Stimulation of calcium flows induced by endothelin in cultured smooth muscle cells

Endothelin is a potent vasoconstrictor peptide isolated from the conditioned medium of porcine aortic endothelial cells. The action of endothelin is thought to be associated with calcium entry via calcium potential channels (Yanagisawa et. al. Nature 1988; 38:411-415). The present study was designed to determine the effect of endothelin on calcium fluxes (influx and efflux) on rat aortic smooth muscle cells in culture. The unidirectional influx of calcium was measured 15, 45, 75 and 105 seconds after the addition of trace amounts of 45Ca++ (5 microCi/ml) to the cells incubated with or without endothelin. Endothelin (50nM) stimulated calcium influx from a basal level of 312 +/- 17 to 537 +/- 12 pmol/mn/10(6) cells. This stimulation was dose-dependent with an EC50 value of about 10 nM. When cells were preincubated with calcium antagonists (nifedipine, dilttiazem, D600, nicardipine and flunarizine) at a final concentration of 1 microM, the endothelin-stimulated calcium influx was not modified. The unidirectional efflux of calcium was measured after an overload of cells with 45Ca++ (5 microCi/ml) for 18 hours, over 10 seconds intervals. In the first 30 seconds after the addition of endothelin (100 nM), the amount of 45Ca++ released was 3 times that in the absence of the peptide. The effect of endothelin was concentration dependent and similar to those observed with other vasoconstrictor peptides (vasopressin and angiotensin II). The results indicate that endothelin does not directly act on voltage-dependent calcium channels. The endothelin-stimulated calcium efflux suggests a mobilization of calcium from intracellular store sites followed by extrusion through an activation of a specific receptor-dependent calcium channel.