SUMMARYBackground and Aims: A G-protein coupled succinate receptor has recently been identified in several tissues, including the liver. The objectives of this work were to determine the hepatic cell types that express this receptor and to determine its physiological role.

Extracellular nucleotides may be important regulators of bile ductular secretion, because cholangiocytes express P2Y ATP receptors and nucleotides are found in bile. However, the expression, distribution, and function of specific P2Y receptor subtypes in cholangiocytes are unknown. Thus our aim was to determine the subtypes, distribution, and role in secretion of P2Y receptors expressed by cholangiocytes. The molecular subtypes of P2Y receptors were determined by RT-PCR. Functional studies measuring cytosolic Ca2+ (Ca) signals and bile ductular pH were performed in isolated, microperfused intrahepatic bile duct units (IBDUs). PCR products corresponding to P2Y1, P2Y2, P2Y4, P2Y6, and P2X4 receptor subtypes were identified. Luminal perfusion of ATP into IBDUs induced increases in Ca that were inhibited by apyrase and suramin. Luminal ATP, ADP, 2-methylthioadenosine 5'-triphosphate, UTP, and UDP each increased Ca. Basolateral addition of adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), but not ATP, to the perifusing bath increased Ca. IBDU perfusion with ATP-gamma-S induced net bile ductular alkalization. Cholangiocytes express multiple P2Y receptor subtypes that are expressed at the apical plasma membrane domain. P2Y receptors are also expressed on the basolateral domain, but their activation is attenuated by nucleotide hydrolysis. Activation of ductular P2Y receptors induces net ductular alkalization, suggesting that nucleotide signaling may be an important regulator of bile secretion by the liver.

Extracellular nucleotides regulate diverse biological functions and are important in the regulation of liver metabolism, hepatic blood flow, and bile secretion. Ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) hydrolyze extracellular nucleotides and are therefore potential regulators of nucleotide-mediated signaling. To examine this, we have contrasted the structural and functional distributions of the 2 characterized membranebound NTPDases NTPDase1 and NTPDase2 within the rat liver. Hepatic expression of NTPDase2 was determined and contrasted to NTPDase1 using confocal immunofluorescence, immunoelectron microscopy, reverse-transcription polymerase chain reaction, Northern blot analysis, Western blot analysis, and functional assays. NTPDase2 was expressed in the periportal region surrounding intrahepatic bile ducts, whereas NTPDase1 was found in hepatic arteries, portal veins, and hepatic central veins, consistent with its known vascular distribution. Functional and molecular expression of NTPDase2 was shown in portal fibroblasts near basolateral membranes of bile duct epithelia. In conclusion, NTPDase2 is expressed in a novel cellular compartment surrounding intrahepatic bile ducts, namely portal fibroblasts. This distribution may represent a previously unrecognized mechanism for regulation of nucleotide signaling in bile ducts and other epithelia. (HEPATOLOGY 2002;36:1135-1144 B ile duct epithelia, or cholangiocytes, secrete fluid and electrolytes in response to extracellular nucleotides through activation of specific P2Y nucleotide receptors. [1][2][3][4] Cholangiocytes express both apical and basolateral P2Y receptors but basolateral cholangiocyte P2Y receptors are activated only by nonhydrolyzable nucleotide analogues, 4 and there is functional evidence for a distinct pathway for degradation of nucleotides at the basolateral membrane. 3 These observations suggest that nucleotide hydrolysis is a primary means to regulate cholangiocyte P2Y receptor activation at the basolateral membrane.Hydrolysis of extracellular nucleotides may largely occur through proteins belonging to a family of recently identified ecto-nucleoside triphosphate diphosphohydrolases (NTPDases). 5-7 Several of these enzymes have been cloned and characterized at the molecular level. NTPDase1 has been identified as a vascular NTPDase expressed at endothelial and leiomyocyte plasma membranes 8,9 and is a critical regulator of platelet aggregation. 10 NTPDase2 is an NTPDase that is expressed by epithelial organs and cell lines, 11,12 but its specific physiologic function is unknown.Here we show that NTPDase2 is expressed in a newly described cellular population of liver cells, that of portal fibroblasts. By interacting with cholangiocyte nucleotide signaling processes, portal fibroblasts may represent a novel functional compartment of liver cells, thus showing a distinct functional interaction between liver epithelia and stromal cells. Materials and MethodsAnimals and Materials. Male Sprague-Dawley rats (180-250 g) were used for al...

Portal fibroblasts (PF) are fibrogenic liver cells distinct from hepatic stellate cells (HSC). Recent evidence suggests that PF may be important mediators of biliary fibrosis and cirrhosis. The cytokine monocyte chemoattractant protein-1 (MCP-1)/CCL2 is upregulated in biliary fibrosis by bile duct epithelia (BDE) and induces functional responses in HSC. Thus we hypothesized that release of MCP-1 may mediate biliary fibrosis. We report that PF express functional receptors for MCP-1 that are distinct from the receptor CCR2. MCP-1 induces proliferation, increase and redistribution of α-smooth muscle (α-SMA) expression, loss of the ectonucleotidase NTPDase2, and upregulation of α1-procollagen production in PF. BDE secretions induce α-SMA levels in PF, and this is inhibited by MCP-1 blocking antibody. Together, these data suggest that BDE regulate PF proliferation and myofibroblastic transdifferentiation in a paracrine fashion via release of MCP-1.

Background & objectivesHepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. Several types of chronic liver disease predispose to HCC, and several different signalling pathways have been implicated in its pathogenesis, but no common molecular event has been identified. Ca2+ signalling regulates the proliferation of both normal hepatocytes and liver cancer cells, so we investigated the role of intracellular Ca2+ release channels in HCC.DesignExpression analyses of the type 3 isoform of the inositol 1, 4, 5-trisphosphate receptor (ITPR3) in human liver samples, liver cancer cells and mouse liver were combined with an evaluation of DNA methylation profiles of ITPR3 promoter in HCC and characterisation of the effects of ITPR3 expression on cellular proliferation and apoptosis. The effects of de novo ITPR3 expression on hepatocyte calcium signalling and liver growth were evaluated in mice.ResultsITPR3 was absent or expressed in low amounts in hepatocytes from normal liver, but was expressed in HCC specimens from three independent patient cohorts, regardless of the underlying cause of chronic liver disease, and its increased expression level was associated with poorer survival. The ITPR3 gene was heavily methylated in control liver specimens but was demethylated at multiple sites in specimens of patient with HCC. Administration of a demethylating agent in a mouse model resulted in ITPR3 expression in discrete areas of the liver, and Ca2+ signalling was enhanced in these regions. In addition, cell proliferation and liver regeneration were enhanced in the mouse model, and deletion of ITPR3 from human HCC cells enhanced apoptosis.ConclusionsThese results provide evidence that de novo expression of ITPR3 typically occurs in HCC and may play a role in its pathogenesis.