sin-1c and linsidomine

We examined the ability of peroxynitrite and other .NO-derived oxidants to inhibit creatine kinase (CK). Peroxynitrite potently inhibited CK activity and depleted protein thiols. The rate constant for this reaction was 8.85x10(5) M(-1) s(-1). Glutathione did not reactivate CK activity nor did it regenerate protein thiol content. In contrast, glutathione reactivated CK, and regenerated protein thiols, after inhibition by either .NO or oxidized glutathione (GSSG). Peroxynitrite did not irreversibly inhibit CK after it had been treated with GSSG to block protein thiols. We conclude that thiol oxidation is a critical event leading to inactivation of CK by peroxynitrite.

Topics: Acetonitriles; Animals; Creatine Kinase; Enzyme Activation; Enzyme Inhibitors; Enzyme Reactivators; Glutathione; Glutathione Disulfide; Kinetics; Molsidomine; Morpholines; Nitrates; Nitric Oxide; Oxidants; Rabbits; Sulfhydryl Compounds; Superoxides

This study was designed to clarify the mechanism of the inhibitory action of a nitric oxide (NO) donor 3-morpholino-sydnonimine (SIN-1) on human neutrophil degranulation. SIN-1 (100-1000 microM) inhibited degranulation (beta-glucuronidase release) in a concentration-dependent manner and concomitantly increased the levels of cGMP in human neutrophils in suspension. However, further studies suggested that neither NO nor increase in cGMP levels were mediating the inhibitory effect of SIN-1 on human neutrophil degranulation because 1) red blood cells or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl added as NO scavengers did not inhibit the effect; 2) inhibitors of cGMP synthesis (methylene blue) or phosphodiesterases (3-isobutyl-1-methylxanthine) did not produce changes in cell function correlating with the changes in cGMP. SIN-1 releases both nitric oxide and superoxide, which together form peroxynitrite. Chemically synthesized peroxynitrite (1-100 microM) did not inhibit, but at high concentrations (1000-2350 microM), it potentiated FMLP-induced beta-glucuronidase release from neutrophils. Thus formation of peroxynitrite from SIN-1 does not explain its inhibitory effects on neutrophil degranulation. The NO-deficient metabolite of SIN-1, SIN-1C (330-1000 microM) inhibited human neutrophil degranulation in a concentration-dependent manner similar to that of SIN-1 and reduced the increase in intracellular free calcium induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine. C88-3934 (330-1000 microM), another NO-deficient sydnonimine metabolite, also inhibited human neutrophil degranulation. In conclusion, the data shows that the NO-donor SIN-1 inhibits human neutrophil degranulation in a cGMP-, NO-, and peroxynitrite-independent manner, probably because of the formation of more stable active metabolites such as SIN-1C. The results demonstrate that studies on the role of NO and/or peroxynitrite carried out with SIN-1 and other NO-donors should be carefully re-evaluated as to whether the effects found are really attributable to NO or peroxynitrite and that in future studies, it will be crucial to carry out control experiments with the NO-deficient metabolites in any studies with sydnonimine NO-donors.

Topics: Acetonitriles; Calcium; Cell Degranulation; Cyclic GMP; Humans; Molsidomine; Morpholines; Neutrophils; Nitrates; Nitric Oxide

In cultured endothelial cells, incubation with TNF-alpha (50 ng/ml) for 72 h markedly reduced viability of endothelial cells. A 6-h pre-incubation with the nitric oxide (NO) donor linsidomine (SIN-1, 10-150 microM) protected endothelial cells in a concentration-dependent manner and increased viability by up to 59% of control. The unmetabolized parent compound molsidomine and the NO-free metabolite of SIN-1 3-morpholinoiminoacetonitrile (SIN-1C) were without cytoprotective effect. Cytoprotection by SIN-1 was completely abolished by the NO scavenger 2-phenyl-4,4,5,5, -tetramethylimidazoline-1-oxyl-3-oxide (PTIO, 30 microM). A cytoprotective effect comparable to SIN-1 was observed when preincubating the cells with dibutyryl cyclic GMP (10-100 microM). Moreover, no protection by SIN-1 occurred in the presence of cycloheximide (1 microM) or 1H--1,2,4-oxadiazole-4, 3-a-quinoxalin-1-one (ODQ, 0.1 microM), a selective inhibitor of soluble guanylyl cyclase. Tin protoporphyrin-IX (SnPP, 25 microM), an inhibitor of heme oxygenase, was found to attenuate SIN-1-induced cytoprotection. Our results demonstrate that SIN-1 produces a long-term endothelial protection against cellular injury by TNF-alpha, presumably via a cyclic GMP-dependent pathway leading to up-regulation of protective proteins such as heme oxygenase.

Topics: Acetonitriles; Animals; Cattle; Cells, Cultured; Cyclic GMP; Drug Interactions; Endothelium, Vascular; Heme Oxygenase (Decyclizing); Molsidomine; Morpholines; Nitric Oxide; Reactive Oxygen Species; Tumor Necrosis Factor-alpha; Vasodilator Agents

To evaluate the effect of intraocular administration of nitric oxide (NO) donors in the rabbit eye on intraocular pressure (IOP), inflammation, and toxicity.. Intravitreal and intracameral injections of two NO donors, SIN-1 and SNAP, and SIN-1C and BSS were performed. Clinical examination, IOP measurements, protein evaluation in aqueous humor, and histologic analysis of the ocular globes were realized. Nitric oxide release was demonstrated by nitrite production in the aqueous humor and in the vitreous using the Griess reaction.. The drastic decrease of IOP, observed after a single NO donor injection, was correlated directly with nitrite production and, thus, to NO release. Injection of inactive metabolite of SIN-1, SIN-1C, which is not able to release NO, did not modulate IOP. When administered in the aqueous humor or in the vitreous, NO did not diffuse from one segment of the eye to another. No inflammation or histologic damage was observed as a result of a single NO donor administration.. Nitric oxide is implicated directly in the regulation of IOP and its acute, and massive release into the rabbit eye did not induce inflammation or other growth toxic effects on the ocular tissues.

Topics: Acetonitriles; Animals; Aqueous Humor; Eye; Eye Proteins; Female; Injections; Intraocular Pressure; Molsidomine; Morpholines; Nitric Oxide; Nitrites; Ocular Hypotension; Penicillamine; Rabbits; S-Nitroso-N-Acetylpenicillamine; Vasodilator Agents; Vitreous Body