nitroarginine and diphenyleneiodonium

NADPH, a substrate for the superoxide-producing enzyme NADPH oxidase, produces vasodilation in the cerebral circulation. However, the mechanisms of the effect have not been fully elucidated. We used a peptide inhibitor of NADPH oxidase (gp91ds-tat) and null mice lacking the gp91phox subunit of NADPH oxidase to examine the mechanisms of the cerebrovascular effects of exogenous NADPH.. Cerebral blood flow (CBF) was assessed by laser-Doppler flowmetry in anesthetized mice equipped with a cranial window. Superfusion with NADPH increased CBF (27% at 100 micromol/L) without affecting the EEG. The CBF increase was attenuated by the free-radical scavenger MnTBAP (-54%, P<0.05) but not by the H2O2 scavenger catalase. The response was also attenuated by gp91ds-tat (-64%, P<0.05) and by the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (-44%, P<0.05). The increase in CBF produced by NADPH was attenuated in gp91-null mice (-41%, P<0.05). NADPH increased production of reactive oxygen species, assessed by hydroethidine microfluorography, an effect blocked by MnTBAP or gp91ds-tat and not observed in gp91-null mice.. These data suggest that the mechanisms of the CBF increases produced by exogenous NADPH are multifactorial and include NADPH oxidase-dependent and -independent factors.

Topics: Animals; Blood Flow Velocity; Cerebrovascular Circulation; Crosses, Genetic; Electroencephalography; Female; Fluorescent Dyes; Free Radical Scavengers; Glycoproteins; Laser-Doppler Flowmetry; Male; Mice; Mice, Inbred C57BL; NADP; NADPH Oxidases; Neurons; Nitric Oxide Synthase; Nitroarginine; Onium Compounds; Phenanthridines; Reactive Oxygen Species; Vasodilation

Reactive oxygen species have been shown to be involved in the mutagenicity, clastogenicity, and apoptosis of mammalian cells treated with arsenic or cadmium. As these endpoints require several hours of cellular processing, it is not clear that reactive oxygen species damage DNA directly or interfere with DNA replication and repair. Using single-cell alkaline electrophoresis, we have detected DNA strand breaks (DSBs) in bovine aortic endothelial cells by a 4-h treatment with sodium arsenite (As) and cadmium chloride (Cd) in sublethal concentrations. As-induced DSBs could be decreased by nitric oxide (NO) synthase inhibitors, superoxide scavengers, and peroxynitrite scavengers and could be increased by superoxide generators and NO generators. Treatment with As also increased nitrite production. These results suggest that As-increased NO may react with O2*- to produce peroxynitrite and cause DNA damage. The results showing that Cd increased cellular H2O2 levels and that Cd-induced DSBs could be modulated by various oxidant modulators suggest that Cd may induce DSBs via O2*-, H2O2, and *OH. Nevertheless, the DSBs in both As- and Cd-treated cells seem to come from the excision of oxidized bases such as formamidopyrimidine and 8-oxoguanine, as the Escherichia coli enzyme formamidopyrimidine-DNA glycosylase (Fpg) increased DSBs in cells treated with As, 3-morpholinosydnonimine (a peroxynitrite-generating agent), Cd, or H2O2.

Topics: Amitrole; Animals; Antioxidants; Aorta; Arsenites; Bacterial Proteins; Cadmium Chloride; Catalase; Cattle; Cells, Cultured; Chromans; Citrulline; Ditiocarb; DNA Damage; DNA-Formamidopyrimidine Glycosylase; Endothelium, Vascular; Enzyme Inhibitors; Escherichia coli Proteins; Free Radical Scavengers; Hydrogen Peroxide; Molsidomine; Mutagens; N-Glycosyl Hydrolases; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroarginine; Onium Compounds; Phenanthrolines; Reactive Oxygen Species; Sodium Compounds; Sodium Selenite; Superoxide Dismutase; Superoxides; Thiomalates; Thiourea; Uric Acid

Rat cerebellar nitric oxide synthase (NOS) purified from transfected human kidney cells catalyzes an NADPHdependent reduction of quinonoid dihydrobiopterin (qBH2) to tetrahydrobiopterin (BH4). Reduction of qBH2 at 25 microM proceeds at a rate that is comparable with that of the overall reaction (citrulline synthesis) and requires calcium ions and calmodulin for optimal activity; NADH has only 10% of the activity of NADPH. The reduction rate with the quinonoid form of 6-methyldihydropterin is approximately twice that with qBH2. 7,8-Dihydrobiopterin had negligible activity. Neither 7,8-dihydrobiopterin nor BH4 affected the rate of qBH2 reduction. Reduction is inhibited by the flavoprotein inhibitor diphenyleneiodonium, whereas inhibitors of electron transfer through heme (7-nitroindazole and N-nitroarginine) stimulated the rate to a small extent. Methotrexate, which inhibits a variety of enzymes catalyzing dihydrobiopterin reduction, did not inhibit. These studies provide the first demonstration of the reduction of qBH2 to BH4 by NOS and indicate that the reduction is catalyzed by the flavoprotein "diaphorase" activity of NOS. This activity is located on the reductase (C-terminal) domain, whereas the high affinity BH4 site involved in NOS activation is located on the oxygenase (N-terminal) domain. The possible significance of this reduction of qBH2 to the essential role of BH4 in NOS is discussed.

Topics: Animals; Arginine; Biopterins; Cell Line; Cerebellum; Enzyme Inhibitors; Humans; Indazoles; Kidney; Kinetics; Methotrexate; NADP; Nitric Oxide Synthase; Nitroarginine; Onium Compounds; Oxidation-Reduction; Rats; Recombinant Proteins; Transfection

Topics: Adenosine Diphosphate; Animals; Arginine; Cell Line; Colonic Neoplasms; Enzyme Inhibitors; Humans; Isoenzymes; Isothiuronium; Kinetics; Macrophages; Mice; Nitric Oxide Synthase; Nitroarginine; omega-N-Methylarginine; Onium Compounds; Tumor Cells, Cultured

The effects of NG-nitro-L-arginine (L-NNA), D-NNA, diphenyleneiodonium and di-2-thienyliodonium on contraction were studied in endothelium-denuded rat aortic rings incubated for 4 h with lipopolysaccharide (10 mu g ml-1) or vehicle. Lipopolysaccharide reduced Emax and increased EC50 of the phenylephrine (10-9-10-5 M) curve. Addition of D-NNA (4, 6 x 10-4 M), L-NNA (1, 10 x 10-6 M) and diphenyleneiodonium (1, 3 x 10-7 M), but not di-2-thienyliodonium (10-7 M), increased Emax and reduced EC50 of the phenylephrine curve of lipopolysaccharide-incubated but not control rings. Therefore, D-NNA, L-NNA and diphenyleneiodonium, but not di-2-thienyliodonium, inhibit inducible NO synthase in vascular smooth muscles.

Topics: Animals; Aorta, Thoracic; Dose-Response Relationship, Drug; Hypoglycemic Agents; Lipopolysaccharides; Male; Muscle Contraction; Muscle, Smooth, Vascular; Nitroarginine; Onium Compounds; Phenylephrine; Rats; Rats, Sprague-Dawley; Thiophenes

1. This study examined the in vitro and in vivo inhibitory effects of diphenyleneiodonium (DPI), a novel inhibitor of nitric oxide (NO) synthase, on endothelium-dependent vasodilatations. 2. DPI (3 x 10(-8)-3 x 10(-6) M) concentration-dependently inhibited acetylcholine (ACh)-induced relaxation in preconstricted rat thoracic aortic rings, with an IC50 of 1.8 x 10(-7) M and a maximal inhibition of nearly 100%. DPI (3 x 10(-6) M) also completely inhibited the relaxation induced by the calcium ionophore, A23187 but not by sodium nitroprusside (SNP). The inhibitory effect of DPI (3 x 10(-7) M) on ACh-induced relaxation was prevented by pretreatment with NADPH (5 x 10(-3) M) and FAD (5 x 10(-4) M) but not L-arginine (L-Arg, 2 x 10(-3) M). Pretreatment with NADPH did not alter the inhibitory effect of NG-nitro-L-arginine on ACh-induced relaxation. 3. The inhibitory effect of DPI on ACh-induced relaxation in the aortae lasted > 4 h after washout. In contrast to pretreatment, post-treatment (1 h later) with NADPH (5 x 10(-3) M) reversed only slightly the inhibitory effect of DPI. 4. In conscious rats, DPI (10(-5) mol kg-1) inhibited the depressor response to i.v. infused ACh, but not SNP. However, it caused only a transient pressor response which was previously shown to be due completely to sympathetic activation. 5. Thus, DPI is an efficacious and 'irreversible' inhibitor of endothelium-dependent vasodilatation in vivo and in vitro. The mechanism of the inhibition may involve antagonism of the effects of FAD and NADPH, co-factors of NO synthase. However, unlike the N0-substituted arginine analogues (another class of NO synthase inhibitors), DPI-induced suppression of endothelium-dependent vasodilatation in vivo does not lead to a sustained rise in blood pressure.

Topics: Acetylcholine; Amino Acid Oxidoreductases; Animals; Arginine; Blood Pressure; Calcimycin; Drug Interactions; Endothelium, Vascular; Flavin-Adenine Dinucleotide; Kinetics; Male; Muscle Relaxation; Muscle, Smooth, Vascular; NADH, NADPH Oxidoreductases; NADP; Nitric Oxide Synthase; Nitroarginine; Nitroprusside; Onium Compounds; Rats; Rats, Sprague-Dawley; Time Factors; Vasodilation