Recovery of cell volume in response to osmotic stress is mediated in part by increases in the Cl1 permeability of the plasma membrane. These studies evaluate the hypothesis that ATP release and autocrine stimulation of purinergic (P2) receptors couple increases in cell volume to opening of Cl-channels. In HTC rat hepatoma cells, swelling Epithelial cells express a variety of plasma membrane proteins that bind solutes in a selective manner and transport them to the cell interior. Although the action of such transport proteins is essential for normal cell function, the resulting increase in intracellular solute concentration leads to an influx of water and cell swelling. However, cell volume is maintained within a relatively narrow physiologic range by adaptive responses that generally involve solute efflux through opening of K+ and Clchannels and restoration of cell volume toward its resting state (1-3). Despite extensive study, the mechanisms that couple increases in cell volume to opening of ion channels remain incompletely understood.In a number of cell types, swelling-induced opening of Clchannels is dependent on the presence of intracellular ATP (4, 5). This ATP dependence of volume-stimulated channel opening is attributable, in part, to the action of intracellular protein kinases on channels or channel regulatory proteins (6, 7). However, other mechanisms are possible as well. Recent experimental evidence suggests that ATP can also function outside of the cell as an autocrine factor to increase membrane Cl-permeability (8). In secretory epithelia, increases in cAMP stimulate cellular release of ATP in concentrations sufficient to activate purinergic (P2) receptors in the plasma membrane, which leads to Cl-channel opening (9). The mechanisms involved in ATP trafficking in secretory cells are not fully defined, with evidence for (9, 10) and against (11) ATP release through the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP-binding cassette (ABC) family of proteins. Other members of the ABC family, including the multidrug resistance P-glycoprotein, are thought to regulate volume-activated channels through cellular release of ATP or other pathways (6, 12).Like secretory epithelia, activation of P2 receptors in liver cells increases membrane Cl-permeability through opening of Cl-channels (13). However, unlike secretory cells, liver cells are not thought to exhibit Cl--dependent secretion (14), and the physiologic importance of purinergic signaling in liver has not been defined. Based on the prominent role of Cl-ions in liver cell volume regulation (15), we have proposed that volume-dependent activation of Cl-channels is mediated by an autocrine mechanism involving ATP release and activation of a P2 receptor. In a model liver cell line that expresses P2 receptors (13, 16), we show that (i) cell swelling increases membrane ATP permeability, and (ii) Cl-channel opening and cell volume recovery from swelling each require stimulation of P2 receptors by extracellular ATP. These...

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Extracellular ATP is a potent signaling factor that modulates a variety of cellular functions through the activation of P 2 purinergic receptors in the plasma membrane. These receptors are widely distributed among different liver cell types, including hepatocytes, cholangiocytes, macrophages, and endothelial cells, but the physiologic roles have not been fully defined. Cells release ATP in response to both osmotic and mechanical stimuli, and one mechanism may involve opening of a channel-like pathway (1, 2). In respiratory epithelia, ATP release stimulated by cytosolic cAMP activates outwardly rectified Cl Ϫ channels coupled to P 2U receptors and enhances Cl Ϫ secretion (3).Recent studies in a model liver cell line support an alternative pathway where increases in cell volume induce conductive ATP efflux. In these cells, removal of extracellular ATP or P 2 receptor blockade prevents both Cl Ϫ channel activation and volume recovery (1). These findings suggest functional interactions between ATP release, P 2 receptor stimulation, and Cl Ϫ channel opening in epithelial secretion and volume regulation.Members of the ATP-binding cassette (ABC) 1 protein family are likely to be relevant to this volume regulatory pathway for two reasons. First, while the molecular basis for the transmembrane ATP conductance has not been established, heterologous expression or up-regulation of ABC family members in some cell models is associated with enhanced electrodiffusional ATP release. In cystic fibrosis respiratory epithelia, cAMP fails to stimulate channel-mediated ATP efflux, a response that is present in native epithelia; CFTR gene transfer restores the ATP conductance (3, 4). In other cell lines, ATP release is proportional to the expression of mammalian and Drosophila Mdr1 P-glycoproteins (5, 6). Second, in some but not all cell types, Mdr1 P-glycoproteins regulate swelling-activated Cl Ϫ currents (I Cl-swell ). Effects include enhancement of I Cl-swell and endowment of Cl Ϫ channel sensitivity to protein kinase C (mdr1 gene transfer) and increase in I Cl-swell for a given hypotonic stress (P-glycoprotein overexpression) (7,8). The cellular mechanisms involved in these responses and the implications for other cell types have yet to be clarified.In hepatocytes, P-glycoproteins transport both amphipathic compounds and phospholipids across canalicular membranes into bile (9, 10). However, the functions of multiple other ABC members present in liver cells are unknown. In light of the putative association of certain ABC proteins with channelmediated ATP and Cl Ϫ transport, we sought to investigate the role of hepatocellular ABC proteins in these processes. Findings in rat HTC hepatoma cells were compared with those in a selected population of HTC cells (HTC-R) that overexpress both endogenous and novel Mdr proteins (11). These studies demonstrate that inhibition of P-glycoprotein transport prevents recovery from swelling and that overexpression of Mdr proteins is associated with enhanced ATP release, volume recovery, and cell surv...