Modulation of aldosterone biosynthesis by adrenodoxin mutants with different electron transport efficiencies.
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Aldosterone biosynthesis is highly regulated on different levels by hormones, potassium, lipid composition of the membrane and the molecular structure of its gene. Here, the influence of the electron transport efficiency from adrenodoxin (Adx) to CYP11B1 on the activities of bovine CYP11B1 has been investigated using a liposomal reconstitution system with truncated mutants of Adx. It could be clearly demonstrated that Adx mutants Adx 4-114 and Adx 4-108, possessing enhanced electron transfer abilities, produce increases in corticosterone and aldosterone biosynthesis. Based on the Vmax values of corticosterone and aldosterone formation, Adx 4-108 and Adx 4-114 enhance corticosterone synthesis 1.3-fold and aldosterone formation threefold and twofold, respectively. The production of 18-hydroxycorticosterone was changed only slightly in these Adx mutants. The effect of Adx 1-108 on the product patterns of bovine CYP11B1, human CYP11B1 and human CYP11B2 was confirmed in COS-1 cells by cotransfection of CYP11B- and Adx-containing expression vectors. It could be shown that Adx 1-108 enhances the formation of aldosterone by bovine CYP11B1 and by human CYP11B2, and stimulates the production of corticosterone by bovine CYP11B1 and human CYP11B1 and CYP11B2 also. (+info)
The role of tyrosine kinases in capacitative calcium influx-mediated aldosterone production in bovine adrenal zona glomerulosa cells.
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In adrenal glomerulosa cells, the stimulation of aldosterone biosynthesis by angiotensin II (Ang II) involves the activation of a capacitative Ca(2+) influx through calcium release-activated calcium (CRAC) channels. In various mammalian cell systems, it has been shown that CRAC channel activation and Ca(2+) entry require tyrosine kinase activity. We have therefore examined in this work whether similar mechanisms contribute to Ang II-induced mineralocorticoid biosynthesis. In fluo-3-loaded isolated bovine glomerulosa cells, two inhibitors of tyrosine kinases, genistein and methyl-2, 5-dihydroxycinnamate (MDHC) (100 microM) prevented capacitative Ca(2+) entry elicited by Ang II (by 54 and 62% respectively), while the inhibitor of epidermal growth factor (EGF) receptor tyrosine kinase, lavendustin A, was without effect. Similar results were observed on Ca(2+) influx triggered by thapsigargin, an inhibitor of microsomal Ca(2+) pumps. The inhibitors blocked Ang II-stimulated pregnenolone and aldosterone production in the same rank order. In addition to its specific effect on capacitative Ca(2+) influx, genistein also affected the late steps of the steroidogenic pathway, as shown by experiments in which the rate-limiting step (intramitochondrial cholesterol transfer) was bypassed with 25-OH-cholesterol (25-OH-Chol), cytosolic calcium was clamped at stimulated levels or precursors of the late enzymatic steps were supplied. In contrast, genistin, a structural analogue of genistein devoid of tyrosine kinase inhibitory activity, was almost without effect on pregnenolone or 11-deoxycorticosterone (DOC) conversion to aldosterone. These results suggest that, in bovine adrenal glomerulosa cells, Ang II promotes capacitative Ca(2+) influx and aldosterone biosynthesis through tyrosine kinase activation. (+info)
1166 A/C polymorphism of the angiotensin II type 1 receptor gene and the response to short-term infusion of angiotensin II.
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BACKGROUND: Previous studies reported an association of the 1166 A/C polymorphism of the angiotensin II (Ang II) type 1 receptor gene with high blood pressure and cardiovascular disease. We tested the hypothesis that this polymorphism affects the blood-pressure, renal hemodynamic, and aldosterone response to infused Ang II. METHODS AND RESULTS: Young, male, white volunteers (n = 116) with normal (n = 65) or mildly elevated (n = 51) blood pressure on a high salt intake were genotyped for the 1166 A/C polymorphism. Two doses of Ang II (0.5 and 3 ng x kg(-1) x min(-1) over 30 minutes each) increased blood pressure, plasma aldosterone, glomerular filtration rate, and filtration fraction and decreased renal blood flow. The blood-pressure, renal hemodynamic, and aldosterone responses were not significantly different between subjects homozygous for the A allele (n = 56) and heterozygous subjects (n = 47) or subjects homozygous for the C allele (n = 13). Comparison of A allele homozygotes with all C allele carriers pooled (n = 60) or restriction of the analysis to normotensive volunteers also revealed no significant differences between genotypes. CONCLUSIONS: The 1166 C variant of the Ang II type 1 receptor does not lead to a greater blood-pressure, aldosterone, or renal vascular response to infused Ang II in young, male, white subjects. We conclude that the 1166 A/C polymorphism does not have a major effect on these actions of Ang II. (+info)
Aldosterone activates Na+/H+ exchange in vascular smooth muscle cells by nongenomic and genomic mechanisms.
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BACKGROUND: In vascular smooth muscle cells (VSMCs), Na+/H+ exchange (NHE) plays an important role in intracellular pH (pHi) regulation. Recently, nongenomic effect of aldosterone (ALDO) on NHE activity has been suggested in VSMCs. However, the nongenomic and genomic effects of ALDO on NHE and the intracellular signaling mechanisms for these effects have not fully been determined in VSMCs. METHODS: The effects of short- (3 hr) and long- (24 hr) term exposure to ALDO on NHE activity were examined in cultured VSMCs from rat thoracic aortae by using single-cell pHi measurement with the pH-sensitive dye 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The NHE activity was calculated from the initial rate of Na+-dependent pHi recovery after acid load. RESULTS: The NHE activity significantly increased after short- and long-term exposure of VSMCs to ALDO (10(-6) M). The inhibitors of gene transcription (actinomycin D) and of protein synthesis (cycloheximide) had no effect on the short-term ALDO effect, but inhibited the long-term ALDO effect. The antagonists of the mineralocorticoid receptor (MR) (spironolactone) and of the glucocorticoid receptor (GR) (RU38486) caused no effect on the short-term ALDO effect, but inhibited the long-term ALDO effect. Two protein kinase C (PKC) inhibitors (staurosporine A and calphostin C) and PKC down-regulation (24 hr pre-exposure to phobol 12-myristate 13-acetate, PMA) inhibited both the short- and long-term ALDO effects. Exposure of VSMCs to PMA for 3 hours mimicked the short-term effect of ALDO on NHE activity. ALDO significantly increased PKC activity in VSMCs. The short-term ALDO effect was inhibited by disruptors of microtubule (colchicine) and of filamentous-actin (cytochalasin B). Long-term exposure of ALDO caused a threefold increase in NHE (NHE-1) mRNA levels. CONCLUSIONS: The short-term effect of ALDO on NHE activity is not mediated through either MR or GR, occurs independent of gene transcription and protein synthesis, and occurs through a mechanism involving the structural elements of cytoskeleton. The long-term effect of ALDO on NHE activity occurs through both MR and GR and requires gene transcription and protein synthesis. Both short- and long-term effects of ALDO are mediated through PKC activation. Therefore, ALDO activates NHE by nongenomic and genomic mechanisms in VSMCs. (+info)
Aldosterone-mediated regulation of ENaC alpha, beta, and gamma subunit proteins in rat kidney.
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Aldosterone stimulates sodium transport in the renal collecting duct by activating the epithelial sodium channel (ENaC). To investigate the basis of this effect, we have developed a novel set of rabbit polyclonal antibodies to the 3 subunits of ENaC and have determined the abundance and distribution of ENaC subunits in the principal cells of the rat renal collecting duct. Elevated circulating aldosterone (due to either dietary NaCl restriction or aldosterone infusion) markedly increased the abundance of alphaENaC protein without increasing the abundance of the beta and gamma subunits. Thus, alphaENaC is selectively induced by aldosterone. In addition, immunofluorescence immunolocalization showed a striking redistribution in ENaC labeling to the apical region of the collecting duct principal cells. Finally, aldosterone induced a shift in molecular weight of gammaENaC from 85 kDa to 70 kDa, consistent with physiological proteolytic clipping of the extracellular loop as postulated previously. Thus, at the protein level, the response of ENaC to aldosterone stimulation is heterogenous, with both quantitative and qualitative changes that can explain observed increases in ENaC-mediated sodium transport. (+info)
Effect of an exercise-heat acclimation program on body fluid regulatory responses to dehydration in older men.
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We examined if an exercise-heat acclimation program improves body fluid regulatory function in older subjects, as has been reported in younger subjects. Nine older (Old; 70 +/- 3 yr) and six younger (Young; 25 +/- 3 yr) male subjects participated in the study. Body fluid regulatory responses to an acute thermal dehydration challenge were examined before and after the 6-day acclimation session. Acute dehydration was produced by intermittent light exercise [4 bouts of 20-min exercise at 40% peak rate of oxygen consumption (VO(2 peak)) separated by 10 min rest] in the heat (36 degrees C; 40% relative humidity) followed by 30 min of recovery without fluid intake at 25 degrees C. During the 2-h rehydration period the subjects drank a carbohydrate-electrolyte solution ad libitum. In the preacclimation test, the Old lost approximately 0.8 kg during dehydration and recovered 31 +/- 4% of that loss during rehydration, whereas the Young lost approximately 1.2 kg and recovered 56 +/- 8% (P < 0.05, Young vs. Old). During the 6-day heat acclimation period all subjects performed the same exercise-heat exposure as in the dehydration period. Exercise-heat acclimation increased plasma volume by approximately 5% (P < 0.05) in Young subjects but not in Old. The body fluid loss during dehydration in the postacclimation test was similar to that in the preacclimation in Young and Old. The fractional recovery of lost fluid volume during rehydration increased in Young (by 80 +/- 9%; P < 0.05) but not in Old (by only 34 +/- 5%; NS). The improved recovery from dehydration in Young was mainly due to increased fluid intake with a small increase in the fluid retention fraction. The greater involuntary dehydration (greater fluid deficit) in Old was accompanied by reduced plasma vasopressin and aldosterone concentrations, renin activity, and subjective thirst rating (P < 0.05, Young vs. Old). Thus older people have reduced ability to facilitate body fluid regulatory function by exercise-heat acclimation, which might be involved in attenuation of the acclimation-induced increase in body fluid volume. (+info)
Renal distal tubular handling of sodium in central fluid volume homoeostasis in preascitic cirrhosis.
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BACKGROUND/AIMS: Patients with preascitic liver cirrhosis have an increased central plasma volume, and, for any given plasma aldosterone concentration, they excrete less sodium than healthy controls. A detailed study of the distribution of sodium reabsorption along the segments of the renal tubule, especially the distal one, is still lacking in preascitic cirrhosis. METHODS: Twelve patients with Child-Pugh class A cirrhosis and nine control subjects (both groups on a normosodic diet) were submitted to the following investigations: (a) plasma levels of active renin and aldosterone; (b) four hour renal clearance of lithium (an index of fluid delivery to the loop of Henle), creatinine, sodium, and potassium; (c) dopaminergic activity, as measured by incremental aldosterone response to intravenous metoclopramide. RESULTS: Metoclopramide induced higher incremental aldosterone responses, indicating increased dopaminergic activity in patients than controls, which is evidence of an increased central plasma volume (+30 min: 160.2 (68.8) v 83.6 (35.2) pg/ml, p<0.01; +60 min: 140.5 (80.3) v 36. 8 (36.1) pg/ml, p<0.01). Patients had increased distal fractional sodium reabsorption compared with controls (26.9 (6.7)% v 12.5 (3. 4)% of the filtered sodium load, p<0.05). In the patient group there was an inverse correlation between: (a) absolute distal sodium reabsorption and active renin (r -0.59, p<0.05); (b) fractional distal sodium reabsorption and sodium excretion (r -0.66, p<0.03). CONCLUSIONS: These data suggest that in preascitic cirrhosis the distal fractional tubular reabsorption of sodium is increased and critical in regulating both central fluid volume and sodium excretion. (+info)
Neuroendocrine activation in heart failure is modified by endurance exercise training.
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OBJECTIVES: The purpose of this study was to determine whether endurance exercise training could buffer neuroendocrine activity in chronic heart failure patients. BACKGROUND: Neuroendocrine activation is associated with poor long-term prognosis in heart failure. There is growing consensus that exercise may be beneficial by altering the clinical course of heart failure, but the mechanisms responsible for exercise-induced benefits are unclear. METHODS: Nineteen heart failure patients (ischemic disease; New York Heart Association [NYHA] class II or III) were randomly assigned to either a training group or to a control group. Exercise training consisted of supervised walking three times a week for 16 weeks at 40% to 70% of peak oxygen uptake. Medications were unchanged. Neurohormones were measured at study entry and after 16 weeks. RESULTS: The training group (n = 10; age = 61 +/- 6 years; EF = 30 +/- 6%) and control group (n = 9; age = 62 +/- 7 years; EF = 29 +/- 7%) did not differ in clinical findings at study entry. Resting levels of angiotensin II, aldosterone, vasopressin and atrial natriuretic peptide in the training and control groups did not differ at study entry (5.6 +/- 1.3 pg/ml; 158 +/- 38 pg/ml; 6.1 +/- 2.0 pg/ml; 37 +/- 8 pg/ml training group vs. 4.8 +/- 1.2; 146 +/- 23; 4.9 +/- 1.1; 35 +/- 10 control group). Peak exercise levels of angiotensin II, aldosterone, vasopressin and atrial natriuretic peptide in the exercise and control groups did not differ at study entry. After 16 weeks, rest and peak exercise hormone levels were unchanged in control patients. Peak exercise neurohormone levels were unchanged in the training group, but resting levels were significantly (p < 0.001) reduced (angiotensin -26%; aldosterone -32%; vasopressin -30%; atrial natriuretic peptide -27%). CONCLUSIONS: Our data indicate that 16 weeks of endurance exercise training modified resting neuroendocrine hyperactivity in heart failure patients. Reduction in circulating neurohormones may have a beneficial impact on long-term prognosis. (+info)