Involvement of c-Src and protein kinase C delta in the inhibition of Cl(-)/OH- exchange activity in Caco-2 cells by serotonin.
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 Ca(2+)-independent protein kinase C delta-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 microM) 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 PKC delta, 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 PKC delta.
Saksena
Seema
S
Section of Digestive Diseases and Nutrition, Department of Medicine, University of Illinois and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois 60612, USA.
Gill
Ravinder K
RK
Tyagi
Sangeeta
S
Alrefai
Waddah A
WA
Sarwar
Zaheer
Z
Ramaswamy
Krishnamurthy
K
Dudeja
Pradeep K
PK
D016227:Benzoquinones; D000081247:CSK Tyrosine-Protein Kinase; D018938:Caco-2 Cells; D027963:Chloride-Bicarbonate Antiporters / Q000037:antagonists & inhibitors*; D002712:Chlorides / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D047029:Lactams, Macrocyclic; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology*; D051745:Protein Kinase C-delta; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011809:Quinones / Q000494:pharmacology; D011985:Receptors, Serotonin / Q000145:classification / Q000502:physiology; D017828:Rifabutin / Q000031:analogs & derivatives; D012701:Serotonin / Q000494:pharmacology*; D012702:Serotonin Antagonists / Q000494:pharmacology; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000502:physiology; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D016227:Benzoquinones; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides; D047029:Lactams, Macrocyclic; D011720:Pyrazoles; D011743:Pyrimidines; D011809:Quinones; D011985:Receptors, Serotonin; D012702:Serotonin Antagonists; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D017828:Rifabutin; D012701:Serotonin; D014443:Tyrosine; D004533:Egtazic Acid; C020754:herbimycin; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D017868:Cyclic AMP-Dependent Protein Kinases; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta