ascorbic-acid and 1-1-diethyl-2-hydroxy-2-nitrosohydrazine

Vascular tolerance to nitroglycerin (GTN) may be caused by impaired GTN bioactivation due to inactivation of mitochondrial aldehyde dehydrogenase (ALDH2). As relaxation to GTN is reduced but still sensitive to ALDH2 inhibitors in ascorbate deficiency, we compared the contribution of ALDH2 inactivation to GTN hyposensitivity in ascorbate deficiency and classical in vivo nitrate tolerance.. Guinea pigs were fed standard or ascorbate-free diet for 2 weeks. Reversibility was tested by feeding ascorbate-deficient animals standard diet for 1 week. Nitrate tolerance was induced by subcutaneous injection of 50 mg x kg(-1) GTN 4 times daily for 3 days. Ascorbate levels were determined in plasma, blood vessels, heart and liver. GTN-induced relaxation was measured as isometric tension of aortic rings; vascular GTN biotransformation was assayed as formation of 1,2- and 1,3-glyceryl dinitrate (GDN).. Two weeks of ascorbate deprivation had no effect on relaxation to nitric oxide but reduced the potency of GTN approximately 10-fold in a fully reversible manner. GTN-induced relaxation was similarly reduced in nitrate tolerance but not further attenuated by ALDH inhibitors. Nitrate tolerance reduced ascorbate plasma levels without affecting ascorbate in blood vessels, liver and heart. GTN denitration was significantly diminished in nitrate-tolerant and ascorbate-deficient rings. However, while the approximately 10-fold preferential 1,2-GDN formation, indicative for active ALDH2, had been retained in ascorbate deficiency, selectivity was largely lost in nitrate tolerance.. These results indicate that nitrate tolerance is associated with ALDH2 inactivation, whereas ascorbate deficiency possibly results in down-regulation of ALDH2 expression.

Topics: Aldehyde Dehydrogenase; Animals; Ascorbic Acid; Ascorbic Acid Deficiency; Biotransformation; Chloral Hydrate; Disease Models, Animal; Dose-Response Relationship, Drug; Down-Regulation; Drug Tolerance; Enzyme Activation; Enzyme Inhibitors; Female; Guinea Pigs; Hydrazines; Injections, Subcutaneous; Isoflavones; Male; Nitric Oxide; Nitric Oxide Donors; Nitroglycerin; Time Factors; Vasodilation; Vasodilator Agents

Bioactivation of nitroglycerin (GTN) into an activator of soluble guanylate cyclase (sGC) is essential for the vasorelaxant effect of the drug. Besides several enzymes that catalyze GTN bioactivation, the reaction with cysteine is the sole nonenzymatic mechanism known so far. Here we show that a reaction with ascorbate results in GTN bioactivation. In the absence of ascorbate, GTN did not affect the activity of purified sGC. However, the enzyme was activated to approximately 20% of maximal NO-stimulated activity by GTN in the presence of 10 mM ascorbate with an EC(50) value of 27.3 +/- 4.9 microM GTN. The EC(50) value of ascorbate was 0.11 +/- 0.011 mM. Activation of sGC was sensitive to oxyhemoglobin, superoxide, and a heme-site enzyme inhibitor. GTN had no effect when ascorbate was replaced by 1000 U of superoxide dismutase per milliliter. Ascorbate is known to reduce inorganic nitrite to NO. In the presence of 10 mM ascorbate, approximately 30 microM nitrite caused the same increase in sGC activity as 0.3 mM GTN. Determination of ascorbate-driven 1,2- and 1,3-glycerol dinitrate formation indicated that a 140 nM concentration of products was generated from 0.3 mM GTN within 10 min, excluding nitrite as a relevant intermediate. Our results suggest that a reaction between GTN and ascorbate or an ascorbate-derived species yields an activator of sGC with NO-like chemical properties. This reaction may contribute to GTN bioactivation in blood vessels under conditions of GTN tolerance and ascorbate supplementation.

Topics: Animals; Ascorbic Acid; Biotransformation; Cells, Cultured; Enzyme Activation; Guanylate Cyclase; Hydrazines; Nitroglycerin; Rats

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

Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate to orotate in the pyrimidine biosynthesis pathway. It is functionally connected to the respiratory chain, delivering electrons to ubiquinone. We report here that inhibition of cytochrome c oxidase by nitric oxide (NO) indirectly inhibits DHODH activity. In digitonin-permeabilized cells, DEA/NO, a chemical NO donor, induced a dramatic decrease in DHO-dependent O(2) consumption. The inhibition was reversible and more pronounced at low O(2) concentration; it was correlated with a decrease in orotate synthesis. Since orotate is the precursor of all pyrimidine nucleotides, indirect inhibition of DHODH by NO may significantly contribute to NO-dependent cytotoxicity.

Topics: Animals; Antioxidants; Ascorbic Acid; Digitonin; Dihydroorotate Dehydrogenase; Electron Transport; Electron Transport Complex IV; Enzyme Inhibitors; Humans; Hydrazines; K562 Cells; Leukemia L1210; Leukemia-Lymphoma, Adult T-Cell; Macrophages; Mice; Mitochondria; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Orotic Acid; Oxidation-Reduction; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Oxygen; Rats; Recombinant Proteins; Tumor Cells, Cultured