Metabolism-Based Inactivation of Neuronal Nitric-Oxide Synthase by Components of Cigarette and Cigarette Smoke

Abstract

This article is available online at http://dmd.aspetjournals.org ABSTRACT:It has been shown that administration of cigarette smoke to rats leads to loss of neuronal nitric-oxide synthase (nNOS) activity and nNOS protein in penile tissue. The exact mechanism for this loss of activity and protein is not known. In the current study, we investigated whether extracts prepared from cigarette smoke or from the cigarette itself could directly inhibit nNOS activity. We discovered that the cigarette smoke extract and the cigarette extract cause a time-, concentration-, and calmodulin-dependent inactivation of nNOS in an in vitro system containing the purified enzyme. LArginine, but not D-arginine, protects nNOS from this time-dependent inactivation, suggesting an active site directed event. The kinetics of inactivation are consistent with the metabolism-based or suicide inactivation of nNOS. Based on studies with other metabolism-based inactivators, this cigarette-mediated inactivation may render nNOS more susceptible to proteasomal degradation and thereby may explain the loss of nNOS protein in vivo. The component(s) responsible for nNOS inactivation is not volatile, is not retained by a 3,000 molecular weight cut-off membrane, binds to activated charcoal, and is highly water-soluble under both acidic and basic conditions. The discovery of a direct inactivation of nNOS by an organic, cationic compound(s) present in tobacco and tobacco smoke provides a basis for further study of not only the mechanisms responsible for the biological effects of tobacco but also a search for a potentially novel inactivator of nNOS.

Adverse Effects of Cigarette Smoke on NO Bioavailability

“…Our findings in regard to the direct actions of CSE on NOS enzymatic activity are consistent with studies that predict that the catalytic domain of human eNOS contains 10 cysteine residues with 2 of them (Cys-94 and Cys-99) being responsible for coordinating an intermolecular interaction between 2 monomers. 35 Although our observations are also consistent with previous reports, 13,[22][23][24] some longer-term studies 13,[22][23][24] suggest that a reduction in NOS activity parallels to some extent a decrease in eNOS content. In our study, we saw a more rapid onset of the inhibitory effect of CSE on NOS activity, using isolated purified eNOS enzyme.…”

“…Previous studies suggest that the effect of CSE may relate in part because of reduced NOS expression and enzymatic activity. 13,[22][23][24] Alternatively, Hutchison et al 5 showed previously that arginine supplementation attenuated the endothelial dysfunction caused by exposure to cigarette smoke, suggesting that substrate availability for NOS may be a key mechanism for CSE-induced endothelial dysfunction.…”

Visually evoked cerebral vasomotor response in smoking and nonsmoking young adults, investigated by functional transcranial Doppler

“…Smoking was reported to inhibit endothelial and neuronal NO synthases, impair NO signaling due to increased release of superoxide anion and subsequent inactivation of nitric oxide, and reduce exhaled NO in man, which is an indicator of decreased endogenous NO production (Raij et al, 2001;Demady et al, 2003;Landmesser, Harrison & Drexler, 2005). Inhibition of neuronal or endothelial nitric oxide synthase was shown to attenuate the stimulus evoked flow response in different animal studies, indicating the substantial role of NO in the coupling of neuronal activation to regional cerebral blood flow (Kharitonov, Robbins, Yates, Keatings & Barnes, 1995;Cholet, Seylaz, Lacombe & Bonvento, 1997;Yang & Iadecola, 1998;Demady et al, 2003). Dysregulation of Ca 2+ -channels was also demonstrated in nicotine treated animals, mimicking the effect of an apparent decrease in bioavailability of endogenous NO (Gerzanich, Zhang, West & Simard, 2001).…”

Effect of nicotine and tobacco administration method on the mechanical properties of healing bone following closed fracture

“…Tobacco extract concentrate was prepared from nicotinefree Quest Cigarettes (Vector Tobacco Inc., Timberlake, NC) as described by Demady et al, 9 and was produced every 10 days to maintain freshness. Tobacco from four cigarettes was ground, mixed with 40 ml of distilled water, then filtered and centrifuged at 5,000 rpm for 20 min.…”