Endothelin binding and receptor down regulation in rat glomerular mesangial cells
Baldi, E.; Dunn, M.J.
Journal of Pharmacology and Experimental Therapeutics 256(2): 581-586
1991
ISSN/ISBN: 0022-3565 PMID: 1847203 Document Number: 378058
Endothelin (RT) exerts various biological actions in mesangial cells, including stimulation of proliferation, contraction and phospholipase C activation. We investigated the presence of specific ET receptors on cultured rat mesangial cells, incubating the cells in the presence of [125I]ET-1 both at 22 and 4.degree. C. ET binding was time- and temperature-dependent and achieved equilibrium at 2 hr at 22.degree. C and at 5 hr at 4.degree. C. Scatchard analyses of equilibrium saturation curves with [125I]ET-1 and homologous competition curves revealed the presence of a single class of high-affinity binding sites (Kd = 31.4 .+-. 7.08 pM). Heterologous competition experiments with ET-3 and sarafotoxin, however, indicated the presence of two binding sites for ET-related peptides in mesangial cells with a Kd for ET-3 of 41.5 .+-. 19.2 and of 374 .+-. 38.5 nM. Nifedipine and arginine-vasopressin failed to compare for ET binding sites. Preincubation of the cells with 1 nM ET-1 caused a dramatic decrease in ET binding capacity (from 0.5-0.02 fmol/100,000 cells) without affecting the Kd for the receptors (38 pM). ET receptor down regulation was not prevented by protein kinase C inhibition with H-7 and sangiovamycin, or after down regulation of protein kinase C induced by 24-hr preincubation with phorbol myristate acetate. ET receptor down regulation also exerts functional effects, as we found a decrease in intracellular-free calcium response to ET-1 after long-term preincubation with the same agonist. Our results are consistent with the presence of two binding sites for ET in rat mesangial cells. These receptors can be down regulated after exposure of the cells to low concentrations of the agonist, inducing a decrease in the posterior signaling, i.e., the intracellular calcium response to ET.