s-nitro-n-acetylpenicillamine and 6-anilino-5-8-quinolinedione

Evidence suggests that the NO/sGC/PKG pathway plays a key role in memory processing but the actual participation of this signaling cascade in the amygdala during memory consolidation remains unknown. Here, we show that when infused in the amygdala immediately after inhibitory avoidance training, but not later, the NO synthase inhibitor L-NNA hindered long-term memory retention without affecting locomotion, exploratory behavior, anxiety state or retrieval of the avoidance response. The amnesic effect of L-NNA was not state-dependent and was mimicked by the soluble guanylyl cyclase inhibitor LY83583 and the PKG inhibitor KT-5823. On the contrary, post-training intra-amygdala infusion of the NOS substrate L-Arg, the NO-releasing compound SNAP or the non-hydrolysable analog of cGMP 8Br-cGMP increased memory retention in a dose-dependent manner. Co-infusion of 8Br-cGMP reversed the amnesic effect of L-NNA and LY83583 but not that of KT-5823. Our data indicate that the NO-induced activation of PKG in the amygdala is a necessary step for consolidation of inhibitory avoidance memory.

Topics: Aminoquinolines; Amygdala; Animals; Anxiety; Arginine; Avoidance Learning; Carbazoles; Cyclic GMP; Enzyme Inhibitors; Exploratory Behavior; Guanylate Cyclase; Male; Maze Learning; Memory; Motor Activity; Nitric Oxide; Nitric Oxide Synthase; Penicillamine; Rats; Rats, Wistar; Signal Transduction

South American (SA) opossum lower esophageal sphincter (LES) circular smooth muscle relaxes by activation of enteric nerves elicited by EFS (electrical field stimulation, 0.5 ms, 48 V, 0.5-8 Hz for 10 s). The identity of the mediator released and the cellular mechanism, however, remain to be fully elucidated. The purpose of this study was to determine the effect of the enzyme soluble guanylate cyclase (cGC) inhibitors, cystamine (100 microM), methylene blue (30 microM), LY 83583 (6-anilino-5,8 quinoledione, 10 microM) and ODQ (H-[1,2,4]oxadiazolo[4,3]quinoxalin-1-one, 1 microM) on the relaxations induced by EFS and by exogenous NO (nitric oxide, 0.5 mM) or NO-donors on SA opossum LES smooth muscle strips. EFS caused frequency-dependent relaxations, which were inhibited by NO-synthase inhibitors and abolished by tetrodotoxin. Cystamine did not affect relaxations caused by EFS and NO or NO-donor. Methylene blue also failed to affect EFS-caused relaxations, although it was capable of inhibiting relaxation induced by NO. LY 83583 inhibited relaxations induced by NO, but did not affect those induced by EFS or by SNAP and HXA. ODQ abolished relaxations caused by EFS at lower frequencies and by HXA (hydroxylamine, 10 microM) and SNAP (S-nitroso-N-acetyl penicillamine, 10 microM). Relaxations at higher frequencies of EFS and induced by SNP (sodium nitroprusside, 30 microM) and NO were only reduced by ODQ. These findings indicate that activation of the cGC can be involved in relaxations induced by EFS at lower frequencies, but other mechanisms can be involved at higher frequencies of EFS and caused by SNP or NO.

Topics: Aminoquinolines; Animals; Cyclic GMP; Cysteamine; Electric Stimulation; Esophageal Sphincter, Lower; Female; Guanylate Cyclase; Hydrazines; Hydroxylamine; In Vitro Techniques; Male; Methylene Blue; Muscle Relaxation; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Opossums; Oxadiazoles; Penicillamine; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase

Nitric oxide (NO) is an important signaling molecule in the CNS, regulating neuronal survival, proliferation and differentiation. Here, we explored the mechanism by which NO, produced from the NO donor S-nitroso-acetyl-d-l-penicillamine (SNAP), exerts its neuroprotective effect in purified cultures of chick retinal neurons. Cultures prepared from 8-day-old chick embryo retinas and incubated for 24 h (1 day in culture, C1) were treated or not with SNAP, incubated for a further 72 h (up to 4 days in culture, C4), fixed, and the number of cells estimated, or processed for cell death estimation, by measuring the reduction of the metabolic dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Experimental cultures were run in parallel but were re-fed with fresh medium in the absence or presence of SNAP at culture day 3 (C3), incubated for a further 24 h up to C4, then fixed or processed for the MTT assay. Previous studies showed that the re-feeding procedure promotes extensive cell death. SNAP prevented this death in a concentration- and time-dependent manner through the activation of soluble guanylate cyclase; this protection was significantly reversed by the enzyme inhibitors 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) or LY83583, and mimicked by 8-bromo cyclic guanosine 5'-phosphate (8Br-cGMP) (GMP) or 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), guanylate cyclase activators. The effect was blocked by the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). The effect of NO was also suppressed by LY294002, Wortmannin, PD98059, KN93 or H89, indicating the involvement, respectively, of phosphatidylinositol-3 kinase, extracellular-regulated kinases, calmodulin-dependent kinases and protein kinase A signaling pathways. NO also induced a significant increase of neurite outgrowth, indicative of neuronal differentiation, and blocked cell death induced by hydrogen peroxide. Cyclosporin A, an inhibitor of the mitochondrial permeability transition pore considered an important mediator of apoptosis and necrosis, as well as boc-aspartyl (OMe) fluoromethylketone (BAF), a caspase inhibitor, also blocked cell death induced by re-feeding the cultures. These findings demonstrate that NO inhibits apoptosis of retinal neurons in a cGMP/protein kinase G (PKG)-dependent way, and strengthens the notion that NO plays an important role during CNS development.

Topics: Adenosine; Aminoquinolines; Analysis of Variance; Animals; Cell Survival; Cells, Cultured; Chick Embryo; Cyclic GMP; Cyclic N-Oxides; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Free Radical Scavengers; Imidazoles; Neurons; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitrites; Penicillamine; Retina; Signal Transduction; Tetrazolium Salts; Thiazoles; Tritium

Pig oocytes matured in vitro were parthenogenetically activated (78%) after treatment with 2 mM nitric oxide-donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP) for 24 h. Inhibition of soluble guanylyl cyclase with the specific inhibitors 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or 6-anilino-5,8-quinolinequinone (LY83583) suppressed the SNAP-induced activation in a dose-dependent manner (23% of activated oocytes after treatment with 400 microM ODQ; 12% of activated oocytes after treatment with 40 microM LY83583). 8-Bromo-cyclic guanosine monophosphate (8-Br-cGMP), a phosphodiesterase-resistant analogue of cGMP, enhances the effect of suboptimal doses (0.1 or 0.5 mM) of the NO donor SNAP. DT3, a specific inhibitor of cGMP-dependent protein kinase (PKG, PKG), is also able to inhibit the activation of pig oocytes after NO donor treatment. Involvement of the cGMP-dependent signalling pathway is specific for NO-induced oocyte activation, because both the guanylyl cyclase inhibitor ODQ and the PKG inhibitor DT3 are unable to inhibit activation in oocytes treated with the calcium ionophore A23187. These data indicate that the activation of pig oocytes with an NO donor is cGMP-dependent and that PKG plays an important role in this mode of oocyte activation.

Topics: Aminoquinolines; Animals; Cell Membrane Permeability; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Digitoxin; Dose-Response Relationship, Drug; Enzyme Activation; Guanylate Cyclase; Nitric Oxide; Nitric Oxide Synthase; Oocytes; Oxadiazoles; Penicillamine; Phosphoric Diester Hydrolases; Protein Kinase Inhibitors; Quinoxalines; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Soluble Guanylyl Cyclase; Swine

We reported that insulin stimulates NAD(P)H oxidase activity but not migration of cultured rat vascular smooth muscle cells (VSMCs). Because angiotensin II (Ang II) increases NAD(P)H oxidase activity in these cells, we wished to determine whether insulin stimulates migration of Ang II-treated VSMCs by synergistically stimulating enzyme activity.. Cultured rat VSMC superoxide anion (O2-) production, cyclic GMP production, and migration were measured by lucigenin luminescence, immunoassay, and wound closure rate, respectively. Nitric oxide (NO) scavenging was measured by inhibition of NO-induced fluorescence of 4-5-diaminofluorescin.. Insulin (1 nmol/L) did not affect and Ang II (100 nmol/L) stimulated VSMC migration by 65% (P < .05), but together stimulated it by 150% (P < .05 versus Ang II) by a mechanism inhibited by the NAD(P)H oxidase inhibitors, diphenyleneiodonium (DPI) or gp91ds-tat. Insulin and Ang II stimulated O2- production by 34% and 35%, respectively (both P < .05), but together synergistically stimulated it by 143% (P < .05 versus insulin or Ang II) in a DPI or gp91ds-tat-sensitive manner. Neither insulin nor Ang II measurably affected NO scavenging, but together reduced NO availability by 46% in a DPI-sensitive manner (P < .05) and significantly inhibited NO-stimulated cyclic GMP production.. Insulin synergestically stimulates NAD(P)H oxidase activity in Ang II-treated cultured rat VSMCs causing increased migration.

Topics: Aminoquinolines; Angiotensin II; Animals; Cell Movement; Cells, Cultured; Cyclic GMP; Enzyme Inhibitors; Glycoproteins; Guanylate Cyclase; Insulin; Male; Muscle, Smooth, Vascular; NADPH Oxidases; Nitric Oxide Donors; Onium Compounds; Penicillamine; Rats; Rats, Sprague-Dawley; Superoxides; Vasoconstrictor Agents

Embryos of Helisoma trivolvis exhibit cilia-driven rotation within the egg capsule during development. In this study we examined whether nitric oxide (NO) is a physiological regulator of ciliary beating in cultured ciliary cells. The NO donor S-nitroso-N-acetylpenicillamine (SNAP; 1-1,000 microM) produced a dose-dependent increase in ciliary beat frequency (CBF). In contrast, the nitric oxide synthase (NOS) inhibitor 7-nitroindazole (10 and 100 microM) inhibited the basal CBF and blocked the stimulatory effects of serotonin (100 microM). NO production in response to serotonin was investigated with 4,5-diaminofluorescein diacetate imaging. Although SNAP (100 microM) produced a rise in NO levels in all cells, only 22% of cells responded to serotonin with a moderate increase. The cGMP analog 8-bromo-cGMP (8-Br-cGMP; 0.2 and 2 mM) increased CBF, and the soluble guanylate cyclase inhibitor LY-83583 (10 microM) blocked the cilioexcitatory effects of SNAP and serotonin. These data suggest that NO has a constitutive cilioexcitatory effect in Helisoma embryos and that the stimulatory effects of serotonin and NO work through a cGMP pathway. It appears that in Helisoma cilia, NO activity is necessary, but not sufficient, to fully mediate the cilioexcitatory action of serotonin.

Topics: Aminoquinolines; Animals; Cells, Cultured; Cilia; Cyclic GMP; Guanylate Cyclase; Nitric Oxide; Nitric Oxide Donors; Penicillamine; Serotonin; Snails

Nitric oxide (NO) exerts neurotrophic and neurotoxic effects on dopamine (DA) function in primary midbrain cultures. We investigate herein the role of glutathione (GSH) homeostasis in the neurotrophic effects of NO. Fetal midbrain cultures were pretreated with GSH synthesis inhibitor, L-buthionine-(S,R)-sulfoximine (BSO), 24 h before the addition of NO donors (diethylamine/nitric oxide-complexed sodium and S-nitroso-N-acetylpenicillamine) at doses tested previously as neurotrophic. Under these conditions, the neurotrophic effects of NO disappeared and turned on highly toxic. Reduction of GSH levels to 50% of baseline induced cell death in response to neurotrophic doses of NO. Soluble guanylate cyclase (sGC) and cyclic GMP-dependent protein kinase (PKG) inhibitors protected from cell death for up to 10 h after NO addition; the antioxidant ascorbic acid also protected from cell death but its efficacy decreased when it was added after NO treatment (40% protection 2 h after NO addition). The pattern of cell death was characterized by an increase in chromatin condensed cells with no DNA fragmentation and with breakdown of plasmatic membrane. The inhibition of RNA and protein synthesis and of caspase activity also protected from cell death. This study shows that alterations in GSH levels change the neurotrophic effects of NO in midbrain cultures into neurotoxic. Under these conditions, NO triggers a programmed cell death with markers of both apoptosis and necrosis characterized by an early step of free radicals production followed by a late requirement for signalling on the sGC/cGMP/PKG pathway.

Topics: Alkaloids; Aminoquinolines; Animals; Antioxidants; Apoptosis; Ascorbic Acid; Buthionine Sulfoximine; Carbazoles; Cell Division; Cells, Cultured; Cyclic GMP-Dependent Protein Kinases; Dopamine; Enzyme Inhibitors; Free Radicals; Glutathione; Glutathione Synthase; Guanylate Cyclase; Homeostasis; Hydrazines; Indoles; Mesencephalon; Methylene Blue; Nerve Tissue Proteins; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Nucleic Acid Synthesis Inhibitors; Parkinson Disease; Penicillamine; Protein Synthesis Inhibitors; Rats; Rats, Sprague-Dawley; Tyrosine 3-Monooxygenase

This study addresses the role of nitric oxide (NO) and its downstream mechanism in mediating the shear-induced increase in hydraulic conductivity (L(p)) of bovine aortic endothelial cell monolayers grown on porous polycarbonate filters. Direct exposure of endothelial monolayers to 20-dyne/cm(2) shear stress induced a 4. 70+/-0.20-fold increase in L(p) at the end of 3 hours. Shear stress (20 dyne/cm(2)) also elicited a multiphasic NO production pattern in which a rapid initial production was followed by a less rapid, sustained production. In the absence of shear stress, an exogenous NO donor, S-nitroso-N-acetylpenicillamine, increased endothelial L(p) 2.23+/-0.14-fold (100 micromol/L) and 4.8+/-0.66-fold (500 micromol/L) at the end of 3 hours. In separate experiments, bovine aortic endothelial cells exposed to NO synthase inhibitors, N(G)-monomethyl-L-arginine and N(G)-nitro-L-arginine methyl ester, exhibited significant attenuation of shear-induced increase in L(p) in a dose-dependent manner. Inhibition of guanylate cyclase (GC) with LY-83,583 (1 micromol/L) or protein kinase G (PKG) with KT5823 (1 micromol/L) failed to attenuate the shear-induced increase in L(p). Furthermore, direct addition of a stable cGMP analogue, 8-bromo-cGMP, had no effect in altering baseline L(p), indicating that the GC/cGMP/PKG pathway is not involved in shear stress-NO-L(p) response. Incubation with iodoacetate (IAA), a putative inhibitor of glycolysis, dose-dependently increased L(p). Addition of IAA at levels that did not affect baseline L(p) greatly potentiated the response of L(p) to 20-dyne/cm(2) shear stress. Finally, both shear stress-induced and IAA-induced increases in L(p) could be reversed with the addition of dibutyryl cAMP. However, additional metabolic inhibitors, 2 deoxyglucose (10 mmol/L) and oligomycin (1 micromol/L), or reactive oxygen species scavengers, deferoxamine (1 mmol/L) and ascorbate (10 mmol/L), failed to alter shear-induced increases in L(p). Our results show that neither the NO/cGMP/PKG pathway nor a metabolic pathway mediates the shear stress-L(p) response. An alternate mechanism downstream from NO that is sensitive to IAA must mediate this response.

Topics: Alkaloids; Aminoquinolines; Animals; Carbazoles; Cattle; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Endothelium, Vascular; Enzyme Inhibitors; Guanylate Cyclase; Indoles; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; omega-N-Methylarginine; Penicillamine; Protein Kinase Inhibitors; Protein Kinases; Stress, Mechanical

We have previously shown that reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) leads to the activation and deposition of complement. In the present study, we investigated whether the terminal complement complex (C5b-9) influences HUVEC nuclear factor-kappaB (NF-kappaB) translocation and vascular cell adhesion molecule-1 (VCAM-1) protein expression after hypoxia/reoxygenation by decreasing endothelial cGMP. Additionally, we investigated the action of anti-human C5 therapy on endothelial cGMP, NF-kappaB translocation, and VCAM-1 protein expression. Reoxygenation (0.5 to 3 hours, 21% O(2)) of hypoxic (12 hours, 1% O(2)) HUVECs in human serum (HS) significantly increased C5b-9 deposition, VCAM-1 expression, and NF-kappaB translocation compared with hypoxic/reoxygenated HUVECs treated with the recombinant human C5 inhibitor h5G1.1-scFv. Acetylcholine (ACh)-induced cGMP synthesis was significantly higher in normoxic HUVECs compared with hypoxic HUVECs reoxygenated in HS but did not differ from hypoxic HUVECs reoxygenated in buffer or HS treated with h5G1.1-scFv. Treatment of hypoxic/reoxygenated HUVECs with h5G1.1-scFv or cGMP analogues significantly attenuated NF-kappaB translocation and VCAM-1 protein expression. Treatment with NO analogues, but not a cAMP analogue, cGMP antagonists, or an NO antagonist, also significantly attenuated VCAM-1 expression. We conclude that (1) C5b-9 deposition, NF-kappaB translocation, and VCAM-1 protein expression are increased in hypoxic HUVECs reoxygenated in HS; (2) reoxygenation of hypoxic HUVECs in HS, but not buffer alone, attenuates ACh-induced cGMP synthesis; and (3) treatment of hypoxic/reoxygenated HUVECs with h5G1.1-scFv attenuates C5b-9 deposition, NF-kappaB translocation, and VCAM-1 expression while preserving ACh-induced cGMP synthesis. C5b-9-induced VCAM-1 expression may thus involve an NO/cGMP-regulated NF-kappaB translocation mechanism.

Topics: Aminoquinolines; Antibodies, Monoclonal; Blotting, Western; Bucladesine; Cell Hypoxia; Cells, Cultured; Complement Membrane Attack Complex; Cyclic GMP; Dibutyryl Cyclic GMP; Endothelium, Vascular; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Glutathione; Humans; Immunotherapy; Intercellular Adhesion Molecule-1; NF-kappa B; NG-Nitroarginine Methyl Ester; Nitric Oxide Donors; Nitroso Compounds; Oxygen; Penicillamine; S-Nitrosoglutathione; Umbilical Veins; Vasculitis; Vasodilation

The role of exogenous nitric oxide (NO) on the expression of interleukin (IL)-2, IL-4, IL-5 and interferon-gamma (IFN-gamma) by freshly isolated human T lymphocytes was investigated. The presence of NO, generated from any of the NO-donor compounds, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), DPTA-nonoate (DPTA) or DETA-nonoate (DETA), added 15 min prior to T-cell stimulation (for 24 hr) with anti-CD3/anti-CD28 monoclonal antibodies (mAbs), resulted in up to 50% inhibition of IL-4, IL-5 and IFN-gamma secretion. In contrast, IL-2 secretion was not inhibited. Using the guanylate cyclase inhibitor, LY83583, it was shown that the inhibition of IL-4 and IL-5 was cGMP dependent, whereas additional mechanisms mediated the inhibition of IFN-gamma. Exposure of T cells to the NO-donor compounds for 24 hr prior to stimulation resulted in a more pronounced inhibition of IFN-gamma secretion by DPTA and DETA (P < 0.01), despite the fact that NO generation could no longer be detected. Under these conditions, IL-4 secretion was not inhibited and IL-5 secretion was inhibited to a lesser extent (P < 0.01 for SNAP and DPTA, P > 0.05 for DETA). IL-2 secretion was inhibited after 24 hr of preincubation with the NO-donor compounds, whereas it was not directly affected by NO. The increased inhibitory effects on IFN-gamma and IL-2 secretion could not be accounted for by the antiproliferative effects of the NO-donor compounds, which were diminished after 24 hr of preincubation relative to 15 min of preincubation. For IFN-gamma, the inhibition was at least partially effected at the transcriptional level as shown by decreased mRNA accumulation. These data show that NO can modulate the balance between the expression, by human T-lymphocytes, of T helper 1- and T helper 2-type cytokines, through selective and persistent inhibition of the expression of IFN-gamma via a cGMP-independent mechanism.

Topics: Aminoquinolines; Cell Division; Cells, Cultured; Cyclic GMP; DEET; Enzyme Inhibitors; Flow Cytometry; Guanylate Cyclase; Humans; Interferon-gamma; Interleukin-2; Interleukin-4; Interleukin-5; Lymphocyte Activation; Nitric Oxide; Nitric Oxide Donors; Penicillamine; RNA, Messenger; T-Lymphocytes

1. The present study was designed to investigate the effects and mechanisms of relaxation induced by the nitric oxide (NO) donor, GEA 3175 (a 3-aryl-substituted oxatriazole derivative) on bovine bronchioles (effective lumen diameter 200-800 microm) suspended in microvascular myographs for isometric tension recording. 2. In segments of bovine bronchioles contracted to 5-hydroxytryptamine, GEA 3175 (10(-8)-10(-4) M) induced concentration-dependent reproducible relaxations. These relaxations were slow in onset compared to other NO-donors such as 3-morpholinosydonimine-hydrochloride (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP). 3. In 5-hydroxytryptamine-contracted preparations the order of relaxant potency (pD2) was: salbutamol (7.80) > GEA 3175 (6.18) > SIN-1 (4.90) > SNAP (3.55). In segments contracted to acetylcholine, the relaxant responses were reduced and GEA 3175 relaxed the bronchioles with pD2 = 4.41 +/- 0.12 and relaxations of 66 +/- 10% (n = 4), while SNAP and salbutamol caused relaxations of 19 +/- 6% (n = 4) and 27 +/- 6% (n = 8) at the highest concentration used, respectively. 4. Oxyhaemoglobin (10(-5) M), the scavenger of nitric oxide, caused rightward shifts of the concentration-relaxation curves to GEA 3175 and NO. 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 3 x 10(-6) M) and LY 83583 (10(-6) M), the inhibitors of soluble guanylate cyclase, also reduced the relaxations induced by GEA 3175 and nitric oxide. However, ODQ did not affect salbutamol-evoked relaxation in the bovine small bronchioles. 5. GEA 3175-induced relaxations were reduced in potassium-rich (60 mmol l(-1) K+) solution. Glibenclamide (10(-6) M) markedly inhibited the relaxations induced by the opener of ATP-sensitive K+ channels, levcromakalim (3 x 10(-8)-10(-5) M), but it did not modify the relaxations induced by GEA 3175 or salbutamol. Apamin (5 x 10(-7) M), a blocker of the small Ca2+-activated K+-channels did not affect the relaxations to GEA 3175. In contrast, blockers of large Ca2+-activated K+-channels, charybdotoxin (3 x 10(-8)-10(-7) M) and iberiotoxin (10(-8) M), did inhibit the relaxations to GEA 3175. The combination of apamin and charybdotoxin did not induce an additional inhibitory effect on the relaxations to GEA 3175 compared to charybdotoxin alone. 6. In preparations where a concentration-response curve to GEA 3175 or NO was first obtained in the presence of LY 83583, incubation with charybdotoxin (10(-7) M) did produce an additional inhibitory eff

Topics: Albuterol; Aminoquinolines; Animals; Apamin; Bronchi; Bronchodilator Agents; Cattle; Charybdotoxin; Female; Glyburide; Male; Molsidomine; Muscle Relaxation; Nitric Oxide; Oxyhemoglobins; Penicillamine; Peptides; Receptors, Adrenergic, beta-2; Superoxide Dismutase; Triazoles

Physiological levels of nitric oxide (NO) regulate vascular tone and protect the microvasculature from injury whereas excessive NO may be harmful. The present study explored the effects of NO on human endothelial cell apoptosis. We found that the NO donor S-nitroso-N-acetylpenicillamine (SNAP) inhibited TNFalpha-induced endothelial apoptosis and that this was mediated partly through the cGMP pathway. In contrast, high SNAP concentration induced endothelial apoptosis via cGMP-independent pathways and the cGMP pathway protected against NO-induced apoptosis. These findings demonstrate that low NO concentrations contribute to human endothelial cell survival, whereas higher NO concentrations are pathological and promote destruction of endothelial cells.

Topics: Aminoquinolines; Apoptosis; Cell Survival; Cyclic GMP; Endothelium, Vascular; Enzyme Inhibitors; Guanylate Cyclase; Humans; Nitric Oxide; Penicillamine; Tumor Necrosis Factor-alpha; Umbilical Veins

Maximum velocity of shortening, Vo, was measured by the method of Edman [J Physiol (Lond) 291:143-159, 1979] on extensor digitorum longus muscles of a mouse in vitro at 20 degreesC. Blockers of nitric oxide synthase, 10 mM nitro-l-arginine or 1 mM 7-nitroindazole, reduced Vo by 18% and 22%, respectively. On removal of the inhibitor, Vo returned to the control value. It was found that 10 mM nitro-d-arginine, an enantiomer of nitro-l-arginine inactive against nitric oxide synthase, did not affect Vo. A donor of nitric oxide, 0.1 mM nitroprusside, increased Vo by 15%. It removed the inhibition caused by nitro-l-arginine. Another donor of nitric oxide, 1 microM (+/-)-S-nitroso-N-acetylpenicillamine (SNAP), increased Vo by 8%. An inhibitor of cGMP synthase, 0.01 mM Ly-83583, decreased Vo by 18%. An analogue of cGMP, 0.1 mM 8-bromo-cGMP, increased Vo by 17%. A general inhibitor of phosphodiesterases, 0.02 mM 3-isobutyl-1-methylxanthine (IBMX), increased Vo by 17%. An inhibitor specific of cGMP phosphodiesterase, 0.01 mM dipyridamole, increased Vo by 8%. The maximal isometric force (F0) was not modified by the drugs, except by 7-nitroindazole and Ly-83583, which depressed F0 by 12%. The cGMP level in tetanized muscles decreased by 12-27% in the presence of blockers of nitric oxide synthase. We conclude that the level of intracellular nitric oxide modulates Vo through the cGMP pathway.

Topics: 1-Methyl-3-isobutylxanthine; Aminoquinolines; Animals; Biomechanical Phenomena; Cyclic GMP; Enzyme Inhibitors; Guanylate Cyclase; Male; Mice; Mice, Inbred C57BL; Muscle Contraction; Muscle, Skeletal; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroarginine; Nitroprusside; Penicillamine; Phosphodiesterase Inhibitors; Stereoisomerism