We hypothesize that nitroglycerin (NTG) causes direct oxidation of multiple cellular sulfhydryl (SH) proteins and that manipulation of SH redox status affects NTG tolerance. In LLC-PK1 cells, we found that nitrate tolerance, as indicated by cGMP accumulation toward NTG, was accompanied by increased protein [35 S]cysteine incorporation, significant S-glutathionylation of multiple proteins, and decreased metabolic activity of several SH-sensitive enzymes, including creatine kinase, xanthine oxidoreductase, and glutaredoxin (GRX). Cells overexpressing GRX exhibited reduced cellular protein S-glutathionylation (PSSG) and absence of NTG tolerance, whereas those with silenced GRX showed increased extent of NTG-induced tolerance. Incubation of LLC-PK1 cells with oxidized glutathione led to several major observations associated with nitrate tolerance, namely, reduced cGMP accumulation, PSSG formation, superoxide accumulation, and the attenuation of these events by vitamin C. Aortic S-glutathionylated proteins increased approximately 3-fold in rats made tolerant in vivo to NTG and showed significant negative correlation with vascular responsiveness ex vivo. NTG incubation in EA.hy926 endothelial cells and LLC-PK1 cells led to increased S-glutathionylation and activity of p21 ras , a known mediator of cellular signaling. These results indicate that the hallmark events of NTG tolerance, such as reduced bioactivation and redox signaling, are associated with GRX-dependent protein deglutathionylation.

We hypothesize that superoxide (O 2 . ) accumulation is not a crucial causative factor in inducing nitroglycerin (NTG) . accumulation may be an effect, rather than an initiating cause, of NTG tolerance.Nitroglycerin (NTG; glyceryl trinitrate) and other organic nitrates have been widely used to treat coronary artery disease in patients with stable and unstable angina, acute myocardial infarction, and congestive heart failure. When administered acutely, NTG relieves ischemic symptoms rapidly without serious side effects. However, its efficacy after repeated dosing is attenuated because of the development of pharmacologic tolerance. This phenomenon was first described by Stewart (1888) in the 19th century, but its underlying mechanism(s) still remained incompletely defined.Nitrate tolerance is a complex phenomenon accompanied by a myriad of events, including decreased pharmacological response, reduced metabolism, increased oxidative stress, altered gene expression, etc. Consequently, several hypotheses have been proposed to account for these various phenomenon, including those of sulfhydryl depletion, impaired biotransformation of mitochondrial aldehyde dehydrogenase (aldehyde dehydrogenase 2; ALDH2), and oxidative stress, as reviewed recently in Fung (2004) and Mü nzel et al. (2005).Mü nzel et al. (1995) found that a prolonged period of NTG exposure in rabbits enhanced superoxide (O 2 . ) production in the blood vessel wall, and they proposed that nitrate tolerance was caused by increased O 2 . formation. Further development of this mechanism, which became known as "the O 2 . -oxidative stress hypothesis," suggested that chronic NTG treatment increases angiotensin II (Ang II) binding to endothelium angiotensin receptors, subsequently stimulating NADPH oxidase (NOX) to produce O 2 . . This oxygen-free radical then reacts with nitric oxide (NO), the putative intermediate of NTG, to form peroxynitrite, an oxidant and a weaker vasodilator than NO. Oxidative stress also brings about uncoupling of endothelial NO synthase (eNOS), resulting in decreased NO availability and further O 2

We hypothesize that superoxide (SO) accumulation is not a critical causative factor in inducing nitroglycerin (NTG) tolerance. Using p47phox−/− and gp91−/− mice vs. their respective wild-type (WT) controls, we showed that aorta from mice null of these critical NADPH oxidase subunits exhibited similar vascular tolerance after NTG dosing (20 mg/kg sc, tid for 3 days), as indicated by their ex vivo pEC 50 and cyclic guanosine monophosphate (cGMP, pmol/mg protein) accumulation upon NTG challenge. In vitro aorta SO production (cpm/mg dry weight) was enhanced by NTG incubation both in p47phox null and WT mice (Table 1 ). Pre-exposure of isolated mice aorta to 100 microM NTG for 1 hr resulted in vascular tolerance toward NTG and increased SO accumulation. Oxypurinol (Oxy, 1mM) reduced SO but failed to attenuate vascular tolerance (Table 2 ). In LLC-PK1 cells, pre-exposure to NTG (1 microM for 4 hours) resulted in increased SO accumulation and reduced cGMP response to 3.16 microM NTG vs. vehicle control. Exposure to 1 microM angiotensin II increased SO but did not reduce cGMP response. Taken together, these results indicate that in vivo vascular NTG tolerance in mice does not require the presence of the p47phox and gp91phox subunits of NADPH oxidase, and that increased SO accumulation may be a consequence, rather than a cause, of NTG tolerance. Table 1 Table 2