repeated observation that it is altered by a variety of pathophysiological conditions. Such alterations of NO production and/or bioavailability have been shown to occur both in experimental animal models and in human subjects, in the setting of such diverse disorders as hypertension, hypercholesterolemia, aging, cigarette smoking, diabetes, and heart failure (1). The mechanisms underlying the alteration of this important function of the endothelium are varied and likely multifactorial. During the past several years an enormous amount of research has been devoted to understanding these abnormalities, which has led to new insights into regulation of vascular tone, redox state, inflammation, growth, and the prothrombotic/antithrombotic properties of the vessel wall. This Perspective will highlight some of these important new observations, as they relate to the pathology of the endothelial cell L -arginine/NO synthase (NOS) system. In addition, future directions of research that may be particularly informative will be indicated. Alterations of the substrate for the NOS enzyme The substrate for NOS is the basic amino acid L -arginine (with a K m of approximately 5 m M, reference 2). L -Arginine is syn

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Cellular and molecular mechanisms of endothelial cell dysfunction.

Nitric oxide synthase (NOS) exists in three established isoforms. NOS I (NOS1, ncNOS) was originally discovered in neurons. This enzyme and splice variants thereof have since been found in many other cells and tissues. NOS II (NOS2, iNOS) was first identified in murine macrophages, but can also be induced in many other cell types. NOS III (NOS3, ecNOS) is expressed mainly in endothelial cells. Whereas NOS II is a transcriptionally regulated enzyme, NOS I and NOS III are considered constitutively expressed proteins. However, evidence generated in recent years indicates that these two isoforms are also subject to expressional regulation. In view of the important biological functions of these isoforms, changes in their expression may have physiological and pathophysiological consequences. This review recapitulates compounds and conditions that modulate the expression of NOS I and NOS III, summarizes transcriptional and posttranscriptional effects that underlie these changes, and-where known-describes the molecular mechanisms leading to changes in transcription, RNA stability, or translation of these enzymes.

Expressional control of the 'constitutive' isoforms of nitric oxide synthase (NOS I and NOS III).

Endothelium-dependent vasodilatation is mediated by release of nitric oxide formed by constitutively expressed endothelial nitric oxide synthase (ecNOS). We explored the distribution of polymorphism ecNOS4a/b in 549 subjects with, and 153 without, coronary artery disease in relation to smoking. In current and ex-cigarette smokers, but not nonsmokers, there was a significant excess of homozygotes for the rare ecNOS4a allele in patients with severely stenosed arteries, compared with those with no or mild stenosis. This genotype was also associated with a history of myocardial infarction. This smoking-dependent excess coronary risk in ecNOS4a homozygotes is consistent with predisposition to endothelial dysfunction.

A smoking-dependent risk of coronary artery disease associated with a polymorphism of the endothelial nitric oxide synthase gene.

Background—Endothelium-derived nitric oxide (NO) is synthesised from l-arginine by endothelial nitric oxide synthase (eNOS) encoded by the NOS 3 gene on chromosome 7. Because reduced NO synthesis has been implicated in the development of coronary atherosclerosis, which has a heritable component, we hypothesised that polymorphisms of NOS 3 might be associated with increased susceptibility to this disorder. Methods and Results—Single-strand conformation polymorphism analysis of NOS 3 identified a G→T polymorphism in exon 7 of the gene which encodes a Glu→Asp amino acid substitution at residue 298 of eNOS. We investigated the relationship between this Glu298→Asp variant and atherosclerotic coronary artery disease (CAD) using 2 independent case-controlled studies. In the first study (CHAOS), cases consisted of 298 unrelated patients with positive coronary angiograms and controls were 138 unrelated healthy individuals ascertained through a population health screen. In the second study (CHAOS II), the cases wer...

A Common Variant of the Endothelial Nitric Oxide Synthase (Glu298→Asp) Is a Major Risk Factor for Coronary Artery Disease in the UK

Essential hypertension has a genetic basis. Accumulating evidence, including findings of elevation of arterial blood pressure in mice lacking the endothelial nitric oxide synthase (eNOS) gene, strongly suggests that alteration in NO metabolism is implicated in hypertension. There are, however, no reports indicating that polymorphism in the eNOS gene is associated with essential hypertension. We have identified a missense variant, Glu298Asp, in exon 7 of the eNOS gene and demonstrated that it is associated with both coronary spastic angina and myocardial infarction. To explore the genetic involvement of the eNOS gene in essential hypertension, we examined the possible association between essential hypertension and several polymorphisms including the Glu298Asp variant, variable number tandem repeats in intron 4 (eNOS4b/4a), and two polymorphisms in introns 18 and 23. We performed a large-scale study of genetic association using two independent populations from Kyoto (n=458; 240 normotensive versus 218 hypertensive subjects) and Kumamoto (n=421; 223 normotensive versus 187 hypertensive subjects), Japan. In both groups, a new coding variant, Glu298Asp, showed a strong association with essential hypertension (Kyoto: odds ratio, 2.3 [95% confidence interval, 1.4 to 3.9]; Kumamoto: odds ratio, 2.4 [95% confidence interval, 1.4 to 4.0]). The allele frequencies of 298Asp in hypertensive subjects were significantly higher than those in normotensive subjects in both groups (Kyoto: 0.103 versus 0.050, P<0.0017; Kumamoto: 0.120 versus 0.058, P<0.0013, respectively). No such disequilibrium between genotypes was significantly associated with any other polymorphisms we examined; the Glu298Asp variant was also not linked to any other polymorphisms. In conclusion, the Glu298Asp missense variant was significantly associated with essential hypertension, which suggests that it is a genetic susceptibility factor for essential hypertension.

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Endothelial Nitric Oxide Synthase Gene Is Positively Associated With Essential Hypertension

Background The basal release of nitric oxide by the endothelium plays an important role in regulating blood flow and pressure and mediates most of the endothelium-dependent vasodilation. Impairment of nitric oxide production by specific inhibitors increases blood pressure in humans, and several reports suggest that hypertensive subjects have a blunted endothelium-dependent vasodilatation that might be secondary to decreased nitric oxide production from the vessel wall. Methods and Results To determine whether the endothelial nitric oxide synthase gene is involved in human essential hypertension, we identified informative biallelic and multiallelic markers of this locus and performed case-control and linkage studies in hypertensive subjects and normotensive control subjects. We used the affected sib pair method to test for potential linkage in 145 hypertensive pedigrees (269 sib pairs, 346 subjects) with a highly polymorphic marker of the nitric oxide synthase gene (polymorphism information content of 92%). There was no evidence for linkage among affected siblings. The 95% upper confidence limit of this value suggests that at most 1% of alleles in excess of expected are shared. We also identified two informative biallelic markers of this gene to perform a case-control study on white hypertensive and normotensive subjects. Similar genotype distributions between the two groups were noted for both markers. Estimated haplotype frequencies by maximum likelihood methods combining the two biallelic markers were also similar in both groups. Conclusions These findings do not suggest that common molecular variants of the endothelial nitric oxide synthase gene are involved in essential hypertension.

Sophie Nadaud

Papers

Lack of Evidence for Linkage of the Endothelial Cell Nitric Oxide Synthase Gene to Essential Hypertension

Functional study of the germinal angiotensin I-converting enzyme promoter.