INDUCTION OF THE CYCLIN-DEPENDENT KINASE INHIBITOR P21(SDI1 CIP1/WAF1) BY NITRIC OXIDE-GENERATING VASODILATOR IN VASCULAR SMOOTH-MUSCLE CELLS/

Nitric oxide-generating vasodilators inhibit vascular smooth muscle ce ll proliferation. To elucidate the mechanism underlying this process, we investigated the effect of S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide-releasing agent, on the smooth muscle cell cycle. When G(0) cells were stimulated with fetal bovine serum and basic fibroblas t growth factor, DNA synthesis assessed by [H-3]thymidine incorporatio n started about 15 h later. SNAP dose-dependently inhibited this incor poration, and this effect was maximal at 100 mu M. This inhibition was attenuated when SNAP was added after 9-12 h. SNAP inhibited the activ ity of cyclin-dependent kinase 2 (Cdk2) and phosphorylation of the ret inoblastoma protein, both of which usually increased from about 9 h, w hereas it did not inhibit the activities of cyclin D-associated kinase (s), Cdk4, and Cdk6, which normally increased from 0-3 h. Although SNA P reduced the mRNA levels of cyclins E and A, it neither reduced their protein levels nor impaired their association with Cdk2. SNAP did not reduce the mRNA levels of cyclins G, C, and D1, Cdk2, Cdk4, and Cdk5, which were normally elevated from 0-3 h. The mRNA and protein levels of the Cdk inhibitor p21 were high in the early G, phase, peaking at 3 h and then rapidly decreasing after 6 h. In the presence of SNAP, how ever, p21 expression was enhanced, and moreover, the later decrease di sappeared. SNAP also increased the amount of Cdk2-associated p21. Thes e results suggested that nitric oxide inhibits the G(1)/S transition b y inhibiting Cdk2-mediated phosphorylation of the retinoblastoma prote in and that p21 induction is involved in the Cdk2 inhibition.