Up-regulation of Na +-dependent Mg 2+ transport by nitric oxide and cyclic GMP pathway in renal epithelial cells

Abstract

A putative, Na +-dependent Mg 2+ transport pathway controls the intracellular free Mg 2+ concentration ([Mg 2+] i) in various mammalian cells. The characteristics of this Mg 2+ transport pathway have not been clarified. Herein, we examined the regulatory mechanism of Na +-dependent Mg 2+ efflux in renal epithelial NRK-52E cells. Mg 2+ removal from the extracellular bathing solution induced an Na +-dependent [Mg 2+] i decrease in Mg 2+ (5 mM)-loaded cells but not in control cells. Amiloride inhibited the [Mg 2+] i decrease in a dose-dependent manner (IC 50=3 μM). Similarly, atomic absorption spectrophotometry showed that Mg 2+ removal decreased intracellular Mg 2+ content, while it increased Na + content. Calphostin C (1 μM), a protein kinase C inhibitor, and genistein, a tyrosine kinase inhibitor (10 μM), blocked the [Mg 2+] i decrease. The [Mg 2+] i decrease was accompanied by an increase in intracellular nitric oxide (NO) and cyclic GMP contents. ( E)-4-methyl-2-[( E)-hydoxyimino]-5-nitro-6-methoxy-3-hexenamide (0.1 mM), an NO donor, and 8-bromo-cyclic GMP (0.1 mM), a membrane-permeable cyclic GMP analogue, accelerated the [Mg 2+] i decrease. In contrast, N G-monomethyl- l-arginine ( l-NMMA, 0.1 mM), an NO competitive inhibitor, and 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxaline-1-one (ODQ, 10 μM), an NO-sensitive guanylate cyclase inhibitor, significantly blocked the [Mg 2+] i decrease. These results indicate that a decrease in extracellular Mg 2+ concentration induces the production of NO and cyclic GMP, which leads to the up-regulation of Na +-dependent Mg 2+ efflux.