Distinct Ca2+- and cAMP-dependent anion conductances in the apical membrane of polarized T84 cells

Didier Merlin1,4, Lianwei Jiang2,4, Gregg R. Strohmeier1,4, Asma Nusrat1,4, Seth L. Alper2,4, Wayne I. Lencer3,4, and James L. Madara1,4

1 Division of Gastrointestinal Pathology, Department of Pathology, Brigham and Women's Hospital, 2 Molecular Medicine and Renal Units, Beth Israel Deaconess Medical Center, 3 Combined Program in Pediatric Gastroenterology and Nutrition, Department of Medicine, Children's Hospital Medical Center, 4 Harvard Medical School and Harvard Digestive Diseases Center, Boston, Massachusetts 02115

    ABSTRACT
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Abstract
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Methods
Results
Discussion
References

Monolayers of the human colonic epithelial cell line T84 exhibit electrogenic Cl- secretion in response to the Ca2+ agonist thapsigargin and to the cAMP agonist forskolin. To evaluate directly the regulation of apical Cl- conductance by these two agonists, we have utilized amphotericin B to permeabilize selectively the basolateral membranes of T84 cell monolayers. We find that apical anion conductance is stimulated by both forskolin and thapsigargin but that these conductances are differentially sensitive to the anion channel blocker DIDS. DIDS inhibits thapsigargin-stimulated responses completely but forskolin responses only partially. Furthermore, the apical membrane anion conductances elicited by these two agonists differ in anion selectivity (for thapsigargin, I- > Cl-; for forskolin, Cl- > I-). However, the DIDS-sensitive component of the forskolin-induced conductance response exhibits anion selectivity similar to that induced by thapsigargin (I- > Cl-). Thus forskolin-induced apical anion conductance comprises at least two components, one of which has features in common with that elicited by thapsigargin.

intestinal epithelium; thapsigargin; forskolin; chloride channels; amphotericin B; human intestinal cell line T84

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

CHLORIDE SECRETION in the intestine requires the activation of specific ion transporters and channels located in apical and basolateral membranes of polarized enterocytes. Basolateral transporters, which include the Na+-K+-ATPase, the Na+-K+-2Cl- cotransporter, and K+ channels, act coordinately to elevate intracellular Cl- concentration to levels above its electrochemical equilibrium potential, so that enterocytes are primed to secrete Cl- through apical anion channels. Agonists that elicit elevations in intracellular concentrations of Ca2+ and cAMP regulate the activities of these transporters and channels and thus regulate secretion (7, 17, 28, 38, 39).

Established human intestinal cell lines, such as T84, maintain a secretory phenotype and have been used widely to examine the physiology and regulation of intestinal Cl- secretion (14). Activation of apical Cl- conductances by cAMP agonists in intact T84 cell monolayers has been well documented. The Cl- effluxes induced by cAMP are presumed to represent Cl- transport through the cystic fibrosis transmembrane conductance regulator (CFTR) (1). CFTR is highly expressed in T84 cells (18). Activation of Cl- secretion by Ca2+-dependent agonists in T84 cells has also been well studied (3-5, 24), but the molecular identity of the channel or channels mediating this response remains undefined (1, 2, 9, 22).

Two biophysically distinct anion conductances have been described in nonpolarized human T84 and parental HT-29 cell lines. Whole cell voltage-clamp experiments have shown that isolated T84 cells grown on glass coverslips contain Ca2+-stimulated Cl- currents that exhibit outward rectification. cAMP-induced currents from the same cells displayed linear current-voltage relationships (9, 32). Ion selectivities of the Ca2+-stimulated Cl- currents in T84 cells differed from the ion selectivities exhibited by channels activated by cAMP (1). Experments using 125I- to trace Cl-secretion similarly indicated that cAMP- and Ca2+-induced Cl- effluxes were mediated by two separate pathways. Experimental measurements of Cl- transport in well-differentiated, polarized T84 and HT-29cl.19A cells grown on permeable supports estimated by the Cl--sensitive fluorophore, 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) (22), microelectrodes (2), or 125I- efflux (16) also support the conclusion that T84 cells express Ca2+-regulated Cl- channels that differ from Cl- channels regulated by cAMP.

Not all studies, however, have found evidence for the expression of Ca2+-sensitive apical membrane Cl- channels on well-differentiated polarized HT-29cl.19A or T84 cells. For example, when basolateral membranes of HT-29cl.19A or T84 cells were permeabilized selectively by nystatin to examine ion conductances across the apical membrane, no evidence for Ca2+-stimulated Cl- conductance was found (1, 12, 34). These authors concluded that Ca2+-stimulated Cl- conductances, although present on nonpolarized cells, were not expressed on apical membranes of well-differentiated and polarized monolayers (1). However, another study using this same approach found an increase of apical Cl- conductance by carbachol, suggesting that Ca2+-sensitive Cl- channels may be present on apical membranes of T84 cells (22). Thus the presence of Ca2+-sensitive Cl- channels in polarized colonic epithelial monolayer cultures has remained controversial.

Our aim in the present study was to reexamine the mechanisms of conductive Cl- transport across apical membranes of polarized T84 cells. For these studies, we selectively permeabilized basolateral membranes of confluent monolayers grown on permeable supports with the ionophore amphotericin B (25). This allowed estimation of apical membrane conductances from measurements of transepithelial conductance. Our data show that well-differentiated human intestinal T84 cells express at least two distinct apical membrane Cl- conductances. Although one can be activated only by the Ca2+ agonist thapsigargin, both appear to be activated by the cAMP agonist forskolin.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell culture. T84 cells (American Type Culture Collection, passages 45-98), a human colonic carcinoma cell line that functionally and morphologically resembles crypt intestinal epithelia, were grown as confluent monolayers in a 1:1 mixture of Dulbecco's Vogt modified Eagle's medium and Ham's F-12 medium supplemented with 15 mM HEPES buffer (pH 7.5), 14 mM NaHCO3, 40 µg/ml penicillin, 90 µg/ml streptomycin, and 5% newborn calf serum. Monolayers were subcultured every 7 days by trypsinization with 0.1% trypsin and 0.9 mM EDTA in Ca2+- and Mg2+-free PBS and grown on collagen-coated permeable supports (area 0.3 cm2, pore size 0.4 µm). All experiments described in this study were performed on cells between passages 65 and 92.

Electrophysiology. Studies were carried out at either 37 or 4°C with confluent monolayers plated on collagen-coated permeable supports (14) and examined 7-16 days postplating as previously described (38). Before all studies, inserts were washed with HCO-3-free medium (see Table 1) warmed to 37°C or cooled to 4°C and transferred to new 24-well tissue culture plates containing the experimental medium. To determine currents, transepithelial potentials, and conductances, a commercial voltage clamp (Bioengineering Dept., University of Iowa, Iowa City, IA) was interfaced with equilibrated pairs of calomel electrodes submerged in saturated KCl and with paired Ag-AgCl electrodes submerged in the experimental medium. Before each experiment, a blank filter was used to compensate for both the fluid resistance and resistance of the filter. In some experiments, transepithelial voltage and short-circuit current (Isc) were continuously recorded with the aid of an analog-to-digital converter (MacLab, World Precision Instruments) and a microcomputer.

                              
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Table 1.   Composition of buffers A-F

Measurement of Cl- conductance of apical plasma membrane. To evaluate the ion conductance of the isolated apical plasma membrane, the polyene ionophore amphotericin B (25, 30, 31, 36) was added to the basolateral solution at a concentration of 100 µM, the lowest concentration that gave a maximal change in steady-state current as determined in preliminary experiments. Only the plasma membrane facing the amphotericin-containing solution (basolateral membrane in this case) incorporates this ionophore, thus electrically isolating the opposing (apical) plasma membrane. To study the Cl- conductance of the apical plasma membrane so isolated, the voltage across the monolayer was then sequentially stepped from a holding voltage of 0 mV to values between -80 and +80 mV over a period of ~20 s. The protocol was performed before and 5 min after addition of forskolin and thapsigargin. The compositions of all solutions used in these experiments are shown in Table 1. Positive currents correspond to anion secretion and/or cation absorption.

cAMP measurement. Forskolin (100 µM) or thapsigargin (10 µM) was added to basolaterally permeabilized monolayers at 4 or 37°C. Seven minutes after stimulation, monolayers (surface area 5 cm2) were removed from their reservoirs and immersed in Hanks' balanced salt solution (HBSS+) at 4°C to terminate the reaction. The monolayers were rapidly cut from their plastic supports and placed in Microfuge tubes containing extraction buffer [66% ethanol, 33% HBSS+, and 1 mM phosphodiesterase inhibitor IBMX (Sigma)] at 4°C. Monolayers were compacted and extracts cleared of cellular debris by centrifugation. An aliquot (100 µl) of cleared cell extract was withdrawn for cAMP RIA (Du Pont-NEN) as previously described (41).

Cytosolic Ca2+ measurement. T84 cells cultured at subconfluent density on prepared collagen-coated 5-cm2 glass coverslips (200 µl of collagen/glass coverslip) were loaded with the fluorescent Ca2+ indicator dye fura 2 by incubation with 5 µM of its AM derivative, fura 2-AM, in HBSS+ at 37°C for 60 min. Extracellular fura 2-AM was removed by washing twice with normal medium (see Table 1). The monolayer was then mounted on a modified Leiden chamber in which the coverslip constituted the bottom. Before the start of the Ca2+ measurements, the coverslip mounted in the Leiden chamber was maintained at either 37 or 0°C. To the chamber was added 1 ml of buffer solution at either 37 or 0°C. The unclamped temperature decreased ~4°C from 37°C and increased 4°C from 0°C during the course of each experiment.

Intracellular free Ca2+ concentration ([Ca2+]i) was measured by fluoresence ratio imaging with an Image-1 digital ratio imaging system (Universal Imaging, West Chester, PA) equipped with an Olympus IMT-2 inverted microscope, a Dage-MTI CCD7 series videocamera, a Genisys image intensifier, a Pinnacle REO-650 optical disk drive, and a color video monitor-printer, as described previously (23). Fura 2 fluorescence images were monitored at 510-nm emission with alternating excitation at 340 and 380 nm. Fura 2-loaded T84 cells were selected by individual cell fluorescence images and were well defined for a region within each cell. Ca2+ response was observed in cells near the center of confluent "islands." Images and 340-to-380-nm ratio values calculated on a pixel-by-pixel basis were collected for data processing. With the same experimental settings for the imaging system, fura 2 ratio values can be calibrated in vitro (19) to Ca2+ concentration. Fura 2 free acid (2 µM) was dissolved in Ca2+-free HEPES buffer solution [110 mM KCl, 10 mM NaCl, 1 mM MgCl2, 25 mM HEPES, and 1.5 mM EGTA (pH 7.0)], and variable total Ca2+ was added in quantities calculated to yield free [Ca2+] in the range of 36 nM to 40 µM. The minimal fluorescence ratio (Rmin) was determined at zero Ca2+ (free Ca2+ < 10 nM), and the maximal fluorescence ratio (Rmax) at 4 mM total Ca2+. The equilibrium constant (Kb) was determined by fitting experimental fluorescence ratio (R) values at various free Ca2+ concentrations by using the following equation (19)
[Ca<SUP>2+</SUP>]<SUB>free</SUB> = <IT>K</IT><SUB>b</SUB> (Sf<SUB>2</SUB>/Sb<SUB>2</SUB>)[(R − R<SUB>min</SUB>)/(R<SUB>max</SUB> − R)]
where the factor Sf2/Sb2 corrects for fura 2 ion sensitivity at 380 nm.

Multipoint in situ calibration could not be completed because of loss of tight junctional integrity and cell adhesion to substratum during the period of cell equilibration in ionophore-Ca2+-EGTA solutions of low free [Ca2+]. Consequently, reported measurements of [Ca2+]i in T84 cells are based on in vitro calibration of the fura 2 fluorescence ratio. However, in situ fluorescence ratio calibration on intact adherent cells was successfully performed at nominal [Ca]i values of 100 and 200 nM. The ratios measured in intact cells at these two concentrations did not differ significantly from those determined by the in vitro calibration procedure at the same values of free [Ca2+] (P > 0.2).

    RESULTS
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Abstract
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Methods
Results
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References

DIDS sensitivity of thapsigargin- and forskolin-stimulated Isc. In intact monolayers, 10 µM thapsigargin added to the basolateral reservoir increases the transepithelial Isc and the conductance by 12.6 ± 1.3 µA/cm2 and 2.8 ± 0.6 mS/cm2 (n = 8), respectively (Fig. 1). As reported in previous studies, the stimulation of adenylate cyclase by forskolin increases Isc and conductance to a greater degree 48.4 ± 5.7 µA/cm2 and 6.1 ± 0.35 mS/cm2 (n = 8), respectively (Fig. 1). These data document stimulation of Isc consistent with electrogenic Cl- secretion but do not discriminate between the apical Cl- efflux pathways activated by thapsigargin on one hand and forskolin on the other.


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Fig. 1.   Transepithelial Cl- secretion was activated by addition of thapsigargin or forskolin. Cells were grown to confluence on collagen-coated permeable supports, with conductances of <1 mS/cm2. Short-circuit current (Isc) was measured under voltage clamp at 0 mV (see METHODS). Arrows indicate addition of 10 µM thapsigargin () or 100 µM forskolin (bullet ) to basolateral compartment. Note that subsequent addition of 100 µM DIDS to apical compartment reduced by ~40% forskolin-dependent Isc (bullet ) and by 100% thapsigargin-dependent Isc (). Data are means of 8 experiments, each performed in triplicate.

Our first approach to distinguish the possible presence of two different channels was to use the Cl- channel inhibitor DIDS. This Cl- channel inhibitor does not affect the function of the CFTR (27). As shown in Fig. 1, DIDS (100 µM added to apical solution) inhibited by nearly 100% the Cl- secretory response induced by thapsigargin (Fig. 1, peak Isc was 13.6 ± 0.8 and 0.4 ± 0.5 before and after addition of DIDS respectively, n = 8). In contrast, DIDS at 100 µM and up to 1 mM (data not shown) reduced by only 40% the Cl- secretion induced by forskolin (Fig. 1, peak Isc was 41.5 ± 6.5 and 25.4 ± 1.7 µA/cm2 before and after addition of DIDS respectively, n = 8). These data indicate that Cl- secretions stimulated by thapsigargin or forskolin exhibit different sensitivities to DIDS.

Basolateral permeabilization does not affect integrity of apical plasma membrane and leaves tight junctions intact. To directly measure conductance changes that occur in the apical plasma membrane, we used amphotericin B to permeabilize selectively basolateral membranes. Only monovalent ions traverse the amphotericin pore. Because of its requirement for cholesterol, amphotericin permeabilizes only the plasma membrane domain in direct contact with the amphotericin B solution as previously demonstrated (25). Thus this procedure leaves the apical membrane and tight junctions intact as judged by measurement of transepithelial conductances (before amphotericin B addition, Gintact = 0.583 ± 0.032 vs. after amphotericin addition, Gapical= 2.1 ± 0.6 mS/cm2, n = 20; see Fig. 2). The observed increase in conductance reflects removal of electrical resistance due to the basolateral membrane and provides indirect evidence that apical membranes and intercellular tight junctions remain intact. As previously reported (30, 31, 36), changes in Cl- conductances of luminal plasma membranes under these conditions are reflected as changes in Isc when an electrochemical gradient is established to drive Cl- across the permeabilized monolayer.


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Fig. 2.   Effect of amphotericin B treatment on intact T84 conductances and effect of thapsigargin and forskolin (+, present; -, absent) on basolaterally permeabilized T84 conductances; n = 20-30 monolayers for each condition.

Several tests were carried out to ensure that basolateral plasma membranes were sufficiently permeabilized by amphotericin B. In these studies, both intact and basolaterally permeabilized monolayers were exposed to an apically directed Cl- gradient (buffer A in basolateral and buffer B in apical reservoirs, Table 1) and voltage clamped at +10 mV. In a first approach, we utilized bumetanide to inhibit basolateral membrane Na+-K+-2Cl- transporters, which represent the rate-limiting step for Cl- uptake in intact T84 cell monolayers. Bumetanide applied to intact monolayers (10 µM) inhibited Cl- currents (Isc) induced by forskolin or thapsigargin by 55 and 65%, respectively. In contrast, when applied to monolayers basolaterally permeabilized by amphotericin B, bumetanide had no effect on Isc (Table 2). These data show that Cl- uptake via Na+-K+-2Cl- transporters in permeabilized monolayers was no longer rate limiting. In a second approach, we utilized Ba2+ (2 mM) or charybdotoxin to inhibit basolateral K+ channels. Basolateral K+ channels are required to establish and maintain membrane potential in Cl- secretory epithelia at rest and during the secretory response (36). Although both reagents inhibited cAMP- and Ca2+-induced Isc in intact monolayers (49% inhibition of forskolin-stimulated Isc by Ba2+ and 46% inhibition of thapsigargin-stimulated Isc by charybdotoxin), neither of these reagents inhibited Isc in basolaterally permeabilized monolayers (Table 2). These data show that selective permeabilization of basolateral membranes with amphotericin B effectively removed any rate-limiting contribution of basolateral membrane K+ transport to the secretory response. Finally, in separate studies, we have recently shown that the K+ channel blocker clotrimazole also had no effect on apical membrane Cl- conductances in basolaterally permeabilized monolayers (36). In T84 cells, clotrimazole blocks both K+ channels activated by cAMP and those activated by Ca2+ (13, 35, 36). Taken together, these data provide direct evidence that basolateral membranes are rendered freely permeant to monovalent ions by treatment with amphotericin B. Thus currents recorded in monolayers treated with basolaterally applied amphotericin B represent the sum of apical membrane conductance and the unknown contribution of conductance through intact tight junctions.

                              
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Table 2.   Paired measurements in absence and presence of each inhibitor were performed in intact basolaterally permeabilized monolayers

Forskolin but not thapsigargin activates an apical conductance at 0-mV membrane potential. We next examined the effects of forskolin and thapsigargin on apical membrane Cl- currents in permeabilized monolayers. After permeabilization of basolateral membranes with amphotericin B, a Cl- gradient was established across the electrically isolated apical membrane by placing "normal Cl- medium" (buffer A) in the basolateral reservoir and "low Cl- medium" (buffer B) in the apical reservoir (Na+ present in buffers A and B at 137 mM). These conditions are essentially identical to those established by Anderson and Welsh (1). When the monolayer was voltage clamped at 0 mV, thapsigargin did not elicit a detectable increase in apical Cl- conductance. In contrast, forskolin stimulated a sizable Cl- current under these same conditions (Fig. 3A). These data reproduce the results of previous studies and show that, at 0-mV membrane potential, only forskolin stimulates a Cl- conductance. However, when the same experiment was performed at +15 mV potential (inside negative), both thapsigargin and forskolin elicited a clear increase in apical anion conductance (Fig. 3, B and C). These data suggest that, under appropriate conditions, apical membranes of T84 cells may exhibit both cAMP- and Ca2+-sensitive Cl- conductances.


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Fig. 3.   A: at 0-mV transmembrane potential (V = 0 mV), forskolin but not thapsigargin stimulates Isc across apical plasma membrane. A defined basolateral-to-lumen Cl- gradient was established across monolayer by substitution of Na-gluconate for NaCl in solution bathing luminal side and addition of 100 µM amphotericin B to basolateral solution (normal Cl- medium). First thapsigargin (10 µM) and later forskolin (100 µM) were added to basolateral solution. Under same conditions but at +15 mV transmembrane potential (V = 15 mV), both 10 µM thapsigargin (B) and 100 µM forskolin (C) stimulate Isc. Each tracing is representative of results for 3 experiments with 4 different filters in each condition.

Thapsigargin and forskolin activate apical Cl- conductances. To test this hypothesis, the basolaterally permeabilized T84 monolayers were studied in symmetrical buffers containing Cl- as the major permeant ion; monovalent cations were in the millimolar range (buffer E, Table 1). Figure 4, A and B, shows that, under symmetrical Cl- concentrations, the relationships between Cl- current induced by either thapsigargin or forskolin were linear. Currents induced by forskolin were much larger than currents induced by thapsigargin. Monolayer conductances induced by thapsigargin were 1 ± 0.2 mS/cm2 and by forskolin 3 ± 0.4 mS/cm2 at +40 mV (mean of 20 independent experiments, see Fig. 2). Same results have been found in buffers containing Cl- as the sole permeant ion (choline chloride, 143 mM; CaSO4, 1.25 mM; thapsigargin and forskolin stimulated a Cl- current under these conditions, 30 and 100 µA/cm2 at +40 mV, respectively). To verify that the thapsigargin- and forskolin-induced currents were specific to Cl- transport, Cl- was replaced by gluconate in the continued presence of low Na+ (buffer F, Table 1). Under these conditions, the currents normally stimulated by thapsigargin and forskolin were inhibited by nearly 100% (Fig. 4, A and B). These experiments suggest that thapsigargin and forskolin stimulate a Cl- current.


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Fig. 4.   Current-voltage relationships in basolaterally permeabilized monolayers of T84 treated with thapsigargin (A) or forskolin (B). Basolateral-to-apical reservoir Cl- concentration ratios (in mM) were 143.50/143.50 mM Cl-, Na+ present in both sides at 0.5 mM (thapsigargin, A, ; forskolin, B, bullet ); with presence of a Cl- gradient between basolateral (143.50 mM Cl-) and apical compartment (6.61 mM Cl-), Na+ present in both sides at 137 mM (thapsigargin, A, ; forskolin, B, open circle ); with a low Cl- concentration in both apical and cytosol basolateral compartment (6.61 mM Cl-), Na+ present in both sides at 0.5 mM (thapsigargin, A, ×; forskolin, B, ×). With presence of a Cl- gradient between basolateral (143 mM Cl-) and apical compartment (43 mM), in absence of Na+ in both sides (thapsigargin, C, ; forskolin, C, open circle ). Ordinates give difference current between stimulated and unstimulated cells. Tracings are representative of results from 10 different filters for each condition.

When shifted from symmetrical to asymmetrical Cl- solutions (see Table 1, buffers A and B; Na+ present at 137 mM), apparent reversal potentials (Vrev) for agonist-induced currents shifted negatively as predicted but by different magnitudes [by 34 ± 5.8 mV (n = 12) for forskolin-stimulated currents (Fig. 4B, open circles), but only by 1.2 ± 0.6 mV (n = 10) for thapsigargin-stimulated currents (Fig. 4A, open squares)]. The observed reversal potentials do not approximate theoretical values in this model system, suggesting that a Cl- conductance in the apical plasma membrane is shunted by one or more pathways (tight junction and/or apical plasma membrane with nonspecific permeability). We examined whether the postulated anion channels had different relative gluconate conductances. We then studied monolayers exposed to symmetrical (apical and basolateral) buffers containing gluconate as the major charge-carrying anion [143 mM N-methyl-D-glucammonium (NMDG) gluconate; 1.25 mM CaSO4 both sides]. We found that, under these conditions, thapsigargin and forskolin had no effect on gluconate currents (results not shown). Thus neither the forskolin- nor thapsigargin-induced currents represent gluconate transport. However, in symmetrical buffers containing Na+ as the major permeant cation (Na+ 143 mM; 1.25 mM CaSO4 both sides), thapsigargin and forskolin stimulated a sizable Na+ current under these conditions [8.2 and 14.2 µA/cm2 (n = 2) at +40 mV, respectively]. Because we still do not know whether the Na+ current is paracellular or transcellular, it will be the focus of further study. We then studied monolayers bathed by asymmetrical Cl- solutions in absence of monvalent cations (apical: 43 mM choline chloride, 100 mM NMDG gluconate, 1.25 mM CaCl2; basolateral: 143 mM choline chloride, 1.25 mM CaCl2); Vrev for agonist-induced currents shifted negatively as predicted but by different magnitudes [by 20 ± 5.2 mV (n = 5) for forskolin-stimulated currents and by 10 ± 2 mV (n = 5) for thapsigargin-stimulated currents (Fig. 4C, open circles, for forskolin; Fig. 4C, open squares, for thapsigargin)]. The observed Vrev levels approximate theoretical values in this model system and demonstrate that thapsigargin and forskolin stimulate a Cl- conductance in the apical plasma membrane.

Together, these results raise the possibility that these macroscopic currents found under Cl- gradient conditions and in presence of monovalent cations (thapsigargin and forskolin dependent) could be the sum of both the cation and Cl- currents (Na+ current < Cl- current), explaining our observed Vrev under these last conditions.

Thapsigargin- and forskolin-activated anion conductances display different ion selectivities. The anion selectivities of apical membrane conductances induced by thapsigargin or forskolin were measured directly in basolaterally permeabilized monolayers. Current-voltage relationships were examined in asymmetrical buffers containing either I- or Cl- as the major permeant anion (buffers A and B or C and D, Na+ present in buffers A-D at 137 mM, Table 1). Figure 5 shows that thapsigargin stimulated a greater increase in Isc (anion conductance) in buffers containing I- [Gapical = 2.4 ± 0.3 mS/cm2 (n = 6); Fig. 5A] than in those containing Cl- [Gapical = 0.8 ± 0.2 mS/cm2 (n = 6); Fig. 5A]. In contrast, forskolin induced a greater increase in Isc when buffers contained Cl- rather than I- as the major permeant ion [Cl-: Gapical = 6 ± 0.9 mS/cm2; I-: 3.3 ± 1.0 mS/cm2 (n = 6)]. Both Cl- and I- currents, whether induced by thapsigargin or forskolin, were linear (Fig. 5, A and B).


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Fig. 5.   Effects of thapsigargin (A) and forskolin (B) on currents in basolaterally permeabilized T84 monolayers in presence of iodide solutions. Cytosol basolateral-to-apical reservoir Cl- concentration ratio was 143.50/6.6.0 mM Cl-, Na+ present in both sides at 137 mM (thapsigargin, ; forskolin, open circle ). Cytosol basolateral-to-apical reservoir I- concentration ratio for thapsigargin and forskolin was 136.89/6.60 mM I-, Na+ present in both sides at 137 mM (thapsigargin, ; forskolin, bullet ). Ordinates show difference current between stimulated and unstimulated cells. Tracings are representative of results from 6 different filters for each condition.

From the directions of the shifts in apparent reversal potentials, it can be deduced that thapsigargin stimulates an anion conductance which displays a greater permeability for I- than for Cl- [I-: Vrev = -6 ± 0.4 mV vs. Cl-: electrode reversal potential (Erev) = -2 ± 0.6 mV (n = 6)] and that forskolin stimulates a conductance which displays greater permeability for Cl- than for I- [Cl-: Vrev = -32 ± 4.2 mV; I-: Erev = -21 ± 3.6 mV (n = 6)]. These results provide direct evidence that anion conductances induced by thapsigargin and forskolin display different anion selectivities and that T84 cell apical membranes exhibit at least two distinct anion conductances.

DIDS affects Cl- conductances stimulated by both thapsigargin and forskolin. As shown in Fig. 1, the addition of DIDS to the apical compartment of intact T84 monolayers inhibited differentially transepithelial Isc stimulated by thapsigargin or forskolin (100 vs. 40%, respectively). We found nearly the same differential inhibition of DIDS on thapsigargin- or forskolin-induced Isc in basolaterally permeabilized monolayers studied in symmetrical buffers containing Cl- as the major permeant ion (buffer E). DIDS inhibited completely Isc elicited by thapsigargin [Delta Gapical = 1.2 ± 0.2 mS/cm2 vs. Delta Gapical = 0.1 ± 0.1 mS/cm2 with DIDS (n = 5); Fig. 6A]. In contrast, DIDS inhibited forskolin-induced Cl- currents by 57% [Delta Gapical = 4.1 ± 0.7 mS/ cm2 vs. Delta Gapical = 1.6 ± 0.8 mS/cm2 with DIDS (n = 5); Fig. 6B]. DIDS had a similar effect on Isc induced by 1 mM 8-bromo-cAMP or 10 mM forskolin applied to permeabilized monolayers (data not shown). This last result verifies the initial analysis using 100 µM forskolin. Thus the anion conductances observed are not attributable to an artifact seen only at 100 µM forskolin concentration. In the presence of transepithelial Cl- gradients in basolaterally permeabilized monolayers, basal Cl- currents were not affected by apical DIDS treatment [-24 ± 4 vs. -20.3 ± 2.7 µA/cm2 at +40 mV (n = 6)]; these results are in accordance with previous studies (8, 40). Thus the effect of thapsigargin on Cl- current cannot easily be attributed to the swelling-activated Cl- channel (29). Taken together, these data confirm results obtained on intact monolayers and provide further evidence that T84 apical membranes exhibit distinct Cl- conductances.


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Fig. 6.   Effect of apical addition of 100 µM DIDS on 10 µM thapsigargin (A)- and 100 µM forskolin (B)-induced conductances in basolaterally permeabilized monolayers of T84 (ICl). In these experiments, apical and cytosol basolateral buffers contained Cl- as major permeant ion, Na+ present in both sides at 0.5 mM (see Table 1). Ordinates show difference currents between stimulated and unstimulated cells in absence (thapsigargin, ; forskolin, bullet ) and presence of DIDS (thapsigargin, ; forskolin, open circle ). Tracings are representative of results from 5 different filters for each condition.

Because DIDS has no or little effect on Cl- transport through CFTR (15, 27), these data suggested that the DIDS-sensitive Isc induced by forskolin may represent Cl- transport through another channel distinct from CFTR, possibly the apical channel activated by thapsigargin. If so, the DIDS-sensitive component of the forskolin-induced current should display anion selectivities characteristic of the thapsigargin-induced channel. To test this hypothesis, we studied the ion selectivities of forskolin-induced currents in the presence and absence of DIDS by applying apically directed Cl- gradients to permeabilized monolayers (buffer A apical and buffer B basolateral). Figure 7 shows the current-voltage relationships for forskolin-induced Cl- conductances in the presence and absence of apical DIDS. In the presence of asymmetrical buffers, a clear shift in Vrev was induced by DIDS, indicating that the residual (DIDS-insensitive) apical membrane conductance displayed greater selectivity for Cl- [Vrev in absence of DIDS = -32 ± 2 mV; in presence of DIDS = -40 ± 5 mV (n = 10); Fig. 7]. These results can be interpreted to indicate that forskolin activates at least two anion conductances. One conductance displays high selectivity for Cl- and insensitivity to DIDS and probably represents CFTR. The other conductance displays lower selectivity to Cl- and complete sensitivity to DIDS. In these properties, it resembles the thapsigargin-regulated Cl- conductance. However, we do know that forskolin is not likely to induce a purinergic receptor-activated DIDS-sensitive current by activating the secretion of ATP via CFTR (37), since the addition of hexokinase (5 U/ml) to apical reservoirs had no effect on forskolin-induced Isc [Isc without hexokinase 43.9 ± 2.6 vs. 47.4 ± 2.3 µA/cm2 with hexokinase (n = 4)].


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Fig. 7.   Effect of apical addition of 100 µM DIDS on 100 µM forskolin-induced conductances in basolaterally permeabilized T84 monolayers. Basolateral-to-apical reservoir Cl- concentration ratio (in mM) was 136.89/6.60, Na+ present in both sides at 0.5 mM. Ordinate shows difference current between stimulated and unstimulated cells in absence (bullet ) and presence of DIDS (open circle ). Tracings are representative of results from 10 different filters for each condition.

Low temperature affects Cl- efflux stimulated by forskolin but not by thapsigargin. Previous studies have shown that activation of cAMP-dependent apical membrane Cl- channels in permeabilized T84 monolayers was markedly attenuated by reducing incubation temperatures to 5°C (11, 21), perhaps via prevention of channel recruitment to the plasma membrane (11). The effects of temperature on Ca2+-sensitive channels have not been similarly studied. Thus, to further compare the apical membrane Cl- conductances in T84 cell monolayers, we examined the effect of temperature on thapsigargin-induced and forskolin-induced Isc in permeabilized monolayers. Permeabilized monolayers were incubated in symmetrical buffers containing Cl- as the major permeant ion; monovalent cations were in the millimolar range (Cl-: 143.25; K+: 0.43; Na+: 0.34 mM; see buffer E, Table 1) at 4 or 37°C for 90 min before treatment with thapsigargin or forskolin. At 4°C, T84 cells maintain intact tight junctions as determined by low transepithelial conductances (0.8 ± 0.4 mS/cm2). The ultimate magnitude of the thapsigargin-induced Cl- secretory response was not affected at 4°C (Fig. 8A). In contrast, the forskolin-induced Cl- secretory response was diminished by incubation at 4°C as previously reported (Fig. 8B and Ref. 11). Thus the activation of Cl- conductances by thapsigargin and forskolin also differ in temperature sensitivity. Interestingly, the magnitude of the residual forskolin-activated current at 4°C was similar to that of the temperature-insensitive thapsigargin-stimulated current, and both currents were inhibited by DIDS to equivalent degree (Fig. 8, A and B). Permeabilized monolayers were incubated in symmetrical buffers containing Cl- or I- (see Table 1, buffer A or C); the ion selectivity of the DIDS-sensitive component of forskolin-induced current at 4°C is I- > Cl- (Fig. 9). These data demonstrate that the fraction of the forskolin-activated conductance which is DIDS sensitive and cold resistant may represent the same Cl- conductance as that stimulated by thapsigargin.


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Fig. 8.   Effect of temperature on thapsigargin (A)- and forskolin (B)-induced Cl- conductances in basolaterally permeabilized T84 monolayers. In these experiments, apical and cytosol basolateral buffers contained Cl- as major permeant anion, Na+ present in both sides at 0.5 mM (see Table 1). Ordinates show difference currents between stimulated and unstimulated cells treated with agonist at 37°C (thapsigargin, ; forskolin, bullet ) and 4°C (thapsigargin, ; forskolin, open circle ) in absence and presence of 100 µM DIDS; addition inhibits completely thapsigargin-stimulated current at 4°C (thapsigargin, A, ×; forskolin, B, ×). Tracings are representative of results from 3 different filters for each condition.


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Fig. 9.   Effects of 100 µM forskolin on currents in basolaterally permeabilized T84 monolayers measured in iodide solutions at 4°C. Basal Na+ present in both sides at 0.5 mM; basolateral-to-apical reservoir Cl- concentration ratio (in mM) was 143.50/6.6.0, Na+ present in both sides at 137 mM (open circle ). Cytosol basolateral-to-apical reservoir I- concentration ratio (in mM) was 136.89/6.60, Na+ present in both sides at 137 mM (bullet ). Ordinate shows difference currents between stimulated and unstimulated cells. Tracings are representative of results from 3 different filters for each condition.

Effects of thapsigargin and forskolin on intracellular cAMP and Ca2+ concentrations. In some epithelial cells, stimulation of cAMP also results in elevated intracellular Ca2+ (10). To investigate the possibility that forskolin may activate a Ca2+-sensitive Cl- conductance by raising cytosolic Ca2+ (in addition to cAMP), the effect of forskolin treatment on [Ca2+]i was examined. T84 cells grown on glass coverslips and loaded with the fluorescent indicator fura 2 were used for these studies (35). Cells incubated at 4 and 37°C were studied. Ca2+ response was observed in cells near the center of confluent islands. As shown in Fig. 10, resting levels of [Ca2+] were between 100 and 150 nM (133 ± 16 nM; n = 28 cells from 3 different coverslips). Application of forskolin at 37 or 4°C caused small but readily detected and consistently observed intracellular Ca2+ elevations (33 ± 3 nM; n = 28 cells from 3 different coverslips). As expected, stimulation with thapsigargin (10 µM) raised [Ca2+]i approximately twofold above resting levels at both temperatures (at 37°C, [Ca2+]i = 165 ± 12 nM vs. [Ca2+]i = 150 ± 14 nM at 4°C; n = 28 cells from 3 different coverslips), although the time required to reach maximal [Ca2+] levels was greater at 4°C. Thapsigargin addition before or after forskolin addition produced similar increases in [Ca2+]i. Thapsigargin had no effect on intracellular cAMP at either temperature (Fig. 11), whereas forskolin increased cAMP 30-fold above resting levels at 37°C and 3-fold above resting levels at 4°C. These data provide direct evidence that forskolin treatment can induce a small but detectable increase in intracellular Ca2+ at both 4 and 37°C. These data are consistent with previous studies in different cell types (10) but differ from previous studies performed in isolated T84 cells (43). The small Ca2+ response that we have found after forskolin addition might be due to the polarized state of T84. Thus the ability of forskolin to produce a small elevation in [Ca2+]i may contribute to its induction of DIDS-sensitive temperature-insensitive anion conductance.


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Fig. 10.   Effects of sequential addition of forskolin (Fors) and thapsigargin (Thap) on cytosolic [Ca2+] ([Ca2+]i) in T84. [Ca2+]i was measured at either 37 () or 4°C () in fura 2-loaded cells on glass coverslips. Ca2+ response was observed in cells near center of confluent "islands." A single representative experiment for each temperature (37 and 4°C) is given, and data are those of 14 cells from same coverslip. Tracings are representative of results from 3 different coverslips.


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Fig. 11.   Effect of temperature on generation of cAMP by basolaterally permeabilized T84 cells treated with forskolin (100 µM) and with thapsigargin (10 µM). Apical and basolateral buffers contained Cl- as major permeant ion. Experiments were carried out at 37 (A) or 4°C (B). Ctrl, resting level of cAMP; Thap and Fors, level of cAMP after addition of thapsigargin and forskolin, respectively. Data are presented as picomoles of cAMP generated per monolayer of T84 cells and represent 3 filters each assayed in duplicate.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The results of these studies show that selective permeabilization of basolateral membranes of T84 cells permits effective electrical isolation of apical membranes for analyses of anion conductances after agonist stimulation, as previously described (30, 31, 36). Such preparations avoid the complexities associated with regulation of various basolateral transporters, pumps, and channels that contribute to agonist-elicited Isc and also permit analyses of apical membrane conductance in the polarized state. We find that apical anion conductance is stimulated by both thapsigargin and forskolin. However, the anion currents induced by forskolin or thapsigargin exhibit different magnitudes and anion selectivities. Furthermore, they are differentially sensitive to the anion channel blocker DIDS and cold temperature, indicating that T84 cells exhibit two forms of apical membrane anion channels. Our results also show that the ion selectivity (Cl- < I-) and temperature dependence of the DIDS-sensitive component of the forskolin-induced Isc are similar to the ion selectivity and temperature dependence of the thapsigargin-induced Isc. Thus the forskolin response appears to represent apical anion conductance of two types, one of which has features common to the response elicited by thapsigargin.

The T84 colon carcinoma cell has frequently been used as a model for Cl- secretion, and considerable efforts have been directed at defining Cl- channels by using different methods such as patch-clamp, microelectrode, or permeabilization techniques. Whole cell voltage-clamp studies with T84 cells reveal two different Cl- conductances, one Ca2+ dependent and the other one cAMP dependent (1, 9). In addition, the anion selectivity of the Ca2+-stimulated Cl- conductance in T84 cells differs from the that of cAMP-dependent Cl- conductance (1). The use of the 125I efflux technique in subconfluent cells and recently on T84 cell monolayers also suggests that Ca2+ and cAMP induce two different anion efflux pathways (5, 16). In contrast, results from studies using a technique in which the basolateral membranes of confluent T84 cells are permeabilized by nystatin are contradictory (1, 22). A speculative conclusion has been made that Ca2+-stimulated Cl- conductances observed in cells grown on permeable supports are absent in cells grown on glass coverslips (1). On the other hand, studies using intracellular microelectrodes or using fluorescent dye (SPQ) have detected evidence favoring the presence of both Ca2+- and cAMP-stimulated Cl- conductances in T84 cells grown to confluence on permeable supports (2, 22).

The present study shows that intracellular elevation of Ca2+ or cAMP activates distinct Cl- conductances in basolaterally permeabilized monolayers. In symmetrical buffers, the conductances stimulated by forskolin or by Ca2+ each have linear relationships over the range ± 80-mV membrane potential. The conductances, however, display different biophysical characteristics. Experiments on the halide permselectivity between Cl- and I- indicate that Ganion(cAMP) has the permeability sequence, Cl- > I-. These results are in accordance with previous studies (1, 6) and suggest that the observed conductance probably represents transport through CFTR (42). In contrast, Ganion(Ca) has the opposite permeability sequence, I- > Cl-. We also show that GCl(cAMP) is only partially inhibited by DIDS in contrast to the GCl(Ca), which is completely inhibited by the addition of DIDS. Thus these data are consistent with the presence of at least two distinct Cl- conductances in T84 monolayers, as also suggested by previous patch-clamp and microelectrode studies (2, 9).

In previous studies, apical Cl- conductances in confluent T84 monolayers grown on permeable supports were measured by permeabilizing basolateral membranes with nystatin and then applying an apically directed gradient of Cl- across the monolayer with the voltage clamped to 0 mV. Under these conditions in the presence of amphotericin rather than nystatin, we observed similar results to those reported, finding that Cl- secretion was activated by the addition of forskolin but not thapsigargin (1). When, however, the transmonolayer potential was clamped at +15 mV, currents were activated both by thapsigargin and forskolin. Our results suggest the possibility that there is less discrimination between Na+ and Cl- currents at 0 mV than at other membrane potentials. Thus, in our studies when higher driving forces were applied, a thapsigargin-induced Cl- conductance was readily apparent. The Cl- conductances induced by thapsigargin, however, were much smaller than those observed for Cl- transport through forskolin-induced conductances. In other words, when stronger driving forces were applied, Cl- transport through the small thapsigargin-induced conductance became detectable.

The finding that thapsigargin induced an elevation of the intracellular Ca2+ without affecting cAMP levels raises the possibility that elevations of intracellular Ca2+ activate apical Cl- conductances in T84 cells. Because low temperatures did not affect the Ca2+-dependent Cl- conductance or the intracellular Ca2+ increase, it is reasonable to suggest that Ca2+ could directly activate this Cl- conductance. In addition, our observation that the cAMP-stimulated Cl- conductance was partially DIDS sensitive in basolaterally permeabilized as well as in intact monolayers raises the possibility that cAMP may stimulate two distinct Cl- conductances. We show that the temperature-insensitive component of forskolin-induced current, like the thapsigargin-induced current, was DIDS sensitive and displayed a permselectivity of I- >=  Cl-. These results are consistent with the possibility that a fraction of the Cl- conductance induced by forskolin represents transport through a Ca2+-activated, DIDS-sensitive Cl- channel. In T84 cells, forskolin can thus activate, in addition to CFTR, the Ca2+-sensitive conductance activated by thapsigargin alone. These results are consistent with previous studies showing that cAMP activates a Cl- conductance which is DIDS sensitive (20, 26) and shows preference for I- over Cl-. Our data, however, do not allow us to distinguish the possibility that T84 cells may contain two distinct DIDS-sensitive conductances, one activated by forskolin and the other by thapsigargin.

Our data do not identify a mechanism by which forskolin might activate the Ca2+-sensitive conductance. Although forskolin induces a small increase in intracellular Ca2+ at both 4 and 37°C, these data do not exclude the possibility that cAMP may activate the DIDS-sensitive conductance directly or through activation of protein kinase(s).

From the results of these and previous studies (2, 5, 16, 22, 33), we propose the following working model of Cl- secretion in T84 cells via two apical conductive pathways (Fig. 12). The first is activated by cAMP and not sensitive to inhibition by DIDS. This conductance exhibits temperature sensitivity (inhibition at 4°C), a large Cl- conductance, ion selectivity of Cl- > I-, and presumably represents CFTR. The second is activated both by thapsigargin and by forskolin. This conductance exhibits little or no temperature sensitivity, a much smaller Cl- conductance, and ion selectivity of I- > Cl-. Forskolin may activate the DIDS-sensitive conductance via cAMP or Ca2+ second messengers. Further studies, however, are needed to identify the mechanism by which forskolin may activate this Ca2+-sensitive conductance.


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Fig. 12.   Cellular model for regulation of Cl- secretion in T84. Ca2+ activates a Cl- channel that is completely inhibited by DIDS and insensitive to cold temperature. Forskolin activates via protein kinase A (PKA), a cold-temperature-sensitive Cl- channel only partially inhibited by DIDS, as well as a second, temperature-insensitive anion channel that is completely DIDS inhibited and of different anion selectivity. Activation of this 2nd channel occurs by an unknown pathway that may involve Ca2+ and/or cAMP and/or other second messengers. PCl and PI, Cl- and I- permeabilities; CFTR, cystic fibrosis transmembrane conductance regulator.

    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-35932 and DK-47622 to J. L. Madara, DK-48106 to W. I. Lencer, DK-51059 to S. L. Alper, and DK-02130 to A. Nusrat. W. I. Lencer is a recipient of the Samuel J. Meltzer Award from the American Digestive Health Foundation. S. L. Alper is an Established Investigator of the American Heart Association. D. Merlin is a recipient of National Institute of Diabetes and Digestive and Kidney Diseases National Research Service Award DK-09800. We also acknowledge the support of the Harvard Digestive Diseases Center (National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-34854).

    FOOTNOTES

Present address and address for reprint requests: D. Merlin, Dept. of Pathology and Laboratory Medicine WMRB, 1639, Emory University, 1639 Pierce Dr., Atlanta, GA 30322.

Received 9 July 1997; accepted in final form 16 April 1998.

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Abstract
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Methods
Results
Discussion
References

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