Role of redox signaling and poly (adenosine diphosphate-ribose) polymerase activation in vascular smooth muscle cell growth inhibition by nitric oxide and peroxynitrite

Nitric oxide and peroxynitrite and smooth muscle cell growth and apoptosis. Smooth muscle cells were exposed to peroxynitrite (ONOO) and S-nitroso-N-acetylpenicillamine (SNAP) as indicated and either (A) cell counts were determined after 48 hours or (B) DNA synthesis was assayed after 18 hours (*P < .05 vs control). C, Cells were treated for 24 hours with the indicated agents, after which the media was replaced with fresh growth media daily and cell counts were determined after 2 and 7 days. (*P < .05 vs day 2 control, **vs day 7 control). Smooth muscle cells were exposed to peroxynitrite and SNAP as indicated for 18 hours and (D) lactate dehydrogenase (LDH) release and (E) DNA fragmentation was determined (*P < .05 vs respective controls). F, Detection of sub-G1 apoptotic nuclei (black arrows) by flow cytometry after peroxynitrite treatment as indicated for 18 hours. All data are mean ± SD of triplicate wells from one of four to eight similar experiments.

Effect of antioxidants on S-nitroso-N-acetylpenillamine (SNAP)-induced growth arrest, cyclic guanosine 3′-5′ monophosphate (cGMP) levels, and vasodilation. Smooth muscle cells were treated with the indicated combinations of SNAP (0.2 mM), ascorbate (0.5 mM), N-acetyl-cysteine (NAC; 0.1 mM), and glutathione (0.1 mM), and assays performed for (A) DNA synthesis after 18 hours, (B) cell proliferation after 48 hours, and (C) cGMP levels after 30 minutes. (*P < .05 vs SNAP alone). Data are mean ± SD of triplicate wells from one of four to eight similar experiments. D, Vasorelaxation in rat aorta treated with increasing concentrations of SNAP in the presence of the antioxidants ascorbate, NAC, and glutathione (all 0.1 mM). Data are mean ± SD of six to nine rings per experimental group. (*P < .05 vs SNAP alone).

Effect of antioxidants on the antiproliferative effect of peroxynitrite. A, Cells were treated with peroxynitrite (ONOO; 270 μM) plus either ascorbate (0.5 mM), N-acetyl-cysteine (NAC; 0.1 mM), or glutathione (0.1 mM), and DNA synthesis was measured. B, Cells were treated with peroxynitrite (180uM) plus either ascorbate (0.5 mM), NAC (0.5 mM) or glutathione (0.5 mM) for 48 hours and cell counts were determined. C, Cells were treated with peroxynitrite (360uM) plus either ascorbate (0.5 mM), NAC (0.5 mM), or glutathione (0.5 mM) for 18 hours, and lactate dehydrogenase (LDH) release was measured as described (*P < .05 vs control). Data are mean ± SD of triplicate samples from one of four to eight similar experiments.

Effect of poly (adenosine diphosphate-ribose) polymerase (PARP) inhibition on growth inhibition by peroxynitrite and S-nitroso-N-acetylpenillamine (SNAP). A, Cells were treated with peroxynitrite (270 μM), SNAP (0.2 mM), and 3-aminobenzamide (3-AB, 1 mM), and DNA synthesis was determined. B, Smooth muscle cells were treated with peroxynitrite (360 μM) with or without 3-AB (1 mM) for 18 hours and lactate dehydrogenase (LDH) release was measured. C, Apoptosis was determined using DNA fragmentation assay after exposure of smooth muscle cells to peroxynitrite (135 μM) with or without 3-AB (1 mM). D, Effect of peroxynitrite on PARP enzyme activity in smooth muscle cells and macrophages (J774) after a 15-minute exposure to the indicated agents. *P < .05 vs control. Data are mean ± SD.

Role of nitric oxide signaling pathways and superoxide in the antiproliferative effect of S-nitroso-N-acetylpenillamine (SNAP). A, Growth arrested smooth muscle cells were treated with growth media containing the experimental agents indicated for 18 hours before analysis of DNA synthesis. SNAP (0.2 mM), PD 98059 (mitogen-activated protein kinase inhibitor, 0.1 mM), and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, guanylate cyclase inhibitor, 1 μM). B, Smooth muscle cells in growth media were treated with SNAP (0.1 mM), putrescine (1 mM) and α-difluoromethylornithine (DMFO, 0.3 mM) in the combinations indicated for 48 hours and cells counts determined. C, Cells were treated with experimental agents as indicated for 18 hours and DNA synthesis measured (*P < .05 vs .05 SNAP, **P < .05 vs 0.2 SNAP). Xanthine (100 μM) and xanthine oxidase (XO, 1 mU/mL), superoxide dismutase (SOD, 50 U/mL). (D) Cells were treated with SNAP (0.2 mM) ± xanthine and XO (100 μM, 1 mU/mL) for 18 hours and lactate dehydrogenase (LDH) release was measured. All data are mean ± SD of triplicate determinations from one of three to six similar experiments.

Detection of nitrotyrosined proteins and cytotoxicity. Cells were treated with experimental agents as indicated and harvested after 18 hours for detection of nitrated proteins. Nitrotyrosine bovine serum albumin (NT-BSA) was used as positive control. A, Blot shown is representative of five independent experiments. Smooth muscle cells were treated with the indicated agents for 90 minutes and membrane function and permeability was assessed as described. SNAP, S-nitroso-N-acetylpenillamine; ONOO, peroxynitrite. A, Control; (B), SNAP, 1.0 mM; (C), peroxynitrite, 280 μM; (D), peroxynitrite, 980 μM; (E), methanol (positive control). Photomicrographs are from one of four similar experiments at ×100 original magnification.