Hepatocellular ATP-binding Cassette Protein Expression Enhances ATP Release and Autocrine Regulation of Cell Volume

Abstract

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...

Liver Cell Volume Regulation: Size Matters

“…17 In contrast, increases in membrane K ϩ and Cl Ϫ permeability provide a powerful counterpoint, and represent the major driving force for water movement out of the cell. 18 While the molecular iden-tity of the K ϩ channels is not known, a portion of the volumesensitive Cl Ϫ conductance appears to be mediated by ClC-2 chloride channels that have been cloned and identified in liver cells at the level of cDNA, mRNA, and functional protein. 19 These are resistant to the Cl Ϫ channel blocker, DIDS, but are inhibited by the nonselective blockers, NPPB and DPC.…”

Mrp2 modulates the activity of chloride channels in isolated hepatocytes

“…While this clearly occurs for the SUR-K IR complex, there may be other possible mechanisms of channel activation as well. Several different ABC proteins have been shown to increase ATP efflux from cells 31,[35][36][37] and extracellular ATP can activate channels in a number of cells. 38 Enhanced ATP efflux resulting from ABC protein overex- Fig.…”

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

“…These cells have been utilized as a liver cell model line to study hepatocyte ATP release, purinergic signaling and the regulation of cell volume [13,28]. HTC cells are derived from rat hepatoma, and the procedures for culturing these cells have been previously described [29].…”

“…There is evidence for ATP release through ATPbinding cassette (ABC) transporters, connexin hemichannels and multiple Cl − channels [12][13][14]. More recent studies have found that pannexin1 hemichannels play a key role in ATP release from many different cells [15][16][17][18].…”