Inhibition of superoxide generation from neuronal nitric oxide synthase by heat shock protein 90: implications in NOS regulation.
Besides NO, neuronal NO synthase (nNOS) also produces superoxide (O(2)(-.) at low levels of L-arginine. Recently, heat shock protein 90 (hsp90) was shown to facilitate NO synthesis from eNOS and nNOS. However, the effect of hsp90 on the O(2)(-.) generation from NOS has not been determined yet. The interrelationship between its effects on O(2)(-.) and NO generation from NOS is also unclear. Therefore, we performed electron paramagnetic resonance measurements of O(2)(-.) generation from nNOS to study the effect of hsp90. Purified rat nNOS generated strong O(2)(-.) signals in the absence of L-arginine. In contrast to its effect on NO synthesis, hsp90 dose-dependently inhibited O(2)(-.) generation from nNOS with an IC(50) of 658 nM. This inhibition was not due to O(2)(-.) scavenging because hsp90 did not affect the O(2)(-.) generated by xanthine oxidase. At lower levels of L-arginine where marked O(2)(-.) generation occurred, hsp90 caused a more dramatic enhancement of NO synthesis from nNOS as compared to that under normal L-arginine. Significant O(2)(-.) production was detected from nNOS even at intracellular levels of L-arginine. Adding hsp90 prevented this O(2)(-.) production, leading to enhanced nNOS activity. Thus, these results demonstrated that hsp90 directly inhibited O(2)(-.) generation from nNOS. Inhibition of O(2)(-.) generation may be an important mechanism by which hsp90 enhances NO synthesis from NOS.
Song
Yao
Y
Department of Medicine, Division of Cardiology, Johns Hopkins University School of Medicine, 5501 Hopkins Bayview Circle, Baltimore, MD 21224, USA.
Cardounel
A J
AJ
Zweier
Jay L
JL
Xia
Yong
Y
D000818:Animals; D001120:Arginine / Q000378:metabolism; D002460:Cell Line; D002956:Citrulline / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D013997:Time Factors; D014969:Xanthine Oxidase / Q000378:metabolism
D018841:HSP90 Heat-Shock Proteins; D011994:Recombinant Proteins; D013481:Superoxides; D002956:Citrulline; D009569:Nitric Oxide; D001120:Arginine; C496320:NOS1 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D014969:Xanthine Oxidase