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• W2015176407 abstract "Serotonin (5-hydroxytryptamine (5-HT)) is an important neurotransmitter and intercellular messenger regulating various gastrointestinal functions, including electrolyte transport. To date, however, no information is available with respect to its effects on the human intestinal apical anion exchanger Cl-/OH- (HCO3−). The present studies were therefore undertaken to examine the direct effects of serotonin on OH- gradient-driven 4,4′-diisothiocyanato-stilbene-2, 2′-disulfonic acid-sensitive 36Cl- uptake utilizing the post-confluent transformed human intestinal epithelial cell line Caco-2. Our results demonstrate that serotonin inhibits Cl-/OH- exchange activity in Caco-2 cells via both tyrosine kinase and Ca2+-independent protein kinase Cδ-mediated pathways involving either 5-HT3 or 5-HT4 receptor subtype. The data consistent with our inference are as follows. (i) The short term treatment of cells with 5-HT (0.1 μm) for 15-60 min significantly decreased Cl-/OH- exchange (50-70%, p < 0.05). (ii) The specific agonists for 5-HT3, m-chlorophenylbiguanide, and 5-HT4, 3-(4-allylpiperazin-1-yl)-2-quinoxaline chloronitrile, mimicked the effects of serotonin. (iii) Tropisetron dual inhibitor for both the 5-HT3/4 receptor subtypes significantly blocked the inhibition, whereas specific 5-HT3 (Y-25130) or 5-HT4 receptor (RS39604) antagonist failed to block the inhibitory effects of 5-HT. (iv) The Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl ester) had no effect on the serotonin-induced inhibition. (v) The specific protein kinase C (PKC) inhibitors chelerythrine chloride or calphostin C completely blocked the inhibition by 5-HT. (vi) The specific inhibitor for PKCδ, rottlerin, significantly blocked the inhibition by 5-HT. (vii) The specific tyrosine kinase inhibitor, herbimycin, or Src family kinase inhibitor, PP1, abolished the 5-HT-mediated inhibition of Cl-/OH- exchange activity. (viii) 5-HT stimulated tyrosine phosphorylation of c-Src kinase and PKCδ. Serotonin (5-hydroxytryptamine (5-HT)) is an important neurotransmitter and intercellular messenger regulating various gastrointestinal functions, including electrolyte transport. To date, however, no information is available with respect to its effects on the human intestinal apical anion exchanger Cl-/OH- (HCO3−). The present studies were therefore undertaken to examine the direct effects of serotonin on OH- gradient-driven 4,4′-diisothiocyanato-stilbene-2, 2′-disulfonic acid-sensitive 36Cl- uptake utilizing the post-confluent transformed human intestinal epithelial cell line Caco-2. Our results demonstrate that serotonin inhibits Cl-/OH- exchange activity in Caco-2 cells via both tyrosine kinase and Ca2+-independent protein kinase Cδ-mediated pathways involving either 5-HT3 or 5-HT4 receptor subtype. The data consistent with our inference are as follows. (i) The short term treatment of cells with 5-HT (0.1 μm) for 15-60 min significantly decreased Cl-/OH- exchange (50-70%, p < 0.05). (ii) The specific agonists for 5-HT3, m-chlorophenylbiguanide, and 5-HT4, 3-(4-allylpiperazin-1-yl)-2-quinoxaline chloronitrile, mimicked the effects of serotonin. (iii) Tropisetron dual inhibitor for both the 5-HT3/4 receptor subtypes significantly blocked the inhibition, whereas specific 5-HT3 (Y-25130) or 5-HT4 receptor (RS39604) antagonist failed to block the inhibitory effects of 5-HT. (iv) The Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl ester) had no effect on the serotonin-induced inhibition. (v) The specific protein kinase C (PKC) inhibitors chelerythrine chloride or calphostin C completely blocked the inhibition by 5-HT. (vi) The specific inhibitor for PKCδ, rottlerin, significantly blocked the inhibition by 5-HT. (vii) The specific tyrosine kinase inhibitor, herbimycin, or Src family kinase inhibitor, PP1, abolished the 5-HT-mediated inhibition of Cl-/OH- exchange activity. (viii) 5-HT stimulated tyrosine phosphorylation of c-Src kinase and PKCδ. The gastrointestinal tract is an important source of the endogenous amine, serotonin, mainly stored in the mucosal enterochromaffin cells and in the enteric nervous system (1Gershon M.D. Erde S.M. Gastroenterology. 1981; 80: 1571Abstract Full Text PDF PubMed Scopus (172) Google Scholar). 5-HT 1The abbreviations used are: 5-HT, 5-hydroxytryptamine (serotonin); BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl ester); PKC, protein kinase C; PKA, cAMP-dependent protein kinase; GI, gastrointestinal; DIDS, 4,4′-diisothiocyanato-stilbene-2, 2′-disulfonic acid; MES, 4-morpholineethanesulfonic acid; PBS, phosphate-buffered saline; pA2, the negative logarithm of the dissociation constant of an antagonist, considered the indirect measure of the antagonist's affinity for its receptors; TBS, Tris-buffered saline; HRP, horseradish peroxidase. is continuously released from the enterochromaffin cells in the intestinal mucosa into the portal circulation and the gut lumen in response to a variety of luminal stimuli such as change in pH or osmolarity and mechanical and chemical stimuli (2Racke K. Schworer H. Pharmacol. Res. 1991; 23: 13Crossref PubMed Scopus (140) Google Scholar). 5-HT is an important neurotransmitter and intercellular messenger to modulate gastrointestinal functions (3Gershon M.D. Takaki M. Tamir H. Branchek T. Experientia (Basel). 1985; 41: 863Crossref PubMed Scopus (33) Google Scholar). 5-HT has been shown to alter gastrointestinal motility (4Cooke H.J. Gastroenterology. 1986; 90: 1057Abstract Full Text PDF PubMed Scopus (141) Google Scholar), alter gastric acid secretion, and enhance intestinal secretions (1Gershon M.D. Erde S.M. Gastroenterology. 1981; 80: 1571Abstract Full Text PDF PubMed Scopus (172) Google Scholar, 5Zinner M.J. McFadden D. Sherlock D. Jaffe B.M. Gastroenterology. 1986; 90: 515Abstract Full Text PDF PubMed Scopus (22) Google Scholar). Most of the previous studies regarding the effects of serotonin on ion transport were mainly based on the electrophysiological data including short circuit current measurements and unidirectional fluxes. These studies showed that 5-HT stimulated electrogenic Cl- secretion and inhibited Na+ and Cl- absorption in the rat colon (6Zimmerman T.W. Binder H.J. Gastroenterology. 1984; 86: 310Abstract Full Text PDF PubMed Scopus (89) Google Scholar), jejunum (7Hardcastle J. Hardcastle P.T. Redfern J.S. J. Physiol. (Lond.). 1981; 320: 41Crossref Scopus (70) Google Scholar), ileum (8Moriarty K.J. Higgs N.B. Woodford M. Warhurst G. Turnberg L.A. Gut. 1987; 28: 844Crossref PubMed Scopus (31) Google Scholar), and guinea pig ileum (9Leysen J. Neuropharmacology. 1984; 23: 247Crossref PubMed Scopus (55) Google Scholar). 5-HT has also been shown to inhibit the NaCl-absorptive process in the rabbit ileum and gall bladder (10Donowitz M. Tai Y.H. Asarkof N. Am. J. Physiol. 1980; 239: G463PubMed Google Scholar) but has no effect in rabbit and guinea pig colon (10Donowitz M. Tai Y.H. Asarkof N. Am. J. Physiol. 1980; 239: G463PubMed Google Scholar, 11Cooke H.J. Carey H.V. Eur. J. Pharmacol. 1985; 111: 329Crossref PubMed Scopus (71) Google Scholar). Moreover, Sundaram et al. (12Sundaram U. Knickelbein R.G. Dobbins J.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6249Crossref PubMed Scopus (32) Google Scholar) have demonstrated that 5-HT inhibits Cl-/HCO-3 exchange activity in villus cells and stimulates HCO-3 secretion in crypt cells of the rabbit ileum. In contrast, 5-HT appears to have no significant effect on coupled NaCl absorption in the human jejunum (13Kellum J.M. Budhoo M.R. Siriwardena A.K. Smith E.P. Jebraili S.A. Am. J. Physiol. 1994; 267: G357Crossref PubMed Google Scholar). 5-HT exerts its effects by binding to 5-HT receptors, which are distributed on the neuronal, muscular, and epithelial structures (14Hansen M.B. Pharmacol. Toxicol. 1995; 77: 3PubMed Google Scholar). Based on pharmacological studies and molecular cloning (15Hoyer D. Clarke D.E. Fozard J.R. Hartig P.R. Martin G.R. Mylecharane E.J. Saxena P.R. Humphrey P.P. Pharmacol. Rev. 1994; 46: 157PubMed Google Scholar), the existence of at least seven 5-HT receptors (5-HT1-7) subdivided into 14 subtypes has been discovered. With the exception of 5-HT5 and 5-HT6, 5-HT1A, 5-HT1P, 5-HT2, 5-HT3, and 5-HT4 have been reported to be expressed in the gut (16Gaginella T.S. Rimele T.J. Wietecha M. J. Physiol. (Lond.). 1983; 335: 101Crossref Scopus (68) Google Scholar). 5-HT1, 5-HT2, and 5-HT4 receptors are coupled via guanine nucleotide binding (G) proteins to their effectors, whereas 5-HT3 receptor is a ligand-gated ion channel permeable to anions and cations (17North R.A. Br. J. Pharmacol. 1989; 98: 13Crossref PubMed Google Scholar). Previous studies in different animal models have shown that 5-HT1 receptors mediate peristaltic reflex and do not seem to play a major role in intestinal fluid transport, whereas 5-HT2, 5-HT3, and 5-HT4 receptors produce a variety of responses in the GI tract such as alterations in intestinal fluid transport (18Burleigh D.E. Borman R.A. Eur. J. Pharmacol. 1993; 241: 125Crossref PubMed Scopus (44) Google Scholar) and modulation of motility (19Bockaert J. Fozard J.R. Dumuis A. Clarke D.E. Trends Pharmacol. Sci. 1992; 13: 141Abstract Full Text PDF PubMed Scopus (239) Google Scholar). 5-HT seems to induce Cl- secretion by stimulating neural 5-HT3 receptors in the distal colon of guinea pig (20Cooke H.J. Wang Y.Z. Frieling T. Wood J.D. Am. J. Physiol. 1991; 261: G833PubMed Google Scholar) and rat small intestine (21Beubler E. Horina G. Gastroenterology. 1990; 99: 83Abstract Full Text PDF PubMed Scopus (116) Google Scholar) and non-neural 5-HT4 receptors in the rat colon (22Budhoo M.R. Kellum J.M. J. Surg. Res. 1994; 57: 44Abstract Full Text PDF PubMed Scopus (22) Google Scholar), human jejunum (13Kellum J.M. Budhoo M.R. Siriwardena A.K. Smith E.P. Jebraili S.A. Am. J. Physiol. 1994; 267: G357Crossref PubMed Google Scholar), and ileum (23Borman R.A. Burleigh D.E. Br. J. Pharmacol. 1993; 110: 927Crossref PubMed Scopus (56) Google Scholar). Recent binding studies have shown the presence of 5-HT4 receptors on the brush border membranes of the rabbit jejunal enterocytes (24Alcalde A.I. Sorribas V. Rodriguez-Yoldi M.J. Lahuerta A. Eur. J. Pharmacol. 2000; 403: 9Crossref PubMed Scopus (8) Google Scholar). Studies have also demonstrated the existence of 5-HT2 receptors on the basolateral membranes of epithelial cells from rabbit ileum (25Salvador M.T. Rodriguez-Yoldi M.C. Alcalde A.I. Rodriguez-Yoldi M.J. Life Sci. 1997; 61: 309Crossref PubMed Scopus (20) Google Scholar), human sigmoid colon (26Borman R.A. Burleigh D.E. Ann. N. Y. Acad. Sci. 1997; 812: 224Crossref PubMed Scopus (25) Google Scholar), and guinea pig small intestinal crypt cells (27Siriwardena A.K. Budhoo M.R. Smith E.P. Kellum J.M. J. Surg. Res. 1993; 55: 55Abstract Full Text PDF PubMed Scopus (31) Google Scholar). Although 5-HT has been known to play a major role in regulating a number of physiological functions in the GI tract, elevated levels of serotonin have been implicated in the patho-physiology of disease states such as carcinoid syndrome (7Hardcastle J. Hardcastle P.T. Redfern J.S. J. Physiol. (Lond.). 1981; 320: 41Crossref Scopus (70) Google Scholar), celiac disease (28Challacombe D.N. Dawkins P.D. Baker P. Gut. 1977; 18: 882Crossref PubMed Scopus (29) Google Scholar), irritable colon (29Kyosola K. Penttila O. Salaspuro M. Scand. J. Gastroenterol. 1977; 12: 363Crossref PubMed Scopus (93) Google Scholar), and dumping syndrome (30Thompson J.H. Res. Commun. Chem. Pathol. Pharmacol. 1971; 2: 687PubMed Google Scholar). Previous studies have shown that 5-HT administration in vivo induces Cl- secretion in the rabbit small intestine (31Donowitz M. Charney A.N. Heffernan J.M. Am. J. Physiol. 1977; 232: E85PubMed Google Scholar) and acts as a mediator of diarrhea in patients with carcinoid syndrome associated with a net secretion of water and electrolytes (32Donowitz M. Binder H.J. Am. J. Dig. Dis. 1975; 20: 1115Crossref PubMed Scopus (64) Google Scholar). Therefore, these studies suggest an important role of 5-HT in modifying electrolyte transport process that could involve either stimulation of Cl- secretion or a decrease in Na+ and Cl- absorption. However, to date, the effects of 5-HT on the human intestinal apical membrane Cl-/OH- exchange activity have not been investigated, and the signal transduction pathways involved in 5-HT-mediated alteration of NaCl absorption have not been delineated. In this regard, most of the effects of 5-HT on the intestinal ion transport have been shown to be mediated through various intracellular mediators such as phosphoinositides, Ca2+, and prostaglandins (33Hansen M.B. Skadhauge E. Comp. Biochem. Physiol A. 1997; 118: 283Crossref PubMed Scopus (66) Google Scholar). Activation of 5-HT4 receptors has been reported to change the levels of cAMP and cGMP (34Hoyer D. Schoeffter P. J. Recept. Res. 1991; 11: 197Crossref PubMed Scopus (196) Google Scholar). However, studies of Eklund et al. (35Eklund S. Brunsson I. Jodal M. Lundgren O. Acta Physiol. Scand. 1987; 129: 115Crossref PubMed Scopus (28) Google Scholar) and Donowitz et al. (31Donowitz M. Charney A.N. Heffernan J.M. Am. J. Physiol. 1977; 232: E85PubMed Google Scholar) have shown that cAMP plays no role in mediating the secretory effects of 5-HT in cat and rabbit intestine. Recent studies (36Stoner M.C. Scherr A.M. Lee J.A. Wolfe L.G. Kellum J.M. Surgery. 2000; 128: 240Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) point out the role of nitric oxide in mediating the effects of 5-HT-induced Cl- secretion in the rat colon. The current studies were therefore undertaken to examine the following: (i) the possible regulation of Cl-/OH- exchange activity in Caco-2 cells (a well established model for the human intestinal transport studies) by serotonin; (ii) to characterize the 5-HT receptor subtype(s) involved; and (iii) the signal transduction pathways involved in this process. Our current studies demonstrate that serotonin inhibits apical Cl-/OH- exchange activity in Caco-2 cells via both tyrosine kinase and protein kinase Cδ-mediated pathways involving either the 5-HT3 or 5-HT4 receptor subtype. Radionuclide 36Cl as HCl was obtained from PerkinElmer Life Sciences. Caco-2 cells were obtained from the ATCC (Manassas, VA). 5-Hydroxytryptamine-creatinine sulfate (5-HT, serotonin), 4,4′-diisothiocyanato-stilbene-2, 2′-disulfonic acid (DIDS), and tropisetron were obtained from Sigma. Specific agonists for 5-HT3, m-chlorophenylbiguanide, and 5-HT4, 3-(4-allylpiperazin-1-yl)-2-quinoxaline chloronitrile, specific antagonists for 5-HT3 (Y-25130) and 5-HT4 (RS39604), were obtained from TOCRIS (Ellisville, MO). BAPTA-AM was obtained from Molecular Probes (Eugene, Oregon). Chelerythrine chloride, calphostin C, herbimycin, and 4-amino-5-(4methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine were obtained from Biomol (Plymouth Meeting, PA). Ro318220, Go6796, and rottlerin were procured from Calbiochem. Affinity-purified rabbit polyclonal antibody against PKCδ, goat anti-rabbit antibody conjugated to horseradish peroxidase, and protein A-agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphorylated p60 (p-Src, Tyr416) and polyclonal anti-Lck were purchased from Cell Signaling (Cell Signaling Technology, Beverly, MA). Anti-phosphotyrosine (4G10) HRP-conjugated antibody, monoclonal anti-Src, anti-Fyn, or polyclonal anti-Yes and anti-Lyn were obtained from Upstate Biotechnology Inc. (Upstate Biotechnology, Lake Placid, NY). All other chemicals were of at least reagent grade and were obtained from Sigma or Fisher. Caco-2 cells were grown in Dulbecco's modified Eagle's medium supplemented with 4.5 g/liter glucose, 2 mm glutamine, 50 units/ml penicillin, 50 μg/ml streptomycin, 10 mm Hepes, 1% essential and nonessential amino acids, and 20% fetal bovine serum, pH 7.4, in 5% CO2, 95% O2 at 37 °C. For the uptake experiments, cells from passages between 20 and 25 were plated in 24-well plates at a density of 2 × 104 cells/ml. Confluent monolayers were used for transport experiments at the10th to the 12th day post-plating (i.e. 5-7 days post confluence). To study the effect of serotonin (5-HT) on Cl-/OH- exchange activity, cells were acutely exposed to 100 nm 5-HT for 15, 30, or 60 min. In a separate set of experiments, cells were pretreated with tropisetron (dual 5-HT3/5-HT4 antagonist, 1 μm) or 5-HT3 receptor antagonist Y25130 (300 nm), or 5-HT4 receptor antagonist RS39604 (300 nm), or Ca2+ chelator BAPTA-AM (20 μm), or the specific PKC inhibitors chelerythrine chloride (2 μm) and calphostin C (200 nm), or the specific PKCα inhibitor Go6976 (5 nm), the specific PKCϵ inhibitor Ro318220 (100 nm), the specific PKCδ inhibitor rottlerin (10 μm), or the general tyrosine kinase inhibitor herbimycin (1 μm) or the Src family kinase inhibitor PP1 (10 μm) for 1 h prior to the addition of serotonin (100 nm). These inhibitors were also co-incubated along with serotonin for another 1 h. In another set of experiments, cells were treated with specific 5-HT3 (m-chlorophenylbiguanide, 1-100 μm) and 5-HT4 (3-(4-allylpiperazin-1-yl)-2-quinoxaline chloronitrile, 0.1-10 μm) agonists for 1 h. Uptake Chloride uptake experiments were performed essentially as described by Olsnes et al. (37Olsnes S. Tonnessen T.I. Ludt J. Sandvig K. Biochemistry. 1987; 26: 2778Crossref PubMed Scopus (36) Google Scholar) with some modifications (38Saksena S. Gill R.K. Syed I.A. Tyagi S. Alrefai W.A. Ramaswamy K. Dudeja P.K. Am. J. Physiol. 2002; 283: C1492Crossref PubMed Google Scholar, 39Saksena S. Gill R.K. Syed I.A. Tyagi S. Alrefai W.A. Ramaswamy K. Dudeja P.K. Am. J. Physiol. 2002; 283: G626Crossref Scopus (28) Google Scholar). Caco-2 cells were incubated with Dulbecco's modified Eagle's base media containing 20 mm Hepes/KOH, pH 8.5, for 30 min at room temperature. The media were removed, and the cells were rapidly washed with 1 ml of tracer-free uptake mannitol buffer containing 260 mm mannitol, 20 mm Tris/MES, pH 7.0. The cells were then incubated with the uptake buffer for a 5-min period. This period was chosen because it falls within the linear range of Cl- uptake in this system (38Saksena S. Gill R.K. Syed I.A. Tyagi S. Alrefai W.A. Ramaswamy K. Dudeja P.K. Am. J. Physiol. 2002; 283: C1492Crossref PubMed Google Scholar, 39Saksena S. Gill R.K. Syed I.A. Tyagi S. Alrefai W.A. Ramaswamy K. Dudeja P.K. Am. J. Physiol. 2002; 283: G626Crossref Scopus (28) Google Scholar). For 36Cl- uptake studies, the uptake buffer was the mannitol buffer containing 1.4 μCi of 36Cl- (2.9 mm) of hydrochloric acid (specific activity-17.12 mCi/g) ± 0.3 mm DIDS. The uptake was terminated by removing the buffer and washing the cells rapidly two times with 1 ml of ice-cold phosphate-buffered saline, pH 7.2 (PBS). Finally, the cells were solubilized by incubation with 0.5 n NaOH for 4 h. The protein concentration was measured by the method of Bradford (40Bradford M.M. Anal. Biochem. 1976; 72: 248Crossref PubMed Scopus (217544) Google Scholar), and the radioactivity was counted by a Packard liquid scintillation analyzer, TRI-CARB 1600-TR (Packard Instrument Co.). 0.3 mm DIDS-sensitive chloride uptake was considered as Cl-/OH- exchange, and the uptake values were expressed as nmol/mg protein/5 min. Caco-2 cells grown to confluence in 6-well plates at a density of 10 × 104 (Corning Glass) were treated with serotonin (100 nm) for the 15-, 30-, and 60-min period or with the specific Src kinase inhibitor PP1 (10 μm). Cells were washed with ice-cold PBS three times and lysed in 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Triton X-100, 1 mm EDTA, 1 mm EGTA, and 1× complete protease inhibitor mixture. The cells were homogenized by passing 10 times through a 26-guage needle. The lysate was centrifuged at 5000 rpm for 5 min at 4 °C. Equal amounts (∼75 μg/sample) of protein, as determined by the Bradford assay, were combined with the SDS sample buffer and boiled for 5 min. Proteins were separated by electrophoresis on 8% SDS-polyacrylamide gels and transblotted to nitrocellulose membranes. The protein-bound nitrocellulose membranes were first incubated for 20 min at room temperature in blocking buffer containing 1× PBS and 3% nonfat dry milk. Nitrocellulose membranes were then incubated with the anti-phosphotyrosine HRP-conjugated antibody (1:1000 dilution) in the blocking buffer overnight at 4 °C. The membranes were rinsed in water and washed with the wash buffer containing 1× PBS and 0.05% Tween 20 for 45 min with agitation, during which time the wash buffer was changed every 5 min. Tyrosine-phosphorylated bands were visualized with ECL detection reagents. After treatment of Caco-2 cells with serotonin (100 nm) for different times or specific Src kinase inhibitor PP1 (10 μm), specific 5-HT3 receptor antagonist Y25130 (300 nm), 5-HT4 receptor antagonist RS39604 (300 nm), or addition of both 5HT3 and 5HT4 receptor antagonists, cells were washed with phosphate-buffered saline, and lysates were prepared as described above. The protein content of the resulting supernatant was adjusted to contain 500 μg in 200 μl of lysis buffer and incubated with the polyclonal antibody against novel PKCδ (2 μg) overnight at 4 °C. After incubation, immune complexes were precipitated by using protein A-agarose beads. In a separate set of experiments, 5-HT-treated lysates at different time points were incubated with monoclonal anti-c-Src or anti-Fyn or polyclonal anti-Yes, anti-Lck, or anti-Lyn overnight at 4 °C. After incubation, the immune complexes were precipitated using protein A- or G-agarose beads. The immunoprecipitates were collected by centrifugation and washed four times with the lysis buffer. Proteins were separated by electrophoresis on 8 or 12% SDS-polyacrylamide gels and transblotted to nitrocellulose membranes. To detect PKCδ phosphorylation, the protein-bound nitrocellulose membranes were incubated with anti-phosphotyrosine-HRP-conjugated antibody, and bands were visualized with the ECL method. To detect the phosphorylation of each of the Src family members, the protein-bound nitrocellulose membranes were first incubated for 60 min at room temperature in blocking buffer containing 1× TBS (Tris-buffered saline), 0.1% Tween 20 with 5% nonfat dry milk. Nitrocellulose membranes were then incubated with the anti-phospho-Src Tyr416 (1:1000 dilution) in the dilution buffer containing 1× TBS, 0.1% Tween 20 with 5% bovine serum albumin overnight at 4 °C. The membranes were washed with the wash buffer containing 1× TBS and 0.1% Tween 20 for 15 min with agitation, during which time the wash buffer was changed every 5 min. Finally, the membranes were probed with HRP-conjugated goat anti-rabbit IgG antibody (1:2000 dilution), and bands were visualized with ECL detection reagents. Subcellular Fractionation—Caco-2 cells grown to confluence in 6-well plates at a density of 10 × 104 (Corning Glass) were treated with serotonin (100 nm) for different times, washed with ice-cold PBS three times, and scraped into 400 μl of the cold homogenization buffer (HB) containing 20 mm Tris-HCl, pH 7.5, 250 mm sucrose, 4 mm EDTA, 2 mm EGTA, and 1× complete protease inhibitor mixture. The cells were homogenized on ice with 25 strokes of a glass tissue homogenizer. The resulting homogenate was ultracentrifuged at 59,000 rpm for 50 min at 4 °C (Optima™ TLX Ultracentrifuge; Beckman Instruments). The supernatant was designated the cytosolic fraction. The pellet was resuspended in 150 μl of the HB containing 0.5% (v/v) Triton X-100 by brief sonication and incubated on ice for 30 min. At the end of the incubation period, the samples were centrifuged at 14,000 rpm for 20 min at 4 °C. The resulting supernatant was designated the membrane fraction. Gel Electrophoresis and Western Blotting—Equal amounts of protein (∼75 μg/sample), as determined by the Bradford assay, were combined with Laemmli's Sample Buffer containing 5% (v/v) β-mercaptoethanol and then boiled for 5 min. Proteins were separated by electrophoresis on 8% SDS-polyacrylamide gels and trans-blotted to nitrocellulose membranes. The protein-bound nitrocellulose membranes were first incubated for 1 h at room temperature in blocking buffer containing 1× PBS, 0.1% Tween 20, and 5% nonfat dry milk. Nitrocellulose membranes were then incubated with the polyclonal antibody specific to PKCδ (1:800 dilution) in the blocking buffer containing 1× PBS, 0.1% Tween 20, and 1% nonfat dry milk for 1 h at room temperature and rinsed for 30 min with a wash buffer containing 1× PBS and 0.1% Tween 20. Finally, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG antibody (1:2000 dilution) for 1 h at room temperature and washed for 45 min with agitation, during which the wash buffer was changed every 5 min. PKC bands were visualized with ECL detection reagent. Results were expressed as mean ± S.E. Each independent set represents mean ± S.E. of data from at least 9 wells used on three separate occasions. Student's t test was used for statistical analysis. p < 0.05 or less was considered statistically significant. To examine the effects of 5-HT on the Cl-/OH- exchange activity, Caco-2 cells were incubated with 5-HT at a concentration of 100 nm for different time points, and the DIDS-sensitive Cl-/OH- exchange activity was assessed. As shown in Fig. 1, a significant decrease (∼60%) in Cl-/OH- exchange activity was observed as early as 15 min, and the inhibition remained about the same at 30 and 60 min. Effect of Tropisetron (Dual 5-HT3/4 Receptor Antagonist)—The receptor subtype mediating the 5-HT-induced inhibition of Cl-/OH- exchange activity was characterized with the use of the 5-HT receptor antagonist tropisetron. When employed at a final concentration of 0.1 μm (concentration at which it is a selective 5-HT3 receptor antagonist), it had no effect on the 5-HT-mediated inhibition of Cl-/OH- exchange activity (data not shown). However, when used at a concentration of 1 μm (1 h), at which it is known to antagonize the 5-HT4 as well as the 5-HT3 receptors, tropisetron caused a significant reversal in the 5-HT-mediated inhibition of Cl-/OH- exchange activity (Fig. 2A). Because tropisetron is characterized as a weak competitive 5-HT4 antagonist (having higher affinity for the 5-HT3 receptor), we therefore used more specific and selective individual receptor antagonists. Effect of 5-HT Receptor Antagonists—The specific 5-HT3 receptor antagonist Y25130 (300 nm) failed to block the 5-HT-induced inhibition of Cl-/OH- exchange activity in Caco-2 cells (Fig. 2B), indicating that if 5-HT3 receptor is blocked, 5-HT can still mediate its effects through the 5-HT4 receptor. Similar results were observed with the selective 5-HT4 receptor antagonist RS39604 (300 nm, 1000-fold selectivity over 5-HT3 receptor) (41Hegde S.S. Bonhaus D.W. Johnson L.G. Leung E. Clark R.D. Eglen R.M. Br. J. Pharmacol. 1995; 115: 1087Crossref PubMed Scopus (55) Google Scholar) (Fig. 2C). Studies were also performed in the presence of the 5-HT3 (Y25130) and 5-HT4 (RS39604) receptor antagonists together on the 5-HT-induced inhibition of Cl-/OH- exchange activity in Caco-2 cells. The inhibitory effects of 5-HT were significantly abolished in the presence of both the 5-HT3 and 5-HT4 antagonists added together (Fig. 2D). Hence, the observed findings with the dual and individual receptor antagonists suggest that either of the 5-HT3 or 5-HT4 receptor subtypes could be sufficient for 5-HT-mediated inhibition of Cl-/OH- exchange activity in Caco-2 cells. When 5-HT3 receptor is blocked, 5-HT could act through the 5-HT4 receptor, and when the 5-HT4 receptor is blocked, 5-HT could act via 5-HT3 receptor. To confirm further that both 5-HT3 and 5-HT4 receptor subtypes are involved in this process, specific 5-HT3, m-chlorophenylbiguanide (1-100 μm, 1 h), and 5-HT4, 3-(4-allylpiperazin-1-yl)-2-quinoxaline chloronitrile (0.1-10 μm, 1 h), agonists were used. Both 5-HT3 (Fig. 3A) and 5-HT4 (Fig. 3B) agonists significantly decreased the Cl-/OH- exchange activity in Caco-2 cells. These results indicate that 5-HT mediates its effects via activation of either 5-HT3 or 5-HT4 receptor as both 5-HT3 and 5-HT4 agonists could mimic the effects of 5-HT. Moreover, no additive effects were observed when both the 5HT3 and 5HT4 agonists were added together (75 ± 2% as compared with control). Previous studies have suggested that the effects of 5-HT on NaCl absorption (10Donowitz M. Tai Y.H. Asarkof N. Am. J. Physiol. 1980; 239: G463PubMed Google Scholar) and Cl- secretion (42Bolton J.E. Field M. J. Membr. Biol. 1977; 35: 159Crossref PubMed Scopus (126) Google Scholar) are mediated by an increase in the intracellular calcium levels. We thus examined the role of calcium in mediating the effects of 5-HT by using the Ca2+ chelator BAPTA-AM (20 μm, 1 h). As shown in Table I, in the presence of BAPTA-AM there was essentially no effect on the 5-HT-mediated inhibition of Cl-/OH- exchange activity. However, previous findings from our laboratory demonstrated that exactly under the same experimental conditions, BAPTA-AM (20 μm, 1 h) significantly blocked the Ca2+-dependent effects of serotonin on Na+/H+ exchange activity (43Gill R.K. Tyagi S. Alrefai W.A. Malakooti J. Ramaswamy K. Dudeja P.K. Gastroenterology. 2005; (in press)PubMed Google Scholar) and the H2O2 effects on Cl-/OH- exchange activity (44Saksena S. Tyagi S. Syed I.A. Alrefai W.A. Ramaswamy K. Dudeja P.K. Gastroenterology. 2003; 124: A143Abstract Full Text PDF Google Scholar) in Caco-2 cells. Collectively, these observations suggest that the inhibitory effects of 5-HT on the Cl-/OH- exchange activity in Caco-2 cells are not related to the intracellular calcium levels and further confirm that BAPTA-AM is an optimal chelating agent of c" @default.
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• W2015176407 title "Involvement of c-Src and Protein Kinase Cδ in the Inhibition of Cl-/OH- Exchange Activity in Caco-2 Cells by Serotonin" @default.
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