Kazuhiro Iki

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.

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.

Gene expression of 11 beta-hydroxysteroid dehydrogenase in the mesenteric arteries of genetically hypertensive rats.

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) modulates the access of corticosteroids to their receptors and plays an important role in controlling blood pressure. We determined 11 beta-HSD activity and mRNA levels in the mesenteric arteries of genetically hypertensive rats, the Dahl salt-sensitive hypertensive rat, and compared them with Dahl salt-resistant and Sprague-Dawley rats. 11 beta-HSD activity was expressed as the percent conversion of [3H]corticosterone to [3H]11-dehydrocorticosterone. 11 beta-HSD activity was significantly decreased in the mesenteric arteries of 8-week-old Dahl salt-sensitive hypertensive rats (11.4 +/- 1.4%) compared with Dahl salt-resistant rats (17.4 +/- 1.4%) or Sprague-Dawley rats (18.0 +/- 1.5%) of the same age (P < .05). There were no significant differences in 11 beta-HSD activity between 4-week-old Dahl salt-sensitive hypertensive and Dahl salt-resistant rats of the same age (15.3 +/- 1.3% and 15.1 +/- 1.9%, respectively). The concentration of 11 beta-HSD mRNA in the mesenteric arteries of 8-week-old Dahl salt-sensitive hypertensive rats was significantly lower than in Dahl salt-resistant or Sprague-Dawley rats of the same age (P < .05). There were no significant differences in the concentration of 11 beta-HSD mRNA in the mesenteric arteries of 4-week-old Dahl salt-sensitive hypertensive rats, Dahl salt-resistant rats, and Sprague-Dawley rats. These results indicate that 11 beta-HSD in the vascular wall may play a role in the pathogenesis of hypertension in this rat model.

Endogenous Renal 11β-Hydroxysteroid Dehydrogenase Inhibitory Factors in Patients With Low-Renin Essential Hypertension

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) modulates the access of corticosteroids to their receptors and is important in blood pressure control. The excretion of renal 11 beta-HSD (ie, NAD(+)-dependent isoform) is thought to protect renal mineralocorticoid receptors from cortisol. To examine whether endogenous renal 11 beta-HSD inhibitory factor(s) may be involved in the pathophysiology of hypertension, we studied the urinary excretion of such inhibitors in 30 patients with low-renin essential hypertension and 20 normotensive control subjects. The effect of sodium restriction on the urinary excretion of the inhibitors wa also evaluated in six normotensive control subjects. Urine was extracted with Sep-Pak cartridges and high-performance liquid chromatography. Endogenous renal 11 beta-HSD inhibitors were measured by the inhibition of 11 beta-HSD bioactivity in microsomes from the human kidney. The urinary excretion of the inhibitors was significantly increased in patients with low-renin essential hypertension (1280 +/- 88 nmol/d, mean +/- SEM) compared with normotensive control subjects (704 +/- 56 nmol/d) (P < .05). Ratios of urinary tetrahydrocortisol+allo-tetrahydrocortisol to tetrahydrocortisone did not differ significantly. Sodium restriction reduced the urinary excretion of the endogenous renal 11 beta-HSD inhibitors but did not affect the ratio of urinary tetrahydrocortisol+allo-tetrahydrocortisol to tetrahydrocortisone. Endogenous renal 11 beta-HSD inhibitory factors may contribute to the pathogenesis of low-renin essential hypertension by modulating the activity of 11 beta-HSD. Sodium intake may directly or indirectly regulate the inhibitory factors.

The activities of 5β-reductase and 11β-hydroxysteroid dehydrogenase in essential hypertension

The activities of 11β-hydroxysteroid dehydrogenase (11β-HSD) and 5β-reductase were analyzed in 39 normotensive controls and 128 patients with essential hypertension. The activity of 11β-HSD was obtained by dividing the 24-hour urinary tetrahydrocortisone by the sum of tetrahydrocortisol (THF) and allotetrahydrocortisol (aTHF), whereas the activity of 5β-reductase was obtained by dividing the 24-hour urinary THF by aTHF. The activity of 5β-reductase was significantly lower in essential hypertensives compared with normotensive controls (P < 0.05). However, the activity of 11β-HSD did not differ between normotensive controls and essential hypertensives. A positive correlation between the activities of 11β-HSD and 5β-reductase was observed in essential hypersentives (r = 0.60, P < 0.01). Neither 11β-HSD nor 5β-reductase activity correlated with indices of renal mineralocorticoid receptor activation, which were assessed by determination of plasma potassium and urinary excretion of sodium as well as potassium. Taken together, these results suggest that disturbances of one of the inactivation pathways of cortisol may contribute to the pathogenesis of hypertension.

Interleukin-2 enhances the release of endothelin-1 from the rat mesenteric artery

We measured the ET-1 concentration in plasma and in the perfusate of the mesenteric arteries of rats treated with a therapeutic dose of IL-2 for 7 days (100000 U/Kg, iv.). The plasma ET-1 concentration in rats given IL-2 was 14.2 +/- 3.2 pg/ml which was significantly greater than that in the controls (2.5 +/- 0.4 pg/ml, P less than 0.05). The mesenteric arteries also released a significantly greater amount of ET-1 (29.5 +/- 1.6 pg/h) than that in controls (16.8 +/- 2.3 pg/h, P less than 0.01). The arterial blood pressure was significantly lower after IL-2 treatment than the pre-dosing level (P less than 0.05). It is concluded that IL-2 induces ET-1 release from the vascular wall, possibly as a result of reversible endothelial dysfunction caused by IL-2.

Decreased activity of 11β-hydroxysteroid dehydrogenase in mesenteric arteries of Dahl salt-sensitive rats

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) activity in mesenteric arteries of 4-week- and 8-week-old Dahl rats was determined to evaluate the relationship between glucocorticoids and norepinephrine in hypertension, 11 beta-HSD activity (expressed as the percentage conversion of 3H-corticosterone (B) to 3H-11-dehydrocorticosterone (A)) significantly decreased in 8-week-old Dahl salt-sensitive (DS) rats (11.4 +/- 1.4%) compared with Dahl salt-resistant (DR) rats (15.3 +/- 0.3%) (p < 0.05) when each group was fed a high-sodium chow (7%) for 4 weeks. There were no significant differences in 11 beta-HSD activity in the 4-week-old DS and DR rats. The enhanced response of mesenteric arteries to norepinephrine was observed only in 8-week-old DS rats. Our results suggest that a decreased 11 beta-HSD activity in the vascular walls contributes to the increased response of norepinephrine by local increment of glucocorticoids in DS hypertensive rats.

Determination of urinary 18-hydroxycortisol in the diagnosis of primary aldosteronism

Urinary excretion of 18-hydroxycortisol (18-OHF), 18-hydroxycorticosterone (18-OHB) and aldosterone 18-glucuronide (Aldo-18-glu) was measured in 10 patients with primary aldosteronism; 5 with aldosterone-producing adenoma (APA) and 5 with idiopathic hyperaldosteronism (IHA), 10 patients with essential hypertension (EHT) and 11 nor-motensive subjects. In EHT patients, urinary 18-OHF (172 ± 15 μ/24h) and 18-OHB (3.1 ± 0.6 μ/24h) values were not significantly different from 18-OHF (142 ± 35 jig/24h) and 18-OHB (3.6 ± 0.5 μg/24h) in the controls. Urinary 18-OHF values were significantly higher in APA (640 ± 213 μg/24h) when compared with controls and EHT, whereas 18-OHB (11.3 ± 1.5 μ/24h) values were only slightly elevated. Both 18-OHF and 18-OHB were significantly increased in APA compared with 18-OHF (232 ± 56 μg/24h) and 18-OHB (4.6 ± 0.3 μ/24h) in IHA. The two urinary steroids, especially 18-OHF proved to be a useful marker for the diagnosis of APA, confirming the previous findings. Aldo-18-glu was not significantly different between APA and IHA. In normal subjects when sodium intake was restricted to 48meq/day for four days the urinary 18-OHF was increased two fold to 383 ± 59 μ/24h (p < 0.01 vs control period) associated with comparable rise in plasma renin activity. This suggests that the biosynthesis of 18-OHF is partly under control of renin-angiotensin axis in normal subjects.

Effect of aging on urinary excretion of 18-hydroxycortisol

The secretion of aldosterone declines with age. We reported that sodium depletion increased urinary excretion of 18-hydroxycortisol (18-OH-F). To elucidate the effect of aging on urinary excretion of 18-OH-F, we measured urinary 18-OH-F in 30 normotensive subjects aged 20-70 yr. There were significant negative correlations between age and urinary excretion of 18-OH-F or of aldosterone (r = -0.49, r = -0.58, P < 0.05, respectively) but not of urinary free cortisol. These results suggest that angiotensin may contribute to chronic regulation of 18-OH-F.

Effect of aging on urinary excretion of 19-noraldosterone and 18,19-dihydroxycorticosterone

19-Noraldosterone, recently shown to be produced in the human adrenal gland, possesses potent mineralocorticoid and hypertensinogenic activity. A possible precursor, 18,19-dihydroxycorticosterone, has been identified in human urine, with both steroids acutely regulated by the renin-angiotensin system. The secretion of aldosterone declines with advancing age. To elucidate the effect of aging on the urinary excretion of 19-noraldosterone and 18,19-dihydroxycorticosterone, we measured their urinary concentrations in 51 normotensive subjects aged 20-70 years. We observed significant negative correlations between age and the urinary excretion of 19-noraldosterone and 18,19-dihydroxycorticosterone (r = -0.69, r = -0.65, P < 0.05, respectively). Urinary and plasma aldosterone and PRA similarly decreased with aging. These results suggest that 19-noraldosterone may be chronically regulated in part by the renin-angiotensin system.