NITRIC OXIDE INDUCES ADP-RIBOSYLATION OF SEVERAL PROTEINS IN MURINE MACROPHAGES

Abstract

Nitric oxide (NO) is a free radical molecule generated during the metabolism of L-arginine that has been implicated in a variety of physiological processes. NO stimulates adenosine diphosphate (ADP)-ribosylation in various cells and tissues. Although the production of NO by murine macrophages is well established (Drapier, et at., 1988; Ding, et al., 1988; Cox, et al., 1990; Narumi, et al., 1990; Belosevic, et al., 1990), the potential effects of NO on macrophage biology are poorly understood. The ability of NO to induce ADP-ribosylation in cytosolic fractions of the murine macrophage cell line ANA-1 was examined. The novel compound diethylamine dinitric oxide (DEA/NO), which releases NO in aqueous solution at neutral pH, was used to demonstrate a specific effect of NO. DEA/NO stimulated the ADP-ribosylation of at least three cytosolic proteins (molecular weights 28,000, 33,000, and 39,000) from ANA-1 macrophages. The effect of DEA/NO on the ADP-ribosylation of the predominant target p39 was dose dependent (E₅₀ = 80 µM). Moreover, the effect of DEA/NO was attributed specifically to released NO rather than diethylamine or nitrite. Sodium nitroprusside (SNP) also stimulated the ADP-ribosylation of cytosolic proteins from ANA-1 mouse macrophages. However, SNP exhibited different time- and dose-dependent effects on the modification of p39. NO synthesized via the activity of interferon-y plus lipopolysaccharide-induced NO synthase also enhanced the ADP-ribosylation of p39, confirming that the effects of DEA/NO and SNP could be attributed to NO or reactive nitrogen oxide species. Neither pertussis toxin nor cholera toxin stimulated the ADP-ribosylation of p39; however, cholera toxin stimulated the ADP-ribosylation of proteins with approximate molecular weights of 28,000 and 33,000. Preliminary amino acid sequencing and western blotting suggest that the identity of p39 and p28 are glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate mutase, respectively. These data suggest that the induced expression of NO synthase in activated macrophages may be associated with autocrine or paracrine effects of NO that include the ADP-ribosylation of various proteins. Moreover, these results indicate that DEA/NO and related compounds may be useful as pharmacologic tools for investigating the effects of NO and reactive nitrogen oxide species on macrophages.