearch Counci7 of Grrat Bntau,
totY-
PUBL ICflTIONS
10333399
014410
1020
owrwn .
with functional effects on secretion . In

this study, we have used radioligand bind-
ing techniques as well as evaluation of

t he functional effects of pharmacologic
agonists on histamine release to test for
the presence of beta-adrenergic, alphaadrenergic, and cholinergic receptors on
dog mastocytoma cells.
Method9
Mmerrats
The radioligands used .vere: ('Hldihydroalprenolol ll'H)DHA), (specific activity, 101
Ci/mmol); I'HJprazosin (specificanivity. 20

Ci/mmoU; f 1-IJquinuclidinyl benzilate
(['HJQNB) (specific aclivity, 33 Ci/mmol) ;
EconoBuoA scintillation-counting fluid : all

were obtained from Ihe New England Nuclear
Corp., Boston, MA .
The drugs and chemicals used were : acetylcholine chloride (ACh) ; arropine sulfate,
bovine serum albumimfracuon V (BSA),
compound 48/B0, ( x )epinephrine hydrochloride, histamine diphosphate. ( m hsoproterenol, ( x )arcerenol (norepinephrine hydrochloride), (x )propnnotol hydrbchloride, penicillin O, streptomycin (Sigma Chemical Co ., St .
Louis, MO), phentolaminr mesylale (CibaGiegy Co., Summit, NJ), terbutaline sulfate
(Astra Pharmaceutical Products, Inc . . Worcester, MA) . (-)pherlylephnne hydrochloride
(Winthrop Laboratories, New York, NY),
shon ragweed (ombrosia arremnifolra) pollen extract (Hollister-Stier Laboratories, Spokane, WA), acepromazine maleate (Averst
Izboratories, Ina, New York, NY), sodium
thiamylal (Bio-Ceutic Laborarories, Inc . St .
Joseph, MO), Dulbecco's modified Eagle
medium / DME), mrnunum essent tal medium
(Eaglel-JOKLIK-modified (MEM-IOKLIK),
4-( 2-hydroxyelhyl)-1-piperazine<I han esulfonic acid (HEPES) (Boehfmger Mannheim
Biochemicals, Indianapolis ; IN) . felal calf serum (Hyclone Sterile Systems, Logan, UT),
amphotericin B with deoxycholate (Fungazone; E. R- Squibb Co., Mirad a, CA ), trypan
blue (GIBCQ. Grand Island, NY), toluidine
blue (Fisher ScientificCa . Pittsburgh, PA),
protease-free cotlagenase (CPLSA, Worthrngton Chemical Co., Freehold . NJ), and deoxyribonuclease (DNAase) (Millipore Corporation, Bedford, MA).
The following reagents were used for
fluorometric histamine analysis ; perch7oric
acid, hydrochtoric acid . mctbanol IJ . 7: Baker
Chemical Ca . Phillipsburg; NJ), n-heptane,
I-butanol alcohol (MCB Manufacturing
Chemists, Inc, Cincinnati, OH) . o-phthalaldehyde (Dionex Co ., Sunnyvale, CA), sodium hydroxide (Maliinckrudr Inc, Paris, KY),
and phosphoric acid (Fisher Scientific Co .,
Pittsburgh, PA) .
The experimenlal procedure followed adhered to Ihe published "Gaiding Principles
in the Care and Use of Animals" approved
by the Council of The American Physiological Society and specific protocols approved
by the Committee on Animal Care of Ihe
University of Califorma, San Francisco
a~wrca
un
pqn
Masmcyrorna Biopsy and Disaggregarion for 20 min ; for saturation binding studies,
Ma-stocytonia nodules were excised from a cnncentrationsrapgedfrom0 .1 to2OnMand
4-yr-oldGerdnanShepherddogthaAhadnrul- forcompcthionsAudiesfrorrilto2nM .No
tiplesutrcutaneouslesionslocatedneainlyover specificbinding .~asmeasuredbyincubatije
the abdomen, chorax, and hindquarters . irstheprosenceoflM(-)propranoloLWhen
These tumors gretv slowly, doubling in size catecholamines ivere used in competition
over the course of I yr . The nodules ranged studies . 100 M ascorbic acid was added to
from 5 to 25 mm in diameter at the time of tbeincubation tubes topreve,nt oxldattorr . AB
biopry. A total of 22 nodules were excised over samples were run in duplicate or triplicate .
a 12-month period . On each occasion, thedog Atpha-adrenergic binding was determined

was frrst anesthetized by a subcutaneous in- byincubationwith0-1 to 5 nM I'Hlprazosin,
jection of 0 .25 mg/lb of acepromazine fol- usingthesameexperimenral conditions . Nonlowed b,v an intravenous infusion of sodium specifSc binding was defined by incubation
Ihiamylal (2a1a wt/vol) until no response to with 10M phentolamineor 100M (-)norpain could be elicited . A mastocytoma nod- epinephrine Cholinergic reeeptor binding was
ule was excised, dissected free of sdbcutane- determined by incubation with 0 .1 to 2 nM
ous tissue, and washed in calcium-free PBS 1'f 1QNB for 60 ririn, and nonspecific bindcontaining penicillin (100 U/ml), streptomy- ing was defined by incubation with 1 .rM atrocin (100 pg/ml), and fungazone (2 .5 ag/mI) pina
to counter any bacterial or fungal contamination.Thenodulewaslhenmincedintofine Dara Analysrs
fragments (approximately I rnm diamrter) Datawasexpressedasmeans m SE . Binding

with scissors (6. 8, 9) . curveswere fitted bya nonlinear least-squares
These fragments were disaggregated enzy- curve fittingcompurer program (IO) . Saturamarically in an Ehrlenmeyer ffask try a solu- lion curves were ilnalyzed by estimating the
lion consisting of 50 U/ml high purity equilibrium dissociation constant, and comprotease-free cullagenase, 2% BSA and 25 petition curves by eslimating the inhibitory
mM HEPES in calcium-free DME,adjusted dissociation constant . Parametcrs were ohotoapHof7-4andanosmolantyof290mos- sentogivethebesAdarafitaideterniinedby
moles .Disaggregationwascarrieddutunder minimalvarianceofexperimenraldataabout
95% O, and 5% CO, at 37 C in a gyrotory thecurve generated by these phirameters . Data
water bath . At 2(}mm intervals supernatant fit was compared at increasing number of
was removcd from the disaggregation solu- parameters (N) thdt resulted inastatisticalt,
tion, which was then replenished with fresh sigmficanr improvement in lit over (N-q .
solut ion . A fter fi0 to 90 min, t he dlsaggrega-
tion was discontinued and the remaining so- Pharma'rologic Studies
lu]ion was filtered through cheesecloth . Cell These studies evaluated
the modulation
of
suspensions obtained from disaggregation h-stamme release by beta-adtenergiq alphawere centrifuged (200 x g for 10 rd .in), and adrenergic, and chblinergic agonisu and anthe resultant cell pellet was washed tvSice with
tagonists. Histamiote release .vas induced eicaldum-free 7yrode's buffer before resuspen- ther nommmunologically by challenge with
sioninanappropriatemedium .Mastocytoma
compound 48/80, or immunologieaUy by
cells were identified by their characteristic challenging passively sensit'urtd cells with angranules. whicl, stained merachromatically tigea For pharmacologic studies requiring
withtoluidineblue(O.2ol.solutionatpH3 .0, compound4g/80

.inducedhistaminerelease,
dilutedll0withthecellsuspension)andwere cellswereresuspendedin7yrode'sbuffercanquantitaled by countingtn a hemacytometer taining 25 mM HEPES and 0 .l wo BSA at a

(American Optical Scientific Instmments,
concentration of approximately 3 x 10'
Buffalo, NY) (cell count ranging fronr 47 to cells/ml . These cells were then reacted with
2)0)_ appropriate concenrrations of compound
48/80 and/or pharmacologic agonist or
Radioligand Binding Studies antagonisl .
Cells were resuspended in 7yrode s buffer con- For studies involving immtdnologic hismtainrng 25 mM HEPES at a concentration of mine release, cells were first sensitized pasapproximately 0 .5 x]0 cells/ml (range, 0.3 sively to ragweed antigen byy resuspending
to 1 0 x 10 cells/m4, then DNAasc (0 .1 them in I ml of MEM-JOKLIK solution and

pg/m1) was added to the cell suspcnsions to incubating them with I ml of dog IgE-rich
prevent clumping . The cell suspension con- hyperimmune antishort mgweed serum . Aftained95 .1 2 .a3cfomastcellsln=7). Cells terpassivesensitization,thecegswerewashed
(approximately I x lo cells/assay tube in twice in calcium-free Tyrode's buffer and
a fmal volume of 0 .25 rtd) were incubated with resuspended in Tyrode s buffer containing 25
radioligand at 25C, and incubations were ter- mM HEPES andO.10lo BSA in a manner ideaminated by dilution with 5 ml ice-cold PBS tical to that used for nonimmunologic chatand filtration under vacuum through What- lrnge and at similar concenlmlions
manGF/Cglasshberblters.Fdterswerethen immunoglobulin E-nch, hyperimmun
.
o
washed twice with 5 ml PBS and, after addi- anti short ragweed serumwas obtained frorn
tion of scimdlahon Buid, were counted by acolonyofdogsthalweresenntizedtoshon
liquid scnntillation spectrometry at an effi- ragweed antigens by repeated parenteral inciency of 5Olo . For rdennficanon of beta)ectionsafterexposuretoliveauenuateddisadrenergic receptors, 1'HJDHA was incubated trmprr virus (11) . Serum used for passive sen-
PuBLrC~iTrONS 014411~
10333400
1021
CHARACTERIHInDN OF PYRIFIHD DDD rrAarnC/TGYA Cr1E5
sitiiation was obtained from dogs that had

been shown to have marke-0 cutaneous and
hronchral reacs ions to short ragweed antigcn
ssc+ciated with high levels of specific IgE
RAST serurn levels (12,97 t 1 .18, mean ~.
SEM) (12).
masched with 41p bes to which only the releasing agent was added . In addition, spontaneous histamine release was also examined in
quadruplicate iri tubes containing eells, but
neither releasing agent nor autonomrcagent .
Hrsramrne Release Jrom Cells
Histamine release was calculated as the

amount of hfssainine present in the supernatant fmctson expressed as a percent of the total histamine present in bot h supernaa ns and
pellet fractions, The effect of an autonomlc
agent on a ragweed antigen or compound
48/80 dose-response curve was analyzed
statisucally fur each experiment bytwo-way
analysis of varlance using replicate .aluhs lo
determine whether significant differences ex-
Histamine release induced by compound

48/$0or ragveed anttgen was carried out by
incubating Ihe cells with the releasing dgent
at a final reaction volume of I ml for 3d min
at 37' C in polypropylene t ubes W hen ph armacologicagonisss were used, the) were first
inclibated wilh the cells for the tollowmg
times : ]-min Incubauonwith ACh, 5-min incubation with isoproterenol . lerbutalina and
phenylephrine . When the pharmacologic antagonists piopranolol, phentolamine, and
anopi ne were used, t hey were incubated wit h
t he cells for 5 min before the addition of the
agohlst.
At the end of the release experiment, the
reaction was stopped in irx, the cells were centrifuged at 450 x g for 10 min, and the supernatanl was separated from the pellet by
aspirat ion . perchlonc acid was added to bot h
cell and supkmatant fractions to a frnal concentrauon of 0 .2 M in order to lysc Ihe cells .
and Ihe samples were stored at -70 C until
assayed for histamine
Experfinenral Desrgn
Pharmacologlc modulaaon of histamine release was evaluated by 2 methods: In one, a
single concentration of the autonomic agent
was examined for its effect on histamir6e release induced by differenl concentrations of
the releasing agent ti .e., a dose-responsecurve
for the histamine-releasrng agent). Concentrationsof ragweed antigen used for a doseresponsecutvemcluded : 1, 5, 10 . 25 .50, 1(Cf,
250 ; and 5f10 trg/mI : concentrations of compound 48/80 included : 0 .01, 0 .02, 0.05, 0 .1,
0.2 . 015 . I, and 10 pg/ml . A few of these concentratlons were omitted in some doseresponse cuFves, but all curves consisted of
at least 6 concentratiuns, each in duplicate
or triplicate. Each dose-response curve always
included control tubes t hat received cells but
no releasing agent so t hal spontaneous histamine release might be determined . For each
espAnment, parallel doseresponse curves for
each histamine releasing agent to which the
autonomic agenl or agents were added were
also analyzed . These dose-response curves
also included tubes to whlch no releasing
agehl was added but which contained the autonomfc agent in order todelermine whether
the agent had any direct effect on histamine
release In the other method of evaluatfon,
the concenttation of the autonomic agent was
varied, white the concentration of the huta
rnrrie-releasing agent was unchanged . Thc
:oncentrauon of the hhs ammeaeleasmg agent
was chosen to cause apprortmately 500?a o(
the maxsmal histamine release observed prcvlnttslv in doseresponu curves wnl
: Ihat
agent . Each concentration of the auronomrc
agent was examined in Uuadruphcate and
I !.~
~
rf
G(ara Analysrs
isted in hislamine release between the dose-
response curve containing the releasing agent

alone and the curve to which the autonomic
agent had also been added . In order to e>:amme statistically all the experiments with a par-
licular autonomic ageni as a group, the histamine release values were normalized by disignatmg the maximal release as 100% and
expressing all other values as a percent of the
maximal release. The extreme ends of the

dose-response curve probably provide art unreliable basis for desermining the inhibitory
or enhancing effect on histamine release of

an autonomic agenl . For this reason, modulation produced by an autonomic agent was
estimated on the basis of its effeclon that
part of the curve at which 50% of maximal
histamine releaae occurred (the ED .n for histamine release) . This effect was expressed in
termsof percent inhibition (or enhancement)
according to the formula :
comrol - eaperi__ental
~a inhibition = control x tm
wherecontrollsthepercent hrstaminereleased
by the releasing agenl and experimental is the
percent histamine released by the releasing
agent in the presenceof the autonomic agent .
The values to be inserted in this formula
were obtained as follows : t he percent release
wit h t he releasing agent cor responding t o the
E Ds was calculated by subtracting rhe spontaneous release from the maximal release obtained and dividing by 2 . In order to dctermtne the corresponding percent release in the
presence of the autonomic agent . dose-response curves were drawn graphically . The
point on rhe dose-response curve fa t he releasing agent corresponding to the ED,o,'alue
was found by adding t he spontaneous release
to the F Dso value. From this poi nl, a perpendicular line was dropped to the abscissa . The
percent telease was noted at the pointat which
this line cut the dose-response curve constructed for the autonuntic agent . The required value for the formula was then calculated b} subtracting the spontaneous release
in the presence of the autonomic agent from
the value obtained from the graph, ai described prevtously. Experiments with different concentrations of the autonomic agents
wcre analyvrd individually by Student'ss test
and s a group by 1woway analysis of varlance using rrplf :ate values .
PUBLICATIQNS
10333401
~.
F 9 1 T,ma Coume Or Epe<inC 1'HIDHA b,nOmg 10 i60

neteG .
nraor oog masrocytoma eells. Bmaenq v.a5 per .
brmea a125 C at a hgnna eonpentranon Of 09 nM
As ~ano was ramtl vnth a L of 25 m,n (-)pro anOlo
1 (r
PM)was added arthe anow and3 d iesooauon r.ol-
lowea for 30 m,n Data Irom i Ya{ponmenr pe .fomrM

in Oplsata, wM1rCn were typatlor 3 such espenmenta .
e .e snswn
Histamine Assay
Histamfne was determined by the o-phthalaldehyde spectrofluorometric procedure of
Shore and coworkers (13) . but modified for
autoanalysis (14) with an automated spectrofluorometric analyzer (Alpkem Corp .,
Clakamas, OR). The autonomic agents, compound 48/80 and ragweed antigen, wereeach
tested in the concentrations used in these experiments for auto0uorescenceand for their
possible effects on the determ-inauon of
known histamine standardsReaulte
Radro(igand Binding
1'HJDHA binding to dog mastocytoma

cells was displaced by I f,M (- )propranolol . Binding was rapid at 25C, reaching equilibrium at 15 min, with a mean
half-ti me of 1-9 min (n = 3), and was partially displaced on addition of 1 ytM
(-)propranolol (figure 1). Scatchard
analysis of propranolol-displaceable
binding revealed a curvilinear plot, and
computer analysis showed that the data
were best fitted by assuming 2 different
bindingsites (figure 2) . The high affinity
bi nd ing site had a mean dissociation constant (Ko) of 0-90 0.17 nM (n=5),
with a maximal concentration of binding sites (Bmax) of 35 .7 t 5 .5 fmol/10
cells, corresponding to 21,500 :t 3,300
high-affinity binding sites/cell (n=S)The second binding site did not appear
to saturate, and it was therefore not possible to estimate Ka or Bmax- Binding
to this nonsaturable site was reduced by
approximately 15%u when 100 M phentolamine was included in the incubation,
as described by others (1S) .
Catecholamines competed for /aH1
DHA binding with the rank order of
potency isoproterenol > epinephrine >
norepinephrinc, indicating binding to
beta-adrenergic receptors (table 1 and figure 3). The beta,-adrenergic agonist had
a relatively low affinity, as noted by
others in receptor-binding studies (16) .
014412
<
1022
nNILLSS . MnNEa, Syq pCXs +
~
~
-~
~ - ~
soi
}0 -
.0
60
ep
D
-e 1
iW
137NTr .e 9o1MU nmdr+06,ab1

F1g 2 SC91Chare analysis Of pMjDHA bmping to Is6
Iated, ,maCl dog masmcytoma L911s. TTIe d,tf6renCe be,
aveen Imel binding antl binding in the presence ol 1

yM (-)pmprarqlol s ITIOnerl- Tne wrve wee ened by
nonGnear Isasf-squarea CUmpuler enalysls . The dota

.were oesl o9scnUee Lry .asSum,ng a mv-sne rmeratoon
The high efen,lY sila 6 rapre59Mr/ by tha steepstra,gnt

nne . Tne slope Of wn,ch,9,yeJ the d050CIaLOn Con9tanl
(Kal. and the i merceprw4h The aU5os9a 9w991he man
Imal CpnpeOireaon pf binding yes ndmaxJ The non.
saturBCle sne ie repte5AMe0 py me nDr aontal line The
date shown afe fmm 1tpenm9la perfnrmep ,n pupln
rate giving a KO of I I nM anC Bmsa of 42 u frnoVtO'

Ce118 (25 :.,ag MnD Fng SuteNC91q TTB was typlta/ pf 5
separeroe.penmentsg,vmgameanKOO1090 x 0.77
nM I s sE)antl emae o12r, .A0 s 3.30Dbrnamg snOSCelI
The (-)isoproterenol displaced approximately 30% of total binding, which is

equivalent to 60Oio of propranolol dis
placeable binding at a .similar concentrationof!'HjDHA~ Competition for /'H[
DHA by (-)propianolol showed a complex curve which was best fitted by assuming 2 binding sites . The first, competed by low concentrations of (-)propranolol had a high d .ffiniry (inhibitory
dissociation constant K, = 1 .6 0 .5 x
10- M ; n- 3) and accounted for approximately 60% of the ['H[DHA binding .
This is similar to the k, values reported
for propranolol in membrane preparaTADLE I
nV ADRENERGIC .AGONISTS
1)ISnprdarenol
1IEOnaphnne
Terbuta4ne
i JNOrepInephr.ne
l-)Proprsnolor
/IOroprendot
. .
07 . t o"
223 06 x t0's
6 A= 06
1 I x D 6
Rnsez
10`
110'
05 n
IU`*
Kl4 2x23x 10-'4

132 06
pI
.3
IM)
1,
109
.10
.0
IANTAGOMSTI
(M]
F,g 3 IMID,tmn ql speMs ('HIDHA Dmeingfi anlated,,ntaCl tlOg maslocyt9rnacens br adrenerg,c agena rho
mncenlrgponofr'NIDNAIntfresestudosrang9tlfrpnO8tolSnM L6npBng/ InhibltqnbYagOmgs ~-Jqoprjte,arpr
(cloBeDCrTJ96J . (-)ep inephnne lopan CllLhs). temutal,ne lOp9n (nanpl96) . (-)norepmephnne (C/oap, mLngys)
Conlrol refers ro,SOpm[arenpl CpmpaGaen AlgMpan9r
Inhibition
by antagonl5la (-Jprppr9nol01(NOSetl squyyp)
and(+proprannlol(Cppnsqu9rsd) Cpmrolsr91prm1-)propranololCOmpehbon .CurveawerelleeohyaMnl,npa,
I6asl-squares C4mputer program . For (-)proP2nptol Me tlala were Llled by assum,ng an o hiDihon ot binding
612 3aes . ThedBla shown are rmm I expenm9nl . perfprmeU m Gupl,cate. which w5s typiCal pf 3 such eapenmenry
tions(17, 18) . The second site had a much

lower affinity, being approximatel)I 1,000
times less than the first . Competition
with (+)propranolol . however, showed
only the single low affinity site . Thls suggests that the high afOnity, site, which is
stereoselective, probably represenlS displacement from beta-receptors . whereas
the low affinity site, which is not sdereoselective, corresponds to the fraction of
binding that is nonsaturable as i)etermi ned by arla[ysis of the sat urat ion eurve.
With ('Hjprazosin . approximately 10

to 15% of total counts bound were displaced by 10 or 100 pM phentolarnine,
but Scatchard analysis revealed no high
affinity binding in 3 experiments, there
was no inhibition of binding to l00 pM
(-)norepinephrine, indicating Ihat alpha-l-adrenergic receptors could not be
detected on these cells .
With I'HIQNB using concentrations

to 2 nM, there was very little binding after 60 m i n a125 C(300 to 1,200 codnts/
l0 cells) and no inhibition of binding
showed that this effect was significant in

each individual experiment (p < 0:01 in
5 experiments and < 0 .02 in I e,iperiment), and also when all experitttents
were taken as a group after normalization of values (p < 0.001) . The percent
inhibition caused by the drug at ED, for
histamine release was 67 .7 :t 15 .4 (mean
SD) . Incubation with (-)propraelolol
(10-' M) blocked the inhibitory effect o
terbutaline and resulted in a dos
response curve l hat was not significantly
different from the dose-response curve
with ragweed antigen (figure 4). ,
The effect of terbutaline (10'' M) on
compound 48/80 dose-response curves
was examined in 6 experiments, I ezample of which is shown in figure 5 . In 5
experiments, terbutaline reduced the response to compound 48/80 at all doses
(p < 0.01). In I experiment, no signifrcant change could be demonstrated by
analysis of variance because of crossover
of the curves in the plateau region, but
suggests an absence of detectable nms-
K, (M)T_
19 z
'.A
(AGONIST)
in the presence of I M atropine, This
INHI131TIOn OF SPECKIC
I'HIDIIavDROALPRENOLOI 61NDIN0 TO
IgOtLATEO DOG M1bASTOCYTOMA CELLS
Agenl
\Y
:5
,6
ie
. 10'
' vaues "s mean u SE v1 3 veon,nx e .o.nmenu
I 4,ne .IOh OR54c .a1Wn (p,gl9ni (I(~ .vM oel.nn~nqe no~n

Ira e,a.alm ìr
.,ne .e ,C .. ..a. nu conOSnnncn u,
' ~a
apen,[eWw(QSpib
.nno,pn01I'MIDrUaMn9. (llvy IM1p
[urcwv, .woo w 1'*IDn . mv ny
.e,p,0~ N 9 nsp- en0 ee
"++~M nean u .ne0c~a,m (enslnrn
~ n . .uuee ~w ,- IO .nn~edvwr m, n .p.. rrwr an ... .q v,en

A qW 1o - ,.In,Ia^,nJV6n9s
tn'M~W ~w r Iam
carinie
cells .
cholinergic
receptors on these
Phprmpcologx Studies
The adrenergic agonist terbutaline was
evaluated for its modulat ion of histamine
release induced by both ragwecd antjgcn
and compound 48/80. In each of 6 experiment s, t erbutahne (10-' M) altered the
ragweed antigen dose-response curves,
one example of which is shown in felgure
4 . The maximal response was reduced
and the concentration of antigen needed
to induce a response that was 50% of
maximum was increased . 7ivo-way analysis of variance using replicate values
-1
!
wa .wn +.ny .n wy~m
F,y 4 Ibetrrasponaa cuTros showrng LM pa '

hgtami ne relea6e in respCn6e to different CanCBnla'
tMna of ragweed ant,gen . with ragwsed ant'qen aNne
(c1psB0 farGas) . m the pres9nce of 1P' M lerqra/me

(opOn m9ng/es), and in the presenCe Uf 10" M tsrelN
Ime ano lD > M propranolol /opm+ Grcks)
PUBLICATIONS "rTmR1T
10333402
CM~WICrERIZ~noM
pr
VeRmFD
DOG
W,SrOLVIOMa
Fig S Uee@reSponse CurveS 5hewing the p9reent

nistamme r916b6e in reaponse ]o Udterenl CpnGemra1ons ol Compoend 48180. ,.ith 1Ompo, .n0 aa/aOalone
(C1p148d C~K'I65) . ~n the pre66nCe of 1e1 M Ierbulalino
(opsn mengrest
. ane ,nrne breaence ai to` M rarUUlaM1ne antl 10" M prop/enolUl (open p/e/9s)
in this experimenl terbutaline did cause

inhibition at the lowerconcentrations of
compound 48/80, with a percent inhibition at the ED,> for histamine release of
66.4% . The percent inhibitic,n for the
group was 57 .7 9.2 . iivo-way analysis
of variance on normalized values for t he
group also showed that terbutaline (10-'
M) signit icam ly aherrd t he dose-response
curve (p < 0 .025). Incubation with
(-)proptanolol (10-' M) again blocked
the inhibitory effect of terbutaline and
esulted in a dose-response curve that was
not significantly different from t he doseresponse curve with compound 48/80
alone (figure 5) .
The beta-adrenergic agonist isoproterenol was also evaluated for its modulat ion of histamine release induced by ragweed antigen . In each of 2 experiments,
isoproterenol (10-' M) markedly inhibited
the ragweed antigen dose-responsecurve,
an example of which is shown in figure
6. The percent inhibition caused by tsoproterenol at ED,/ for histamine release
was 85 .2 ~ 12 .5 . In 4 additional expcriments, each in quadruplicate, the effect
of different concentrations of isoproterenol on histaminerelease induced by 50
pg/ml ragweed antigen was examined
(figure 7) . In each experiment, all concentrations of isoproterenol significantly
inhibited release of histamine (p < 0 .01)
when compared with their preassigned
controls, and similar significant differences were also found when all 4 experiments were considered as a group . Incubation with (- )propranolol (10" M) par
tially blocked the inhibition of
'coproterenol (10" M) on ragweedntigen-induced histamine release (figure 7)
. ln 2 of the 4 experimems, histamine release in the presence of
( - )plopranolol and isoproterenol was
not significant ly different from histamine
CEris
NY,ew,o F.y.9r
release obtained in the presence of antiIt. om .uu
gen alone, and when a11 4 experiments
,oor
were considered as a group, no significant differences were found . In a sepae
rate experiment, the use of (+)propranolol failed to alter the inhibitory effect
of isoproterenol on antigen-in'duced
histamine release .
The alpha-adrenergic agonist pheny6
ephrine ( IO-' M) was evaluated for modulation of histamine release induced by
ragweed antigen in 5 experiments and
modulation of histamine release induced
o . . nw)
by compound 48/80 in 2 experirments .
rig 7 HiSlam~ne .9leasetnryyceLlqrpgn,,a6eanl
Phenylephrine (10" M) inhibited hista(50 regrmr) m me presenee bt mnyrent tmnpentrn'
OI ~dOpml9renol (541W Dar3) }iipre3aee a5 B p9ree
mine release induced by antigen by 18 .3
a malChed eGMml wtnGYl Imeprotprenol ?Te BHe
m 23.7% and enhanced histamine release
,nCUbaeon w .th botE 10" M prApranolel aOE lD-i N
induced .by compound 48/80 by 6.3 m
proterenol bebre the atl6i(Icin ol fagweEd y,4gr
4 .0%u at the ED,o for each mast cell actishpWn q Ine hal[rlatl pa1 . The rd5ul16 are eepM :
as mean z SD TTe aponreniwus release w99 3vator, respectively. Neifher overal0, nor
a 1394 H1stam,ne releese was induced Gy 50,rgrr
in an individual experintent, could Jphenragw64tl anl .gen cakulffied to g.vo spprOamatnly I
ylephrine be demonstrated to cause a sigmal,mal hlstaamme releasB (adunl releaYe achle
nificant change in the dose-response
2ar s 004%)
curve.
The etlolinergic agonist ACh (1d- M)
was evaluated for its effect on histamine
ragweed antigen (1 to, 100 pg/ml) w
release induced by ragweed antigen in 5
examined and no significant effect of
experiments and on histamine release int her agonist was obser.+ed, suggesting r
duced by compound 48/80 in 2 experihancement of nonimmunologic or i
ments . Overall- ACh caused enhancemunologic activation of Ihe mast cel
ment of histamine release at ED,e of 11 .4
Incubation times with ACH and betl
- 7-6% (or ragweed-an[igen-induced renecol were varied frorh I to 5 min .
lease exp'eriments and 8 .5 x 4 .74'u for
In these series of experiments, t
compourld-48/80-induced release . Howmean histamine content of the cells w
ever, in rtio experiment did Ach cause a
6.5 x 3 .6 pg/cell and the mean spom
significant change in the dose-response
neous release was 4.3% (range, 0.6
curve by two-way analysis of variance .
8 .5oJo) . The mean maxiunalhistamine .
In another experiment, the molar conlease induced by ragweed antigen w
centration ofACh was varied from 10-10
3fi-4t7n (ra nge, 17 .R to 68', 3%) and by coi
M to 10- M in tenfold increments and
pound 48/80was 65 .5% (range, 47-8
its effect on histamine release induced by
74 .40/0) . Cell suspensions yielded a hi,
compound 48/80 (0 .2 pg/ml) was exampercentage of mast cells (93 .8 m 3-4ined . No significant differences were oband cell viability, as assessed by trypi
served in histamine release in the presblue staining, was always in excess
ence of ACh at any concentration com92% . None of the auto I nomic agents
pared to the release with compound
releasing agents caused autofluorescen
48/80alone. The effect of both bethaneat the concentrations used, affected I
col (2 experiments) and ACh (2 experimeasurement of known histamine sta
ment s) over fu l I dose-response cu : ves for
dards with the autoanalyter, or alterl
compounds 48/80 (0 .01 to 1)<g/ml) and
significantly the rate of spontaneohistamine release
. There was no diffe
ence in histamine released by antigen
preparations from which amphoteric
B(fungazone) was removed by washil
or from preparations in which no ar
photericin B was added (n=5). indica
ing that amphotericin B not removed I
washing had no effect on histamine r
lease itt these cells .
~
~
Fig 6 Oose-response-c .nves`show.ng the pemem

ngtam .ne release n responga to dArerenl COneentrarions of mgweeo am
.gen . -rth ragwaee anogen alonp
tcrose0 nrcros I ana ~n me vresenCe ol to' M~soprpre~ena lopen rnangres)
PUBLICATION$
10333403
Diacussion
The presence of autonomic recepto

in purified mast cell preparations hi
014414
wowos. rwwee . ua oota
1024
proved tb be a contentious issue In this

study, we demonstmtea the presence of
beta-adrenergic receptors but no alphaadrenergic or cho6nergic receptors . These
findings are based on both radioligand
studies and pharmacologic studies that
+.vere in accord with each other . In comrnotd with all purified mast cell preparations, rhk results are subject to the criticisrri that the prooedute used for the isolatidn of the cells may have caused
damage to cell membrane receptors . We
helieve that this potential hazard has been
minimized in this study by the use of
a gentle disaggregation process, using
highly purified collagenase, and more importantlN by the fact that purifrcation did
hot require density centrifugation, a techniqtie known to result in a loss of 50 to
80% of IgE-receptorbinding activity
(19) . We have also shown, in another
study, a close similarity in the morphologic characteristics of mastocytoma cells
in situ inn the tumor and those in culture
aftet disaggregation (20) . These results
conl irm those of other workers (3) who
compared the effect of collagenase and
pronase disaggregation procedures on
dog lung and mcsenteric tissue, and
found that collagenase had advantages
over pronase and produced a high yield
of mast cells with ultrnstructures always
intar_t .
We believe these mastocytoma cel ls are
similar to, and reasonably representative
of, thormal mast cells on the following
grounds : (!) They are morphologically
and histochemically very similar to normal dog mast cells (20) . (2) They appear
functionally viable and similar to other
species in that the cell histaminecontent
is similar to that of mast cells in dogs
and other species (20) ; they bind IgE and
can be triggered to release histamine by
immunologic and nonimmunologic stimuli (8, 9) ; they are capable of generating
leukotrienes (8), and the autonomic
receptors and pharmacologic modulation
of secretion is similar to that described
for dog lung in vitro (21) and in vivo (22)
and to isolated human lung mast cells
(23).
The presence of beta-adrenergic n-cepiors on mast cells was first inferred from
studies of heterogeneous lung tissue in
which beta-adrenergic agonists caused inhibitionof mediator release . However, it
cannot be assumed that beta-adrenergic
receptors on mast cells that have been purified and are from other species necessanly modulate mediator release. Indeed,
studies of purified rat peritoneal mast
cells and murine mastocytoma eel ls have
failed to show such a link . Although t here
is one report that beta-adrenergic stimulation of rat peritoneal cells in vivo appeared to inhibit mediator secretion (24),
most other studies using cells that had
been purified by centrifugation through
density gradients failed to demonstrate
inhibition of mediator telease (25), afthough increases in cyclic AMP may occur (26). It appears that in rat peritonea]
mast cells the adenylate cyclase stimulated by beu-adrenergic agonists may not
activate the cyclic-AMP-dependent protein kinases that affect mediator release
(27) . Furthermore, Sullivan and coworkers (26) found that the effects of a varfety of catecholamines on rat peritoneal
mast cell cyclic AMP were inconsistent
and did not fit into the generally accepted
pattern of alpha-adrenergic and betaadrenergic receptors . Thus, epinephrine
and norepinephrine stimulated cyclic
AMP production and inhibited mediator release, whereas isoproterenol had little effect on either cyclic AMP levels or
compound-48/80-induced histamine release. Nevertheless, there is evidence from
radioligand binding studies that betaadrenergic receptors are present on rat
mast cells (28-3 1) . Thus, Marquardt and
Wasserman (31) demonstrated the presence of these adrenergic receptors on rat
serosal mast cells, and showed that betaadrenergic agonists caused a rise in cyclic AM P levels, but were unable to demonstrate anyeffect on IgE-mediated mast
cell histamine release
-fhus, pharmacologic studies alone, or
radiol igand b inding studies alone arc inadequate for characterization of receptors on mast cells- In addition to radioligand binding studies, we therefore
undertook pharmacologic studies to examine the modulation of mediator release. In this study, we have shown that
beta-adrenergic agonists can inhibit immunologically and nonimmunologically
induced histamine release and that this
action is affected through a stereoselective site that can be blocked by (-)proptanolol but not by (+)propranoloL This
finding indicates the presence of functional beta-adrenergic receptors and substantiates the conclusions drawn from
studies of heterogeneous human lung tissue and confirms reports recently made
on purified human mast cells (32, *3) .
It is of interest that dog mastocytoma
cells in this respect appear similar tohuman mast cells . in contradistinction to
rat peritoneal mast cells or murine
mastocyioma cells .
The demonstration of beta-adrenergic
receptors by radioligand binding assays
in our study correlated with the inhibi-
PUBLICRTICNs
10333404
tion of histamine release by beta-adsenergic receptor agonists . ['HJDHA boufg

to intact dog mastocytoma cells and
displaced by (- . )propranolol. Scatch:6
analysis of displaceable bi nding revealed
a component that had an affinity similar to that e.xpected for 1'HJDHA binding to beta-receptors in broken membrane preparations (l7, 18) and other isolated cells (34)- There was a second
binding site, which was not satorable.
Similar nonreceptor binding that is displaceable by propranolol has also been
described in isolated fat cells (15) and in
rat peritoneal mast cells (28) and nSay be
due to the solubility of ['HJDHA in the
cell membrane. Further e .idence lhat the
high affinity binding represents bihding
to beta-receptors is provided by the
demonstration that high affinity,binding accounts for approximately 60v/o of
displaceable binding and isoprorerenol
competes for a similar proportion of
binding (60Io of propranolol displaceable binding;. Funhermore, the proportion of binding that is stereoselective is
also similar to the component of displaceable bindingthat is of high af6inity.
All this evidence suggests that high affrnity ['HJDHA binding to dog mastocytoma cells labels beta-receptors . Agonis~
competed for binding in the rank ord
of potency isoproterenol > epinephrine
> norepinephrine, suggesting that the
predominant beta-adrenergic receptor
subtype on these dog mastocytoma cells
is beta, . The affinities of adrenergic
agonistswere lower than expected for interaction with beta-adrenergic receptors,
as determined by competition studies in
the cell membrane preparations. However, this discrepancy has been described
in other isolated cell preparations (34)
and may be due to lhe presence of guanosine triphosphate in whole cells, which
reduces agonist affinity, either because
of desensit ization of beta-adrenergic receptors during incubation with agonists
(35) or because of metabolic breakdown
or uptake of catecholamines . The beta,selective agonist terbutaGne similarty had
low potency, but a low affinity and absence of beta,-selectivity of this agonist
have been described previously. These
findings suggest that the selectivity that
is seen physiologically may be due to
differential activation of adenylate cycl ase (16) . The af finity of binding of propranolol to t he stereoselective binding s
was in good agreement with that det
mined from competition experi4nents
with whole cells (34) and membrane
preparations (17, IS) .
The density of beta-adrenergic recep-
I
tors (21,500 33UD receptors/cell) was
high, although greater numbers of bind
g sites havp been reported in other inact cells including fat cells (15), cultured
mllsclecells (36),and tat peritoneal mast
cells (28 . 31) . The high density of betaadrenergic receptors on these cells may
indicate the presence of spare receptors,
which are a means by which a cell
achieves a high sensitivity to agomsts . as
stimulation of only a fraction of Ihe
receptors results in a maximal response .
This might also account for the dis
crepancy between thecffect of isoproterenolon binding, and its funnional effecL
The presence of alpha-adrenergic
receptors on mast cells was initially
postulated in studies of heterogeneous
human lung tissne in which alphaadrenergic stimulation by phenylephrine
or norepi nephrine (n the pmsence of propmnolol) resulted in enhancement of
anligcn-induced mediator release (37) .
Opposite results were obtained by Nm
and Bloom (38) in rat pentoneal mast
cells . They found that norepinephrine in
the presence of the beta-adrenergic antagonists propranolol or practolol inhibited histamine release, and this effect
was dimimshed by phentolaminc {ndiing that alpha-adrenergic stimulation
~'nhib{ted mediator releasa We were unable to find any evidence for alphaadrenergic receptors in these dog
mastocyloma cells based on radioliga.rd
binding asszys or pharmacologic effects.
lb our knowledge, radioligand bindinR
assays have not been used before to look
for alpha-adtenergic receptors in purified
mast cell preparations .
The presence of cholinergic receptors
on mast cells was also first inferred from
studies on human lungussue (37) . Most
studies on purified cells were based on
the rat peritoneal mast cell, and once
again confliaing results exist . Some
workers (39, 40) reported cho6nergicinduced histamine release in W istar rats,
but others (41-43), using dif feren l strains
of mts, could not demonstrate arry such
ef fe<u. Masini and ooworkers (44) later
reported variable results even within the
same species, and could find no correlation between ACh-induced histamine release and the high af6ruty binding of
/'H]QNB, a specific cholinergic muscarinic ligand . to rat mast cell membranes. These discrepancies might be due
o o diffcn:nces m the species, to differcnces
n purification lechniques, or, in hcterogeneous tissue such as lung, t he choliuer .
gic agonist may be acting indirectly
though other cdl types
. This laaer possibility is thought to he the case in the
autonomic regulation of type 11 pneu-
mocyles w here chotinergic agonisu cause

secretion of surfactant but not in virro
(45 . 46) . An alternative source of varia-
tion in rat mast cells was suggested by

Schmutz)er and coworkers (47), who
found that cholinesgic-induced histamine

release occurred only sn sensitized cells .
A similar rffect was noted in dispersef] ;
dog lung cells, wherechoGnergioinduced
histamine release could oNy be demonstmted in cells from actively sensitized

dogs (3)- These effects, however, appeat
to be relatively small . In our study, with
dog mastocytoma cells taken from an an-
imal that had not been actively sensitized,

we were unable to fi nd any evidence for
cholinergic receptors based on radioligand binding assays or pharmacologic
effects.
It is apparent that a confusing picture

exists witb regard to the autonomic coMtrol of mast cells. Some of Ihis confresion may be attributable to studies that
were performed on heterogeneous tissue
or cell suspensions . While the need for
pure mast cell preparations has been appreciated, it has proved difficult to
achieve thls goal without extensive puri frcation procednres that may, in themselves, be deleterious to the cells- The
most readily available pure mast cell
preparationscome from rodent sources .
Most rodent mastocytoma cell lines suffer from limitations in their histamine
content or the releasability of their histamine, while the rat peritoneal mast cell
appears to differ significantly from the
reporled characteristies of human mast
cells . Thus a further element of confusion may be due to species differences .
More recently, Bienenstock and coworkers (48) suggested that mast cells may
have marked differences in morphology,
histochemistry, and function within the
same tissue, further compGcating attempts to characterize mast cells of even
the sarne species . If this is the case, it is
important to have spenfrc knowledge
about the behavior of mast cells in the
laboratory animals being studied, and
even the behavior of mast cells within
specific tissues . Knowledge about dos
mast cells is relevant because dogs have
been used widely to study bronchial ro~
anivity. The dog mastocytoma cells caami ned in this study are morphologic2lly
simtlar to normal dog mast cells and have
the advantage of being readily availablc
in a relatively pure form . Our frndings
that these cells possessed beta-adrMergic
receptors but no alpha-adrenergic, or
eholinerg7c receptors was Strengthened by
concordant results from radio6gllnd
binding assays and pharmaeologic

studiect, whereas many previous invtstigators have relied on evidence from one
source olily.
Acknowlcdgmenr
The vnters wish to thank Ryckie ttragner/
Hall foi her assistance in preparing thi9 mau
uscript, Mary Helen Briseoe for the illussrations, and Denise Bcnkek Roben L StueldsAndnw D. KeOer and Lassrence McCabe for
their technical 25sutaslce.
References
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1977; 3b.I676-g3 .
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by anu8m tn pa.ssMly senssliitd human lu ng . Na .
rure 1969: 22naOlb-9 .
3 . Hc4manns l . Brerenat H
. Sctmruvla W Comyar~bve nudies of maat ceUr from normel (nonrmmiuiitN) and acvvely uanuud dogs . Agcnts
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Pruzarnky 1J . Aespwrory mml cHa and basopnilmd cef]s. I . E+.dence t hat t hq are secreted mto rhe
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16 :2231-3 .
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masra :poma cens (ab .arun. An- Rev Respv Dt6
1992. 125 .63 .
l . Taurog JR Mepdov GR Hoot WA
. MeVger H . Nonrytutavc IgE-metluted
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release of tunamrnr nnd sermmun from munne
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deperkdens and .onophorr.mduced genaralion of
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PUBLICBTIOVS
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014416
rozs
-IMPbannamlogral speoRarS-olbaa-1 and bere .2
adreaergic rvoeptnn in rn hean and lung in vrma
Mot Phumacol 1979 . 16di-H.
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Gold WM . An ullrastrvnutal analyers of dog
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21 . Ftef MJ, Cbcsrown SE, Rmi BR-er of- Im .

munological release of hummme Gom dog lungenmpanson of,n vrvo end
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RR. Winrs SC . Gold WM . E/fecx of hetaatlnnergie stimulation on eapmmenrnl ~nr
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23. Schlnmer RP. MacGlau .an OW Jr . Peten SP,
NaG Inflammetarym[d:etonmdmaRensmsoi
mleasr from purified human basophils and ma .W
ee8s. J Allergy Chn Immunol 1984 . 74 <v3-81 .
24 . Koopman WI, Omoge RP. Auuen KF. Immunoehenumi and broloetc popenies of r IgE .
ill. Modolanon of the IgE-nsemaled nkax of slow

reaettog subtanee of ansphylam hy agenu mRurnong ihe level of cycbc 3 .5 ''-adenoasne monophospAate / Immunol 19]0; 105 :1096-102 .
25 . Jonmon AR . Moran NC InWbruon of the

rNW a of bnmmme from ra mau cdls : me effeot
uf cold and etlrenerg,e drugson t6e rNeaseof ]ustaminebycompoundGB/80anGanugeu J Pharma .
col Eap Tha l9"JR 175 :632ao.
26 . Sulhvan Tl . Parker KL . Srevsoo W. Parker
CW. MadWwon o( cy>:bc AMP m panfscd ral mast
[eRS I . Ropousef to Pharcnambjae memuwe and
physical stimug- J tmmunol 1975 : 1I4 :1a93-9. stana of anapMlanu from human h.ag, ly. P,ar
22 . HolgartST-W-m1ov .CM,LewisRA-'Auatm haattmmt by choghergec and alpha-adreptrglig

KF. Ff7erta of prnsmgaodirn D, and theophyWne stimulatson . J F~P Med 19]2; 13&556-7 .
on ra, icroul mast cella : d .yicordantz brrwern .n- 38- Alm PE. Bloom G11 What-if asy-y
creaud cellular Icvels of cyclic AMP and activa role of adrenergic medunigms ip hiaumin<
uonofpcbcAMP4ryen .dcnAPrurrfnlinaw_J Im- fmm mau sas' Agcucs Amions 1981, 11 :6p-6&
munol 1981 : 121:1536-3 .
.
39. Fmtory R . Mworu F, Masmi E, Blandin. g
28- Donlon M- Hunr WA-Cavavaa GN, Ka3inee Menaawm PE Modulation of the spuntaneouy
M. A eRaraeunration of bua-ad .energic reC<plors huumtne releasc by,aNenergic and chobnape
onccVularandpengranularmembrenaofrAtpen- dmgs Agenis Aaions l9]8 : 8'3a-f_Sg.
toneal maat cells . Lrfe Sc . 1982: 31 :411-6 . 40 Bmndina P. Fanrdm R . Mannaioni PF, Masmf
29 Mar9uardl DL. MorulskyH], Waurmian SI . E . CTaractersration o(histartineteleaseevoked by

Rat lung <holmergu rKeptor: chanaennbon and acrtylchuline in isolatxW rat man a1La J Phydol
reguletion by roaicosremrdz J APpI Physros 1982 ; 198Q 3012 81-9 3.
53-231-46
.
<I . Rieman JA . Effect of knwn and suspamd
30. Maa~ni L-Fanm~ R, glànd .na P. Hnl Pres- nesunennsmraleo subsunecs and of some nudeoenm of funaqmng anrve bcta-adrenoccpsors m tsdes on isolated man ttlls . Eapenrmia 19/2 ;
mr mast celh . Conelauon berween (-)PHldr 2gb53-5.
Aydroalpnnolol binding and inhibition of htsta- 12 . f(ansai~k W . Adamas a MauliMks Ci,
mme release Naunyn Srhmtedcbcrgs Arch PhmFadur< of aKlycholine lo release huumioe fmm
macol 1982 ; }21
:p1-0- tal mast calls- Agents Actions 198P 101-3 .
31 . MarquardtDL .WassermanSlChamcrenn- 43 EriavecR-Histamdnereleaeefmmmancds
uon of mt masr ceu beuadrencrgsc rxtptor rn rcM- bypllysrologlnRy oaudring subsunca Agmu pam8 nnd sttmulated arllsby mdioliyand bmdmg . J sions 1981 : 11 :71-2 .
Im.nunol 1982; 129:2122-'1
.
m. Masmi E, Blanditu P, Famom R Brmelle .a3d
33 . Pcten SP ScFUlman ES. MarGlashan DW Jr- ,5~ Mannaioni PF . ConKlasionbetueea[hnhnerpe
SchlermnRP,Ne*haBHH,L :.chtensusnLM . Dis- hsstaminereleaseandpmnudidsnyi-bennlaletFH}
pened Ruman lung mast cells : pharmarologrc QNB3bir
.dingmulceB<brspaAgntfC)ecuadomprnslhvaugsctrom198 ; 11 :55-9.
ftagmasts . Am Rev Resmr Du 198k 126.ID34-9
. 45 . Bmav 1 a G LonBmore WJ Adrtnag4 and
33 . Sahu3manE5 .MacGlashanDWJr,PetcnSP . choboergsregulauondflongsWfaeunfsxrrtrnn
ScMmmerRP,NmvhallHH . LirhtcmtnnLM Hv- intheisolatedperfusedratlungandiotheelveolar
man lung masl erJls : purif.caNuon and charactcr- 1ype 11 cegmculture J B&olChern 1981 :255:66-TL
srannn . J Immunol 1982; 1292662-`7 .
.6, Massaro D. Clerch L . Maadno GD. Sur(a[34 . InSel PA . Stoolman LM . Ratlrobgand bind- tam senesson : evidena that chobnerpe mmWarng to beta odrenergi<recePtors of mtast cullured uon of secreuon is mdireee Am I Phlstol 198s
5a9 cclls . Mul Pharmacol 19"J8 : 1<Sa9-61 .
243 :39-45 .
35 . Toews ML. Hard[n TK . PRrkuu 1P. Iletec4'l . SchmmrJer W, Po4lete-Frrundl G, Raucb K
non of lugh-affSniry agon6l, binding tU bela- $choenftld W. ResPOGSr ro i111mnnOlO,Wl or
ad .energicreceptorson~mm~ceRa .FadProcf98Z chohn[rgsestiauulauomufieplatedmasice8sfrom
91 :1535
an,
gutoea-Piga
sad
ra,te . CIA sympoaunk Ne .
36 . Atlas D, Ha.uki E. Lec-nzta A. Eghty Ihou- [Jrleans . MoetoB ABtryy 1978 ; 14W9-56,
sand betaa atlrenorecepton in a S+nglr B . Nature 48 . BitoconocY J, Befya Aq Penroe F, Dm6uq
1977 . 268 :1au-6 .
1-GoodaneR .Mastce4hetc.rogcndty.denvaaon
37 . Kal,nerM .OrangeRP,AussenKF.lmmunp andNncnon .wieRcmpquuontneiotutinalAC
Io8ical ydcax of Ruramloc and s/ow reaenng suD- lergy Cho lmmunol 19g2 ; 7D:407-12 .
PUBLICRTIONS
10333406
014417
~ cAMP immunocytochemistry provides evidence

for functional VIP receptors in trachea
STEPHEN C. LAZARUS . CAROL B . BASBAUM, PETER J- BARNES.
AND WARREN M :GOLD
Cardiouascalar Reseorch Institute and Deportmenta of Medictne andAnamrny,
Untvers+ry of Cohforrirn. San Franc+sco- CaliJomia 94143
LAZARU$ . STEPHEN C . . CAROL B . BASRAUM . PETER JBARNES, AND WARREN M . GOLD . eqAfh imrnunotytoclyrmstry
promdes evidence )or funcrional VIP receptora in trachea Am .
J . Physiol . 251 (Cell Physiol . 20)e C115-C119, 5986 .-Vasoat .
tive mtrstinal pepdde (V1P)- fvst isolated from porcine mtestine (S . 1 . Said and V . Mutt . Scirna-e Was), DC 169: 1217-12181970), has been identified in postgenglionic autonomic axons
of many ti .c.nrs. VIP has potent regulamry effrots on the
function of various cell typfs within these ti58ues, ranging from
relaxation of smooth muscle to ion transport . Recently, VIP
has been implicated in the regulation of mucus secretion in the
respiratory tract, a process involvmg releas .e of macromolecules
from exocrine cells and transport of ions and water across the
airway mucoss . However, because airway glands and mucose
both consist of mixed cell populattons- it was unclear which
specific cells contained VIP receptors and contributed to VIPevoked responses. We idenufied these specific ce0s by using
immunoeywehemical techniques to monitor concentration
changes in odenosine 3'-5'-cyclic monophosphate (cAMP) . the
intracellular compound known to mediate VIP reaponaea . Serous and mucous cells of ferret trecheal submucosal glands and
cihated and basal cells of dog trsebeal mucose all increused
cAMP in resportse to VIP stimulation . We conclude that these
cell types nossess VIP receptors and thus participate in VIPstimulated responses. In contrast ferret tracheal epitbe(ium
and dog epPtbelial goblet cells sbowed little or no reactivity
after VIP, and tbuswe believe that these rells lack VIP receptors .
ion transport : mucus secretion ; cilisted epitbelial cell ; submueosal gland: dog: ferret
The biologic effocts of VIP in the respiratory tract

appear to occur at sites close to nerve fibera containing
immunoreactive VIP. VIP produces dilatation of blood
vessels in the nnsal mucosa (14) and lung (15), causes
relaxation of guinea pig tracheal smooth muscle in vitro
(19, 25, 35, 36), protects against histamine and prostaglandin Fye-induced hronchocnnstriction in dogs in vivo
(24), and increases short-circuit current and ion transport in dog tracheal epithelium (I6) . It has also been
reported to both stimulate (18) and inhibit (4) the release
of radiolabeied macromolecules from ferrM and human
tracheal esplants, respectively .
It is believed that VIP-evoked effects are mediated via
adenosine 3',5'-cyclic monopbosphate (cAMP), since

V IP stimulates adenylate cyclase in a number of systems
(2, 3, 7, 9, 11, 22, 27) . In rat intestinal epithelium, VIP
not only stimulates adenylate cyclase but also at.imulates

ion transport (6) . binds to specific receptors (21) . and
stimulates cAMP-dependent protein kinases I1 . 9, 101 .
Furthermore, cAMP phosphorylates specific proteins of
rat intestinal membranes associated with ion transport
(28) .
Many investigatnts have now identified aod character-
ized receptors for VIP on cell membranes (2) . However,
there is presently no method for detecting functionally

coupled VIP receptors on individual cells in a heterogeneous tiasue such as the trachea. Even if the various cell
types could be successfully isolated. disaggregation techniques might alter the characteristics of receptors . We
have taken advantage of the physiological coupling between VIP and cAMP and have used intracellular cAMP
VASOACTIVE INTESTINAL pErrlnE (VIP), thought to be

as a marker for VIP receptor modulation of specific cells .
responsible for nonadrenergic, noncholinergic neuroBecause of the recent studies at ou> institution by Natransmission, has been found not only in the gastrointhanson et al . (16) and Peatfield et al . (18) showing
tcstinal t .rart but in a number of additlonal sitvs in the
physiological effectsa of VIP in dog and fernt trachea, we
central and peripheral nervous system of many species .
examined tracheal tissue from these species .
Polak et al . (20) frrst used itnmunocytocbernical techniques to demonstrate immunoreactive VIP in specific MErHODS
cell types . Nerve f5bers containing immunoreactive VIP
have since been demonstrated in the respiratory tract of These esperiments were conducted according to the
guinea pig3- rabbits- cats, dogs, and humans (5 . 33 . 34) . published gttidelines of the American f'hysiological SoThese VIP nerves are distributed around submucosal ctery for the care and use of laboratory animals . The
gland acini in the nasal and tracheobronchia] mucosa, in specific protocols were approved by the Animal Research
the subepithelial connective tissue of the nose, and Committee of the University of California . San Franaround blood vessels and airway smooth muscle of both cisco .
intra- and eatrapulmonary mrways . Dogs and ferrets were anesthetized with pentobarbital
U3Gt-6143/86 31 .50 Copyngbt &~ 1988 tbe Amencm PhyetolocsJ Sooery C71 S
http://legacy.library.ucsf.edu/tid/tfz49c00/pdf
PUBL IC19TIONS
10333325
014336
Clio
VEP
RECEPTORS
QJ
DOG
wND
PsRItEr
sodium (25-35 mg/kg)- Although nerves containing immunoreactive VIP have been demonstrated in the respiratory tract of many ~pecies including dngs, no studies
1Rwctnrw
have described the distribution in ferret trachea- To
and sectioned at thicknesses of 6-8 um . Cryoatat

were mounted on gelatinized glass slidea . These sectie
were stained for cAMP using the unlabeled an
enzyme (horseradish perozidase-antiborseradish pero>D
ret tracheae were rewoved, intmersed in frsative, and

processed for immunocytochemistry to localize VIP (see
viously (12) . Anti-cAMP antiserum was produced

immunizing New Zealand White rabbits with 2'-O-mon-
demonstrate the presence of ViP-containing nerves, fer-
dase. PAP) method (30, 32), as we have described
below) . Dog and ferret tissues for itnmunocytochemistry

to localize cAMP were imme-qed in oxygenated Ham's
F-12 medium containing penicillin/streptomycin (1 U/
osuccinyl-cAMP (Sigma Chemical) conjugated to humaa
stimulation of intrackllular cAMP by cycloosygenase
passed through a 22-Nm nitmcellulose filter (Millipore)
100 ml) . Indomethacin (10- M) was included to prevent
products of arachidonic acid generated as a result of the

mechanical traume associated with dissection (13) . In-
domethacin does not interfere with the subsequent tnaa-
imal response of the tissue to direct pbarmacological

stimulation (29) . Bacitracin (1 mg/ml), a protease irthibitor, was used to prevent breakdown of VIP during incubations (16. 18) . After equilibration for 60 min, tissues
were transferred to beakers containing fresh culture medium, isoproterenol (10~ M) . VIP (3 x 10-r M), or
pmpranolol (10' M), phentolamine 110~ M), atropine

(30's M), and tetrodotoain (3 x 10'' M) for 15 min,
followed by VIP (3 x 10' M) . After 5 min, tissues were
froten in liquid Freon and liquid N~ and processed for
cAMP immunocytochemistry .
VIP immunoeyrochemiatry . Antiserums to VIP were

obtained from Drs . S. Said and J . Walsh . These antisemms have been characterized extensively and show negligible or no cross-reactivity with other peptides (S . 23) .
Ferret tracheal rings were fixed in 4% parafortneldehyde in 0 .1 M phosphate buffer, pH 74 (2 h, 4C) then
cryoprotected by incubation for 18 b in 30% sucrose .
Tissues were then frozen in embedding molds containing
Optimal Cutting Temperature compound (OCT) (LabTek Products), Sections (10 pm thick) were made using
a Bright cryostat and inelted onto gelatinized glass slides .
Slides were briefly washed in pbosphatr-buffered saline
(PBS) to remove OCC and were blocked in PBS containing 3''ro normal goat srrum (NGS. Antibodies) and 0 .3%
7titon . VIP antiserum was diluted 1 :1,000 with PBS
containing 1% NGS and 0 .3% Triton . The diluted senun
was applied to the seccfons for 48 h at 4'C . Sections were
washed in PBS containing 1% NGS and 0 .3% Triton.
They were then blocked as above and incubated with
goat anti-rabbit immdmoglobulin (Ig) G fluorescein isothiocyanate (Cappel Laboratoriea ; 1 :40) for 30 tnin at
room temperature . After that they were washed in PBS
and covered with glycerin-PBS (3:1) and glass cover afips .
Slides were viewed with a Zeiss fluorescence nvcroacope .
The absorption control was prepared as descnbed above,
e :cept that anti-VIP serum waa incubated with 50 ug/
ml pure synthetic VIP (Peninsula Laboratories) for 18 h
and was centrifuged before use for stainingP cAMP imntunocytochemiacry
. For cAMP immunocytoebernintry, ferret tracheae were divided into rings 1 .5
cm ia length . To obtain samplee of dog tracheal epitbelium, the posterior membranaus portion of trachea was
dissected free of its 4artilaginous attachments and was
divided into pieces 1 .5 cm in length . Froren tissues were
embedded in OCT, equilibrated in a cryostat at -20C,
setun albumin, Fraction V (US Biochemical)- Tlre

fraction of this antiserum was incubated for 12 h with;
human serum albutnin (I%), centrifuged at 10,000g,an
Testing of this antiserum in a radioimmunoassay (31)
and by Ouchterlony agar double diffusion (17) demoa
strared high affinity for cAMP without significant crossreactivity with other nucleotides or with the carrier pretein, human setum albumin . Cryostat sections were
washed for 10 m.in at room temperature in PBS (pH 7 .4)
then incubated for 30 min with 3% normal goat aetuaL
The slides were blotted to remove excess goat serum, and
tissues were incubated at 4C for iS h with anti-cAMP
antiserum diluted (1 :800) in PBS containing 1% NGS .
After thorough washing with PBS containing 1% NGB ;
sections were incubated for 30 min with goat anti-tab
IgG (Cappel l.eboratoriea ; 1 :20), washed again, and
cubated for 30 tnin with horseradish peroaidaesan

horseradish peroridase complex (Cappel Lsboratori
1 :300) . Sections were washed with PBS
tris(hydrnsymetbyl)aminomethane buffer (0 .05 M.
7 .6) and reacted with a mixture of diaminobenzi
(Sigma Chemical; 0 .05'k) and hydrogen peroxide (0
for 5 min- Slides were examined using conven

bright-field miernacopy . Staining controls included
substitution of preimmune for immune serum, o
of first or second antiserum, and liquid phase absorptt

of anti-cAMP serum with cAMP . No sr, iniug was
with any controls. Wben necessary to confirm the
tity of immtmoreactive cell types, aome tissues
counterstained with Alcian blue-periodic acid-Schifr

described previously (12)REBVLTS
Studies were performed in duplicate on tiesuea
tained from two ferrets and two dogs. Immun

ical localization of VIP revealed nerve fibers con
immunoeeactive VIP surrounding submucoeal g]ande
ferret trachea (Fig . 1) . In ferret trachea, incubation

VIP (3 x 10' M) increased immunoreactive
both serous and mutous cells in submucosal glands

2) . The iucrease in staining aftrr VIP was com
with that produced by isoproterenol (10-a M) and

not inhibited wheu tissues were preincubated with,
pranolol (10,M).phentolamine (10' M), atropine (1
M), and tetrodotoxin (3 x 10-' M) . In contrast
pronounced effect on tracheal submucoeal glanda,
produced little if any incteease in immunoreactive
in ciliated epithelial celle of the ferret trachea In the
trachea, incubation with VIP produced a marked in

in immunoreactive cAMP in ciliated and baeal oelhr
the epithelium, and, as in the ferret, this incre,ee
PUBL ICHTINS
10333326
014337
C117
VIP RECEPTORS IN DOG AND PERRET 7RiwCHEA
ne . 2. Bnght-6eld phnrnm.crogrepho of ferret trubeal eubmucoeal

glands etawed for immuooreetu'r tM1P- A : cuotrol tusue- B
. C . VlP (3 x]0-' M) . D: VIP (3 x 10-'
:uoprtel(10'M) M) .
proprmo)ol (10~ M) + phentelamme 00-~7) . euopme 110~ M) +
iewJuwun 13 x 10'' M). Note thnt VIP 'u .ervnue.l nutuunureeetiw
cAMP m aubmueoeal glend mW oad lhat ttus mereme m" not in .
h.bned by bloc4ere restad . Boe, 50,m.
CV-
P3G . I . A' Ouoreatence pbotoovcrog}-aph of ferret tracbesl eubmu .

rnssl glsnde sratned mtb annVI}' eePUm, ehoaemg unmunorracave
VIP rn ner,res Lo~/ surroundtng submucosal glends (SG)- H : sen+oo
naned mth ennVIP aervm preatlsarbed mth pure VIP (50 rg/ml)_
Bar. 40 emm
not blocked by propranolol, pheninlam3ne, atropine, and

tetrodototin (Fig . 3) . Goblet cells in the dog trachea
contained little Immunoreactive cAMP under baaal conditions and did not respond to exogenous VIPOISCUSSIOT:
In this study . we utilized iminunocyt

VIP in ferret tracheal
.ochemialtnquodematr
tissues and
to localize cAMP wlthin specific cells in these tissues .
We have demonstrated immunoreactive nerves near fer-
ret tracheal glande and have localized cAMP in specific
cells in thesetiues . The submucoaal glands of ferrets

and the epithelium of dog tratheae have heen shown
previously to secrete sulfated macromolecules and chlo-
nde, respectively, in response to VIP (16, 18) . We now

report tbat serous and mucous gland cells in the fenet
trachea and ciliated epithehal 'cells in the dog trachea
show elevated cAMP levels in response to VIP . Thus

immunocytochemtatry enables us to attnbute specific
functions to specific cells . Because the rombrnation of
propranolol, phentnlamine, atropine, and tetvodotoxin
did not mhlbit the mcrease in cAMP produced by VIP,
rlo . 3 . Bnght5eld pbotomtcrogrephe of dog tracbeel epithelium

slaad for unmunoreective cAMP A' controL & rsoptoterenol (10M) C: VIP (3 x 10-' M) . D- VIP 13 x 10" M 1+ propnnolot (10-' M)
a Pbenwlam .oe ( l0- M) + stropme (10 ' '-N ) atetrodorna .n (3 x 10M) Nole that ViP increesed unmunortact .ve cAMP in eprtSrlium and
wnt tbu mereaee w not mhubited by blochera teeted Bos, 30 ..m
we believe that VIP was acting directly by way of apecifrc
V IP receptors on individual secret .ory target cella, rather
than by causing release of other mediators or activation
of neural pathways .
The dietribution of innaunoreactive VIP in theee ferret
tracheal tissues waa similar In that previously described
in studies of other respiratory tract tissues (5, 33, 34) .
Nerves containing VIP bavebeen identified around bronchial submucosal glands of the cat, dog, rabbit, and
pUBLICRTIONS
10333327
014338
'Clt8
VIP
RECEPTORS
IN
DOG
AND
FSRREP
human : around submucosal glands in the 6asa1 mucoss

and trachea of the human, rabbit, and catl and close to
the epithelium in the subepitbelial connective tissue of
the human nose and the cat and rabbit broncbus . In our
studies, the nerves containing immunoreaetive V IP terminated sufficiently close to the cells activated by esogenous VIP to suggest that endogenously released VIP
might easily reach these sites by diffusion .
The Increase in cAMP staining ohserved in tissues
stimulated with VIP was qualitatively siinilar to that
produced by isoproterenol (10-e M) . Although in these
studies no attempt was made to quantitate the increase
in cAMP observed comparison with a ptevious study
(12) may allow a semiquantitative estimate . In that
study, tissues incubated with isoproterenol (10-6 M) increased total tissue levels of cAMP approximately fourfold and could be stained for cAMP by the PAP method
using anti-cAMP antiserums at a dilution of 1 :1,000. In
the current study . immunoreactive cAMP in stimulated
tissue was visualized using the same antiserum diluted
1 :800, suggesting that cAMP levels may have been in
approximately the same range as in the previous study .
Recent studies have clearly established d role for VIP
in the regulation of mucus secretion in the respiratory
tract. However, even the newly developed techniques for
detection of VIP receptors do not permit tlssessment of
receptor function on individual cells in a beterogeneous
tissue such as the trachea . By taking advantage of the
physiological coupling between V1P and cAMP, immunocytochemical localization of cAMP permits identification of cells activated by VIP and serves as a marker
for the presence of VIP receptors on these cells . Our
results are consistent with studies which demonstrate
that V IP stimulates ion transport in dog tracheal epitheliuen (16) and release of macromolecules from ferret
trachea (18), and suggest that ciliated epithelial ceUs are
involved in the former and that both serous and mucous
submucosal gland cells are involved in the latter . Our
results also support the observation that VIP does not
stimulate ion transport in ferret trachea (18) .
We thsn4 Dts Samr Smd endJobn Walsh for their generoue gina
of snti-VlP anueenmu
This study wee supporud .n pan by National Heart, Lung, and
Blood Institute Pmgam prolu-t Grent HL24138 end by Grant 1327
from the Council for Tobacco Reee.erchUSA S. C . lararue aae the
tenpieat of Natioeal Hean, Ltmg, and Blood lnenmrae New Inveeu
getor Award FIIr29877 and grmte 6nm the Ftance S . North, Sr .,
Found.uon and the Strobel Medical Research Flmd of the Amencan
Lung Awaorieuon of San Francisco . P . J . Bamee wes supported by a
Medical Heseerch Counrd TYaveBing FeBome6rp .
Current addrese of P . J- Henoes The Candouroranc lneutvte.
London SW36HP- UK.
The raaulu of tha amdy were preeented m prelimwery form et the
Annual Meetmg of the Amenean Thoraoc Society . May 10 . 1983.
Kanses Gry . MO.
Received 31 August 1984 : atzwpt4d in final form 11 Pebrnary 1986 .
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Bwphya. Arrn 544 : 474 481, 197&
2. AMIIiANdPP, B- . AND G . Ro8861 .IN . VIP MCepeVre and coetrol of
1TtAC11EA
cyclic AMP produetion . Ip : Voswcm :! Lnrurinal Peptid.,

& t Said New York: Beven . 1982 . p . 3a7-3'22.
3. BA7Aa.18, D F . PeaI.ON, J . BassoN . AND G . Rosen.a .
-an .vr mteaunal peptide (VIP): recApleun speetfrquea at
de Yedeoylete eycleea dms une t, .mkur bypophyeeire bvmpy

proluaoa . C R Amd Sr. Panr 1E8: 1315-1317 1979.
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mtawnel pepude of glymcoWugate end lyeoryme seerelion
humeo e.r.oaye ,n v.uo. An. Ren. Redpp. . Dis. 124: 531S3B, igey'
5. D6Y, R D., W. A. SNANNON, JR . . AND 5. f . SAm . 1 . ..ItN .Vlp .mmlmoreanive nervea in wirweye and pulmonary vesy
doga. cetsm and humen eublecte . Ceff Twae Ree. 22R 231-Sig, lW
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,v. .voective intestiml pepude on mreeaml ehfonde secmnnn . fo :
Vvmmve lacettioo( PePnde . edited by S . I . Sai1 New York
Raven, 1982.p - 211-222.
7. KrrAUUaA~ S . . Y . IsanultA, AND S. 1. SA1D . EBea of VIP, pb,
norybenmmrne and predaieolooe on cyclic nucleotide mot4ar of
ieulaud guineap.g lung and trachea . 6ur. J. Phornwcut 67: 2W
223.1988.
& KRws, G . J- . J- H . W AteN . S. G . MoaAwssr. Areu J . S . Foaaawta .
IatractaDle diarrhea : int<eunal per[upion scudiee and pleema VIP
tenerotreuons in peuenta witL paocratic cholera syndrmne .ad
rrrptiuoue ingesnon of Isaativee and diuretiee . Am . J. D'y. Di.
22 : 2ga292, 1977 .
9. L..etmrNS, M ., P . btwccnr, G . M.u+atts-MouasN . wso G. ROSseuN . Activation of cyclic AMP-0epeadent protein kiaeses by
vaw.eaw inteatinel peptide ( VIP) in uolaud inteatmal epitb .~Wl
celle fmm rat Le Sc. 25 : 1931-1938, 1979.
10. Lt9neifm . M . . J . C . Paxero . B . AaqWVOn. C. DuPOnr, D . ytN
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pepnde a.th ieoleted mteetmel epahelisl eells from ret- 2 Chsraeterlraaoo and evucnnal requvemlot of the simlulewry eBect
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malsaon m gm canlnome cell linn m cvltwe . P~ NatL Aoa1
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of cAhff u1 dog and cat nschen eHecte of S-edtanerc ageaista
Am J PhyewL 241 tCeR PhpioL 161 :,C327-C334 . 1984.
13 . L.aneus. S. G. C. B . BASaAum . AND W . M. GOLD . Pmstagleodin
and ~ntmceWiler cyclic AMP in reepimtory secretory callw An .
Rev . Rz.pv. Du . 130. 262-266 . 198i.
14 . MAta . L, . P, SONDLan, AND R Uoauwn . Effecu of vanacta .
mtmmel polypeptide (VIPI on rnmuace and eapecitana .roewb
in the nsesl mucoeo . Arm OcobryngoL tStockA .l 90: 3M-808, 1999.
15 . MaASAD. M . . S . I . SAm. AraD A . L. HrnrAN . Vanoecuv<intenitW
pepude /VIPI dilatee pulmonery vessels in anesthetized eets tAbb
etrseU . C4n Rra 28 : 89M, 19g0.
16 . NAreAecsoN . 1.,J. H . Wmmcora[ . AND P . J . BARNES. 69ettd
vasoattrve mteaetnal pepude on ,on trmsport ecroea dog uached
epitbeBum. J Appl. PhyamL 55: 18f4-IBI8. 1963
17 . Oucmwrt .onr, 0 . Diff.loo-io-gel nmthode for immunelopeJ

cnelyua . Im Prog.ea. m ARergy. Soel Harger. 1968 . voL S . p . 17b .
18. PaArPmtu, A . C . P . J. BAaxas. C . Baxrc~ J- A. N .Dal ., AND
B- DAVIS . Vaaeetteve intenunal pepade stimulates tracheal abmumeal gland aenetlon m ferret A ... Reo. Reapv Du l2& 8B93, 1983
19 . Pam P. J . . S . 1 . SAm, AND J . R VANt Bffecte on emooth mmdr
pieparemne of amdeatifial vaaWecve pepridee from inta .4ne and
lung. Nmure Innd 22S 1144-1118 .197D.
20 . Pous, J. M. . A. G . E Pacase, J . C. GARAw. AND S. R Bl.oar.
CaBNer locafvetion uf a vaeoacnve mtritiml peptide m the m .mloelim and evien psuptateattual tmec Car 15 : 72o-724, 1974.
21 . Patsro. J . C. . M . LASunrtts: AND G . Rosela.m . luteractien of
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from ra6 1 . Cberartenaauoa Quaotiunve aepecte aud acmcrort .l
n~uirtmenb of bmdlvg utea. Eur. J. RiacAern. 96 : 229-'Li7. 197A
22 RnaaaaacNt, P, T. P . CONLON. AND J . D. GMUNVt. Iau~etVOa
of poteine vesoective mteetmel pepude wnb diaperad psnme .ltc
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of vaeoectivt mleetiael ixptide snd seoetion on cellular ederueine
3' :5' .monophoapNte . d BwL CAem 251 : 4635-46i39. 1976
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of the wetery durrhen eyndrome . Wor)d J Surg. 3: 559-563 . 1979 .
24 . SAOD . 5. 1 ., A . Gauwel: uvo N . HAUA. 8roncboiiiletor effect of
VW in vivo : proteMton against broncbocvnetric#ion induced by
butamine or proetagleedio Ft . . In : Voroanur InuannoE Pepnde,
edited by S. L Said New York: Raven, 1982.
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HDt.DSrw . Humoral raotrol of euweye. Ann NY Acad Sci 22t :
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S. SAID. VLa4acGve intGft1O91 peptide ltimulatjon of edenyllra
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Chloride secrttion by cenme trechea) epitbelYUm . I. Role of intre
telluler cAMP levele . J Memhr Biot 70 : 217-226. 1962.
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_ immunochemietry. In : Aduancn m Cyrlic Nuc4otde Reuarch .
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7, 1976.
31 . STSINBn. A . L, C . W. PutRElt. r,ND D. M . K.vrna Radioimmunoeaey for cycEc nucleotidea 1 . Prepetauon of epubodiee and
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its use in idenciSauon of apirochetee. J . Hietochern Cyto-Aen . 18:
315--333, 1970 .
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33 UDDrawrv . R., J . Ar_UMerg . O . Derrsem, R . HArtwrrsnx. AND P.
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443i48. 1978 .
34 .
UDOHAN. R, AND F . SUNDLM VBeOtetnY intestinal pOtypepdde
Derva in humen upper respiratory trecr_ ORL 41 : 221-226, 1979.

35 . VstrucopAww, C . S, S. 1 . S .aD, AND J . M . Dx .zan 8ffeeta of
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36. W t,sssrWArv, M . A . . R . L GtetlnN, AND P. E. Mwtu . Comperetive
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Revet4 1984 p. 177-154 .
'
PUBL I CAl' I ON S 014340

10333329
Copyright @ERS Journals Ltd 1995

Eur Respir J, 1995, a, 1458-1464 European Respiratory Journal
Pnnted in UK - all rights reserved ISSN 0903 - 1936
(M)
Localization of neutral endopeptidase (NEP)
mRNA in human bronchi
J .N . Baraniuk*, K . Ohkubo**, O .J . Kwon+, J . Mak+, M . Ali*, R . Davies,
C. Twort*, M . Kaliner##, M . Letartet, P.J . Barnes
Localization of neutral endopeptidase (NEP) mRNA in human 6ronchi . J.N. Baraniuk,
K. Ohkubo, 03. Kwon, J. Mak, M. Ali, R. Davies, C Twort, M. Kaliner, M. Lerarte,
P .J. Barnes. (PERS Journals Ltd 1995 .
ABSTRACT : Neutral endopeptidase (NEP) may regulate peptide-induced inflammation in the respiratory tract . It is of interest to determine which respiratory resident cells express NEP .
Trachea and bronchi from seven nonsmoking, nonasthmatic subjects were examined . NEP messenger ribonucleic acid (mRNA) was characterized by Northern blot
hybridization of cultured human tracheobronchial epithelial and smooth muscle
cells, and reverse traascriptase-polymerase chain reaction (RT-PCR) in trachea and
bronchi. ln situ hybridization with biotin- and 35S-labelled antisense complementary ribonucleic acid (cRNA) probes was used to determine the distribution of NEP
mRNA in human bronchial mucosa . NEP-immunoreactive material was detected
using MEK10 murine monoclonal antibodies and the immunogold method with silver enhancement.
NEP mRNA was 4 .5 kb in size in the cultured human smooth muscle and epithelial cells by Northern blot analysis . No evidence was found by RT-PCR for truncated, alternatively spliced NEP mRNAs, such as del exon 16 or del exons 5-18 in
human bronchus . NEP mRNA was detected by in situ hybridization in epithelial
cells, submucosal glands, bronchial smooth muscle and endothelium . NEP-immunoreactive material was identified in the epithelium, submucosal glands, bronchial
smooth muscle, and endothelium, demonstrating an excellent correlation between
the distribution of NEP mRNA and the cell surface protein . NEP mRNA and
immunoreactive material were excluded from epithelial goblet cell and submucosal gland mucous cell vacuoles .
We conclude that the various sites of NEP protein and mRNA expression correlate with the locations of peptide receptors and NEP enzyme function, and are consistent with the hypothesis that NEP may regulate peptide-induced inflammation in
human bronchi .
Eur Respir J., 1995, 8, 1458-1464 .
Neutral endopeptidase (NEP) is a 749 amino acid, zinccontaining, membrane-bound enzyme, which may play
a key role in regulating peptide-induced inflammatory
events [1-3] . NEP, also known as E.C.3 .4.24.11, enkephalinase, common acute lymphoblastic leukaemia antigen
(CALLA), CD10, and gplOO, has been cloned from
human, rat and rabbit tissues [4-9] . Functional studies
indicate that NEP activity is present on epithelial, glandular, smooth muscle and vascular cells which possess
peptide receptors [1, 2] . Loss of NEP activity may significantly contribute to the hypersecretion, vascular permeability, bronchoconstriction and other pathological
changes seen in respiratory inflammation by permitting
unopposed, prolonged actions of inflammatory peptides,
such as tachykinins and bradykinin .
In the present study, in situ hybridization was used to
determine the distribution of NEP gene expression in
http://legacy.library.ucsf.edu/tid/tyg16d00/pdf
Division of Rheumatology, Immunology

and Allergy, Georgetown Medical Center,
Wacttington, DC, USA . 'Ikpt of Otolaryogology, Nippon Medical School, Tokyo,
Japan. 'Dept of Thorac:ic Medicane, National
Heart and Lung Institute, London, UK .
-Department of Respiratory Medicine . St .
Banholotnew's Hospital, Lordon, UK . 'Deq
of Pulmonary Medicine, SL Thomas Hospital, London, UK . "Asthma and Allergy
Institute, Washington Hospital Center,
Washington. DC, USA. loivision of
Immunology . Hospital for Sick Children,
Toronto, Ontario, Canada
Correspondence : J .N . Baraniuk, Division
of Rheumatology, Immunology and Allergy
GL-020 Gorman Building, Georgetown
University Medical Center, Washington,
D .C . 20007-2197, USA
Keywords : .4sthma, CD10, enkephalinase,

neurogenic inflammation, neuropeptides,
neutral endopeptidase
Received : October 4 1994
Accepted after revision April 11 1995

The research was supported by ScheringPlough Research, Kenilworth, NJ, USA,
Boehringer-Ingelheim, Bracknell, UK, and
the National Asthma Campaign, London,
UK . JNB has been awarded the Edward
Livingston Trudeau Scholar Award by the
American Lmg Association, and is a Tobacco
Council for Research Scholar .
-
IF riboi
ing :
phos
emb
celi
Hi
cle c
NEF
Abase
com
[6,7
genf
regi(
acid
A
sis 1
ing '
poly
gion
preF
with
the
for
urid
UK)
tion
shar
biot
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nucl
PH E
im
sodi
UTI
(opt
human bronchial mucosa, whilst the identity of NEP messenger ribonucleic acid (mRNA) was confirmed by
Northern blotting . NEP immunoreactive material was
detected by immunohistochemistry .
Nor
Methods
apir
acid
elec
RN,
filte
stan
ligh
50%
g't
tota
anti
Human Tissue
Human trachea and large, cartilaginous bronchi were

obtained from four organ specimens collected at heart
transplantation and at autopsy of three victims of motor
vehicle accidents. None of the subjects were known to
be smokers or asthmatic . Tissue was transported in 4C
Krebs solution, and : 1) frozen in liquid nitrogen for
R
fron
acid
tion
NEP mRNA IN HUMAN BRONCHI
ribonucleic acid (RNA) extraction and cryostat sectioning ; and 2) fixed in 4% paraformaldehyde in pH 7 .4
phosphate buffered saline (PBS) for 4 h at 4C before
embedding in paraffin.
1459
Cell culture
mL hybridization buffer) was added, incubated 16 h at

50C, and washed in 0 .5xSSC at 55C before exposure
to radiographic film . The filter was stripped in 50% formamide, pH 6 .5, 10 mM Na phosphate for 60 min at
65C . After prehybridization, the filter was hybridized
with sense NEP cRNA and washed using the same conditions .
Human bronchial epithelial cells [10] and smooth muscle cells [11] were cultured as described previously .
In situ hybridization
NEP complementary ribonucleic acid (cRNA)

An NEP insert [7] coding for bases 642 to 2223 (1,581
bases) was obtained by Eco RI digestion of human NEP
complementary deoxyribonucleic acid (cDNA) clones
[6,7] and inserted into a M13+ Bluescript plasmid (Stratagene, San Diego, CA, USA) . This insert codes for the
region stretching from amino acid 210 in exon 8 to amino
acid 735 in exon 24 [6, 81 .
Antisense NEP cRNA probes for Northern blot analysis and in situ hybridization were prepared by linearizing the plasmid with Ava II and transcribing with RNA
polymerase T3 . The antisense cRNA coded for the region from base 2029 to 2223 . Sense cRNA probes were
prepared by linearizing with Pvu l1 and transcribing
with RNA polymerase T7 . The sense cRNA coded for
the region between bases 642 and 1099 . cRNA probes
for Northern blots were labelled by incorporating 32Puridine triphosphate (UTP) (Amersham, Inc ., Amersham,
UK) during transcription. Probes for in situ hybridization were labelled by incorporation of 35S-UTP (Amersham, Inc ., Amersham, UK) for radioactive detection, or
biotin-5-UTP (Sigma, Poole, UK) for nonradioactive
detection . Probes were purified from unincorporated
nucleotides using G-50 Sephadex columns eluted with
pH 8, 10 mM Tris (hydmxymethyl) aminomethane (TRIS),
l mM ethylenediamine tetra-acetic acid (EDTA), 0 .1%
sodium dodecyl sulphate (SDS) . The yield of biotin-5UTP-labelled probe was determined from the OD26o,
(optical density at 260 nm) .
Northern blots
RNA samples for Northern blot analysis were prepared

from cultured cells scraped from culture flasks using the
acid, guanidinium thiocyanate, phenol-chloroform extraction method [12] . RNA integrity was confirmed by the
appearance of the 28S and 18S recombinant ribonucleic
acid (rRNA) bands [13] after denaturing 1% agarose gel
electrophoresis in TAE (Tris-Acetic acid-EDTA) . The
RNA was transferred from the gel to Hybond nylon
filters (Amersham, UK) by capillary action with 20x
standard sodium citrate (SSC), and fixed by ultraviolet
light (UV) for 4 min . The filters were prehybridized in
50% formamide, SxSSC, 0 .1% SDS, 5 mM EDTA, 100
pgmL-1 denatured salmon sperm DNA, 0.5 pgmL-t yeast
total RNA, 5x Denhardt's solution for 16 h at 50C . 32Pantisense NEP cRNA probe (106 cpm per 10 cmz in I
Cryostat tissue sections (10 pm) from seven subjects

were thaw mounted onto gelatin-coated slides, allowed
to dry for 12 h at 37C, and then post-fixed in freshly
prepared 4% paraformaldehyde in pH 7 .4, 0 .1 M PBS
for 30 min . Paraffin sections were dewaxed and hydrated in PBS for 5 min . Cryostat and paraffin sections were
subsequently treated in identical fashion [14, 15] . Sections
were permeabilized in 0.3% Triton X-100 in PBS for 10
min and then proteinase K (l pg mL'i) in 0.1 M TRIS,
50 mM EDTA for 30 min at 37C . After treatment in
4% paraformaldehyde in PBS for 5 min, nonspecific
binding sites were blocked in 0 .25% acetic anhydride,
0 .1 M triethanolamine for 10 min . Slides were prehybridized in 0 .3 M NaCl, 30 mM Na citrate (2xSSC), 50%
formamide for 30 min at 50C . Slides were hybridized
for 16 h at 50C . 35S-cRNA probe in hybridization buffer
was added at 106 cpm per slide . Biotin-cRNA probes
were added at 200 ngmL-J . After hybridization, slides
were washed in 4xSSC. Unincorporated, single-stranded cRNAs were degraded in 20 pg mL I ribonuclease
(RNase) A, 0 .5 M NaC1 . 10 mM TRIS, and I mM EDTA
for 30 min at 42C . Washing was continued in decreasing concentrations of SSC to 0 .2xSSC at 50'C .
35S-labelled slides were dehydrated in 70% ethanol,
air dried, and coated with Ilford K-5 photographic emulsion melted at 42C . Coated slides were exposed for
14-21 days, developed in Kodak reagents, and stained
with haematoxylin .
Biotin-labelled slides were immersed in Lugol's iodine
for 2 min, decolourized in 2 .5% Na thiosulphate, washed
in PBS [15, 16], and nonspecific binding sites blocked
with 0.8% bovine serum albumin (BSA), 0 .1% gelatin,
5% nonimmune goat serum, 2 mlvl Na azide in PBS for
1 h at room temperature . Anti-biotin goat serum labelled
with I nm colloidal gold particles (Amersham, Amersham,
UK) diluted 1/10 with blocking solution was added, and
the slides incubated overnight at 4C . Slides were washed
twice for 5 min in PBS followed by distilled water . Silver
enhancing solution (Amersham, Amersham, UK) was
added to pairs of sense and antisense slides and stain
development observed under darkfield illumination . Slides
were washed in water for 5 min, 2.5% Na thiosulphate
for 3 min, and dehydrated .
Primers
NEP primers coding for mRNA splice sites were

identified from published cDNA sequences [5, 6, 8] and
were synthesized by the Lombardi Cancer Center Core
1460
J .N . BARANIUK
Laboratory of Georgetown University, Washington, DC .

NEP primers were chosen to identify mRNAs containing deletions of exon 16 (del 16) [17] and deletion of
exons 5-18 (del 5-18) [18] . A single antisense primer
was used that coded for the exon 19-exon 20 splice site
(5'GTTTCCGCrsPUCECATTGTCATCGAA) . When paired
with a sense primer coding for the exon 3-exon 4 splice
site (5'ATGCAACCTACGATGSPWMATGGTAT), several possible NEP mRNA RT-PCR products could be
generated, including one from full length mRNA (1,641
nucleotides), one for del 16 (1,563 nucleotides), and one
for del5-18 (177 nucleotides) . When paired with a sense
primer coding for the exon 14-exon 15 splice site
(5'AGTAAACATGTC,/sPUCE~GTCGAGGAT), full length
NEP mRNA would generate a RT-PCR product 477
nucleotides long, and 399 nucleotides long for the del
16 variant. Deletion of exons 5-18 would generate no
product. The annealing temperatures (55C) were calculated according to MenaKOnt and WAFn. [19] .
(3-actin [20, 21 J primers were purchased from Clontech
(Palo Alto, CA, USA) . They generated an RT-PCR product 661 bases long from mRNA, but 867 bases long
from genomic DNA because of the presence of a short
intron .
Reverse transcriptase-polymerase chain reaction
RNA (5 pg), reverse transcriptase/antisense primers,

and Perkin Elmer RT-PCR reagents (Norwalk, CN, USA)
were mixed according to manufacturer's recommendations at 4C in a Perkin Elmer thermocycler . Mineral
Microliter oil (70 pL) was added, and then the temperature increased to 42C to permit annealing of the RT/
antisense primer to specific mRNA sequences . After 60
min at 42C, the solution was denatured at 99C for 5
min, and then cooled to 60C . Perkin Elmer taq, other
PCR reagents, and sense primers were preheated to 60C
and added to each reversely transcribed tube . In this
way, "Hot-Start" conditions that reduce nonspecific priming were produced . To permit efficient annealing and
extension for this first cycle of PCR, the temperature was
then appropriately adjusted to the annealing temperature
(55C) [19], and maintained for 5 min . This was followed by 2 min at 70C . Then the thermocycler was
set to cycle for a total of 45 cycles at 94C for I min,
55C for I min, and 70C for 1 min . In preliminary
experiments, it was found that most samples generated
positive bands after 35 cycles, but some samples with
minimal RNA required 45 cycles . Since the additional
10 cycles did not cause generation of superfluous bands,
45 cycles was adopted as a standard method .
PCR products were mixed with TRIS-borate-EDTA
(TBE) loading buffer and run in 0 .5xIBE on 2% agarose
(FMC, Rockland, ME, USA) 1 .5 h at 120 V . Clon-Tech
(Palo Alto, CA, USA) DNA molecular weight standards (1353, 1078, 872, 603, 310, 281, 271, 234, 194,
118 and 72 nucleotides) were run on each gel. Gels
were stained with ethidium bromide, bands visualized by
UV-fluorescence, and photographed with Polaroid 667
film .
Immunohistochemistrv
Paraffin embedded sections from seven human large

bronchi were dewaxed, rehydrated in PBS, and incubated in blocking solution [15] . Murine immunoglobulin
G(IgG) monoclonal antibody to NEP (MEKIO, courtesy
of S . Shak, Genentech, South San Francisco, CA, USA)
diluted 1 :200 with blocking solution was aliquoted onto
the slides and incubated for 20 h at 4C . After washing in PBS, the monoclonal antibody was detected by
the immunogold method with silver enhancement [15] .
Nonspecific staining was determined by preadsorption of
the antibody with 200 pgmL-t recombinant human NEP
(courtesy of S . Shak, Genentech, South San Francisco,
CA, USA) .
Results
Northern blot analysis
Cultured human tracheal smooth muscle cells and cultured human bronchial epithelial cells contained a single
NEP mRNA band at 4 .5 kb (fig . 1) .
RT-PCR
A single NEP mRNA band was identified indicating
the presence of full length mRNA coded by exons 4-20
(fig . 2) . No variant NEP mRNA could be amplified,
suggesting that truncated mRNA (del exon 16 or del
exons 5-18) were not present in human tracheobronchial
tissues from nonasthmatic, nonsmoking subjects .
In situ hybridization
The biotin-labelled antisense NEP cRNA probe detected NEP mRNA in epithelial cells and submucosal glands
(fig . 3) . Staining was less intense, but still present, over
bronchial smooth muscle and endothelial cells of subepithelial capillaries/postcapillary venules and deeper,
larger venous vessels . This may suggest that the NEP
mRNA concentration was lower in smooth muscle and
endothelial cells than epithelium and glands .
ic
20 10 5
Smooth Muscle
20 10 5
Epithelium
Fig. 1 . - Northem blot of 32P-antisense NEP cRNA binding to 20,

10 and 5 pg of total RNA obtained from cultured human smooth muscle [111 and epithelial cells [ 10] . The bands are at 4 .5 kb . The large
aevowhead identifies the location of the 28S rRNA band (4 .8 kb), and
the small arrowhead the 18S rRNA band (1 .8 kb) [131 . NEP : neutral endopeplidssc ; RNA : ribonucleic acid; cRNA : complementary
RNA; rRNA : recombinant RNA.
1461
NEP mRNA IN HUMAN BRONCHI
A B C
r r
. .
. ' . ..
Y
>
Fig . 2 . - Reverse transcriptase-polmeritation chain reaction (RTPCR) for NEP mRNA in human bronchus total RNA . The eaons
19-20 splice site antisense (RT) primer and e.waa 14-15 splice site
sense primer generated a band at 477 nucleolides corresponding to the
full length mRNA coded from the exons 14-20 (t .anes A) . No truncated variant NEP mRNAs were found. Lane B shows markers at
1353 (top), 1078, 872, 603 and 310 (bottom) nucleotides . Lane C
shows the ~-actin mRNA RT-PCR product (661 nucleotides) . Lanes
A : NEP ; B : markers ; C : (S-actin . mRNA: messenger ribonucleic acid . For further abbreviations see legend to figure 1 .
A
100Nm r.
Fig . 3 . - Nonradioactive in situ hybtidiTation of NEP in human

bronchus . A) Biotin-labelled antisense NEP mRNA was detected by
the immunogold method with silver enhancement as the intense black

stain over the epithelium (e) and submucosal glands (g) . Slightly less
intense staining was detected over bronchial smooth muscle (m),

endothelium of subepithelial capillarieslpost-capillary venules and larg-
er vessels (arrowheads), and chondrocytes (c) . B) Biotin-labelled sense

NEP eRNA did not bind to the sections . For abbreviations see legends to figures I and 2 . (Internal scale bar=100 m) .
This distribution of NEP mRNA-containing cells was

confirmed using the 35S-labelled cRNA probes . The
epithelium, vessels, smooth muscle, and glands contained
NEP mRNA . The epithelium had a high silver grain
density. The nuclei of basal and other cells, and the
vacuoles of goblet cells were devoid of NEP mRNA
(fig . 4) . The serous cells of submucosal glands were
positive (fig. 5) . The vacuoles of mucous cells contained
no NEP mRNA, but the cytoplasmic rims of mucous
cells appeared to contain some silver grains suggesting
the presence of NEP mRNA . Endothelial cells of subepithelial capillary/post-capillary venules contained NEP
mRNA (figs 3 and 4), as did the smooth muscle of a
Fig . 4 . - NEP in sim hybridization in epithelium . Brightlield (A and

C and darkfield (B and D) images of 35S-antisense NEP binding to
epithelium shows dense silver grains over epithelial cell cytoplasm
excluding nuclei (small arrowheads) and goblet cell vacuoles (G) .
Superficial vessel endothelial cells (large arrowheads) have a low density of silver grains when viewed with darkfield illumination . The
bronchial lumen (L) is shown . Serial sections treated with sense probes
had no binding (not shown) . Bar Iine=50 pm .
deeper periglandular artery (not shown) . NEP mRNA

was detected in bronchial smooth muscle . The distribution was the same in all specimens .
Neither the biotin- nor the 'SS-labelled sense probes

hybridized with the tissue (figs 3 and 5) indicating the
specificity of the antisense probe .
Immunoh isrochemistn,
NEP immunoreactive material was detected in epithelial cells, submucosal glands, smooth muscle and endothelium of human bronchus (figs 6 and 7) . In submucosal
1462
1 .N . BARANIUK
: :.
t_
serr t ,
dt
~.
rt
. I
Fig. 6. - Neutral endopeptidase (NEP) immunohistochemistry . A)

NEP immunoreaclive material was detected with MEK10 antibodies
and the immunogold method, and appears as the densely stained black
material . Goblet cells and glandular cells appear grey with the methyl
pyronin green counterstain . NEP was detected in the basal region of
the epithelium (B), and the outer rims of submucosal gland cells (G) .
Vacuoles of goblet cells and submucosal gland mucous cells did not
contain the black stain indicative of NEP, but still appear grey from
the counterstain . Smooth muscle (Sm) also contained NEP. The NEP
appeared to be localized to the surfaces of these cells in a rim pauem .
B) MEKIO antibodies adsorbed with recombinant human NEP did not
bind to this serial tissue sectiooa Only methyl pyronin green counterstained cells can be seen . (Internal scale bar-100 m) .
Fig . 5 . -!n situ hybridization using antisense neutral endopeptidase

(NEP) 35S-cRNA probe in human bronchial submucosal glands . A)
Darkfield showing silver grains over glandular cells . Serous cells, possibly mucous cell cytoplasm, but not mucous cell vacuoles, demonstrate silver grains . B) Brightfield image showing submucosal gland
acini stained with haematoxylin . C) The sense probe did not bind to
the gland, confirming the specificity of the antisense binding seen in
(A). cRNA : complementary ribonucleic acid . Bar line=50 pm .
glands, the NEP appeared to be at the edges of gland

cells rather than in secretory granules, suggesting that
the NEP was membrane-associated . The same impression is obtained for bronchial smooth muscle . Endothelium of vessels immediately below the epithelium were
positive (fig . 6A) . The cellular distribution of NEP
immunoreactive material was the same as that of NEP
mRNA . The distribution was identical in all specimens .
Adsorbed NEP antibodies did not stain the tissue,
indicating the specificity of the immunolocalization
method.
Fig . 7. - Neutral endopeptidase (NEP) immunohistochemisny . A)

NEP immunoreactive material (black stain, immunogold method) is
shown with methyl pyronin green counterstain. NEP immunore.artive
material was detected in the outer rims of glandular acini (G) in semus
cells, mucous cells, and possibly mycepithelial cells. Smooth muscle
(Sm) and the endothelium of a venule (V) contained NEP . B) MEK10
antibodies adsorbed with recambinant human NEP did not bind to this
serial tissue section . Only the methyl green pyronin counterstain (grey)
is apparent. (Internal scale bar-40 m) .
NEP
mRNA
Discussion
Cell surface NEP may limit the actions of many of the
peptides that are active in human tracheobronchial mucosa
[23-25], including tachykinins and calcitonin gene-related peptide (CGRP) released by axon response mechanisms from nociceptive sensory netuones during neurogenic
inflammation, vasoactive intestinal peptide (VIP) released
by parasympathetic reflexes, bradykinin generated in
many types of allergic and nonallergic inflammation, and
circulating peptides, such as endothelin and atrial natriuretic peptide (ANP) [26-28] . Decreases in NEP activity
[22] may underlie the increased responses of respiratory mucosa found during viral infections [29-31], after
exposure to cigarette smoke [32], ozone [33], hypochlorous acid, [34], and high doses of toluene diisocyanate
[35] . The release of peptides into areas with reduced
NEP activity could lead to enhanced peptide-induced
epithelial cell function, glandular secretion, vascular permeability and smooth muscle contraction [1-3, 26, 27] .
Each of these proinflammatory processes occurs in sites
where NEP mRNA and protein are found : epithelial cells,
submucosal gland cells, bronchial smooth muscle, endothelium and arterial smooth muscle (table 1) . Apparent differences in the numbers of gland and epithelial cells
containing NEP mRNA and immunoreactive materials
between specimens were due to different proportions of
goblet and mucous gland cells (figs . 3-7). These mucous
cell vacuoles did not contain NEP . Other epithelial cells
and glandular serous cells did contain NEP mRNA and
immunoreactive material .
These locations also correlate with the sites of NEP
enzyme activity detected in guinea-pig tracheal epithelium, glands, and vessels by fluorescent zymographic
microscopy [36] . Enzyme activity was also detected in
the perichondrium and chondrocytes, but was not detected in guinea-pig tracheal smooth muscle cells [361 .
Expression of NEP in lung parenchyma has been investigated by JotwsoN et al . [371 .
Whilst changes in NEP distribution or expression or
decreased activity have been postulated to contribute to
-changes in airway reactivity to selected stimuli in vivo
[ 1, 2], there are as yet few data from humans to confirm
this contention . In fact, RotsMAN et al . [38] suggested
an increase in NEP activity in lungs of asthmatic subjects . Modulation of NEP activity in disease or after
Table 1 . - Distribution and relative intensity of expression of NEP mANA and immunoreactive material in human
trachea and large bronchi
Site
Epithelium*
Endothelium
Submucosal glands*
Smooth muscle
In situ
hybridization
Immunoreactive
material
++
+
++
+
++
+
++
+
* : mRNA and irtununoreactive material were excluded from

epithelial goblet cell and submucosal gland mucous cell vacuoles. NEP: neutral endopeptidase ; mRNA : messenger
ribonucleic acid . + : present ; ++: intense stain .
IN
HUMAN
BRONCHI
1463
oxidant exposure may be more complex in humans in

vivo than animal and in vitro models [l-3] would lead
us to believe . The current investigation of NEP distribution was not designed to determine whether destruction
of NEP activity is a primary event in airway inflammation that permits exaggerated neurogenic inflammation,
but does indicate that changes in expression by resident
cells is a feasible hypothesis .
An additional level of complexity in NEP expression
is the regulation of NEP gene transcription . The NEP
gene is complex, with 24 miniexons and multiple polyadenylation sites [6, 8] . NEP mRNAs of several sizes
are generated by post-transcriptional processing . The use
of alternate polyadenylation sites accounts for some of
the variation [6] . However, alternate splicing with the
deletion of certain exons may also lead to mRNA size
and protein product diversity . ItmrtA et al. [17] have
detected a 3 .2 kb mRNA which lacks exon 16 . RT-PCR
(fig . 2) identified mRNA coding for exons 14-20, but
did not identify any for the del exon 16 variant . Another
truncated NEP gene product detected by PCR in rat thyroid, intestine and whole brain [18] excludes exons 5-18,
and is postulated to generate a 255 amino acid protein .
No evidence was found to support the expression of this
variant (data not shown) .
These data indicate that NEP mRNA and immunoreactive materials are widely distributed on airway epithelium, glands, vessels and smooth muscle, sites known
to possess receptors for many peptide mediators . The
distribution of NEP indicates its critical role in limiting
the effects of peptides in human airways in vivo .
Acknowledgements : The authors wish to thank
L Roman, 1 . Tabachnik and I . Adcock for their support. Recombinant NEP and MEK-10 antibodies were
kindly donated by S . Shak of Genentech, South San
Francisco, CA. USA .
References
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* 16 Comments that are particularly useful examples .

PROFESSOR PETER J . BARNES

Professor of Thoracic Medicine, University of London

MA, DM, DSC, Fellow of the Royal College of Physicians

Consultant Physician, Royal Brompton Hospital,

PROFESSOR PETER J . BARNES

Pulmonary Diseases and Conditions :

Anna-Lisa Fisher (Secretary)

Welcome and Introduction - RB welcomed all members . The objectives of this

The previous work performed by BAT on the effect of humectants on lung

Following publication of a paper on ammonia/nicotine chemistry by Pankow et al,

BAT Position Papers on Smoking and Health

Smoking and respiratory disease,

It was recommended that SA would make a presentation of the proposed protocols to

Graham Read/Southampton/GB/BATCo@BAT, Graham R .

LONDON SW3 6LY

Dear Professor Barnes

My contact details are :

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Professor Peter J Barnes MA, DM, DSc, FRCP

OF SCIENCE, TECHNOLOGY AND MEDICIN E

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Imperial College School of Medicine

Professor Peter J Barnes MA, DM, DSc, FRCP

Southampton SO15 8TL

Peter J Barnes MA DM DSc FRCP

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Grant Application of Dr R P Youn g

history in emphysema and normal subjects .

Preliminary data to show the existence of polymorphisms in the MMP/MMP

Characterization of Beta Adrenoceptor Subtypes in Canine

Using selective beta antagonists, w a detennined the ratio of

of.. 1975: Omini ft a/.. 1979: lakovidiH vt <if. 1980: Jnhnnamm

AMHEVIS-TION: DHO o.nyofoaOfenoiol

Airway BUM Receptor Subtype*

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Cauauc H. R , BAI D M S . C. 4. AKnTrrtMm*.

MI-MA. C NAOXL. J. A. AN R o i c m , J M^ TJ .fencarinnrmtotHi a.