Nitric oxide synthase generates superoxide and nitric oxide in arginine-depleted cells leading to peroxynitrite-mediated cellular injury.

Abstract

Besides synthesizing nitric oxide (NO), purified neuronal NO synthase (nNOS) can produce superoxide ( O-) at lower L-Arg concentrations. By using electron paramagnetic resonance spin-trapping techniques, we monitored NO and°2-formation in nNOS-transfected human kidney 293 cells. In control transfected cells, the Ca2+ ionophore A23187 triggered NO generation but no -was seen. With cells in L-Arg-free medium, we observed *°2 formation that increased as the cytosolic L-Arg levels decreased, while NO generation declined.°2 formation was virtually abolished by the specific NOS blocker, N-nitro-L-arginine methyl ester (L-NAME). Nitrotyrosine, a specific nitration product of peroxynitrite, accumulated in L-Arg-depleted cells but not in control cells. Activation by A23187 was cytotoxic to L-Argdepleted, but not to control cells, with marked lactate dehydrogenase release. The cytotoxicity was largely prevented by either superoxide dismutase or L-NAME. Thus, with reduced L-Arg availability NOS elicits cytotoxicity by generating°O2 and NO that interact to form the potent oxidant peroxynitrite. Regulating arginine levels may provide a therapeutic approach to disorders involving°2 /NO-mediated cellular injury.Nitric oxide (NO), a gaseous free radical, regulates vascular tone, platelet aggregation, leukocyte adhesion, synaptic transmission and cytostatic/cytotoxic actions of macrophages (1, 2). NO arises from the guanidino group of L-Arg in an NADPHdependent reaction catalyzed by a family of NO synthases (NOSs) (3). Three distinct isoforms of NOS, derived from separate genes, are neuronal NOS (nNOS, type I), inducible NOS (type II), and endothelial NOS (type III) (4,5). The three isoforms are similar in structure and function, utilizing L-Arg, oxygen, and NADPH as substrates and requiring FAD, FMN, calmodulin, and tetrahydrobiopterin as cofactors (6). NOS is a cytochrome P450 reductase-like hemoprotein containing FAD,-FMN, NADPH, calmodulin, and ferroprotoporphyrin IX (heme) binding sites (7). The catalytic mechanism of NOS involves flavin-mediated electron transport from NADPH to the terminal heme, where oxygen is bound and incorporated into NO and citrulline (8,9).Besides synthesizing NO, purified porcine nNOS generates hydrogen peroxide (H202) at low concentrations of L-Arg (10, 11). Purified rat nNOS produces superoxide (°O2) in an NADPH and Ca2+/calmodulin-dependent manner (12). Ques-tions not yet answered include (i) whether NOS-mediated°2 generation occurs in intact cells, (ii) how cytosolic NOS is regulated to produce either NO or°, and (iii) whether in intact cells NO and O2 generated by NOS combine to form the cytotoxic oxidant peroxynitrite (ONOO-). Furthermore, the biological significance of the°O2 generated by NOS has not been established. In the present study, we directly measure and characterize the formation of both NO and°2J in neuronal constitutive NOS-transfected human kidney 293 cells by using electron paramagnetic resonance (EPR) spin-trapping techniques. The effects of intracellular L-Arg depleti...

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

“…Consistent with this proposal was the observation that, in the absence of H % Bip, NOS monomerization was inhibited by SOD (1 k-unit\ml ; Figure 7B), implicating the involvement of destabilizing O # − d. Importantly, NOS seemed to be the source of O # − d, because NO # Arg (1 mM), which inhibits the formation of O # − d from NOS-I [49,50], prevented monomerization (Table 2). Taken together, these observations suggest a protective role for H % Bip by a novel mechanism involving the suppression of auto-damaging O # − d.…”

“…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. a situation in which O # − d is known to be formed [21,37,49], SOD was discovered to inhibit NOS monomerization. This finding provides a mechanistic insight into the stabilizing effects of H % Bip.…”

“…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].…”

“…Interestingly, when pig brain NOS-I was added in the absence of -arginine, i.e. to uncouple catalysis fully and increase the endogenous generation of O # − d [49][50][51], total H % Bip recovery (enzyme-bound and free in solution) became increasingly incomplete ( Figure 8B). Most notably, H % Bip (in the absence of NOS) was found to be chemically stable under these assay conditions ( Figure 8A), in accord with previous observations [25].…”

Regulation of smooth muscle by inducible nitric oxide synthase and NADPH oxidase in vascular proliferative diseases

“…Increased expression of arginase during vascular injury may shunt L-arginine metabolism to ornithine and polyamine synthesis, inhibit iNOS-derived NO production, and promote cell proliferation [37]. Similarly, the requirement of iNOS activity for tetrahydriobiopterin (H 4 B) is well established and the production of superoxide and peroxynitrite by iNOS in vitro under suboptimal conditions has been described [58,164]. H 4 B deficiency has been recently investigated as a cause of eNOS dysregulation and endothelial dysfunction in several clinical settings including artherosclerosis [150] and the implications of changes in H 4 B bioavailability for iNOS activity and vascular injury have yet to be described.…”