This Article Abstract Figures Only Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by McGrath, L. T. Articles by McVeigh, G. E. Search for Related Content PubMed PubMed Citation Articles by McGrath, L. T. Articles by McVeigh, G. E.

EXPERIMENTAL STUDY

Production of 8-epi prostaglandin F₂ in human platelets during administration of organic nitrates

^* Department of Therapeutics and Pharmacology, Queen’s University Belfast, Belfast, Ireland

Manuscript received January 28, 2002; revised manuscript received April 24, 2002, accepted May 7, 2002.

^* Reprint requests and correspondence: Dr. Lawrence McGrath, Department of Therapeutics and Pharmacology, Whitla Medical Building, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, Ireland.
l.mcgrath{at}qub.ac.uk

	Abstract

Top Abstract Methods Results Discussion References

 
OBJECTIVES: The objective of this study was, using isolated platelets as a surrogate for vascular cells, to examine the effect of nonintermittent organic nitrate administration on 8-epi prostaglandin F₂ (8-epi PGF₂) content and the effect of concurrent oral ascorbate administration.

BACKGROUND: The long-term efficacy of organic nitrates is hampered by hemodynamic tolerance, which develops during continuous administration. This has been associated with altered production of superoxide and nitric oxide, as well as oxidative stress. This effect may be ameliorated by the co-administration of antioxidants.

METHODS: Ten healthy male subjects received nitroglycerin (NTG) transdermally at a dosage of 0.4 mg/h for 3 days with ascorbate or lactose (1.2 g/day). After two weeks washout, the treatment was repeated with reversed ascorbate/lactose. Platelets were prepared by centrifugation and esterified 8-epi PGF₂ measured at the start and finish of each treatment by immunoassay.

RESULTS: Nitroglycerin, in the absence of supplemental ascorbate, was associated with a significant increase in platelet-esterified 8-epi PGF₂, from 32.9 (95% confidence interval [CI] 11.8 to 54.0) to 51.0 (95% CI 16.3 to 85.7) pg/mg protein (p < 0.05). Co-administration of ascorbate with NTG resulted in a significant decrease in 8-epi PGF₂ production, from 38.8 (95% CI 24.9 to 52.7) to 19.0 (95% CI 13.5 to 24.5) pg/mg protein (p < 0.05).

CONCLUSIONS: Continuous NTG administration results in an increase in platelet-esterified 8-epi PGF₂, a free radical and cyclooxygenase-dependent compound. This is reversed by co-administration of the free radical scavenger ascorbate. Whether this increase is merely a marker for increased oxidative stress or a mediator of oxidative injury contributing to the hemodynamic changes observed in nonintermittent organic nitrate treatment has yet to be resolved.

Abbreviations and Acronyms   AT1   angiotensin II type 1 receptor   CI   confidence interval   EDTA   ethylenediamine tetraacetic acid   8-epi PGF2   8-epi prostaglandin F2   NADPH   nicotinamide adenine dinucleotide phosphate reduced form   NO   nitric oxide   NOS   nitric oxide synthase   NTG   nitroglycerin   O2·–   superoxide anion

In addition to scavenging NO, O₂^·– represents a source of oxidative stress/damage to the vascular cell membrane. Vitamin C supplementation, which can directly scavenge O₂^·– and peroxynitrite, a prooxidant product of NO and O₂^·–, can delay the development of tolerance and correct endothelial vasomotor dysfunction associated with increased oxidative stress in a number of diseases (5–12). 8-Epi prostaglandin F₂ (8-epi PGF₂) was initially shown to be a specific marker of lipid peroxidation produced in situ in membrane phosphoplipids in a nonenzymatic manner by direct interaction with free radicals (13). More recently, 8-epi PGF₂ has been demonstrated to be produced, at least in part, by the action of cyclooxygenase on arachidonic acid (14). 8-Epi PGF₂, in addition to being a reliable marker of free radical damage, is a potent bioactive compound in its own right, with an ability to induce vasoconstriction (13).

In this study, we used platelets, which possess many components of NO and O₂^·– pathways in common with vascular cells, to examine the effect of nonintermittent organic nitrate treatment on the production of 8-epi PGF₂, and from this, we inferred changes in vascular cells. We also examined the effect of oral supplementation with the free radical scavenger ascorbate on this process.

	Methods

Top Abstract Methods Results Discussion References

 
Subjects.   Ten healthy male volunteers aged 23 to 48 years were recruited from within the Queen’s University School of Medicine. The Ethical Committee of the Queen’s University of Belfast approved the procedures. All subjects gave written, informed consent and underwent a full examination, including an electrocardiogram; their histories were also taken. No subjects were taking any drugs or vitamin supplements before or at the time of the study, and they refrained from consuming alcohol, tobacco, or caffeine for 12 h before each study day.

Study design.   All studies were performed in the Department of Therapeutics, with the subject supine. On the first study day, subjects had their blood sampled for laboratory analysis. Subjects were randomized to receive 1.2 g ascorbate or lactose (placebo) daily, in a double-blinded fashion. Nitro-Dur transdermal NTG patches (Schering-Plough, Dublin, Ireland) (0.4 mg/h) were applied over the deltoid area and replaced at 12-h intervals. Each subject received the medications and wore the patches for three days when blood sampling was repeated. After a washout period of two weeks, subjects had this protocol repeated, but with the oral medication reversed.

Sample collection and preparation.   Blood (23 ml) was withdrawn from the antecubital fossa, through an indwelling intravenous catheter. Blood (9.0 ml) was added to each of two polypropylene tubes containing 1.0 ml sodium citrate (3.18% wt/vol) and 300 µl acetyl salicylic acid (100 µmol/l). Blood (5.0 ml) was added to an ethylenediamine tetraacetic acid (EDTA)-containing tube for analysis of plasma ascorbate. The citrated blood was centrifuged at 160 g for 17 min at 22°C, and the platelet-rich plasma was removed. Platelet-rich plasma was centrifuged at 960 g for 10 min, and platelet-poor plasma was discarded. The platelet pellet was washed twice with Tyrode’s HEPES buffered saline of the following composition (mmol/l): NaCl 140, HEPES 6.0, Na₂HPO₄ 2.0, MgSO₄ 2.0 and dextrose 5.6 (pH 7.40) and finally resuspended in 300 µl homogenization buffer containing 320 mmol/liter sucrose, 10 mmol/l HEPES, 0.1 mmol/liter EDTA, 1 mmol/liter DL-dithiothreitol, 10 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor and 2 µg/ml aprotinin adjusted to pH 7.2. The suspension was flash frozen in liquid nitrogen and stored at –80°C until analyzed for 8-epi PGF₂ content. EDTA-containing blood was centrifuged at 3,000 g for 10 min, and the plasma (500 µl) was mixed with 500 µl 10% (wt/vol) metaphosphoric acid. This was centrifuged at 3,000 g for 10 min, and the supernatant was stored at –80°C until analyzed for ascorbate.

8-epi PGF₂.   Esterified 8-epi PGF₂ was measured in platelets using a commercially available immunoassay kit (Cayman Chemicals, Ann Arbor, Michigan). Total 8-epi PGF₂ (free and esterified) was extracted into chloroform/methanol (1:2). The free 8-epi PGF₂ was then extracted into water. The organic phase, containing 8-epi PGF₂ esterified in phospholipid, was then dried, and the 8-epi PGF₂ was liberated by saponification with 15% KOH. The sample was then purified by passing through a C18 solid-phase extraction column (SEP-PAK, Waters Corp., Milford, Massachusetts) and quantified by enzyme immunoassay. The result was standardized for protein and expressed as pg/mg protein. The recovery of 8-epi PGF₂ from each sample was calculated by adding a trace quantity of (³H) PGF₂ to each sample immediately after extraction of free 8-epi PGF₂.

Vitamin C.   Vitamin C concentrations in plasma were measured using a high-performance liquid chromatography method, as described by Speek et al. (15).

Data analysis.   Data were analyzed using the SPSS version 8.0 statistical package (SPSS Inc., Chicago, Illinois). Comparisons before and after treatment for the active and placebo treatment groups were made using the paired samples t test. The mean changes from baseline within the NTG/placebo versus NTG/ascorbate-treated group and the two baseline measurements for each subject were compared using the independent samples t test. Results are expressed as the mean value (95% confidence interval [CI]). A p value of 0.05 was considered significant.

	Results

Top Abstract Methods Results Discussion References

 
Plasma vitamin C.   All individuals receiving NTG and ascorbate showed an increase in plasma ascorbate, from 12.2 (CI 11.0 to 13.4) to 21.7 (CI 15.9 to 27.5) µg/ml (p < 0.01) (Fig. 1). Treatment with NTG and placebo caused no change from baseline (11.3 [CI 9.3 to 13.3] vs. 10.0 [CI 8.0 to 12.0] µg/ml). The mean change from baseline in the group receiving NTG and ascorbate was significantly different from that observed in the group receiving NTG and placebo (9.5 [CI 4.4 to 14.6] vs. –1.3 [CI –1.6 to –1.0] µg/ml, p < 0.01). There was no difference between both baseline values. This was independent of the order of treatment, suggesting the washout period was adequate.

View larger version (12K): [in this window] [in a new window]   Figure 1 Plasma ascorbate concentrations at baseline and after 72 h of nonintermittent transdermal nitroglycerin (NTG) (10 mg/24 h) with co-administration of placebo (NTG/placebo) or ascorbic acid (NTG/ascorbate). Data are presented as the mean value (95% CI). *p < 0.01.

8-epi PGF₂.

2

2

View this table: [in this window] [in a new window]   Table 1 Individual Values of Platelet 8-epi PGF2 Content for Each Subject

View larger version (16K): [in this window] [in a new window]   Figure 2 Platelet membrane esterified 8-epi prostaglandin2 (isoprostane) content at baseline and after 72 h of nonintermittent transdermal nitroglycerin (NTG) (10 mg/24 h) with co-administration of placebo (NTG/placebo) or ascorbic acid (NTG/ascorbate). Data are presented as the mean value (95% CI). *p < 0.05.

View larger version (13K): [in this window] [in a new window]   Figure 3 Change in platelet membrane esterified 8-epi prostaglandin F2 (isoprostane) content after 72 h of nonintermittent transdermal nitroglycerin (NTG) (10 mg/24 h) with co-administration of placebo (NTG/placebo) or ascorbic acid (NTG/ascorbate). Data are presented as the mean value (95% CI). *p < 0.05.

	Discussion

Top Abstract Methods Results Discussion References

2

2

2

Background.   Although organic nitrates remain a cornerstone in the treatment of acute and chronic ischemic syndromes and congestive heart failure (16), their usefulness is compromised by the development of hemodynamic tolerance. Nitric oxide and O₂^·– will rapidly interact, leading to the formation of peroxynitrite, which can oxidize a variety of biomolecules and alter vascular reactivity (17–19). Thus, activity or bioavailability of NO and O₂^·– will be related to the amount remaining after their mutual destruction and resultant peroxynitrite formation, rather than the absolute rate of production. Removal of the liberated and endogenous NO by increased vascular O₂^·– activity has been proposed as a major mechanism in the development of tolerance (3). This is supported by the moderation of tolerance by the administration of ascorbate, which scavenges superoxide and peroxynitrite (7).

Superoxide may be derived from a number of sources, including xanthine oxidase, mitochondrial respiration, cyclooxygenase, glycosylated proteins, and the membrane-bound, reduced form of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidases (3,20,21). Recent work by Munzel et al. (22) in the setting of tolerance to organic nitrates in rats demonstrated that nitric oxide synthase (NOS) could become uncoupled, generating O₂^·– itself. More recently, work has shown that peroxynitrite derived from the interaction between NO and O₂^·– can exhaust intracellular tetrahydrobiopterin, a critical co-factor for NOS, subsequently uncoupling the enzyme to produce O₂^·– instead of NO (23). This increases both the production and bioavailability of O₂^·–.

In addition to scavenging NO, O₂^·– can be metabolized to the hydroxy radical and cause oxidative damage to structural lipid and protein components in the membrane and within the cell, with consequent endothelial dysfunction (24).

Oxidative stress and NTG administration.   Oxidative stress in NTG treatment appears to be related to continuous, long-term administration. Short-term administration can reduce the evidence of oxidative stress, with vascular tissue still retaining the ability to release antioxidant NO from NTG (25,26). When NTG was administered to animals in a 12 h-on/12 h-off manner, endothelial dysfunction was corrected and vascular O₂^·– decreased (27). The evidence for oxidative stress related to NTG treatment in humans is not consistent. Jurt et al. (28) demonstrated a parallel increase in plasma aldehydes and 8-epi PGF₂ in continuous NTG administration. In contrast, Milone et al. (29) failed to show any change in plasma 8-epi PGF₂ in response to short- or long-term NTG administration or any affect of vitamin C co-administration. In the present study, the changes we have demonstrated have been related to esterified 8-epi PGF₂ in platelets.

Limitations of available technologies.   The ability to assess free radical damage or activity has always been compromised by limitations of the assays available. One approach has been to assess peroxidative damage to lipids. This has been assessed, most commonly, by measuring malondialdehyde, a breakdown product of lipid hydroperoxides. Original methods depended on the reaction with thiobarbituric acid, measuring thiobarbituric acid-reacting substances, and have been replaced by methods specific for malondialdehyde (30). F₂ isoprostanes, isomers of prostaglandin discovered as oxidative artifacts in stored plasma, have been proposed as a specific and reliable marker of oxidative stress in vivo (31). They were initially believed to be the result of noncyclooxygenase oxidative modifications of arachidonic acid, resulting from a free radical attack on cell membrane phospholipids (32). One of the main products of F₂ isoprostane synthesis in vivo is 8-iso PGF₂ (33).

Justification for the use of platelets to infer changes in vascular cells.   A major drawback of markers in the peripheral circulation is that they reflect total activity and cannot address the question of the site of origin. The physiologic consequences of nonintermittent NTG administration are brought about by changes in the behavior of vascular cells—the endothelial and smooth muscle cells. It is obviously not possible to directly examine these cells in humans. We used platelets to study the effects of NTG, as these cells serve as an easily accessible, compartmentalized model, and they are exposed to the drug in vivo and share a number of relevant chemical systems. Platelets contain well-defined NO- and superoxide-generating pathways similar to those found in vascular cells (34–36). They also share similar contractile regulation systems with vascular smooth muscle cells (37,38). Platelets express and have the capacity for dynamic upregulation and downregulation of angiotensin II type 1 (AT₁) receptors, which are known to be important in superoxide generation (39,40). Platelets are also capable of cyclooxgenase-mediated generation of superoxide (41). In animal studies, the response of platelets has been shown to match that of vascular cells in response to insulin resistance and hydroxymethyl glutaryl coenzyme A inhibition (37,42). The short half-life of platelets means that the adaptive responses by megakaryocytes are reflected in the platelet population within a few days. These factors suggest that changes in circulating platelets may reflect the consequences of altered NO/O₂^·– free radical activity in vascular cells. Platelets have been used as an accessible alternative to vascular myocytes for functional studies involving the effect of circulating levels of angiotensin II and preeclampsia on AT₁ receptor expression in humans (43,44).

Sources and functions of 8-epi PGF₂.   Recent work has shown that 8-epi PGF₂ is not produced exclusively by free radicals, but, to a lesser extent, is a result of cyclooxygenase activity (41). This problem can be minimized by either inhibiting cyclooxygenase activity in vivo or ex vivo or analyzing esterified 8-epi PGF₂. In contrast to classic prostaglandins, which are formed by the action of PGH synthase isoenzymes on free arachidonic acid, F₂ isoprostanes are formed in situ from the fatty-acid backbone esterified in membrane phospholipids (45). They are released through a phospholipase-mediated mechanism in response to cellular activation and circulate in the plasma in the free or esterified form. In this study, co-administration of ascorbate, which can scavenge superoxide and peroxynitrite, reduced platelet 8-epi PGF₂, supporting oxidative stress as the source of this compound in organic nitrate treatment (46).

Nitric oxide and 8-epi PGF₂ interact in a number of ways. Nitric oxide synthase has been shown to be directly involved, at least partly, in 8-epi PGF₂ production in human vasculature (47). Nitric oxide, acting as an antioxidant in a cell-free system, can decrease 8-epi PGF₂ production (48). In contrast, high levels of NO, possibly acting as the superoxide anion/NO reactant peroxynitrite, can increase 8-epi PGF₂ production (48,49).

8-Epi PGF₂ also exerts potent biologic activity that alters vascular tone, giving it significance beyond that as a marker of free radical activity (32). 8-epi PGF₂ can enhance coronary vasoconstriction in animal experiments (50–52). Work in rats suggests that 8-epi PGF₂ may induce vasoconstriction through the thromboxane receptor, but it can also cause relaxation through a distinct receptor inducing NO release (53).

Studies have also indicated that 8-epi PGF₂ activates platelets by inducing platelet adhesion and reducing the inhibitory activity of NO. It antagonizes the action of NO through increases in intraplatelet calcium (54). Concentrations of 8-epi PGF₂ in the range 1 nmol/liter to 1 µmol/liter induce a dose-dependent increase in calcium release from intracellular stores in inositol phosphate. It causes a dose-dependent change in platelet shape, platelet aggregation and vasoconstriction (55). However, this is contradicted by more recent work reporting 8-epi PGF₂ and other isoprostanes to be anti-aggregatory (56).

Conclusions.   This study addressed the effect of nonintermittent organic nitrate administration in relatively young, healthy individuals. These individuals had no obvious existing cardiovascular pathology and were unlikely to have upregulated NADPH oxidase-dependent vascular O₂^·– activity. The advantage is that the effects of NTG can be examined without being confounded by existing derangements of superoxide metabolism. Older individuals with existing cardiovascular pathology are likely to have increased bioavailability of vascular O₂^·–, although the prooxidant/antioxidant balance of their vascular cells is likely to be shifted toward oxidation, suggesting that the effect of nonintermittent organic nitrates in these individuals would be more pronounced.

We have shown that nonintermittent organic nitrate administration increases the platelet content of 8-epi PGF₂, and this can be reversed by co-administration of ascorbate. This is consistent with increased oxidative stress driving the production of 8-epi PGF₂, either directly by the action of free radicals or indirectly through cyclooxygenase, NOS or some other mechanism. It has yet to be resolved whether this increase is merely a marker of increased oxidative stress or the result of 8-epi PGF₂ operating as a transduction mechanism linking oxidative stress to specialized forms of cellular activation.

Appendix.   The samples for this study were obtained during a separate study on the effect of nonintermittent organic nitrates on human hemodynamics and nitric oxide/superoxide chemistry. This study has been published in Circulation. McVeigh GE, Hamilton P, Wilson M, et al. Platelet nitric oxide and superoxide release during the development of nitrate tolerance: effect of supplemental ascorbate. Circulation 2002;106:208–13.

	References

Top Abstract Methods Results Discussion References

 
1. Molina CR, Andresen JW, Rapoport RM, Waldman S, Murad F. Effect of in vivo nitroglycerin therapy on endothelium-dependent and -independent vascular relaxation and cyclic GMP accumulation in rat aorta. J Cardiovasc Pharmacol. 1987;10:371–378[Medline]

2. Pagani ED, VanAller GS, O’Connor B, Silver PJ. Reversal of nitroglycerin tolerance in vitro by the cGMP-phosphodiesterase inhibitor zaprinast. Eur J Pharmacol. 1993;43:141–147

3. Munzel T, Sayegh H, Freeman BA, Tarpey MM, Harrison DG. Evidence for enhanced vascular superoxide anion production in nitrate tolerance: a novel mechanism of tolerance and cross tolerance. J Clin Invest. 1995;95:187–194[Medline]

4. Munzel T, Kurz S, Rajagopalan S, et al. Hydralazine prevents nitroglycerin tolerance by inhibiting activation of a membrane-bound NADH oxidase: a new action for an old drug. J Clin Invest. 1996;98:1465–1470[Medline]

5. Fink B, Bassenge E. Exogenous NO in contrast to endogenous NO is subject to tolerance due to enhanced superoxide radical formation resulting from impaired NO scavenging. Pflugers Arch. 1995;429:R108–112

6. Bassenge E, Fink B. Tolerance to nitrates and simultaneous upregulation of platelet activity prevented by enhancing antioxidant state. Naunyn Schmiedebergs Arch Pharmacol. 1996;353:363–367[CrossRef][Medline]

7. Bassenge E, Fink B. Suppression of nitrate induced tolerance by vitamin C and other antioxidants. Moncada S, Stamler J, Gross S, Higgs EA. The Biology of Nitric Oxide, Part 5. London: Portland Press Ltd; 1996. p. 198

8. Ting HH, Timimi FK, Boles KS, Creager SJ, Ganz P, Creager MA. Vitamin C improves endothelium-dependent vasodilation in patients with non–insulin-dependent diabetes mellitus. J Clin Invest. 1996;97:22–28[Medline]

9. Levine GN, Frei B, Koulouris SN, Gerhard MD, Keaney JFJ, Vita JA. Ascorbic acid reverses endothelial vasomotor dysfunction in patients with coronary artery disease. Circulation. 1996;93:1107–1113[Abstract/Free Full Text]

10. Solzbach U, Hornig B, Jeserich M, Just H. Vitamin C improves endothelial dysfunction of epicardial coronary arteries in hypertensive patients. Circulation. 1997;96:1513–1519[Abstract/Free Full Text]

11. Ting HH, Timimi FK, Haley EA, Roddy MA, Ganz P, Creager MA. Vitamin C improves endothelium-dependent vasodilation in forearm resistance vessels of humans with hypercholesterolaemia. Circulation. 1997;95:2617–2622[Abstract/Free Full Text]

12. Heitzer T, Just H, Munzel T. Antioxidant vitamin C improves endothelial dysfunction in chronic smokers. Circulation. 1996;94:6–9[Abstract/Free Full Text]

13. Morrow JD, Roberts LJ. The isoprostanes: unique bioactive products of lipid peroxidation. Prog Lipid Res. 1997;36:1–21[CrossRef][Medline]

14. Pratico D, Lawson JA, Fitzgerald GA. Cyclooxygenase-dependent formation of the isoprostane, 8-epi prostaglandin F₂. J Biol Chem. 1995;17:9800–9808

15. Speek AJ, Schrijver J, Schreurs WH. Fluorometric determination of total vitamin C in whole blood by high-performance liquid chromatography with pre-column derivatization. J Chromatogr. 1984;305:53–60[Medline]

16. Glasser SP. Prospects for therapy of nitrate tolerance. Lancet. 1999;353:1545–1546[CrossRef][Medline]

17. Huie RE, Padmaja S. The reaction of NO with superoxide. Free Radic Res Commun. 1993;18:195–199[Medline]

18. Kojda G, Harrison D. Interactions between NO and reactive oxygen species: pathophysiological importance in atherosclerosis, hypertension, diabetes and heart failure. Cardiovasc Res. 1999;43:562–571[CrossRef][Medline]

19. Harrison DG. Endothelial function and oxidant stress. Clin Cardiol. 1997;20(Suppl II):II.11, 17

20. Munzel T, Bassenge E. Long-term angiotensin-converting enzyme inhibition with high-dose enalapril retards nitrate tolerance in large epicardial arteries and prevents rebound coronary constriction in vivo. Circulation. 1996;93:2052–2058[Abstract/Free Full Text]

21. Rajagopalan S, Kurz S, Munzel T, et al. Angiotensin II–mediated hypertension in the rat increases superoxide production via membrane NADH/NADPH oxidase activation: contribution to alterations of vasomotor tone. J Clin Invest. 1996;97:1916–1923[Medline]

22. Munzel T, Li H, Mollnau H, et al. Effects of long-term nitroglycerin treatment on endothelial nitric oxide synthase (NOS III) gene expression, NOS III–mediated superoxide production, and vascular NO bioavailability. Circ Res. 2000;86:E7–12[Medline]

23. Laursen JB, Somers M, Kurz S, et al. Endothelial regulation of vasomotion in apoE-deficient mice: implications for interactions between peroxynitrite and tetrahydrobiopterin. Circulation. 2001;103:1282–1288[Abstract/Free Full Text]

24. Moro MA, Russell RJ, Cellek S, et al. cGMP mediates the vascular and platelet actions of nitric oxide: confirmation using an inhibitor of the soluble guanylyl cyclase. Proc Natl Acad Sci USA. 1996;93:1480–1485[Abstract/Free Full Text]

25. Rahman A, Ahmed S, Khan N, et al. Glyceryl trinitrate, a nitric oxide donor, suppresses renal oxidant damage caused by potassium bromate. Redox Report. 1999;46:263–269

26. Laight DW, Kengatharan KM, Gopaul NK, et al. Investigation of oxidant stress and vasodepression to glyceral trinitrate in obese Zucker rats in vivo. Br J Pharmacol. 1998;125:895–901[CrossRef][Medline]

27. Munzel T, Mollnau H, Hartmann M, Effects of a nitrate-free interval on tolerance, vasoconstrictor sensitivity and vascular superoxide production. J Am Coll Cardiol 2000;36:628–34

28. Jurt U, Gori T, Ravandi A, Babaei S, Zeman P, Parker JD. Differential effects of pentaerythritol tetranitrate and nitroglycerin on the development of tolerance and evidence of lipid peroxidation: a human in vivo study. J Am Coll Cardiol. 2001;38:854–859[Abstract/Free Full Text]

29. Milone SD, Pace-Asciak CR, Reynaud D, et al. Biochemical, hemodynamic, and vascular evidence concerning the free radical hypothesis of nitrate tolerance. J Cardiovasc Pharmacol. 1999;33:685–690[CrossRef][Medline]

30. Slater TF. Overview of methods used for detecting lipid peroxidation. Methods Enzymol. 1984;105:283–293[Medline]

31. Morrow JD, Harris TM, Roberts JL. Noncyclooxygenase oxidative formation of a series of novel prostaglandins: analytical ramifications for measurement of eicosanoids. Anal Biochem. 1990;184:1–10[CrossRef][Medline]

32. Morrow JD, Hill KE, Burk RF, Nammour TM, Badr KF, Roberts LJ II. A series of prostaglandin F₂–like compounds are produced in-vivo in humans by a non–cyclo-oxygenase free radical catalysed mechanism. Proc Natl Acad Sci USA. 1990;87:9383–9387[Abstract/Free Full Text]

33. Morrow JD, Minton TA, Badr KF, Roberts LJ. Evidence that the F₂-isoprostane, 8-epi prostaglandin F₂ alpha, is formed in vivo. Biochim Biophys Acta. 1994;1210:244–248[Medline]

34. Moro MA, Russell RJ, Cellek S, et al. cGMP mediates the vascular and platelet actions of nitric oxide: confirmation using an inhibitor of the soluble guanylyl cyclase. Proc Natl Acad Sci USA. 1996;93:1480–1485[Abstract/Free Full Text]

35. Kojda G, Harrison D. Interactions between NO and reactive oxygen species: pathophysiological importance in atherosclerosis, hypertension, diabetes and heart failure. Cardiovasc Res. 1999;43:562–571[CrossRef][Medline]

36. Seno T, Inoue N, Gao D, Okuda M, et al. Involvement of NADH/NADPH oxidase in human platelet ROS production. Thromb Res. 2001;103:399–409[CrossRef][Medline]

37. Trovati M, Anfossi G. Insulin, insulin resistance and platelet function: similarities with insulin effects on cultured vascular smooth muscle cells. Diabetologia. 1998;41:609–622[CrossRef][Medline]

38. Erne P, Bolli P, Burgisser E, et al. Correlation of platelet calcium with blood pressure: effect of antihypertensive therapy. N Engl J Med. 1984;310:1084–1088[Medline]

39. Morgan LS, Crawshaw PM, Baker R, Edwards F, Broughton-Pipkin, Kalsheker N. Functional and genetic studies of angiotensi II type 1 receptor in pre-eclamptic and normotensive pregnant women. J Hypertens. 1997;15:1389–1396[CrossRef][Medline]

40. Nickenig G, Bäumer AT, Temur Y, Kebben D, Jockenhövel F, Böhm M. Statin-sensitive dysregulated AT₁ receptor function and density in hypercholesterolaemic men. Circulation. 1999;100:2131–2134[Abstract/Free Full Text]

41. Pratico D, Lawson JA, Fitzgerald GA. Cyclooxygenase-dependent formation of the isoprostane, 8-epi prostaglandin F₂. J Biol Chem. 1995;270:9800–9808[Abstract/Free Full Text]

42. Laufs U, Gertz K, Huang P, et al. Atorvastatin upregulates type III nitric oxide synthase in thrombocytes, decreases platelet activation, and protects from cerebral ischaemia in normocholesterolemic mice. Stroke. 2000;31:2437–2449[Abstract/Free Full Text]

43. Moore TJ, Taylor T, Williams GH. Human platelet angiotensin II receptors: regulation by the circulating angiotensin level. J Clin Endocrinol Metab. 1984;58:778–782[Abstract/Free Full Text]

44. Masse J, Forest JC, Moutquin JM, et al. A prospective longitudinal study of platelet angiotensin II receptors for the prediction of preeclampsia. Clin Biochem. 1998;31:251–255[CrossRef][Medline]

45. Smith WL, Garvito RM, DeWitt DL. Prostaglandin endoperoxide H synthases (cyclooxygenases) 1 and 2. J Biol Chem. 1996;271:33157–33160[Free Full Text]

46. Stocker R. Lipoprotein oxidation: mechanistic aspects, methodological approaches and clinical relevance. Curr Opin Lipidol. 1994;5:422–433[Medline]

47. Jourdan KB, Mitchell JA, Evans TW. Release of isoprostanes by human pulmonary artery in organ culture: a cyclo-oxygenase and nitric oxide–dependent pathway. Biochem Biophys Res Commun. 1997;233:668–672[CrossRef][Medline]

48. Laskey RE, Mathews WR. Nitric oxide inhibits peroxynitrite-induced production of hydroxyeicosatetraenoic acids and F₂-isoprostanes in phosphotidylcholine liposomes. Arch Biochem Biophys. 1996;330:193–198[CrossRef][Medline]

49. Marnett LJ, Wright TL, Crews BC, Tannenbaum SR, Morrow JD. Regulation of prostaglandin biosynthesis by nitric oxide is revealed by targeted deletion of inducible nitric-oxide synthase. J Biol Chem. 2000;275:13427–13430[Abstract/Free Full Text]

50. Wilson SH, Best PJ, Lerman LO, Holmes DR Jr., Richardson DM, Lerman A. Enhanced coronary vasoconstriction to oxidative stress product, 8-epi prostaglandin F₂ alpha, in experimental hypercholesterolaemia. Cardiovasc Res. 1999;44:474–476[Free Full Text]

51. Garcia-Cohen EC, Marin J, Diez-Picazo LD, Baena AB, Salaices M, Rodriguez-Martinez MA. Oxidative stress induced by tert-butyl hydroperoxide causes vasoconstriction in the aorta from hypertensive and aged rats: role of cyclooxygenase-2 isoform. J Pharmacol Exp Ther. 2000;293:75–81[Abstract/Free Full Text]

52. Sametz W, Prasthofer S, Wintersteiger R, Juan H. Vascular effects of isoprostanes after endothelial damage. Prostaglandins Leukot Essent Fatty Acids. 1999;61:369–372[CrossRef][Medline]

53. Jourdan KB, Evans TW, Curzen NP, Mitchell JA. Evidence for a dilator function of 8-iso-prostaglandin F₂ alpha in rat pulmonary artery. Br J Pharmacol. 1997;120:1280–1285[CrossRef][Medline]

54. Minuz P, Andrioli G, Degan M, et al. The F₂-isoprostane 8-epiprostaglandin F₂ increases platelet adhesion and reduces the antiadhesive and antiaggregatory effects of NO. Arterioscler Thromb Vasc Biol. 1998;18:1248–1256[Abstract/Free Full Text]

55. Patrono C, Fitzgerald GA. Isoprostanes: potential markers of oxidant stress in atherothrombotic disease. Arterioscler Thromb Vasc Biol. 1997;17:2309–2315[Abstract/Free Full Text]

56. Cranshaw JH, Evans TW, Mitchell JA. Characterisation of the effects of isoprostanes on platelet aggregation in human whole blood. Br J Pharmacol. 2001;132:1699–1706[CrossRef][Medline]

This article has been cited by other articles:

				P. J. Fadel, M. Farias III, K. M. Gallagher, Z. Wang, and G. D. Thomas Oxidative stress and enhanced sympathetic vasoconstriction in contracting muscles of nitrate-tolerant rats and humans J. Physiol., January 15, 2012; 590(2): 395 - 407. [Abstract] [Full Text] [PDF]

				T. Munzel, A. Daiber, and T. Gori Nitrate Therapy: New Aspects Concerning Molecular Action and Tolerance Circulation, May 17, 2011; 123(19): 2132 - 2144. [Full Text] [PDF]

				S A Wright, F M O'Prey, M T McHenry, W J Leahey, A B Devine, E M Duffy, D G Johnston, M B Finch, A L Bell, and G E McVeigh A randomised interventional trial of {omega}-3-polyunsaturated fatty acids on endothelial function and disease activity in systemic lupus erythematosus Ann Rheum Dis, June 1, 2008; 67(6): 841 - 848. [Abstract] [Full Text] [PDF]

				S. A. Wright, F. M. O'Prey, D. J. Rea, R. D. Plumb, A. J. Gamble, W. J. Leahey, A. B. Devine, R. C. McGivern, D. G. Johnston, M. B. Finch, et al. Microcirculatory Hemodynamics and Endothelial Dysfunction in Systemic Lupus Erythematosus Arterioscler Thromb Vasc Biol, October 1, 2006; 26(10): 2281 - 2287. [Abstract] [Full Text] [PDF]

				T. Munzel, A. Daiber, and A. Mulsch Explaining the Phenomenon of Nitrate Tolerance Circ. Res., September 30, 2005; 97(7): 618 - 628. [Abstract] [Full Text] [PDF]

				F. Krotz, H.-Y. Sohn, and U. Pohl Reactive Oxygen Species: Players in the Platelet Game Arterioscler Thromb Vasc Biol, November 1, 2004; 24(11): 1988 - 1996. [Abstract] [Full Text] [PDF]

				L. J. Dixon, D. R. Morgan, S. M. Hughes, L. T. McGrath, N. A. El-Sherbeeny, R. D. Plumb, A. Devine, W. Leahey, G. D. Johnston, and G. E. McVeigh Functional Consequences of Endothelial Nitric Oxide Synthase Uncoupling in Congestive Cardiac Failure Circulation, April 8, 2003; 107(13): 1725 - 1728. [Abstract] [Full Text] [PDF]

This Article Abstract Figures Only Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by McGrath, L. T. Articles by McVeigh, G. E. Search for Related Content PubMed PubMed Citation Articles by McGrath, L. T. Articles by McVeigh, G. E.