Cell cycle effects of nitric oxide on vascular smooth muscle cells

We characterized the cell cycle block induced by nitric oxide (NO) on smooth muscle cells (SMC). We hypothesized that the inhibition of SMC proliferation by NO was due to a specific block in cell cycle progression. Treatment of cultured rat aortic SMC with the NO donors S-nitroso-N-acetylpenicillamine or S-nitrosoglutathione (0.1 mM for 48 h) resulted in a 50% decrease (P < 0.05) in the fraction of cells in the S and G2 + M phases and a corresponding increase in the G1 fraction, suggesting that NO inhibits entry into S phase, causing accumulation of cells in G1 phase. Application of both NO donors to cycling SMC resulted in a short-term, concentration-dependent (0.06-0.3 mM) inhibition of ongoing DNA synthesis as measured by radiothymidine incorporation, demonstrating that NO causes an S-phase arrest. The S-phase arrest by NO was not mimicked by exogenous guanosine 3',5'-cyclic monophosphate (cGMP, 10 mM) and was associated with, but not due to, a 20% inhibition of RNA synthesis. The S-phase block was completely reversed within 2 h of removal of the NO donors, similar to inhibition by the ribonucleotide reductase inhibitor hydroxyurea. Prolonged treatment of SMC with either NO donor (0.1 mM) did not synchronize cells at the G1-S boundary as expected after a prolonged S-phase arrest, but instead induced a quiescent G0-like state characterized by a 12- to 18-h lag before DNA synthesis returned to normal levels after NO removal. These findings demonstrate that NO inhibition of SMC proliferation is associated with two distinct and reversible cell cycle arrests, an immediate cGMP-independent S-phase block followed by a shift back in the cell cycle from the G1-S boundary to a quiescent G0-like state.