Extracellular ATP represents an important autocrine/paracrine signaling molecule within the liver. The mechanisms responsible for ATP release are unknown, and alternative pathways have been proposed, including either conductive ATP movement through channels or exocytosis of ATP-enriched vesicles, although direct evidence from liver cells has been lacking. Utilizing dynamic imaging modalities (confocal and total internal reflection fluorescence microscopy and luminescence detection utilizing a high sensitivity CCD camera) at different scales, including confluent cell populations, single cells, and the intracellular submembrane space, we have demonstrated in a model liver cell line that (i) ATP release is not uniform but reflects point source release by a defined subset of cells; (ii) ATP within cells is localized to discrete zones of high intensity that are ϳ1 m in diameter, suggesting a vesicular localization; (iii) these vesicles originate from a bafilomycin A 1 -sensitive pool, are depleted by hypotonic exposure, and are not rapidly replenished from recycling of endocytic vesicles; and (iv) exocytosis of vesicles in response to cell volume changes depends upon a complex series of signaling events that requires intact microtubules as well as phosphoinositide 3-kinase and protein kinase C. Collectively, these findings are most consistent with an essential role for exocytosis in regulated release of ATP and initiation of purinergic signaling in liver cells.Extracellular ATP functions within liver as a key autocrine/ paracrine signaling molecule. The purinergic signaling cascade is initiated by release of ATP from intracellular stores, but the mechanisms involved are poorly understood. However, P2 receptors are expressed on all liver cell types, and once outside of the cell ATP has multiple effects, including (i) coordination within the liver lobule of cell-to-cell [Ca 2ϩ ] i signaling (1), (ii) maintenance of cell volume within a narrow physiological range (2), and (iii) coupling of the separate hepatocyte and cholangiocyte contributions to bile formation and stimulation of biliary secretion (3). Specifically, cellular ATP release leads to increased concentrations of ATP in bile sufficient to activate P2 receptors in the apical membrane of targeted cholangiocytes, resulting in a robust secretory response through activation of Cl Ϫ channels in the apical membrane. Moreover, multiple signals including intracellular calcium, cAMP and bile acids appear to coordinate ATP release, which has been recognized recently as a final common pathway responsible for biliary secretion (3-5). Accordingly, definition of the mechanisms involved in ATP release represents a key focus for efforts to modulate liver function and the volume and composition of bile.Previous studies indicate that increases in cell volume serve as a potent stimulus for physiologic ATP release in many epithelia and in liver cells increase extracellular nucleotide concentrations 5-10-fold (6). Two broad models for ATP release by nonexcitatory cells have been...
Bile formation by the liver is initiated by canalicular transport at the hepatocyte membrane, leading to an increase in ductular bile flow. Thus, bile duct epithelial cells (cholangiocytes), which contribute to the volume and dilution of bile through regulated Cl(-) transport, are exposed to changes in flow and shear force at the apical membrane. The aim of the present study was to determine if fluid flow, or shear stress, is a signal regulating cholangiocyte transport. The results demonstrate that, in human and mouse biliary cells, fluid flow, or shear, increases Cl(-) currents and identify TMEM16A, a Ca(2+)-activated Cl(-) channel, as the operative channel. Furthermore, activation of TMEM16A by flow is dependent on PKCα through a process involving extracellular ATP, binding purinergic P2 receptors, and increases in intracellular Ca(2+) concentration. These studies represent the initial characterization of mechanosensitive Cl(-) currents mediated by TMEM16A. Identification of this novel mechanosensitive secretory pathway provides new insight into bile formation and suggests new therapeutic targets to enhance bile formation in the treatment of cholestatic liver disorders.
Bile acids stimulate a bicarbonate‐rich choleresis, in part, through effects on cholangiocytes. Because Cl− channels in the apical membrane of cholangiocytes provide the driving force for secretion and transmembrane member 16A (TMEM16A) has been identified as the Ca2+‐activated Cl− channel in the apical membrane of cholangiocytes, the aim of the present study was to determine whether TMEM16A is the target of bile‐acid–stimulated Cl− secretion and to identify the regulatory pathway involved. In these studies of mouse, rat, and human biliary epithelium exposure to ursodeoxycholic acid (UDCA) or tauroursodeoxycholic acid (TUDCA) rapidly increased the rate of exocytosis, ATP release, [Ca2+]i, membrane Cl− permeability, and transepithelial secretion. Bile‐acid–stimulated Cl− currents demonstrated biophysical properties consistent with TMEM16A and were inhibited by pharmacological or molecular (small‐interfering RNA; siRNA) inhibition of TMEM16A. Bile acid–stimulated Cl− currents were not observed in the presence of apyrase, suramin, or 2‐aminoethoxydiphenyl borate (2‐APB), demonstrating that current activation requires extracellular ATP, P2Y, and inositol 1,4,5‐trisphosphate (IP3) receptors. TUDCA did not activate Cl− currents during pharmacologic inhibition of the apical Na+‐dependent bile acid transporter (ASBT), but direct intracellular delivery of TUDCA rapidly activated Cl− currents. Conclusion: Bile acids stimulate Cl− secretion in mouse and human biliary cells through activation of membrane TMEM16A channels in a process regulated by extracellular ATP and [Ca2+]i. These studies suggest that TMEM16A channels may be targets to increase bile flow during cholestasis. (Hepatology 2018;68:187‐199).
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.