Endothelin increases NO-dependent cGMP production in isolated glomeruli but not in mesangial cells

Abstract

The ability of endothelins (ETs) to modulate nitric oxide-dependent glomerular guanosine 3'-5'-cyclic monophosphate (cGMP) production has recently been reported. The aim of this study was to directly confirm, using an antagonist, the involvement of the ETB receptor subtype and to investigate the potential role of mesangial cells (MC) in this ET-induced cGMP production. In glomeruli freshly isolated from rats, endothelin-3 (ET-3) induced a dose-dependent increase in cGMP content. This increase was inhibited by NG-monomethyl-L-arginine (L-NMMA) and methylene blue and was calcium dependent. Moreover, the effect of ET-3 was prevented by two ETB-selective receptor antagonists, BQ-788 and IRL-1038, but not by BQ-123, an ETA-selective receptor antagonist. It therefore appeared that ET-3 stimulates the glomerular constitutive NO pathway through activation of the ETB receptor subtype. In contrast, ET-3 and calcium ionophore had no effect on cGMP formation in cultured MC, whereas incubation with sodium nitroprusside resulted in an approximately 50-fold increase in the intracellular content of cGMP. However, ET-3 induced a dose-dependent rise in free MC cytosolic calcium that was abolished by an ETB antagonist. Moreover, both ETA and ETB receptors mRNA were expressed in primary cultures of MC. Finally, we failed to detect the presence of constitutive NO synthase (NOS), as demonstrated by the absence of L-citrulline forming activity and of the mRNA encoding for endothelial NOS, whereas they were present in isolated glomeruli. These data indicate that MC, despite the fact that they express ETB receptors, are not involved in glomerular NO production induced by exposure to ET-3, because they do not express constitutive NO synthase.