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.
Department of Medicine, Division of Cardiology, Johns Hopkins University School of Medicine, 5501 Hopkins Bayview Circle, Baltimore, MD 21224, USA.
Yao
Y
Song
A J
AJ
Cardounel
Jay L
JL
Zweier
Yong
Y
Xia
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