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

5'-AMP-activated kinase (AMPK) plays a key role in the regulation of cellular lipid metabolism. The contribution of vesicular exocytosis to this regulation is not known. Accordingly, we studied the effects of AMPK on exocytosis and intracellular lipid content in a model liver cell line. Activation of AMPK by metformin or 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) increased the rates of constitutive exocytosis by about 2-fold. Stimulation of exocytosis by AMPK occurred within minutes, and persisted after overnight exposure to metformin or AICAR. Activation of AMPK also increased the amount of triacylglycerol (TG) and apolipoprotein B (apoB) secreted from lipid-loaded cells. These effects were accompanied by a decrease in the intracellular lipid content indicating that exocytosis of lipoproteins was involved in these lipid-lowering effects. While AMPK increased the rates of fatty acid oxidation (FAO), the lipid-lowering effects were quantitatively significant even after inhibition of FAO with R-etomoxir. These results suggest that hepatic AMPK stimulates constitutive exocytosis of lipoproteins, which may function in parallel with FAO to regulate intracellular lipid content.

Extracellular ATP acts as a potent signaling molecule in many different tissues including the immune system, neurons, endothelial cells, and secretory epithelia by activation of purinergic receptors in the plasma membrane (1). Cells release ATP in response to physiologic stimuli such as shear stress, stretch, osmotic swelling, and hypoxia (2-5). One mechanism for ATP release involves movement of ATP through transporters or channel proteins in the plasma membrane. There is evidence for ATP release through ATP-binding cassette (ABC) transporters, connexin and pannexin hemichannels, P2X7-pannexin1 receptorchannel complex, and multiple Cl Ϫ channels (6 -11). In addition, there is evidence for exocytic vesicular release of ATP (4, 12). Under basal conditions, the concentration of ATP in extracellular medium is in the low nanomolar range. Vesicles store ATP in the millimolar range, and exocytosis of these ATP-enriched vesicles increases local ATP concentrations. It has been difficult to study the contribution of exocytosis in ATP release because many cells are capable of releasing ATP through more than one mechanism. For example, ATP release from astrocytes is mediated by both vesicular exocytosis and transport proteins (8,(13)(14)(15). Thus, the role of vesicular exocytosis in ATP release is still poorly understood.5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) 2 is widely used as an inhibitor of many different Cl Ϫ channels and has been reported to inhibit ATP release mediated by Cl Ϫ channels (16,17). In some cells, NPPB also inhibits ATP release mediated by mechanosensitive and pannexin1 channels, and other channels that have not been defined (2,10,18,19). Thus, NPPB is an inhibitor of channel-mediated ATP release.The purpose of these studies was to assess the role of vesicular exocytosis in cellular ATP release. Using FM1-43 fluorescence to measure exocytosis and bioluminescence assay to measure ATP release in real time, we found that exposure to NPPB under basal conditions potently stimulates ATP release. These previously unknown effects of NPPB appear to be mediated through stimulation of exocytosis of a pool of ATP-enriched vesicles. EXPERIMENTAL PROCEDURESCell Models-Studies of ATP release were performed in HTC and Mz-Cha-1 cells. Both cell lines have been utilized as models for cellular ATP release, degradation, and purinergic signaling in secretory epithelia (7,20). HTC cells are derived from rat hepatoma, and Mz-Cha-1 cells are derived from human adenocarcinoma of the gall bladder. The procedures for culturing these cells have been previously described (21,22). Cells were used within 48 h after plating.Measurement of ATP Release-Cellular release of ATP was measured using the luciferin-luciferase assay as previously described (23,24). All cells were grown to confluence in 35-mm Petri dishes. Prior to study, cells were gently washed twice with 1 ml of OptiMEM (Invitrogen) and then 800 l of OptiMEM containing 2 mg/ml firefly luciferin-luciferase (Sigma cat. num.

Extracellular ATP regulates many important cellular functions in the liver by stimulating purinergic receptors. Recent studies have shown that rapid exocytosis of ATP-enriched vesicles contributes to ATP release from liver cells. However, this rapid ATP release is transient, and ceases in~30 s after the exposure to hypotonic solution. The purpose of these studies was to assess the role of vesicular exocytosis in sustained ATP release. An exposure to hypotonic solution evoked sustained ATP release that persisted for more than 15 min after the exposure. Using FM1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl)pyridinium dibromide) fluorescence to measure exocytosis, we found that hypotonic solution stimulated a transient increase in FM1-43 fluorescence that lasted~2 min. Notably, the rate of FM1-43 fluorescence and the magnitude of ATP release were not correlated, indicating that vesicular exocytosis may not mediate sustained ATP release from liver cells. Interestingly, mefloquine potently inhibited sustained ATP release, but did not inhibit an increase in FM1-43 fluorescence evoked by hypotonic solution. Consistent with these findings, when exocytosis of ATP-enriched vesicles was specifically stimulated by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), mefloquine failed to inhibit ATP release evoked by NPPB. Thus, mefloquine can pharmacologically dissociate sustained ATP release and vesicular exocytosis. These results suggest that a distinct mefloquinesensitive membrane ATP transport may contribute to sustained ATP release from liver cells. This novel mechanism of membrane ATP transport may play an important role in the regulation of purinergic signaling in liver cells.

Purinergic P2Y 2 G-protein coupled receptors play a key role in the regulation of hepatic Ca 2+ signaling by extracellular ATP. The concentration of copper in serum is about 20 μM. Since copper accumulates in the liver in certain disease states, the purpose of these studies was to assess the effects of copper on P2Y 2 receptors in a model liver cell line. Exposure to a P2Y 2 agonist UTP increased [Ca 2+ ] i by stimulating Ca 2+ release from thapsigargin-sensitive Ca 2+ stores. Pretreatment of HTC cells for several minutes with copper did not affect cell viability, but potently inhibited increases in [Ca 2+ ] i evoked by UTP and thapsigargin. During this pretreatment, copper was not transported into the cytosol, and inhibited P2Y 2 receptors in a concentration-dependent manner with the IC 50 of about 15 μM. These results suggest that copper inhibits P2Y 2 receptors through the effects on thapsigargin-sensitive Ca 2+ stores by acting from an extracellular side. Further experiments indicated that these effect of copper may lead to inhibition of regulatory volume decrease (RVD) evoked by hypotonic solution. Thus, copper may contribute to defective regulation of purinergic signaling and liver cell volume in diseases associated with the increased serum copper concentration.

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RATIONALE: X-linked inhibitor of apoptosis protein (XIAP) deficiency is a primary hyperinflammatory immunodeficiency typically diagnosed following an episode of hemophagocytic lymphohistiocytosis (HLH) or in the setting of early onset severe inflammatory bowel disease (IBD). Hematopoietic cell transplant (HCT) is the definitive treatment. We present an infant with prenatal hepatosplenomegaly and hepatic calcifications who was subsequently diagnosed with XIAP deficiency in infancy. METHODS: Whole exome sequencing (WES) for patient and parents was performed. Clinical course and immunophenotyping were reviewed. RESULTS: Hepatosplenomegaly, hepatic calcifications and large placenta were noted on prenatal ultrasound at 35 weeks performed for maternal pre-eclampsia. Postnatal evaluation revealed thrombocytopenia and direct hyperbilirubinemia, raising concerns for congenital infection, chromosomal abnormality, or a metabolic disorder. However, the abnormalities normalized over 7 days and he had a negative congenital infection work up, and karyotype, microarray, and metabolic evaluation were normal. WES showed a single likely pathogenic variant, a de novo c.1021_1022delAA; p.Asn341Tyr-fs*8 hemizygous deletion in XIAP, predicted to be null. Flow cytometry revealed <1.5% XIAP expression in all peripheral blood mononuclear lineages. After hospitalization at 10 mo for fever and hives, IgG and prophylactic medications were started. At 14 mo he was again hospitalized for fever, neutropenia, and elevated ferritin that spontaneously resolved. Otherwise, his development is progressing normally and HCT is planned. CONCLUSIONS: XIAP deficiency presented with prenatal hepatic inflammation and newborn findings mimicking congenital infection. Fever, hives and incomplete HLH occurred before 15 mo. The early diagnosis of XIAP deficiency has allowed for definitive HCT to be undertaken electively.

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