DETECTION OF ENDOTHELIN-1 AND ITS RECEPTORS IN RAT-LIVER ENDOTHELIAL-CELLS BY IN-SITU REVERSE TRANSCRIPTASE-POLYMERASE CHAIN-REACTION

Background/Aims: Endothelin (ET)-1 is a potent vasoconstrictor, also i n the liver; it increases intrahepatic resistance by targeting hepatic stellate cells acting via the ET-A receptor, The role of the expressi on and regulation of ET-1 and its receptors in endothelial liver cells , an important site of ET production in normal liver, is not yet well documented, In the present study, we have developed an in situ reverse transcriptase (RT)-polymerase chain reaction method to elucidate the presence of prepro-ET-l, ET-A and ET-B receptors in isolated rat liver endothelial cells, Methods: After ill situ collagenase-pronase liver perfusion, endothelial cells were isolated using a Nycodenz gradient, RT-polymerase chain reaction and in situ RT-polymerase chain reaction were performed using specific primers for prepro-endothelin, ET-A and ET-B. Results: Trypan blue exclusion test showed a viability of more t han 90% in the freshly isolated non-parenchymal cells, RT-polymerase c hain reaction showed expression of prepro-endothelin, ET-A and ET-B in RNA isolated from the endothelial cells, After in situ RT-polymerase chain reaction, we detected cytoplasmatic staining representing presen ce the of mRNA in the endothelial cells, with all sets of primers, Abo ut 60-80% of cells were positive, Negative controls, in which the RT s tep was omitted, did not show any cytoplasmatic staining, Conclusions: Endothelin 1 and both ET-A and ET-B receptors are expressed in rat en dothelial liver cells, The expression of ET and its receptors in liver endothelial cells could be important in the development of liver dise ases and the regulation of microvascular exchange.