Ca2+/calmodulin-dependent formation of hydrogen peroxide by brain nitric oxide synthase

Abstract

L-Arginine-derived nitric oxide (NO) acts as an inter- and intra-cellular signal molecule in many mammalian tissues including brain, where it is formed by a flavin-containing Ca2+/calmodulin-requiring NO synthase with NADPH, tetrahydrobiopterin (H4biopterin) and molecular oxygen as cofactors. We found that purified brain NO synthase acted as a Ca2+/calmodulin-dependent NADPH:oxygen oxidoreductase, catalysing the formation of hydrogen peroxide at suboptimal concentrations of L-arginine or H4biopterin, which inhibited the hydrogen peroxide formation with half-maximal effects at 11 microM and 0.3 microM respectively. Half-maximal rates of L-citrulline formation were observed at closely similar concentrations of these compounds, indicating that the NO synthase-catalysed oxygen activation was coupled to the synthesis of L-citrulline and NO in the presence of L-arginine and H4biopterin. N omega-Nitro-L-arginine, its methyl ester and N omega-monomethyl-L-arginine inhibited the synthesis of L-citrulline from L-arginine (100 microM) with half-maximal effects at 0.74 microM, 2.8 microM and 15 microM respectively. The N omega-nitro compounds also blocked the substrate-independent generation of hydrogen peroxide, whereas N omega-monomethyl-L-arginine did not affect this reaction. According to these results, activation of brain NO synthase by Ca2+ at subphysiological levels of intracellular L-arginine or H4biopterin may result in the formation of reactive oxygen species instead of NO, and N omega-nitro-substituted L-arginine analogues represent useful tools to effectively block NO synthase-catalysed oxygen activation.

Effect of Nitric Oxide Blockade by NG-Nitro-l-Arginine on Cerebral Blood Flow Response to Changes in Carbon Dioxide Tension

“…This suggests that the in crease in NO hypothesized above to occur with CO2 should be expected to act on the pHcsensitive com ponent of the CO2 responsiveness. This hypothesis fits with the interesting in vitro study of Heinzel et al (1992) showing that NO snythase activity from the brain tissue (cerebellum) is quite markedly in fluenced by pH in that acid pH increases NO pro duction. With acetazolamide, pHi is essentially un changed, while with CO2, pHi becomes acid and NO synthesis might therefore be increased.…”

Plasma amino acids in anorexia nervosa

“…The low arginine concentration might also have other consequences, because this amino acid is a precursor for the synthesis of creatine and nitric oxide. In the presence of suboptimal concentrations of arginine, nitric oxide synthase forms superoxide (Heinzel et al, 1992), that reacts avidly with nitric oxide to form peroxinitrite (Heales et al, 1996). Excess free radical production by this and other mechanisms might be a further factor causing cellular damage in anorexia (Roediger, 1995).…”

Allosteric regulation of neuronal nitric oxide synthase by tetrahydrobiopterin and suppression of auto-damaging superoxide

“…It is known that in the absence of either -arginine or H % Bip, NOS becomes enzymically uncoupled and reduces molecular oxygen at the expense of NADPH to form reactive oxygen species such as O # − d [48][49][50] and H # O # [22,41]. Moreover, we have recently shown that NOSderived uncoupled catalysis products can accumulate during -arginine turnover and influence enzyme activity [41].…”

“…During catalysis, NOS can produce significant quantities of reactive oxygen species (O # − d and H # O # ) [21,22,41,50,51] ; H % Bip markedly attenuates this [21,22,37] by a largely unknown mechanism that might involve the haem domain [51]. Importantly, given that NOS-derived reactive oxygen species contribute to enzyme inactivation [41], we considered the possibility that H % Bip stabilizes and protects NOS by interfering with autodamaging O # − d. In agreement with this hypothesis, during enzyme catalysis and in the absence of H % Bip, i.e.…”

“…However, the current consensus in the literature does not support such a classical catalytic role [13][14][15][16][17], although some studies can be found to the contrary [18]. Alternatively, protective and stabilizing effects of H % Bip have been described, including (1) the stabilization of the NOS mRNA product [19], (2) protection of the NOS catalytic centre from oxidative damage [16,20], (3) prevention of the formation of superoxide (O # − d) [21] and H # O # [22] by coupling reductive oxygen activation to -arginine oxidation, and (4) promotion of subunit assembly from monomers and stabilization of dimers in the presence of chemical denaturants [23,24] or under native conditions during -arginine turnover [17,25]. Interestingly, the stabilizing effects of H % Bip might be isoform-dependent.…”