Nitric oxide derived from L-arginine impairs cytoplasmic pH regulation by vacuolar-type H+ ATPases in peritoneal macrophages

The ability of macrophages (Mos) to function within an acidic environment has been shown to depend on cytoplasmic pH (pHi) regulation by vacuolar-type H+ ATPases. Mos metabolize L-arginine via an oxidative pathway that generates nitric oxide, nitrate, and nitrite. Since each of these products could potentially inhibit vacuolar-type H+ ATPases, we investigated the effect of L-arginine metabolism on Mo pHi regulation in thioglycolate-elicited murine peritoneal Mos. H+ ATPase- mediated pHi recovery from an imposed cytoplasmic acid load was measured fluorometrically. When Mos were incubated with L-arginine (0.25-2.0 mM), their rate of pHi recovery declined progressively from 2 to 6 h of incubation. By contrast, the recovery rate of cells incubated in arginine-free medium remained stable over the same period. The impairment of pHi recovery was specific for L-arginine, and was blocked competitively by NG-monomethyl-L-arginine, demonstrating its dependence on L-arginine metabolism. In addition, the inhibition of pHi recovery was enhanced by lipopolysaccharide, an agent known to stimulate L- arginine metabolism by Mos. Scavenging the L-arginine metabolite nitric oxide with either ferrous sulphate or ferrous myoglobin prevented the inhibition of pHi recovery, implying that L-arginine-derived nitric oxide was the species responsible for the inhibition. This concept was supported by the finding of elevated nitrite levels in the supernatant of cells incubated in L-arginine. Furthermore, incubation of Mos with sodium nitroprusside mimicked the L-arginine-dependent inhibition of H+ ATPase activity. Treatment with the cyclic GMP analogue, 8- bromoguanosine 3':5'-cyclic monophosphate, similarly impaired Mo pHi recovery, suggesting that a nitric oxide-stimulated elevation of cyclic GMP may contribute to the L-arginine-dependent inhibition of pHi regulation.