Increasing evidence suggests that angiotensin II (AngHI) acts as a modulator for ventricular remodeling after myocardial infarction. Using competitive reverse-transcriptase polymerase chain reaction, nuclear runoff, and binding assays, we examined the regulation of AngHI type la and lb (ATla-R and ATlb-R) and type 2 receptor (AT2-R) expression in the infarcted rat heart as well as the effects of AngiH receptor antagonists. ATla-R mRNA levels were increased in the infarcted (4.2-fold) and noninfarcted portions (2.2-fold) of the myocardium 7 d after myocardial infarction as compared with those in sham-operated controls, whereas ATlb-R mRNA levels were unchanged. The amount of detectable AT2-R mRNA increased in infarcted (3.1-fold) and noninfarcted (1.9-fold) portions relative to that in the control. The transcription rates for ATla-R and AT2-R genes, determined by means of a nuclear runoff assay, were significantly increased in the infarcted heart. The Angil receptor numbers were elevated (from 12 to 35 fmol/mg protein) in the infarcted myocardium in which the increases in AT1-R and AT2-R were 3.2-and 2.3-fold, respectively, while the receptor affinity was unchanged. Therapy with AT1-R antagonist for 7 d reduced the increase in AT1-R and AT2-R expressions in the infarcted heart together with a decrease in blood pressure, whereas therapy with an AT2-R antagonist did not affect mRNA levels and blood pressure. Neither ATi-R nor AT2-R antagonists affected the infarct sizes. These results demonstrated that myocardial infarction causes an increase in the gene transcription and protein expression of cardiac ATla-R and AT2-R, whereas the ATlb-R gene is unaffected, and that therapy with an AT1-R antagonist, but not with an AT2-R antagonist, is effective in reducing the increased expression of AngII receptor subtypes induced by myocardial infarction. (J. Clin. Invest. 1995.95:46-54.)

Although both rat cardiac nonmyocytes (mostly fibroblasts) and cardiomyocytes have a functional angiotensin II (AngII) receptor, the regulation mechanism of its subtype expression in the rat heart remains unknown. In this study, by using a binding assay and a competitive reverse-transcriptase polymerase chain reaction, we examined the regulation of AngII types la and lb (ATla-R and AT1b-R) and type 2 receptor (AT2-R) expression in embryonal day 19 (E19) and neonatal (1-d) rat cardiac fibroblasts and cardiomyocytes. The number of AT2-R in E19 fibroblasts was dramatically decreased (from 305 to 41 fmol/mg protein) in 1-d fibroblasts, whereas that of AT1-R and the mRNA levels remained unchanged. The ratio of ATla-R to AT1b-R mRNA in both E19 and 1.4 fibroblasts was 9:1. The number of AT2-R in E19 cardiomyocytes was also significantly decreased (from 178 to 87 fmol/mg protein) in 1-d cardiomyocytes, whereas the magnitude was less prominent compared with that in fibroblasts. AT1-R expression remained unaltered in E19 and 1-d cardiomyocytes. In E19 and l-d cardiomyocytes, the ATlb-R mRNA level was 1.5-fold higher than that of AT1a-R mRNA. Dexamethasone induced significant increases in AT1a-R mRNA (2.1-fold) and numbers (1.8-fold) without changing the affinity, whereas neither AT1b-R mRNA nor the number of AT2-R was affected by dexamethasone. The ATla-R gene transcription rate, determined by means of a nuclear run-off assay, was increased (2-fold) by dexamethasone. The half-life of ATla-R mRNA (18 h) was unchanged by dexamethasone. These data indicate that AngI1 receptor subtype expression in the rat heart is regulated in a cell-and subtype-specific manner. (J. Clin. Invest. 1994.

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We examined the relationship between flow-mediated dilation (FMD) of the brachial artery and the extent and severity of coronary artery disease (CAD). Using high-resolution ultrasonography, we measured FMD and nitroglycerin-induced brachial artery dilation. We studied 121 patients (77 men, 44 women; mean age 64+/-11 years, range 25-79 years) who underwent coronary arteriography. The extent and severity of CAD were assessed by the coronary stenosis index (CSI). The adjusted FMD correlated inversely with CSI (rs=-0.63, p<0.0001). Multivariate analysis demonstrated that the adjusted FMD was an independent predictor of CSI. The adjusted FMD was 10.2+/-4.8% in patients without CAD (n=32), 7.7+/-6.0% in patients with single-vessel disease (n=31), 5.2+/-5.5% in patients with double-vessel disease (n=29), and 2.0+/-3.9% in patients with triple-vessel disease (n=29). The adjusted FMD was significantly lower in the double- (p<0.01) and triple-vessel (p<0.0001) disease groups than in patients without CAD. The adjusted FMD was significantly lower in the triple-vessel disease group than in the single-vessel disease group (p<0.001). Based on our results, as coronary atherosclerosis becomes more severe, the adjusted brachial artery FMD becomes more severely impaired.

To elucidate the molecular mechanism of the stimulatory effect of thyrotropin on the gene regulation of alB adrenergic receptor in functioning rat thyroid (FRTL-5) cells, we established a competitive reverse-transcriptase (RT) polymerase chain reaction (PCR) and nuclear run-off assay to quantify changes in mRNA levels and transcription rates. A binding assay showed that FRTL-5 cells predominantly expressed alB adrenergic receptor and that thyrotropin increased its expression sevenfold. By means of RT-PCR, we found that thyrotropin induced an 11-fold increase in alB receptor mRNA abundance. The nuclear run-off assay demonstrated that thyrotropin caused a ninefold increase at the gene transcriptional level, which occurred in the presence of the protein synthesis inhibitor cycloheximide. The half-life of the alB receptor mRNA in cells incubated with thyrotropin for 1 h increased 1.5-fold but returned to the original value after 12 h. Dibutyryl cAMP and forskolin mimicked the stimulatory effects of thyrotropin on the gene transcriptional level. The 5 '-flanking region of the rat alB receptor gene contained a putative cAMP responsive element (CRE) at nucleotide -438 relative to the translation start site. The promoter analysis using the reporter gene indicated that the CRE motif confers the cAMP sensitivity to the transcription of the rat alB receptor gene. These results demonstrated that a CRE-mediated mechanism is involved in the transcriptional regulation of the alB receptor gene by thyrotropin without requiring new protein synthesis. (J. Clin. Invest. 1994. 94:2245-2254