fk-409 and linsidomine

Treatment of PC12 cells with fungus-derived alkaloid neoechinulin A for more than 12 h renders the cells resistant to subsequent superoxide (O₂⁻)/nitric oxide (NO) insults derived from 3-morpholinosydnonimine (SIN-1). However, the underlying mechanism(s) remains largely unclear. To elucidate the mechanism(s), we assessed the specificity of the cytoprotection afforded by neoechinulin A treatment using other cytocidal stressors and also clarified the resulting cellular alterations, focusing on the antioxidant and metabolic enzymes systems. Neoechinulin A treatment for more than 12 h endowed PC12 cells with significant resistance to transient NO toxicity, but not persistent NO toxicity, bolus H₂O₂ toxicity, or oxidative insult from the redox cycling quinone menadione. Cellular antioxidant system profiling revealed no substantial potentiation of the activity of any antioxidant enzyme in lysate from the neoechinulin A-treated cells excluding glutathione (GSH) content, which was significantly decreased (>50%), resulting in a proportional compromise in the thiol-reducing activity of the intact cells. In addition, no differences were observed in the activity for any nicotinamide adenine dinucleotide (phosphate) reduced form (NAD(P)H)-generating enzyme, steady-state NAD(P)H/nicotinamide adenine dinucleotide (phosphate) oxidized form (NAD(P)⁺) ratios, or the levels of total NAD(P)H. Nevertheless, the neoechinulin A-treated intact cells exhibited increased NAD(P)H redox turnover when driven by extracellular tetrazolium. The structurally inactive analog preechinulin failed to protect cells against NO toxicity or induce these alterations, suggesting their link with the cytoprotective mechanism. These results suggest that neoechinulin A, despite disabling the GSH defense system, confers cytoprotection against nitrosative stresses by elevating the cellular reserve capacity for NAD(P)H generation, which could offset crippling of energy-supplying systems due to nitrosative stress.

Topics: Animals; Cell Survival; Cytoprotection; Glutathione; Indole Alkaloids; Molsidomine; Nitric Oxide; Nitro Compounds; Oxidation-Reduction; Oxidative Stress; Oxidoreductases; PC12 Cells; Piperazines; Rats; Sodium-Potassium-Exchanging ATPase; Transferases

Nitric oxide (NO) releasing drugs (e.g., glyceryl trinitrate) were successfully used in the treatment of cutaneous leishmaniasis in man. In the present study, the effect of NO donors on the catalytic activity of the cysteine proteinase from promastigotes of Leishmania infantum, an agent of Old World visceral and cutaneous leishmaniases, is reported. In particular, one equivalent of NO, released by the NO donors S-nitrosoglutathione, glyceryl trinitrate, (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide, 3-morpholinosydnonimine, S-nitrosoacetylpenicillamine and sodium nitroprusside, inhibited one equivalent of the parasite cysteine proteinase. As expected, NO-deprived compounds did not affect the catalytic activity of the parasite cysteine proteinase. Furthermore, the absorption spectrum of the (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide-treated inactive L. infantum enzyme displayed a maximum in the 330-350 nm wavelength range. The reducing agents dithiothreitol and L-ascorbic acid completely prevented parasite cysteine proteinase inhibition by NO, fully restored the catalytic activity, and reversed the NO-induced absorption spectrum of the inactive enzyme. Moreover, S-nitrosoacetylpenicillamine displayed a leishmanicidal effect, inhibiting the cysteine proteinase activity in vivo. As expected, the NO-deprived compound N-acetylpenicillamine did not affect significantly the parasite viability and the enzyme activity in vivo. These data suggest that the L. infantum cysteine proteinase undergoes NO-mediated S-nitrosylation, thereby representing a possible mechanism of antiparasitic host defence.

Topics: Animals; Ascorbic Acid; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dithiothreitol; Glutathione; Kinetics; Leishmania infantum; Leupeptins; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Nitro Compounds; Nitroglycerin; Nitroprusside; Nitroso Compounds; Penicillamine; Protozoan Proteins; S-Nitrosoglutathione

Nitric oxide (NO) is a pluripotent regulatory molecule possessing, among others, an antiparasitic activity. In the present study, the inhibitory effect of NO on the catalytic activity of falcipain, the papain-like cysteine protease involved in Plasmodium falciparum trophozoite hemoglobin degradation, is reported. In particular, NO donors S-nitrosoglutathione (GSNO), (+/-)-(E)-p6ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenami de (NOR-3), 3-morpholinosydnonimine (SIN-1), and sodium nitroprusside (SNP) inhibit dose-dependently the falcipain activity present in the P. falciparum trophozoite extract, this effect likely attributable to S-nitrosylation of the Cys25 catalytic residue. The results represent a new insight into the modulation mechanism of falcipain activity, thereby being relevant in developing new strategies for inhibition of the P. falciparum life cycle.

Topics: Animals; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dithiothreitol; Glutathione; Kinetics; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Nitro Compounds; Nitroprusside; Nitroso Compounds; Plasmodium falciparum; S-Nitrosoglutathione

ODQ, (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase) inhibits vasorelaxant responses to nitric oxide (NO)-donor drugs, but the extent of the inhibition varies depending on the NO donor studied. The purpose of this study was to test the hypothesis that these variations in the effects of ODQ reflect differences in the mechanisms whereby each NO donor generates NO. On pulmonary artery preparations pre-contracted submaximally with phenylephrine, ODQ (3 microM) almost abolished the relaxant responses to glyceryl trinitrate, isosorbide dinitrate and nitroprusside; each of these drugs requires activation in the tissue (by enzymes or reducing agents) to generate NO. In contrast, ODQ (3 microM) caused a parallel shift in the concentration-relaxation curves to linsidomine (SIN-1), FK409, MAHMA NONOate and spermine NONOate (1.63 to 2.54 log units) with no depression in maximum response; each of these NO donors generates NO in the physiological bathing solution without requiring tissue activation. For the four drugs in this group, the effects of 10 microM ODQ were not significantly greater than the effects of 3 microM ODQ; thus there was an ODQ-resistant component to the response suggesting that part of the response involved a mechanism that was independent of soluble guanylate cyclase. NO donors that require tissue activation probably generate NO within the smooth-muscle cell, whereas those that do not require tissue activation generate NO outside the cell. Hence it is concluded that the site of NO generation (intra- or extracellular) might determine whether or not there is an ODQ-resistant component in the relaxation response.

Topics: Animals; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanylate Cyclase; Hydrazines; In Vitro Techniques; Male; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Nitro Compounds; Nitrogen Oxides; Oxadiazoles; Pulmonary Artery; Quinoxalines; Rats; Rats, Wistar; Spermine; Vasodilation

Studies were conducted to clarify the effects of nitric oxide donors NOR 3 ((+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide, FK409), SIN-1 (3-morpholinosydnonimine) and SNAP (S-nitroso-N-acetylpenicillamine) on the accumulation of cGMP and cAMP and Ca2+ mobilization as well as ketogenesis from oleate in isolated rat hepatocytes. NOR 3 caused inhibition of ketogenesis from oleate along with stimulation of cGMP accumulation in rat hepatocytes, whereas SIN-1 and SNAP exerted no effect on ketogenesis despite their marked stimulation of cGMP accumulation. Although the nitric oxide trapping agent, carboxy-PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide), antagonized the stimulation by NOR 3 of cGMP accumulation, it failed to modulate the anti-ketogenic action of NOR 3. Furthermore, neither 8-bromoguanosine-3',5'-cyclic monophosphate nor N2,2'-O-dibutyrylguanosine-3',5'-cyclic monophosphate mimicked the anti-ketogenic action of NOR 3. It is concluded in the present study that NOR 3-induced inhibition of ketogenesis in rat hepatocytes is not mediated by cGMP. The present study revealed that the remaining structure of NOR 3 from which nitric oxide had been spontaneously released had no anti-ketogenic action. We first and clearly demonstrated that nitrite production was dramatically enhanced when NOR 3 was incubated in the presence of rat hepatocytes. The mechanism whereby NOR 3 inhibits ketogenesis in rat hepatocytes will be discussed.

Topics: Adenosine Triphosphate; Animals; Benzoates; Calcium; Cells, Cultured; Cyclic GMP; Imidazoles; Lactic Acid; Liver; Male; Molsidomine; Nitric Oxide; Nitro Compounds; Oleic Acid; Penicillamine; Rats; Rats, Wistar; S-Nitroso-N-Acetylpenicillamine; Sodium Nitrite; Vasodilator Agents

We tested the effects of several nitric oxide (NO) generating compounds on the activity of sodium-potassium adenosine 5'-triphosphatase [(Na+,K+)-ATPase] purified from porcine cerebral cortex. Sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine (SIN-1) and (d1)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (NOR 3) inhibited the (Na+,K+)-ATPase activity dose dependently. Superoxide dismutase, a NO scavenger, and sulfhydryl (SH) compounds, reduced-form glutathione (rGSH) and dithiothreitol (DTT), prevented the inhibitory action of SNAP, SIN-1 and NOR 3 but not of SNP, when applied simultaneously with NO generating compounds, and this enzyme inhibition could be reactivated by the incubation with these SH compounds but not with SOD. The inhibitory action by SNP was magnified by simultaneous application of DTT. These results suggest that NO generating compounds, SNAP, SIN-1 and NOR 3 but not SNP, may release NO or NO-derived products and may inhibit (Na+,K+)-ATPase activity by interacting with a SH group at the active site of the enzyme.

Topics: Animals; Cerebral Cortex; Dithiothreitol; Enzyme Inhibitors; Molsidomine; Nitric Oxide; Nitro Compounds; Nitroprusside; Penicillamine; S-Nitroso-N-Acetylpenicillamine; Sodium-Potassium-Exchanging ATPase; Sulfhydryl Reagents; Superoxide Dismutase; Swine