2-2--(hydroxynitrosohydrazono)bis-ethanamine and peroxynitric-acid

The purpose of this study was to determine the effects of sodium nitroprusside (SNP), 2,2'-(hydroxynitrosohydrazino)bis-ethanamine (DETA/NO) and 3-morpholinosydnonimine (SIN-1), NO donors which yield different NO reactive species (NO+, NO* and peroxynitrite, respectively), as well as exogenous peroxynitrite, on gall bladder contractility. Under resting tone conditions, SNP induced a dose-dependent contraction with a maximal effect (10.3 +/- 0.7 mN, S.E.M.) at 1 mM. Consistent with these findings, SNP caused a concentration-dependent depolarization of gall bladder smooth muscle. The excitatory effects of SNP were dependent on extracellular calcium entry through L-type Ca2+ channels. Furthermore, the contraction and depolarization were sensitive to tyrosine kinase blockade, and an associated increase in tyrosine phosphorylation was detected in Western blot studies. DETA/NO induced dose-dependent relaxing effects. These relaxations were sensitive to the guanylyl cyclase inhibitor 1H-[1,2,4]oxidiazolo[4,3-a]quinoxaline-1-one (ODQ, 2 microM) but they were not altered by treatment with the potassium channel blockers tetraethylammoniun (TEA, 5 mM) and 4-aminopyridine (4-AP, 5 mM). When tested in a reducing environment (created by 2.5 mM 1,4-dithiothreitol, DTT), SNP caused a relaxation of gall bladder muscle strips. Similarly, the SNP-induced contraction was converted to a relaxation, and associated hyperpolarization, when DTT was added during the steady state of an SNP-induced response. SIN-1 (0.1 mM), which has been shown to release peroxynitrite, induced relaxing effects that were enhanced by superoxide dismutase (SOD, 50 U ml(-1)). The relaxations induced by either SIN-1 alone or SIN-1 in the presence of SOD were strengthened by catalase (1000 U ml(-1)) and abolished by ODQ pretreatment. However, exogenous peroxynitrite induced a concentration-dependent contraction, which was dependent on activation of leukotriene (LT) metabolism and extracellular calcium. The peroxynitrite-induced contraction was abolished in the presence of the peroxynitrite scavenger melatonin. These results suggest that SIN-1 behaves as an NO* rather than a peroxynitrite source. We conclude that, depending on the redox state, NO has opposing effects on the motility of the gall bladder, being a relaxing agent when in NO * form and a contracting agent when in NO+ or peroxynitrite redox species form. Knowledge of the contrasting effects of the different redox forms of NO can clarify our und

Topics: Acetylcholine; Animals; Arachidonic Acid; Atropine; Calcium; Gallbladder; Guinea Pigs; Male; Molsidomine; Muscle Contraction; Muscle, Smooth; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Nitroso Compounds; Oxidants; Oxidation-Reduction; Parasympatholytics; Vasodilator Agents

Leukocyte accumulation has been shown to be increased in sepsis. Moreover, in inducible nitric oxide synthase (iNOS) knockout mice, a further increase in leukocyte accumulation has been observed during sepsis, suggesting that nitric oxide (NO) may affect leukocyte/endothelial interaction. Accelerated peroxynitrite formation also occurs during sepsis. In the present study, the effect of peroxynitrite or NO on leukocyte adhesion to nitric oxide synthase (NOS)-inhibited or endotoxin-treated endothelium was examined. Bovine aortic endothelial cells were treated with either L-NAME or lipopolysaccharide (LPS) and interferon-gamma for 4 hr and subsequent leukocyte adhesion was measured. Both L-NAME and LPS treatment resulted in increased leukocyte adhesion compared with control. Neither a peroxynitrite donor, SIN-1, nor a direct NO donor, DETA-NO, had any effect on leukocyte adhesion to untreated endothelium. However, when the L-NAME or LPS-treated endothelial cells were treated simultaneously with either SIN-1 or DETA-NO, there was a significant reduction in leukocyte adhesion. Moreover, at the concentrations used in the present study, neither peroxynitrite nor NO showed harmful effects on normal cultured endothelial cells. These data demonstrating inhibition of leukocyte adhesion to endotoxin-treated endothelium suggest that peroxynitrite or NO may exert a beneficial effect during sepsis.

Topics: Animals; Cattle; Cell Adhesion; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Enzyme Inhibitors; Interferon-gamma; Leukocytes; Lipopolysaccharides; Molsidomine; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Triazenes

The survival of skeletal muscle myoblasts in culture after exposure either to a donor of NO, sodium nitroprusside (SNP), or ethanamine, 2,2'-(hydroxynitrosohydrazono)bis-(DETA NONOate), or to a donor of both NO and O(-)(2), 3-morpholinosydnonimine hydrochloride (SIN-1), was investigated. SIN-1 reduced clonogenic survival markedly but donors of NO alone did not. The injurious effect of SIN-1 was prevented by oxyhemoglobin or by uric acid but not by superoxide dismutase. The exposure of myoblasts to authentic peroxynitrite (ONOO(-)) or to DETA NONOate in the presence of an O(-)(2)-generating system did not reduce their survival. The results show that NO or ONOO(-) alone is not detrimental to myoblast survival and suggest that SIN-1 toxicity is, at least in part, mediated by H(2)O(2) in this myoblast culture system.

Topics: Animals; Cell Line; Cell Survival; Molsidomine; Muscle, Skeletal; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Oxyhemoglobins; Rats; Superoxide Dismutase; Superoxides; Uric Acid; Xanthine Oxidase

Protein tyrosine phosphatases (PTPs) contain an essential thiol in the active site which may be susceptible to attack by nitric oxide-derived biological oxidants. We assessed the effects of peroxynitrite, nitric oxide, and S-nitrosoglutathione on the activity of three human tyrosine phosphatases in vitro. The receptor-like T-cell tyrosine phosphatase (CD45), the non-receptor-like tyrosine phosphatase PTP1B, and leukocyte-antigen-related (LAR) phosphatase were all irreversibly inactivated by peroxynitrite in less than 1 s with IC(50) values of

Topics: Aniline Compounds; Anions; Dithiothreitol; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glutathione; Humans; Leukocyte Common Antigens; Models, Molecular; Molsidomine; Nitrates; Nitric Oxide; Nitroso Compounds; Organophosphates; Organophosphorus Compounds; Protein Tyrosine Phosphatases; Receptor-Like Protein Tyrosine Phosphatases, Class 4; Receptors, Cell Surface; S-Nitrosoglutathione; Superoxide Dismutase; Tyrosine