1-1-diethyl-2-hydroxy-2-nitrosohydrazine and 6-anilino-5-8-quinolinedione

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

The direct effects of nitric oxide (NO) donors and sulfhydryl-modifying agents on the GABA(A) receptor function were examined by perforated patch, whole-cell and single channel recordings in cultured frog melanotrophs. In amphotericin B-perforated cells incubated with the soluble guanylyl cyclase inhibitors LY 83583 and ODQ (10-4 M each), the NO donor sodium nitroprusside (SNP) (10(-3) M) reversibly increased the current evoked by GABA (5 x 10(-6) M). In the whole-cell configuration, internal application of the oxidizing agent H2O2 (0.05%) potentiated the GABA-evoked current while the reducing agent 2-mercaptoethanol (5 x 10(-3) M) slightly decreased the current amplitude. In inside-out patches, GABA (2 x 10(-7) M) triggered single channel bursts of openings. Incubation with the NO donors SNP or DEA/NO (10(-4) M each) enhanced the open probability of the GABA(A) receptor channel but did not modify the chloride reversal potential and did not affect the conductance states. The oxidizing agents H2O2 (0.05%) or DTNB (10-4 M) mimicked the stimulatory effect of the NO donors on the open probability while the reducing compounds 2-mercaptoethanol (5 x 10(-3) M) or DTT (10(-4) M) markedly attenuated the channel activity. Potentiation of the GABA-induced single channel activity by SNP or H2O2 was blocked by 2-mercaptoethanol. Similarly, the potentiating effect produced by DEA/NO or DTNB on the open probability was reversed by DTT. In outside-out patches, incubation with SNP also significantly enhanced the open probability of single channels activated by GABA (10(-6) M). These data indicate that, in frog pituitary melanotrophs, NO potentiates the GABA-evoked current independently of the cGMP/protein kinase pathway. The effect of NO can be accounted for by S-nitrosylation/oxidation of thiol groups either directly on the GABA(A) receptor subunits or on a regulatory protein tightly associated with the GABA(A) receptor.

Topics: Aminoquinolines; Animals; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dithionitrobenzoic Acid; Electric Conductivity; Enzyme Inhibitors; gamma-Aminobutyric Acid; Hydrazines; Ion Channel Gating; Male; Melanocytes; Membrane Potentials; Mercaptoethanol; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Nitroprusside; Oxadiazoles; Oxidation-Reduction; Patch-Clamp Techniques; Pituitary Gland; Quinoxalines; Rana ridibunda; Receptors, GABA-A; Sulfhydryl Reagents

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

Intracellular Ca(2+) mobilization and release into mammal CSF plays a fundamental role in the etiogenesis of fever induced by the proinflammatory cytokine interleukin-1beta (IL-1beta) and other pyrogens. The source and mechanism of IL-1beta-induced intracellular Ca(2+) mobilization was investigated using two experimental models. IL-1beta (10 ng/ml) treatment of rat striatal slices preloaded with (45)Ca(2+) elicited a delayed (30 min) and sustained increase (125-150%) in spontaneous (45)Ca(2+) release that was potentiated by l-arginine (300 microm) and counteracted by N-omega-nitro-l-arginine methyl ester (l-NAME) (1 and 3 mm). The nitric oxide (NO) donors diethylamine/NO complex (sodium salt) (0.3 and 1 mm) and spermine/NO (0.1 and 0.3 mm) mimicked the effect of IL-1beta on Ca(2+) release. IL-1beta stimulated tissue cGMP concentration, and dibutyryl cGMP enhanced Ca(2+) release. The guanyl cyclase inhibitors 1H-[1,2, 4]oxadiazole[4,3-a] quinoxalin-1-one (100 microm) and 6-[phenylamino]-5,8 quinolinedione (50 microm) counteracted Ca(2+) release induced by 2.5 but not 10 ng/ml IL-1beta. Ruthenium red (50 microm) and, to a lesser extent, heparin (3 mg/ml) antagonized IL-1beta-induced Ca(2+) release, and both compounds administered together completely abolished this response. Similar results were obtained in human astrocytoma cells in which IL-1beta elicited a delayed (30 min) increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) (402 +/- 71.2% of baseline), which was abolished by 1 mm l-NAME. These data indicate that the NO/cGMP-signaling pathway is part of the intracellular mechanism transducing IL-1beta-evoked Ca(2+) mobilization in glial and striatal cells and that the ryanodine and the inositol-(1,4,5)-trisphosphate-sensitive Ca(2+) stores are involved.

Topics: Aminoquinolines; Animals; Arginine; Astrocytoma; Calcium; Corpus Striatum; Cyclic GMP; Dibutyryl Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanylate Cyclase; Heparin; Humans; Hydrazines; In Vitro Techniques; Interleukin-1; Intracellular Fluid; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Oxadiazoles; Quinoxalines; Rats; Rats, Sprague-Dawley; Ruthenium Red; Spermine; Tumor Cells, Cultured