Proc. Nati. Acad. Sci. USAVol. 89, pp. 1438-1442, February 1992Physiology

Carbachol-activated calcium entry into HT-29 cells is regulated byboth membrane potential and cell volume

(secretory epithelium/colon carcinoma cells/ftra-2 imaging)

HORST FISCHER*t, BEATE ILLEK*, PAUL A. NEGULESCU*, WOLFGANG CLAUSSt, AND TERRY E. MACHEN**Department of Molecular and Cell Biology, Division of Cell and Developmental Biology, 231 Life Science Addition, University of California, Berkeley, CA94720; and tInstitut fur Veterinarphysiologie, Freie Universitut Berlin, Koserstrasse 20, 1000 Berlin 33, Federal Republic of Germany

Communicated by Jared M. Diamond, November 1, 1991

ABSTRACT Intracellular Ca2+ ([Ca2+fj) was measured insingle Cl1-secretory HT-29/B6 colonic carcinoma cells withthe Ca2+ probe fura-2 and digital imaging microscopy. Resting[C:a2i was 63 ± 3 nM (a = 62). During treatment with themuscarinc agonist carbachol, [Ca2+J rapidly increased to 901± 119 nM and subsequently reached a stable level of 309 :± 23OM, which depended on Ca2+ entry into the cells from theextracellular solution. The goal of this study was to characterizethe Ca+ entry pathway across the cell membrane with respectto its dependence on membrane potential and cell volume.Under resting conditions [Ca2i+] showed no apparent depen-dence on either potential or cell volume. After stimulating Ca2+entry with carbachol (100 jEM), [Ca2J1 increased with hyper-polarization (low-K+ or valinomycin treatment) and decreasedwith depolarization (high-V+ or gamlcdin treatment) of thecell, as expected from changes w driving force for Ca2+ entry.In stimulated cells, hypotonic solutions caused LCa2+1 toIncrease, whereas hypertonic solutions blocked Ca2+ entry.The shrinkage-induced decreases-in [Ca2+1 were only slightlyaffected when the membrane potential was increased withvalinomycin, suggesting that shnkage directly affects thecarbachol-activated Caz+ conductance. In contrast, the swell-ing-induced increase in [Ca2 was significantly reduced invalinomycin-treated cells, suggesting an indirect dependenceon a swelling-activated K+ conductance. Thus, carbachol-stimulated Ca2+ entry is under the dual control of membranepotential and cell volume. This mechanism may serve as aregulatory influence that determines the extent of Ca2+ influxduring cholinergic stimulation.

Many nonexcitable cells, including epithelial cells, respondto stimulation by muscarinic agonists with a biphasic eleva-tion of intracellular Ca2+ concentration ([Ca2+]j). The initialtransient phase (peak) is due to release of intracellular Ca2+stores, whereas a secondary, sustained elevation of [Ca2 ]i(plateau) is maintained by Ca2+ entry from the extracellularspace into the cytosol (1, 2). Ca channels responsible for Ca2+entry into epithelial cells are, in contrast to their relatives inexcitable cells, characterized by their insensitivity to voltageand- the organic dihydropyridine blockers (3). In epithelialcells, Ca2+ increases mediated by these channels can regulatesecretory responses (e.g., refs. 1 and 4). Thus, regulation ofthis Ca2+ entry pathway is important in controlling secretion.We investigated the effects of membrane potential and cell

volume on Ca2+ entry because these parameters are knownto change with the activation of secretion. For example,epithelial cell potential is dominated by a K+ conductance,which is regulated to accommodate transcellular ion trans-port while maintaining the intracellular ionic milieu (5). InCl--secreting cells, [Ca2+]J directly activates both K+ andCl- conductance's (6), and carbachol induces a transient

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depolarization in Cl--secreting HT-29 cells (7). Becausechanging the membrane potential would affect the drivingforce for Ca2l entry (8), the possibility of a regulatoryfeedback between [Ca2+]j and ionic conductances exists.With regard to cell volume, the activation of ion channels

for K+ and Cl- could lead to cell shrinkage due to the loss ofK+, Cl-, and osmotically obliged water. Indeed, it hasrecently been shown that carbachol-stimulation of salivaryepithelial cells with carbachol decreases cell volume by10-20o (9). Volume can affect K+ and Cl- conductances incultured (10) and intact (11) colonic epithelia, and there is avolume-sensitive, Ca2+-permeable channel in the epithelia ofthe kidney and choroid plexus (12, 13). These findingssuggest that the membrane conductances to K+, Cl-, andCa2+, cell potential, and cell volume are tightly interrelatedand could provide a regulatory circle to control Ca2+ entryand, thus, the level of [Ca2+]i during stimulus. The purposeof our work was to characterize these relationships in theCl--secretory epithelial cell line HT-29/B6.

MATERIALS AND METHODSCell Culture. The B6 subclone (14) of the colon carcinoma

cell line HT-29 was advantageous due to its homogeneity inhormonal responses. Cells were grown on glass coverslips inDulbecco's minimal essential medium, supplemented with10%o fetal calf serum, 4 mM glutamine, penicillin at 100units/ml, and streptomycin at 0.1 mg/ml. Experiments weredone on cells from passages 17 to 26 at the second to seventhdays after seeding, when cells were still in a subconfluentstate. Cell [Ca2.]i responses were qualitatively independentof growth state, but single cells or cells in clumps showedamplified [Ca2+l responses after stimulation compared withcells grown to confluent monolayers.Measurement of [Ca2+J1. Cytoplasmic [Ca2+]i was mea-

sured according to the procedures described by Negulescuand Machen (15) and Tsien and Poenie (16). Briefly, cellswere loaded with 5 gM of the acetoxymethylester of theCa2+-sensitive dye fura-2 (Molecular Probes) at room tem-perature for 30 min. Then the coverslips were mounted in aperfusion chamber at 370C on the stage of a microscope.Because the cells were subconfluent, 'perfusion solutionsbathe both apical and basolateral sides of the cells simulta-neously. Single cells or cells in small islands were alternatelyexcited at 350 and 385 nm through a40x objective (Nikon, 1.3NA), and fluorescence was measured with a TV camera(SIT-66DX, Dage-MTI, Michigan City, IN); the signal wasprocessed (Gould FD5000 and DEC clone computer), and350/385 fluorescence ratios were collected and averagedfrom 16 to 20 single cells every 4 to 12 sec. Every trace shownin the figures is one experiment from at least three similar

Abbreviations: [Ca2+]i, intracellular Ca2+ concentration; [K+]0,extracellular K+ concentration.tTo whom reprint requests should be addressed.

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Proc. Natl. Acad. Sci. USA 89 (1992) 1439

repetitions. [Ca2+], was calculated from the fluorescenceratios according to Grynkiewicz et al. (17). Maximum andminimum ratios were obtained from fura-2-loaded cells byeither Ca2' depletion (no added Ca2+, 2 mM EGTA) for 30min or by addition of the Ca2+ ionophore ionomycin (5 AM)with 10 mM CaCl2 added to the solutions. A Kd of thefura-2-Ca2+ complex of 300 nM was assumed, based on thevalue measured in intact gastric epithelial cells (18). Allvalues are given as mean ± SEM. Number of experimentscorresponds to separate runs with 16-20 cells each.

Solutions. Standard solution was 141 mM NaCl/4 mMKCl/1 mM KH2PO4/0.9 mM MgSO4/1.7 mM CaCl2/10 mMHepes/25 mM glucose, titrated to pH 7.4 with -5 mMNaOH. In Ca2+-free solution CaCl2 was substituted by 0.1mM EGTA. In solutions of high or low K+ concentrations,Na+ and K+ replaced each other equimolarily to yield finalK+ concentrations of 0.5-151 mM. Carbachol was used at100 ,uM in all experiments shown. All chemicals and the cellculture reagents were from Sigma.

RESULTS AND DISCUSSIONEffects of Carbachol on [Ca21]1. In resting HT-29/B6 cells

average [Ca2+]i was 63 + 3 nM (mean + SE, n = 62).Stimulation with 100 ,uM carbachol caused a sudden increaseof [Ca2+]i to 901 + 119 nM (Fig. 1). Experiments to bereported elsewhere have shown that the spike is primarilydue to Ca2+ release from intracellular stores. This initial spikewas followed by a stable and prominent plateau phase (309 +23 nM), which depended on extracellular Ca2+. Exchangingthe Ca2+ in the solution with 0.1 mM EGTA depressed theplateau to baseline levels, indicating that the [Ca2+]i plateauis solely due to Ca2+ entry from the extracellular space intothe cell. The plateau was fully recovered when Ca2+ wasadded back to the perfusion solution. Removal of carbacholfrom the solution caused [Ca2+]1i to return to control within15-20 sec, suggesting that the Ca2+ entry pathway remainedactive until the agonist was removed. The [Ca2+]i plateaucould be maintained for >30 min without significant inacti-vation. The present work deals exclusively with this carba-chol-stimulated plateau, which is also completely blocked by250 nM La (B.I., H.F., P.A.N., and T.E.M., unpublishedwork).

Effects of Depolarization and Hyperpolarization on [Ca2+]1.The membrane potential of resting HT-29 cells is highlydependent on extracellular K+ concentration [K+]0 (7), de-polarizing by 35 mV when [K+]o is increased from 5 to 50mMor hyperpolarizing by 4 mV due to stepping [K+]o from 5 to1 mM. Because carbachol-treated HT-29 cells have both K+and Cl- conductances (7), changes of [K+]h will similarly

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FIG. 1. Effect of carbachol (100 AuM) on [Ca2+]j in single HT-29/B6 cells with and without extracellular Ca2'. Vertical axes showthe measured Ca-sensitive fluorescence ratios (at right) and respec-tive calculated [Ca2+]i values (at left). In Ca2+-free solution (O Ca2+;no added Ca2+, 0.1 mM EGTA) the [Ca2+]j plateau was suppressed.Removal of carbachol in the presence of Ca2+-containing solutionrestored [Ca2+]i rapidly to resting prestimulated levels.

affect membrane potential of stimulated cells. In Fig. 2 weshow the effects of cell depolarization on [Ca2']i, by ex-changing the standard solution ([K+]. = 5 mM) for a 95 mMK+ solution or by addition of the cation ionophore gramici-din. There was no measurable effect on [Ca2+]i due to highK+ depolarization in resting cells (Fig. 2A). However, thecarbachol-induced [Ca2+], plateau was clearly diminished byK+ depolarization (Fig. 2B), and the plateau was fullyreversible when stepped back to the standard solution.[Ca2+] depression was linearly related to the logarithm of[K+]o over a [K+]0 range of 5-151 mM (Fig. 2B Inset), asexpected were Ca2+ entry governed by the equilibrium(Nernst) potential for K+. The simplest interpretation ofthese data was that carbachol opened Ca2+ conductance(s),and Ca2+ entry was driven mainly by the membrane poten-tial.

In a complementary approach, cells were treated withgramicidin (2 MM), which depolarizes cells by increasing Na'permeability (18). Gramicidin treatment of HT-29 cells alsorapidly decreased the carbachol-induced [Ca2+], plateau (Fig.2C). Similar effects on [Ca2W] were also seen when cells weredepolarized by blocking the K+ conductance of the cells withthe K+ channel blockers Ba2+ (1 mM), lidocaine (1 mM), or5-nitro-2-(3-phenylpropylamino)-benzoate (0.3 mM) (datanot shown).During the carbachol-stimulated plateau phase, [Ca2+]i is

determined by the rates ofCa2+ entry and Ca2+ efflux. [Ca2+]jdecreased rapidly and approximately at the same rates wheneither (i) carbachol or (ii) extracellular Ca2+ was removedfrom solutions or during (iii) the depolarizing treatments withhigh [K+]o (Fig. 2B) and gramicidin (Fig. 2C). These resultsindicate that during the carbachol-induced plateau phase,Ca2+ influx and efflux both occur at rapid rates and also thatdepolarizing membrane potential reduces [Ca2+]i by decreas-ing Ca2+ entry not by increasing Ca2+ pumping.

If cell depolarization reduced Ca2+ influx, then hyperpo-larization would be expected to increase it. To test thishypothesis, cells were hyperpolarized either by reducing[K+]o from 5 mM to 0.5 mM or by adding the K+-selectiveionophore valinomycin. Fig. 3 shows that low [K+]o treat-ment did not affect [Ca2+], in resting cells (Fig. 3A), but therewas a rapid [Ca2+]i increase in carbachol-stimulated cells. Onaverage, [Ca2+] increased by 67 + 13 nM, n = 5. Treatmentof resting cells with valinomycin (20 AM) had no effect on[Ca2+], (Fig. 3D), whereas in carbachol-treated cells thistreatment significantly increased [Ca2+], (Fig. 3C). On aver-age, [Ca2+]i was elevated by 57 + 14 nM, n = 3. Thus,increasing membrane potential by decreasing [K+]. or in-creasing the absolute K+ conductance by adding valinomycinincreased Ca2+ entry into carbachol-stimulated cells but notinto resting cells.

Interestingly, the [Ca2+] responses after the differentmodes of hyperpolarization differed somewhat. For example,low [K+]O-induced increase of [Ca2+], was only transientdespite maintenance of the hyperpolarizing conditions,whereas the valinomycin-induced [Ca2+]i response did notinactivate as long as it stayed present (compare Fig. 3 B andC). Also, when cells were preincubated with valinomycin andsubsequently hyperpolarized with low [K+]0 (Fig. 3D),[Ca2+], increased and remained elevated. Therefore, theinserted valinomycin conductance appeared to maintain astable hyperpolarization-induced response of [Ca2+],whereas the normal response of the cell to low [K+]o ap-peared to inactivate. This inactivation may be from hyper-polarization effects on Ca2+-activated K+ channels, whichhave been shown to be voltage-dependent (see, e.g., refs. 19and 20). Low [K+]0 could also depolarize the cells byinactivating the Na+/K+-ATPase and reducing intracellular[K+].

Physiology: Fischer et al.

Proc. Natl. Acad. Sci. USA 89 (1992)

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FiG. 2. Effects of depolarization on [Ca2+]i. Depolarization of resting cells (A) and carbachol-stimulated cells (B) with a solution containing95 mM K+. (Inset) Dose-dependency of K+ depolarization of 11 separate experiments; numbers ofexperiments at each concentration are given.Vertical axis is normalized to plateau level [[Ca2+]j(plateau) - [Ca2+]j(base1ine) is defined as 100%]. (C) Addition ofgramicidin (2 ,uM) in presenceof carbachol.

Effects ofCell Volume Changes on [Ca2+f1. Because changesof [Kk]o and treatment with valinomycin may affect cellvolume (21), we addressed the question whether the changesof [Ca2+]i were due to changes in membrane potential, in cellvolume, or in both.

Cell swelling was elicited by diluting the solutions withwater (hypotonic = 0.8x isotonic, decreasing osmolalityfrom 310 to 242 mosm/kg), and cell shrinkage was elicited byadding sucrose (10-500 mM) to the standard solutions (there-by increasing osmolality to 817 mosm/kg). Fig. 4 summarizesthe effects of changes in extracellular osmolarity on [Ca2+]i.In resting cells (Fig. 4A) treatment with the hypotonic solu-tion had no significant effect on [Ca2W]i. Also, when cellswere treated with an isosmotic solution containing 80% oftheions of the standard solution (osmolarity balanced withsucrose), there were no effects on [Ca2W]i in either resting orcarbachol-stimulated cells (data not shown). However, whenresting cells were shrunken by adding 500 mM sucrose (butnot during addition of <250 mM sucrose, data not shown) tothe solution, [Ca2+]i increased, probably due to both theconcentrating effect on [Ca2+]1j (caused by decreased cellvolume) and perhaps also the release of Ca2+ from internalstores.

In contrast to resting cells, cell volume changes had largeeffects on [Ca2+]J in carbachol-treated cells. Fig. 4B showsthe effect of hypotonic treatment. [Ca2+]i first rapidly in-creased and then decreased back to plateau level. Similarresults came from experiments in which hypotonicity was

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obtained by removing sucrose from an isotonic solution (inwhich sucrose replaced an equivalent amount of NaCl). Theshape of the swelling-activated transient was not affected byvalinomycin treatment (data not shown), indicating that thedecrease in [Ca2W]i during hypotonic treatment is not afeature of a K+ channel inactivation with time but rather aninactivation of the Ca2+ entry itself.Hyperosmolarity caused [Ca2+]J to decrease rapidly (Fig.

4C), indicating that Ca2+ entry into carbachol-stimulatedcells is rapidly reduced by hypertonicity. This effect wasdose-dependent and completely reversible (Fig. 5). Addingincreased amounts of sucrose decreased the plateau step-wise. On average, addition of 34.8 mM sucrose (correspond-ing to 343 mosm/kg) reduced plateau [Ca2+]i by 50%o. Thus,assuming the cells behave like perfect osmometers, a rela-tively small shrinkage (=10-15%), which can be expectedunder physiological conditions (9), reduced plateau Ca2+]iby 50%o. This change indicates an exquisite sensitivity ofCa2+entry to shrinkage. Maximal effects were seen with 250 mMsucrose (equals 565 mosm/kg). In preshrunken cells (solu-tions containing 250 mM sucrose) carbachol caused charac-teristic peaks of [Ca2]i,, but the plateau was absent, indicat-ing that the carbachol receptor and release of the internalstore were not altered by hypertonicity (data not shown) butthe entry of Ca2+ from outside was blocked. Additionalhypertonicity increased [Ca2+]i, which might be from anoverlap of the shrinkage-induced decrease in Ca2+ entry andan increase of ICa2eli due to the concentrating effect ofdecreased cell volume.

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reducing [K+] to 0.5 mM (A, B, and D) or by addition of valinomycin (Val; 20 IzM; C and D). Note that valinomycin did not affect the initiallarge rise in [Ca2+]i due to Ca2+ release from internal stores.

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1440 Physiology: Fischer et al.

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Proc. Natl. Acad. Sci. USA 89 (1992) 1441

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FIG. 4. Effects of volume changes on [Ca2+]i. Swelling (swell)(0.8x isotonic solution) and shrinkage (shr) (addition of 500 mMsucrose) of resting cells (A) and carbachol-stimulated cells (B and C)are shown.

Distinguishing Effects of Volume and Potential on [Ca2+]J.Because volume changes might affect membrane potentialthrough volume-dependent K+ and Cl- conductances, wetested for independent effects of potential and volume onCa2+ entry. We clamped K+ conductance by adding valino-mycin and then tested the effects of altered osmolarity.Under these conditions changes in [Ca2+]j are likely to resultfrom effects on Ca2+ conductance rather than from influenceson membrane potential. Valinomycin itself seemed to haveno untoward effects on Ca2+ metabolism because [Ca2+4] wasunaffected by the drug in resting cells (Fig. 3D).

Fig. 6A summarizes experiments testing the effects ofsucrose-dependent shrinkage of carbachol-stimulated cellsunder control and valinomycin-pretreated conditions (com-pare with Fig. 5). Under both conditions [Ca2+]i decreasedwith increased sucrose concentrations. The presence ofvalinomycin (to maintain high K+ conductance) reducedshrinkage effects somewhat compared with control. There-fore, we concluded that carbachol-activated Ca2+ entry wasdirectly inhibited by hyperosmolarity. A possible shrinkage-induced inhibition of K+ channels and subsequent depolar-ization of the membrane potential may have an additionalseparate inhibitory effect, as seen from the difference be-tween the valinomycin-treated and control cells.

Swelling effects on [Ca24], in stimulated cells with andwithout valinomycin treatment are shown in Fig. 6B. Con-trols with standard 5 mM [K4]O (compare with Fig. 4B)showed pronounced increases of [Ca2+]i during hypotonicchallenge, and these increases in [Ca24]1 were diminishedwhen the cells were perfused with increased [K+]0. Inexperiments using valinomycin-pretreated cells, swellingevoked much smaller [Ca24]i responses that were indepen-dent of [K+]0 depolarization and coincided at high [K+]o withthe control swelling responses. It should be noted thatswelling effects on [Ca24], were nearly abolished (within 10%)by maneuvers that would be expected to reduce changes ofmembrane potential due to changes of K4 conductance (i.e.,

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FIG. 6. Volume and potential effects on [Ca24]i. (A) Shrinkageeffects on [Ca2+]i depend only slightly on potential. Dose-dependentdepression of carbachol-stimulated [Ca24]i plateau with increasedsucrose concentrations. o, control; n, cells in presence of 20 /AMvalinomycin (valino). y axes give the normalized [Ca24]i levels[[Ca2+]i(plateau) - [Ca24]i(basal) defined as 100%]. Six independentexperiments were done for each group; numbers at each concentra-tion are given. (B) Swelling effects on [Ca2+]i are potential depen-dent. [Ca24]i increase induced by swelling (hypotonic = 0.8x iso-tonic) vs. [K+]. in carbachol + valinomycin (20 ,uM)-treated cells.Ten independent control and three valinomycin experiments wereconducted.

high [K4]0 or valinomycin treatment). Also, pronouncedswelling-induced increases in [Ca2+] were seen only underconditions where the potential could be controlled by thecellular K+ conductance. Thus, the effects of swelling on[Ca2+], in carbachol-treated cells were mainly due to swellingactivation of a K+ conductance, which, in turn, hyperpolar-ized the cell and increased Ca2+ entry due to the increaseddriving force. The small (<10%) increases in [Ca2+], thatoccurred at high [K+]0 or in valinomycin-treated cells wereprobably due to a swelling activation of an apparent Ca2+conductance.Although we have not measured cell volume or potential

directly, the results from this study suggest that cell volume,membrane potential, K+ conductance, and [Ca2+], are allintimately interrelated (see Fig. 7). During carbachol treat-ment a Ca2+ conductance in the cell membrane is activated,allowing Ca2+ entry into the cell from the extracellular space.In the resting state Ca2+ conductance appeared relativelysmall because [Ca2+], was unaffected by our treatments. Incarbachol-stimulated cells, Ca2+ entry and, thus, [Ca2+], aredirectly or indirectly under the control ofmembrane potentialand cell volume as follows.

Let us first consider the feedback relationship among[Ca2+]j, K+ conductance, and membrane potential. Carba-

Carbachol

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Cell Cellvolume Ca. potential

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FIG. 7. Model showing relations among cell volume, cell poten-tial, and [Ca2+], in secretory epithelia. Positive coupling (+) ornegative coupling (-) signs appear between connected variables.Volume effects were exerted by hypoosmolar (swell) or hyperosmo-lar (shrink) solutions. Different letter sizes depict the influence of thisvariable with respect to [Ca2+] .

Physiology: Fischer et al.

Proc. Natl. Acad. Sci. USA 89 (1992)

chol-induced increases in [Ca2+]i activate a K+ conductance(5) and the cell hyperpolarizes, which, in turn, increases Ca2lentry due to the increased driving force (see also ref. 22). Thispositive feedback among [Ca2+]j, K+ conductance, and po-tential would be limited by a number of factors not shown inthe figure: (i) the presence of a Ca2'-activated Cl- conduc-tance (6), (ii) the magnitude of the maximal K+ equilibriumpotential, and (iii) effects of voltage on the K+ conductance(see e.g., refs. 19 and 20).

In addition to effects on membrane potential and Ca2'entry, increases in K+ conductance will also lead to cellshrinkage, which directly inhibits Ca2" entry (see also Fig.6A). In this way, activation of the K+ conductance controlsboth positive (cell potential) and negative (cell shrinkage)influences on Ca2+ entry. Carbachol stimulation of salivaryepithelial cells decreases cell volume by 10-20% (9). Becausethe carbachol-activated Ca2+ entry into HT-29 cells washighly sensitive to shrinkage exactly in this volume range(Fig. 6A), shrinkage-induced decreases in Ca2" entry may actas an important physiological regulator during carbachol-stimulation. (As shown in the results of Fig. 6A, a smallportion of the hyperosmotic effect on Ca2` entry is due toshrinkage-induced reduction of K+ conductance and cellpotential.) Other interactions between volume and Ca2'entry may be mediated through volume effects on the K+ andCa2+ conductances. For example, when carbachol-treatedcells are exposed to hypotonic solutions, the volume-sensitive K+ channels open, and Ca2+ entry increases. [Cellswelling in the presence of carbachol caused small potential-independent increases in Ca2+ entry (Fig. 6B), indicating thatswelling has a small direct effect on Ca2' conductance.]

In summary, during carbachol stimulation Ca2+ entry isdetermined by interactions among K+, Cl-, and Ca2+ chan-nels and, thus, the membrane potential, cell volume, and[Ca2+]i itself. The membrane potential will be determinedprimarily by the K+ and C1- conductances and the trans-membrane gradients for these ions. The magnitude of the K+equilibrium potential will be the physical limit as far as theeffect of the membrane potential on Ca2+ entry. The Ca2+conductance appears very sensitive to cell shrinkage in thephysiological range, so Ca2+ entry and [Ca2W]i decrease whenthe K+ and Cl- channels of the cell have been activated andthe cell shrinks. Thus, during carbachol stimulation, [Ca2+]iregulates secretion through effects on K+ and Cl- conduc-

tances, which feed back on Ca2l entry through their effectson membrane potential and cell volume.

We gratefully thank Prof. Dr. U. Hegel and Dr. K.-M. Kreusel(Berlin) for providing the HT-29 cell clone. This work was supportedby Deutsche Forschungsgemeinschaft Grant Fi 414/2-1 to H.F., theDeutsche Akademische Austauschdienst (B.I.), National Institutesof Health Grants DK 08238 to P.A.N. and DK 19520 to T.E.M., andthe Cystic Fibrosis Foundation.

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