Abstract 21538: Tetrahydrobiopterin Enhances Hydrogen Peroxide Production by Nitric Oxide Synthase While Suppressing Superoxide Generation: Implications for Cardiovascular Disease
Research into the involvement of NOS in cardiovascular disease has focused on two products of the enzyme, nitric oxide (NO•) and superoxide (•O2-). Some therapeutic regiments have endeavored to regulate NOS function by modulating its substrate L-arginine (L-arg) and cofactor (BH4) in an effort to restore the balance between NO and •O2-. However, NOS can also produce hydrogen peroxide (H2O2), either indirectly via the dismutation of •O2- or directly through a two electron reduction of O2, and this fact has largely been unaccounted for in models of how NOS is involved in the pathogenesis of cardiovascular disease. In this work we investigated the mechanisms involved in each of these H2O2 production pathways. We used direct electrochemical detection of H2O2, coupled with spin-trapping, to determine the direct and indirect production of H2O2 from iNOS. Additionally, we simultaneously measured NADPH oxidation and O2 consumption using UV/Visible spectroscopy and a Clark electrode, respectively. In the absence of BH4 and L-arg, iNOS produced only •O2- and the addition of L-arg or the endogenous NOS inhibitor ADMA increased the rate of its production by an ∼3-fold increase. On the other hand, addition of BH4 (∼75% BH4-saturated iNOS) in the absence of substrate or in the presence of ADMA shifted the enzyme toward the direct production of H2O2 (>60% of the electron flux directly formed H2O2) with a concomitant decrease in •O2- production. In the presence of substrate, the BH4 bound enzyme produced NO• (∼800 nmol min-1 mg-1 at 25oC). The addition of imidazole, which competes with oxygen for the heme site, blocked the direct production of H2O2 in BH4-bound iNOS (>90% decrease) and attenuated the arginine-induced increase in superoxide production from the BH4-free iNOS. Identical results were found with the addition of L-NAME, which prevents electron transfer to the heme and thus inhibits oxygen binding. Therefore, the direct production of H2O2 in iNOS is via the heme site, and although •O2- can be produced by the reductase domain, production of •O2- from the heme site is more efficient. Finally, addition of either L-arg or BH4 alone, will not restore NO• production from NOS damaged by exposure to oxidants. Indeed, under some conditions the addition of L-arg alone could be detrimental.
- © 2010 by American Heart Association, Inc.