Bile salts potentiate adenylyl cyclase activity and cAMP-regulated secretion in human gallbladder epithelium

Nicolas Chignard1, Martine Mergey1, Danielle Veissière1, Raoul Poupon1,2, Jacqueline Capeau1,3, Rolland Parc4, Annick Paul1, and Chantal Housset 1,2,3

1 Institut National de la Santé et de la Recherche Médicale, 2 Service d'Hépatologie, Hôpital Saint-Antoine, and 4 Service de Chirurgie Générale et Digestive, Hôpital Saint-Antoine, 75012 Paris; and 3 Service de Biochimie, Hôpital Tenon, 75020 Paris, France


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fluid and ion secretion in the gallbladder is mainly triggered by the intracellular second messenger cAMP. We examined the action of bile salts on the cAMP-dependent pathway in the gallbladder epithelium. Primary cultures of human gallbladder epithelial cells were exposed to agonists of the cAMP pathway and/or to bile salts. Taurochenodeoxycholate and tauroursodeoxycholate increased forskolin-induced cAMP accumulation to a similar extent, without affecting cAMP basal levels. This potentiating effect was abrogated after PKC inhibition, whereas both taurochenodeoxycholate and tauroursodeoxycholate induced PKC-alpha and -delta translocation to cell membranes. Consistent with a PKC-mediated stimulation of cAMP production, the expression of six adenylyl cyclase isoforms, including PKC-regulated isoforms 5 and 7, was identified in human gallbladder epithelial cells. cAMP-dependent chloride secretion induced by isoproterenol, a beta -adrenergic agonist, was significantly increased by taurochenodeoxycholate and by tauroursodeoxycholate. In conclusion, endogenous and therapeutic bile salts via PKC regulation of adenylyl cyclase activity potentiate cAMP production in the human gallbladder epithelium. Through this action, bile salts may increase fluid secretion in the gallbladder after feeding.

beta -adrenergic agonist; chenodeoxycholic acid; chloride channels; ursodeoxycholic acid


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE GALLBLADDER, WHICH STORES and concentrates bile through fluid absorption during fasting, secretes a bicarbonate-rich fluid after feeding (24, 34). The reversal of absorption to secretion is mainly triggered by the intracellular second messenger cAMP (31) in response to beta -adrenergic stimulation (17) and to stimulation by the vasoactive intestinal polypeptide (VIP) (34) or by secretin (24). The cellular effects of beta -adrenergic agonists, of VIP and of secretin, are mediated by G protein-coupled receptors (13, 36). After activation of these receptors by their ligands, the G protein salpha -subunit is released and stimulates an adenylyl cyclase (AC) enzyme, which converts ATP into cAMP. Adenylyl cyclase enzymes form a superfamily of nine isoforms termed AC1-9. Whereas stimulation through the Gsalpha subunit is the major mechanism by which all adenylyl cyclases are activated, individual isoforms have different regulatory properties, which allow complex signal integration. AC1, -3, and -8 may be stimulated or inhibited by intracellular free calcium and by calmodulin, whereas AC2, -5, and -7 are stimulated by PKC, AC4 is stimulated by the beta gamma -subunit, AC6 is inhibited by low concentrations of calcium, and AC9 is insensitive to either calcium, PKC, or beta gamma -subunit (12).

The major driving force for both fluid and bicarbonate secretion across the gallbladder epithelium, is the extrusion of chloride ions through the apical cAMP-dependent chloride channel, CFTR (9, 31). Current evidence indicates that bile salts, the main constituents in bile, contribute to the regulation of biliary epithelial cell secretory functions (2, 8, 33). The apical sodium-dependent bile salt transporter is expressed in human gallbladder epithelial cells, and bile salt uptake affects chloride and mucin secretion in these cells (8). We have also previously shown that the tauroconjugate of ursodeoxycholic acid, a hydrophilic bile salt used in the treatment of biliary disorders (4), may generate distinct regulatory events compared with taurochenodeoxycholate (TCDC), a more hydrophobic endogenous bile salt (8). The purpose of the present study was to examine the action of TCDC and of tauroursodeoxycholate (TUDC) on the cAMP-dependent secretory pathway in the human gallbladder epithelium, including potential regulating effects on AC isoforms expressed in these cells.


    MATERIALS AND METHODS
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Reagents. DMEM/Ham's F-12 (1:1) mixture was purchased from Life Technologies (Cergy Pontoise, France), Ultroser G, from Biosepra (Villeneuve-la-Garenne, France), and human type IV collagen, from Tebu (Le Perray-en-Yvelines, France). Protease type XIV from Streptomyces griseus, forskolin, and diphenylamine carboxylic acid (DPC) were provided by Sigma (Saint-Quentin Fallavier, France). TCDC, TUDC, and glycochenodeoxycholate (GCDC) (99% pure), PMA, DIDS, BAPTA/AM, Bordetella pertussis toxin, GF 109203X, CPT-cAMP, and IBMX were obtained from Calbiochem (Meudon, France). 36Cl was obtained from Amersham (Les Ulis, France). Ribonuclease inhibitor RNazine was purchased from Promega (Charbonnières, France), Moloney murine leukemia virus RT, from Life Technologies (Cergy Pontoise, France), and Taq DNA polymerase, from PerkinElmer (Les Ulis, France).

Cell culture. Gallbladder samples were obtained from patients who underwent liver or pancreas surgery. The procedure was in accordance with current French legislation. The samples displayed no significant histological abnormality. Epithelial cells were isolated from gallbladders by incubation in 0.075% (wt/vol) protease type XIV for 12 h at 4°C, as described (8, 23). The cells were suspended in DMEM/Ham's F-12 containing 1 mmol/l Ca2+, supplemented with 2% (wt/vol) Ultroser G, 7 g/l D-glucose, 14 mmol/l NaHCO3, and 200,000 international units of 200 mg/l penicillin G-streptomycin, pH 7.4, and plated in 12-well culture dishes coated with human type IV collagen. The cells were incubated under 95% air-5% CO2, at 37°C. Culture medium was renewed every 48 h. All experiments were performed at days 5-6 of primary culture when the gallbladder epithelial cells form a confluent monolayer. Incubations with bile salts were performed at concentrations that did not induce cytotoxicity as ascertained by lactate dehydrogenase release (14).

cAMP assay. Gallbladder epithelial cells at days 5-6 of primary culture were incubated for 10 min at 37°C, with forskolin (10 µmol/l) or isoproterenol (10 µmol/l) and/or with TCDC (0.5 mmol/l), TUDC (0.5 mmol/l), GCDC (0.5 mmol/l), or PMA (0.16 µmol/l) in DMEM/Ham's F-12, whereas the controls were incubated with plain culture medium. Part of the cells underwent preincubation with either PMA (1.6 µmol/l) for 24 h, B. pertussis toxin (100 µg/l) for 18 h, or GF 109203X (1 µmol/l) for 30 min. All incubations were performed at 37°C. At the end of experiments, the cells were transferred on ice. DMEM/Ham's F-12 containing digitonin (40 µmol/l) and IBMX (1 mmol/l) was added. cAMP was assayed in the supernatant by a commercial radioimmunoassay kit (RIA; New England Nuclear-Life Science Products, Paris, France). The protein content of cell samples was determined by BCA-protein assay (Pierce, Bezon, France). Incubations with inhibitors were performed at concentrations that did not induce cytotoxicity, as ascertained by lactate dehydrogenase release.

RT-PCR. Total RNA was extracted from freshly isolated and cultured cells, using RNA plus lysis solution (Quantum, Montreuil-sous-Bois, France), according to Chomczynski and Sacchi's method (10). Total RNA from human brain tissue was provided by Clontech (Palo Alto, CA). One microgram of total RNA was denatured by heating at 72°C for 10 min, and then incubated in 25 µl of a reaction buffer containing 10 mmol/l DTT, 0.5 mmol/l dNTP, 20 units of RNazine, 5 µmol/l random hexamers, and 200 units of Moloney murine leukemia virus RT. Reverse transcription was allowed to proceed for 1 h at 37°C.

The following primers were designed on the basis of adenylyl cyclase human cDNA sequences, except for AC4 and -5 sequences, which were from Rattus norvegicus: 5'-CTG CGA GTC TAC ACA CCA TG-3' (sense) and 5'-CCT GTG CTA TCC ATC CGA CT-3' (antisense) that amplify a 870-bp cDNA fragment (nucleotides 1331-2200) of AC1 (GenBank accession no. LO5500); 5'-CTG AAC GAG ATC ACG GCT GA-3' (sense) and 5'-CGG TTG GCG AGC TAC CAT AT-3' (antisense) that amplify a 900-bp cDNA fragment (nucleotides 901-1800) of AC2 (GenBank accession no. L21993); 5'-GGG GAG TTT GAT GTG GAG CC-3' (sense) and 5'-GTC CCG TGT AGT ACT GGA GA-3' (antisense) that amplify a 805-bp cDNA fragment (nucleotides 1543-2347) of AC3 (GenBank accession no. AF033861); 5'-CAG AGA GAA GGA GAT GGA GA-3' (sense) and 5'-GCA GCC AGT GCA ACA TCT TG-3' (antisense) that amplify a 248-bp cDNA fragment (nucleotides 1801-2048) of AC4 (GenBank accession no. M80633); 5'-ATC GCC AAG ATG AAC CGC CA-3' (sense) and 5'-GCA GCT GAT CTG CAG GAA CA-3' (antisense) that amplify a 822-bp cDNA fragment (nucleotides 2402-3223) of AC5 (GenBank accession no. M96159); 5'-ATG CTG GCC AAG CTG CAG CG-3' (sense) and 5'-GGA CAC CAA GCA GCA GGT CA-3' (antisense) that amplify a 922-bp cDNA fragment (nucleotides 1814-2735) of AC6 (GenBank accession no. I29958); 5'-CCC TTT GCA CAT CTC AAC CA-3' (sense) and 5'-GAG AGG TTG AAG AGC ACC AG-3' (antisense) that amplify a 800-bp cDNA fragment (nucleotides 1751-2550) of AC7 (GenBank accession no. NM001114); 5'-CTA CCA GCG CTA TTT CTT GG-3' (sense) and 5'-CTC TTT ACC ATG GCC CTC TT-3' (antisense) that amplify a 1210-bp cDNA fragment (nucleotides 2601-3810) of AC8 (GenBank accession no. NM001115); 5'-CCG CAG GAG CAC CTG CAG AT-3' (sense) and 5'-TGA CCA CAT AAC ACC ACG TC-3' (antisense) that amplify a 793-bp cDNA fragment (nucleotides 3929-4720) of AC9 (GenBank accession no. AJ133123). Primers of human beta -actin cDNA used as an internal standard were previously selected to generate a 631-bp product (28). Amplifications were achieved using 0.5 µmol/l of primers, 1.25 units of Taq DNA polymerase, and annealing temperatures of 60°C. PCR products obtained after the completion of 38 cycles were separated by electrophoresis through a 2% agarose gel stained with ethidium bromide. The authenticity of all AC isoforms PCR products obtained from gallbladder epithelial cells was verified by sequencing.

PKC immunoassays. Gallbladder epithelial cells at days 5-6 of primary culture were incubated for 10 min, with or without PMA (0.16 µmol/l), TCDC (0.5 mmol/l), or TUDC (0.5 mmol/l) in Ultroser G-free DMEM/Ham's F-12. Cells were then harvested and sonicated in ice-cold buffer containing 10 mmol/l Tris · HCl (pH 7.5), 0.25 mol/l sucrose, 0.2 mmol/l CaCl2, and protease inhibitor cocktail (from Roche, Meylan, France). After the addition of EDTA at a final concentration of 1 mmol/l, the samples were centrifuged at 100,000 g for 60 min. The crude membrane pellet was suspended in a buffer containing 20 mmol/l Tris · HCl (pH 7.5), 0.25 mol/l sucrose, 1 mmol/l EDTA, 1 mmol/l EGTA, 10 mmol/l 2-mercaptoethanol, protease inhibitor cocktail, and 1% Triton X-100. After sonication on ice, and centrifugation at 100,000 g for 60 min, insoluble material was discarded and the supernatant was collected as a solubilized membrane fraction. Membrane proteins (10 µg) were then subjected to electrophoresis through a 7.5% SDS polyacrylamide gel, and transferred to polyvinylidene difluoride membranes. Immunoblotting was performed using monoclonal antibodies raised against alpha -, beta -, gamma -, delta -, and epsilon -isoforms (Transduction Laboratories, Le Pont-de-Claix, France), at concentrations of 0.25-1 mg/l. Immunoreactivity was revealed by enhanced chemiluminescence (Amersham, Les Ulis, France).

Chloride efflux assay. Chloride efflux was measured as described (15). Gallbladder epithelial cells at days 5-6 of primary culture were loaded with 36Cl (5 µCi/ml) in efflux buffer containing (in mmol/l) 140 NaCl, 4 KCl, 1 KH2PO4, 2 MgCl2, 1 CaCl2, 10 glucose, and 10 HEPES, pH 7.4, for 1 h at 37°C. Cells were washed rapidly three times with 1 ml of isotope-free buffer, which was then replaced at 2-min intervals, before stimulating agents were added in 1 ml of efflux buffer, whereas efflux buffer alone was added to the controls. The following agents were tested: isoproterenol (10 µmol/l) and CPT-cAMP (0.2 mmol/l) alone or combined with TCDC (0.5 mmol/l) and TUDC (0.5 mmol/l). Where indicated, the efflux buffer at all time points contained DPC (1 mmol/l) or DIDS (1 mmol/l) chloride channel blockers, or BAPTA/AM (50 µmol/l), a chelator of intracellular calcium. At the end of experiments, the cells were solubilized in 1 mol/l NaOH, and samples were counted for radioactivity. The efflux was calculated as the ratio of radioactivity in the efflux sample at a given time to the total radioactivity present in the cells during the previous 2-min interval. Increases in chloride efflux expressed in percent were calculated as the ratio between the peak of chloride efflux occurring 2-4 min after stimulation and the chloride efflux measured immediately before stimulation (basal level).

Statistical analysis. Comparisons were made using the Student's t-test and the paired Wilcoxon signed-rank test. n = Number of human samples; P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Bile salt regulation of cAMP production in gallbladder epithelial cells. In gallbladder epithelial cells exposed to TCDC or TUDC alone, the cAMP content showed no significant change, whereas in cells exposed to forskolin, which causes AC activation, cAMP content rose from 1.2 ± 0.1 to 166 ± 38 pmol/mg protein (n = 11; P < 0.002) (Fig. 1), and in those exposed to IBMX, which causes phosphodiesterase inhibition, cAMP content rose from 1.4 ± 0.1 to 2.7 ± 0.1 pmol/mg protein (n = 3; P < 0.05). These results indicated that bile salts alone affected neither AC nor phosphodiesterase activities in gallbladder epithelial cells. By contrast, as shown in Fig. 1, when TCDC or TUDC were added in combination with forskolin, the cAMP content rose to levels (242 ± 55 and 241 ± 63 pmol/mg protein, respectively), which were significantly higher than those elicited by forskolin alone (Fig. 1). The production of cAMP was increased to a similar extent by both tauroconjugates and also by the glycoconjugate GCDC. GCDC augmented the rise in cAMP elicited by forskolin from 152 ± 17 to 216 ± 28 pmol/mg protein (n = 3; P < 0.05). The physiological relevance of these results was supported by the findings that bile salts elicited the same potentiation of forskolin-induced cAMP accumulation in freshly isolated cells as in cultured cells (not shown). Because these effects of bile salts on cAMP production required concomitant AC stimulation, we inferred that they potentiated AC activity. To provide support to this possibility, we analyzed the pattern of AC expression in human gallbladder epithelial cells.


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Fig. 1.   Bile salt regulation of cAMP production in gallbladder epithelial cells. Taurochenodeoxycholate (TCDC; 0.5 mmol/l) or tauroursodeoxycholate (TUDC; 0.5 mmol/l) and forskolin (10 µmol/l) were added to gallbladder epithelial cells in primary culture either separately or simultaneously, whereas the controls remained in plain culture medium. cAMP content was measured by RIA and reported to cell protein content. Data represent means ± SE of experiments performed in duplicate using 11 different human samples. *P < 0.01 vs. forskolin alone.

Pattern of AC expression in gallbladder epithelial cells. Transcripts of six AC isoforms, 3-7 and 9, were detected by RT-PCR in all freshly isolated gallbladder epithelial cells issued from twelve different donors (Fig. 2A), whereas in all these preparations, the transcripts of AC1, -2, and -8 were undetectable (Fig. 2C). All AC amplification products obtained from gallbladder epithelial cells were authenticated by sequencing. Those of AC4 and -5 showed >80% homology with Rattus norvegicus sequences. In cultured cells, sustained expression of all the isoforms identified in freshly isolated cells, was recorded up to at least 6 days of primary culture (Fig. 2B).


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Fig. 2.   Pattern of adenylyl cyclase (AC) expression in gallbladder epithelial cells. RT-PCR analysis of AC3-7, and -9 in freshly isolated cells (A), in cells at day 6 of primary culture (B), and of AC1, -2, and -8 in human brain tissue used as a positive control (C, lanes 1), in freshly isolated cells (lanes 2) and in cultured cells (lanes 3), was performed. AC amplification products are visualized as unique bands distinct from beta -actin. beta -actin PCR products were diluted (1:4) before electrophoresis. Gels are representative of results obtained from the analysis of 12 different human samples.

Signaling pathways mediating bile salt regulation of cAMP production in gallbladder epithelial cells. The pattern of AC expression in gallbladder epithelial cells pointed to PKC and to Gi protein beta gamma -subunit as potential intracellular signals regulating cAMP production in these cells, via the stimulation of AC5 and -7, and of AC4 and -7 isoforms, respectively. Confirmation that PKC regulation of cAMP production occurs in gallbladder epithelial cells was provided by testing the effect of PKC activation, in cells exposed to the phorbol ester PMA (0.16 µmol/l). In cells exposed to PMA, cAMP levels elicited by forskolin increased from 112 ± 25 to 172 ± 28 pmol/mg protein (n = 5; P < 0.05) (Fig. 3). PKC downregulation was then achieved in cells incubated with PMA at high concentration (1.6 µmol/l) for 24 h, and resulted in the suppression of TCDC potentiating effect on cAMP production (Fig. 4A). By contrast, in cells treated with B. pertussis toxin, despite effective inhibition of Gbeta gamma release from Gi protein as ascertained by an increase in forskolin-stimulated cAMP formation (Fig. 4B), the potentiating effect of TCDC remained significant (Fig. 4B). Therefore, whereas TCDC regulation of cAMP production required PKC activation, no evidence was found to indicate that Gi protein beta gamma -subunit contributes to this regulation.


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Fig. 3.   Effect of PKC activation on cAMP production in gallbladder epithelial cells. PMA (0.16 µmol/l) and forskolin (10 µmol/l) were added to gallbladder epithelial cells either separately or simultaneously, whereas the controls remained in plain culture medium. cAMP content was measured by RIA and reported to cell protein content. Data represent means ± SE of experiments performed in duplicate using 5 different human samples. *P < 0.05.



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Fig. 4.   Effect of PKC and Gi protein signaling pathways on cAMP production in gallbladder epithelial cells. A: effect of PKC downregulation on TCDC potentiating effect. Gallbladder epithelial cells were pretreated or not with PMA (1.6 µmol/l) for 24 h and were then exposed to forskolin (10 µmol/l) alone or in combination with TCDC (0.5 mmol/l). B: effect of Gi protein inhibition on TCDC potentiating effect. Gallbladder epithelial cells were pretreated or not with B. pertussis toxin (100 µg/l) for 18 h, and were then exposed to forskolin (10 µmol/l) alone or in combination with TCDC (0.5 mmol/l). Data normalized during pairing are expressed as a percentage of cAMP content in cells exposed to forskolin alone. They represent means ± SE of experiments performed in duplicate using 3 different human samples. *P < 0.05.

Further investigations of the PKC-dependent pathway showed that, on forskolin stimulation, the elevations in cAMP levels triggered by PMA, TCDC, or TUDC were significantly reduced by the PKC inhibitor GF 109203X at 1 µmol/l (Fig. 5). These inhibitions were effective, whereas the cAMP response to forskolin alone was not significantly different in GF 109203X-treated cells (148 ± 71 pmol cAMP/mg protein) compared with controls (136 ± 61 pmol cAMP/mg protein, n = 4, P = not significant). GF 109203X at 1 µmo/l inhibits PKC-alpha , -beta , -gamma , -delta , and -epsilon isoforms. Among these isoforms, only PKC-alpha , -delta and -epsilon were detected by immunoblot analyses in cultured gallbladder epithelial cells. The addition of PMA, TCDC, or TUDC to these cells caused an increase in the amounts of membrane-bound PKC-alpha and -delta (Fig. 6), whereas no membrane-bound PKC-epsilon was detectable in these cells, irrespective of whether or not they were exposed to PMA, TCDC, or TUDC (data not shown). These data suggest that both TCDC and TUDC potentiate the production of cAMP in gallbladder epithelial cells, through AC regulation by PKC-alpha and/or -delta isoforms.


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Fig. 5.   Effect of PKC signaling pathway in bile salt regulation of cAMP production. Gallbladder epithelial cells were pretreated or not with GF 109203X (1 µmol/l) for 30 min, exposed to forskolin (10 µmol/l) alone or in combination with PMA (0.16 µmol/l) and then used as a positive control of PKC activation with TCDC (0.5 mmol/l) or with TUDC (0.5 mmol/l). cAMP content was measured by RIA and reported to cell protein content. Data normalized during pairing are expressed as a percentage over cAMP content in cells exposed to forskolin alone. Data represent means ± SE of experiments performed in duplicate using 4 different human samples. *P < 0.05 vs. similar stimulus in the absence of GF 109203X.



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Fig. 6.   Membrane translocation of PKC isoforms. Gallbladder epithelial cells were exposed to either PMA (0.16 µmol/l), TCDC (0.5 mmol/l), or TUDC (0.5 mmol/l) for 10 min. Controls were maintained in plain medium. Membrane proteins (10 µg) prepared from 3 different cell preparations were pooled and subjected to PKC-alpha and -delta immunoblot-enhanced chemiluminescence analysis.

Bile salt regulation of cAMP-dependent chloride secretion in gallbladder epithelial cells. To test the influence of bile salts on cAMP-dependent secretion, chloride efflux was measured in cells exposed to isoproterenol, a beta -adrenergic agonist, and to bile salts, either separately or simultaneously. In cells exposed to isoproterenol, the cAMP content rose from 1.3 ± 0.3 to 518 ± 153 pmol/mg protein and further to 635 ± 143 pmol/mg protein when isoproterenol was combined with TCDC (n = 5; P < 0.05 vs. isoproterenol alone). Both isoproterenol and bile salts (TCDC or TUDC), when added separately, stimulated chloride secretion in the cells (Fig. 7, A and B). When isoproterenol and TCDC or TUDC were added simultaneously, chloride secretion was significantly higher than in cells exposed to either isoproterenol or bile salts alone (Fig. 7, A-C). The chloride channel blocker DIDS significantly reduced chloride secretion stimulated by TCDC or TUDC (Fig. 7B). Moreover, BAPTA/AM, a chelator of intracellular calcium, caused 80 and 75%-inhibitions in TCDC- or TUDC-induced secretions, respectively, providing further evidence that calcium-dependent chloride channels were involved. By contrast, in cells exposed to isoproterenol alone (although DPC, a nonselective chloride channel blocker, significantly decreased chloride secretion) DIDS had no effect (Fig. 7A), consistent with cAMP-dependent chloride secretion in response to beta -adrenergic stimulation. Similar inhibition profiles were observed when chloride secretion was triggered by isoproterenol in conjunction with TCDC, TUDC (Fig. 7C), or PMA (not shown). Furthermore, the potentiating effect of TCDC on chloride secretion induced by isoproterenol was abrogated by the PKC inhibitor GF 109203X. The effect of isoproterenol combined with TCDC, compared with that of isoproterenol alone, decreased from 147 ± 13 (n = 4; P < 0.05) to 107 ± 45% (n = 4; P = 0.9), on inhibition by GF 109203X, whereas the response to isoproterenol alone, was not significantly modified by GF 109203X (not shown). These results suggest that on beta -adrenergic stimulation, rather than additional activation of calcium-dependent channels, further activation of the cAMP production by means of AC potentiation through PKC is the major mechanism by which bile salts increase chloride secretion. In support of this conclusion, TCDC was unable to further enhance chloride secretion elicited by the cAMP analog CPT-cAMP, which elicits cAMP-dependent chloride secretion without activating AC (30 ± 5 vs. 27 ± 4% over basal n = 3; P = not significant).


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Fig. 7.   Chloride channel activities in bile salt-regulated chloride secretion. Chloride efflux was measured in response to isoproterenol (10 µmol/l) alone (A), TCDC or TUDC (0.5 mmol/l) alone (B), or a combination of both (C) in gallbladder epithelial cells treated or not with the chloride channel blockers diphenylamine carboxylic acid (DPC) or DIDS. Data are expressed as increases in chloride efflux over basal levels and represent means ± SE of experiments performed in duplicate using 5 different human samples. *P < 0.05 vs. the same stimulus in the absence of chloride channel blocker. #P < 0.05 vs. isoproterenol alone.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In previous investigations, we have shown that bile salts, below critical micellar concentrations (32), regulate secretory functions in the human gallbladder epithelium (8, 14). The present study provides evidence that bile salts potentiate cAMP-regulated secretion in this epithelium. Both TCDC and TUDC increased forskolin-induced cAMP accumulation without affecting basal levels, consistent with a regulation of adenylyl cyclase activity. The analysis of AC expression in a large number of preparations allowed us to show for the first time that human gallbladder epithelial cells express six isoforms of AC, namely AC3-7 and -9, whereas AC1, -2, and -8 are not expressed. We deduced from this analysis that some of the isoforms expressed in gallbladder epithelial cells could be positively regulated by PKC (AC5 and -7) (22, 26) or by the beta gamma -subunit of B. pertussis toxin-sensitive Gi proteins (AC4 and -7) (21, 39). The potentiating effect of TCDC was insensitive to B. pertussis toxin (18), making unlikely the possibility that bile salts modulate cAMP synthesis through the release of beta gamma -subunit from Gi protein in gallbladder epithelial cells. By contrast, in support to PKC regulation, the potentiating effects of bile salts on cAMP production were mimicked by PMA and were abrogated by GF 109203X, which acts as a competitive inhibitor of ATP-binding sites for PKC-alpha , -beta , -gamma , -delta , and -epsilon isoforms (35). The potentiation of cAMP production by bile salts was also suppressed by downregulation of the same isoforms after long-term exposure to PMA (7, 14). Immunoanalyses showed that, among PMA and GF 109203X inhibitable isoforms, only PKC-alpha and -delta translocated to the membranes of gallbladder epithelial cells exposed to bile salts. TCDC and TUDC were both effective in inducing PKC-alpha and -delta translocation, in keeping with similar effects on cAMP formation. The signals induced by TUDC were more intense, in agreement with what we previously found by measuring membrane-bound PKC activity (8), although the difference with the effect of TCDC was not significant. The pattern of AC expression in gallbladder epithelial cells raise the possibility that bile salts may have additional effects on AC and cause inhibition of isoforms, such as AC3 or -6, expressed in these cells. AC6 is inhibited by free intracellular Ca2+ (38), at concentrations (IC50 of 200 nmol/l) within the range of those triggered by TCDC in gallbladder epithelial cells (approx 260 nmol/l) (14), whereas AC3 is inhibited by calmodulin-dependent protein kinase II (37), an enzyme activated by TCDC and by TUDC in gallbladder epithelial cells (8, 14). We found that the pattern of AC isoforms expressed in human gallbladder epithelial cells is different from that in human hepatocytes (unpublished observation). Because a distinct pattern of AC expression may result in a different balance between the positive and negative regulatory signals generated by bile salts, this may be part of the reason why bile salts exert inhibitory instead of potentiating effects on cAMP formation in hepatocytes (5).

The switch between fluid absorption and secretion that occurs in the gallbladder after feeding is triggered by intracellular cAMP. cAMP accumulates in gallbladder epithelial cells in response to VIP (16), secretin (20), and beta -adrenergic stimulation as shown in both present and previous reports (29, 30). Higher levels of cAMP were elicited in the present study when isoproterenol was used instead of forskolin, which may be because isoproterenol via beta -adrenergic receptors activates all AC isoforms, including AC9 expressed at high levels in gallbladder epithelial cells, whereas forskolin stimulates all isoforms with the exception of AC9. In keeping with a potentiation of cAMP production, TCDC and TUDC potentiated the stimulation of chloride secretion by isoproterenol. In the absence of any other stimulus, bile salts also stimulated chloride secretion. On the basis of DIDS and BAPTA/AM inhibiting effects and of previous demonstration that TCDC induces a rapid rise of intracellular Ca2+ concentration in gallbladder epithelial cells (14), we may postulate that the secretory response to bile salts alone was mediated by calcium-dependent chloride channels (25). By contrast, in the presence of concomitant beta -adrenergic stimulation, bile salts increased chloride secretion mainly via a cAMP-dependent pathway. Chloride secretion in this setting was inhibited by DPC but not by DIDS, consistent with an effect on the cAMP-dependent chloride channel, CFTR (3, 6, 9, 11, 15, 19, 27). It was previously shown that bile salts increase secretin-induced cAMP levels and secretin receptor gene expression in rat cholangiocytes (1), and that ductal bile secretion is increased in bile acid-fed rats (2). Because cAMP-dependent chloride secretion promotes bicarbonate and fluid secretion in the gallbladder (9, 31), the present findings suggest that bile salts potentiate hormonal and neurogenic stimulation of fluid secretion that will facilitate the progression of bile in bile ducts and assist gallbladder emptying after feeding. Given that TUDC induces lower mucin secretion than TCDC (8), the present findings showing similar effects of TCDC and TUDC on cAMP-dependent anion secretion reinforce the concept that ursodeoxycholic acid may alter the ratio between mucin and hydroelectrolytic secretion in a direction that will increase bile fluidity.


    ACKNOWLEDGEMENTS

The authors are very grateful to Jacques Hanoune and Joëlle Masliah of Institut National de la Santé et de la Recherche Médicale for helpful discussion.


    FOOTNOTES

This work was supported by a grant from the Association "Vaincre la mucoviscidose."

Address for reprint requests and other correspondence: C. Housset, INSERM U402, Faculté de Médecine Saint-Antoine, 27 rue de Chaligny, 75571 Paris Cedex 12, France (E-mail: chantal.housset{at}st-antoine.inserm.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published November 6, 2002;10.1152/ajpgi.00292.2002

Received 19 July 2002; accepted in final form 31 October 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastrointest Liver Physiol 284(2):G205-G212
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