ABSENCE OF ENDOTHELIN RECEPTORS AND RECEPTOR MESSENGER-RNA IN MAMMALIAN FIBROBLASTS TRANSFORMED WITH SV40 OR RAS ONCOGENE

Endothelin-1 (ET-1), a peptide isolated from the culture medium of end othelial cells, mediates a variety of physiological and pathological r esponses including mitogenesis. We have compared the expression of ET receptors in untransformed versus ras-transformed NIH-3T3 murine fibro blasts and in untransformed versus SV40-transformed W138 (VA13) human fibroblasts by ligand binding and Northern analysis. NIH-3T3 and W138 cells displayed high affinity (200 and 220 pM) and high density (23,00 0 sites/cell and 14,000 sites/cell for NIH-3T3 and W138 cells, respect ively) ET receptors, Competition binding experiments using subtype-sel ective ligands identified these receptors as the ET, subtype. Addition of ET-I to the cells produced a concentration-dependent increase in i ntracellular calcium release. Both ras-transformed NIH-3T3 cells and S V40-transformed W138 cells (VA13) completely lacked [I-125]ET-1 bindin g and failed to release calcium when exposed to ET-1. Northern analysi s of the polyadenylated RNA (polyA RNA) isolated from untransformed an d transformed cells revealed that the steady-state level of ET, recept or RNA was 90-95% less in transformed cells compared to untransformed cells, Thus, the loss of ET receptors as well as the receptor-mediated responses in transformed cells can be explained by down-regulation of ET receptor mRNA.