CHARACTERIZATION OF RECOMBINANT ENDOTHELIN RECEPTOR-TYPE-B OVEREXPRESSED IN CHINESE-HAMSTER OVARY CELLS

The recombinant B type of endothelin (ET) receptor (ET(B) receptor) was expressed at high levels in Chinese hamster ovary (CHO) cells. The recombinant ET(B) receptor possessed biochemical characteristics of the endogenous ET(B) receptor with regard to affinities, specificities for ET isopeptides, and molecular mass. In the transfected CHO cells, high concentrations of ET (10(-10) M less than or equal to) caused a rapid, transient increase in intracellular free Ca2+ concentrations ([Ca2+](i)) followed by a lower, sustained phase. In contrast, low concentrations of ET (less than or equal to 10(-11) M) induced only the sustained [Ca2+](i) phase. Removal of extracellular Ca2+ and the addition of nicardipine, an antagonist of L-type Ca2+ channels, completely blocked and significantly decreased the sustained [Ca2+](i) response, respectively. These results in conjunction with the findings that ET-induced inositol triphosphate production was observed at only high concentrations of ET (10(-10) M less than or equal to) suggest that the ET(B) receptor preferentially couples with the induction of Ca2+ channel opening at low ligand concentrations (less than or equal to 10(-11) M), whereas that the receptor causes both activation of phospholipase C and Ca2+ channel opening at high ligand concentrations (10(-10) M less than or equal to). Rapid internalization of the ET-ET(B) receptor complex with a half time of 15 min was detected by affinity labeling, presenting the necessity for studying correlation between the internalization and ET(B) receptor-mediated generation of intracellular signals. Finally, this study demonstrates the usefulness of our transfected CHO cells stably expressing the recombinant ET(B) receptor, with which signal transduction pathways through this receptor and internalization of the ET-ET(B) receptor complex can be analyzed.