Nitric oxide (NO) is produced in almost all tissues and organs, exerting a variety of biological actions under physiological and pathological conditions. NO is synthesized by three different isoforms of NO synthase (NOS), including neuronal, inducible, and endothelial NOSs. Because there are substantial compensatory interactions among the NOS isoforms, the ultimate roles of endogenous NO in our body still remain to be fully elucidated. Here, we have successfully developed mice in which all three NOS genes are completely deleted by crossbreeding singly NOS ؊/؊ mice. NOS expression and activities were totally absent in the triply NOS ؊/؊ mice before and after treatment with lipopolysaccharide. Although the triply NOS ؊/؊ mice were viable and appeared normal, their survival and fertility rates were markedly reduced as compared with the wild-type mice. Furthermore, these mice exhibited marked hypotonic polyuria, polydipsia, and renal unresponsiveness to an antidiuretic hormone, vasopressin, all of which are characteristics consistent with nephrogenic diabetes insipidus. In the kidney of the triply NOS ؊/؊ mice, vasopressin-induced cAMP production and membranous aquaporin-2 water channel expression were reduced associated with tubuloglomerular lesion formation. These results provide evidence that the NOS system plays a critical role in maintaining homeostasis, especially in the kidney.

Objective-Asymmetric dimethylarginine (ADMA) is widely believed to be an endogenous nitric oxide synthase (eNOS) inhibitor. However, in this study, we examined our hypothesis that the long-term vascular effects of ADMA are not mediated by inhibition of endothelial NO synthesis. Methods and Results-ADMA was infused in wild-type and eNOS-knockout (KO) mice by osmotic minipump for 4 weeks. In wild-type mice, long-term treatment with ADMA caused significant coronary microvascular lesions. Importantly, in eNOS-KO mice, treatment with ADMA also caused an extent of coronary microvascular lesions that was comparable to that in wild-type mice. These vascular effects of ADMA were not prevented by supplementation of L-arginine, and vascular NO production was not reduced by ADMA treatment. Treatment with ADMA caused upregulation of angiotensin-converting enzyme (ACE) and an increase in superoxide production that were comparable in both strains and that were abolished by simultaneous treatment with temocapril (ACE inhibitor) or olmesartan (AT 1 receptor antagonist), which simultaneously suppressed vascular lesion formation. Key Words: asymmetric dimethylarginine Ⅲ arteriosclerosis Ⅲ nitric oxide Ⅲ endothelial nitric oxide synthase Ⅲ mice E ndothelium-derived nitric oxide (NO), synthesized from L-arginine by endothelial NO synthase (eNOS), has several important antiatherogenic actions. 1-5 Indeed, reduction of endothelial NO synthesis (endothelial dysfunction) predisposes the blood vessel to arteriosclerosis, 1-5 and the eNOS-deficient (eNOS-KO) mice exhibit accelerated vascular lesion formation. 6,7 As pharmacological tools to inhibit endothelial NO synthesis, synthetic L-arginine analogues have been used in vitro and in vivo. Among them, N -nitro-L-arginine methyl ester (L-NAME) is the most frequently used agent. [1][2][3][4][5] Long-term treatment with L-NAME is known to cause arteriosclerotic coronary lesions, especially at microvascular levels, in experimental animals. 8,9 This model with L-NAME is regarded as a useful animal model for examining the protective roles of endothelium-derived NO in the pathogenesis of arteriosclerosis. 8,9 See cover However, it is controversial whether these vascular effects of L-NAME are caused primarily by the inhibition of endothelial NO synthesis for the following reasons: first, the importance of endothelium-derived NO decreases as the vessel size becomes smaller, 10 whereas L-NAME-induced vascular lesions are prominent at microvascular levels; 8 second, long-term treatment with L-NAME does not reduce eNOS activity; 11 third, multiple actions of L-NAME other than simple inhibition of NO synthesis have been reported. 12,13 The most appropriate way to address this issue is to use mice that are deficient in the eNOS gene and to examine whether long-term treatment with L-NAME causes coronary vascular lesions in those mice. We have recently shown that treatment with L-NAME causes a comparable extent of Conclusions-These

Background-The roles of nitric oxide (NO) in the cardiovascular system have been investigated extensively in pharmacological studies with NO synthase (NOS) inhibitors and in studies with NOS isoform-deficient mice. However, because of the nonspecificity of the NOS inhibitors and the compensatory interactions among NOS isoforms (nNOS, iNOS, and eNOS), the ultimate roles of endogenous NO derived from the entire NOS system are still poorly understood. In this study, we examined this point in mice deficient in all 3 NOS isoforms (triply n/i/eNOS Ϫ/Ϫ mice) that we have recently developed. Methods and Results-The triply n/i/eNOSϪ/Ϫ mice, but not singly eNOS Ϫ/Ϫ mice, exhibited markedly reduced survival, possibly due to spontaneous myocardial infarction accompanied by severe coronary arteriosclerotic lesions. Furthermore, the triply n/i/eNOS Ϫ/Ϫ mice manifested phenotypes that resembled metabolic syndrome in humans, including visceral obesity, hypertension, hypertriglyceridemia, and impaired glucose tolerance. Importantly, activation of the renin-angiotensin system was noted in the triply n/i/eNOS Ϫ/Ϫ mice, and long-term oral treatment with an angiotensin II type 1 receptor blocker significantly suppressed coronary arteriosclerotic lesion formation and the occurrence of spontaneous myocardial infarction and improved the prognosis of those mice, along with ameliorating the metabolic abnormalities. Conclusions-These results provide the first direct evidence that genetic disruption of the whole NOS system causes spontaneous myocardial infarction associated with multiple cardiovascular risk factors of metabolic origin in mice in vivo through the angiotensin II type 1 receptor pathway, demonstrating the critical role of the endogenous NOS system in maintaining cardiovascular and metabolic homeostasis.

Oxidative stress has been implicated as an important mechanism of vascular endothelial dysfunction induced by aging. Previous studies suggested that tetrahydrobiopterin (BH 4), an essential cofactor of endothelial NO synthase, could be a molecular target for oxidation. We tested the hypothesis that oxidative stress, in particular oxidation of BH 4, may contribute to attenuation of endothelium-dependent relaxation in aged mice. Vasomotor function of isolated carotid arteries was studied using a video dimension analyzer. Vascular levels of BH 4 and its oxidation products were measured via HPLC. In aged mice (age, 95 Ϯ 2 wk), endothelium-dependent relaxation to ACh (10 Ϫ5 to 10 Ϫ9 M) as well as endothelium-independent relaxation to the NO donor diethylammo-Ϫ5 to 10 Ϫ9 M) were significantly reduced compared with relaxation detected in young mice (age, 23 Ϯ 0.5 wk). Incubation of aged mouse carotid arteries with the cell-permeable SOD mimetic Mn(III)tetra(4-benzoic acid)porphyrin chloride normalized relaxation to ACh and DEA-NONOate. Furthermore, production of superoxide anion in aorta and serum levels of amyloid P component, which is the murine analog of C-reactive protein, was increased in old mice. In aorta, neither the concentration of BH 4 nor the ratio of reduced BH4 to the oxidation products were different between young and aged mice. Our results demonstrate that in mice, aging impairs relaxation mediated by NO most likely by increased formation of superoxide anion. Oxidation of BH 4 does not appear to be an important mechanism underlying vasomotor dysfunction in aged mouse arteries. endothelial dysfunction; nitric oxide; superoxide anion; reactive oxygen species; C-reactive protein AGING IS A RISK FACTOR for vascular disease; however, the role of aging, either as a process or as the result of longer exposure to other risks, is not well defined (16,17). In murine vascular tissue, the age-dependent changes in vasomotor function have not been characterized. Studies on rats have shown impairment of endothelium-dependent relaxation due to increased production of superoxide anions, but the source of the superoxide anions has also not been characterized (16,30). Reactive oxygen species (ROS) have been implicated in endothelial dysfunction associated with aging, hypertension, hypercholesteremia, diabetes, and cigarette smoking (3). ROS can interfere with endothelium-dependent relaxation particularly by the scavenging of NO by superoxide anion (O 2 Ϫ ⅐; Refs. 1, 3, 34).