Time-dependent inhibition and tetrahydrobiopterin depletion of endothelial nitric-oxide synthase caused by cigarettes.
Smoking causes a dysfunction in endothelial nitric-oxide synthase (eNOS), which is ameliorated, in part, by administration of tetrahydrobiopterin (BH(4)). The exact mechanism by which the nitric oxide deficit occurs is unknown. We have previously shown that aqueous extracts of chemicals in cigarettes (CE) cause the suicide inactivation of neuronal NO synthase (nNOS) by interacting at the substrate-binding site. In the current study, we have found that CE directly inactivates eNOS by a process that is not affected by the natural substrate l-arginine and is distinct from the mechanism of inactivation of nNOS. We discovered that CE causes a time-, concentration-, and NADPH-dependent inactivation of eNOS in an in vitro system containing the purified enzyme, indicating a metabolic component to the inactivation. The CE-treated eNOS but not nNOS was nearly fully reactivated upon incubation with excess BH(4), suggesting that BH(4) depletion is a potential mechanism of inactivation. Moreover, in the presence of CE, eNOS catalyzed the oxidation of BH(4) to dihydrobiopterin and biopterin by a process attenuated by high concentrations of superoxide dismutase but not catalase. We speculate that a redox active component in CE, perhaps a quinone compound, causes oxidative uncoupling of eNOS to form superoxide, which in turn oxidizes BH(4). The discovery of a direct inactivation of eNOS by a compound(s) present in tobacco provides a basis not only for further study of the mechanisms responsible for the biological effects of tobacco but also a search for a potentially novel inactivator of eNOS.
Department of Pharmacology, University of Michigan Medical School, 1301 Medical Science Research Building III, Ann Arbor, MI 48109-0632, USA.
Ezra R
ER
Lowe
Andrew C
AC
Everett
Anthony J
AJ
Lee
Miranda
M
Lau
Anwar Y
AY
Dunbar
Vladimir
V
Berka
Ah-Lim
AL
Tsai
Yoichi
Y
Osawa
D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000494:pharmacology; D007313:Insecta; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D051381:Rats; D012907:Smoking / Q000378:metabolism; D013638:Tars / Q000302:isolation & purification / Q000494:pharmacology*; D013997:Time Factors
D004791:Enzyme Inhibitors; D013638:Tars; C024746:tobacco tar; D001708:Biopterins; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; C003402:sapropterin