Fluid flow induces mechanosensitive ATP release, calcium signalling and Cl- transport in biliary epithelial cells through a PKCzeta-dependent pathway.
ABSTRACT ATP in bile is a potent secretogogue, stimulating cholangiocyte Cl- and fluid secretion via binding to membrane P2 receptors, though the physiological stimuli involved in biliary ATP release are unknown. The goal of the present studies was to determine the potential role of fluid flow in biliary ATP release and secretion. In both human Mz-Cha-1 biliary cells and normal rat cholangiocyte monolayers, exposure to flow increased relative ATP release which was proportional to the shear stress. In parallel studies, shear was associated with an increase in [Ca2+]i and membrane Cl- permeability, which were both dependent on extracellular ATP and P2 receptor stimulation. Flow-stimulated ATP release was dependent on [Ca2+]i, exhibited desensitization with repetitive stimulation, and was regulated by PKCzeta. In conclusion, both human and rat biliary cells exhibit flow-stimulated, PKCzeta-dependent, ATP release, increases in [Ca2+]i and Cl- secretion. The finding that fluid flow can regulate membrane transport suggests that mechanosensitive ATP release may be a key regulator of biliary secretion and an important target to modulate bile flow in the treatment of cholestatic liver diseases.
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.
Article: Rat cholangiocytes absorb bile acids at their apical domain via the ileal sodium-dependent bile acid transporter.[show abstract] [hide abstract]
ABSTRACT: Although bile acid transport by bile duct epithelial cells, or cholangiocytes, has been postulated, the details of this process remain unclear. Thus, we performed transport studies with [3H]taurocholate in confluent polarized monolayers of normal rat cholangiocytes (NRC). We observed unidirectional (i.e., apical to basolateral) Na+-dependent transcellular transport of [3H]taurocholate. Kinetic studies in purified vesicles derived from the apical domain of NRC disclosed saturable Na+-dependent uptake of [3H]taurocholate, with apparent Km and Vmax values of 209+/-45 microM and 1.23+/-0.14 nmol/mg/10 s, respectively. Reverse transcriptase PCR (RT-PCR) using degenerate primers for both the rat liver Na+-dependent taurocholate-cotransporting polypeptide and rat ileal apical Na+-dependent bile acid transporter, designated Ntcp and ASBT, respectively, revealed a 206-bp product in NRC whose sequence was identical to the ASBT. Northern blot analysis demonstrated that the size of the ASBT transcript was identical in NRC, freshly isolated cholangiocytes, and terminal ileum. In situ RT-PCR on normal rat liver showed that the message for ASBT was present only in cholangiocytes. Immunoblots using a well-characterized antibody for the ASBT demonstrated a 48-kD protein present only in apical membranes. Indirect immunohistochemistry revealed apical localization of ASBT in cholangiocytes in normal rat liver. The data provide direct evidence that conjugated bile acids are taken up at the apical domain of cholangiocytes via the ASBT, and are consistent with the notion that cholangiocyte physiology may be directly influenced by bile acids.Journal of Clinical Investigation 01/1998; 100(11):2714-21. · 15.39 Impact Factor