ArizonaStateUniversity

Na+FLUXRESPONSETOCHANGESINNa+CONCENTRATIONANDADDITION
OFAMILORIDEANDOUABAINTOXINS

Bio361Lab

By

LovenderPhiri,
AngelinaLedesma,
AdlinurLamAliffLam
CelestineLeNoir
INTRODUCTION
Maintaining homeostatic ion balance is one of the most basic mechanisms living

organismsdoautomatically. Theconcentrationofwateriscomparedtotheconcentrationsof

solutes within and outside of acell. Water may passively flow through cellular membranes

(despite being nonlipid soluble) through pores called aquaporins. Water diffuses through

aquaporinsthroughaprocessknownasosmosis.

Unchargedmolecules(suchascarbondioxideandoxygen)havetheabilitytosimply

diffuse down their concentration gradients, due to the fact that the cellular membrane is

permeabletothem.However,chargedmolecules,suchasions,cannotsimplydiffusethrougha

cellmembrane.Ionsrequiretheaidofeitherchannelproteinsorcarrierproteins.Thechannel

and carrier proteins are incorporated in a process known as passive transport. This kind of

transportrequiresnoenergy.Moleculesaretransporteddowntheirconcentrationgradients,from

an area of high concentration to an area of low concentration, to establish electrochemical

equilibrium.

Whentransportationrequiresenergy,intheformofATP,theprocessisreferredtoas

eitherprimaryactivetransportorsecondaryactivetransport.Primaryactivetransportdirectly

utilizes ATP in order to pump molecules across a cell membrane, against a concentration

gradient.Secondaryactivetransportrequirestransportationofamoleculeusingprimaryactive

transport first, then utilizing the energy released to transport another molecule against its

concentrationgradient.ThisformofactivetransportiscommonlyseenwhentransportingNa+

(sodium)ionswithglucosemolecules.
 Primary active transport can be observed in the Na+/K+ATPase pump in cell

membranes.ThispumpmaintainsconcentrationsofNa+andK+innearlyeverycell.Sincemost

cellmembranesarenotpermeabletoNa+,ATPmustbeutilizedtobringNa+intothecell.Three

Na+ionsarepumpedinforeverytwoK+ionspumpedout.Thisisduetophosphorylationof

ATPwhichcausesachangeinthemembraneinfavorofNa+.Dephosphorylationofthepump

thenleadsthecelltothrowK+out.

Frogsfaceanongoingstruggleofsodiumlosstotheirenvironmentthroughdiffusion.

Since frogs live in freshwater conditions, sodiums electrochemical gradient is in

disequilibrium.Inordertomaintainequilibrium,theskinoffrogsmustincorporateprimary

active transport for the recovery of Na+ ions from the mucosal to the serosal side of the

epithelium.

Similartomanyotherorganismswhichincorporateepithelialtransportpumps,thefrog

skinepithelialcellscontaintightjunctions.Thisensuresthatmovementofcertainmoleculesis

throughtheepithelium,andnotinbetween.Themucosalandserosalsidesofthefrogskinare

asymmetricalwhenitcomesdowntomembranepermeability.Themucosalsideofthefrogskin

ispermeabletoNa+,andimpermeabletoK+.Theserosalsideofthefrogskinistheexact

oppositeitispermeabletoK+andimpermeabletoNa+.Theflowofionsintoandoutofthe

epitheliumwhenutilizingtheNa+/K+ATPasepumpisinaantiportsystem.

TheflowofionsthroughtheNa+/K+ATPasepumpalsogivesoffanelectricalcharge.

AccordingtoOhmsLaw,anelectricalcurrentisproportionaltothevoltagegivenoff,and

inversely proportional to the resistance the cell maintains. In this experiment, Na+
concentrations,currentsduetoappliedvoltages,andeffectsofAmilorideandOuabainpoisoning

onNa+flux,wasmeasured.

ThefrogskinpatchwasplacedinanUssingchamber,andkeptmoistinasalinesolution

(to mimic a frogs everyday environment). When a Ringers solution containing no sodium

slowlyreplacedthesalinesolution,whatdidweexpecttohappentothemagnitudeofthecurrent

forNa+?ManipulatingtheNa+concentrationoneithersideoftheepitheliumwillaltertheNa+

flux.Wepredictedthatiftheconcentrationofsalinesolutionwasloweredthroughtheconstant

additionoftheRingerssolutionthentheNa+fluxwoulddecreaseaswell.

WhentheAmiloridepoisonwasaddedtoreplacethesalinesolution,weexpectedthatif

placed on the mucosal side, Na+ net flux would decrease. This is because Amiloride is an

inhibitorofcarriermediatedpassivetransport(amucosalsideprotein),responsibleformoving

Na+fromtheapicalsidetothebasalsideoftheepithelium.Wepredictedthatifweplaced

Amilorideontheserosalsideoftheepithelium,therewouldbelittletonochange.Whenthe

Ouabainpoisonwas addedtoreplacethesalinesolution,weexpectedthatifplacedonthe

serosalside,Na+fluxwoulddecrease.ThisisbecauseOuabainisaNa+/K+ATPase(aserosal

side)inhibitor,andwouldpreventNa+fromcrossingoverthebasalsideoftheepithelium.We

predictedthatifweplacedOuabainonthemucosalsideoftheepithelium,therewouldbelittle

tonochange.
PROCEDURE

Toshortcircuitthefrogskin,astimulatingvoltagewasappliedtotheUssingchamber.

Thisvoltagewasslowlyandsteadilyincreaseduntilthemembraneelectricalpotentialwasread

aszero.Atamembranepotentialofzero,thecurrentreadingisthemeasureoftheactivityofthe

Na+/K+ATPasepumps.

Todeterminehowtheexternalconcentrationofsodiumaffectsthesodiumtransport,the

salinesolutiononthemucosalsideoftheskinwasdilutedinincrementsof75%,50%,25%and

0%withRingerssolution.Eachtime,thevoltagewasadjustedandthenewshortcircuitcurrent

wasfound.

Todeterminetheeffectsofamilorideonsodiumtransportandmembranepotential,0.5

mlofamiloridewasaddedtotheRingerssolutionontheserosalsideoftheskin.Thepotential

wasthenbroughtbacktozeroandthenewshortcircuitcurrentwasrecordedandcomparedto

theoriginalbaselinecurrent.Thesalinesolutionwasthenreplacedonbothsidesofthechamber

andasecondbaselinecurrentwasrecorded.Then,0.5mlofamiloridewasaddedtothemucosal

sideoftheskinandthepotentialwasbroughtbacktozeroandthatnewshortcircuitcurrentwas

recordedandcomparedtothesecondbaselinecurrent.
 Todeterminetheeffectsofouabainonsodiumtransportandmembranepotential,0.5ml

ofouabainwasaddedtotheRingersonthemucosalsideoftheskin.Thepotentialwasbrought

tozeroandthenewshortcircuitcurrentwasrecordedandcomparedtothebaselinecurrent.The

salinewasthenreplacedonbothsidesontheUssingchamberwithfreshbuffer.Then,0.5mlof

ouabainwasaddedtotheserosalsideoftheskin.Thepotentialwasbroughtbacktozeroandthe

newshortcircuitcurrentwasrecordedandcomparedtothesecondbaselinecurrent.

RESULTS
Table1:Determiningtheshortcircuitcurrent,theelectricalpotential,andresistanceacrossfrog
skin.

AppliedVoltage(mV) CorrectedCurrent( A )

0 0.4

300 5.2

310 5.3

320 5.4

330 5.6

+
Table2:Determiningtheeffectofaltered Na onsodiumtransport

Concentration(%) Concentration(mM) Isc( A ) + flux ( moles sec 1 cm2 ) (1011)

Na

100 110 4 6.52

75 82.5 4 6.52

50 55 3.3 5.78

25 27.5 2.96 4.73

10 11 2.5 4.07

0 0 2.2 3.58

Table3:Determiningtheeffectofthedrugsonsodiumtransport

Amiloride Ouabain

Treatment Isc( A ) + flux ( moles sec 1 Isc(

cm2) (10
A ) 11) + flux ( moles sec 1 cm2 ) (1

Na Na

Premucosal 7.2 0.117 1.9 3.1

baseline

Drugonmucosal 1.9 3.1 1.9 3.1

side

Preserosal 5.5 8.96 2 3.26

baseline

Drugonserosal 6.1 9.94 2.8 4.56

side

Figure1:DeterminingtheeffectofalteredNa+onSodiumTransport
 Effect of Altered (Na+ ) on Sodium Transport

f(x) = 1.66x - 0.04

Figure2:Determiningtheeffectofamilorideandoubainonsodiumtransport

Determining the effect of amiloride and ouabain on sodium transport

12
9.94
10 8.96

6
4.56
4 3.1 3.1 3.1 3.26
2
2 1
0.12
0

Pre-mucosal Drug on mucosal Pre-serosal

Drug on serosal

+
Activetransportand Na leakchannelimpactedtheresultsthatwereobtainedas

+
showninFigure1and2.Forfigure1,itshowsthatastheconcentrationof Na lowers,the
 +
currentand Na fluxdecreases.Forfigure2,theTrial1signifiestheeffectsofamilorideon

sodiumtransport,whereasTrial2showstheeffectofouabainonsodiumtransport.Asseenin

+
thegraph,itcanbeseenthatamilorideincreasesthe Na fluxonthemucosalandtheserosal

+
side.However,theouabainonlyaffectthe Na fluxonserosalside.

DISCUSSION
Fig1supportsourpreviouslystatedhypothesis,thatmanipulationoftheNa+concentration

affectsthefluxofNa+ionsacrossthefrogskinandthepredictionthatiftheconcentrationis

loweredthenflowofionsdecreaseswhichdecreasesthecurrent.Thegraph(fig1)showsa

positivecorrelationbetweenconcentrationandthecurrent(fluxofNa+ions),thisisbecausethe

Na+ionsaremovingacrosstheskinfromthemucosalsideintothecellsviapassivetransport

downitsconcentrationgradient.Hencewhentheconcentrationisloweredonthemucosalside

concentrationgradientisreduced,causinglessionstoflowintothecellsthusdecreasingthe

currentwhichisproducedbythesystem.

Thesecondsetofresultssupportedoursecondhypothesisthatintroductionoftoxinstothe

systemwillinhibitthefluxofbothNa+andK+ionsacrossboththemucosalandtheserosalside
andthepredictionthatifAmilorideisaddedtoboththemucosalandserosalsidethenitwill

inhibittheNa+transportproteincausinglowNa+fluxandwillhavenoeffectontheserosalside

layer.WhereasifOuabainwasaddedtoboththemucosalandserosalsidethenitwillinhibitthe

Na+/K+pumpwhichhasnoeffectonthemucosalside,butincreasethefluxofK+acrossthe

serosallayer.Fig2supportsourhypothesesandpredictions,Amilorideaddedtothemucosal

sidedecreasedcurrentgreatly,butincreasedthecurrentbyasmallchangeontheserosalside.

ThisisbecauseNa+proteinchannelsareonlyfoundonthemucosalsideandnottheserosal

(Henry2017)henceAmiloridedenaturedNa+proteinchannelthusloweringthecurrentand

potentialdifference.Therecordedcurrentchangeontheserosalsidecouldhaveoccurreddueto

increasedflowofK+intotheserosalwhichcouldhavebeenstimulatedbytheAmiloridetoxin

ontheK+channelprotein.OuabainshowednoeffectonthecurrentandfluxofNa+ionsonthe

mucosalside,butcausedanincreaseinK+ionsfluxandcurrentrecordedontheserosalside.

ThiswasbecauseitinhibitedthefunctioningoftheNa+/K+pumpwhichisonlyontheserosal

sideonlyandnotonthemucosalside(Henry2017)makingitimpermeabletoanyNa+ionsinto

theserosalsideandanyK+leavingviaactivetransport.Theincreaseincurrentontheserosal

sidewasbecauseofthepassivetransportofK+ionsintotheserosalsidethusapositivecharge

wascreated.

Thedrivingquestionofthislabwasbasedonhowtodeterminetheevidencewhichverifies

thetransportofNa+ acrossfrogskinoccursbyactivetransport.Theaboveresults(Fig2)

supportandanswerthisquestion,theouabaininhibitedthepumpontheserosalsidegaveazero

currentchange(Na+flux)whichverifiesthatNa+fluxisprimarilyactivelytransportedacross

transepithelialfrogskinusingATPfromtheATPasepump.Howevertofurtherimproveonthis
labandanswertotheposedquestiondifferentcellscontainingthesamepumpsandchannel

proteinscouldbeincludedtotheexperimentandusedascomparisonbaseontheeffectsofthe

manipulatedvariables.