PGE2 Activation of Apical Membrane Cl Channels in A6 Epithelia

15 Pages·2001·0.19 MB·English

852 BiophysicalJournal Volume81 August2001 852–866
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PGE Activation of Apical Membrane Cl Channels in A6 Epithelia:
2
Impedance Analysis
Teodor G. Pa˘unescu and Sandy I. Helman
DepartmentofMolecularandIntegrativePhysiology,UniversityofIllinoisatUrbana-Champaign,Urbana,Illinois61801USA
ABSTRACT Measurementsoftransepithelialelectricalimpedanceofcontinuouslyshort-circuitedA6epitheliaweremadeat
audio frequencies (0.244 Hz to 10.45 kHz) to investigate the time course and extent to which prostaglandin E (PGE )
2 2
modulates Cl2 transport and apical membrane capacitance in this cell-cultured model epithelium. Apical and basolateral
membraneresistancesweredeterminedbynonlinearcurve-fittingoftheimpedancevectorsatrelativelylowfrequencies(,50
Hz) to equations (Pa˘unescu, T. G., and S. I. Helman. 2001. Biophys. J. 81:838–851) where depressed Nyquist impedance
semicircles were characteristic of the membrane impedances under control Na1-transporting and amiloride-inhibited con-
ditions.Inalltissues(control,amiloride-blocked,andamiloride-blockedandfurosemide-pretreated),PGE causedrelatively
2
small (,;3 mA/cm2) and rapid (,60 s) maximal increase of chloride current due to activation of a rather large increase of
apical membrane conductance that preceded significant activation of Na1 transport through amiloride-sensitive epithelial
Na1 channels (ENaCs). Apical membrane capacitance was frequency-dependent with a Cole-Cole dielectric dispersion
whoserelaxationfrequencywasnear150Hz.Analysisofthetime-dependentchangesofthecomplexfrequency-dependent
equivalentcapacitanceofthecellsatfrequencies.1.5kHzrevealedthatthemean9.8%increaseofcapacitancecausedby
PGE was not correlated in time with activation of chloride conductance, but rather correlated with activation of apical
2
membraneNa1transport.
INTRODUCTION
In experiments designed to study hormonal regulation of persionsataudiofrequencies(Awaydaetal.,1999;Liuand
Na1transportinepithelialtissues,theactivationofchloride Helman, 1998; Pa˘unescu and Helman, 2000) that compli-
transportinadditiontosodiumtransportimposescomplex- cate measurement of plasma membrane capacitance. The
itiesinthedesignofexperimentsandinterpretationofdata theoretical principles and considerations relevant to the
unlike those encountered in studies of frog skin and toad present studies have been discussed elsewhere (Pa˘unescu
urinary bladder where adenosine 39,59-cyclic monophos- andHelman,2001)whereweexaminedthecontributionsof
phate(cAMP)selectivelyactivatesapicalmembraneamilo- Maxwell-Wagner-like and Cole-Cole dielectric dispersions
ride-sensitive epithelial Na1 channels (ENaCs) (Els and tothetransepithelialimpedancelocusoftheseriesarrange-
Helman, 1981, 1997; Schlondorff and Satriano, 1985). To ment of apical and basolateral membrane impedances that
understandthecomplexitiestobeencounteredinstudiesof are paralleled electrically by paracellular shunt resistances.
cell-cultured A6 epithelia where cAMP activates both Na1 Evaluationofthetime-dependentchangesofshort-circuit
and Cl2 channels, we turned to noninvasive methods of currentandtransepithelialimpedancehasledustoconclude
impedance analysis to determine the time course and mag- that PGE maximally activates a rather large apical mem-
2
nitude of change of apical membrane conductance to chlo- brane chloride conductance in ,1 min. Activation of chlo-
rideandapicalmembranecapacitanceinresponsetoeleva- ride channels preceded activation of the apical membrane
tion of intracellular cAMP by forskolin and prostaglandin sodium conductance from basal states where chloride con-
E (PGE )thatareknowntoelevatecAMPintargettissues ductancewasimmeasurablysmallorabsentinA6epithelia
2 2
(Chalfant et al., 1993; Hall et al., 1976; Sonnenburg and grown on Transwell-clear inserts. Because the time-depen-
Smith, 1988; Yanase and Handler, 1986; Noland et al., dent increases of apical membrane capacitance paralleled
1992). the increases of sodium transport and were completely
Although the measurement of transepithelial impedance dissociated from those of activation of chloride conduc-
is in principle straightforward, previous studies from our tance,weconcludedthattheslightlydelayedandrelatively
laboratory had indicated that the dielectric properties of slow increases of capacitance were associated with activa-
epithelial plasma membranes may exhibit a-dielectric dis- tion of apical membrane sodium conductance.
Received for publication 19 September 2000 and in final form 26 April MATERIALS AND METHODS
2001.
Tissues, solutions, and drugs
AddressreprintrequeststoDr.SandyI.Helman,Dept.ofMolecularand
Integrative Physiology, University of Illinois at Urbana-Champaign, 524
A6 cells at passages 109 to 114 were used in the present studies. After
BurillHall,407S.GoodwinAve.,Urbana,IL61801-3796.Tel.:217-333-
growthon75cm2plasticcultureflasksat28°Cinahumidifiedincubator
7913;Fax:[phone];E-mail:[email protected].
containing1%CO ,thecellsweresubculturedonTranswell-clearcluster
2
© 2001bytheBiophysicalSociety inserts(Costar,Cambridge,MA)foratleast14daystoachieveconfluence
0006-3495/01/08/852/15 $2.00 anddevelopmentoftheirtransepithelialtransportcharacteristics(Helman
PGE -ActivatedEpithelialCl2Channels 853
2
FIGURE 1 (A)Compositevoltagecommandsignal,V ,consistedofthevectorialsumof43sinusoidswithrelativeamplitudes(B)andphaseangles
cmd
(C)andwithtimeperiods,T,determinedbytheirfundamentalfrequencies(0.244,3.91,and50.0Hz).
andLiu,1997).ThetissueswerefedtwiceweeklywithaCl2-richgrowth free, gluconate Ringer’s solution containing 100 mM sodium gluconate,
medium that was based on a mixture of equal parts of Ham’s nutrient 2.4 mM KHCO, and 2.0 mM CaSO was perfused through apical and
3 4
mixtureF-12withL-glutamineandwithoutsodiumbicarbonate(N-6760, basolateralchambersinthoseexperimentswheretissueswereexposedfor
SigmaChemicalCo.,St.Louis,MO)andL-15Leibovitzmedium(L-4386, 1 h during control periods and chronically thereafter to chloride-free
Sigma Chemical Co.). This mixture was supplemented with 10% fetal solution.
bovine serum (FBS) (SH0070, HyClone, Logan, UT), 2.57 mM sodium
bicarbonate,3.84mML-glutamine(SigmaChemicalCo.),96U/mlpeni-
cillin,and96mg/mlstreptomycin(BioWhittaker,Walkersville,MD). Impedance analysis
The tissues were transferred to edge damage-free chambers (Abram-
checketal.,1985;Awaydaetal.,1999)andshort-circuitedfortheduration Transepithelialimpedancewasmeasuredundervoltageclampconditions
oftheexperimentsusingaverylow-noise,four-electrode(Ag/AgCl,4.5M using three overlapping bands of frequencies (low, medium, and high)
NaCl,3%agar)voltageclampwhilebeingperfusedcontinuouslyatflow between0.244Hzand10.45kHz(Fig.1)andusingessentiallythesame
ratesof;7ml/minthroughchambervolumesof;0.5mlwiththegrowth approach described previously (Awayda et al., 1999), but with several
medium without FBS and glutamine. Na1, K1, Cl2, Ca21, and Mg21 modifications.Thevoltagecommandsignals(V )consistedofthevec-
cmd
concentrationswere103.0,3.34,106.8,0.59,and0.91mM,respectively. torialsumof43frequencieswheretheabsoluteamplitudeofeachsinusoid
Short-circuitcurrents(I )wereallowedtostabilizeforatleast2hbefore decreasedwithincreasingfrequency(Fig.1B)andwiththephaseangle
sc
onset of experimental periods. Characteristically, the I increased tran- indicatedinFig.1C.Thisdesignofthecompositevoltagecommandsignal
sc
sientlywhentissueswereinitiallyshort-circuited,returningtonearsteady- servedtoassurethatthecapacitivecurrentsateachfrequencywouldbe
state values within 1 to 2 h (Pa˘unescu et al., 1997); 100 mM amiloride closer in magnitude than would occur if the amplitudes of the voltage
addedtotheapicalperfusionsolutionwasusedtoinhibitblocker-sensitive command sinusoids were of equal amplitude at all frequencies. With
Na1currents(Ibs). fundamentalperiods(T)of4.098s(lowfrequencies),255.7ms(medium
Na
To elevate intracellular cAMP, forskolin (Sigma Chemical Co.) or frequencies),and20ms(highfrequencies),correspondingtofundamental
prostaglandinE (PGE)(SigmaChemicalCo.)wasaddedtothebasolat- frequencies of 0.244, 3.91, and 50.0 Hz, the relative amplitudes of the
2 2
eralperfusionsolutionatfinalconcentrationsof25mMor1mM,respec- voltagecommandsinusoidsinallthreefrequencybandscanbecompared
tively.StocksolutionsofforskolinandPGE weredissolvedinethanolat (Fig.1B),especiallyintheoverlappingrangesoffrequencybetweenbands
2
1022 M and stored at 220°C. Furosemide (Sigma Chemical Co.) was (3.91to51.02Hzand50.0to816.4Hz).Consequently,impedanceinthe
addeddirectlytothebasolateralsolutionat1mMtoinhibitelectroneutral overlappingbandsoffrequencywasmeasuredwithsinusoidsofmarkedly
chloride transport at the basolateral membranes of the cells (Brazy and differing voltage and current amplitudes at each time point of measure-
Gunn,1976;LambertandLowe,1980;Stoddardetal.,1985).Achloride- ment, thereby providing a built-in check for testing and assuring that
BiophysicalJournal81(2)852–866
854 Pa˘unescuandHelman
impedancewasindependentofthemagnitudeofV .Themagnitudesof
cmd
V (Fig.1A)wereadjustedsothatthepeak-to-peakchangesoftrans-
cmd
epithelialvoltagewerenear2mV.Consequently,itcouldbeassumedthat
impedancewasmeasuredforallpracticalpurposesinlinearrangesofthe
current-voltagerelationshipsofthechannels.
An IBM-compatible computer containing a DSP2200 board (16 bit
ADCs,16bitDACs,NationalInstruments,Austin,TX)wasprogrammed
using LabWindows for DOS to output the analog V signals and to
cmd
simultaneously digitize the measured transepithelial voltage and current
signalsafteramplificationandfiltrationattheNyquistfrequencies.Low,
medium,andhighfrequencybandswereeachoutputinsequenceoverfour
timeperiodsanddatacollectionforanalysiswasretainedforonlythelast
periodofeachband.Accordingly,thetotaltimefordataacquisitionwas
near 5.2 s [4.098 1 (4 3 0.2557) 1 (4 3 0.020)]. These signals were
Fourier-transformed to yield the voltage and current vectors. Z was
meas
calculatedasthequotientofvoltageandcurrentvectorsattherespective
frequencies. Where appropriate, data were normalized to planar surface
areaandplottedasNyquist(ReZ vs.ImZ )orBodeplots(uZ uand
meas meas meas
phaseangleversusfrequency).SeeResultsforcalculationsofapicaland
basolateralmembraneresistancesandcapacitances.
Data are summarized as means 6 SE. Statistical analyses were per-
formedwithSigmaStat(JandelScientificSoftware,SanRafael,CA)using
paired or unpaired t-tests where appropriate. A p value , 0.05 was
consideredsignificant.Allexperimentswerecarriedoutatroomtemper-
ature.
RESULTS
Activation of amiloride-insensitive chloride
current by forskolin and PGE
2
In agreement with the observations of others (Yanase and
Handler, 1986; Perkins and Handler, 1981; Chalfant et al.,
FIGURE 2 Typicalstripchartrecordingsareshownoftheshort-circuit
1993; Niisato and Marunaka, 1997), increases of intracel- current responses to forskolin or PGE in control (A) and amiloride-
2
lular cAMP lead to activation of chloride currents in A6 pretreated tissues (B and C). The tissue in A was treated with 25 mM
epithelia. As indicated in the strip chart recording of Fig. 2 forskolin.
A,wehaveobservedcharacteristically,followingtreatment
witheitherforskolinorPGE ,thattheI increasesabruptly
2 sc
within tens of seconds to peak or quasi-plateau values sitive short-circuit currents increased abruptly within 30 to
(Pa˘unescu, 1999). After what appears to be a short delay, 40 s to peak values, followed by relaxation of the currents
theI increasesmarkedlyandrelativelyslowlytosubstan- in,10mintostablebutelevatedvaluesthatweresustained
sc
tially elevated plateau values within 20 to 30 min that are forthedurationofobservation(1to2h).Intheabsenceof
sustained for several hours. Addition of amiloride to for- PGE furosemide was without effect on the amiloride-in-
2
skolin-orPGE -stimulatedtissuesattheendsof2-hexper- sensitive currents (Fig. 2 C). In PGE -stimulated tissues
2 2
iments (not shown) characteristically resulted in large inhi- furosemide addition to the basolateral solution inhibited
bitions of the I . Compared to untreated tissues where the reversibly, but not completely, the amiloride-insensitive
sc
steady-state amiloride-insensitive currents, Iamil, normally current (Fig. 2, B and C). Peak current values averaged
sc
average in the range of 0.1–0.3 mA/cm2 (Pa˘unescu et al., 3.32 6 0.17 mA/cm2 (amiloride, n 5 3) and 2.26 6 0.23
1997, 2000b) and that averaged 0.16 6 0.03 mA/cm2 (n 5 mA/cm2 (amiloride 1 furosemide, n 5 3) at 37.9 6 2.7 s
6)inthepresentseriesofexperiments(Fig.2,BandC),the (n 5 6) following exposure of the tissues to PGE . The
2
IamilofforskolinandPGE -treatedtissuesaveraged1.916 currents decayed thereafter with a time constant of 2.48 6
sc 2
0.18 mA/cm2 (n 5 12) and 1.66 6 0.09 mA/cm2 (n 5 11), 0.14 min (n 5 6) to plateau values that averaged 1.20 6
respectively. Because the responses to forskolin, PGE , 0.11 mA/cm2 (amiloride, n 5 3) and 0.51 6 0.12 mA/cm2
2
exogenous cAMP, and theophylline caused similar in- (amiloride1furosemide,n53).Afteradditionaltreatment
creases of the amiloride-insensitive short-circuit currents of amiloride-blocked tissues with furosemide, the currents
(Pa˘unescu,1999;Pa˘unescuandHelman,1998,2000),PGE decreased to 0.38 6 0.04 mA/cm2 (n 5 3) (Fig. 2 B).
2
was used exclusively in the experiments reported below. Shown in expanded form in Fig. 3 are representative
When amiloride-blocked tissues were challenged with changes of I (DI ) from basal levels caused by PGE
sc sc 2
PGE as indicated in Fig. 2, B and C, the amiloride-insen- within7mininacontroltissue,anamiloride-blockedtissue
2
BiophysicalJournal81(2)852–866
PGE -ActivatedEpithelialCl2Channels 855
2
FIGURE 3 Time courses of change of the short-circuit current, DI ,
sc
caused by PGE within the first 7 min in a control tissue and a tissue
2
pretreatedwithamiloridethatwerebathedwiththechloride-richsolution.
Also shown is the time course of change of the DI of a control tissue FIGURE 4 (A)TypicalNyquistplotsofthemeasuredimpedance,Z ,
sc meas
bathedwiththechloride-freesolution.Thedelayinonsetofincreaseofthe are illustrated for a control tissue that was treated with amiloride. The
sodiumcurrentwas;30s,atwhichtimestimulationofchloridecurrent frequencies of the impedance vectors are indicated at the apices of the
wasnearmaximal. semicircles and at 300 Hz. An expanded view of the impedance vectors
between300Hzand10,450HzisshowninB.ThesolidlinesshowninA
weredeterminedbynonlinearcurve-fittingoftheimpedancevectorstoEq.
1(seetextandFig.6).
bathedinthechloride-richperfusionsolution,andacontrol
tissue bathed in chloride-free solution. Whereas currents in
tissues with functional ENaCs exhibit relatively large sec- additionallytreatedwithfurosemide.Neglectingthecontri-
ondaryincreasesofamiloride-sensitivecurrent(notshown), bution of amiloride-insensitive Na1 currents to the Iamil of
sc
the delayed increases of current are absent in amiloride- furosemide-treated tissues, furosemide inhibited between
pretreated tissues but are observed in chloride-free media. ;70–100%ofthechloridecurrentactivatedbyPGE .Ifthe
2
Notably, the abrupt increases of current are completely IamilofPGE -treatedtissuesbathedinchloride-freesolution
sc 2
absent in chloride-free media, thereby providing evidence is taken as an estimate of the amiloride-insensitive Na1
that the initial response to PGE and forskolin is due to current (0.47 6 0.08 mA/cm2, n 5 5) then furosemide
2
activation of a chloride conductance that precedes full ac- inhibited nearly 100% of the steady-state PGE -activated
2
tivationofNa1transport.Intheseregardsourobservations chloride current. In the absence of step changes of Iamil in
sc
are the same as those reported by Chalfant et al. (1993). response to a high concentration of basolateral furosemide,
The amiloride-insensitive currents following PGE or it would be reasonable to conclude that furosemide acts to
2
forskolin represent the maximal increases of chloride cur- inhibit basolateral membrane chloride entry into the cells
rentattheirpeakandduringthesubsequentsteadystatesof viaanelectroneutralmechanism(s)oftransportbecausestep
transport.Inthisregarditisrelevanttonotethattheamilo- changes of transcellular resistance, if they occurred, would
ride-insensitive basal short-circuit currents prior to PGE / cause step changes of short-circuit current that were never
2
forskolin are due principally to amiloride-insensitive Na1 observed.
currents(Baxendale-Coxetal.,1997).Itisunknowntowhat
extent PGE /forskolin may activate amiloride-insensitive
2 Impedance of control and
Na1 current together with the chloride current. Conse-
amiloride-treated tissues
quently, the specific current due to chloride would be less
than that measured by Iamil if amiloride-insensitive Na1 TypicalNyquistplotsofthemeasuredimpedance(Z )of
sc meas
currents are increased by PGE /forskolin. Thus, for tissues control,unstimulatedtissuesbeforeandaftertreatmentwith
2
continuously short-circuited, the steady-state chloride cur- amilorideareillustratedinFig.4.I averaged7.4160.60
sc
rents activated by PGE /forskolin are rather small in mag- mA/cm2 and was decreased to 0.46 6 0.06 mA/cm2 within
2
nitude,averagingatmost,2mA/cm2inamiloride-blocked 6minafteramiloride(Table1).Theimpedancelocus(Fig.
tissues and at most near 0.44 mA/cm2 when tissues are 4 A) conformed to a depressed semicircle at frequencies
BiophysicalJournal81(2)852–866
856 Pa˘unescuandHelman
TABLE 1 Effectof100mMamilorideonthebasalelectricalparametersofshort-circuitedA6epithelia
I R R R Cfit ffit gfit
sc T p cell eq
(mA/cm2) (kVzcm2) (kVzcm2) (kVzcm2) (mF/cm2) (Hz)
Control 7.4160.60 6.1160.77 10.7461.83 15.8361.73 1.3660.04 21.763.4 0.9660.012
6minafterAmiloride 0.4660.06 10.6161.81 — 262629 1.3960.04 13.563.1 0.9660.003
45minafterAmiloride 0.2760.01 9.7661.58 10.0361.65 419622 1.4060.03 14.263.1 0.9660.003
Valuesaremeans6SE(n56)andwhereapplicablearenormalizedtoplanarsurfacearea.R werecalculatedbythemethodofYonathandCivan(1971).
p
,;100HzandcouldbefittoanequationintheformofEq. existsincontroltissues,itsvaluemustbeextremelylowand
1, thereby permitting estimation of the dc resistance. Al- immeasurably small.
though not apparent when viewed this way, the data points
Capacitance of control and
at frequencies .100 Hz did not conform to this equation
amiloride-treated tissues
(see below) and so the apparent resistance of apical and
basolateral solutions, Rapp, calculated this way (156.6 6 Frequency-dependent capacitances can arise from Max-
sol
36.7 V z cm2, n 5 6) overestimated the actual series resis- well-Wagner and/or Cole-Cole dielectric dispersions
tance R (47.5 6 1.5 V z cm2, n 5 6). The R could be (Pa˘unescu and Helman, 2001). At sufficiently high fre-
sol sol
estimated by eye to within ;1–2 V z cm2 by extrapolation quencies where the apical and basolateral membrane
of the ImZ to the real axis. In practice, R was deter- capacitive reactances are considerably less in value than
meas sol
mined by nonlinear curve-fitting of the ReZ to infinite theirrespectivemembraneresistances,theequivalentcell
meas
frequency using TableCurve (SPSS Inc., Chicago, IL). The capacitance,C 5C C /(C 1C ),wouldbeconstantif
eq a b a b
transepithelialresistance(R )wasdeterminedasthediffer- the C and C were frequency-independent. If, however,
T a b
ence between the dc value of Z (Zdc ) and the R . the capacitances exhibit audio frequency a-dispersions,
meas meas sol
C would appear as a complex frequency-dependent
Rfit eq
Zmeas511~jvRfitCefiqt!gfit1Rsaoplp (1) capAatcaitlalnfrceequCe*enqc.iesC*eq[[jv(Za1Zb)]21(Pa˘unescuand
Helman, 2001). Accordingly, the transepithelial impedance
In these and all subsequent experiments, R was not
sol is given by Eq. 2 and C* can be determined from the
changed by amiloride nor PGE . Control R averaged eq
6.1160.77kVzcm2andat6and245minwasiTncreasedto measuredvaluesofZTandRp.ShowninFig.5Aaretypical
means of 10.61 and 9.76 kV z cm2, respectively, at these resultsforatissueinitscontrolandamiloride-treatedstates.
With increasing frequency, uC*u decreased from values
time points (Table 1). With the values of transepithelial eq
conductance (G 5 R21) and short-circuit currents before near 1.5 mF/cm2 to near 0.9 mF/cm2 at 10.45 kHz. Amilo-
and after amilorTide, theTshunt resistance R 5 G21 and the ride did not change the uC*equ.
p p
The´venin emf of the cellular pathway for Na1 transport R
Z 5 p (2)
(E ) were calculated using the method of Yonath and T 11jvR C*
Na p eq
Civan (1971) where G 5 I /E 1 G . R averaged 10.74
T sc Na p p
and10.03kVzcm2at6and45minafteramiloride(Table1) The mean uC*u of amiloride-treated tissues is summa-
eq
andtheE averaged112.564.3mV(n56),whichisquite rized in Fig. 5 C. The solid line fitted manually defines an
Na
typical for A6 and other tight epithelial tissues that transport approximate Cole-Cole a-dispersion with a characteristic
Na1 exclusively through apical membrane ENaCs (Helman frequency near 150 Hz. The deviations of uC*u and phase
eq
andLiu,1997;Koeppenetal.,1980;HelmanandThompson, anglefofC* frompureCole-Colebehaviorshownbythe
eq
1982;MacchiaandHelman,1979;YonathandCivan,1971). manually fitted lines in Fig. 5, C and D at frequencies
From the differences between R and R , the series re- ,;10 Hz are expected due to the Maxwell-Wagner-like
T p
sistanceofapicalandbasolateralplasmamembranesofthe dispersion (Pa˘unescu and Helman, 2001). It should be
cells,R 5R 1R ,wascalculatedtohavebeenincreased stressedthatmanualfittingofasingleCole-Colerelaxation
cell a b
byamiloridefrom15.83kVzcm2to.260kVzcm2(Table processtothedataisnotexact,andsothesolidlinesshown
1). To the extent that control tissues express amiloride- in Fig. 5, B and C should be taken as reasonable but
sensitive ENaCs and amiloride-insensitive Na1 currents imperfect approximations.
that are small, the amiloride-insensitive resistance of the ShownalsoinFig.5BaretypicalNyquistplotsofC*
eq
apicalmembraneisexpectedtobeintherangeof250kVz for the control and amiloride-treated states of the tissues.
cm2ifapicalmembranevoltageis100mVandtheblocker- Significant deviations from single ideal depressed semi-
insensitiveNa1current,Ibi,is0.4mA/cm2.Thus,incontrol circles were observed, as expected at the lower frequen-
sc
tissues, apical membranes are predominantly permeable to ciescorrespondingtothoseassociatedwiththeMaxwell-
Na1. If a finite apical membrane chloride conductance Wagner dispersions (Pa˘unescu and Helman, 2001).
BiophysicalJournal81(2)852–866
PGE -ActivatedEpithelialCl2Channels 857
2
FIGURE 5 Apicalmembranecapacitanceisfrequency-dependent.ShowninAaretypicalplotsofthedependenceonfrequencyoftheabsolutevalues
oftheequivalentcellcapacitance,uC*u,inthecontrolstateofatissueandafterinhibitionofNa1transportbyamiloride.Whenthesedataareplottedas
eq
NyquistplotsasindicatedinB,thecapacitancevectorsappeartoconformtoadepressedsemicircle(dielectricincrement’0.58mF/cm2;Cole-Colepower
lawcoefficient’0.65;characteristicfrequency’110Hz)atthehigherfrequencies.Significantdeviationsfromapuredepressedsemicircleatthelower
frequenciesareexpectedandobservedduetotheMaxwell-Wagnereffectatthelowerfrequencies(Pa˘unescuandHelman,2001).Asummaryofthemean
uC*uisshowninCwithselectedstandarderrorbarsatthelowerfrequenciesandatseveralhigherfrequenciesforthepurposeofclarity.Thecorresponding
eq
meanphaseanglesofC* aresummarizedinD.ToillustratetheapproximatecontributionandlocationoftheCole-ColerelaxationprocessinCandD
eq
(solidlines),theparametersoftheprocesswereadjustedmanually(dielectricincrement50.54mF/cm2;powerlawcoefficient50.625;characteristic
frequency5150Hz;limitingstaticcapacitance50.89mF/cm2)accordingtoEq.5inPa˘unescuandHelman(2001)toobtainanapproximatefitofthe
linestothemeandatapoints.Morerobustmethodswerenotattemptedduetothelessthanadequatenumberandreliabilityofdatapointsatthelowest
frequencies.
Nevertheless, it was apparent, at least to a first approxi- Capacitancecalculatedattheapexofthefittedsemicircle
mation, that an a-relaxation process was responsible for yieldedmeanvaluesnear1.36and1.40mF/cm2forcontrol
thefrequencydependenceofC* .BecauseC ,,C (see andamiloride-treatedtissues,respectively(Table1)atmean
eq a b
below), it follows that this dispersion arises predomi- frequencies near 22 and 14 Hz, respectively. The corre-
nantly from the apical membranes of the cells. Conse- spondingmeanvaluesofuC*uatthesefrequenciesreported
eq
quently, apical membrane capacitance could not be as- in Fig. 5 C are 1.33 6 0.03 and 1.36 6 0.03 mF/cm2 at 22
sumed to be constant in the audio range of frequencies. and 14 Hz, respectively, and are quite similar to the fitted
Provided that the bandwidth of impedance vectors was valuesexpectedatthesefrequencies(Pa˘unescuandHelman,
limitedtoamaximumof;50Hzorlessforthepresentset 2001).Itshouldbenotedthatthecapacitancescalculatedat
ofexperiments,itwaspossibletoobtainsatisfactoryfitsof theapicesofthefitteddepressedsemicirclesarenotunique,
theimpedancevectorsinthislimitedrangeoffrequenciesto but will depend not only on the frequency dependence of
Eq. 1, which is an equation of a depressed semicircle. C* but also on the values of Rfit ’ R because the fre-
eq p
Typically, as illustrated in Fig. 6 and viewed as Nyquist quency at the apices ffit 5 (2pRfituC*u)21. Consequently,
eq
(Fig.6,AandB)orBode(Fig.6,CandD)plots,thefitted differences in the tightness or leakiness of the paracellular
lines deviated significantly from symmetrical depressed shunt pathways will result in differences in ffit and thus
semicircles at frequencies .100 Hz. Rapp significantly un- differencesincapacitancecalculatedthisway,eventhough
sol
derestimated the values of R as noted above (Fig. 6 B). C* has not changed.
sol eq
BiophysicalJournal81(2)852–866
858 Pa˘unescuandHelman
FIGURE 6 Impedancedataofcontrolandamiloride-treatedtissueswerefittoEq.1atfrequencies,50HzasshownbythesolidlinesinNyquist(A
and B) and Bode plots (C and D). (A) The fitted solid lines were depressed from ideal semicircles (dashed line). (B) Expanded view of A at higher
frequencies.Thesolidfittedlinedeviatedfromthedatapointsatfrequencies.50HzandintersectedtherealaxisatvaluesofRappsignificantlylessthan
sol
thoseofR ,indicatingthattheimpedancelocusisskewedtowardlowfrequencies.
sol
PGE activates a large apical membrane TheimpedancevectorsnormalizedtothevaluesofZdc
2 meas
conductance to chloride (Fig. 7 B) more clearly indicate that the impedance locus
waschangedbyPGE fromasingledepressedsemicircleat
PGE2causesnotonlyadramaticdecreaseintransepithelial frequencies,50Hzi2ntheamiloridecontrolstatetoalocus
impedancebutalsoamarkedchangeoftheimpedancelocus
consistent with a large decrease of apical membrane resis-
as indicated for a typical experiment, illustrated in Fig. 7.
tance. Under these latter circumstances where apical mem-
Thechangeofimpedancewasclearlymaximalat2min(not
brane resistance, R , is decreased by PGE into a range of
shown) at frequencies .5 Hz. The impedance at the very a 2
values comparable in magnitude to the basolateral mem-
low frequencies ,;3–4 Hz could not be resolved during
brane resistance, R , and where in addition the time con-
thetransientrelaxationofcurrentsthatviolatedtherequire- b
stants of apical and basolateral membranes are sufficiently
ment of steady-state currents and voltages to measure im-
differentfromeachothertoresultinobservableinteracting
pedance. It should be noted that these experiments were
semicircles in Nyquist plots, the measured impedance is
carried out in the presence of amiloride to block Na1
quite generally:
currents so that the responses could be attributed to an
increase of Cl2 conductance. R of the amiloride-treated ~Z 1Z !R
tissue in Fig. 7 was decreased froTm near 13 to 4 kV z cm2. Zmeas5Z 1a Z 1b Rp 1Rsol (3)
From control values of 0.27 6 0.01 mA/cm2, the Iamil a b p
sc
averaged at 16 and 45 min after PGE 2.01 and 1.93 where apical (Z ) and basolateral (Z ) membrane imped-
2 a b
mA/cm2, respectively. The mean R was decreased from ances are:
T
amiloridecontrolvaluesnear10kVzcm2(Table1)to3.52
and 3.39 kV z cm2 (Table 2), respectively, at these same Z 5 Rafit (4)
timepoints.Within2minPGE2causedmaximaldecreases a 11~jvRafitCafit!gafit
of impedance that were sustained. The changes of imped-
ance were reversible after withdrawal of PGE although Rfit
2 Z 5 b (5)
rceovmerpslaeltewaats 4co5nsmidine.raFblryomslotwheerP(GFiEg.-7stiCm)ulaantdednoIatmqiluiotef b 11~jvRbfitCbfit!gbfit
2 sc
1.8260.07mA/cm2(n56),theIamildecreasedafterPGE It should be emphasized here, as above, that the values of
sc 2
washout at 2, 6, and 45 min to 1.02 6 0.07, 0.74 6 0.06, CfitandCfitarethoseattheuniquefrequenciesattheapices
a b
and 0.39 6 0.03 mA/cm2, respectively. of the semicircles and where the semicircles would be
BiophysicalJournal81(2)852–866
PGE -ActivatedEpithelialCl2Channels 859
2
FIGURE 8 PGE causes decrease of both apical (R) and basolateral
2 a
membrane (R) resistance of amiloride-pretreated tissues as shown in A.
FIGURE 7 PGE causesadramaticchangeofimpedanceanditslocus, b
2 WhereasthedecreaseofR ismaximalwithin2min,thedecreaseofR is
asindicatedinA.ThedataofAwerenormalizedtothevaluesofZmdceasto relatively slow, but essentaially complete within ;6–10 min. The corbre-
betterillustratethechangeofimpedancelocusasshowninB.Theimped-
spondingchangesofthetranscellularThe´veninemf,E ,areshowninB
ancevectorsatthelowerfrequencies,;200HzofPGE-treatedtissues cell
2 (seetext).
requiredthefittingofatleasttwodepressedsemicircles(solidline)toEq.
3(seetext).TheeffectsofPGE werepartiallyreversiblewithin45min,
2
as indicated in C. In comparison with the onset of PGE effects on
2
impedancethewashoutwasrelativelyslow,asindicatedbythechangesof that were evident by inspection of the impedance plots. Ca
impedancelocusat2,6,and45min. and C were estimated at frequencies corresponding to the
b
approximate apices of the impedance loci. We will refer to
theapicalandbasolateralmembraneresistancesandcapac-
depressed if the power law dependencies gfit and gfit were itancesbelowsimplyasR ,R ,C ,andC ,recognizingthat
a b a b a b
less than unity due to capacitance being frequency-depen- these parameters were determined by fitting of the imped-
dent (Pa˘unescu and Helman, 2001). ance loci to Eq. 3 at the very low audio frequencies.
To determine apical and basolateral membrane resis- During steady-state periods of transport and at time
tances and capacitances, impedance data were fit by non- pointsof16and45min,R averaged1.42and1.70kVzcm2
a
linear curve-fitting (Scientist for Windows, MicroMath, andR averaged4.02and3.90kVzcm2,respectively(Table
b
Inc.,SaltLakeCity,UT)toEq.3atfrequencies,50Hzfor 2). R averaged near 10 kV z cm2 (Table 2) and was
p
the amiloride control data, as was indicated above, and at essentiallyunchangedbyPGE .Althoughitwasnotpossi-
2
frequencies,200HzwhentissuesweretreatedwithPGE bletodeterminethevaluesofR beforePGE incontrolor
2 b 2
(Fig. 7). Starting values of R were determined indepen- amiloride-blockedtissues,thePGE -relatedtime-dependent
p 2
dently before treatment of the tissues with PGE . Starting changesofR andR couldbeassessedasindicatedinFig.
2 a b
values of R and R were estimated with the values of R 8 A. R was maximally decreased from greater than a few
a b cell a
and the fractional transcellular resistances (R /(R 1 R )) hundred kV z cm2 within 2 min by PGE and remained
a a b 2
BiophysicalJournal81(2)852–866
860 Pa˘unescuandHelman
essentiallyconstantforthe45minperiodofobservation.In frogskinandtoadurinarybladder,wheregenerallyE 5
cell
contrast, it became apparent following PGE that R de- E averages above 100 mV and as indicated above, 112.5
2 b Na
creased from .5 kV z cm2 to steady-state values near 4.0 mV for the A6 epithelia of the present experiments.
kVzcm2within6min.R incontroltissuesdeterminedby For tissues like A6, where cAMP activates both Na1
b
a different method was found to average .6.6 kV z cm2 and Cl2 channels and where chloride channels are rela-
(Pa˘unescu,1999)and9.0kVzcm2(Pa˘unescuetal.,2000b), tively close to electrochemical equilibrium, the E after
a
which would be consistent with the time-dependent de- PGE mustmovetowardtheNernstequilibriumpotential
2
creaseofR measuredherebyimpedanceoftissuestreated difference of Cl2, d . With amiloride-blocked tissues
b Cl
with PGE . Accordingly, whereas the PGE -related de- whereapicalmembranesexpressblocker-insensitiveNa1
2 2
crease of R was relatively small and relatively slow, the channels (Rbi ), E 5 (E Rbi )/(Rbi 1 R ). Because R
b Na a Cl Na Na Cl Cl
major and very rapid effect of PGE was to decrease the ,, Rbi , after PGE E can practically be equated with
2 Na 2 a
apicalmembraneresistancetovaluesconsiderablylessthan d . Thus, with reference to a grounded apical solution,
Cl
those of the basolateral membranes of the cells. E 5 E 2 d 5 Iamil(R 1 R ) so that E can be
cell b Cl sc a b cell
calculatedfromtheIamilandtranscellularsloperesistance
sc
(R 1 R ). As indicated in Fig. 8 B, E decreased
a b cell
Transcellular emfs dramaticallywithin2minfrom112.5mVtomeanvalues
of 20.4 mV, and then to 10.9 and 10.7 mV at 16 and 45
Themagnitudesofthetranscellularcurrentbeforeandafter
min, respectively, after PGE . Consequently, despite a
PGE willbedeterminednotonlybyR andR ,butalsoby 2
chang2esofthetranscellulardrivingforcaeE wbhereE 5 rapid opening of a large apical membrane Cl2 conduc-
cell cell
E 1E andwhereE andE are,respectively,theThe´venin tance within 2 min, the initial peak magnitude of the
emafsofbapicalandbaasolaterbalmembranes.Accordingly,Isc chloride current (ICl 5 (Va 2 dCl)/RCl) was compensated
5whe(Erea 1apicEabl)/(mReam1braRnbe)s5arEecpelol/p(Ruala1tedRpbr)i.nBciepfaolrleyPifGnEo2t, Cfolr2bcyuarrecnotrreenstpeornsdtihnegclyelllasrigneitdiaelclyre,aVsea .ofdECcel.ll.WBiethcaluosses
solelybyamiloride-sensitiveand-insensitiveNa1channels of Cl2 from the cells and increase of dCl, Ecell declines
a2pn0adl0l1yw)b,hyeIsrtcehe5EcaoEinsbda/(utRcoatra1nvceerRyobn)f.ebIaafrsotzhelaertoeRr(baPlai˘smundeemestceburrmaaninendeKdH1eplcrmihnaacnni--, thoewnTcaheredtahpsetiecCaadll2ym-csetumartrbeernavtnaecluoNemsap,1oacnsuerndrtoeneostfcItosahcmmeilp.Voane2ntsdoCflIaamndil
sc
nels,thenatzerocurrentflowthroughthebasolateralmem- duringthesetimesareIbi 5V /Rbi ,sothatIamil5Ibi 1
Na a Na sc Na
brane,E 5ud 1I R u(HelmanandThompson,1982), I .BecauseRbi ..R ,therapidinitialincreasesofIamil
b K pump b Cl Na Cl sc
whered istheNernstequilibriumpotentialdifferenceand must be due to I , with I relaxing to its steady-state
K Cl Cl
I isthecurrentgeneratedbytheNa,K-ATPase.Accord- value where chloride secretion is determined principally
pump
ingly, relatively fast decreases of R that occur before ap- by the rate of basolateral membrane Cl2 entry (see Ap-
b
preciablechangesofd orI wouldbeexpectedtocause pendix). If the basolateral membrane chloride entry were
K pump
increasesofE ,whichtogetherwithdecreasesofR would zero, then chloride must come to electrochemical equi-
b b
leadtostimulationofI .AlthoughdecreasesofR willlead librium at the apical membranes of the cells where at the
sc b
toincreasesofI ,E wouldbeexpectedtoincreaseifthe steadystateI 50orchloridesecretionwouldbeabsent
sc cell Cl
channels expressed at apical membranes were exclusively despite high values of apical membrane conductance to
Na1 channels, as would be the case for tight epithelia like Cl2.
TABLE 2 Electricalparametersofamiloride-pretreatedA6epitheliastimulatedbyPGE
2
Iamil R R Rfit Rfit R
sc T p a b cell
(mA/cm2) (kVzcm2) (kVzcm2) (kVzcm2) (kVzcm2) (kVzcm2)
16minafter 2.0160.09 3.5260.34 10.5361.64 1.4260.04 4.0260.27 5.4560.26
PGE
2
45minafter 1.9360.11 3.3960.36 9.3261.66 1.7060.04 3.9060.24 5.5960.24
PGE
2
Cfit Cfit Cfit ffit ffit gfit gfit
a b eq a b a b
(mF/cm2) (mF/cm2) (mF/cm2) (Hz) (Hz)
16minafter 1.3860.03 20.160.7 1.2960.03 81.262.1 2.060.12 0.8560.003 0.8960.008
PGE
2
45minafter 1.3960.03 20.260.8 1.3060.03 67.660.7 2.160.10 0.8660.004 0.9260.005
PGE
2
Valuesaremeans6SE(n55)andwhereapplicablearenormalizedtoplanarsurfacearea.
BiophysicalJournal81(2)852–866
PGE -ActivatedEpithelialCl2Channels 861
2
FIGURE 9 ThechangesofimpedanceanditslocuscausedbyPGE aredependentonchloride.InA,gluconatereplacedallchlorideintheapicaland
2
basolateralsolutionsbathingatissuethathadalreadybeentreatedwithPGE andamiloride.Theeffectofchlorideremovalwascompletelyreversibleupon
2
returntothechloride-richsolution.TheimpedancevectorsinAnormalizedtothevaluesofZdc areshowninB.InCthetissuewastreatedwithPGE
meas 2
whileitwasbathedwiththechloride-freegluconatesolution,causingaminusculechangeofimpedance.Exposuretothechloride-richsolutioncauseda
typicalchangeofimpedanceanditslocus.ThenormalizedimpedancevectorsinCareshowninD.
Depressed semicircles of impedance loci tissuescausedreversibledecreasesoftheIamilto0.4760.08
sc
mA/cm2 from 1.75 6 0.08 mA/cm2 (n 5 5). The R and
Because capacitance is frequency-dependent, it is expected T
impedancelocuswereunchangedbyPGE whentissueswere
that the impedance locus would consist of depressed semi- 2
bathed in chloride-free solution (Fig. 9 C). Subsequent expo-
circles (Pa˘unescu and Helman, 2001). Nonlinear curve-
sure to the chloride-rich solution resulted in a decrease of R
fitting of the impedance vectors as indicated above yielded T
and a change of the impedance locus (Fig. 9, C and D).
meanpowerlawdependenciesg andg near0.85and0.90,
a b Removal of chloride from tissues treated first with PGE
respectively(Table2).Theabsolutevaluesofcapacitanceat 2
resulted in reversible increases of R and changes of the
the apices of the depressed semicircles uC u and uC u aver- T
a b impedancelocus(Fig.9A).Thesespecificchloride-dependent
aged near 1.38 and 20.1 mF/cm2, respectively, in PGE -
2 changes of current, resistance, and impedance locus provide
treated tissues. The frequencies at the apices averaged
compelling evidence to support the view that PGE acting
;70–80 Hz for uC u and ;2.0 Hz for uC u (Table 2). 2
a b through cAMP activates a large chloride conductance at the
apicalmembranesofthecellsthatprecedessignificantactiva-
PGE -related changes of impedance in tion of apical membrane ENaCs, and hence the amiloride-
amilo2ride–blocked tissues require Cl2 sensitiveapicalmembraneNa1 conductance.Thus,theR of
a
amiloride- and PGE -treated tissues can be equated with the
2
To ensure that PGE activated a chloride conductance in the
2 apicalmembraneresistancetochloride,R .Totheextentthat
Cl
tissues we studied, two experimental protocols were used to incontroltissuesPGE stimulatesNa1transportaboutthree-
2
test for chloride dependence of the PGE2-related changes of fold (Pa˘unescu and Helman, 1997; Pa˘unescu, 1999) to ;20
impedance in amiloride-pretreated tissues. In experiments il- mA/cm2,theamiloride-sensitiveNa1resistance,Rbs,wouldbe
Na
lustrated in Fig. 9, A and B, tissues bathed in chloride-rich ;5.6 kV z cm2 (112.5 mV/20 mA/cm2) or about three to
solution were treated first with PGE and then exposed to a
2 fourfoldlargerthanR inPGE -treatedA6epithelia.
Cl 2
chloride-freegluconatesolution.Intheexperimentsillustrated
inFig.9,CandD,tissueswerebathedfirstwithchloride-free
Time-dependent change of capacitance
solution,treatedwithPGE intheabsenceofchloride,andthen
2
exposed to a chloride-rich solution. Regardless of the se- In the face of a frequency-dependent apical membrane
quence, removal of chloride in the PGE -treated states of the capacitanceandchangesofresistance,wecouldnotrelyon
2
BiophysicalJournal81(2)852–866