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Absence of endothelin receptors and receptor mRNA in mammalian fibroblasts transformed with SV40 or ras oncogene

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Abstract

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 Wl38 (VA13) human fibroblasts by ligand binding and Northern analysis. NIH-3T3 and Wl38 cells displayed high affinity (200 and 220 pM) and high density (23,000 sites/cell and 14,000 sites/cell for NIH-3T3 and Wl38 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 Wl38 cells (VA13) completely lacked [125I]ET-1 binding and failed to release calcium when exposed to ET-1. Northern analysis of the polyadenylat ed 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.

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Authors and Affiliations

  1. Department of Renal Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA, 19406, USA

    Ponnal Nambi, Hsiao-Ling Wu & Mark Pullen

  2. Department of Biomolecular Discovery, SmithKline Beecham Pharmaceuticals, King of Prussia, PA, 19406, USA

    Michael R. Mattern & Glenn A. Hofmann

  3. Department of Molecular Genetics, SmithKline Beecham Pharmaceuticals, King of Prussia, PA, 19406, USA

    Parvathi Nuthulaganti & Chandrika Kumar

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  1. Ponnal Nambi
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  6. Glenn A. Hofmann
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  7. Chandrika Kumar
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Nambi, P., Mattern, M.R., Wu, HL. et al. Absence of endothelin receptors and receptor mRNA in mammalian fibroblasts transformed with SV40 or ras oncogene. Mol Cell Biochem 175, 29–35 (1997). https://doi.org/10.1023/A:1006827007251

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