nitric oxide synthase-independent generation of nitric oxide in muscle ischemia–reperfusion injury

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NITRIC OXIDE: Biology and ChemistryVol. 4, No. 6, pp. 541–545 (2000)doi:10.1006/niox.2000.0308, available online at http://www.idealibrary.com on

BRIEF REVIEW

Nitric Oxide Synthase-Independent Generation of NitricOxide in Muscle Ischemia–Reperfusion Injury

D. A. Lepore1

Bernard O’Brien Institute of Microsurgery, St. Vincent’s Hospital, Fitzroy, 3065, Melbourne, Australia

Received January 25, 2000, and in revised form June 22, 2000

Nitric oxide (NO) is an important molecule inmany physiological or pathophysiological pro-cesses including ischemia–reperfusion injury. Theenzymatic nitric oxide synthase (NOS)-dependentpathway was universally accepted as the source ofNO in ischemia–reperfusion injury. However, gen-eration of NO that is independent of NOS has alsobeen identified in ischemia–reperfusion injury toboth cardiac and skeletal muscle. This review sum-marizes the evidence for the generation NOS-independent NO in ischemia–reperfusion injury tocardiac and skeletal muscle. © 2000 Academic Press

Nitric oxide (NO)2 is considered to be a key mole-ule in many physiological and pathophysiologicalrocesses. In normal vascular physiology, NO regu-ates blood pressure (1, 2), maintains vasodilatorone (3), maintains a nonthrombotic endothelial sur-ace (4), prevents platelet aggregation (5), preventseukocyte–endothelial cell interactions (6), inhibitsascular smooth muscle cell proliferation (7), and is

1 Address correspondence to author at Bernard O’Brien Insti-tute of Microsurgery, St. Vincent’s Hospital, Victoria Parade,Fitzroy, 3065, Melbourne, Australia. Fax: 0613 94160926. E-mail:[email protected].

2 Abbreviations used: NO, nitric oxide; NOS, nitric oxide syn-
thase; L-NAME, N-nitro-L-arginine methyl ester; SMT,
-methylisothiourea.

1089-8603/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

a neurotransmitter of the central nervous system(8–10).

Pathologically, NO is involved in edema (11, 12),is believed to mediate the tumoricidal and antimi-crobial activities of macrophages (13), can behave asboth a pro- and anti-inflammatory compound (7, 14),and appears to be responsible for the hypotensivevascular collapse in endotoxemia (15).

The role of NO in ischemia–reperfusion injury iswell documented (16). The consequences ofischemia–reperfusion injury limit the survival ofmuscle involved in tissue trauma or transfers andcardiac infarcts (17). The sources of NO generationduring this process are important in understandingthe pathophysiological mechanisms of ischemia–reperfusion injury.

HOW IS NITRIC OXIDE FORMED?

The Nitric Oxide Synthase Pathway

L-arginine ™™™3n

NO•

NOSL-citrulline (Reaction [1])

NO can be produced from the multistep conver-sion of the amino acid L-arginine to L-citrulline viathe enzymatic nitric oxide synthase (NOS) pathway(Reaction [1]) (18). This pathway requires oxygen(O2) and the cofactors nicotinamide adenine dinucle-
otide phosphate (NADPH) and tetrahydrobiopterin(BH4) (19). The physiological cellular concentration
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of NO is reportedly between 1 and 10 mM, but theconcentration can vary depending on the environ-ment (20, 21). Significant amounts of oxygen arerequired to maintain synthesis of NO in micromolarconcentrations. NOS requires two molecules of oxy-gen per molecule of NO produced and the half-life ofNO is approximately 1 s (20). Hence, to maintain aconcentration of 1 mM NO, 170 nmol O2 min21 isrequired per gram of tissue.

DIFFERENT FORMS OF NITRIC OXIDE SYNTHASEARE BIOCHEMICALLY DISTINGUISHABLE

There are two known forms of NOS, the constitu-tive and inducible forms. Constitutive NOS is foundin neurons and endothelial cells and the NOS foundin each cell type is encoded by distinguishable genes.Constitutive NOS is Ca21-dependent and thus isstimulated by agents that alter intracellular levelsof Ca21. For endothelial cells, these are acetylcho-ine, bradykinin, tachykinins, serotonin, platelet-ctivating factor, and histamine (22). Neuronal NOSs also sensitive to increases in intracellular Ca21

and is particularly responsive to the excitatory neu-rotransmitter glutamate (22). Inducible NOS isCa21-independent (22, 23) and is found in inflamma-ory cells, endothelial cells, smooth muscle cells, andther cell types. It responds to specific stimuli suchs lipopolysaccharide (24) or the cytokinesnterferon-gamma (IFN-g), tumour necrosis factor-

alpha (TNF-a), and interleukin-1-alpha (IL-1-a)(25). An exception is the respiratory epithelium thatexpresses inducible NOS continuously (26).

NOS-INDEPENDENT NITRIC OXIDE FORMATION

It has been known for some time that bacteria inthe oral cavity reduce dietary nitrate (NO3

2) to ni-trite (NO2

2), in a similar way to bacteria in infectedurine, using the enzyme nitrate reductase (27). NOand other nitrogen oxides are then generated fromNO2

2 in the acidic/reducing environment of the stom-ch (28). At low pH, NO2

2 is converted into nitrousacid and then nitrogen oxides including NO. Thepresence of a reducing agent such as ascorbic acidfacilitates NO production by rapidly reducing ni-

542 D. A

trous acid (29). A similar type of NO production hasbeen demonstrated on the skin surface. In sweat

Copyright © 2000 by Academic Press. All right

glands, NO32 is converted to NO2

2 and the acidicsurface of the skin converts NO2

2 to NO (27).Recently yet another pathway for the generation

of NO has been implicated. Activated neutrophilscan convert NO2

2 via a myeloperoxidase-dependentpathway into the inflammatory oxidants nitryl chlo-ride (NO2Cl) and nitrogen dioxide (NO2) (30). Thesenitrogen species may act as a source of NO.

NOS-DEPENDENT NITRIC OXIDE GENERATIONIN ISCHEMIA–REPERFUSION INJURY

Studies using inhibitors of NOS have indicatedthat NOS-dependent NO is formed during ischemia–reperfusion injury and that it can be either protec-tive or detrimental. In a skeletal muscle model ofhind limb ischemia in the rat, inhibition of NOS, byN-nitro-L-arginine methyl ester (L-NAME) and byNG-monomethyl-L-arginine (L-NMMA), resulted in asignificant reduction in vascular permeability (31).In our laboratory, the inhibitors of NOS, L-NAME,S-methylisothiourea (SMT), and the glucocorticoiddexamethasone, improved muscle survival and re-duced neutrophil activity in the same model (32).Attenuation of skeletal muscle ischemia–reperfusion injury was also observed in a hind limbskeletal muscle model in mice lacking the gene en-coding inducible NOS. These mice showed an in-crease in muscle viability and a decrease in edemaand in neutrophil infiltration compared with theirwild-type littermates (33). Furthermore, in a rabbitmodel the NOS inhibitor nitroiminoethyl-L-rnithine (L-NIO) was shown to increase skeletal

muscle survival, to reduce neutrophil accumulation,to reduce the hyperemic response, and to marginallyreduce edema (34). In cardiac muscle, infusion ofL-NAME has resulted in the protection of ex vivo ratischemic–reperfused hearts (35) and in protectionfrom coronary artery ligation in vivo in rabbits (36)and piglets (37).

On the other hand, a lack of protection againstcardiac infarct size was observed in a rabbit modelusing a different inhibitor of NOS, L-nitroarginine(L-NA) (38). In a rat cardiac ischemia–reperfusionmodel, L-NAME significantly increased infarct size(39). Further evidence for the exacerbation of

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ischemia–reperfusion injury by inhibitors of NOShas been observed in skeletal muscle (40), liver (41,

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42), gut mesentery (43), small bowel (12), lung (44),and pancreas (45).

NOS-INDEPENDENT NITRIC OXIDE GENERATIONIN ISCHEMIA–REPERFUSION INJURY

The generation of NO during ischemia–reperfusion injury was universally accepted as de-riving from a NOS-dependent pathway until Zweierand colleagues identified a nonenzymatic pathwayfor the production of NO in cardiac muscleischemia–reperfusion injury. An increase in NO for-mation from 15NO2

2 correlated with increased dura-tion of ischemia and a decrease in tissue pH (46). Ina subsequent study the same group showed that thereduction of NO2

2 to NO was increased 100-fold,nder the acidic conditions of ischemic tissue (Reac-ion [2]) (47). In addition to acidic pH it was foundhat ischemic tissue contained nonenzymatic reduc-ng equivalents that reduce NO2

2 to NO, increasingNO production a further 40- to 4000-fold which isabove the maximum possible physiological valuesgenerated by NOS (47). The generation of NOS-independent NO during the reducing conditions ofischemia has been mapped in cardiac muscle usingelectron paramagnetic resonance imaging and wasshown to occur throughout the myocardium (48).Studies on the generation of NOS-independent NOin cardiac muscle have been reviewed in detail byZweier et al. (49).

3NO22 1 2H13 2NO 1 NO3

2 1 H2O (Reaction [2])

Our laboratory has recently reported that inischemia–reperfusion injury to rat skeletal musclein vivo, generation of NO that was insensitive toinhibitors of NOS (L-NAME or SMT) was detected in

icromolar amounts at 24 h postreperfusion (50). Athis time of reperfusion in this model there is poorerfusion of the muscle and extensive necrosis (32),roviding an environment of low oxygen tension, lowADPH levels, and acidosis (50). NOS is known to

ose activity under conditions of low oxygen tension19) and low pH (51). Thus, in poorly perfused mus-le the conditions favor the production of NOS-ndependent NO (35, 47).

NOS-INDEPENDENT NO GENER

There is evidence to suggest that NOS-ndependent NO generation plays a role in


ischemia–reperfusion injury. In cardiac muscle, in-fusion of NO2

2 prior to ischemia increased not onlyNO levels but also the severity of muscle injury (46).When infused with the NOS inhibitor L-NAME, NO2

2

increased the levels of muscle NO and prevented theprotective effect of L-NAME on muscle function. Fur-thermore, oxyhemoglobin (a scavenger of NO irre-spective of its mode of production) was superior toL-NAME in decreasing the levels of muscle NO andat improving cardiac muscle function afterischemia–reperfusion (46). Taken together, thesefindings are consistent with an injurious role forNOS-independent NO in ischemia–reperfusion in-jury to cardiac muscle, but whether it has a role inskeletal muscle ischemia–reperfusion injury re-mains a subject for further investigation.

SUMMARY

During ischemia–reperfusion injury to muscle,NO can be generated from two pathways, NOS-dependent and NOS-independent. Significant gen-eration of NOS-independent NO has been demon-strated in models of cardiac and skeletal muscle.NOS-independent NO is the predominant form ofNO in models exhibiting extensive necrosis and poorreperfusion. There is some evidence to suggest thatNOS-independent NO contributes to injury duringcardiac muscle ischemia–reperfusion but its role inskeletal muscle has not yet been investigated.

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NOS-INDEPENDENT NO GENER

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nitric oxide synthase-independent generation of nitric oxide in muscle ischemia–reperfusion injury

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