Role of multidrug resistance P-glycoprotein in the secretion of aldosterone by human adrenal NCI-H295 cells.

We determined the role of the multidrug resistance (MDR1) gene product, P-glycoprotein (PGP), in the secretion of aldosterone by the adrenal cell line NCI-H295. Aldosterone secretion is significantly decreased by the PGP inhibitors verapamil, cyclosporin A (CSA), PSC-833, and vinblastine. Aldosterone inhibits the efflux of the PGP substrate rhodamine 123 from NCI-H295 cells and from human mesangial cells (expressing PGP). CSA, verapamil, and the monoclonal antibody UIC2 significantly decreased the efflux of fluorescein-labeled (FL)-aldosterone microinjected into NCI-H295 cells. In MCF-7/VP cells, expressing multidrug resistance-associated protein (MRP) but not PGP, and in the parental cell line MCF7 (expressing no MRP and no PGP), the efflux of microinjected FL-aldosterone was slow. In BC19/3 cells (MCF7 cells transfected with MDR1), the efflux of FL-aldosterone was rapid and it was inhibited by verapamil, indicating that transfection with MDR1 cDNA confers the ability to transport FL-aldosterone. These results strongly indicate that PGP plays a role in the secretion of aldosterone by NCI-H295 cells and in other cells expressing MDR1, including normal adrenal cells.

Primary aldosteronism.

Primary aldosteronism occurs much more commonly than generally appreciated. Diagnosis is facilitated by measuring urinary aldosterone excretion in conjunction with determination of the level of plasma aldosterone suppression after infusion of 2,000 mL of normal saline over 4 hours. Multiple diagnostic procedures are required to determine the specific type of primary aldosteronism. Patients with aldosterone-producing tumors, unilateral adrenal hyperplasia, and primary adrenal hyperplasia are treated surgically, whereas those with bilateral zone glomerulosa hyperplasia and glucocorticoid-remediable aldosteronism are treated medically.

Aldosterone synthase gene regulation by angiotensin.

Excessive aldosterone secretion in some hypertensive patients may result from abnormal aldosterone synthase (AS) gene regulation in response to changes in dietary sodium intake. We have utilized NCI-H295 cells, which exhibit stable angiotensin-induced aldosterone secretion, for transient transfections with murine AS/human growth hormone reporter constructs. An angiotensin response element increasing AS gene transcription during angiotensin stimulation appears to reside within the initial 425 nt of the murine AS promoter. We also noted the possible presence of a negatively-acting cis element between nt -425 and -1500. These studies provide an initial step toward characterizing molecular mechanisms by which angiotensin regulates AS gene transcription.

Angiotensin increases aldosterone synthase mRNA levels in human NCI-H295 cells.

Understanding the regulation of aldosterone secretion has been hampered by the lack of a cell culture system that remains chronically responsive to angiotensin stimulation. NCI-H295 cells, cultured from a human adrenocortical tumor, express the three major pathways of adrenal steroidogenesis and produce small amounts of aldosterone during basal culture. We have determined changes in aldosterone production and in aldosterone synthase (AS, P45011B2) mRNA levels in these cells in response to angiotensin II (AII) and forskolin. Culture of NCI-H295 cells with 10(-7) M AII or with 10(-5) M forskolin stimulated aldosterone production and increased AS mRNA levels, though the effect of AII was greater. When cells were cultured with increasing concentrations of AII from 10(-11) through 10(-8) M, a dose-dependent increase in AS mRNA levels paralleled increases in aldosterone production. In view of these findings, these human adrenocortical cells should be useful for exploring mechanisms regulating aldosterone production.

Modulation of aldosterone synthase messenger ribonucleic acid levels by dietary sodium and potassium and by adrenocorticotropin.

Recent evidence suggests that an aldosterone synthase (AS) separate from the 11 beta-hydroxylase (11 beta-OHase) mediates the final step(s) in aldosterone synthesis in the rat. We have compared changes in AS and 11 beta-OHase mRNA levels with experimental maneuvers known to stimulate or suppress aldosterone secretion. In Exp 1, male rats were fed regular rat chow (group 1), a low sodium, high potassium diet (group 2), or a high sodium, low potassium diet (group 3). Northern analysis of adrenal capsular (zona glomerulosa) and decapsulated adrenal core (fasciculata-reticularis) tissues was performed with specific oligonucleotide probes for AS and 11 beta-OHase mRNAs, normalized with a cDNA probe for 18S ribosomal RNA (rRNA). There was a marked increase in capsular AS mRNA in group 2 rats compared to levels in group 1 (P < 0.0001) and group 3 (P < 0.0001) rats. Capsular As mRNA decreased (P < 0.05) in group 3 compared to that in group 1 rats. Adrenal core AS mRNA levels were quite low with all three diets. In contrast, capsular and core 11 beta-OHase mRNA levels did not change significantly with diet. In Exp 2, two groups served as controls (groups 1 and 3), and two groups received sc injections of repository ACTH (groups 2 and 4). Control and ACTH-treated rats were killed 3 h (groups 1 and 2) or 24 h (groups 3 and 4) after initial ACTH administration. Capsular and core AS and 11 beta-OHase mRNA levels were evaluated with and without normalization with 18S rRNA, cyclophilin, and glyceraldehyde phosphate dehydrogenase housekeeping probes. Capsular AS mRNA increased at 3 h (P < 0.05), but decreased at 24 h (P < 0.01). Nonnormalized adrenal capsular and core 11 beta-OHase mRNA levels were unchanged at 3 h, but increased significantly at 24 h (P < 0.05) in capsular tissue of ACTH-treated rats. However, in ACTH-treated rats, 18S rRNA and cyclophilin mRNA levels increased at 24 h in both capsular and core tissue (P < 0.01), and glyceraldehyde phosphate dehydrogenase mRNA levels increased in capsular tissue (P < 0.00001), resulting in a lack of change in normalized 11 beta-OHase mRNA. Changes in cholesterol side-chain cleavage were similar to those in 11 beta-OHase mRNA. These data provide further evidence that a separate AS plays a significant role in modulating aldosterone secretion in rodents.

Systemic and regional hemodynamic effects of 1,25-dihydroxyvitamin D3 administration.

OBJECTIVE: To evaluate the cardiovascular effects of 1,25-dihydroxyvitamin D3 (1,25-D). DESIGN: Recent studies suggest that Ca-regulating hormones may contribute to the genesis of hypertension. We determined systemic and regional hemodynamics 24 h after administration of 1,25-D or vehicle to normal conscious Sprague-Dawley rats. In addition, to dissociate the vascular effects of 1,25-D from changes in serum ionized Ca2+, 1,25-D and vehicle were administered to rats maintained for 3 days on a low-Ca diet. To evaluate the effect of the slight rise in serum ionized Ca2+ with 1,25-D administration, we infused CaCl or vehicle over 1 h into normal rats to raise the serum Ca2+ to near that of rats treated with 1,25-D. METHODS: The radioactive microsphere technique was used. RESULTS: Systemic hemodynamics (blood pressure, heart rate, cardiac output, total peripheral resistance and stroke volume) did not differ between the two groups receiving a normal-Ca diet. In these rats 1,25-D significantly decreased renal blood flow (RBF), increased renal vascular resistance (RVR) and slightly increased serum ionized Ca2+. Similarly, in rats receiving a low-Ca diet, 1,25-D administration decreased renal blood flow, increased renal vascular resistance and caused only a minimal increase in serum ionized Ca2+. A low-Ca diet also increased heart rate, cardiac blood flow and renal blood flow. Although CaCl infusion raised serum ionized Ca2+, blood pressure, renal blood flow and renal vascular resistance did not change significantly. CONCLUSION: 1,25-D may constrict the renal vasculature directly or indirectly by enhancing the vascular sensitivity to circulating vasoconstrictors.

Drug-induced hypokalaemia. A cause for concern.

Drug-induced hypokalaemia is a widespread problem in the elderly that can be caused by many therapeutically useful substances, the most common of which are diuretics. In certain classes of patients (e.g. those with acute myocardial infarction, with congestive heart failure receiving digitalis, or with cirrhosis), iatrogenic hypokalaemia is an established risk factor. In patients with hypertension who have no underlying heart disease or liver disease, the use of diuretics may lead to worsened glucose tolerance and cardiac arrhythmias. There is also evidence for an increased risk of sudden cardiac death.

Decreased spinal cord content of calcitonin gene-related peptide in the spontaneously hypertensive rat.

Calcitonin gene-related peptide (CGRP), produced by alternative processing of the primary transcript of the calcitonin gene, is a potent vasodilator. We have shown that dietary calcium deficiency accompanied by decreased serum ionized calcium significantly decreases the neuronal content of CGRP in laminae I and II of the dorsal horn of the spinal cord in the growing rat. The spontaneously hypertensive rat (SHR) is characterized by decreased serum ionized calcium levels and is thought to most closely resemble human essential hypertension. To determine if the neuronal content of CGRP is decreased in the SHR compared to the Wistar-Kyoto (WKY) parent strain, CGRP was localized immunocytochemically in the dorsal horn of the spinal cord. The density of immunocytochemical staining was quantitated by computer-assisted image processing of laminae I and II of the upper thoracic spinal cord of 12-14 week old male SHR (n = 4) and WKY (n = 4) normotensive, control rats. The SHR had significantly decreased neuronal CGRP content compared to the WKY rats (107 +/- 5 vs 121 +/- 6 arbitrary units, P less than 0.01). In contrast, the neuronal density of substance P (SP), which frequently co-exists with CGRP in this neuronal population, was not different between the two groups (SHR, 91 +/- 6 (n = 4) vs WKY, 88 +/- 3 arbitrary units (n = 4)).

Effects of calcium channel blockade on calcium homeostasis in mild to moderate essential hypertension.

Calcium channel blockers may alter parathyroid hormone secretion in vitro, which would alter calcium homeostasis. To determine the chronic effect of calcium channel blockade in vivo, we conducted a randomized, double blind, 16 week study comparing the effects of two pharmacologic antihypertensive agents, the calcium channel blocker diltiazem and the angiotensin-converting enzyme inhibitor captopril on parameters of calcium homeostasis. Both diltiazem and captopril lowered blood pressure to a similar degree. Neither drug produced any significant change in blood levels of total and ionized calcium, magnesium, or phosphorus, which affect the regulation of parathyroid hormone and vitamin D. In addition, at eight or 16 weeks following initiation, neither drug altered the serum levels of parathyroid hormone (PTH) or 1,25-(OH)2-vitamin D3 (1,25-D). Chronic calcium channel blockade with diltiazem does not alter serum parameters of calcium homeostasis and, thus, should not affect bone mineralization.

Humoral factors determining the blood pressure response to converting enzyme inhibition and calcium channel blockade.

Renin and catecholamine levels were determined in patients with mild to moderate hypertension before and after treatment with sustained release diltiazem or captopril and were correlated with the blood pressure response to these antihypertensives. Eight weeks of treatment with either agent led to equal decreases in both systolic and diastolic blood pressure. Pretreatment plasma renin activity (PRA) and plasma norepinephrine did not predict the blood pressure response to either agent. Diltiazem significantly increased both PRA and supine norepinephrine levels. However, in the diltiazem treated patients, there was no correlation between the change in plasma norepinephrine and the change in systolic or diastolic blood pressure. In contrast, there was a negative correlation (P less than .05) between the reactive rise in PRA and the decrease in systolic blood pressure. Thus, the antihypertensive response to a calcium channel blocker may be determined, in part, by the reactive response of pressor systems.

Combination of converting enzyme inhibitor with diuretic for the treatment of hypertension.

In recent years there has been increased discussion about goals of antihypertensive therapy other than blood pressure reduction. The development of angiotensin-converting enzyme inhibitors has provided a class of drugs with a very low side-effect profile. However, single-drug therapy is effective in only about half of hypertensive patients. In the past, diuretics have traditionally been used as the initial or second antihypertensive. Increasingly, diuretic therapy is being avoided, and other antihypertensive combinations are being used. In controlled trials, combination converting enzyme inhibitor-diuretic therapy is effective in about 85% of patients. This synergistic combination allows the diuretic dose to be reduced so that the adverse effects and metabolic complications are minimized. At this time, the combination of converting enzyme inhibitor and diuretic provides an ideal choice in terms of efficacy, compliance, side effects, and cost.

Effect of dietary calcium supplementation on blood pressure and calciotropic hormones in mineralocorticoid-salt hypertension.

In order to determine the effect of dietary calcium supplementation on blood pressure and calciotropic hormones, we studied two groups (n = 12 each) of mineralocorticoid [deoxycorticosterone (DOC)]-salt hypertensive rats, one receiving a normal-calcium diet (0.6% calcium, as calcium carbonate) and the other a high-calcium diet (2.5% calcium), over an 8-week period. Dietary calcium supplementation significantly attenuated the rise in blood pressure. Serum ionized calcium concentrations were significantly decreased from baseline levels in both groups but tended to be higher among the calcium-supplemented rats. Serum concentrations of parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 (1,25-D) were significantly higher in the DOC-salt rats than in normotensive rats fed normal rat chow [PTH: 49 +/- 4 versus 15 +/- 0.9 pg/ml (means +/- s.e.m.); 1,25-D: 108 +/- 7 versus 73 +/- 13 pg/ml, in DOC-salt and normotensive rats, respectively]. In the DOC-salt rats, dietary calcium supplementation did not significantly lower the elevated serum concentration of PTH (from 49 +/- 4 to 40 +/- 4 pg/ml; NS), but did significantly reduce that of 1,25-D (from 108 +/- 7 to 66 +/- 8 pg/ml; P less than 0.01). Since 1,25-D may increase vascular smooth muscle calcium uptake, dietary calcium supplementation may lower blood pressure in DOC-salt hypertension, in part, by suppressing 1,25-D.

Importance of blood glucose concentration in regulating lipolysis during fasting in humans.

The importance of the decline in blood glucose concentration on lipolysis and the lipolytic effect of epinephrine was evaluated during short-term fasting. Lipolytic rates were determined by infusing [2H5]glycerol and [1-13C]palmitic acid. Five volunteers were studied after 12 h of fasting before and during epinephrine infusion and after 84 h of fasting, before and during glucose infusion when plasma glucose was restored to postabsorptive values, and during glucose plus epinephrine infusion. In another protocol, five volunteers were given glucose intravenously throughout fasting to maintain plasma glucose at postabsorptive levels and isotopic studies were performed after 12 and 84 h of fasting before and during epinephrine infusion. Glucose infusion after 84 h of fasting restored glucose and insulin concentrations and lipolytic rates toward 12-h fasting values. When euglycemia was maintained throughout fasting, plasma insulin still declined (P less than 0.05) and lipolytic rates still increased (P less than 0.05). Despite similar glucose concentrations, the lipolytic response to epinephrine infusion was greater after 84 h than after 12 h of fasting in both protocols (P less than 0.05). These studies demonstrate that the decline in plasma glucose contributes to, but is not required for, the increase in lipolysis during fasting. The increase in epinephrine-stimulated lipolysis that occurs during fasting is not dependent on a decrease in plasma glucose concentration.

Effect of short- and long-term beta-adrenergic blockade on lipolysis during fasting in humans.

Stable isotope tracers and indirect calorimetry were used to evaluate the importance of beta-adrenergic stimulation of lipolysis and triglyceride-fatty acid cycling during fasting in healthy human volunteers. Each subject was studied after 12 and 84 h of fasting both with and without propranolol infusion (protocol 1) and when oral propranolol treatment was given throughout fasting (protocol 2). In protocol 1, the rates of appearance of glycerol and palmitic acid increased from 3.04 +/- 0.19 and 1.78 +/- 0.17 mumol.kg lean body mass-1.min-1, respectively, after 12 h of fasting to 5.28 +/- 0.31 and 3.47 +/- 0.15 mumol.kg lean body mass-1.min-1, respectively, after 84 h of fasting (P less than 0.005). The rate of triglyceride-fatty acid cycling increased from 97 +/- 8 to 169 +/- 5 mumol/min (P less than 0.005). Intravenous propranolol infusion decreased the rate of lipolysis after both 12 and 84 h of fasting, but the magnitude of the antilipolytic effect was much greater after 84 h (P less than 0.005). In protocol 2, the rate of lipolysis and triglyceride-fatty acid cycling was still increased by fasting despite beta-adrenergic blockade with oral propranolol. This study demonstrates that beta-adrenergic stimulation contributes to the mobilization of fat during fasting. However, other mechanism(s) can increase lipolysis and triglyceride-fatty acid cycling when beta-adrenergic receptors are continuously blocked.

18-oxocortisol: effect of dexamethasone, ACTH and sodium restriction.

The urinary excretion of 18-oxocortisol in 37 normal subjects consuming a normal sodium diet was 1.2 +/- 0.9(SD) microgram/24 h. Dexamethasone administration to 5 normal individuals suppressed the excretion of 18-oxocortisol from 1.16 +/- 0.5 micrograms/24 h to 0.6 +/- 0.2 micrograms/24 h. While they still received dexamethasone, ACTH administration raised the 18-oxo-cortisol excretion to 3.82 +/- 1.2 micrograms/24 h. Seven normal subjects were placed on a sodium restricted diet, and the urinary excretion of 18-oxocortisol rose from 1.5 +/- 1.21 micrograms/24 h to 8.54 +/- 5.08 micrograms/24 h and aldosterone from 6.6 +/- 2.0 micrograms/24 h to 39.7 +/- 14.6 micrograms/24 h. Two of the seven individuals showed minimal increases in the excretion of 18-oxocortisol, but in all cases aldosterone increased with sodium restriction. The urinary excretion of 18-oxocortisol correlated significantly with the excretion of aldosterone, 18-hydroxycortisol, cortisol, and 19-nordeoxycorticosterone. These studies indicate that 18-oxocortisol secretion is under ACTH regulation, but since sodium restriction also increases the excretion of 18-oxocortisol, the renin-angiotensin system must also participate in its regulation. However, some individuals do not increase their excretion of 18-oxocortisol with sodium restriction, although aldosterone excretion increases as expected, suggesting that additional factors participate in the regulation of 18-oxocortisol production.

Dose-dependent systemic and regional hemodynamic effects of calcitonin gene-related peptide.

The dose-response effects of infused calcitonin gene-related peptide (CGRP), a potent vasodilator, on systemic and regional hemodynamics in the conscious rat remain incompletely defined. The radioactive microsphere technique provided these determinations before and after the intravenous administration of vehicle or 22, 65, 220, and 2200 pmol of CGRP. Neither vehicle nor 22 pmol of CGRP significantly changed any systemic or regional hemodynamic parameter. Starting at the 65-pmol dose, CGRP significantly decreased mean blood pressure and total peripheral resistance, while increasing heart rate without changing cardiac output. CGRP produced selective regional vasodilatory effects, with the coronary circulation being unusually sensitive. In contrast, CGRP caused significant increases in blood flow to the mesenteric and cutaneous circulations only at the two highest doses. CGRP increased plasma norepinephrine, epinephrine, and renin activity significantly at only the 2200-pmol dose. In conclusion, CGRP decreases blood pressure by peripheral vasodilation, with a threshold dose occurring between 22 and 65 pmol. In addition, the coronary circulation appears to be particularly sensitive to the vasodilatory properties of CGRP.

Systemic and regional hemodynamic effects of dietary calcium supplementation in mineralocorticoid hypertension.

This study was undertaken to determine the systemic and regional hemodynamic effects of long-term dietary calcium supplementation in mineralocorticoid (DOC)-salt hypertension. Systemic and regional hemodynamic measurements were determined by the radioactive microsphere technique in conscious and unrestrained rats (kidneys intact) with DOC-salt-induced hypertension that were pair-fed either a normal calcium (0.6% by weight, n = 12) or a calcium-supplemented (high-calcium) diet (2.5% by weight, n = 12). After 7 to 8 weeks, there were no differences in weight, heart rate, or cardiac output between the two groups. In contrast, the high-calcium rats had a significantly lower mean blood pressure (125 +/- 4 mm Hg, mean +/- SEM) than the normal calcium rats (143 +/- 5 mm Hg); this finding appeared to result predominantly from a reduction in total peripheral resistance. The high-calcium rats had a higher renal blood flow (7.8 +/- 0.5% vs. 6.2 +/- 0.4% cardiac output; p less than 0.05) and lower renal (14.3 +/- 1 vs. 19.3 +/- 2 mm Hg/min/ml/g tissue; p less than 0.05) and jejunal vascular resistance than did the normal calcium rats. Two additional identical groups of normal calcium-and high-calcium-DOC-salt rats (n = 12 each) were also studied. In these rats, serum-ionized calcium decreased significantly (p less than 0.05) from baseline in both groups. Urinary sodium increased in both groups but did not differ significantly. In conclusion, dietary calcium supplementation attenuates the rise in peripheral vascular resistance that accompanies DOC-salt hypertension. This attenuated resistance appears to be relatively selective and is noted particularly in the renal vasculature.

Dietary calcium modulates spinal cord content of calcitonin gene-related peptide in the rat.

This study was undertaken to determine if calcium status modulates calcitonin gene-related peptide (CGRP) neuronal content. In two separate experiments, young, growing rats and mature rats were placed on low, normal, and high calcium diets for four weeks. CGRP immunostaining was localized immunocytochemically in laminae I and II of the upper thoracic spinal cord in young rats and in the upper thoracic and lumbar spinal cord in mature rats. Low calcium intake decreased dorsal horn CGRP content in young, growing rats, while high calcium diet significantly increased CGRP content, as determined by computer-assisted image processing, in both young and adult rats. A significant positive correlation was found between the serum ionized calcium and CGRP content in laminae I and II. Thus, calcium balance appears to modulate neuronal CGRP content.

Metabolic effects of hydrochlorothiazide and enalapril during treatment of the hypertensive diabetic patient. Enalapril for hypertensive diabetics.

To determine the effect of enalapril maleate and low-dose hydrochlorothiazide therapy on blood pressure and glucose and lipid homeostasis in hypertensive type II diabetic patients, we treated nine of these patients sequentially with placebo, hydrochlorothiazide (25 mg/d with supplemental potassium chloride), and enalapril (10 to 20 mg/d). Sitting blood pressure fell significantly and to comparable levels with both hydrochlorothiazide and enalapril monotherapy. Enalapril monotherapy was associated with a slight, but not significant, fall in fasting blood glucose levels and with a significant fall in hemoglobin A1c levels. This improved glucose homeostasis could not be explained satisfactorily by changes in peripheral insulin sensitivity or hepatic glucose output, determined with the euglycemic clamp technique, or by changes in fasting serum insulin levels or monocyte insulin binding. In these low doses, hydrochlorothiazide did not worsen glucose homeostasis. Serum total cholesterol levels were significantly lower with enalapril therapy than with hydrochlorothiazide therapy or with placebo, and serum high-density lipoprotein cholesterol and triglyceride levels did not change significantly with either treatment. Thus, by providing effective blood pressure control and beneficial metabolic effects, enalapril therapy appears ideal for treatment of hypertension in diabetic patients. Similarly, low-dose hydrochlorothiazide therapy appears to have fewer metabolic complications in these patients and is, therefore, a logical alternative to substitute for, or add to, enalapril therapy.