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Mouse Models of Nitric Oxide Synthase Deficiency

Cardiovascular Research Center and Cardiology Division, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts.

Correspondence to Dr. Paul L. Huang, Cardiovascular Research Center and Cardiology Division, Massachusetts General Hospital, Harvard Medical School, Mail Code 149-4201, 149 13th Street, Charlestown, MA 02129-2060.

	Abstract

Top Abstract Introduction NO in Stroke, a... Endothelial NO in Vascular... NO and Cardiac Function NO in the Autonomic... Summary References

 
Abstract. Knockout mice for each of the three nitric oxide (NO) synthase (NOS) genes have been generated. Their phenotypes reflect the roles of each NOS isoform in physiologic and pathologic processes. This article reviews how neuronal NOS (nNOS) and endothelial NOS (eNOS) knockout mice have contributed to our knowledge of the roles of NO in cerebral ischemia, cardiovascular processes, and the autonomic nervous system. In some instances, the effects of NO produced by one isoform antagonize the effects of NO produced by another isoform. For example, after cerebral ischemia, the nNOS isoform is involved in tissue injury, whereas the eNOS isoform is important in maintaining blood flow. All three isoforms are expressed in the respiratory tract, but only the nNOS isoform appears to be involved in modulating airway responsiveness and only the inducible NOS isoform appears to respond to antigen stimulation. In the cardiovascular system, endothelial NO is important for vascular tone, systolic and diastolic cardiac function, vascular proliferative responses to injury, platelet aggregation, and hemostasis.

	Introduction

Top Abstract Introduction NO in Stroke, a... Endothelial NO in Vascular... NO and Cardiac Function NO in the Autonomic... Summary References

 
Nitric oxide (NO) is an important signaling molecule in the cardiovascular system. NO is responsible for the activity of endothelium-dependent relaxing factor (EDRF), which was first described by Furchgott and Zawadzki (1). The vasodilatory effects of NO are mediated by the relaxation of vascular smooth muscle. In addition, NO modulates other important aspects of vascular function, including leukocyte activation, platelet aggregation, interactions between the endothelium and circulating cells, and vascular smooth muscle cell proliferation (2,3). In hypertension, hypercholesterolemia, atherosclerosis, diabetes, and aging, the amount of bioavailable NO generated by the endothelium is decreased. This condition, termed endothelial dysfunction, may be thought of as a relative deficiency of endothelial NO production. It has been hypothesized that this NO deficiency leads to vasoconstriction, leukocyte activation, a propensity to thrombosis, and increased vascular smooth muscle cell proliferation.

NO is also produced by nonvascular cells, including neurons, skeletal myocytes, and monocytes/macrophages. Most tissues contain NO, because they contain vasculature and innervation, in addition to NO generated by parenchymal cells in the tissues themselves. Our understanding of the enzymes that produce NO, i.e., the NO synthases (NOS), has increased dramatically in the past 10 years, since neuronal NOS (nNOS) protein was first purified and its gene was cloned (4,5). We now know that there are three major isoforms of NOS (6), encoded by separate genes. Type I, or nNOS, is found in specific populations of neurons in the brain and in the nonadrenergic, noncholinergic, autonomic nervous system. Type II, or inducible NOS (iNOS), can be induced in macrophages in response to infection or tumor immunity. Type III, or endothelial NOS (eNOS), is found in endothelial cells and is likely the isoform responsible for EDRF activity. The nNOS and eNOS isoforms are similar, in that they are generally constitutively produced and their activity is regulated by intracellular calcium concentrations. In contrast, iNOS is regulated not by calcium concentrations but by induction of its expression by stimuli such as tumor necrosis factor or interferon.

Targeted disruption of each of the NOS genes has been achieved, resulting in viable and fertile knockout mice for each NOS isoform (7,8,9,10,11). These animals serve as useful animal models for NOS deficiencies, and their phenotypes reflect the functions of each NOS isoform. nNOS knockout mice display enlarged stomachs because of abnormalities in pyloric relaxation (7). eNOS knockout mice lack EDRF activity and are hypertensive (8). iNOS knockout mice are more sensitive to certain infections, whereas they are resistant to sepsis-induced hypotension (9,10,11). This article reviews some of the information obtained from studies with nNOS and eNOS knockout animals.

	NO in Stroke, a Double-Edged Sword

Top Abstract Introduction NO in Stroke, a... Endothelial NO in Vascular... NO and Cardiac Function NO in the Autonomic... Summary References

 
Brain NO levels (measured using a porphyrinic microsensor) increase dramatically after interruption of cerebral blood flow (12). Studies that blocked the production of NO by using NOS inhibitors such as L-n-monomethylarginine or L-nitroarginine produced disparate results, leading to speculation that NO may play both protective and harmful roles after cerebral ischemia (13). Indeed, production of NO by the neuronal isoform is stimulated by calcium influx after N-methyl-D-aspartate receptor binding to the excitatory amino acid neurotransmitter glutamate. Excessive N-methyl-D-aspartate receptor stimulation, which leads to excitotoxicity, is likely related to excessive NO production. Potential mechanisms for NO toxicity include the reaction of NO with superoxide to form peroxynitrite anion, activation of poly-ADP-ribose synthase, or direct effects of NO on mitochondrial enzyme complexes. NO is important in the regulation of cerebral blood flow and in coupling cerebral blood flow to metabolic activity. Therefore, NO is likely to play a protective role in preserving cerebral blood flow under conditions of ischemia. The nNOS and eNOS knockout mice offer the opportunity to directly test the hypothesis that the toxic effects of NO are attributale to excessive production of NO by the nNOS isoform and the protective effects of NO are attributable to eNOS.

Middle cerebral artery occlusion (MCAO) with filaments results in well defined and reproducible strokes in mice. When subjected to MCAO, nNOS knockout mice develop smaller cerebral infarctions than do control wild-type animals, indicating that the absence of nNOS decreases infarction size (14). Laser Doppler measurements of cerebral blood flow indicate that filament occlusion reduces blood flow to the same extent in wild-type and nNOS knockout mice. Therefore, nNOS knockout mice do not develop smaller strokes because they have better blood flow; they do so despite the same reduction in blood flow, suggesting that nNOS is normally involved in tissue damage.

In contrast, eNOS knockout mice develop larger strokes after MCAO (15). Laser Doppler measurements indicate that the effect of filament occlusion is more pronounced in eNOS knockout mice, confirming that eNOS is normally important in maintaining blood flow in the presence of ischemia. This point was elegantly demonstrated by Lo et al. (16), who used temporal correlation mapping and functional computed tomography. The areas at risk in eNOS knockout and wild-type mice are the same but, within those areas, a substantially larger region demonstrates no blood flow in eNOS knockout mice, compared with wild-type mice. The rim of ischemic but still perfused tissue is correspondingly larger in wild-type mice, compared with eNOS knockout mice.

In contrast to focal ischemia, which is a model for stroke and transient ischemic attacks, global ischemia is a model for cerebral hypoperfusion secondary to cardiac arrest. Using models of global ischemia (which damages selectively vulnerable neurons in the hippocampus), nNOS knockout mice are protected, compared with wild-type mice (17), whereas eNOS knockout mice experience worse outcomes. Therefore, in both focal and global ischemia, the data indicate that nNOS is involved in the pathogenesis of tissue damage and its absence in nNOS knockout mice is protective. In contrast, eNOS is important in maintaining cerebral blood flow, and its absence leads to more pronounced detrimental effects of ischemia.

	Endothelial NO in Vascular Function

Top Abstract Introduction NO in Stroke, a... Endothelial NO in Vascular... NO and Cardiac Function NO in the Autonomic... Summary References

 
Aortic rings from eNOS mutant mice do not relax in response to acetylcholine, providing genetic evidence that eNOS is required for EDRF activity (8). The vessels are able to relax in response to sodium nitroprusside and papaverine. Furthermore, eNOS knockout mice are hypertensive, with mean arterial BP values that are 20 to 30 mmHg greater than values observed in wild-type animals. These results confirm a role for basal eNOS activity in BP regulation and vascular tone.

Endothelial production of NO is important for other aspects of vessel function. Intimal proliferation, which is a common response to arterial injury, plays a critical role in the development of atherosclerotic lesions. To test the hypothesis that eNOS-derived NO suppresses vascular smooth muscle cell proliferation, eNOS knockout mice have been subjected to a cuff model of vessel injury (18). eNOS knockout mice develop significantly more neointimal proliferation after cuff injury than do wild-type mice, consistent with a physiologic role for eNOS in suppressing these responses. In eNOS knockout mice as well as wild-type animals, there is less intimal proliferation in female animals, compared with male animals. These gender differences are thought to reflect the protective effects of estrogens. Although estrogen has been postulated to suppress atherosclerosis by increasing eNOS expression, this cannot be the case in eNOS knockout mice. Therefore, estrogens are involved in protective mechanisms in addition to modulation of eNOS gene expression. Experiments using eNOS knockout mice bred into an apoE knockout background to study the effects of eNOS gene deletion on diet-induced atherosclerosis are underway.

Freedman et al. (19) have studied the role of platelet-derived NO in vivo by using eNOS knockout mice. Although surface expression of P-selectin is not altered in these mice, bleeding times are markedly decreased, compared with wild-type animals. To determine the relative contributions of endothelium-derived and platelet-derived NO, those authors have transfused either eNOS knockout or wild-type platelets into thrombocytopenic eNOS knockout mice. Bleeding times are decreased in mice treated with eNOS knockout platelets, compared with those treated with wild-type platelets. Therefore, platelet-derived NO is important in the regulation of hemostasis.

	NO and Cardiac Function

Top Abstract Introduction NO in Stroke, a... Endothelial NO in Vascular... NO and Cardiac Function NO in the Autonomic... Summary References

 
Using a Millar catheter to measure left ventricular pressure, Gyurko et al. (20) have determined the contractile responses of eNOS knockout mice to the -agonist isoproterenol. The inotropic response is significantly increased, compared with wildtype mice, consistent with a role for eNOS-derived NO in blunting the cardiac inotropic response to sympathetic stimuli. L-Nitroarginine treatment of wild-type mice interferes with diastolic relaxation, whereas eNOS knockout mice exhibit normal diastolic relaxation patterns. This indicates that compensatory mechanisms mediate ventricular relaxation despite the absence of eNOS. One such mechanism involves upregulation of pre-pro-atrial natriuretic peptide, which is observed in eNOS knockout mice. This may be a response to increased afterload in the mutant mice, or it may be an adaptive response to maintain diastolic relaxation.

	NO in the Autonomic Nervous System

Top Abstract Introduction NO in Stroke, a... Endothelial NO in Vascular... NO and Cardiac Function NO in the Autonomic... Summary References

 
The stomachs of nNOS knockout mice are variably enlarged, in some instances to several times the normal size (7). The myenteric plexus, which is located between the longitudinal muscle layer and the circular muscle layer, contains nNOS (as determined in immunohistochemical analyses) in wild-type mice but not nNOS knockout mice. These results demonstrate that the same gene encodes nNOS in the central nervous system and in the myenteric plexus. The stomach enlargement observed in nNOS knockout mice is consistent with an important role for NO in gastrointestinal motility and pyloric relaxation. The absence of nNOS in the knockout mice results in gastric outlet obstruction and secondary enlargement of the stomach.

Mashimo et al. (21) studied the response of gastric smooth muscle to electric field stimulation. Nonadrenergic, noncholinergic, autonomic neurotransmission in the gastrointestinal tract includes an excitatory junction potential mediated by substance P and an inhibitory junction potential (IJP). The IJP response consists of two overlapping components, i.e., a slow component mediated by NO and vasoactive intestinal peptide and a fast component mediated by ATP binding to purinergic receptors and activating calcium-dependent potassium channels. Stomach strips from nNOS knockout mice display only the fast component of the IJP. ATP desensitization or apamin treatment, which blocks fast IJP, results in the abolition of all IJP in nNOS knockout mice.

NO causes tracheal smooth muscle relaxation in cats and guinea pigs, and inhibition of NOS produces enhanced airway responsiveness. NO levels are also increased in patients with asthma. It is not known whether NO is a marker for inflammatory responses associated with asthma or whether NO may be involved in bronchoconstrictor responses. De Sanctis et al. (22) studied the contribution of nNOS to expired NO by using nNOS knockout mice. Those authors observed that expired NO levels are reduced in nNOS knockout mice, to approximately 60% of the levels observed in wild-type mice, suggesting that 40% of expired NO is derived from nNOS (with the assumption that there are no other changes in NO production secondary to gene deletion).

Under basal conditions, similar NOS activities are observed in the lungs of nNOS, eNOS, or nNOS/eNOS knockout mice and wild-type mice (23). Approximately 80% of the NO originates from iNOS and 20% originates from other NOS isoforms. After ovalbumin challenge, the NOS activity increases dramatically, with 99% of NOS activity being attributable to iNOS. These results are consistent with reports of iNOS induction after antigen sensitization in other animal systems. In iNOS knockout mice, the baseline NOS activity is decreased and there is no increase with ovalbumin sensitization and challenge, as expected.

Despite the marked differences between wild-type and iNOS knockout mice with respect to total NOS and iNOS levels, there is no difference in airway responsiveness, as measured using methacholine challenge assays or plethysmography. These date indicate that there is no significant difference between wild-type and iNOS knockout mice with respect to airway responsiveness. Furthermore, total and differential cell counts for bronchoalveolar lavage (BAL) fluid and specific IgE levels were the same for these two groups. Therefore, despite the marked increase in iNOS activity after antigen exposure, iNOS-derived NO is not required for increased BAL cell counts, eosinophilia, or increased IgE levels. Furthermore, the presence or absence of iNOS-derived NO does not seem to affect airway responsiveness.

In contrast, the airway responsiveness of nNOS and nNOS/eNOS knockout mice was reduced, compared with wild-type mice. The response of eNOS knockout mice was similar to that of wild-type mice. These results suggest that, despite the modest contribution of nNOS-derived NO to expired NO, nNOS plays a key role in the bronchoconstriction mediated by antigen challenge. In the absence of nNOS, ovalbumin-sensitized and -challenged mice are hyporesponsive to methacholine challenges. The mechanism of action of nNOS is not known. However, the degree of airway inflammation, the levels of ovalbumin-specific IgE, and the recruitment of inflammatory cells into BAL fluid were no different in the nNOS knockout, nNOS/eNOS knockout, and wild-type cells, suggesting that nNOS is not involved in mediating inflammation.

	Summary

Top Abstract Introduction NO in Stroke, a... Endothelial NO in Vascular... NO and Cardiac Function NO in the Autonomic... Summary References

 
With three genes being responsible for three separately expressed NOS isoforms, the roles of NO in physiologic processes are complex. NOS gene knockout mice are useful tools to study the contribution of each NOS isoform to specific physiologic and pathologic processes. Some key findings obtained using NOS knockout mice can be summarized as follows. In cerebral ischemia, nNOS appears to contribute to tissue damage after stroke, whereas eNOS is important in preserving cerebral blood flow. All three NOS isoforms are present in the respiratory tract, with iNOS being responsible for the majority of NOS activity under basal conditions and in response to antigen challenge. However, it is the nNOS isoform that is important in determining airway responsiveness, despite its less dramatic contribution to total NOS activity. Endothelial NO production is important for vascular function, not only in terms of vascular tone and BP regulation but also in terms of its effects on smooth muscle cell proliferation, responses to vessel injury, and platelet aggregation. These effects are likely important in modulating susceptibility to atherogenesis. These examples demonstrate how the genetic approach of knocking out each NOS gene provides useful information on the complex roles of NO in physiologic processes.

	References

Top Abstract Introduction NO in Stroke, a... Endothelial NO in Vascular... NO and Cardiac Function NO in the Autonomic... Summary References

 

1. Furchgott RF, Zawadzki JV: The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature (Lond) 288:373 -376, 1980[Medline]
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23. De Sanctis GT, MacLean JA, Hamada K, Mehta S, Scott JA, Jiao A, Yandava CN, Kobzik L, Wolyniec WW, Fabian AJ, Venugopal CS, Grasemann H, Huang PL, Drazen JM: Contribution of nitric oxide synthases 1, 2, and 3 to airway hyperresponsiveness and inflammation in a murine model of asthma. J Exp Med 189:1621 -1630, 1999.[Abstract/Free Full Text]

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