Nitric oxide synthase (NOS) is a highly regulated enzyme that produces nitric oxide, a critical messenger in many physiological processes. In this perspective, we explore the role of proteolytic degradation of NOS, in particular the inducible and neuronal isoforms of NOS, as a mechanism of regulation of the enzyme. The ubiquitin-proteasome and calpain pathways are the major proteolytic systems identified to date that are responsible for this regulated degradation. The degradation of NOS is affected by diverse agents, including glucocorticoids, caveolin, neurotoxic compounds, and certain NOS inhibitors. Some irreversible inactivators of NOS enhance the proteolytic degradation of the enzyme, and this property may be of great importance in understanding the biological effects of these inhibitors, some of which are being developed for clinical use. Analogies with the regulated degradation of liver microsomal cytochromes P450, which are related to NOS, provide a framework for understanding these processes. Finally, a new perspective on the regulation of NOS by hsp90-based chaperones is presented that involves facilitated heme insertion into the enzyme.Nitric oxide, the radical metabolite formed from the metabolism of L-arginine by nitric oxide synthase (NOS), has been shown to be involved in a variety of physiological processes, including neurotransmission, vasorelaxation, platelet aggregation, and penile erection, as well as in a variety of pathological conditions including septic shock, reperfusion injury, arthritis, atherosclerosis, diabetes, and graft rejection (Moncada et al., 1991;Burnett et al., 1992;Forstermann et al., 1994;Schmidt and Walter, 1994). There have been several reviews, one of them very recent (Alderton et al., 2001), on the structure, function, and inhibition of the three major isoforms of NOS: the neuronal (nNOS), inducible (iNOS) and endothelial (eNOS). Although various aspects of the regulation of these enzymes have also been reviewed, the focus of the current perspective on the regulated proteolytic degradation of NOS has not. Since certain NOS inhibitors selectively enhance the degradation of NOS protein, this regulatory process is likely to be of importance in understanding the biological actions of NOS inhibitors, some of which are currently being tested for human use. We also present a perspective on the role of hsp90-based chaperones in affecting the proteolysis of NOS by regulating heme insertion and formation of the active dimeric form. Proteolysis of NOSSelective proteolytic degradation of NOS is a mechanism for regulation of the enzyme. For example, transforming growth factor-␤ enhances the degradation of iNOS in interferon-␥-treated mouse peritoneal macrophages and causes suppression of NO release from these cells (Vodovotz et al., 1993). The glucocorticoid-mediated suppression of iNOS expression in IL-1␤-treated rat glomerular mesangial cells (Kunz et al., 1996) and interferon-␥-treated murine macrophage cell line RAW 264.7 (Walker et al., 1997) is due, in part, to increas...

ABSTRACT: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.Cigarette smoking is dose dependently associated with impairment of endothelial-dependent dilation in humans (Adams et al., 1997). The role of NO deficit in the genesis of vascular disease has been reviewed (Cooke and Dzau, 1997b); moreover, it is known that acute as well as chronic smoking decreases exhaled NO in humans (Kharitonov et al., 1995). A variety of mechanisms have been postulated to explain this NO deficit (Cooke and Dzau, 1997a), including the enhanced formation of superoxide, which would rapidly react with NO and thereby decrease its bioactivity (Kelm et al., 1997). More recently, Heitzer et al. (2000) reported that administration of tetrahydrobiopterin (BH 4 ) to chronic smokers improves endothelium-dependent vasodilation as determined by forearm blood-flow measurements. Administration of tetrahydroneopterin, which has the same antioxidant properties as BH 4 but is not a cofactor for NO synthase, did not improve vascular function and further supports the notion of a specific dysfunction in NOS rather than a general enhancement of oxidative stress (Heitzer et al., 2000). In addition, the vasodilator response due to sodium nitroprusside is not affected in these smokers irrespective of BH 4 administration, indicating that the responsiveness to NO is not changed. Thus, taken together, these observations strongly implicate a dysfunctional NO synthase in smokers (Heitzer et al., 2000). This is consistent with an earlier r...

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.

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