Endothelin (ET) has been implicated in the regulation of hepatic microcirculation

Endothelin (ET) has been implicated in the regulation of hepatic microcirculation and development of portal hypertension. (SECs) and hepatic stellate cells (HSCs) and ETAR was scantily expressed. These findings were confirmed by Western blot and ISH. In cirrhotic liver tissue overexpression of ETBR was demonstrated by Western blot and ISH. Morphometric analysis showed significant increase of ETBR expression on HSCs and SECs in cirrhotic liver particularly on HSCs. ETAR expression was increased but remained low. Enhanced ETBR expression in cirrhosis may intensify the effect of endothelin on HSCs and increase hepatic microvascular tone. A number of vasoactive substances have been implicated as potential mediators of intrahepatic portal hypertension. BIIB-024 1 The precise sites of action of these compounds remain controversial but in theory could be at any level within the liver; presinusoidal sinusoidal or postsinusoidal. 2-4 Endothelin (ET)-1 is a potent vasoconstrictive peptide composed BIIB-024 of 21 amino residues originally isolated from the supernatant of cultured porcine endothelial cell. 5 Two other endothelins ET-2 and ET-3 6 were subsequently isolated. The mature peptides each 21 amino acid residues in length are cleavage products of larger precursor proteins. 7 Their major function appears to be control of local vascular tone 8 but broad effects on growth and development have also been suggested. 9 Two types of ET receptors have been identified and studied; endothelin A (ETAR) and B receptors (ETBR) both signal BIIB-024 via GTP-binding proteins. 10 Another type of endothelin receptor ETCR has been found but its function and distribution remains unknown. ETAR is found predominantly on vascular smooth muscle cells and the affinities for endothelins are in the order ET-1 > ET-2 > ET-3; the affinity for ET-1 is more than 100-fold that of ET-3. 11 The primary response mediated by ETAR is vasoconstriction. 11 ETBR stimulation leads to Rabbit Polyclonal to Cyclin H (phospho-Thr315). diverse responses depending in part on cell type. A study has proposed that ETBR-dependent relaxation and smooth muscle vasoconstriction are mediated by distinct ETBRs termed ETB1 and ETB2 respectively. 11 While ET receptors are detectable on all cell types in rat liver they are far more numerous on hepatic stellate cells (HSCs) than on other hepatic cells such as sinusoidal endothelial cells (SECs) Kupffer cells and hepatocytes. 12 13 When ET-1 is perfused into the rat liver its BIIB-024 localization is consistent with binding to HSCs and SECs. 14 Rat HSCs express both ETAR and ETBR as established by mRNA analysis and competitive binding assays. 13 Functional studies indicate that both receptors mediate biological effects. 15-17 The ETBR receptor appears to be involved additionally in regulating growth of human myofibroblast-like stellate cells. 16 While the data of ETR have largely been obtained using rat liver there is relatively little information on human liver. No study so far has localized ETAR and ETBR in intact human liver tissue. Our present report is the first to investigate the expression and distribution of ETR subtypes in human normal liver and cirrhotic liver BIIB-024 tissues at protein (immunohistochemistry Western blot immunoelectron microscopic study) and mRNA (hybridization) levels. Materials and Methods Materials As controls wedge biopsy specimens from normal portions of the liver were obtained from 5 patients (4 males and 1 female; aged from 44 to 73 years with a mean of 57.3 years) who underwent surgical resection for metastatic liver carcinoma (4 colonic carcinomas and 1 gastric carcinoma). Cirrhotic liver specimens were obtained BIIB-024 from gross cirrhotic portions surgically resected from 5 patients (5 males; aged from 62 to 75 years with a mean of 67.4 years) who had hepatocellular carcinoma combined with hepatitis C-related cirrhosis. Immunoperoxidase Staining Liver tissues (approximately 5 × 5 × 5 mm) were fixed in periodate-lysine-paraformaldehyde (PLP) rinsed in 0.01 mol/L phosphate buffer (pH 7.4) 18 containing 15 to 30% sucrose embedded in Tissue-Tek OCT-compound (Sakura Finetek Inc. Torrance CA) and frozen at ?80°C until use. Semithin sections 5 μm in thickness were cut using a cryostat and incubated two days at 4°C with 1:50 dilution of anti-ETAR or anti-ETBR rabbit polyclonal antibodies (Immunobiological Lab. Fujioka Japan). 19 Then the sections were incubated with the EnVision system (DAKO Glostrup Denmark) 20 at room temperature for 120 minutes. After repeated washes with phosphate-buffered saline (PBS) the sections were reacted with.