DUAL CELL CYCLE-SPECIFIC MECHANISMS MEDIATE THE ANTIMITOGENIC EFFECTSOF NITRIC-OXIDE IN VASCULAR SMOOTH-MUSCLE CELLS

Objective To determine the cell cycle specificity and intracellular me chanisms involved in inhibition by nitric oxide (NO) of vascular smoot h muscle cell mitogenesis, Methods Cultured rat aortic smooth muscle c ells 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 measur ed 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 a ntagonists of free NO to reverse the effects of NO donors was also eva luated, Bypass of ribonucleotide reductase by use of exogenous deoxynu cleosides was attempted to determine whether inhibition of this S-phas e enzyme was the mechanism by which NO inhibited DNA synthesis during the S phase, Results Vascular smooth muscle cell mitogenesis was inhib ited by cyclic GMP (cGMP) up to late G(1) phase of the cell cycle, whi ch 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 p oint 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 antag onists and was not observed with non-NO analogs of the donors, Inhibit ion by NO donors in S phase was neither reversed by the guanylate cycl ase inhibitor methylene blue nor mimicked by exogenous cGMP, The S-pha se inhibition by all three NO donors was reversed partially by bypass of ribonucleotide reductase, establishing this enzyme as an S-phase ta rget of NO, Conclusions These findings demonstrate that NO inhibits sm ooth muscle mitogenesis by cGMP-dependent and -independent mechanisms acting at distinct points in the cell cycle, NO is the first endogenou s substance to have been shown to inhibit mitogenesis beyond the restr iction point in late G(1) phase, suggesting that it plays a role in re gulation of cells that have lost normal mechanisms of G(1) growth cont rol, such as the hyperproliferative smooth muscle cells noted in hyper tension and restenosis.