Extracellular nucleotides stimulate Cl- currents in biliary epithelia through receptor-mediated IP3 and Ca2+ release.
ABSTRACT 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.
Article: p38 MAP kinase modulates liver cell volume through inhibition of membrane Na+ permeability.[show abstract] [hide abstract]
ABSTRACT: In hepatocytes, Na+ influx through nonselective cation (NSC) channels represents a key point for regulation of cell volume. Under basal conditions, channels are closed, but both physiologic and pathologic stimuli lead to a large increase in Na+ and water influx. Since osmotic stimuli also activate mitogen-activated protein (MAP) kinase pathways, we have examined regulation of Na+ permeability and cell volume by MAP kinases in an HTC liver cell model. Under isotonic conditions, there was constitutive activity of p38 MAP kinase that was selectively inhibited by SB203580. Decreases in cell volume caused by hypertonic exposure had no effect on p38, but increases in cell volume caused by hypotonic exposure increased p38 activity tenfold. Na+ currents were small when cells were in isotonic media but could be increased by inhibiting constitutive p38 MAP kinase, thereby increasing cell volume. To evaluate the potential inhibitory role of p38 more directly, cells were dialyzed with recombinant p38alpha and its upstream activator, MEK-6, which substantially inhibited volume-sensitive currents. These findings indicate that constitutive p38 activity contributes to the low Na+ permeability necessary for maintenance of cell volume, and that recombinant p38 negatively modulates the set point for volume-sensitive channel opening. Thus, functional interactions between p38 MAP kinase and ion channels may represent an important target for modifying volume-sensitive liver functions.Journal of Clinical Investigation 12/2001; 108(10):1495-504. · 15.39 Impact Factor
Article: Nucleotide receptors activate cation, potassium, and chloride currents in a liver cell line.[show abstract] [hide abstract]
ABSTRACT: By use of whole cell patch-clamp techniques, the effects of extracellular ATP on membrane ion currents of HTC cells from a rat liver tumor line were evaluated. ATP (500 microM) or the nonhydrolyzable analogue adenosine 5'-O-(3-thiotriphosphate) caused sequential activation of three currents: Icat (-1,325 +/- 255 pA at -80 mV) occurred early, was due to increased Na+ and K+ permeability, was present in 56% of 64 consecutive cells, and rapidly inactivated; IK (274 +/- 45 pA at 0 mV) was present in 59% of cells and also inactivated; and ICl (1,172 +/- 237 pA at +60 mV) was present in 94% of studies, was sustained, and exhibited outward rectification of the current-voltage relation. All three currents were present in 39% of cells. Increasing intracellular Ca2+ concentration ([Ca2+]i) by exposure to the 5'-nucleotide receptor agonist UTP (500 microM) or to thapsigargin activated Icat and IK but not ICl, whereas increasing ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in the pipette (> or = 5 mM) inhibited ATP-dependent activation of Icat and IK but not ICl. A P2x-preferring agonist alpha, beta-methylene ATP (500 microM) did not activate currents; a P2y-preferring agonist 2-methylthioadenosine triphosphate activated Icat and IK at concentrations of 500 microM but not 50 microM. In perforated patch recordings, ATP produced triphasic changes in membrane potential with initial depolarization due to Icat, subsequent hyperpolarization due to IK, and a later sustained depolarization due to ICl. These findings indicate that ATP modulates HTC cell ion permeability through initial activation of Icat and IK mediated by 5'-nucleotide receptors which mobilize [Ca2+], and sustained activation of ICl through a separate Ca(2+)-independent mechanism.The American journal of physiology 05/1994; 266(4 Pt 1):G544-53.