The mechanisms of vasorelaxation elicited by N -hydroxy-Larginine (L-NOHA) and other compounds bearing a CϭNOH function and the structural determinants governing this effect were investigated in rat aorta. L-NOHA, formamidoxime, five aromatic monosubstituted amidoximes, and one aromatic monosubstituted ketoxime elicited relaxation in endothelium-denuded rings. N-Hydroxyguanidine and substituted N-hydroxyguanidines were markedly less active. Relaxations induced by L-NOHA and by the most active studied compound, 4-chlorobenzamidoxime (ClBZA), were unmodified by the presence of endothelium. In endothelium-denuded rings, they were blunted by the NO scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (300 M) and by the inhibitor of guanylylcyclase activation 1H[1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one (1 M). In addition, L-NOHA-and ClBZA both caused cGMP accumulation. L-Arginine, but not D-arginine (1 mM), antagonized the effect of L-NOHA but not ClBZA. Both L-NOHA-and ClBZA-induced relaxations were inhibited by the NAD(P)H-dependent enzymes inhibitor diphenyliodonium (30 M) and the NAD(P)H-dependent reductases inhibitor 7-ethoxyresorufin (10 M), but they were unmodified by the cytochrome P450 (P450) inhibitor proadifen (10 M) and by the NO synthase inhibitor N -nitro-L-arginine methyl ester (L-NAME, 300 M). These results show that L-NOHA and other compounds with a CϭNOH function can cause endothelium-independent relaxation in the rat aorta. They suggest that activation of guanylyl cyclase and NO formation is implicated in relaxation and that a 7-ethoxyresorufin-sensitive NAD(P)H-dependent pathway is involved. On one hand, L-NOHA and amidoximes may be useful tools for characterizing this pathway in blood vessels and, on the other, may offer a novel approach for treating vascular diseases with impaired endothelial NO activity.
Nitric oxide (NO) production from exogenous NG-hydroxy-L-arginine (OH-L-Arg) was investigated in rat aortic smooth muscle cells in culture by measuring nitrite accumulation in the culture medium. As well, the interaction between OH-L-Arg and L-arginine uptake via the y+ cationic amino acid transporter was studied. In cells without NO-synthase activity, OH-L-Arg (l-1000 ,uM) induced a dose-dependent nitrite production with a half-maximal effective concentration (EC,,) of 18.0 f 1.5 PM (n = 4-7). This nitrite accumulation was not inhibited by the NO-synthase inhibitor No-nitro+arginine methyl ester, L-NAME (300 PM). In contrast, it was abolished by miconaxole (100 pM), an inhibitor of cytochrome PJ5@ Incubation of vascular smooth muscle cells with LPS (10 &ml) induced an L-NAME inhibited nitrite accumulation, but did not enhance the OH-L-Arg induced nitrite production. OH-L-Arg and other cationic amino acids, L-lysine and L-ornithine, competitively inhibited [3H]-L-arginine uptake in rat aortic smooth muscle cells, with inhibition constants of 195 f 23 ,uM (a = 12), 260 & 4OpM (n = 5) and 330 f 10pM (n = 5), respectively. These results show that OH-L-Arg is recognized by the cationic ~-amino acid carrier present in vascular smooth muscle cells and can be oxidized to NO and nitrite in these cells in the absence of NO-synthase, probably by cytochrome P4M or by a reaction involving a cytochrome P4r,, byproduct.
The nitrogen oxide (NO), a free radical molecule, plays a key role in the regulation of mammalian physiology and pathophysiology, e.g., in cardiovascular, nervous or immune systems. This molecule is produced from guanidino moiety of amino acid L-arginine with NG-hydroxy-L-arginine as intermediate and L-citrulline as a co-product of this reaction. This conversion is catalyzed by an ezyme called NO-synthase. The NO-synthase belongs to the cytochrome P450 superfamily and four its isoenzymes are known so far. Two (denoted NOS-I and III) are constitutive, generate lower amounts (pmol) of NO and are regulated by Ca++/calmodulin system. The others (NOS-II and IV) are inducible, produce a larger quantity of NO (nmol) and are regulated at a transcriptional level. The constitutive form is present, for example, in endothelial and neuronal cells whereas the inducible form is de novo synthesized as a consequence of certain stimuli (including cytokines) in macrophages, vascular smooth muscle cells and other cells. There are several target molecules for NO depending on cells. The most frequent target is supposed to be the soluble guanylate cyclase. However, superoxide anion is a very important target for NO, too. The reaction between these two molecules leads to the production of peroxynitrite, the fate of which depends on environmental conditions. Therefore, the importance of this reaction is still debated. This review deals with the nature of NO, the mechanisms of its production, the role of intermediate NG-hydroxy-L-arginine and summarizes the biology of superoxide anion with respect to its reaction with NO. A review with 218 references.
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