tion of biliary secretion through apical purinergic receptors in cultured rat cholangiocytes. Am. J. Physiol. 273 (Gastrointest. Liver Physiol. 36): G1108-G1117, 1997.-To evaluate whether ATP in bile serves as a signaling factor regulating ductular secretion, voltage-clamp studies were performed using a novel normal rat cholangiocyte (NRC) model. In the presence of amiloride (100 µM) to block Na ϩ channels, exposure of the apical membrane to ATP significantly increased the shortcircuit current (I sc ) from 18.2 Ϯ 5.9 to 52.8 Ϯ 12.7 µA (n ϭ 18). The response to ATP is mediated by basolateral-to-apical Cl Ϫ transport because it is inhibited by 1) the Cl Ϫ channel blockers 4,4Ј-diisothiocyanostilbene-2,2Ј-disulfonic acid (1 mM), diphenylanthranilic acid (1.5 mM), or 5-nitro-2-(3-phenylpropylamino)benzoic acid (50 or 100 µM) in the apical chamber, 2) the K ϩ channel blocker Ba 2ϩ (5 mM), or 3) the Na ϩ -K ϩ -2Cl Ϫ cotransport inhibitor bumetanide (200 µM) in the basolateral chamber. Other nucleotides stimulated an increase in I sc with a rank order potency of UTP ϭ ATP ϭ adenosine 5Ј-O-(3)-thiotriphosphate, consistent with P 2u purinergic receptors. ADP, AMP, 2-methylthioadenosine 5Ј-triphosphate, and adenosine had no effect. A cDNA encoding a rat P 2u receptor (rP 2u R) was isolated from a liver cDNA library, and functional expression of the corresponding mRNA in Xenopus laevis oocytes resulted in the appearance of ATP-stimulated currents with a similar pharmacological profile. Northern analysis identified hybridizing mRNA transcripts in NRC as well as other cell types in rat liver. These findings indicate that exposure of polarized cholangiocytes to ATP results in luminal Cl Ϫ secretion through activation of P 2u receptors in the apical membrane. Release of ATP into bile may serve as an autocrine or paracrine signal regulating cholangiocyte secretory function.

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Biliary epithelial cells contribute to bile formation through absorption and secretion of fluid and electrolytes. Recent studies indicate that membrane Cl- permeability is regulated in part by increases in intracellular Ca2+ concentration. The purpose of these studies was to evaluate the effects of intracellular Ca2+ on channel activity, using the human Mz-ChA-1 cholangiocarcinoma cell line as a model, and to assess the possible roles of Ca(2+)-dependent kinases in channel regulation. Exposure to ionomycin (1 microM) activated ion channels in the cell-attached configuration in 63 of 74 attempts, increasing open probability (NPo) from 0 to 0.26 +/- 0.15 (n = 17). Multiple channels were present in each patch, and the effects of ionomycin were reversed by subsequent addition of ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'- tetraacetic acid (2 mM) to the bath. With Cl(-)-containing solutions, channels had a slope conductance of 14 +/- 4 pS (n = 11), and the mean open time was estimated to be 5.3 +/- 1.8 ms. These channels were anion selective, and currents were carried by efflux of Cl- at the resting potential. Exposure to the Ca2+/calmodulin-dependent protein kinase II (CaMKII) antagonist calmidazolium (100 microM) decreased NPo in ionomycin-stimulated cells to 0.02 +/- 0.06 (n = 19). The protein kinase C antagonist chelerythrine (50 microM) was without effect. In parallel studies in subconfluent cell monolayers, CaMKII antagonists were also potent inhibitors of ionomycin-stimulated 125I efflux. These findings indicate that Ca(2+)-dependent increases in membrane Cl- permeability are related in part to opening of 14.pS anion channels through a mechanism that depends on both Ca2+ and CaMKII. These channels represent a potential target for pharmacological modulation of biliary cell transport and function.