Extracellular ATP regulates bile formation by binding to P2 receptors on cholangiocytes and stimulating transepithelial Cl(-) secretion. However, the specific signaling pathways linking receptor binding to Cl(-) channel activation are not known. Consequently, the aim of these studies in human Mz-Cha-1 biliary cells and normal rat cholangiocyte monolayers was to assess the intracellular pathways responsible for ATP-stimulated increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) and membrane Cl(-) permeability. Exposure of cells to ATP resulted in a rapid increase in [Ca(2+)](i) and activation of membrane Cl(-) currents; both responses were abolished by prior depletion of intracellular Ca(2+). ATP-stimulated Cl(-) currents demonstrated mild outward rectification, reversal at E(Cl(-)), and a single-channel conductance of approximately 17 pS, where E is the equilibrium potential. The conductance response to ATP was inhibited by the Cl(-) channel inhibitors NPPB and DIDS but not the CFTR inhibitor CFTR(inh)-172. Both ATP-stimulated increases in [Ca(2+)](i) and Cl(-) channel activity were inhibited by the P2Y receptor antagonist suramin. The PLC inhibitor U73122 and the inositol 1,4,5-triphosphate (IP3) receptor inhibitor 2-APB both blocked the ATP-stimulated increase in [Ca(2+)](i) and membrane Cl(-) currents. Intracellular dialysis with purified IP3 activated Cl(-) currents with identical properties to those activated by ATP. Exposure of normal rat cholangiocyte monolayers to ATP increased short-circuit currents (I(sc)), reflecting transepithelial secretion. The I(sc) was unaffected by CFTR(inh)-172 but was significantly inhibited by U73122 or 2-APB. In summary, these findings indicate that the apical P2Y-IP3 receptor signaling complex is a dominant pathway mediating biliary epithelial Cl(-) transport and, therefore, may represent a potential target for increasing secretion in the treatment of cholestatic liver disease.
[Show abstract][Hide abstract] ABSTRACT: Purines appear to be the most primitive and widespread chemical messengers in the animal and plant kingdoms. The evidence for purinergic signalling in plants, invertebrates and lower vertebrates is reviewed. Much is based on pharmacological studies, but important recent studies have utilized the techniques of molecular biology and receptors have been cloned and characterized in primitive invertebrates, including the social amoeba Dictyostelium and the platyhelminth Schistosoma, as well as the green algae Ostreococcus, which resemble P2X receptors identified in mammals. This suggests that contrary to earlier speculations, P2X ion channel receptors appeared early in evolution, while G protein-coupled P1 and P2Y receptors were introduced either at the same time or perhaps even later. The absence of gene coding for P2X receptors in some animal groups [e.g. in some insects, roundworms (Caenorhabditis elegans) and the plant Arabidopsis] in contrast to the potent pharmacological actions of nucleotides in the same species, suggests that novel receptors are still to be discovered.
[Show abstract][Hide abstract] ABSTRACT: In the liver, adenosine triphosphate (ATP) is an extracellular signaling molecule that is released into bile and stimulates a biliary epithelial cell secretory response via engagement of apical P2 receptors. The molecular identities of the ion channels involved in ATP-mediated secretory responses have not been fully identified. Intermediate-conductance Ca(2+)-activated K(+) channels (IK) have been identified in biliary epithelium, but functional data are lacking. The aim of these studies therefore was to determine the location, function, and regulation of IK channels in biliary epithelial cells and to determine their potential contribution to ATP-stimulated secretion. Expression of IK-1 mRNA was found in both human Mz-Cha-1 biliary cells and polarized normal rat cholangiocyte (NRC) monolayers, and immunostaining revealed membrane localization with a predominant basolateral signal. In single Mz-Cha-1 cells, exposure to ATP activated K(+) currents, increasing current density from 1.6 +/- 0.1 to 7.6 +/- 0.8 pA/pF. Currents were dependent on intracellular Ca(2+) and sensitive to clotrimazole and TRAM-34 (specific IK channel inhibitors). Single-channel recording demonstrated that clotrimazole-sensitive K(+) currents had a unitary conductance of 46.2 +/- 1.5 pS, consistent with IK channels. In separate studies, 1-EBIO (an IK activator) stimulated K(+) currents in single cells that were inhibited by clotrimazole. In polarized NRC monolayers, ATP significantly increased transepithelial secretion which was inhibited by clotrimazole. Lastly, ATP-stimulated K(+) currents were inhibited by the P2Y receptor antagonist suramin and by the inositol 1,4,5-triphosphate (IP3) receptor inhibitor 2-APB. Together these studies demonstrate that IK channels are present in biliary epithelial cells and contribute to ATP-stimulated secretion through a P2Y-IP3 receptor pathway.
[Show abstract][Hide abstract] ABSTRACT: Bicarbonate secretion from cholangiocytes is required for appropriate adjustment of primary canalicular bile along the biliary tract. In human and rat cholangiocytes, bicarbonate secretion is mediated by anion exchanger (AE) 2, an electroneutral Na(+)-independent Cl(-)/HCO(3) (-) AE also involved in intracellular pH (pH(i)) regulation. In Ae2(a,b)-deficient mice, pH(i) is increased in lymphocytes and fibroblasts, whereas it is surprisingly normal in cholangiocytes. Here, we analyze the mechanisms for HCO(3) (-) secretion in cultured Ae2(a,b) (+/+) and Ae2(a,b) (-/-) mouse cholangiocytes by microfluorimetric measurement of pH(i) changes upon established perfusion maneuvers. Cl(-) withdrawal by isethionate-based perfusions showed that Ae2(a,b) (+/+) but not Ae2(a,b) (-/-) mouse cholangiocytes can display Cl(-)/HCO(3) (-) exchange, which is therefore entirely mediated by Ae2. Nevertheless, simultaneous withdrawal of Cl(-) and Na(+) revealed that mouse cholangiocytes possess an additional transport activity for HCO(3) (-) secretion not observed in control rat cholangiocytes. Propionate-based maneuvers indicated that this supplemental Na(+)-driven HCO(3) (-)-secreting activity is Cl(-)-independent, consistent with a Na(+)-HCO(3) (-) cotransport (NBC). NBC activity is greater in Ae2(a,b) (-/-) than Ae2(a,b) (+/+) mouse cholangiocytes, and membrane-depolarization experiments showed that it is electrogenic. Consistent with the potential role of Slc4a4/Nbc1 as the involved transporter, Ae2(a,b) (-/-) mouse cholangiocytes exhibit up-regulated expression of this electrogenic NBC carrier. Whereas Ae2-mediated Cl(-)/HCO(3) (-) exchange in Ae2(a,b) (+/+) mouse cholangiocytes is stimulated by cyclic adenosine monophosphate (cAMP) and acetylcholine, the NBC activity is down-regulated by cAMP and adenosine triphosphate (ATP) in Ae2(a,b) (-/-) mouse cholangiocytes. Polarized Ae2(a,b) (-/-) mouse cholangiocytes placed in Ussing chambers show decreased (but not abolished) cAMP-dependent Cl(-) current and increased ATP-dependent/Ca(2+)-activated Cl(-) secretion, which run in parallel with decreased cystic fibrosis transmembrane conductance regulator messenger RNA expression and increased intracellular Ca(2+) levels. Conclusion: Bicarbonate secretion in mouse cholangiocytes involves two differentially regulated activities: Ae2-mediated Cl(-)/HCO(3) (-) exchange and Na(+)-HCO(3) (-) cotransport.
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