JOURNAL OF EXPERIMENTAL ZOOLOGY 286:97–100 (2000)

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Aplysia Gut Epithelial Cells: Luminal MembraneChloride Channel Activity

GEORGE A. GERENCSER*Department of Physiology, College of Medicine, University of Florida,Gainesville, Florida 32610

ABSTRACT The Cl– energy gradient across the luminal membrane of Aplysia foregut epithe-lial cells is directed downhill from the lumen to the cellular cytosol. No primary or secondaryactive transporters had been shown to be involved in Cl– translocation across the luminal mem-brane. Cl– channel blockers impeded the movement of Cl– from the lumen into the foregut cellularcytosol. It was concluded that the primary means of Cl– transport across the luminal membranewas via Cl– conductance. J. Exp. Zool. 286:97–100, 2000. © 2000 Wiley-Liss, Inc.

Cl– absorption by the Aplysia californica (sea-hare) foregut is active and mediated by a baso-laterally localized, electrogenic, Cl–-stimulatedATPase (Gerencser and Zelezna, ’93; Gerencser,’96). This Cl– pump accounts for an electrochemi-cal “well” existing for Cl– in the cytosol of the fore-gut epithelial cells (Gerencser, ’93). In addition,D-galactose, a relatively nonmetabolizable mono-saccharide, is actively accumulated by the Aplysiaforegut in the presence of Na+ (Gerencser, ’78, ’96).This luminal-membrane localized Na+-dependentD-galactose transport enhanced the net mucosal-to-serosal Cl– flux across the Aplysia foregut(Gerencser, ’78, ’96). However, no transport mecha-nism for Cl– has been rigorously defined for the lu-minal membrane of the Aplysia foregut epithelium.In view of a potential luminal membrane couplingof Na+, D-galactose, and Cl–, the present study wasundertaken to discern possible mechanisms of Cl–

transport across the luminal membrane.Adult seahares (Aplysia californica) were ob-

tained from Marinus (Westchester, CA) and weremaintained at 25°C in circulating filtered sea wa-ter. The animals were sacrificed by decapitation,and their posterior foreguts were removed, slit lon-gitudinally, rinsed, and then positioned betweentwo halves of a Lucite chamber described previ-ously (Gerencser, ’93), which allowed measure-ment of transepithelial electrical potential (ψms)and, simultaneously, the introduction of microelec-trodes into the surface epithelial cells. The cham-ber exposed the tissue to an oxygenated seawatermedium. The formula for the seawater mediumwas (in mM): NaCl, 462.0; MgSO4 7H2O, 2.4; KCl,10.0; KHCO3, 2.4; MgCl2, 9.8; CaCl2, 11.4. The to-tal osmolality of the bathing medium was 1000

mosmol/liter, and the final pH was 7.8. TrisCl re-placed NaCl, mole for mole, in the Na-free bath-ing medium. Microelectrodes for measurements ofmucosal membrane potential (ψm) and the elec-trochemical potential (µ– Cl) were constructed andutilized as previously described (Gerencser, ’93).Briefly, the experimental protocol was as follows:after the excised tissue was placed in the Lucitechamber, microelectrodes were advanced into cellslining the gut villi to obtain an independent esti-mate of ψm, after which, Cl– selective microelec-trodes were passed into the villus epithelial cellsto measure the intracellular µ– Cl. The intracellu-lar Cl– activity (ai

Cl) was calculated using

aiCl = a′′Cl/e2.303[(ψi – ψm) – ψ′′]/S (1)

where ψi is the potential of the Cl– electrode inthe cell; ai

Cl, the activity of Cl–; ψ′′, the potentialof the Cl– electrode in 500 mM NaCl; a′′Cl, the ac-tivity of Cl– in 500 mM NaCl; ψm, the mean mu-cosal membrane potential; and S, the slope of theelectrode response defined as described previously(White, ’80; Gerencser, ’93). The data obtainedwere analyzed statistically by both a two-wayanalysis of variance (the two grouping factors be-ing those experimental designs that stimulatedor inhibited Cl– conductance as measured bychanges in ai

Cl) and the “new multiple range test”developed by Duncan (Duncan, ’55) which ana-lyzed the multigroup comparisons including the

Grant sponsor: Eppley Foundation for Research, Inc.*Correspondence to: George A. Gerencser, Ph.D., Department of

Physiology, P.O. Box 100274, College of Medicine, University ofFlorida, Gainesville, FL 32610.

Received 13 January 1999; Accepted 25 June 1999

98 G.A. GERENCSER

Tris Cl data. The empirically determined F valuefor the ratio of in and between sample varianceswas 3.27 at an F.95 = 5.19.

As demonstrated in the present study (Table1), mucosally applied D-galactose (15 and 30mM) significantly depolarized ψm, significantlyhyperpolarized ψms, and enhanced ai

Cl in a con-centration-dependent fashion. However, 30 mMD-galactose-induced changes were not observedin the absence of Na+ (Table 2) or in the pres-ence of either mucosal 4-acetamide-4′-isothio-cyano-stilbene-2,2′-disulphonate (SITS) at 10µM, bumetanide (10 µM), or chlorothiazide (10µM) (Table 1). Both 9-carboxyanthracene (9-AC)or thiocyanate (mucosally applied) at 1 µM con-centrations inhibited the increase in ai

Cl, whichwas stimulated by mucosal 30 mM D-galactoseboth in the presence and absence of Na+ (Tables1 and 2). In fact, both these agents caused asignificant decrease in ai

Cl when 30 mM D-ga-lactose was not present (Tables 1 and 2). Phlo-rizin (1 mM) added to the mucosal bathing

solution also prevented any increases in aiCl or

decreases in ψm in the presence of mucosal 30mM D-galactose (Table 1).

Mucosally applied D-galactose caused a depolar-ization of ψm, a hyperpolarization of ψms, and anincrease in ai

Cl in the foregut cells of Aplysiacalifornica bathed in Na+ (Table 1). These obser-vations can be explained as follows: If an activelytransported sugar such as D-galactose is Na+-coupled at the luminal membrane (Gerencser, ’78),this would depolarize the ψm because of the elec-trogenic nature of the symport mechanism (Roseand Schultz, ’70; Gerencser and White, ’80;Gerencser, ’96). The findings that mucosal phlo-rizin, or the absence of Na+, prevents these elec-trical or electrochemical changes from occurringsupports this hypothesis since phlorizin is a com-petitive inhibitor of sugar transport (Schultz, ’77),and the absence of Na+ would prevent the mecha-nism from working since the mechanism is a Na+-driven symport process (Gerencser, ’78, ’81, ’96).The depolarization of ψm by the electrogenic

TABLE 1. Potential profiles and intracellular Cl– activities in Aplysia californica foregutbathed in NaCl or Tris Cl seawater media1

ψm (mV) ψs (mV) ψms (mV) aiCl (mM) n

NaCl (control) –70.2 ± 1.3 (37) +69.5 ± 1.1 (37) –0.7 ± 0.1 (37) 10.1 ± 0.5 (17) 11NaCl + D-galactose (15 mM) –65.1 ± 1.1 (37) +59.1 ± 1.3 (37) –6.0 ± 1.2 (37) 12.8 ± 0.7 (15) 11

P < 0.05 P < 0.05 P < 0.05 P < 0.05NaCl + D-galactose (30 mM) –60.8 ± 1.4 (37) +56.3 ± 1.3 (37) –4.5 ± 1.3 (37) 14.8 ± 0.5 (14) 11

P < 0.05 P < 0.05 P < 0.05 P < 0.05NaCl + 30 mM D-galactose + phlorizin –70.8 ± 2.6 (11) +68.8 ± 2.1 (11) –2.0 ± 1.5 (11) 10.4 ± 0.7 (10) 6

N.S. N.S. N.S. N.S.NaCl + 9-AC –60.1 ± 1.2 (15) +56.3 ± 1.4 (15) –3.8 ± 1.4 (15) 8.0 ± 0.4 (12) 5

P < 0.05 P < 0.05 P < 0.05 P < 0.05NaCl + 30 mM D-galactose + 9-AC –59.1 ± 1.4 (8) +56.0 ± 1.1 (8) –3.1 ± 1.2 (8) 7.1 ± 0.8 (5) 4

P < 0.05 P < 0.05 P < 0.05 P < 0.05NaCl + SCN– –59.0 ± 2.1 (9) +56.1 ± 2.0 (9) –2.9 ± 2.0 (9) 7.6 ± 0.05 (8) 5

P < 0.05 P < 0.05 P < 0.05 P < 0.05NaCl + 30 mM D-galactose + SCN –61.3 ± 3.0 (5) +58.1 ± 2.8 (5) –3.2 ± 2.6 (5) 7.4 ± 0.07 (4) 4

P < 0.05 P < 0.05 P < 0.05 P < 0.05NaCl + chlorothiazide –71.4 ± 1.5 (8) +70.8 ± 1.1 (8) –0.4 ± 1.0 (8) 10.9 ± 0.7 (7) 5

N.S. N.S. N.S. N.S.NaCl + 30 mM D-galactose + chlorothiazide –62.3 ± 3.1 (4) +62.1 ± 3.5 (4) –0.2 ± 2.5 (4) 12.3 ± 0.5 (4) 4

P < 0.05 P < 0.05 N.S. P < 0.05NaCl + bumetanide –70.9 ± 1.8 (7) +70.5 ± 1.0 (7) –0.4 ± 0.8 (7) 10.2 ± 0.4 (7) 5

N.S. N.S. N.S. N.S.NaCl + 30 mM D-galactose + bumetamide –60.3 ± 2.0 (5) +58.1 ± 2.0 (5) –2.2 ± 2.4 (5) 12.9 ± 1.0 (5) 4

P < 0.05 P < 0.05 N.S. P < 0.05NaCl + SITS –69.8 ± 0.4 (5) +69.6 ± 0.4 (5) –0.2 ± 0.4 (5) 10.1 ± 0.6 (4) 5

N.S. N.S. N.S. N.S.NaCl + 30 mM D-galactose + SITS –60.1 ± 1.5 (5) +58.1 ± 2.1 (5) 2.0 + 1.6 (5) 12.3 ± 0.5 (5) 4

P < 0.05 P < 0.05 N.S. P < 0.05Tris Cl –73.2 ± 1.0 (10) +69.8 ± 2.2 (10) –3.4 ± 1.9 (10) 7.3 ± 0.4 (9) 4

P < 0.05 N.S. P < 0.05 P < 0.051Values are means ± S.E. Numbers in parentheses are numbers of observations; n is the number of animals. Polarity of ψm and ψms is relativeto the mucosal solution. Polarity of calculated ψs is relative to cytoplasm. ai

Cl was calculated by means of Eq. 1; P, probability; N.S. notsignificant.

APLYSIA GUT ION TRANSPORT 99

cotransport of Na+ and D-galactose would then in-crease the electrochemical gradient, favoring in-tracellular Cl– entry from the mucosal bathingsolution (Gerencser and White, ’80). This wouldthen allow Cl– to move across the mucosal mem-brane, down its electrochemical gradient (Gerenc-ser and White, ’80), into the intracellular spacewith greater ease via Cl– conductance pathwaysthat were further opened by the ψm depolariza-tion induced by the electrogenic transport of Na+

and D-galactose (Table 1). These ideas are sup-ported by the finding that D-galactose significantlydepolarized ψm and significantly enhanced ai

Cl ac-cording to its maximally stimulating concentra-tion of 30 mM (Gerencser, ’78; Table 1), and 9-ACand thiocyanate, both specific Cl– channel blockers(Gerencser, ’96), prevented increases in ai

Cl in thepresence of a D-galactose–induced depolarizationof ψm. Also, the finding that 9-AC and SCN– de-creased the ai

Cl in the absence of Na+ further sup-ports the hypothesis that luminal membranetransport of Cl– is via Cl– conductance pathways(Table 2). The basolaterally located electrogenicCl– pump or Cl–-ATPase (Gerencser and Zelezna,’93) would then accommodate the increased ther-modynamic activity of Cl– (Cl– transport pool) byincreasing its rate of work, similar to a variableoutput device that was described by Michaelis-Menten kinetics in a isolated Cl– pump proteinsystem (Gerencser and Purushotham, ’96). In ad-dition, this was previously demonstrated both byan increase in short-circuit current (SCC) and uni-directional mucosal-to-serosal Cl– flux after mu-cosal D-glucose addition to the voltage-clamped,isolated foregut (Gerencser, ’78). Since it hasbeen shown that the major portion of the SCCbefore and after sugar addition to the mucosalsolution is a Cl– current, there would be a de-crease in the negativity of ψs in the presence ofD-galactose by both the increased electrogenicCl– pump activity and the linkage of ψm to ψs

TABLE 2. Potential profiles and intracellular Cl– activities in Aplysia californica foregut bathed in Tris Cl seawater media1

ψm (mV) ψs (mV) ψms (mV) aiCl (mM) n

Tris Cl (control –73.2 ± 1.0 (10) +69.8 ± 2.2 (10) –3.4 ± 1.9 (10) 7.3 ± 0.4 (9) 4Tris Cl + 30 mM D-galactose –72.9 ± 2.2 (10) +69.9 ± 2.0 (10) –3.0 ± 2.5 (10) 7.5 ± 0.5 (8) 4

N.S. N.S. N.S. N.S.Tris Cl + 9-AC –64.4 ± 1.5 (8) +60.2 ± 1.7 (8) –4.2 ± 1.4 (8) 5.3 ± 0.3 (8) 4

P < 0.05 P < 0.05 N.S. P < 0.05Tris Cl + SCN– –62.9 ± 1.6 (7) +59.1 ± 1.1 (7) –3.8 ± 1.0 (7) 5.8 ± 0.3 (7) 5

P < 0.05 P < 0.05 N.S. P < 0.051Values are means ± S.E. Numbers in parentheses are numbers of observations; n is the number of animals. Polarity of ψm and ψms is relativeto the mucosal solution. Polarity of calculated ψs is relative to cytoplasm. ai

Cl was calculated by means of Eq. 1; P, probability; N.S., notsignificant.

through a low resistance extracellular shunt(Gerencser and Loughlin, ’83) which would leadto a greater serosally negative ψms (Schultz, ’77)as shown in Table 1.

The findings that neither SITS, bumetanide, norchlorothioazide had any effect on ψm, ψms or ai

Cl(Table 1) suggests that Cl–/anion antiport, Na+/K+/2Cl– symport or Na+/Cl– symport are not me-chanisms that transport Cl– from the mucosal so-lution into the cytosol of the Aplysia foregutepithelial cells since these compounds are inhibi-tors of these respective transport processes (Ger-encser, ’96). It has been previously documented thatno primary active transporter for Cl– exists in theluminal membrane of Aplysia foregut columnar cells(Gerencser, ’96). Coupling that observation withthe present observations (Table 1), which indicatethe absence of secondary active transporters forCl–, the possibility cannot be excluded that Cl– isprimarily moved from the foregut lumen into theforegut absorptive cell interior by Cl– channels.

In summary, there appear to be Cl– channelsin the luminal membrane of Aplysia californicaforegut absorptive cells. These channels seemto be the primary means by which Cl– movesfrom the extracellular space to the foregut’s epi-thelial intracellular space. The electrochemicalpotential gradient for Cl– is directed from theextracellular to the intracellular space (Geren-cser, ’96). Additionally, there is suggestive evi-dence that these Cl– channels respond to changesin ψm as shown in Table 1 where ai

Cl increasedwith incremental depolarizations of ψm. However,further studies are needed to define the regula-tion of the Cl– channels found localized in the lu-minal membrane of Aplysia foregut absorptivecells. Physiologically, any increase in Cl– chan-nel activity could fuel secondary, electrophoretic(or electroneutral) transport processes such asthe nutritional uptake of sugars and/or aminoacids (Gerencser, ’96).

100 G.A. GERENCSER

ACKNOWLEDGMENTSMy thanks to Dr. William Mc D. Armstrong for

helpful discussions during the course of the study.I acknowledge the excellent technical assistanceof C. Burgin.

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