Metabolism-based inactivation of nitric oxide synthase by constituents of tobacco and related plants

Cigarette smoking is strongly associated with endothelial dysfunction and impotence. Nitric oxide synthases (NOS) are hemoprotein enzymes that catalyze the conversion of L-arginine to nitric oxide (NO), an important intracellular signaling molecule for many physiological processes including vasodilation and penile erection. Smoking causes a dysfunction in endothelial NOS (eNOS), which is ameliorated, in part, by administration of tetrahydrobiopterin (BH4). In addition, the administration of cigarette smoke leads to loss of neuronal NOS (nNOS) activity and nNOS protein in penile tissue. The exact mechanism that causes the dysfunction in eNOS or nNOS is unknown. I aim to determine in this thesis if a component in cigarettes directly inactivates NOS.; I discovered that water-soluble extracts of cigarette smoke and cigarettes (CE) cause a time-, concentration-, NADPH-, and calmodulin (CAM)-dependent inactivation of nNOS and eNOS in an in vitro system containing the purified enzymes. The kinetics of inactivation are consistent with the metabolism-based inactivation of NOS. Interestingly, the inactivation of eNOS occurs at low levels of exogenous BH4, but at higher concentrations, the BH4 protects eNOS from inactivation by CE. BH4 could slowly reactivate eNOS after treatment with CE, which strongly suggests that BH4-depletion is the mechanism of inactivation. Consistent with this notion, superoxide dismutase protected against eNOS catalyzed oxidation of BH4, suggesting that a redox active component in CE, perhaps a quinone compound, is involved in the inhibition.; In contrast, BH4 does not affect CE-mediated inactivation of nNOS. L-arginine, but not D-arginine, protects nNOS from CE-mediated inactivation. This suggests that CE inactivates nNOS by an active site-directed process. Inactivation is not unique to tobacco, as extracts prepared from other members of the Solanaceae family, such as white petunia and tomato, also inactivate nNOS. I have established that the inactivator(s) of eNOS and nNOS are stable, non-volatile, and have a low molecular mass.; Overall, this thesis is the first to show that chemicals in cigarettes directly inactivate NOS. This work may lead to further understanding of disorders related to smoking.