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 progres sion. Treatment of cultured rat aortic SMC with the NO donors S-nitros o-N-acetylpenicillamine or S-nitrosoglutathione (0.1 mM for 48 h) resu lted in a 50% decrease (P < 0.05) in the fraction of cells in the S an d G(2)+M phases and a corresponding increase in the GL fraction, sugge sting that NO inhibits entry into 8 phase, causing accumulation of cel ls in G(1) phase. Application of both NO donors to cycling SMC resulte d in a short-term, concentration-dependent (0.06-0.3 mM) inhibition of ongoing DNA synthesis as measured by radiothymidine incorporation, de monstrating that NO causes an S-phase arrest. The S-phase arrest by NO was not mimicked by exogenous guanosine 3',5'-cyclic monophosphate (c GMP, 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 ribonucleot ide reductase inhibitor hydroxyurea. Prolonged treatment of SMC with e ither NO donor (0.1 mM) did not synchronize cells at the G(1)-S bounda ry as expected after a prolonged S-phase arrest, but instead induced a quiescent G(0)-like state characterized by a 12- to 18-h lag before D NA synthesis returned to normal levels after NO removal. These finding s demonstrate that NO inhibition of SMC proliferation is associated wi th two distinct and reversible cell cycle arrests, an immediate cGMP-i ndependent S-phase block followed by a shift back in the cell cycle fr om the G(1)-S boundary to a quiescent G(0)-like state.