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(Stroke. 1997;28:1283-1288.)
© 1997 American Heart Association, Inc.

Articles

Nitric Oxide Synthase in Models of Focal Ischemia

Amer F. Samdani, MD; Ted M. Dawson, MD, PhD; Valina L. Dawson, PhD

From the Departments of Neurology (A.F.S., T.M.D., V.L.D.), Neuroscience (T.M.D., V.L.D.), and Physiology (V.L.D.), Johns Hopkins University School of Medicine, Baltimore, Md.

Correspondence to Valina L. Dawson, PhD, Department of Neurology, Johns Hopkins University School of Medicine, 600 N Wolfe St, Path 2-210, Baltimore, MD 21287. E-mail: valina_dawson{at}qmail.bs.jhu.edu

	Abstract

Top Abstract Introduction NOS Isoforms nNOS Mediates Early Neuronal... eNOS Is Neuroprotective Targets of NO Conclusions References

 
Background and Purpose Cessation of blood flow to the brain, for even a few minutes, sets in motion a potential reversible cascade of events resulting in neuronal cell death. Oxygen free radicals and oxidants appear to play an important role in central nervous system injury after cerebral ischemia and reperfusion. Recently, divergent roles for the newly identified neuronal messenger molecule and oxygen radical, nitric oxide (NO), have been identified in various models of cerebral ischemia. Because of the chemical and physical properties of NO, the numerous physiological activities it mediates, and the lack of specific agents to modulate the activity of the different isoforms of NO synthase (NOS), reports regarding the role of NO in focal cerebral ischemia have been confounding and often conflicting. Recent advances in pharmacology and the development of transgenic knockout mice specific for the different isoforms of NOS have advanced our knowledge and clarified the role of NO in cerebral ischemia.

Methods Animal models of focal ischemia employ occlusion of nutrient cerebral vessels, most commonly the middle cerebral artery. Primary cortical cultures are exposed to excitotoxic or ischemic conditions, and the activities of NOS isoforms or NO production are evaluated. Transgenic mice lacking expression of either the neuronal isoform of NOS (nNOS), the endothelial isoform of NOS (eNOS), or the immunologic isoform of NOS (iNOS) have been examined in models of excitotoxic injury and ischemia.

Results Excitotoxic or ischemic conditions excessively activate nNOS, resulting in concentrations of NO that are toxic to surrounding neurons. Conversely, NO generated from eNOS is critical in maintaining cerebral blood flow and reducing infarct volume. iNOS, which is not normally present in healthy tissue, is induced shortly after ischemia and contributes to secondary late-phase damage.

Conclusions Pharmacological and genetic approaches have significantly advanced our knowledge regarding the role of NO and the different NOS isoforms in focal cerebral ischemia. nNOS and iNOS play key roles in neurodegeneration, while eNOS plays a prominent role in maintaining cerebral blood flow and preventing neuronal injury.

Key Words: cerebral ischemia • free radicals • nitric oxide • glutamates • neurotoxins • oxidants

	Introduction

Top Abstract Introduction NOS Isoforms nNOS Mediates Early Neuronal... eNOS Is Neuroprotective Targets of NO Conclusions References

 
Loss of blood flow to the brain results in neuronal injury due to both the cessation of blood flow leading to oxygen and nutrient deprivation and the initiation of a cascade of secondary mechanisms (Figure).¹ This neurotoxic cascade involves derangements in normal metabolic and physiological functions as well as recruitment of cell death processes. Thus, both restoration of blood supply and control of secondary neurotoxic cascades are necessary to limit ischemic neuronal damage. Numerous transmitter and second messenger pathways are inappropriately activated after the initial ischemic event.² A major pathway leading toward neuronal injury involves elevation of extracellular glutamate and activation of glutamate receptors, with a subsequent increase in intracellular calcium, resulting in generation of free radicals and NO.³ Glutamate initiates its actions postsynaptically by binding to four major types of receptors: metabotropic receptors, NMDA receptors, AMPA receptors, and kainate receptors. NMDA receptor activation mediates, in large part, glutamate excitotoxicity and neuronal damage after focal ischemia.⁴ Glutamate-stimulated NMDA receptors flux calcium and activate a variety of intracellular calcium–dependent enzymes and processes, of which activation of neuronal NOS (nNOS) plays a prominent role. Thus, overproduction of NO from excessive or inappropriate stimulation of nNOS appears to mediate a major component of excitotoxic damage.

View larger version (38K): [in this window] [in a new window]   Figure 1. Postulated mechanisms of neuronal death after focal ischemia. CBF indicates cerebral blood flow; VSCC, voltage-dependent calcium channels; NMDA-R, N-methyl-D-aspartate receptor; PLA2, phospholipase A2; PKC, protein kinase C; CaMK II, calcium-calmodulin–dependent protein kinase II; ROS, reactive oxygen species, and ICE, interleukin-1ß converting enzyme.

	NOS Isoforms

Top Abstract Introduction NOS Isoforms nNOS Mediates Early Neuronal... eNOS Is Neuroprotective Targets of NO Conclusions References

 
There are three NOS isoforms that are named after the tissue from which they were first cloned and numbered in the order in which they were cloned^{5 6} (Table 1). nNOS (type I) and endothelial NOS (eNOS) (type III) are constitutively expressed and are calcium dependent. Immunologic NOS (iNOS) (type II) is expressed after immunologic challenge and neuronal injury and is calcium independent under most circumstances.⁷

View this table: [in this window] [in a new window]   Table 1. NOS Isoforms

NOS catalyzes the stoichiometric conversion of L-arginine to NO and citrulline in the presence of oxygen and NADPH. There are multiple sites on all three isoforms for cofactor and substrate interactions that provide potential targets for pharmacological regulation of NOS catalytic activity.^{7 8}

	nNOS Mediates Early Neuronal Injury

Top Abstract Introduction NOS Isoforms nNOS Mediates Early Neuronal... eNOS Is Neuroprotective Targets of NO Conclusions References

 
nNOS is primarily expressed in a small population of neurons throughout the nervous system and a variety of other tissues throughout the body (Table 1). It is neuronally produced NO that is believed to mediate synaptic plasticity and neuronal signaling and, after ischemic damage, neurotoxicity.

In primary cortical neuronal cultures, in which nNOS is expressed at levels equivalent to in vivo expression, glutamate and NMDA neurotoxicity are mediated largely by excess formation of NO. Coexposure of primary cortical neurons to NMDA and arginine analogue NOS inhibitors, flavoprotein inhibitors, calmodulin antagonists or agents that bind calmodulin, and calcineurin inhibitors, all of which decrease NOS catalytic activity, results in neuroprotection against NMDA neurotoxicity.^{3 7 9} NO is toxic and appears to activate death pathways similar to those activated by NMDA, since compounds that directly release NO are neurotoxic and exhibit the same kinetics of death as NMDA neurotoxicity. Elimination of nNOS through transgenic technology results in primary neuronal cultures that are resistant to NMDA neurotoxicity, indicating that nNOS neurons are the source of neurotoxic NO.¹⁰ Neuronal injury after combined oxygen-glucose deprivation of neuronal cultures, which may more accurately reflect in vivo ischemic conditions, is reversed by NOS inhibitors. Additionally, neuronal cultures from nNOS null transgenic mice are markedly resistant to combined oxygen-glucose deprivation compared with wild-type cultures.¹⁰ These in vitro studies indicate that NO is not only a major mediator of glutamate excitotoxicity but also contributes in a significant way to the neuronal damage after oxygen-glucose deprivation. While these observations have been replicated in a variety of cell culture systems as well as slice preparations, there are conflicting reports in the literature in which NO-mediated neurotoxicity was not observed.^{3 7 9} In many cases these differences can be attributed to insufficient expression of nNOS or differences in experimental paradigms altering the reaction pathways of NO. For instance, recent studies indicate that an NO component to NMDA neurotoxicity is critically dependent on the number of nNOS neurons and the level of nNOS protein.¹¹ The expression of nNOS is critically dependent on the culture condition used, in that neurons grown on glial feeder layers contain relatively low levels of nNOS, whereas neurons grown on a polyornithine matrix tend to contain high levels of nNOS. A variety of neurotrophins markedly increase the number of nNOS neurons, nNOS protein, and NOS catalytic activity and enhance NMDA neurotoxicity through NO-dependent mechanisms when neurons are grown on glial feeder layers. In contrast, when rat or mouse primary cortical neurons are grown on a polyornithine matrix, neurotrophins have no influence on nNOS neuronal number or NOS catalytic activity and reduce NMDA neurotoxicity. Primary neuronal cultures from mice lacking nNOS grown on a glial feeder layer fail to respond to neurotrophin-mediated enhancement of neurotoxicity. Thus, nNOS expression and NMDA, NO-mediated neurotoxicity are dependent on the culture paradigm, and neurotrophins regulate the susceptibility to NMDA neurotoxicity through modulation of nNOS.

In animal models of focal ischemia, inhibition of nNOS with concentrations of NOS inhibitors that do not perturb eNOS activity reduces infarct volume after MCA occlusion in mice, rats, and cats.^{12 13 14} Additionally, selective nNOS inhibitors, including 7-nitroindazole^{15 16} and ARL 17477,¹⁷ which do not influence eNOS activity but effectively diminish nNOS activity in vivo, are consistently neuroprotective in models of focal ischemia. Although many investigators have observed a reduction in infarct volume with a variety of nonselective NOS inhibitors, other investigators have observed either no effect or exacerbation of injury. Exacerbation of injury seems to occur at higher doses of nonselective NOS inhibitors through inhibition of eNOS, resulting in deleterious alterations of cerebral blood flow and subsequent increased infarction volume.^{12 13 14}

Clarification of the role of the different NOS isoforms in focal ischemia was obtained from transgenic mice studies in mice lacking nNOS, eNOS, or iNOS (Table 2). Despite the usefulness of these models, one needs to be cognizant that transgenic animals have lacked the respective enzyme since birth and that compensatory processes may have occurred that enabled the animal to reach adulthood. It is conceivable that these compensatory processes account for phenotypic differences in transgenic versus wild-type mice. Thus, it is important to recognize these potential limitations and confirm observations obtained with the use of transgenic technology with conventional pharmacological approaches.

View this table: [in this window] [in a new window]   Table 2. Phenotypes of NOS Null Mice

After permanent focal MCA occlusion, nNOS null mice have reduced infarct volumes compared with age-matched wild-type controls.¹⁸ The reduction of infarct volume in nNOS null transgenic mice could be reversed by administration of nonspecific NOS inhibitors at concentrations sufficient to inhibit NO-dependent relaxation of pial vessels. Under these conditions, infarct volumes were equivalent to those of wild-type mice. Genetic deletion of nNOS also conferred dramatic resistance to focal ischemic injury in a reperfusion model of transient MCA occlusion,¹⁹ and hippocampal damage was reduced in a model of global cerebral ischemia.²⁰ Selective nNOS inhibitors may need to be used cautiously, since inhibition of nNOS can activate nuclear factor-B (NF-B), leading to induction of iNOS, which could exacerbate neuronal injury in the later stages following cerebral ischemia.²¹

Although nNOS is constitutively expressed, after certain pathological insults nNOS can be induced in some cells through new protein synthesis. For instance, after MCA occlusion in the rat, a rapid upregulation of nNOS mRNA as well as nNOS protein and NADPH-diaphorase positive staining has been observed in the ischemic lesion.²² It is possible that the increase in nNOS expression contributes to the spread of neuronal damage after ischemic injury. However, recent studies indicate that upregulation of nNOS may subserve a restorative function through NO's activation of the Ras extracellular signal–regulated protein kinase pathway, leading to long-term changes in neuronal plasticity.²³

Anatomic localization of nNOS reveals a distribution for the enzyme that does not correlate with any one neurotransmitter but is coincident with the histochemical stain NADPH-diaphorase.^{24 25} NADPH-diaphorase–positive neurons are of extreme interest because they are relatively spared from neuronal cell death after vascular stroke and excitotoxicity and in Huntington's and Alzheimer's diseases.²⁶ Recent studies suggest that the mitochondrial Mn-SOD accounts for the selective resistance of nNOS neurons to toxic insults.²⁷ nNOS neurons are selectively enriched in Mn-SOD. Antisense knockdown of Mn-SOD renders nNOS neurons susceptible to NMDA neurotoxicity but does not influence the overall susceptibility of non-nNOS cortical neurons to NMDA toxicity. Furthermore, overexpression of Mn-SOD by adenoviral-mediated gene transfer provides dramatic protection against NMDA and NO toxicity in cortical cultures.

iNOS Contributes to Late Neuronal Injury
iNOS is not detectable in healthy tissue (Table 1). In addition to macrophages and microglia, iNOS under pathological conditions can be expressed in most tissues, including neurons, astrocytes, and endothelial cells.²⁸ Induction of iNOS in vitro results in delayed neuronal cell death^{29 30 31} and can also exacerbate glutamate excitotoxicity.³² In human disease, iNOS expression may play a role in demyelination in multiple sclerosis³³ and may contribute to neuronal injury in severe AIDS dementia.³⁴ In rat brain, iNOS protein and catalytic activity are detectable 12 hours after cerebral ischemia, peak at 48 hours, and return to baseline in 7 days.³⁵ The relatively selective iNOS inhibitor aminoguanidine, administered 24 hours after the ischemic insult, results in reduced infarct volumes compared with vehicle-treated controls.³⁶ Neuroprotection by aminoguanidine is reversed by excess substrate L-arginine but not the inactive stereoisomer D-arginine. Clarification of the role of iNOS in late neuronal injury accompanying cerebral ischemia is provided by the observation that mice lacking the gene for iNOS have significantly reduced infarct volumes compared with wild-type controls (Table 2).³⁷ Whether upregulation of iNOS in endothelial cells could play a protective role after stroke through beneficial effects on cerebral blood flow is not known.

	eNOS Is Neuroprotective

Top Abstract Introduction NOS Isoforms nNOS Mediates Early Neuronal... eNOS Is Neuroprotective Targets of NO Conclusions References

 
Attempts to understand the vasodilatory effects of organic nitrates and the mechanisms of acetylcholine-induced relaxation of vascular smooth muscle led to the discovery that endothelially derived NO is critical in the regulation of vascular hemodynamics.³⁸ eNOS is constitutively expressed and briefly activated by increases in intracellular calcium (Table 1). In addition to expression in endothelial cells, a small population of neurons in the central nervous system express eNOS.³⁹

NO is a major regulator of vascular hemodynamics and is the primary messenger molecule mediating blood vessel relaxation.⁴⁰ Even partial inhibition of eNOS can result in large changes in cerebral blood flow. In initial reports examining ischemic outcomes after inhibition of NOS, investigators based their conclusions regarding the role of NO in cerebral ischemia on results obtained with nonspecific NOS inhibitors.^{12 13 14} These nonselective agents inhibit both eNOS and nNOS. Administration of nonspecific NOS inhibitors results in constriction of pial arterioles, reduction in cerebral blood flow, and subsequent increases in infarct volume. In contrast, NO donors or intra-arterial administration of L-arginine increases regional cerebral blood flow and decreases infarct volume distal to MCA occlusion in rats.^{12 13 14} Consistent with the notion that eNOS subserves a protective role in cerebral ischemia by maintaining regional cerebral blood flow is the observation that transgenic mice that lack eNOS experience increased infarct volumes after MCA occlusion (Table 2).⁴¹ Furthermore, the nonspecific NOS inhibitor, nitro-L-arginine, decreases the infarct size in eNOS null mice but does not affect infarct size in wild-type mice. The only target for nitro-L-arginine in eNOS null mice during the acute phase is nNOS, and therefore these studies highlight the dual actions of NO in focal ischemia. Overproduction of NO from activation of nNOS leads to neurotoxicity; however, production of NO from eNOS protects brain tissue by maintaining regional cerebral blood flow. The development of selective pharmacological tools and the development of transgenic mice have allowed the dissection of the divergent roles for NO in focal ischemia (Table 2). eNOS protein and catalytic activity, like nNOS, are also upregulated during the acute phase of ischemia.⁴² Perhaps upregulation of eNOS subserves a protective role by facilitating the maintenance of cerebral blood flow in the setting of ischemia.

	Targets of NO

Top Abstract Introduction NOS Isoforms nNOS Mediates Early Neuronal... eNOS Is Neuroprotective Targets of NO Conclusions References

 
The exact pathways by which excessive NO formation results in neuronal cell death are not known. Disruption of any of the normal physiological processes mediated by NO could have deleterious effects on neuronal survival. Because of an unpaired electron, NO is by definition a free radical. The best known biological targets of NO include oxygen, transition metals, iron-sulfur–containing proteins, and heme-containing proteins⁴³ (Table 3 ). An important and well-studied transition metal target of NO is the iron in the heme moiety of guanylate cyclase. NO alters the conformation of guanylate cyclase, which leads to the formation of cGMP. cGMP plays little if any role in the toxicity of NO; instead, it may be neuroprotective.⁴⁴ NO readily reacts with several iron-sulfur cluster–containing proteins, including the mitochondrial NADH-ubiquinone oxidoreductase and NADH:succinate oxidoreductase. NO inhibits both enzymes, which may contribute to neuronal injury by inhibiting oxidative phosyphorylation.⁷ NO also inhibits cis-aconitase, another iron-sulfur cluster–containing protein, leading to inhibition of glycolysis, and it reversibly inhibits mitochondrial respiration by competing with oxygen at cytochrome oxidase.⁴⁵ NO reacts with the thiols of many proteins, resulting in S-nitrosylation. NO inhibits creatine kinase activity through nitrosothiol modification.⁴⁶ Inhibition of creatine kinase leads to decrement in ATP due to the inhibition of phosphoryl transfer between phosphocreatine and ATP, which could contribute to neuronal injury by decreasing the availability of ATP. An established pathway of NO-mediated neuronal cell death is NO activation of the nuclear enzyme PARS.⁴⁷ NO activates PARS by damaging DNA. PARS catalyzes the transfer of ADP-ribose units from NAD to nuclear proteins. For every mole of ADP-ribose transferred, one mole of NAD is consumed, and four free energy equivalents of ATP are necessary to regenerate NAD. Therefore, overactivation of PARS can rapidly deplete cellular energy stores. If mitochondrial enzymes are simultaneously impaired from exposure to NO, the cell's ability to replace NAD and ATP is compromised, leading to energy failure and cell death (Figure). Recently, targeted disruption of PARS provided compelling evidence for participation of PARS in NO-mediated toxicity.⁴⁸ Mutant mouse islet cells lacking PARS do not show DNA damage–induced NAD depletion and are more resistant to NO toxicity. However, NO-mediated cell death was not completely abolished in the mutant mice islet cells, suggesting the existence of alternative pathways for NO-mediated toxicity not involving PARS-mediated NAD depletion.

View this table: [in this window] [in a new window]   Table 3. Targets of NO

In biological systems, the most permissive reaction of NO is with O₂·^- to produce the potent oxidant ONOO^-. Some investigators have stressed that direct toxic effects of NO are modest⁴⁹ and that NO may have neuroprotective properties.¹³ Recent investigations suggest that the majority of the toxic effects attributed to NO, as well as O₂·^-, are due to ONOO^-.⁵⁰ Interestingly, nNOS in the setting of decreased substrate L-arginine availability, which would occur during ischemia, is capable of producing both NO and O₂·^-.⁵¹ Although both transgenic mice that lack nNOS¹⁸ and transgenic mice that overexpress the O₂·^- cytosolic scavenging enzyme Cu²⁺/Zn-SOD⁵² have reduced infarct volumes after permanent MCA occlusion, double transgenic mice lacking nNOS and overexpressing Cu²⁺/Zn-SOD have significantly smaller infarct volumes then either genetic manipulation alone.⁵³ Thus, it is the combination of the O₂·^- produced simultaneously with NO leading to ONOO^- formation that accounts for the majority of toxicity after cerebral ischemia. Activation of NMDA receptors mediates the majority of damage in focal ischemia. Interestingly, NMDA, but not non-NMDA, receptor activation increases mitochondrial reactive oxygen species,⁵⁴ and NMDA, but not non-NMDA, receptor activation is the major stimulator of nNOS activity. Perhaps it is through the simultaneous production of NO and O₂·^- that the majority of the neurotoxic actions of glutamate are mediated. One of the major targets of ONOO^- is mitochondrial Mn-SOD. Thus, ONOO^- inactivation of Mn-SOD could initiate a self-propagating cascade of neural injury through failure of mitochondrial scavenging of O₂·^-.⁵⁵

NO-mediated cell death occurs through both necrotic and apoptotic cell death pathways. Although NMDA-mediated neuronal death is thought to be largely necrotic, the mode of cell death may depend on the intensity of toxic insults of NMDA and NO and mitochondrial function.^{56 57} In cortical cultures, mild excitotoxic or free radical insults lead to delayed neuronal death dominated by apoptotic features, whereas intense exposure to a high concentration of NMDA or ONOO^- induces necrotic cell death. Activation of iNOS in primary neuronal cultures causes a slowly progressive neuronal cell death that is dominated by apoptotic features (K. Kopnisky et al, unpublished data, 1997). Recent studies in nonneuronal systems suggest that iNOS-mediated toxicity occurs through activation of interleukin-1ß converting enzyme–like proteases and P53-dependent pathways.⁵⁸

	Conclusions

Top Abstract Introduction NOS Isoforms nNOS Mediates Early Neuronal... eNOS Is Neuroprotective Targets of NO Conclusions References

 
It is now clear that NO plays major roles in modulating brain injury after ischemic events. The development of selective pharmacological tools as well as the development of transgenic mice lacking each NOS isoform has greatly advanced our understanding of the diverse roles of NO in the central nervous system as well as the roles of NO in response to ischemic injury. Current studies indicate that NO plays a dual role in focal cerebral ischemia. Depending on its source, NO may be toxic or protective to the brain under ischemic conditions. Overproduction of NO from either nNOS or iNOS leads to neurotoxicity; however, NO production from endothelial NOS protects brain tissue by maintaining regional cerebral blood flow. These studies emphasize the necessity of developing truly selective inhibitors for nNOS and iNOS to adequately protect the brain from ischemic injury due to overproduction of NO yet simultaneously maintain or enhance regional cerebral blood flow.

	Selected Abbreviations and Acronyms

 

AMPA = -amino-3-hydroxy-5-methyl-4-isoxazolepropionate eNOS = endothelial NOS iNOS = immunologic NOS MCA = middle cerebral artery NMDA = N-methyl-D-aspartate nNOS = neuronal NOS NO = nitric oxide NOS = nitric oxide synthase O2·- = superoxide anion ONOO- = peroxynitrite PARS = poly(ADP-ribose) synthetase SOD = superoxide dismutase

	Acknowledgments

 
This study was supported in part by US Public Health Service grants NS 33277 and NS 01578 and by the International Life Sciences Institute (T.M.D.), US Public Health Service grant NS 33142, and the American Heart Association (V.L.D.). The authors thank Ann Schmidt for secretarial assistance and our colleagues who provided preprints of their work. Under an agreement between the Johns Hopkins University and Guilford Pharmaceuticals, T.M.D. and V.L.D. are entitled to a share of sales royalty received by the University from Guilford Pharmaceuticals. T.M.D. and the University also own Guilford stock, which is subject to certain restrictions under University policy. The terms of this arrangement have been reviewed and approved by the University in accordance with its conflict of interest policies.

Received January 21, 1997; revision received April 9, 1997; accepted April 10, 1997.

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