Stimulation of phosphatidylinositol hydrolysis, diacylglycerol release, and gene expression in response to endothelin, a potent new agonist for fibroblasts and smooth muscle cells

Endothelin is a potent vasoconstructive peptide recently isolated by Yanagisawa, M., Kurihara, H., Kimura, S., Tamobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K., and Masaki, T. (1988) Nature 332, 411-415). In In order to understand the mechanism of action of endothelin in various cell types we have examined the effects of endothelin on second messenger levels in Rat-1 fibroblasts and A10 smooth muscle cells. Endothelin stimulated a 15-fold increase in the accumulation of inositol triphosphate in Rat-1 cells, with half-maximal stimulation observed at 0.5 nM endothelin. In the A10 vascular smooth muscle cell line, endothelin stimulated phosphatidylinositol turnover more than 3-fold, comparable to the stimulation produced by serum. Concurrent with the increase in inositol phosphate release, endothelin increased diacyl-glycerol levels by 50% in A10 cells and by more than 3-fold in Rat-1 cells. The increase in diacylglycerol levels in response to endothelin was equal to or greater than the response to serum. Stimulation of phosphatidylinositol turnover by endothelin did not require the presence of extracellular calcium and was not blocked by treatment with EGTA or cobalt. Furthermore, endothelin did not stimulate Ca²⁺ uptake by either cell type and actually reduced Ca²⁺ uptake below control levels with increased duration of preincubation. Endothelin stimulated an increase in intracellular Ca²⁺ levels, from 100 to over 750 nM in Rat-1 cells and from 150 to over 350-nM in A10 cells as measured by fura-2 microspectrofluorimetry. The rise in intracellular Ca²⁺ concentration was not inhibited by the presence of EGTA or cobalt. These data indicate that endothelin did not act to open Ca²⁺ channels ineither Rat-1 fibroblasts or A10 smooth muscle cells. Cytoplasmic levels of VL30 RNA, a gene independently induced by protein kinase C and by epidermal growth factor, were increased following endothelin treatment, even in protein kinase C-depleted cells. We conclude that endothelin is a very potent stimulus for phosphatidylinositol turnover, diacylglycerol release, and gene transcription. These data may have wide-ranging implications for a number of disease states.