Dissociation between Superoxide Accumulation and Nitroglycerin-Induced Tolerance

Abstract

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

Role of Glutaredoxin-Mediated ProteinS-Glutathionylation in Cellular Nitroglycerin Tolerance

“…production was observed in both NTG-tolerant- (Tsou et al, 2008) and GSSG-treated cells. In both cases, O 2 .…”

“…A possible concern of the applicability of this work may arise because we used LLC-PK1 cells, a porcine kidney epithelial cell line, to examine the cellular mechanisms of nitrate tolerance. However, these cells have been used for studying the pathways of nitrate-induced activation and desensitization of cGMP response in several studies Schröder, 1998, 1999;Tsou et al, 2008) and have shown many parallel hallmarks of vascular nitrate tolerance, including the lack of tolerance and cross-tolerance toward strict NO donors such as SNAP, dose-dependent tolerance induction, and tolerance attenuation by vitamin C. We have attempted to examine cellular NTG tolerance in cultured human vascular smooth muscle cells. However, consistent with literature reports (Bennett et al, 1989), these cells, when cultured, were poorly responsive to NTG.…”

“…Thus, increased protein SH oxidation and S-glutathionylation, brought about by either NTG or GSSG, can lead to increased intracellular O 2 . production without the obligatory participation of the angiotensin II (Ang II)/NOX pathway (Tsou et al, 2008). PSSG formation may proceed through several mechanisms (Shelton et al, 2005), namely, 1) thiol-disulfide exchange between cysteine groups in proteins with GSSG; 2) sulfenic acid intermediates; 3) S-nitrosothiol intermediates; and 4) thiyl radical intermediates, e.g., PS ⅐ or GS ⅐ .…”

“…These investigators therefore viewed oxidative stress in nitrate tolerance to precede ALDH2 inactivation. We showed recently (Tsou et al, 2008) that O 2 . generation, particularly through the NADPH oxidase (NOX) system, cannot account for nitrate tolerance initiation.…”

“…Thus, SH oxidation of ALDH2 and other metabolizing enzymes for NTG will lead to reduced bioactivation of the organic nitrate to NO (Beretta et al, 2008), whereas oxidation of the xanthine oxidoreductase (XOR) system leads to the activation of xanthine oxidase (XO), resulting in increased O 2 . accumulation (Tsou et al, 2008). Glutathionylation of SH-sensitive signaling proteins such as p21 ras , we surmise, may account for the downstream effects of nitrate tolerance, such as withdrawal rebound and vascular gene regulation.…”