© 2001 American Heart Association, Inc.
Editorials |
Inducible NO Synthesis in the Vasculature
Molecular Context Defines Physiological Response
From the Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, Boston, Mass.
Correspondence to Dr Joseph Loscalzo, Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, 80 East Concord, W507, Boston, MA 02118. E-mail jloscalz{at}bu.edu
Key Words: Editorials • endothelial NO synthase • inducible NO synthase • vasculature
Nitric oxide synthesized by endothelial NO synthase (eNOS) is a principal mediator of smooth muscle relaxation in the normal blood vessel. With inflammation, however, inducible NO synthase (iNOS) is expressed in the vessel wall, including the adventitia,1 and is associated with impaired endothelium-dependent vasorelaxation2 and impaired vasoconstriction.3,4 The molecular mechanism(s) underlying these alterations in vasomotor function accompanying inflammation remains unknown, although in vitro studies suggest that proinflammatory stimuli reduce the expression of eNOS, agonist-mediated cytosolic calcium mobilization, and NO production.5
See page 1281
In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology, Gunnett et al6 attempt to address the basis for impaired vasomotor responses in inflammation by transferring the iNOS gene to blood vessels. They argue that all prior studies suffer from the lack of selectivity of the pharmacological inhibitors of iNOS used and from the concomitant changes in the expression of other vascular gene products accompanying the inflammatory response and, thus, do not permit an unequivocal assessment of the role of iNOS in these vasomotor abnormalities. They used an adenoviral vector construct containing the mouse iNOS gene to transfer the gene to rabbit carotid arteries in vitro and in vivo. They observed that iNOS gene transfer in vitro led to impaired contractile responses and impaired relaxation responses to endothelium-dependent (acetylcholine and ADP) and endothelium-independent (A23187) agonists, as well as to a direct nitrovasodilator (sodium nitroprusside); NO-independent responses to 8-bromo-cGMP and papaverine were normal. In vivo, they observed that iNOS gene transfer had no effect on contractile responses but led to impaired endothelium-dependent relaxation. They conclude from these studies that vascular expression of iNOS alone impairs NO-dependent relaxation.
Although the conclusions of this elegantly conducted study6 are clearly supported by the results, the fundamental question (ie, why does vascular expression of iNOS impair vasomotor responses?) remains unanswered. The authors have excluded some possible explanations by showing no effect of indomethacin (cyclooxygenase-mediated endothelium-derived contracting factors) or allopurinol (xanthine/xanthine oxidase-derived superoxide anion) on vasomotor responses. They also used cell-permeable superoxide dismutase (PEG-SOD) and 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron) in an effort to exclude superoxide anion derived from any source as a mediator of impaired vasomotor responses; however, they point out that the results of these studies were inconsistent (ie, not statistically different from controls), with improved responses in only 5 of 14 PEG-SOD experiments and 5 of 18 Tiron experiments. Results using control virus (AdBgIII), which can induce a mild inflammatory response in infected vessels, were not reported in this set of experiments.
To understand the implications of these findings, it is important to review the known functions and consequences of the induction of vascular iNOS by inflammatory mediators. As with eNOS, iNOS not only oxidizes L-arginine to L-citrulline and NO but also reduces molecular oxygen to superoxide anion at the flavin-binding sites in the absence of sufficient L-arginine.7 Superoxide anion derived from iNOS, in fact, has been shown to contribute to the hyperreactivity of small mesenteric arterioles in a chronic heart failure model.8 In inflammatory states, abundant reactive oxygen species generated by leukocytes likely play a role in promoting the induction of iNOS, inasmuch as an increase in oxygen tension and a concomitant decrease in intracellular glutathione levels lead to an increase in iNOS expression that can be attenuated by N-acetyl-L-cysteine.9 In addition, the synthesis of another cofactor critical for the production of NO by iNOS, tetrahydrobiopterin (BH4), is increased by inflammatory stimuli, including endotoxin and cytokines, owing to an increase in the expression of the rate-limiting enzyme in BH4 synthesis, GTP cyclohydrolase I.10 Moreover, inhibiting BH4 synthesis with 2,4-diamino-6-hydroxypyrimidine leads to a reduction in BH4 levels and an accompanying loss of L-arginine-induced vasorelaxation in endotoxin-treated rat aortas.10
The expression of another enzyme essential for the maintenance of vascular cell redox state and for the generation of reductive cofactors essential for iNOS-dependent NO generation, glucose-6-phosphate dehydrogenase (G6PD), is also increased with inflammation and oxidant stress.11,12 In vascular smooth muscle cells and endothelial cells, G6PD is a principal source of NADPH, which is a required cofactor for NO synthesis, for glutathione disulfide reduction by glutathione reductase, and for BH4 synthesis from the direct and salvage pathways. Inhibiting G6PD activity or expression in vascular cells leads to the suppression of bioactive NO production by iNOS and eNOS and to enhanced NOS- derived superoxide anion generation.11,12 In addition to these changes in gene expression that accompany oxidative and inflammatory stresses, the expression of iNOS and the generation of NO induced by endotoxin is directly coupled to an increase in the expression of Cu/Zn superoxide dismutase, thereby limiting superoxide flux, peroxynitrite formation, and oxidative inactivation of NO.13
These numerous changes in oxidoreductase and antioxidant enzymes that accompany inflammatory states indicate the importance of studying the consequences of expressing iNOS in the appropriate vascular context. The exclusive expression of iNOS by adenoviral vector administration, by definition, is not associated with coexpression of coordinately regulated vascular genes. Although the need to eliminate the coordinated expression of other, potentially confounding, genes served as the rationale for the study design of Gunnett et al,6 the lack of induction of the expression of these genes may lead to cofactor limitations (NADPH, glutathione, and BH4), promote superoxide production, and thereby limit bioactive NO production by iNOS as well as eNOS. The inability of PEG-SOD or Tiron to reverse the abnormal vasomotor responses consistently may be a reflection of interindividual differences in inflammatory response to the adenovirus, which may lead to variable degrees of endogenous (adventitial) iNOS expression or superoxide production. Measuring the antioxidant capacity of vessels treated with PEG-SOD or Tiron may provide some insight into the basis for these variable responses, as would the use of inhibitors that suppress superoxide anion production from specific sources, including NAD(P)H oxidase and the NO synthases themselves.
Without direct measurement of vascular NO (or NOx) production, NO synthase activities, eNOS expression, iNOS expression and localization, superoxide production, and levels of BH4, glutathione, and NADPH, meaningful mechanistic interpretation of the results of the study of Gunnett et al6 is limited. Parallel studies using more selective iNOS inhibitors, eg, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine or N-[3-(aminomethyl)-benzyl]acetamidine hydrochloride,14 or cotransfection of selective, but essential, genes regulating iNOS cofactors may offer additional insights that complement the information presented by Gunnett et al and may, ultimately, provide a rational mechanism by which the expression of iNOS and other gene products in inflammatory states impairs vasomotor responses.
References
1. Zhang H, Du Y, Cohen RA, Chobanian AV, Brecher P. Adventitia as a source of inducible nitric oxide synthase in the rat aorta. Am J Hypertens. 1999; 12: 467–475.[Medline] [Order article via Infotrieve]
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Parker JL, Adams HR. Selective inhibition of endothelium-dependent vasodilator capacity by Escherichia coli endotoxemia. Circ Res. 1993; 72: 539–551.
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Beasley D, Cohen RA, Levinsky NG. Endotoxin inhibits contraction of vascular smooth muscle in vitro. Am J Physiol. 1990; 258: H1187–H1192.
4. Gunnett CA, Chu Y, Hiestad DD, Loihl A, Faraci FM. Vascular effects of LPS in mice deficient in expression of the gene for inducible nitric oxide synthase. Am J Physiol. 1998; 275: H416–H421.
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Graier WF, Myers PR, Rubin LJ, Adams HR, Parker JL. Escherichia coli endotoxin inhibits agonist-mediated cytosolic Ca2+ mobilization and nitric oxide biosynthesis in cultured endothelial cells. Circ Res. 1994; 75: 659–668.
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Gunnett CA, Lund DD, Chu Y, Brooks RM, II Faraci FM, Heistad DD. Nitric oxide-dependent vasorelaxation is impaired after gene transfer of inducible NO synthase. Arterioscler Thromb Vasc Biol. 2001; 21: 1281–1287.
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Xia Y, Roman LJ, Masters BS, Zweier JL. Inducible nitric-oxide synthase generates superoxide from the reductase domain. J Biol Chem. 1998; 273: 22635–22639.
8. Miller AA, Megson IL, Gray GA. Inducible nitric oxide synthase-derived superoxide contributes to hyperreactivity in small mesenteric arteries from a rat model of chronic heart failure. Br J Pharmacol. 2000; 131: 29–36.[Medline] [Order article via Infotrieve]
9. Miralles C, Busquets X, Santos C, Togores B, Hussain S, Rahman I, MacNee W, Agusti AG. Regulation of iNOS expression by glutathione levels in rat liver by oxygen tension. FEBS Lett. 2000; 476: 253–257.[Medline] [Order article via Infotrieve]
10. Shimizu S, Ishii M, Kawakami Y, Kiuchi Y, Momose K, Yamamoto T. Presence of excess tetrahydrobiopterin during nitric oxide production from inducible nitric oxide synthase in LPS-treated rat aorta. Life Sci. 1999; 65: 2769–2779.[Medline] [Order article via Infotrieve]
11. Leopold JA, Loscalzo J. Cyclic strain modulates resistance to oxidant stress by increasing glucose-6-phosphate dehydrogenase expression in vascular smooth muscle cells. Am J Physiol. 2000; 279: H2477–H2485.
12. Leopold JA, Cap A, Scribner AW, Stanton RC, Loscalzo J. Glucose-6-phosphate dehydrogenase deficiency promotes endothelial oxidant stress and decreases endothelial nitric oxide bioavailability. FASEB J. In press.
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Frank S, Zacharowski K, Wray GM, Thiemermann C, Pfeilschifter J. Identification of copper/zinc superoxide dismutase as a novel nitric oxide-regulated gene in rat glomerular mesangial cells and kidneys of endotoxemic rats. FASEB J. 1999; 13: 869–882.
14. Rudd MA, Trolliet M, Hope S, Scribner AM, Daumerie G, Toolan G, Cloutier T, Loscalzo J. Salt-induced hypertension in Dahl salt-resistant and salt-sensitive rats with NOS II inhibition. Am J Physiol. 1999; 277: H732–H739.
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