Absence of endothelin receptors and receptor mRNA in mammalian fibroblasts transformed with SV40 or ras oncogene.
Endothelin-1 (ET-1), a peptide isolated from the culture medium of endothelial cells, mediates a variety of physiological and pathological responses including mitogenesis. We have compared the expression of ET receptors in untransformed versus ras-transformed NIH-3T3 murine fibroblasts 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 (23,000 sites/cell and 14,000 sites/cell for NIH-3T3 and W138 cells, respectively) ET receptors. Competition binding experiments using subtype-selective ligands identified these receptors as the ETA subtype. Addition of ET-1 to the cells produced a concentration-dependent increase in intracellular calcium release. Both ras-transformed NIH-3T3 cells and SV40-transformed W138 cells (VA13) completely lacked [125I]ET-1 binding and failed to release calcium when exposed to ET-1. Northern analysis of the polyadenylated RNA (polyA RNA) isolated from untransformed and transformed cells revealed that the steady-state level of ETA receptor 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.
Nambi
P
P
Department of Renal Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA.
Mattern
M R
MR
Wu
H L
HL
Pullen
M
M
Nuthulaganti
P
P
Hofmann
G A
GA
Kumar
C
C
D016475:3T3 Cells; D000818:Animals; D001667:Binding, Competitive; D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism; D002461:Cell Line, Transformed / Q000378:metabolism*; D015536:Down-Regulation; D019332:Endothelin-1 / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism*; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D051379:Mice; D012333:RNA, Messenger / Q000378:metabolism*; D017466:Receptors, Endothelin / Q000378:metabolism*; D013539:Simian virus 40 / Q000235:genetics
D019332:Endothelin-1; D012333:RNA, Messenger; D017466:Receptors, Endothelin; D002118:Calcium