nitroarginine and etiron

Nitric oxide (NO) produced by NO synthase (NOS) modulates fetal pulmonary vascular tone and contributes to the fall in pulmonary vascular resistance (PVR) at birth. Although the inducible (type II) NOS isoform is present in human and rat fetal lungs, it is uncertain whether type II NOS activity contributes to vascular NO production in the fetal lung. To determine whether type II NOS is present in the ovine fetal lung and to study the potential contribution of type II NOS on the regulation of basal PVR in the fetus, we measured the hemodynamic effects of three selective type II NOS antagonists: aminoguanidine (AG), 2-amino-5,6-dihydro-6-methyl-4H-1,3 thiazine (AMT), and S-ethylisothiourea (EIT). Studies were performed after at least 72 h of recovery from surgery in 19 chronically prepared fetal lambs (133+/-3 d; 147 d, term). Brief intrapulmonary infusions of AG (140 mg), AMT (0.12 mg), and EIT (0.12 mg) increased basal PVR by 82, 69, and 77%, respectively (P < 0.05). The maximum increase in PVR occurred within 20 min, but often persisted up to 80 min. These agents also increased mean aortic pressure but did not alter the pressure gradient between the pulmonary artery and aorta, suggesting little effect on tone of the ductus arteriosus. Acetylcholine-induced pulmonary vasodilation remained intact after treatment with selective type II NOS antagonists, but not after treatment with the nonselective NOS blocker, nitro-L-arginine. Using Northern blot analysis with poly(A)+ RNA, we demonstrated the presence of two mRNA transcripts for type II NOS (4.1 and 2.6 kb) in the fetal lung. We conclude that the type II NOS isoform is present in the ovine fetal lung, and that selective type II NOS antagonists increase PVR and systemic arterial pressure in the late-gestation fetus. We speculate that type II NOS may play a physiological role in the modulation of vascular tone in the developing fetal lung.

Topics: Acetylcholine; Animals; Aorta; Blotting, Northern; Enzyme Inhibitors; Female; Gestational Age; Guanidines; Hemodynamics; Isothiuronium; Lung; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Pulmonary Artery; Rats; Sheep; Thiazines; Vascular Resistance

Nitric oxide synthases (NOS) have a bidomain structure comprised of an N-terminal oxygenase domain and a C-terminal reductase domain. The oxygenase domain binds haem, (6R)-5,6,7,8-tetrahydro-l-biopterin (tetrahydrobiopterin) and arginine, is the site where nitric oxide synthesis takes place and contains determinants for dimeric interactions. A novel scintillation proximity assay has been established for equilibrium and kinetic measurements of substrate, inhibitor and cofactor binding to a recombinant N-terminal haem-binding domain of rat neuronal NOS (nNOS). Apparent Kd values for nNOS haem-domain-binding of arginine and Nomega-nitro-L-arginine (nitroarginine) were measured as 1.6 microM and 25 nM respectively. The kinetics of [3H]nitroarginine binding and dissociation yielded an association rate constant of 1.3x10(4) s-1.M-1 and a dissociation rate constant of 1.2x10(-4) s-1. These values are comparable to literature values obtained for full-length nNOS, suggesting that many characteristics of the arginine binding site of NOS are conserved in the haem-binding domain. Additionally, apparent Kd values were compared and were found to be similar for the inhibitors, L-NG-monomethylarginine, S-ethylisothiourea, N-iminoethyl-L-ornithine, imidazole, 7-nitroindazole and 1400W (N-[3-(aminomethyl) benzyl] acetamidine). [3H]Tetrahydrobiopterin bound to the nNOS haem domain with an apparent Kd of 20 nM. Binding was inhibited by 7-nitroindazole and stimulated by S-ethylisothiourea. The kinetics of interaction with tetrahydrobiopterin were complex, showing a triphasic binding process and a single off rate. An alternating catalytic site mechanism for NOS is proposed.

Topics: Animals; Arginine; Binding Sites; Binding, Competitive; Biopterins; Enzyme Inhibitors; Heme; Indazoles; Isothiuronium; Kinetics; Nitric Oxide Synthase; Nitroarginine; Oxidoreductases; Oxygenases; Peptide Fragments; Protein Binding; Rats; Recombinant Fusion Proteins

The inhibitory potency of different classes of nitric oxide synthase (NOS) inhibitors (amino acid-based substances, guanidines, isothioureas, imidazoles and indazoles) versus peripheral neuronal NOS in the pig gastric fundus was investigated by studying their influence on electrically induced relaxations in non-adrenergic noncholinergic conditions. Circular muscle strips were mounted for isotonic registration in the presence of atropine and guanethidine, and tone was raised with 5-hydroxytryptamine. Electrical field stimulation (40 V, 0.1 ms, 4 Hz, 10 s) induced short-lasting relaxations. The inhibitory effect of 1-phenylimidazole could not be evaluated because it nearly abolished the 5-hydroxytryptamine-induced tone of the tissues. 7-Nitroindazole, imidazole, 2-iminobiotin and aminoguanidine did not inhibit the electrically induced relaxations, while the other 9 substances tested were able to do so. The influence of the incubation period was tested by studying the inhibitory effect after incubation for 10 up to 60 min. For N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-nitro-L-arginine (L-NNA), L-N5-(1-iminoethyl)-ornithine (L-NIO), L-N6-(1-iminoethyl)-lysine (L-NIL), S-methyl-L-thiocitrulline and S-isopropyl isothiourea there was a moderate increase in the inhibitory effect up to 30 min of incubation so that they were incubated for 30 min to study their inhibitory potency. For L-thiocitrulline, S-methyl isothiourea and S-ethyl isothiourea, an incubation period of 60 min was used. The 9 substances concentration-dependently inhibited the electrically induced relaxations with a maximal inhibitory effect of approximately 80% except for S-methyl isothiourea (Emax of 53%). The overall order of potency was: S-isopropyl isothiourea > S-ethyl isothiourea > or = S-methylL-thiocitrulline > or = L-NNA > L-NIO > L-NAME > S-methyl isothiourea > L-thiocitrulline > L-NIL. While the potency for S-isopropyl isothiourea (EC50: 3.1 x 10(-5) M, n = 6) to S-methyl isothiourea (EC50: 11.5 x 10(-5) M, n = 5) was in the same range, the potency of L-thiocitrulline and L-NIL was clearly lower. This study showed several compounds to be potent inhibitors of peripheral neuronal NOS in the pig gastric fundus while some compounds, that were reported to inhibit brain neuronal NOS were not effective. The EC50 values found for the effective substrates in this functional study may be a guideline for the concentrations required to evaluate the role of NO in NANC neurotransmission in gas

Topics: Animals; Biotin; Citrulline; Electric Stimulation; Enzyme Inhibitors; Gastric Fundus; In Vitro Techniques; Isothiuronium; Lysine; Male; Muscle Contraction; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitroarginine; Ornithine; Swine; Thiourea

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

We have identified two novel potent and selective inhibitors of inducible nitric oxide synthase, S-ethylisothiourea and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine. Ki values of 14.7 nM for S-ethylisothiourea and 4.2 nM for 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine were obtained with partially purified preparations of inducible nitric oxide synthase. These compounds demonstrate about 1000-fold greater potency than prototypical inhibitors, and the inhibitions are 10-40-fold more selective for murine inducible nitric oxide synthase, compared with the rat neuronal and bovine endothelial isoforms of nitric oxide synthase. These compounds also potently inhibit the nitric oxide synthase activity in intact J774 mouse macrophages. The inhibition is competitive with the substrate L-arginine and reversible in both enzymatic and intact cell assays. These potent and selective inhibitors of inducible nitric oxide synthase may have potential therapeutic applications in the treatment of inflammatory and autoimmune diseases.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Enzyme Induction; Isoenzymes; Isothiuronium; Mice; Nitric Oxide Synthase; Nitroarginine; omega-N-Methylarginine; Rats; Thiazines