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Role of Nitric Oxide in Endotoxic Shocka

An Overview of Recent Advances

CSABA SZABÓb

Division of Critical Care, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA

INTRODUCTION

The overproduction of nitric oxide (NO) in endotoxic shock and various otherforms of circulatory shock is well documented.1,2 However, there is a considerablecontroversy regarding the beneficial versus detrimental effects of NO synthesisinhibitors for the experimental therapy of circulatory shock. The reasons for thesecontroversial findings, such as the choice of the inhibitor, the choice of the exper-imental model, the timing of the intervention, and related issues have recentlybeen overviewed.3 This chapter focuses on recent developments in the field ofnitric oxide in circulatory shock. Specifically, this chapter overviews (1) some ofthe recent developments with genetically engineered animals lacking theinducible isoform of NO synthase (iNOS); (2) the interactions of NO with someother mediators of shock, namely superoxide anion and cyclooxygenase; and (3)some of the recent developments with iNOS-selective inhibitors and with antiin-flammatory agents with combined modes of actions.

RESULTS ON THE ROLE OF iNOS IN SHOCK USING GENETICALLYENGINEERED ANIMALS

Three groups have independently succeeded in constructing genetically engi-neered animals lacking iNOS.4–6 These animals are fertile and viable and do notdemonstrate detectable pathological alterations. The iNOS knockout animals, ingeneral, appear to be protected against several types of inflammatory insults.However, the degree of protection against shock and inflammation provided bythe lack of iNOS gene is widely variable among different investigators, and itappears to be dependent on the experimental model used. For instance, in a shockmodel induced by i.v. injection of bacterial lipopolysaccharide (LPS) in anes-thetized mice, the iNOS knockouts are protected against the hypotension.4

However, no difference between wild-type and iNOS knockouts was found inrespect to LPS/C. parvum–induced hepatic injury. 4 Another group has reported

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aThe work was supported by a grant from the National Institutes of Health(R29GM54773).

bAdditional correspondence information: Telephone: (513) 636-8714; Fax: (513) 636-4892.

100% four-day survival in LPS-challenged iNOS knockout animals versus 50%survival in the respective wild-type animals.5 This finding, however, was not con-firmed by a third group.6 Taken together, it is clear that additional work is neededto define the role of iNOS in LPS-induced shock, but from the data so far, itappears that the lack of iNOS at least does not exacerbate the course of shock. Thisfinding is in marked contrast to findings with non-iNOS-selective NOS inhibitors,which frequently worsen the outcome of endotoxic shock.3 One obvious reserva-tion against the use of knockout animals is that these animals are likely to developcompensatory mechanisms after genetic ablation of a given gene, and thereforethe responses seen in the knockout animals may or may not be relevant for theintact animal. Such disappointing experiences have recently been obtained, forexample, with the knockout animals lacking the two isoforms of cyclooxygenase.

INTERACTIONS OF NO WITH OTHER INFLAMMATORY SPECIESDURING ENDOTOXIC SHOCK

It is clear that NO is only one of many terminal mediators of shock and inflam-mation. In this respect, it is important to point out that NO interacts with severalother final common pathways of injury. One important interaction is the reactionof NO with superoxide. In contrast to previous proposals, which suggested thatthis pathway reduces the biological effects of NO, more recent investigationsdemonstrated that this reaction yields peroxynitrite, which is a highly cytotoxicspecies.7,8 Prevention of the generation of peroxynitrite, by the use of cell-perme-able superoxide dismutase mimetics, provides protection in terms of vascular fail-ure and cellular energetic alterations in rat models of endotoxic shock: Thisprotection is comparable with the protection provided by iNOS inhibition.8

Peroxynitrite can mediate tissue injury via a number of independent pathways.Several studies employing inhibitors of the nuclear enzyme poly (ADP-ribose)synthetase (PARS) have suggested that the oxidative injury in response to oxyrad-icals and peroxynitrite during circulatory shock is, at least in part, related to DNAsingle-strand breakage, and consequent activation of PARS, a nuclear enzyme.9,10

Massive ADP-ribosylation of nuclear proteins by PARS then results in cellularenergy depletion and injury, reminiscent of necrosis. This pathway is important inthe vascular failure and mortality associated with endotoxic shock.10–12

Another important interaction occurs between NO and cyclooxygenase,whereby NO directly increases the catalytic activity of cyclooxygenase and thusincreases the production of cytotoxic prostaglandins in shock.13 NO synthaseinhibitors in shock, therefore, are inhibiting not only NO, but alsoprostaglandin formation.14

NOVEL PHARMACOLOGICAL APPROACHES

Recent developments in the field of NO synthase inhibitors have resulted in thediscovery of novel inhibitors, with marked selectivity toward the inducible iso-form.15,16 Using these inhibitors, marked protection can be seen against the vascu-lar dysfunction, organ injury, and mortality associated with endotoxic shock.15,16

SZABÓ: NITRIC OXIDE AND ENDOTOXIC SHOCK 423

Another novel approach for drug development is the design of multifunctionalantiinflammatory compounds. One of these compounds is mercaptoethylguani-dine, a combined iNOS inhibitor, peroxynitrite scavenger, and cyclooxygenaseinhibitor.17–19 This agent exerts remarkable beneficial effects in various models ofinflammation and also in endotoxic shock.17,20,21 The property of this agent as a per-oxynitrite scavenger is especially noteworthy, since peroxynitrite can also beformed independent of iNOS: Such peroxynitrite would still be expected to exertcytotoxic effects even in an organism in which iNOS is completely blocked.7,18 Infact, formation of peroxynitrite can be still detected in iNOS knockout animalssubjected to LPS.22

CONCLUSIONS

Taken together, important developments are continuing in the area of basicresearch and drug development in relation to NO and circulatory shock. One ofthe prototypical NOS inhibitors, NG-methyl-L-arginine has entered phase III clin-ical trials.3 It is expected that this nonisoform-selective inhibitor will be followedby second- and third-generation inhibitors that may have higher iNOS selectivityand/or combined antiinflammatory modes of action.

REFERENCES

1. SZABÓ, C. 1995. Alterations in the production of nitric oxide in various forms of circula-tory shock. New Horizons 3: 3–32.

2. KILBOURN, R. G., D. TRABER & C. SZABÓ. 1997. Role of nitric oxide in the vascular failurein shock. Disease-A-Month 43: 277–348.

3. KILBOURN, R. G., D. TRABER & C. SZABÓ. 1997. Beneficial versus detrimental effects ofnitric oxide synthase inhibitors in circulatory shock: Lessons learned from experi-mental and clinical studies. Shock 7: 235-246.

4. MACMICKING, J. D., et al. 1994. Altered responses to bacterial infection and endotoxicshock in mice lacking inducible nitric oxide synthase. Cell 81: 641–650.

5. WEI, X. Q., et al. 1995. Altered immune responses in mice lacking inducible nitric oxidesynthase. Nature 375: 408–411.

6. LAUBACH, V. E., et al. 1995. Mice lacking inducible nitric oxide synthase are not resistantto lipopolysaccharide-induced death. Proc. Natl. Acad. Sci. USA 92: 10688–10692.

7. SZABÓ, C. 1996. The role of peroxynitrite in the pathophysiology of shock, inflammationand ischemia-reperfusion injury. Shock 6: 79–88.

8. ZINGARELLI, B., et al. 1997. The potential involvement of peroxynitrite in the pathogene-sis of endotoxic shock. Br. J. Pharmacol. 120: 259–267.

9. SZABÓ, C., B. ZINGARELLI & A. L. SALZMAN. 1996. Role of poly-ADP ribosyltransferaseactivation in the nitric oxide- and peroxynitrite-induced vascular failure. Circ. Res. 78:1051–1063.

10. SZABÓ, C. 1996. DNA strand breakage and activation of poly-ADP ribosyltransferase: Acytotoxic pathway triggered by peroxynitrite. Free Rad. Biol. Med. 21: 855–869.

11. SZABÓ, C., et al. 1997. Endothelial dysfunction in endotoxic shock: Importance of theactivation of poly (ADP ribose) synthetase (PARS) by peroxynitrite. J. Clin. Invest.100: 723–735.

12. SZABÓ, C., et al. 1997. Regulation of components of the inflammatory response by 5-iodo-6-amino-1,2-benzopyrone, and inhibitor of poly (ADP-ribose) synthetase andpleiotropic modifier of cellular signal pathways. Int. J. Oncol. 10: 1093–1101.

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13. SALVEMINI, D. & J. L. MASFERRER. 1996. Interactions of nitric oxide with cyclooxygenase:In vitro, ex vivo, and in vivo studies. Meth. Enzymol. 269: 12–25.

14. SALVEMINI, D., et al. 1997. Regulation of prostaglandin production by nitric oxide; an invivo analysis. Br. J. Pharmacol. 114: 1171–1178.

15. SOUTHAN, G. J. & C. SZABÓ. 1996. Selective pharmacological inhibition of distinct nitricoxide synthase isoforms. Biochem. Pharmacol. 51: 383–394.

16. GARVEY, E. P., et al. 1997. 1400W is a slow, tight binding, and highly selective inhibitor ofinducible nitric-oxide synthase in vitro and in vivo. J. Biol. Chem. 272: 4959–4963.

17. SOUTHAN, G. J., et al. 1996. Spontaneous rearrangement of aminoalkylguanidines intomercaptoalkylguanidines—a novel class of nitric oxide synthase inhibitors with selec-tivity towards the inducible isoform. Br. J. Pharmacol. 117: 619–632.

18. SZABÓ, C., et al. 1997. Mercaptoethylguanidine and related guanidine nitric oxide syn-thase inhibitors react with peroxynitrite and protect against peroxynitrite-inducedoxidative damage. J. Biol. Chem. 272: 9030–9036.

19. ZINGARELLI, B., et al. 1997. Mercaptoalkylguanidines are direct inhibitors of cyclooxyge-nase activity. Br. J. Pharmacol. 120: 357–366.

20. CUZZOCREA, S., et al. 1998. Carrageenan-induced local inflammation: Effect of mercap-toethylguanidine, a selective inhibitor of the inducible nitric oxide synthase and ascavenger of peroxynitrite. Free Rad. Biol. Med. 24: 450–459.

21. BRAHN, E., et al. 1997. Beneficial effects of mercaptoethylguanidine, an inhibitor of nitricoxide synthase and a scavenger of peroxynitrite, in collagen-induced arthritis:Inhibition of synovitis and suppression of TNF-α, collagenase and stromeolysin.FASEB J. 11: A530.

22. ZINGARELLI, B., et al. 1998. Oxidation, nitration and cytostasis induction in the absenceof inducible nitric oxide synthase. Int. J. Mol. Med. In press.

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