Dual cell cycle-specific mechanisms mediate the antimitogenic effects of nitric oxide in vascular smooth muscle cells

Objective To determine the cell cycle specificity and intracellular mechanisms involved in inhibition by nitric oxide (NO) of vascular smooth muscle cell mitogenesis, Methods Cultured rat aortic smooth muscle cells were synchronized by serum withdrawal, treated with the NO donor S-nitroso-N-acetylpenicillamine and the cyclic GMP analog 8-Br-cGMP at various times during cell cycle progression, and DNA synthesis measured during the S phase, Two additional NO donors, 5-nitroso-glutathione and diethylamine NONOate, were used to confirm the inhibition of DNA synthesis by S-nitroso-N-acetylpenicillamine, and the ability of two antagonists of free NO to reverse the effects of NO donors was also evaluated, Bypass of ribonucleotide reductase by use of exogenous deoxynucleosides was attempted to determine whether inhibition of this S-phase enzyme was the mechanism by which NO inhibited DNA synthesis during the S phase, Results Vascular smooth muscle cell mitogenesis was inhibited by cyclic GMP (cGMP) up to late G(1) phase of the cell cycle, which corresponded to the point of greatest sensitivity to exogenous NO, In contrast to cGMP, three different NO donors inhibited DNA synthesis when added to cells synchronized in S phase, beyond the restriction point of cell cycle control in late G(1) phase. This S-phase inhibition was reversible by removal of the NO donor or addition of two NO antagonists and was not observed with non-NO analogs of the donors, Inhibition by NO donors in S phase was neither reversed by the guanylate cyclase inhibitor methylene blue nor mimicked by exogenous cGMP, The S-phase inhibition by all three NO donors was reversed partially by bypass of ribonucleotide reductase, establishing this enzyme as an S-phase target of NO, Conclusions These findings demonstrate that NO inhibits smooth muscle mitogenesis by cGMP-dependent and -independent mechanisms acting at distinct points in the cell cycle, NO is the first endogenous substance to have been shown to inhibit mitogenesis beyond the restriction point in late G(1) phase, suggesting that it plays a role in regulation of cells that have lost normal mechanisms of G(1) growth control, such as the hyperproliferative smooth muscle cells noted in hypertension and restenosis.