1-3-dihydroxy-4-4-5-5-tetramethyl-2-(4-carboxyphenyl)tetrahydroimidazole and linsidomine

We previously reported that both nitric oxide (NO) generated from NO synthase by bombesin and NO generated from SIN-1 (NO donor) activate the brain cyclooxygenase (COX) (COX-1 for bombesin), thereby eliciting the secretion of both catecholamines (CA) from the adrenal medulla by brain thromboxane A(2)-mediated mechanisms in rats. NO exerts its effects via not only soluble guanylate cyclase, but also protein S-nitrosylation, covalent modification of a protein cysteine thiol. In this study, we clarified the central mechanisms involved in the bombesin-induced elevation of plasma CA with regard to the relationship between NO and COX-1 using anesthetized rats. Bombesin (1 nmol/animal, i.c.v.)-induced elevation of plasma CA was attenuated by carboxy-PTIO (NO scavenger) (0.5 and 2.5 μmol/animal, i.c.v.), but was not influenced by ODQ (soluble guanylate cyclase inhibitor) (100 and 300 nmol/animal, i.c.v.). The bombesin-induced response was effectively reduced by dithiothreitol (thiol-reducing reagent) (0.4 and 1.9 μmol/kg/animal, i.c.v.) and by N-ethylmaleimide (thiol-alkylating reagent) (0.5 and 2.4 μmol/kg/animal, i.c.v.). The doses of dithiothreitol also reduced the SIN-1 (1.2 μmol/animal, i.c.v.)-induced elevation of plasma CA, but had no effect on the U-46619 (thromboxane A(2) analog) (100 nmol/animal, i.c.v.)-induced elevation of plasma CA even at higher doses (1.9 and 9.7 μmol/kg/animal, i.c.v.). Immunohistochemical studies demonstrated that the bombesin increased S-nitroso-cysteine-positive cells co-localized with COX-1 in the spinally projecting neurons of the hypothalamic paraventricular nucleus (PVN). Taken together, endogenous NO seems to mediate centrally administered bombesin-induced activation of adrenomedullary outflow at least in part by S-nitrosylation of COX-1 in the spinally projecting PVN neurons in rats.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adrenal Medulla; Animals; Benzoates; Bombesin; Brain; Catecholamines; Cysteine; Dithiothreitol; Dose-Response Relationship, Drug; Ethylmaleimide; Imidazoles; Injections, Intraventricular; Male; Molsidomine; Nitric Oxide Donors; Oxadiazoles; Paraventricular Hypothalamic Nucleus; Quinoxalines; Rats; Rats, Wistar; S-Nitrosothiols; Sulfhydryl Reagents

The effect of (+/-)cis-2-methylspilo(1,3-oxathiolane-5,3')quinuclidine (SNI-2011) on the secretory pathway of amylase in parotid tissues was investigated. SNI-2011-induced exocytosis was inhibited by a cell-permeable Ca(2+) chelator or inhibitors of calmodulin kinase II, neuronal nitric oxide synthase (nNOS), soluble guanyl cyclase, cyclic GMP-dependent protein kinase (PKG), and myosin light chain kinase, suggesting that these enzymes were coupled with the exocytosis. Stimulation with SNI-2011 of isolated rat parotid acinar cells loaded with 4,5-diaminofluorescein/diacetate (DAF-2/DA) induced a fast increase in DAF fluorescence corresponding to an increase in the NO production. SNI-2011-induced amylase secretion from parotid tissues in nNOS knockout mice has not been observed yet in spite of the expression of muscarinic M(3) receptors and the maintenance of secretory response to isoproterenol in the tissues. These results indicate the implication of the activation of Ca(2+)- and calmodulin-dependent enzymes and NOS-PKG signaling pathway in SNI-2011-induced amylase secretion from parotid acinar cells.

Topics: Alkaloids; Amylases; Animals; Azepines; Benzoates; Benzylamines; Calcium; Calcium Channel Blockers; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Carbachol; Carbazoles; Chelating Agents; Cyclic GMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Egtazic Acid; Enzyme Activation; Enzyme Inhibitors; Estrenes; Gallic Acid; Genotype; Guanylate Cyclase; Imidazoles; In Vitro Techniques; Indoles; Male; Mice; Mice, Knockout; Molsidomine; Muscarinic Agonists; Myosin-Light-Chain Kinase; NG-Nitroarginine Methyl Ester; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Oxadiazoles; Parotid Gland; Penicillamine; Phosphodiesterase Inhibitors; Pilocarpine; Protein Kinase Inhibitors; Pyrroles; Pyrrolidinones; Quinoxalines; Quinuclidines; Rats; Rats, Wistar; Sulfonamides; Thiophenes; Type C Phospholipases

Vasorelaxation mediated by peroxynitrite (ONOO-) and 3-morpholinosydnonimine (SIN-1) were investigated in isolated bovine intramammary arteries. Both ONOO- and SIN-1 relaxed U 46619-precontracted rings in a dose-dependent, endothelium-independent manner. Pretreatment with an adenylyl cyclase inhibitor, SQ 22536 [(9-tetrahydro-2-furyl)adenine], resulted in an enhanced ONOO--mediated relaxation, but did not modulate the response to SIN-1. ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), a potent and selective inhibitor of soluble guanylyl cyclase (sGC), did not significantly affect relaxant actions of ONOO-, but ODQ markedly attenuated SIN-1-elicited relaxation with a rightward shift in the dose-response curve and an unaltered maximal response. In the presence of carboxy-PTIO (2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide), a putative nitric oxide scavenger and ONOO- inactivator, the relaxant response to ONOO- was abolished, while relaxant actions of SIN-1 appeared to be unaffected. The results reveal a difference between ONOO- and SIN-1-mediated relaxation with regards to the role of the sGC and suggest that ONOO--evoked relaxation may not be associated with sGC activity, but rather depends on an sGC-independent mechanism triggered by ONOO- and/or NO itself. It also re-emphasizes that SIN-1 induces a vasorelaxant response, in part, via stimulation of sGC.

Topics: Adenine; Animals; Benzoates; Cattle; Dose-Response Relationship, Drug; Drug Combinations; Drug Interactions; Enzyme Inhibitors; Female; Guanylate Cyclase; Imidazoles; In Vitro Techniques; Mammary Arteries; Molsidomine; Muscle Relaxation; Muscle, Smooth, Vascular; Oxadiazoles; Oxazines; Oxidants; Peroxynitrous Acid; Vasodilation; Vasodilator Agents

A characteristic physiological property of the neuromuscular junction between giant motor neurones (MoGs) and fast flexor muscles in crayfish is synaptic depression, in which repetitive electrical stimulation of the MoG results in a progressive decrease in excitatory junction potential (EJP) amplitude in flexor muscle fibres. Previous studies have demonstrated that l-arginine (l-Arg) modulates neuromuscular transmission. Since l-Arg is a precursor of nitric oxide (NO), we examined the possibility that NO may be involved in modulating neuromuscular transmission from MoGs to abdominal fast flexor muscles. The effect of a NO-generating compound, NOC7, was similar to that of l-Arg, reversibly decreasing the EJP amplitude mediated by the MoG. While NOC7 reduced the amplitude of the EJP, it induced no significant change in synaptic depression. In contrast, a scavenger of free radical NO, carboxy-PTIO, and an inhibitor of nitric oxide synthase, l-NAME, reversibly increased the EJP amplitude mediated by MoGs. Synaptic depression mediated by repetitive stimulation of MoGs at 1 Hz was partially blocked by bath application of l-NAME. Bath application of a NO scavenger, a NOS inhibitor and NO-generating compounds had no significant effects on the depolarisation of the muscle fibres evoked by local application of l-glutamate. The opposing effects on EJP amplitude of NOC7 and of carboxy-PTIO and l-NAME suggest that endogenous NO presynaptically modulates neuromuscular transmission and that it could play a prominent role at nerve terminals in eliciting MoG-mediated synaptic depression in the crayfish Procambarus clarkii.

Topics: Animals; Arginine; Astacoidea; Benzoates; Enzyme Inhibitors; Female; Glutamic Acid; Hydrazines; Imidazoles; Male; Molsidomine; Motor Neurons; Neuromuscular Junction; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Superoxide Dismutase; Synaptic Transmission

The purpose of this study was to examine whether tetrahydrobiopterin (BH4), one of the cofactors of nitric oxide (NO) synthase, attenuates endothelial cell death induced by 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1), which is known to produce both superoxide and NO. Endothelial cell death was assessed by the release of intracellular lactate dehydrogenase (LDH). Addition of SIN-1 (500, 1,000 microM) to endothelial cells induced cell death from 6 h after its addition. The SIN-1-induced endothelial cell death was strongly reduced by treatment with carboxy-PTIO, a NO scavenger, or superoxide dismutase (SOD). Iron chelators and hydroxyl radical scavengers also reduced the SIN-1-induced endothelial cell death. Interestingly, the SIN-1-induced endothelial cell death was also reduced by treatment with catalase. Thus NO, superoxide, hydroxyl radical, and hydrogen peroxide are likely to be implicated in SIN-1-induced endothelial cell death. Moreover, pretreatment with sepiapterin, a precursor of BH4 synthesis, reduced the SIN-1-induced endothelial cell death and increased the intracellular BH4 content. Both the protective effect of sepiapterin and the increase in intracellular BH4 content were prevented by co-pretreatment with N-acetylserotonin (NAS), an inhibitor of BH4 synthesis. The protective effect of sepiapterin also was observed when up-take of trypan blue was used as another marker of cell death. These findings suggest that BH4 has a protective effect against endothelial cell death caused by the presence of NO and superoxide. The protective effect of BH4 may at least partly involve scavenging of superoxide or hydrogen peroxide or both, because we and other groups previously found that BH4 has a scavenging activity for reactive oxygen species.

Topics: Animals; Antioxidants; Aorta; Benzoates; Biopterins; Catalase; Cattle; Cell Survival; Cells, Cultured; Endothelium, Vascular; Free Radical Scavengers; Imidazoles; Iron Chelating Agents; L-Lactate Dehydrogenase; Molsidomine; Nitric Oxide; Plant Extracts; Pteridines; Pterins; Reactive Oxygen Species; Superoxide Dismutase; Trypan Blue

Nitric oxide (NO) is an unstable free radical that functions as a cytotoxic agent secreted by macrophages to kill cancer cells. Here we report the effect of NO on the expression of intercellular adhesion molecule-1 (ICAM-1) on cancer cells. NO donors such as SNP, SNAP and SIN-1 up-regulated the expression of ICAM-1 on NA cells, a squamous cell carcinoma cell line. Northern blot analysis showed that the induction of ICAM-1 might be due to transcriptional induction of ICAM-1 mRNA. Up-regulation of ICAM-1 mRNA by NO donors was inhibited by carboxy-PTIO, a NO scavenger. Although NF-kappaB activity was induced by NO donors, AP-1 was not induced by them. Staurosporin, a protein kinase C (PKC) inhibitor, inhibited the induction of ICAM-1 on NA cells by NO, whereas genistein, a protein tyrosine kinase inhibitor, did not. These findings indicate that NO up-regulates ICAM-1 expression on cancer cells by a regulatory mechanism involving PKC and suggest that NF-kappaB, but not AP-1, might be involved in induction of ICAM-1 by NO in cancer cells.

Topics: Benzoates; Carcinoma, Squamous Cell; Cell Adhesion Molecules; Free Radical Scavengers; Genistein; Humans; Imidazoles; Intercellular Adhesion Molecule-1; Molsidomine; NF-kappa B; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Penicillamine; Phosphorylation; Promoter Regions, Genetic; Protein Kinase C; RNA, Messenger; S-Nitroso-N-Acetylpenicillamine; Staurosporine; Tongue Neoplasms; Transcription Factor AP-1; Tumor Cells, Cultured; Up-Regulation

Guanidinosuccinic acid (GSA) is noted for its nitric oxide (NO) mimicking actions such as vasodilatation and activation of the N-methyl-D-aspartate (NMDA) receptor. We have reported that GSA is the product of argininosuccinate (ASA) and some reactive oxygen species, mainly the hydroxyl radical. We tested for GSA synthesis in the presence of NO donors. ASA (1 mM) was incubated with NOR-2, NOC-7 or 3-morpholinosydomine hydrochloride (SIN-1) at 37 degrees C. GSA was determined by HPLC using a cationic resin for separation and phenanthrenequinone as an indicator. Neither NOR-2 or NOC-7 formed GSA. SIN-1, on the other hand, generates NO and the superoxide anion which, in turn, generated peroxynitrite which was then converted to the hydroxyl radical. Incubation of ASA with SIN-1 leads, via this route, to GSA. When ASA was incubated with 1 mM SIN-1, the amount of GSA produced depended on the incubation time and the concentration of ASA. Among the tested SIN-1 concentrations, from 0.5 to 5 mM, GSA synthesis was maximum at 0.5 mM and decreased with increasing concentrations of SIN-1. Carboxy-PTIO, a NO scavenger, completely inhibited GSA synthesis. SOD, a superoxide scavenger, decreased GSA synthesis by 20%, and catalase inhibited GSA synthesis only by 12%; DMSO, a hydroxyl radical scavenger completely inhibited GSA synthesis in the presence of SIN-1. These data suggest that the hydroxyl radical derived from a combination of NO and the superoxide anion generates GSA, a stable NO mimic. Meanwhile, synthesis of GSA by NO produces reactive oxygen and activates the NMDA receptor that generates NO from GSA, suggesting a positive feed back mechanism.

Topics: Argininosuccinic Acid; Benzoates; Chromatography, High Pressure Liquid; Dimethyl Sulfoxide; Feedback; Free Radical Scavengers; Free Radicals; Guanidines; Hydrazines; Hydroxyl Radical; Imidazoles; Molsidomine; Nitrates; Nitric Oxide; Nitric Oxide Donors; Reactive Oxygen Species; Receptors, N-Methyl-D-Aspartate; Succinates; Superoxide Dismutase

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

The central effect of 3-morpholinosydnonimine, a nitric oxide donor, on the sympatho-adrenomedullary system was investigated in urethane-anesthetized rats. Intracerebroventricular administration of 3-morpholinosydnonimine (100, 250 and 500 microg/animal) induced a marked elevation of adrenaline levels and a slight elevation of noradrenaline levels in the plasma. These 3-morpholinosydnonimine (250 microg/animal)-induced elevations of catecholamines were abolished by intracerebroventricular treatments with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl 3-oxide (750 microg/animal), a nitric oxide scavenger, and indomethacin (500 microg/animal), a cyclo-oxygenase inhibitor, but not with superoxide dismutase (250 units/animal), a superoxide anion scavenger. Furthermore, the 3-morpholinosydnonimine (250 microg/animal)-induced elevation of plasma adrenaline levels was abolished by intracerebroventricular treatments with thromboxane A2 synthase inhibitors [furegrelate (100, 250 and 1000 microg/animal) and carboxyheptyl imidazole (500 microg/animal)], and also with thromboxane A2 receptor blockers [(+)-S-145 (100, 250 and 1000microg/animal) and SQ29548 (8microg/animal)]. The elevation of noradrenaline levels was, however, not attenuated by these thromboxane A2-related test agents. The present results indicate that nitric oxide but not peroxynitrite markedly activates central adrenomedullary outflow. Thromboxane A2 in the brain is probably involved in this central activation of adrenomedullary outflow.

Topics: Animals; Benzoates; Cyclooxygenase Inhibitors; Epinephrine; Imidazoles; Indomethacin; Injections, Intraventricular; Male; Medulla Oblongata; Molsidomine; Nitric Oxide; Norepinephrine; Rats; Rats, Wistar; Receptors, Thromboxane; Superoxide Dismutase; Thromboxane A2; Thromboxane-A Synthase

Carboxy-PTIO reacts rapidly with NO to yield NO2 and has been used as a scavenger to test the importance of nitric oxide (NO) in various physiological conditions. This study investigated the effects of carboxy-PTIO on several NO- and peroxynitrite-mediated reactions. The scavenger potently inhibited NO-induced accumulation of cGMP in endothelial cells but potentiated the effect of the putative peroxynitrite donor SIN-1, Carboxy-PTIO completely inhibited peroxynitrite-induced formation of 3-nitrotyrosine from free tyrosine (EC50 = 36 +/- 5 microM) as well as nitration of bovine serum albumin. Peroxynitrite-mediated nitrosation of GSH was stimulated by the drug with an EC50 of 0.12 +/- 0.03 mM, whereas S-nitrosation induced by the NO donor DEA/NO (0.1 mM) was inhibited by the scavenger with an IC50 of 0.11 +/- 0.03 mM. Oxidation of NO with carboxy-PTIO resulted in formation of nitrite without concomitant production of nitrate. Our results demonstrate that the effects of carboxy-PTIO are diverse and question its claimed specificity as NO scavenger.

Topics: Animals; Benzoates; Cattle; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Free Radical Scavengers; Imidazoles; Kinetics; Molsidomine; Nitrates; Nitric Oxide; Nitrites; Serum Albumin, Bovine; Swine; Tyrosine