Tetrahydrobiopterin protects against guanabenz-mediated inhibition of neuronal nitric-oxide synthase in vitro and in vivo.
It is established that guanabenz inhibits neuronal nitric-oxide (NO) synthase (nNOS) and causes the enhanced proteasomal degradation of nNOS in vivo. Although the time- and NADPH-dependent inhibition of nNOS has been reported in studies where guanabenz was incubated with crude cytosolic preparations of nNOS, the exact mechanism for inhibition is not known. Moreover, even less is known about how the inhibition of nNOS triggers its proteasomal degradation. In the current study, we show, with the use of purified nNOS, that guanabenz treatment leads to the oxidation of tetrahydrobiopterin and formation of a pterin-depleted nNOS, which is not able to form NO. With the use of 14C-labeled guanabenz, we were unable to detect any guanabenz metabolites or guanabenz-nNOS adducts, indicating that reactive intermediates of guanabenz probably do not play a role in the inhibition. Superoxide dismutase, however, prevents the guanabenz-mediated oxidation of tetrahydrobiopterin and inhibition of nNOS, suggesting the role of superoxide as an intermediate. Studies in rats show that administration of tetrahydrobiopterin prevents the inhibition and loss of penile nNOS due to guanabenz, indicating that the loss of tetrahydrobiopterin plays a major role in the effects of guanabenz in vivo. Our findings are consistent with the destabilization and enhanced degradation of nNOS found after tetrahydrobiopterin depletion. These studies suggest that drug-mediated destabilization and subsequent enhanced degradation of protein targets will likely be an important toxicological consideration.
Dunbar
Anwar Y
AY
Department of Pharmacology, University of Michigan Medical School, 1301 Medical Science Research Building III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0632, USA.
Jenkins
Gary J
GJ
Jianmongkol
Suree
S
Nakatsuka
Mikiya
M
Lowe
Ezra R
ER
Lau
Miranda
M
Osawa
Yoichi
Y
D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004795:Enzyme Stability; D006143:Guanabenz / Q000494:pharmacology*; D008297:Male; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010413:Penis / Q000187:drug effects* / Q000201:enzymology; D051381:Rats; D017208:Rats, Wistar; D013482:Superoxide Dismutase / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D025801:Ubiquitin / Q000378:metabolism
D004791:Enzyme Inhibitors; D025801:Ubiquitin; D001708:Biopterins; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D013482:Superoxide Dismutase; C003402:sapropterin; D006143:Guanabenz