Abstract-Recently a point mutation of guanine to thymine at nucleotide position 1917 in the endothelial nitric oxide synthase (eNOS) gene has been reported to be associated with coronary artery spasm. In addition, a significant association of the 4a/b polymorphism in intron 4 of the eNOS gene with coronary artery disease has been reported. However, the implications of these polymorphisms with respect to acute myocardial infarction (AMI) remain to be established. We conducted a case-control study of 226 patients with AMI and 357 healthy gender-and age-matched control subjects. In the former group, coronary angiograms were evaluated according to angiographic criteria based on the number of diseased vessels (Ն75%) and the number of stenotic lesions (Ն50%). Homozygosity for the Glu-Asp298 polymorphism existed in 5 of 226 patients with AMI (2.2%) but not in any of the 357 control subjects (Pϭ.0085). However, when we evaluated the coronary angiograms of 226 case patients, there was no difference in the number of diseased vessels or the number of stenotic lesions between the patients with this homozygote and those without it. By contrast, there was no evidence of a significant increase in the risk of AMI or the severity of coronary atherosclerosis among individuals with the a/a genotype of the eNOS4a/b polymorphism. Our results imply that patients who are homozygous for the Glu-Asp298 polymorphism may be genetically predisposed to AMI; however, this mutation apparently is not related to the severity of coronary atherosclerosis. Further studies are needed to confirm our results and characterize the molecular mechanisms by which eNOS is involved in susceptibility to AMI. (Hypertension. 1998;32:521-526.)Key Words: endothelium-derived relaxing factor Ⅲ genes Ⅲ myocardial infarction Ⅲ atherosclerosis Ⅲ angiography S ince the identification of nitric oxide (NO) as an important endothelium-derived relaxing factor, there has been an explosion of new information on the physiological and pathophysiological roles of NO. NO is synthesized from the amino acid L-arginine by a family of enzymes, referred to as NO synthase (NOS). Three distinct isoforms of NOS have been identified to date.1 The inducible NOS is expressed in vessel walls and macrophages by certain cytokines and endotoxin lipopolysaccharides in pathological conditions. 2The constitutive neuronal NOS is expressed in the central and peripheral nervous systems as well as in macula densa of kidney. It plays important roles in physiological 3 and pathophysiological 4 conditions. The constitutive endothelial NO synthase (eNOS) is expressed in the endothelium, where it produces NO from L-arginine. NO diffuses from the endothelium to the vascular smooth muscle cells, where it increases the concentration of cGMP by stimulating soluble guanylate cyclase, leading to vascular relaxation.

Several lines of evidence suggest that patterns of genetic variability in the human angiotensinogen gene (AGT) contribute to phenotypic variability in human hypertension. The A(-6) promoter variant of AGT is associated with higher plasma angiotensinogen levels and increased risk of essential hypertension. The geographic distribution of the A(-6) variant leads to the intriguing hypothesis that the G(-6) promoter variant has been selectively advantageous outside Africa. To test these hypotheses, we investigated the roles of population history and natural selection in shaping patterns of genetic diversity in AGT, by sequencing the entire AGT gene (14400 bp) in 736 chromosomes from Africa, Asia, and Europe. We found that the A(-6) variant is present at higher frequency in African populations than in non-African populations. Neutrality tests found no evidence of a departure from selective neutrality, when whole AGT sequences were compared. However, tests restricted to sites in the vicinity of the A(-6)G polymorphism found evidence of a selective sweep. Sliding-window analyses showed that evidence of the sweep is restricted to sites in tight linkage disequilibrium (LD) with the A(-6)G polymorphism. Further, haplotypes carrying the G(-6) variant showed elevated levels of LD, suggesting that they have risen recently to high frequency. Departures from neutral expectation in some but not all regions of AGT indicate that patterns of diversity in the gene cannot be accounted for solely by population history, which would affect all regions equally. Taken together, patterns of genetic diversity in AGT suggest that natural selection has generally favored the G(-6) variant over the A(-6) variant in non-African populations. However, important localized effects may also be present.

Abstract-Elements of a renin-angiotensin system expressed along the entire nephron, including angiotensinogen secreted by proximal tubule and renin expressed in connecting tubule, may participate in the regulation of sodium reabsorption at multiple sites of the nephron. The response of this tubular renin-angiotensin system to stepwise changes in dietary sodium was investigated in 2 mouse strains, the sodium-sensitive inbred C57BL/6 and the sodium-resistant CD1 outbred. Plasma angiotensinogen was not affected by sodium regimen, whereas plasma renin increased 2-fold under low sodium. In both strains, the variation in urinary parameters did not parallel the changes observed in plasma. Angiotensinogen and renin excretion were significantly higher under high sodium than under low sodium. Water deprivation, by contrast, induced significant activation in the tubular expression of angiotensinogen and renin. C57BL/6 exhibited significantly higher urinary excretion of angiotensinogen than did CD1 animals under both conditions of sodium intake. The extent to which these urinary parameters reflect systemic or tubular responses to challenges of sodium homeostasis may depend on the relative contribution of sodium restriction and volume depletion. Key Words: angiotensinogen Ⅲ renin Ⅲ sodium Ⅲ mouse Ⅲ genetics Ⅲ urine W e have advanced the hypothesis that a paracrine tubular renin-angiotensin system operates along the entire nephron. 1 Although angiotensinogen (AGT) is not filtered across the glomerular membrane, the protein 2 and its mRNA 3,4 have been detected in proximal tubule (PT), the protein is secreted to the apical side of PT cell monolayers, 1 has been detected in final urine under normal physiological conditions, 5 and was detected in luminal fluid of PT epithelium collected by micropuncture. 6 Systemic renin is filtered and reabsorbed in the PT. 7 Although not detected in situ, it may be expressed at low level in the PT. 8,9 We have found that renin was also synthesized and secreted in connecting tubule (CNT). 1 ACE and angiotensin (Ang) II receptors are expressed along the nephron. 10,11 High luminal Ang II has been observed in the PT, 12,13 where it stimulates sodium reabsorption. 14 Some observations support a similar role in terminal segments of the nephron. 15 The potential significance of this tubular renin-angiotensin system in blood pressure regulation is underlined by the observation that double transgenic animals overexpressing human renin systemically and human AGT in the PT develop hypertension. 16 The impact of dietary sodium on the expression of renin and tubular AGT and the significance of their urinary excretion as indicators of the activity of this tissue system were tested in the mouse. Two strains were investigated, C57BL/6 and CD1. The C57BL/6 inbred differs from other inbred lines in its response to dietary sodium 17 ; its sodium sensitivity has been demonstrated 18,19 and exploited in an attempt to map genetic determinants of the arterial pressure response to dietary sodium. 19 We have verified...

Variation in the angiotensinogen gene, AGT, has been associated with variation in plasma angiotensinogen levels. In addition, the T235M polymorphism in the AGT product is associated with an increased risk of essential hypertension in multiple populations, making AGT a good example of a quantitative-trait locus underlying susceptibility to a common disease. To better understand genetic variation in AGT, we sequenced a 14.4-kb genomic region spanning the entire AGT and identified 44 single-nucleotide polymorphisms (SNPs). Forty-two SNPs were observed both in 88 white and in 77 Japanese unselected subjects. Six major haplotypes accounted for most of the variation in this region, indicating less allelic complexity than in many other genomic regions. Although the two populations were found to share all of the major AGT haplotypes, there were substantial differences in haplotype frequencies. Pairwise linkage disequilibrium (LD), measured by the D', r(2), and d(2) statistics, demonstrated a general pattern of decline with increasing distance, but, as expected in a small genomic region, individual LD values were highly variable. LD between T235M and each of the other 39 SNPs was assessed in order to model the usefulness of LD to detect a disease-associated mutation. Among the Japanese subjects, 13 (33%) of the SNPs had r(2) values >0.1, whereas this statistic was substantially higher for the white subjects (occurring in 35/39 [90%]). LD between a hypertension-associated promoter mutation, A-6G, and 39 SNPs was also measured. Similar results were obtained, with 33% of the SNPs showing r(2)>0.1 in the Japanese subjects and 92% of the SNPs showing r(2)>0.1 in the white subjects. This difference, which occurs despite an overall similarity in LD patterns in the two populations, reflects a much higher frequency of the M235-associated haplotype in the white sample. These results have important implications for the usefulness of LD approaches in the mapping of genes underlying susceptibility to complex diseases.

A close relationship between obesity and hypertension has been recognized, and plasma angiotensinogen concentrations (p-AGT) have been reported to correlate with blood pressure (BP). However, little is known about AGT in obese patients with hypertension. To define the role of AGT in obese hypertension, we measured p-AGT in obese patients. The subjects were 42 obese patients diagnosed on the basis of a body mass index (BMI) of more than 25 kg/m2, and 21 sex- and age-matched nonobese patients, whose BMI was less than 25 kg/m2. The hypertensive patients had not previously received antihypertensive drugs. P-AGT (P < .05) and mean BP (P < .0001) was increased in the obese patients as compared with the nonobese patients. Positive correlations were observed between BMI and p-AGT, mean BP and p-AGT, and BMI and mean BP (all P < .05). However, after adjustment for blood pressure, p-AGT was not different between groups, and after adjustment a positive correlation remained only between BMI and mean BP. These results suggested the possible involvement of increased p-AGT in hypertension in obese patients, although this may be a secondary change to hypertension or obesity.

reported counterpart. Variant 13-A precludes the formation of a transcript encoding a full-length Ca 2ϩ -dependent lipid-binding (C2) domain with very high evolutionary conservation among NEDD4L orthologs. A similar C2 domain in the paralogous NEDD4 gene plays a significant role in the transfer of its product to the apical membrane of epithelial cells. Differential function of NEDD4L isoforms could prove significant in blood pressure regulation through an effect on ENaC-dependent sodium reabsorption.

Activation of angiotensin II (Ang II) type 1 receptor (AT1R) signaling is reported to play an important role in cardiac hypertrophy. We previously cloned a novel molecule interacting with the AT1R, which we named ATRAP (for Ang II type 1 receptor-associated protein). Here, we report that overexpression of ATRAP significantly decreases the number of AT1R on the surface of cardiomyocytes, and also decreases the degree of p38 mitogen-activated protein kinase phosphorylation, the activity of the c-fos promoter and protein synthesis upon Ang II treatment. These results indicate that ATRAP significantly promotes downregulation of the AT1R and further attenuates certain Ang II-mediated hypertrophic responses in cardiomyocytes.

Abstract-We cloned a novel molecule interacting with angiotensin II type 1 receptor, which we named ATRAP (for angiotensin II type 1 receptor-associated protein). Previous in vitro studies showed that ATRAP significantly promotes constitutive internalization of the angiotensin II type 1 receptor and further attenuates angiotensin II-mediated hypertrophic responses in cardiomyocytes. The present study was designed to investigate the putative functional role of ATRAP in cardiac hypertrophy by angiotensin II infusion in vivo. We first examined the effect of angiotensin II infusion on endogenous ATRAP expression in the heart of C57BL/6J wild-type mice. The angiotensin II treatment promoted cardiac hypertrophy, concomitant with a significant decrease in cardiac ATRAP expression, but without significant change in cardiac angiotensin II type 1 receptor expression. We hypothesized that a downregulation of the cardiac ATRAP to angiotensin II type 1 receptor ratio is involved in the pathogenesis of cardiac hypertrophy. To examine this hypothesis, we next generated transgenic mice expressing ATRAP specifically in cardiomyocytes under control of the ␣-myosin heavy chain promoter. In cardiac-specific ATRAP transgenic mice, the development of cardiac hypertrophy, activation of p38 mitogen-activated protein kinase, and expression of hypertrophy-related genes in the context of angiotensin II treatment were completely suppressed, in spite of there being no significant difference in blood pressure on radiotelemetry between the transgenic mice and littermate control mice. These results demonstrate that cardiomyocyte-specific overexpression of ATRAP in vivo abolishes the cardiac hypertrophy provoked by chronic angiotensin II infusion, thereby suggesting ATRAP to be a novel therapeutic target in cardiac hypertrophy. (Hypertension. 2010;55:1157-1164.)Key Words: basic science Ⅲ receptors Ⅲ gene expression/regulation Ⅲ hypertrophy/remodeling Ⅲ angiotensin receptors E vidence suggests that the activation of angiotensin II (Ang II) type 1 receptor (AT 1 R) through the tissue renin-angiotensin system may play an important role in the development of cardiac hypertrophy. The carboxyl-terminal portion of AT 1 R is involved in the control of AT 1 R internalization independent of G protein coupling, and it plays an important role in linking receptor-mediated signal transduction to the specific biological response to Ang II. 1,2 We previously cloned a novel AT 1 R-associated protein (ATRAP) that specifically interacts with the carboxylterminal domain of AT 1 R. [3][4][5][6] We showed that ATRAP is broadly expressed in many tissues, as is AT 1 R, and suppresses Ang II-mediated pathological responses in cardiomyocytes and vascular smooth muscle cells by promoting the constitutive internalization of AT 1 R. 7-9 However, the function of ATRAP in cardiac hypertrophy in vivo still remains to be demonstrated. Thus, the present study was carried out to investigate whether there is a role for ATRAP in the cardiac hypertrophy induced by chronic Ang II ...