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

The recombinant B type of endothelin (ET) receptor (ET(B) receptor) wa s expressed at high levels in Chinese hamster ovary (CHO) cells. The r ecombinant ET(B) receptor possessed biochemical characteristics of the endogenous ET(B) receptor with regard to affinities, specificities fo r ET isopeptides, and molecular mass. In the transfected CHO cells, hi gh concentrations of ET (10(-10) M less than or equal to) caused a rap id, transient increase in intracellular free Ca2+ concentrations ([Ca2 +](i)) followed by a lower, sustained phase. In contrast, low concentr ations of ET (less than or equal to 10(-11) M) induced only the sustai ned [Ca2+](i) phase. Removal of extracellular Ca2+ and the addition of nicardipine, an antagonist of L-type Ca2+ channels, completely blocke d and significantly decreased the sustained [Ca2+](i) response, respec tively. These results in conjunction with the findings that ET-induced inositol triphosphate production was observed at only high concentrat ions of ET (10(-10) M less than or equal to) suggest that the ET(B) re ceptor preferentially couples with the induction of Ca2+ channel openi ng at low ligand concentrations (less than or equal to 10(-11) M), whe reas that the receptor causes both activation of phospholipase C and C a2+ 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, presenti ng the necessity for studying correlation between the internalization and ET(B) receptor-mediated generation of intracellular signals. Final ly, this study demonstrates the usefulness of our transfected CHO cell s 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.