Role of redox signaling and poly (adenosine diphosphate-ribose) polymerase activation in vascular smooth muscle cell growth inhibition by nitric oxide and peroxynitrite.
OBJECTIVE
The vascular mediator, nitric oxide regulates vascular smooth muscle cell proliferation and can react with superoxide to form peroxynitrite, a highly reactive free radical. The intracellular mechanisms by which nitric oxide and peroxynitrite inhibit smooth muscle cell growth remain undefined, as is the potential role of peroxynitrite formation in the antiproliferative effects of nitric oxide. We sought to define the intracellular effects and signaling mechanisms of nitric oxide and peroxynitrite in smooth muscle cells.


METHODS
Cultured rat aortic smooth muscle cells were treated with exogenous nitric oxide or peroxynitrite and inhibitors of nitric oxide and redox signaling pathways. Cell growth, DNA synthesis, apoptosis, cyclic guanosine 3'-5' monophosphate (cGMP) levels, poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) activity, and cytotoxicity were assayed. Peroxynitrite formation was determined by nitrotyrosine immunoblotting. Vasoreactivity was assessed in isolated rat aortic rings after treatment with nitric oxide/peroxynitrite and redox agents.


RESULTS
Both exogenous nitric oxide and peroxynitrite decreased cell growth and DNA synthesis of cultured rat aortic smooth muscle cells, but peroxynitrite-induced growth arrest was irreversible and associated with apoptosis and cytotoxicity. Inhibition of guanylate cyclase, PARP activity, mitogen-activated protein kinase, or bypass of ornithine decarboxylase did not reverse growth arrest by nitric oxide. The antioxidants N-acetylcysteine, ascorbate, and glutathione selectively reversed growth inhibition by nitric oxide but not by peroxynitrite. Antioxidants did not impair nitric oxide-induced cGMP generation in smooth muscle cells or nitric oxide-induced vasodilatation of isolated aortic rings. Nitric oxide treatment did not result in peroxynitrite formation and augmentation of superoxide levels did not induce peroxynitrite-like effects. Peroxynitrite-induced cytotoxicity and apoptosis were not reversed by antioxidants or PARP inhibition, because peroxynitrite activated PARP in J774 macrophages but failed to activate PARP in smooth muscle cells.


CONCLUSIONS
Exogenous nitric oxide induces reversible cytostasis in smooth muscle cells by a redox-sensitive mechanism independent of peroxynitrite formation and distinct from the nitric oxide vasodilating mechanism. Peroxynitrite does not activate PARP selectively in smooth muscle cells and induces redox-independent smooth muscle cell cytotoxicity and apoptosis. Thus, the antiproliferative effects of nitric oxide and peroxynitrite on smooth muscle cells use divergent intracellular pathways with distinct redox sensitivities. These findings are relevant to the pathogenesis of vascular disease and the potential application of nitric oxide-based therapy for vascular disease.


CONCLUSIONS
Vascular smooth muscle cell proliferation is an important component of atherosclerosis, vein graft failure, and arterial restenosis, and is known to be regulated by the vascular signaling molecule nitric oxide. Nitric oxide can combine with the free radical superoxide to form the unstable metabolite peroxynitrite, which has been detected in human vascular lesions. This study examines the role of peroxynitrite in mediating the antiproliferative effects of nitric oxide. We identify important differences in the effects and intracellular mechanisms of nitric oxide and peroxynitrite in regulating vascular smooth muscle cell proliferation and programmed cell death. Defining the differential effects of these free radicals in vascular cells is important to our understanding of the pathogenesis of vascular disease and the development of novel therapy aimed at treating proliferative vascular lesions.
Huang
James
J
Department of Surgery, San Francisco Veteran's Affairs Medical Center, Division of Vascular Surgery, University of California, San Francisco, USA.
Lin
Stephanie C
SC
Nadershahi
Afshin
A
Watts
Stephanie W
SW
Sarkar
Rajabrata
R
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D004261:DNA Replication; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000378:metabolism*; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D013481:Superoxides / Q000378:metabolism; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D014664:Vasodilation
D000975:Antioxidants; D004791:Enzyme Inhibitors; D020030:Nitric Oxide Donors; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D013481:Superoxides; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D011065:Poly(ADP-ribose) Polymerases; D006152:Cyclic GMP