Hepatocyte lipoapoptosis induced by saturated free fatty acids (FFA) contributes to hepatic inflammation in lipotoxic liver injury, and the cellular mechanisms involved have not been defined. Recent studies have shown that apoptosis in nonhepatic cells stimulates ATP release via activation of pannexin1 (panx1), and extracellular ATP functions as a proinflammatory signal for recruitment and activation of the inflammatory cells. However, it is not known whether lipoapoptosis stimulates ATP release in liver cells. We found that lipoapoptosis induced by saturated FFA stimulated ATP release in liver cells that increased extracellular ATP concentration by more than fivefold above the values observed in healthy cells. This sustained pathophysiological ATP release was not dependent on caspase-3/7 activation. Inhibition of c-Jun NH(2)-terminal kinase (JNK), a key mediator of lipoapoptosis, with SP600125 blocked pathophysiological ATP release in a dose-dependent manner. RT-PCR analysis indicated that panx1 is expressed in hepatocytes and multiple liver cell lines. Notably, inhibition of panx1 expression with short hairpin (sh)RNA inhibited in part pathophysiological ATP release. Moreover, lipoapoptosis stimulated uptake of a membrane impermeable dye YoPro-1 (indicative of panx1 activation), which was inhibited by panx1 shRNA, probenecid, and mefloquine. These results suggest that panx1 contributes to pathophysiological ATP release in lipoapoptosis induced by saturated FFA. Thus panx1 may play an important role in hepatic inflammation by mediating an increase in extracellular ATP concentration in lipotoxic liver injury.

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.

In the past decade, there has been remarkable progress in understanding of the roles of Cl(-) channels in the development of human diseases. Genetic studies in humans have identified mutations in the genes encoding Cl(-) channels which lead to a loss of Cl(-) channel activity. These mutations are responsible for the development of a variety of deleterious diseases in muscle, kidney, bone and brain including myotonia congenita, dystrophia myotonica, cystic fibrosis, osteopetrosis and epilepsy. Recent studies indicate that some diseases may develop as a result of Cl(-) channel activation. There is growing evidence that the progression of glioma in the brain and the growth of the malaria parasite in red blood cells may be mediated through Cl(-) channel activation. These findings suggest that Cl(-) channels may be novel targets for the pharmacological treatment of a broad spectrum of diseases. This review discusses the proposed roles of abnormal Cl(-) channel activity in the pathogenesis of human diseases.

Ionotrophic purinergic (P2X) receptors function as receptor-gated cation channels, where agonist binding leads to opening of a nonselective cation pore permeable to both Na ؉ and Ca 2؉ . Based on evidence that extracellular adenosine 5-triphosphate (ATP) stimulates glucose release from liver, these studies evaluate whether P2X receptors are expressed by hepatocytes and contribute to ATP-dependent calcium signaling and glucose release. Studies were performed in isolated hepatocytes from rats and mice and hepatoma cells from humans and rats. Transcripts and protein for both P2X4 and P2X7 were detectable, and immunohistochemistry of intact liver revealed P2X4 in the basolateral and canalicular domains. H epatocytes exhibit regulated release of adenosine 5Ј-triphosphate (ATP) into the extracellular space. 1 Once outside the cell, these nucleotides function as potent autocrine/paracrine signaling molecules that modulate liver function through activation of purinergic receptors in the plasma membrane. Initially discovered in neurons, purinergic receptors are implicated in the control of a spectrum of essential physiologic mechanisms ranging from vascular tone to programmed cell death. In the liver, nanomolar concentrations of ATP, or its breakdown products, activate Cl Ϫ channels, decrease cell volume, and stimulate bile flow. 2,3 Moreover, exposure of perfused isolated livers to ATP stimulates a large release of glucose. 4 Collectively, these coordinated pathways of ATP release, receptor binding, and degradation constitute a versatile mechanism for paracrine regulation of liver function.These diverse effects of ATP are not readily explained by a single receptor type. Previous studies indicate that hepatocytes express several subtypes of G-protein coupled P2Y receptors, including P2Y1, P2Y2, P2Y4, and P2Y6. Selective stimulation of P2Y2 receptors activates plasma membrane chloride channels and stimulates ductular bile secretion. 5 P2Y receptors also appear to play a role in the activation of glycogen phosphorylase, the rate-controlling enzyme in hepatic glycogenolysis, 6 and regulate multiple signaling pathways with changes in cellular [Ca 2ϩ ], eicosanoid production, and cyclic adenosine monophosphate

The present studies of cholangiocytes used complementary histological, biochemical, and electrophysiological methods to identify a dense population of subapical vesicles, quantify the rates of vesicular trafficking, and assess the contribution of the actin cytoskeleton to membrane trafficking. FM 1-43 fluorescence measured significant basal rates of total exocytosis (1.33 +/- 0.16% plasma membrane/min) in isolated cholangiocytes and apical exocytosis in cholangiocyte monolayers. Cell surface area remained unchanged, indicating that there was a concurrent, equivalent rate of endocytosis. FM 1-43 washout studies showed that 36% of the endocytosed membrane was recycled to the plasma membrane. 8-(4-Chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP; cAMP analog) increased exocytosis by 71 +/- 31%, whereas the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS; protein kinase A inhibitor) diminished basal exocytosis by 53 +/- 11%. A dense population of 140-nm subapical vesicles arose, in part, from apical membrane endocytosis. Phalloidin staining showed that a subpopulation of the endocytosed vesicles was encapsulated by F-actin. Furthermore, membrane trafficking was inhibited by disrupting the actin cytoskeleton with cytochalasin D (51 +/- 13% of control) or jasplakinolide (58 +/- 9% of control). These studies indicate that there is a high rate of vesicular trafficking at the apical membrane of cholangiocytes and suggest that both cAMP and the actin cytoskeleton contribute importantly to these events.