Detection of endothelin-1 and its receptors in rat liver endothelial cells by in situ reverse transcriptase-polymerase chain reaction

Abstract

Background/Aims: Endothelin (ET)-1 is a potent vasoconstrictor, also in the liver; it increases intrahepatic resistance by targeting hepatic stellate cells acting via the ET-A receptor. The role of the expression 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-1, ET-A and ET-B receptors in isolated rat liver endothelial cells. Methods: After in 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 than 90% in the freshly isolated non-parenchymal cells. RT-polymerase chain 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 the presence of mRNA in the endothelial cells, with all sets of primers. About 60–80% of cells were positive. Negative controls, in which the RT step was omitted, did not show any cytoplasmatic staining. Conclusions: Endothelin 1 and both ET-A and ET-B receptors are expressed in rat endothelial liver cells. The expression of ET and its receptors in liver endothelial cells could be important in the development of liver diseases and the regulation in microvascular exchange.