Vesicular exocytosis contributes to volume-sensitive ATP release in biliary cells

Abstract

Extracellular ATP is a potent autocrine/paracrine signal that regulates a broad range of liver functions through activation of purinergic receptors. In biliary epithelium, increases in cell volume stimulate ATP release through a phosphoinositide 3-kinase (PI3-kinase)-dependent mechanism. Because PI3-kinase also regulates vesicular exocytosis, the purpose of these studies was to determine whether volume-stimulated vesicular exocytosis contributes to cellular ATP release. In a human cholangiocarcinoma cell line, exocytosis was measured by using the plasma membrane marker FM1-43, whereas ATP release was assessed by using a luciferase-luciferin assay. Under basal conditions, cholangiocytes exhibited constitutive exocytosis at a rate of 1.6%/min, and low levels of extracellular ATP were detected at 48.2 arbitrary light units. Increases in cholangiocyte cell volume induced by hypotonic exposure resulted in a 10-fold increase in the rate of exocytosis and a robust 35-fold increase in ATP release. Both vesicular exocytosis and ATP release were proportional to cell volume, and both exhibited similar regulatory properties including: 1) dependence on intact PI3-kinase, 2) attenuation by inhibition of PKC, and 3) potentiation by activation of PKC before hypotonic exposure. These findings demonstrate that increases in cholangiocyte cell volume stimulate ATP release and vesicular exocytosis through similar regulatory paradigms. Functional interactions among cell volume, PKC, and PI3-kinase modulate exocytosis, thereby regulating ATP release and purinergic signaling in cholangiocytes. It is hypothesized that PKC is involved in the recruitment of a volume-sensitive vesicular pool to a readily releasable state.

P2X4 Activation Modulates Volume-sensitive Outwardly Rectifying Chloride Channels in Rat Hepatoma Cells

“…The main ones proposed comprise exocytosis (42) and ATP 4Ϫ translocation via VSOR Cl Ϫ channels at high positive voltages (50). However, as no ATP release was detected in cells stimulated with 200 M H 2 O 2 (a concentration twice the EC 50 for VSOR Cl Ϫ channel activation (22)), our results suggest that ATP is not released through VSOR Cl Ϫ channels.…”

“…Because the amount of ATP released from swollen cells has been found to be dependent on the magnitude of cell swelling (33,42), we explored the role of P2X4 in VSOR Cl Ϫ channel activity at a smaller hypotonic challenge. As depicted in Fig.…”

Evidence for sustained ATP release from liver cells that is not mediated by vesicular exocytosis

“…Thus, it is not known whether vesicles mobilized by hypotonic solution contain functional ATP transporters in their membranes. It is interesting to note that by using FM1-43 fluorescence, recent studies have shown that inhibition of PKC or PI 3-kinase in biliary cells blocks both vesicular exocytosis and ATP release [21]. These studies indicate that vesicular exocytosis may contribute to ATP release from biliary cells.…”

“…More recent studies have found that pannexin1 hemichannels play a key role in ATP release from many different cells [15][16][17][18]. Vesicular exocytosis may also contribute to ATP release [19][20][21]. Under basal conditions, the concentration of ATP in extracellular media is in a low nanomolar range.…”

“…Thus, FM1-43 fluorescence provides a measure of cellular exocytic activity resulting from exocytosis of all vesicles including ATP-enriched vesicles. Using FM1-43 fluorescence, it has been shown that phosphoinositide 3-kinase (PI 3-kinase) and protein kinase C (PKC) are required for stimulation of both vesicular exocytosis and ATP release from biliary cells [21]. These findings indicate that vesicular exocytosis may mediate ATP release from biliary cells.…”