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Réimpressions et Biochem. Cell Biol. 72(1): 8–11 (1994) | doi:10.1139/o94-002 |
autorisations © 1994 NRC Canada

Cell-swelling-induced taurine release


from isolated perfused rat liver
H. S. Brand, A. J. Meijer, L. A. Gustafson, G. G. A. Jörning,

A. C. J. Leegwater, M. A. W. Maas, and R. A. F. M.


Chamuleau

Abstract: Astrocytes and lymphocytes are able to release


significant amounts of taurine during periods of hypotonicity to
reduce the increase in cell volume. To investigate this mechanism
in the liver, we studied the release of free amino acids from
isolated perfused rat liver during hypotonicity. The osmolarity of
the perfusion medium was reduced from 305 to 255 or 205 mosM
by decreasing the NaCl concentration 25 or 50 mM, respectively.
This induced an 6–8% increase in liver mass and was associated
with a specific 1.7-fold (−50 mosM) and 14-fold (−100 mosM)
increase of the taurine release. None of the other amino acids
measured showed a significant increase in their concentration in
the effluent. The increase in taurine release occurred within 30 s
after exposure to hypotonicity (maximal after 1–1.5 min) and
followed closely the changes in liver mass. The taurine release
declined gradually during successive exposures of the isolated liver
to −100 mosM. This release was 29 and 17% of the original during
the second and third exposure, respectively.

Key words: cell swelling, liver, taurine.

Résumé : Une quantité appréciable de taurine est libérée des


astrocytes et des lymphocytes dans des conditions hypotoniques
afin de réduire l'augmentation du volume cellulaire. Pour étudier ce
mécanisme dans le foie, nous avons mesuré la libération d'acides
aminés libres du foie de rat perfusé dans des conditions
hypotoniques. L'osmolarité du milieu de perfusion a été réduite de
305 à 255 ou à 205 mosM en diminuant la concentration du NaCl
respectivement de 25 ou de 50 mM. Cela engendre une
augmentation de 6–8% du volume du foie et celle-ci est associée à
une augmentation spécifique de la libération de taurine de 1,7 fois
(−50 mosM) et de 14 fois (−100 mosM). La concentration d'aucun
autre acide aminé mesuré dans l'effluent n'augmente de façon
significative. L'augmentation de la libération de taurine se produit
en dedans de 30 s après l'exposition à un milieu hypotonique, elle
est maximale après 1–1,5 min et elle suit étroitement les
changements de volume du foie. La quantité de taurine libérée
diminue graduellement lors d'expositions consécutives du foie isolé
à −100 mosM. Lors de la deuxième et de la troisième exposition, il
y a libération respectivement de 29 et de 17% de la quantité de
taurine libérée lors de la première exposition.

Mots clés : gonflement cellulaire, foie, taurine.

[Traduit par la rédaction]


 
8

Cell-swelling-induced taurine release from isolated perfused rat Iiver


1
H.S. BRAND
1. van Goal Laboratory for Experimental In/ernal Medicine, Academie Medical een/re, Meibergdreef 9, 1105 AZ
Amsterdam Z. 0., The Neth erlands
A.l. MElJER AND L.A. GUSTAFSON
E. C. Sla/er hlSli/ute for Biochemical Research, Academic Medical een/re, Meibergdreef 9, 1105 AZ Ams/erdam
Z. 0., The Ne/herlands
AND
G.G.A. JÖRNING, A.C.l. LEEGWATER, M.A .W. MAAS, AND R.A.F.M. CHAMULEAU
J. van Goal Laboratory for Experimen/allnternal Medicine , Academic Medical eentre, Meibergdreef 9, 1105 AZ
Amsterdam Z.O .. The Netherlands
Received August 24, 1993

BRAND, H.S., MEIJER , A.l. , GUSTAFSON, L.A ., JÖRNING, G.G.A. , LEEGWATER, A.C.J. , MAAS, M.A.W., and CHAMULEAU,
R.A.F.M. 1994. Cell-swelling-induced taurine release from isolated perfused rat liver. Biochem. Cell Biol. 72:
8-1 J.
Astrocytes and Iymphocytes are ab Ie to release significant amounts of taurine during periods of hypotonicity to
reduce the increase in cell volume. To investigate this mechanism in the Iiver, we studied the release of free amino
acids from isolated perfused rat liver during hypotonicity. The osmolarity of the perfusion medium was reduced
from 305 to 255 or 205 mosM by decreasing the NaCI concentration 25 or 50 mM , respectively. This induced an 6-8%
increase in Iiver mass and was associated with a specific 1.7-fold (- 50 mosM) and 14-fold (-100 mosM) increase
of the taurine release. None of the other amino acids measured showed a significant increase in their concentration
in the effluent. The increase in ta urine release occurred within 30 s after exposure to hypotonicity (maximal after
1- 1.5 min) and followed closely the changes in liver mass. The taurine release declined gradually during successive
exposures of the isolated Iiver to -100 mosM . This release was 29 and 17% of the original during the seco nd and
third ex pos ure, respecti vely.
Key words: cell swellin b , liver, taurine.

BRAND, H.S., MEIlER, A.J ., GUSTAFSON, L.A ., JÖRNING, G.G.A., LEEGWATER, A.C.J., MAAS, M.A.W., et CHAM ULEAU,
R.A.F.M. 1994. Cell-swelling-induced taurine release from isolated perfused rat Iiver. Biochem. Cell Biol. 72 :
8-11.
Une quantité appréciable de taurine est Iibérée des astrocytes et des Iymphocytes dans des conditions hypotoniques
afin de réduire I'augmentation du volume cellulaire. Pour étudier ce mécanisme dans Ie foie, nous avons mesuré la
libération d'acides aminés Iibres du foie de rat perfusé dans des conditions hypotoniques. L'osmolarité du milieu de
perfusion a été réduite de 305 à 255 ou à 205 mosM en diminuant la concentration du NaCJ respectivement de 25
ou de 50 mMo Cela engendre une augmentation de 6-8% du volume du foie et celle-ci est associée à lIne aug­
mentation spécifique de la Iibération de taurine de 1,7 fois (- 50 mosM) et de 14 fois ( - 100 mosM). La concentration
d'aucun autre acide aminé mesuré dans I'effluent n 'augmente de façon significative. L'augmentation de la libération
de taurine se produit en dedans de 30 s après I'exposition à un milieu hypotoniqu e, elle est maximale après
1-1,5 min et elle suit étroitement les changements de volume du foie . La quantité de taurine libérée diminue graduelJe­
ment lors d'expositions consécutives du foie isolé à -100 mosM. Lors de la deuxième et de la troisième exposition ,
il y a libération respectivement de 29 et de 17% de la quantité de taurine libérée lors de la première exposition.
Mots c/és : gonflement cellulaire, foie, taurine.
[Traduit par la rédaction]

Introduction during RVD (JeslIs Garcia et al. 1991; Kimelberg et al.


In hypoosmotic conditions, cells rapidly swell because of 1990). To study the possible contriblltion of the release of free
their high permeability to water. Swelling is followed by a amino acids during RVD in hepatocytes, we studied the
recovery phase in which cells reduce their volume. This release of a number of free amino acids, including taurine ,
process is known as regulatory volume decrease and is medi­ from the isolated perfused rat liver during hypotonic-induced
ated by a reduction in the internal solute content. RVD has cell swelling.
been described in many cells including epithelial cel Is, red
Materials and rnethods
blood cells, astrocytes, Iymphocytes , and hepatocytes
(Chamberlin and Strange 1989; Hoffman and Simonsen Livers from 24-h-starved male Wistar rats (225-250 g) we re
1989). DUI'ing RVD, hepatocytes are able to release large used. Following pentobarbital anesthesia (60 mg ip/kg body
weight) , the portal vein was cannulated. The liver was perfused
amollnts of ions, especially potassium and chloride (Lang
with bicarbonate-buffered Krebs-Henseleit saline containing
et al. 1989; Häussinger et al. 1990b; Haddad et al. 1991). l.3 mM Ca2+ and 10 mM sodium HEPES (pH 7.4) at a constant
Astrocytes and lymphocytes, 011 the other hand, release sig­ flow of 3 mLl(min·g liver) (Boon et al. 1990). After cannulation
nificant amounts of free amino acids, especially taurine, of the inferior caval vein, the Iiver was transferred to an open non­

recirculating system (Sies 1978). In isoosmotic perfusion, the

ABBREVIATIONS: RVD, regulatory volume decrease; HPLC, osmolarity of the medium was 305 mosM. Hypoosmotic media

high-performance liquid chromatography. were prepared by decreasing the NaCl concentrarion 25 or 50 mM,

lAuthor to whom all correspondence should be addressed. resulting in osmolarity values of 255 or 205 mosM , respectively.

Pnnlcd in Calillda l Imprim é all Canada


BRAND ET AL. 9

TABLE I. Concentration of amino acids in effluent (f.LM), during


the first 5 min of hypoosmotic perfusion
5~------~==~------1

4
1 -0 mosM 1
Treatment 3

Hypoosmotic :2 2

Isoosmotic , ::i
Preperfusion 305 mosM 255 mosM 205 mosM

(isoosmotic) (n = 4) (n = 5) (n = 5)

f- o
AJa J.58±0.\2 1.28±0.07 0.96±0.06 1.23±0.05 Z
Arg 0.30±0.01 0.31±0.02 0.24±0.06 0.26±0.02 W 4
~
Asn 1.20±0.07 1.38±0.17 1.09±0.10 1.09±0.10 ~
Cit 0.81±0.04 0.86±0.05 0.72±0.10 0.89±0.03 IJ... 3
Gin 20.6±2.37 14.5±0.86 17.6±1.l4 18.6±3.69 IJ...
Glu 17.0±2.83 16.5±2.34 18.8±4.80 19.8±1.17 W
Gly 29.5±2.22 26.0±1.50 21.3± 1.66 27.4±2.26 Z
His 4.11±0.27 3.07±0.24 3.71±0.24 4.04±0.31
Ile 7.52±0.26 7.31±0.13 7.84±0.27 7 .88±0.72 W
Leu 12.7±0.44 12.3±0.27 13.8±0.50 13 .5±1.24 Z
Lys
Met
9.16±0.89
3.00±0.11
8.47±0.74
2.92±0.07
6. I 5±0.22
3.01±0.13
7.28±0.37
3.10±0.28
cr
~
Om
Phe
2.65±0.83
6.13±0.33
2.82±0.66
5.96±0.25
2.94±0.90
6.61±0.23
2.72±1.05
6.83±0.84
«

Ser 3.02±0.28 2.77±0.26 1.69±0.11 2.53±0.49
Tau 0.88±0.ll 0.89±0.11 2.36±0.15a.h 13.9±0.95,,·b.c
Thr 3.39±0.2\ 3.07±0.14 2.62±0.13 3.02±0.30
Trp 1.37±0.03 1.31±0.03 1.48±0.14 1.47±0.16
Tyr 1.68±0.08 1.52±0.09 1.73±0.12 1.67±0.19
5 10 15 20 25 30 35 40
Val 10.5±0.46 9.90±0.29 10.8±0.39 10.9±0.90
NOTE: Dala are means ± SEM . Analysis of varianee: a. P < 0.005 versus preperfusion;
b. P < 0.005 versuS isoosmolie control: c. P < 0.01 versus -50 mosM. Cil. eilrulline; FRACTIONS ( MIN )
Om, ornithine: Tau. taurine.
FIG. I. Effect of - 50 or - 100 mosM hypoosmotic stress on
taurine release by isolated perfused rat liver. Boxes indicate
The osmolarity of the media was verified with an Osmomat 030 period and ex tent of hypotonicity. Data are means ± SEM.
cryoscopic osmometer (Gonotec, Berlin, Germany). The perfusion Controls, n = 4; -50 mosM, n = 5; - 100 mosM, 11 = 5.
medium was gassed with 02-C02 (19: I); the temperature was
3rc.
A similar perfusion protocol was used in all experiments. 15
After 15 min of equilibration with isoosmotic buffer, the medium
was switched to hypoosmotic (255 or 205 mosM) or isoosmotic
medium (305 mosM = control) for 10 min. I­ 10
Unless otherwise stated , the effluent was collected in 5-min Z
fractions and the free amine acids in these fractions were deter­ ~
--.J
mined using an HPLC technique (van Eijk et al. 1988), after l..L
l..L
the fractions had been deproteinized with sulfosalicylic acid W
5
(4 mg/lOO f.LL). ~
In a separate series of isolated rat liver perfusions , liver mass
during the perfusion was monitored continuously with a specific ~
balance pan on a Sartorius L 610 D balance. The liver mass ~ 0
before the switch from isotonicity to hypotonicity was set to f!: 5 15 25 35 45 55 65 75 85
100% in each individual perfusion experiment and the liver mass FRACTIONS ( MIN )
changes recorded during the experiment were expressed as per­
FIG . 2. Effect of successive exposUI'es to -100 mosM hypo­
centage liver mass increase (Häussinger et al. 1990a). Effluent
osmotic stress on taurine release by isolated perfused rat li ver.
perfusate in these perfusions were collected in fractions of 30 s
Boxes indicate periods of hypotonicity. Data are means ± SEM
and analyzed for free amino acids as described above.
(11 = 4).
Animal welfare was in accordance with institutional guide­
lines of the University of Amsterdam.
Data are expressed as means ± SEM (n = number of perfusion and was specifie for taurine in that no significant extra
experiments). ANOVA was performed for statistica I analysis release was observed for any of the other amino aeids under
using the spss/pc+ statistical software package (vers ion 3.0). these eonditions . Ouring the seeond half of the hypotonie
period, the coneentration of taurine was less, but still sig­
Results nifieantly increased (Fig. I). When the perfusion medium
Hypotonie stress induced an inerease in Iiver mass and a was returned to isotonic medium after the hypotonie stress,
fast, significant inerease in the eoneentration of taurine in the the taurine eoneentration in the effluent returned to its eon­
perfusate effluent during the first half of the hypotonie trol values and remained at a steady state.
period (TabIe I). This inerease was 1.7-fold during We observed th at with sueeessive exposures of isolated
-50 mosM and 14-fold during -100 mosM hypotonicity, rat livers to hypotonie media, there was a deereased release
10 BIOCHEM. CELL BIOL. VOL. 72, 1994

110
...J
0 (A)
a:

Ö
() 105
t5
0~

(j)
(j) 100
«
2

cr:

UJ
>
::ï

(8)
2 20
::l.

0
~ 15
«
UJ
cd
a: 10
~

~ 5

«

0
0 10 20 30 40 50 60

FRACTIONS MIN
FIG, 3, Effect of -100 mosM hypoosmotic stress on mass of isolated perfused rat liver (A), Liver mass before hypotonicity was
set to 100% in each individual perfusion experiment. Effluent perfusate was colJected in 30-s fractions and analyzed for taurine
(B), Box indicates period of hypotonicity, Data are means ± SEM (n = 3),

of ta urine (Fig. 2), The amount of taurine released during the ing in a 40-50% decrease of the intracellular concentration
second and third exposure to -100 mosM was 29 ± land of ch,l oride (MeUer et al. 1992), Our results show that during
17 ± 1%, respectively, of the release of taurine during the first hypotonie stress the amino acid taurine is also released,
exposure, The RVD was not decreased during the second This increased release of taurine from the liver was not due
or third exposure to -100 mosM, to damage of liver cells for two reasons, First, the release into
Simultaneous measurement of taurine release and changes the effluent was specific for taurine, Second, the release of
in liver mass revealed th at the release of taurine c10sely lactate dehydrogenase from the isolated perfused rat li ver
followed swelling, Changes in liver volume showed a rapid during the period of hypotonicity is very low (H,S, Brand,
initial liver swelling, followed by RVD which was completed unpublished observations; see also Häussinger et al. 1990a),
after about 5 min, Upon reexposure to isotonicity, liver During isoosmotic perfusions, some amino acids (e,g"
volume restored to its original volume (Fig, 3A), Maximum Gly, Gin, Ser, His, Lys) showed a smalI, but not statistieally
cell swelling and maximum taurine release both occurred significant, tendency to deerease during the perfusion,
1-1,5 min after the switch from isotonic to hypotonic Under our experimental conditions, the tota! amount of
medium , After this period, during RVD, the release of taurine taurine released into the effluent during lO-min hypotonie
declined (Fig, 3B), stress was about 50 and 200 nmol/g liver for -50 and
- 100 mosM hypotonici ty, respeeti vely, Häussinger et al.
Discussion (l990b) showed that during perfusion with - 80 mosM hypo­
Exposure of isolated perfused livers to hypotonic perfusion tonie medium, isolated rat liver relea sed 12,6 /-Lmol K + tg
medium induces water uptake, To restore normal cell volume, liver, indicating that the quantitative contribution of released
the liver has several mechanisms for water extrusion, During taurine to the RVD is very small eompared with the efflux
hypotonicity, the bile flow is increased (Bruck et al. 1992: of KCI.
Hallbrucker et al. 1992), However, the water efflux into Two possible meehanisms of the increased taurine release
bile represents only a minor fraction of the total water efflux during cell swelling in the isolated perfused rat liver seem
from the liver during RVD (Brand et al. 1993), feasible, First, there is an energy-dependent release of taurine
Therefore, reduction of the increased cell volume must as described reeently for isolated skate hepatocytes (Ballatori
mainly depend on the extrusion of osmotically active sub­ and Boyer 1992), However, this is unlikely since the release
stances, During hypotonicity large amounts of potassium of taurine from isolated perfused rat liver is not decreased at
and chloride are released (Häussinger and Lang 199 1), result­ 23°C (H.S, Brand, unpublished observation) , Therefore, it
BRAND ET AL. II

seems more likely th at cell swelling, induced by hypo­ sensitive taurine transport in fish erythrocytes. J. Membr. Biol.
tonicity, increased plasma membrane permeability, and con­ 96: 45-56 .
sequently, intracellular taurine may leak to the extracellular Haddad, P., Beek, J .S., Boyer, J .L. , and Graf, J. 1991. Role of
space. Activation of such passive, gradient-dependent taurine chloride ions in liver cell volume regulation. Am. J. Physiol.
261: G340-G348.
Ieak pathways during hypotonicity have also been described
Hallbrucker, c. , Lang, F., Gerok , w. , and Häussinger, D. 1992.
for Ehrlich ascites tumour cells (Hoffmann and Lambert
Cell swelling increases bile flow and taurocholate excretion
1983) and skate erythrocytes (Fincham et al. 1987). Two into bile in isolated perfused rat liver. Biochem. J. 281:
pools of intracellular taurine exist: a large slowly exchange­ 593-595.
able pool and a small rapidly exchangeable po;)1 (Sturman Häussinger, D., and Lang, F. 1991. Cell volume regulation of
et al. 1975) . The observed reduction in taurine release from hepatic function: a mechanism for metabolic contro!. Biochim.
the isolated perfused rat liver after successive exposures to Biophys. Acta, 1071: 331-350.
-100 mosM may result from a decreased gradient after Häussinger, D., Hallbrucker, C. , vom Dahl, S., Lang, F., and
partial depletion of the rapidly exchangeable pool. Gerok, W. 1990a. Cell swelling inhibits proteolysis in perfused
Our data are the first to show that the intact isolated per­ rat liver. Biochem. J. 272: 239-242.
Häussinger, D., Stehle, T., and Lang, E 1990b. Volume regulation
fused liver releases taurine in a rather specific way during cell
in liver: further characterization by inhibitors and ionic sub­
swelling . The taurine release seems not to be involved in
stitutions. Hepatology (Baitimore) , 11: 243-254.
RVD, but rather the consequence of membrane stretching Hoffmann, E.K., and Lambert , LH . 1983. Amino acid transport
as recently described for astrocytes (O'Connor and Kimelberg and cell volume regulation in Ehrlich ascites tumour cells. 1.
1993). Physiol. (London), 338: 613-625.
Hoffman, E.K., and Simonsen, L.O. 1989. Membrane mechanisms
Acknowledgements
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1991. Taurine release associated to volume regulation in rabbit
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