Time-Dependent Inhibition and Tetrahydrobiopterin Depletion of Endothelial Nitric-Oxide Synthase Caused by Cigarettes

Abstract

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...

ACE2–angiotensin-(1–7)–Mas axis and oxidative stress in cardiovascular disease

“…[30][31][32] Interestingly, BH 4 is believed to be deficient in conditions associated with altered endothelial function, 30 such as hypercholesterolemia, 33 diabetes, 34 high blood pressure 35 and cigarette smoking. 36,37 Both NO and O 2 À are radicals. These molecules react rapidly with each other to generate peroxynitrite, ONOO À38 (Figure 2), which is an important lipid peroxidation mediator.…”

The eNOS cofactor tetrahydrobiopterin improves endothelial dysfunction in livers of rats with CCl4 cirrhosis

“…7 In addition, O 2 Ϫ may interact with NO, leading to the formation of peroxinitrite, further decreasing NO bioavailability. 8,9 eNOS uncoupling, has been demonstrated to modulate endothelial dysfunction in other vascular disorders such as atherosclerosis, 10 diabetes, 11,12 hypertension, 13 hypercholesterolemia, 14 or smoking, 15 and supplementa-tion with BH 4 was able to reverse the reduced NO bioavailability and enhanced O 2 Ϫ production. 14,[16][17][18] This study investigates whether BH 4 deficiency and the ensuing eNOS uncoupling are involved in the reduced NO bioavailability and in the endothelial dysfunction of livers with cirrhosis.…”

Nitric Oxide Pathways and Evidence-Based Perturbations in Acute Pancreatitis

“…Secondly, we anticipated that AP in normal mice would lead to BH 4 oxidation and endothelial dysfunction. Surprisingly, we found that mice with AP had a 5-fold increase over baseline in pancreatic tissue BH 4 concentrations (quantitated by an HPLC fluorescence method [9] ) and a …”

“…Antioxidant effects of BH 4 do not explain these observations because a chemically related pteridine family member, tetrahydroneopterin, has antioxidant properties but no effect on the vasodilatory response in smokers because it cannot couple eNOS to NO synthesis. Recent in vitro studies aimed at chemically identifying the NOS inactivator(s) in cigarettes and cigarette smoke showed that extract prepared from cigarette smoke or unsmoked, ground cigarettes (but not nicotine) irreversibly inhibited nNOS activity by suicide inactivation and reversibly inhibited eNOS enzyme activity by depleting BH 4 within 30-60 min, but nicotine had no effect [9] . Relevant to the pancreas, nNOS inactivation may disrupt neural-mediated smooth muscle relaxation of gastrointestinal tract sphincter muscles, including the sphincter of Oddi, and pose a risk for pancreatitis after endoscopic retrograde cholangiopancreatography (ERCP).…”

“…1 ). A major cause of endothelial dysfunction is BH 4 depletion by cigarette smoking [8][9][10] and alcohol consumption [7] , major risk factors for pancreatic pathology. Impaired NO production by eNOS occurs early in the development of vascular diseases, including diabetes, hypertension, atherosclerosis, myocardial or cerebral ischemia and possibly pancreatic diseases.…”