Hepatic effects of endothelin. Receptor characterization and endothelin-induced signal transduction in hepatocytes.
Endothelin, a potent vasoactive peptide originally isolated from the vascular endothelial cells, exerts glycogenolytic and vasoconstrictive actions in the perfused rat liver. In this paper we demonstrate high-affinity binding sites for endothelin-1 (ET-1) on rat hepatocytes. Upon incubation at 37 degrees C, association of ET-1 with hepatocytes occurred in a time-dependent manner, was maximal between 3 and 6 h, and subsequently declined; at this temperature ET-1 was rapidly internalized with the internalized ligand exceeding the surface-bound ligand at all time points. The rate of association of 125I-ET-1 with hepatocytes was much slower when the binding assay was performed at 4 degrees C; sequestration of ET-1 in hepatocytes was also substantially reduced at this temperature. ET-1 was extremely potent in stimulating phosphoinositide metabolism in hepatocytes, with significant activation of this signal transduction process occurring at ET-1 concentrations as low as 0.1 pM, with an EC50 of 1 pM. The effect of ET-1 was coupled via a pertussis toxin-sensitive G-protein. Cholera toxin did not affect ET-1-mediated phosphoinositide metabolism and neither toxin influenced the association of 125I-ET-1 with hepatocytes. PAGE of hepatocyte membranes following exposure of the cells to 125I-ET-1 and cross-linking revealed labelling of three major proteins with apparent molecular masses of 32, 49 and 72 kDa. 125I-ET-1 labelling of each of these proteins was inhibited by unlabelled ET-1, whereas unlabelled ET-3 inhibited the labelling of only the 32 and 49 kDa proteins. 125I-ET-3 labelled the 49 kDa protein and this labelling was inhibited by both unlabelled ET-1 and ET-3. Each of these receptors appears to be functional, since both ET-1 and ET-3 stimulated phosphoinositide metabolism in hepatocytes. Down-regulation of ET-1 association and desensitization of ET-1-induced phosphoinositide metabolism occurred upon incubation of hepatocytes with the homologous ligand. Following down-regulation, the ET-1 receptor was restored to the surface of the hepatocyte by prolonged incubation, although the ET-1-stimulated phosphoinositide response remained inhibited even after complete recovery of the ET-1 association capability. These results demonstrate the presence of multiple high-affinity receptors for ET-1 on hepatocytes and the direct action of this peptide on hepatic parenchymal cells via the phosphoinositide signal transduction pathway.
Gandhi
C R
CR
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Behal
R H
RH
Harvey
S A
SA
Nouchi
T A
TA
Olson
M S
MS
D000818:Animals; D001667:Binding, Competitive; D002478:Cells, Cultured; D015536:Down-Regulation; D016232:Endothelins / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000378:metabolism*; D008297:Male; D010716:Phosphatidylinositols / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017466:Receptors, Endothelin / Q000378:metabolism*; D015398:Signal Transduction
D016232:Endothelins; D008024:Ligands; D010716:Phosphatidylinositols; D017466:Receptors, Endothelin; D019204:GTP-Binding Proteins