The purpose of this study was to determine the involvement of the p42͞p44 mitogen-activated protein kinase (MAPK) pathway and induction of p21 waf1/cip1 in the antiproliferative effects of nitric oxide (NO) on rat aortic smooth muscle cells (RASMC). NO, like ␣-difluoromethylornithine (DFMO), interferes with cell proliferation by inhibiting ornithine decarboxylase (ODC) and, therefore, polyamine synthesis. S-nitroso-N-acetylpenicillamine or (Z)-1-[N-(2-aminoethyl)-N-(2-aminoethyl)-amino]-diazen-1-ium-1,2-diolate inhibited RASMC growth at concentrations as low as 3 M, and DFMO elicited effects at concentrations of 100 M or greater. The cytostatic effect of NO and DFMO was prevented by the MAPK kinase 1͞2 inhibitors PD 098,059 or U0126. This finding suggests that the p42͞p44 MAPK pathway is involved in the inhibition of RASMC proliferation by NO. Western blot analysis revealed that treatment of RASMC with NO or DFMO leads to activation of p42͞p44 MAPK and induction of p21 waf1/cip1 . This effect was prevented by MAPK kinase 1͞2 inhibitors, suggesting that induction of p21 waf1/cip1 depended on activation of p42͞p44. Moreover, activation of p42͞p44 and induction of p21 waf1/cip1 were prevented by exogenous putrescine but not ornithine, suggesting this effect was due to the inhibition of ODC by NO or DFMO. Finally, activation of p42͞p44 MAPK and induction of p21 waf1/cip1 were cGMPindependent. Neither 1H-(1,2,4)oxadiazolo[4,3-␣]quinoxalin-1-one nor zaprinast influenced the cytostatic effect of NO or DFMO or their ability to activate these signal transduction pathways. These observations suggest that inhibition of ODC and accompanying putrescine production are the underlying mechanisms by which NO and DFMO activate the MAPK pathway to promote induction of p21 waf1/cip1 and consequent inhibition of cell proliferation.polyamines ͉ ornithine decarboxylase ͉ atherosclerosis ͉ MAPK V ascular smooth muscle cells (VSMC) are normally quiescent in the adult, rarely exhibiting mitogenic activity in their contractile state (1). However, vascular injury stimulates VSMC growth by disrupting the physiological balance between inhibition and stimulation. Over-proliferation of VSMC contributes to many clinical vascular pathologies, the causes of which vary from mechanical (restenosis after balloon angioplasty), to immunological (transplant arteriosclerosis), to multifactorial (atherosclerosis). Increased VSMC proliferation has been attributed to dysfunction of or damage to the endothelial lining in the area of intimal thickening and, more specifically, to decreased nitric oxide (NO) production. This effect is made evident by several studies in which NO donor agents or NO synthase gene transfer were shown to inhibit neointimal formation after balloon angioplasty in animal models (2-4). The effect inverse has been shown in studies where animals were subjected to balloon angioplasty and then treated with inhibitors of NO synthase (4). In these studies, intimal formation was enhanced by the inhibition of NO production.The mechanism by whi...