Yoshiyu Takeda

Vascular aldosterone. Biosynthesis and a link to angiotensin II-induced hypertrophy of vascular smooth muscle cells.

Mineralocorticoids have been suggested to act on blood vessels, leading to increased vasoreactivity and peripheral resistance. However, the site of their production has so far been believed to be only the adrenal cortex. Here, we show direct evidence that vascular cells per se are aldosteronogenic, possessing their own system that responds to the steroid. Using polymerase chain reaction after reverse transcription, the CYP11B2 mRNA encoding the key enzyme for the biosynthesis of aldosterone was detected in both endothelial cells and smooth muscle cells cultivated from human pulmonary artery. The aldosterone receptor (type 1 mineralocorticoid receptor) gene was also found to be expressed in smooth muscle cells and, to a lesser extent, in endothelial cells. CYP11B2 gene expression in smooth muscle cells was stimulated by angiotensin II, the effector peptide of the renin-angiotensin system. Furthermore, the angiotensin II-induced increase in [3H]leucine incorporation in smooth muscle cells was significantly enhanced by aldosterone but inhibited by ZK 91587, a type 1 mineralocorticoid receptor antagonist. This may indicate that vascular aldosterone participates in the angiotensin II-induced hypertrophy of vascular smooth muscle cells. The present study therefore provides the starting point for a novel understanding of the molecular basis of vascular remodeling and hypertension.

Production of Aldosterone in Isolated Rat Blood Vessels

Angiotensin I (Ang I), Ang II, angiotensinogen, and renin are formed locally in the vasculature. We undertook this study to determine whether the rat mesenteric artery produces aldosterone and to investigate the effects of adrenalectomy, an angiotensin-converting enzyme inhibitor, Ang II, or potassium on aldosterone production in vascular tissue. Isolated rat mesenteric arteries were perfused with Krebs-Ringer solution for 4 hours. The perfusate was collected and chromatographed in a reversed-phase high-performance liquid chromatographic (HPLC) system. The fraction corresponding to synthetic aldosterone was collected and analyzed by mass spectrometry. The aldosterone concentration in the perfusate from the adrenalectomized rats and rats treated with an angiotensin-converting enzyme inhibitor was measured using radioimmunoassay after HPLC separation. The mass spectra of synthetic aldosterone and aldosterone isolated from the perfusate of rat mesenteric arteries were identical. Aldosterone production in the mesenteric arteries of adrenalectomized rats was increased and of rats treated with an angiotensin-converting enzyme inhibitor was reduced compared with that of controls. Ang II (1.9 x 10(10) mol/L) and potassium (6.0 mmol/L) increased aldosterone production in mesenteric arteries. This study shows that the rat mesenteric artery produces aldosterone and that the intravascular renin-angiotensin-aldosterone system may contribute to vascular tone.

Cardiac Aldosterone Production in Genetically Hypertensive Rats

Aldosterone is synthesized in extra-adrenal tissues, both blood vessels and brain. We undertook the present study to determine whether the rat heart produces aldosterone and to investigate the effects of adrenalectomy, ACE inhibition, and angiotensin II on aldosterone synthesis in the heart. To clarify the pathophysiological role of cardiac aldosterone in the hypertensive heart, we compared the synthesis of aldosterone in the hearts of stroke-prone spontaneously hypertensive rats (SHRSP) with that in Wistar-Kyoto rats. The effects of the aldosterone antagonist spironolactone on myocardial hypertrophy in adrenalectomized SHRSP were also studied. Isolated rat hearts were perfused for 2 hours, and the perfusate was analyzed with HPLC and mass spectrometry. The activity of aldosterone synthase was estimated on the basis of the conversion of [14C]deoxycorticosterone to [14C]aldosterone. The levels of aldosterone synthase gene (CYP11B2) mRNA were determined with competitive polymerase chain reaction. Aldosterone production, the activity of aldosterone synthase, and the expression of CYP11B2 mRNA were increased in hearts from adrenalectomized rats and rats treated with angiotensin II. ACE inhibitors decreased cardiac aldosterone synthesis. Cardiac aldosterone, aldosterone synthase activity, and CYP11B2 mRNA levels in hearts from 2- and 4-week-old SHRSP were significantly greater than those of age-matched Wistar-Kyoto rats. Spironolactone prevented cardiac hypertrophy in adrenalectomized SHRSP. These results suggest that the rat heart produces aldosterone and that endogenous cardiac aldosterone may affect cardiac function and hypertrophy in hypertension in rats.

Vascular Aldosterone in Genetically Hypertensive Rats

We have reported that aldosterone is synthesized and cytochrome P450aldo mRNA exists in the vasculature. To clarify the pathophysiological role of vascular aldosterone in hypertension, we compared aldosterone production in the mesenteric arteries of stroke-prone spontaneously hypertensive rats (SHRSP) with that in Wistar-Kyoto rats (WKY). The expressions of mRNA of cytochrome P450aldo, mineralocorticoid receptor, and alpha 1, Na,K-ATPase in the mesenteric arteries were compared between the two groups. Aldosterone concentration in the perfusate of the vasculature was measured by radioimmunoassay after purification with high-performance liquid chromatography. Cytochrome P450aldo and mineralocorticoid receptor mRNA levels were quantified by Southern blot analysis of the products of reverse-transcribed polymerase chain reaction. Levels of alpha 1 Na,K-ATPase mRNA were measured by Northern blot analysis. Vascular aldosterone and cytochrome P450aldo mRNA levels of 2-week-old SHRSP were significantly increased compared with those of age-matched WKY. However, vascular aldosterone in 4- and 9-week-old SHRSP did not differ from that in age-matched WKY. Expression levels of mineralocorticoid receptor mRNA in the vasculature of 4- and 9-week-old SHRSP were significantly increased compared with those in age-matched WKY. Concentrations of vascular alpha 1 Na,K-ATPase mRNA of 2-, 4-, and 9-week-old SHRSP also were significantly higher than those in age-matched WKY. These results suggest that vascular aldosterone contributes to the pathophysiology of hypertension in SHRSP in the early stage.

Production of endothelin-1 from the mesenteric arteries of streptozotocin-induced diabetic rats

Release of endothelin-1 (ET-1) from the mesenteric arteries of Wistar rats with streptozotocin-induced diabetes (STZ-DM) rats and nondiabetic rats was measured by a specific enzyme immunoassay following purification using an immunoaffinity column. The mesenteric arteries from STZ-DM rats released a significantly higher amount of ET-1 as compared to control rats (35.8 +/- 2.8 vs 14.9 +/- 2.0 pg/1hr, p less than 0.05). The plasma level of ET-1 in STZ-DM rats was also elevated to a significant extent as compared to controls (5.1 +/- 0.4 vs 3.0 +/- 0.4 pg/ml, p less than 0.05). The systolic blood pressure of STZ-DM rats was significantly higher than of the controls (p less than 0.05). The increased level of plasma ET-1 as well as its release from the mesenteric artery of STZ-DM rats may suggest its release following damage to the endothelium caused by diabetes and/or by associated changes in blood pressure.

Effects of high sodium intake on cardiovascular aldosterone synthesis in stroke-prone spontaneously hypertensive rats.

Objectives Aldosterone is synthesized in extra-adrenal tissues such as the vasculature, heart and brain. The mechanisms underlying the effect of high salt intake on the development and acceleration of vascular injury and cardiac hypertrophy in the stroke-prone spontaneously hypertensive rats (SHRSP) are still not clear. The goal of this study was to determine whether high salt intake increases cardiovascular aldosterone synthesis in SHRSP. Methods Four-week-old SHRSP were given tap water or 0.9% NaCl solution for hydration for 4 weeks in addition to a normal salt diet. Isolated rat mesenteric arteries and hearts were perfused for 2 h, and the perfusate was analysed by high-performance liquid chromatography. The concentrations of aldosterone synthase gene (CYP11B2) mRNA and angiotensin II receptor (AT1R) mRNA were determined by competitive polymerase chain reaction. Results Salt-loaded SHRSP had higher blood pressures than SHRSP with normal salt intake. Plasma aldosterone concentrations and plasma renin activity were decreased by high salt intake. Aldosterone production, the expression of CYP11B2 mRNA and AT1R mRNA in mesenteric arteries and hearts were significantly increased by high salt intake. Conclusions These results suggest that high salt intake increases aldosterone production and expression of the AT1R mRNA in the cardiovascular tissue in SHRSP, which may contribute to the development of malignant hypertension in salt-loaded SHRSP.

Aldosterone biosynthesis and action in vascular cells

In view of the hypothetical possibility that the vascular renin-angiotensin system (RAS) might include aldosterone biosynthesis and action in the vasculature, we have undertaken a study to identify aldosterone released into the perfusion circuit from the rat mesenteric artery, and to investigate the effects of an angiotensin converting enzyme inhibition (ACEI) on aldosterone production from the vasculature. After 30 min equilibration, 240 mL of perfusate was collected and subjected to reverse-phase HPLC and subsequent mass spectrometry. Mass spectra corresponding to authentic corticosterone and aldosterone were obtained from the samples of mesenteric artery perfusate. Production of aldosterone in the mesenteric artery was not changed by adrenalectomy, although it was reduced in the arterial perfusate from rats pretreated with ACEI. By RT-PCR the expression of CYP 11B2 and mineralocorticoid receptor genes were demonstrated in both vascular endothelial and smooth muscle cells. These studies constitute indirect evidence supporting our hypothesis that locally produced aldosterone in the vascular tissue acts on vascular tone and remodeling via a paracrine or autocrine manner.

Impacts of Visceral Adipose Tissue and Subcutaneous Adipose Tissue on Metabolic Risk Factors in Middle‐aged Japanese

Regional fat distribution rather than overall fat volume has been considered to be important to understanding the link between obesity and metabolic disorders. We aimed to evaluate the independent associations of visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) with metabolic risk factors in apparently healthy middle‐aged Japanese. Participants were 1,119 men and 854 women aged 38–60 years who were not taking medications for diabetes, hypertension, or dyslipidemia. VAT and SAT were measured by use of computed tomography (CT) scanning. VAT and SAT were significantly and positively correlated with each other in men (r = 0.531, P < 0.001) and women (r = 0.589, P < 0.001). In multiple regression analyses, either measure of abdominal adiposity (VAT or SAT) was positively associated with blood pressure, fasting plasma glucose, and log triglyceride (P < 0.001) and inversely with high‐density lipoprotein (HDL)‐cholesterol (P < 0.001). When VAT and SAT were simultaneously included in the model, the association of VAT with triglycerides was maintained (P < 0.001) but that of SAT was lost. The same was true for HDL‐cholesterol in women. For fasting plasma glucose, the association with VAT was strong (P < 0.001) and the borderline association with SAT was maintained (P = 0.060 in men and P = 0.020 in women). Both VAT and SAT were independently associated with blood pressure (P < 0.001). Further adjustment for anthropometric indices resulted in the independent association only with VAT for all risk factors. In conclusion, impacts of VAT and SAT differed among risk factors. VAT showed dominant impacts on triglyceride concentrations in both genders and on HDL‐cholesterol in women, while SAT also had an independent association with blood pressure.

Comparison of waist circumference with body mass index for predicting abdominal adipose tissue

AIMS To compare waist circumference (WC) with body mass index (BMI) for the prediction of abdominal adipose tissues in Japanese men and women. METHODS 1432 men and 1038 women aged 38-60 years were recruited. WC, BMI, visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) using CT scans were measured. RESULTS Women had a lower mean VAT than men (79.3 cm(2) vs. 132.3 cm(2); p<0.001) and a higher mean SAT (196.2 cm(2) vs. 139.7 cm(2); p<0.001). The correlation with WC or BMI was greatest for total adipose tissue (TAT), followed by SAT, and least for VAT. The correlation coefficients were not significantly different between WC and BMI for any adipose tissue except for VAT in men (p<0.05). Age was positively correlated with VAT in both genders (p<0.001). Using multiple regression analyses on VAT, R(2) values using WC and age were 0.45 in men and 0.48 in women. For SAT, the values were 0.57 in men and 0.59 in women. CONCLUSIONS The relationship with WC or BMI was greatest for TAT and SAT, and least for VAT. WC and BMI provided essentially similar estimates of TAT, VAT, and SAT in both genders.

Mechanisms of FK 506–Induced Hypertension in the Rat

-Tacrolimus (FK 506) is a powerful, widely used immunosuppressant. The clinical utility of FK 506 is complicated by substantial hypertension and nephrotoxicity. To clarify the mechanisms of FK 506-induced hypertension, we studied the chronic effects of FK 506 on the synthesis of endothelin-1 (ET-1), the expression of mRNA of ET-1 and endothelin-converting enzyme-1 (ECE-1), the endothelial nitric oxide synthase (eNOS) activity, and the expression of mRNA of eNOS and C-type natriuretic peptide (CNP) in rat blood vessels. In addition, the effect of the specific endothelin type A receptor antagonist FR 139317 on FK 506-induced hypertension in rats was studied. FK 506, 5 mg. kg-1. d-1 given for 4 weeks, elevated blood pressure from 102+/-13 to 152+/-15 mm Hg and increased the synthesis of ET-1 and the levels of ET-1 mRNA in the mesenteric artery (240% and 230%, respectively). Little change was observed in the expression of ECE-1 mRNA and CNP mRNA. FK 506 decreased eNOS activity and the levels of eNOS mRNA in the aorta (48% and 55%, respectively). The administration of FR 139317 (10 mg. kg-1. d-1) prevented FK 506-induced hypertension in rats. These results indicate that FK 506 may increase blood pressure not only by increasing ET-1 production but also by decreasing NO synthesis in the vasculature.