Nongenomic Effect Of Aldosterone Research Articles

Rapid stimulation of cyclic AMP production by aldosterone in rat inner medullary collecting ducts.

Aldosterone stimulates sodium transport in the inner medullary collecting duct (IMCD) via the classic genomic pathway, but it is not known whether it also acts via a rapid, non-conventional pathway in this part of the nephron. The IMCD regulates the final sodium content of urine and expresses vasopressin receptors coupled to adenylate cyclase. The recently reported rapid, non-genomic actions of aldosterone have been associated mainly with an increase in intracellular Ca(2+); however, it has also been shown to stimulate camp generation. Thus the aim of this study was to determine whether aldosterone stimulates rapid generation of cAMP in isolated IMCD segments. IMCD segments were microdissected from Sprague-Dawley rat kidneys and incubated at 37 degrees C for 4 min with aldosterone (10(-12) to 10(-6) M), vasopressin (10(-12) to 10(-6) M), or a combination of hormones in the presence of a phosphodiesterase inhibitor. cAMP was measured by radioimmunoassay. While corticosterone and dexamethasone were ineffective, aldosterone stimulated a dose-dependent increase in cAMP within 4 min (P<0.05). This action of aldosterone was not inhibited by the MR antagonist spironolactone. Co-incubation of aldosterone with vasopressin resulted in a further increase in cAMP generation above that induced by the neurohypophysial hormone alone. Aldosterone-mediated cAMP generation was not inhibited by a vasopressin V(1) or V(2) receptor antagonist. These data support a novel and rapid, non-genomic effect of aldosterone in IMCD. Aldosterone does not apparently interact with the vasopressin receptor to stimulate cAMP generation.

Effect of Spironolactone and Its Metabolites on Contractile Property of Isolated Rat Aorta Rings

Spironolactone and its active metabolites canrenone and potassium canrenoate are normally used as antihypertensive drugs. Although they are classified as antagonists of aldosterone, their mechanism of action cannot be ascribed solely to the regulation of ion transport in the distal tubule of nephrons. Here we have evaluated the effects of spironolactone, canrenone, and potassium canrenoate on contractile properties of isolated rat aorta rings. Spironolactone (1-300 microM), canrenone (1-300 microM), and potassium canrenoate (0.01-10 mM), in a concentration-dependent manner, relaxed rat aorta rings precontracted with phenylephrine (1 microM) or KCl (40 mM). These relaxant effects were not affected by prior treatment with either aldosterone (100 microM), glibenclamide (10 microM), or tetraethylammonium (10 mM), excluding the possibility that these drugs can be involved in either the nongenomic effect of aldosterone or on activation of potassium channels. Spironolactone and canrenone at concentrations of 30 and 100 microM, but not at 10 microM, and potassium canrenoate at concentrations of 0.3 and 1 mM, but not at 0.1 mM, significantly inhibited the phenylephrine (0.001-3 microM) concentration-response curve. Conversely, all tested concentrations of spironolactone (10, 30, and 100 microM), canrenone (10, 30, and 100 microM), and potassium canrenoate (0.1, 0.3, and 1 mM) significantly inhibited the concentration-response curve induced by cumulative concentrations of KCI (10-80 mM). Because both phenylephrine- and KCl-induced contractions imply an intracellular Ca2+ influx, we suggest that these drugs could act through an inhibition of voltage-dependent Ca2+ channels.

Role of 11beta-hydroxysteroid dehydrogenase in nongenomic aldosterone effects in human arteries.

The aim of the present study was to demonstrate rapid effects of aldosterone on the Na(+)-H(+) exchanger in strips of human vascular vessels and to determine whether 11beta-hydroxysteroid dehydrogenase enzyme (11beta-HSD) could play a protective role in this response, such as that described for the classic type I mineralocorticoid receptor (MR). The activity of 11beta-HSD isoforms 1 and 2 were measured in fetal and adult arteries. Both isoforms are present in adult and fetal vessels. However, a significant difference in the proportion of each isoform was found. Isoform 1 activity (in pmol x min(-1) x 100 mg(-1) protein) was 42+/-5 in fetal vessels and 29+/-2 in adult arteries, and isoform 2 activity was 78+/-7 in fetal and 12+/-2 in adult tissue. The nongenomic effect of aldosterone on Na(+)-H(+) exchanger activity was measured in strips of chorionic and radial uterine arteries loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein. Recordings of intracellular pH (pH(i)) were made by videofluorescence microscopy. Aldosterone (0.5 nmol/L) rapidly increased pH(i), with a half-maximal effect between 2 and 3 nmol/L in both fetal and adult vessels. Ethylisopropylamiloride, a specific inhibitor of the Na(+)-H(+) exchanger, inhibited this effect. The hormone-mediated increase in pH(i) was unaffected by spironolactone, a classic antagonist of MR, but was completely blocked by RU28318. Cortisol (up to 1 micromol/L) had no effect on pH(i), but when applied in the presence of carbenoxolone, a dramatic increase in Na(+)-H(+) exchanger activity was evident. The increments on pH(i) for each cortisol concentration were similar to those observed for aldosterone. These findings suggest that vascular 11beta-HSD plays an active role in maintaining the specificity of the rapid effects of aldosterone.

Aldosterone activates Na+/H+ exchange in vascular smooth muscle cells by nongenomic and genomic mechanisms

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. 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. 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. 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.

Rapid responses to aldosterone in human distal colon

Aldosterone at normal physiological levels induces rapid increases in intracellular calcium and pH in human distal colon. The end target of these rapid signaling responses are basolateral K + channels. Using spectrofluorescence microscopy and Ussing chamber techniques, we have shown that aldosterone activates basolateral Na/H exchange via a protein kinase C and calcium-dependent signaling pathway. The resultant intracellular alkalinization up-regulates an adenosine triphosphate (ATP)-dependent K + channel (K ATP) and inhibits a Ca 2+-dependent K + channel (K Ca). In Ussing chamber experiments, we have shown that the K ATP channel is required to drive sodium absorption, whereas the K Ca channel is necessary for both cyclic adenosine monophosphate and calcium-dependent chloride secretion. The rapid effects of aldosterone on intracellular calcium, pH, protein kinase C and K ATP, K Ca channels are insensitive to cycloheximide, actinomycin D, and spironalactone, indicating a nongenomic mechanism of action. We propose that the physiological role for the rapid nongenomic effect of aldosterone is to prime pluripotential epithelia for absorption by simultaneously up-regulating K ATP channels to drive absorption through surface cells and down-regulating the secretory capacity by inhibiting K Ca channels involved in secretion through crypt cells.

Rapid effects of corticosteroids on cytosolic protein kinase C and intracellular calcium concentration in human distal colon

Recent studies from our laboratory have reported rapid (<1 min) non-genomic activation of potassium recycling, Na +–H + exchange, protein kinase C (PKC) activity and PKC-sensitive Ca 2+ entry by mineralocorticoids in mammalian distal colonic epithelium. Previous studies from other laboratories have described stimulation of the Na +–H + exchanger by PKC activation. Here a rapid non-genomic effect of aldosterone on PKC activity and intracellular free calcium [Ca 2+] i is demonstrated in human distal colonic epithelium. Rapid activation (after 15 min incubation) of basal PKC activity was observed in cytosolic fractions of human colonic epithelium by aldosterone, fludrocortisone and deoxycorticosterone acetate (DOCA). PKC activation was inhibited by the specific PKC inhibitor bisindolylmaleimide (GF109203X). The glucocorticoid hydrocortisone failed to activate PKC activity. Aldosterone induced a rapid increase in [Ca 2+] i in isolated human colonic crypts. This stimulatory effect on [Ca 2+] i was inhibited by the PKC inhibitor chelerythrine chloride. Hydrocortisone and dexamethasone similarly failed to increase [Ca 2+] i. These results indicate that intracellular signalling for aldosterone involves changes in [Ca 2+] i via activation of PKC. Since stimulation of PKC activity and increase in [Ca 2+] i are apparent at normal circulating levels of aldosterone, our findings may have important physiological implications and prompt a reassessment of mineralocorticoid effects on electrolyte homeostasis.

Rapid effects of steroid hormones on free intracellular calcium in T84 colonic epithelial cells.

Studies from our laboratory have demonstrated rapid (< 1 min) nongenomic activation of K+ recycling and Na+/H+ exchange by mineralocorticoids in human colonic epithelium, and studies from other laboratories have demonstrated rapid effects of aldosterone on intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells. Here a rapid nongenomic effect of aldosterone on [Ca2+]i is demonstrated in the human colonic epithelial cell line T84. Aldosterone induced a rapid increase in [Ca2+]i within approximately 2 min. The rise in [Ca2+]i after aldosterone appears to result from the activation of a Ca2+ influx pathway, inasmuch as 1) no increase in [Ca2+]i was observed with aldosterone when cells were bathed in Ca(2+)-free Krebs solution and 2) emptying of the intracellular Ca2+ stores by thapsigargin was not enhanced by addition of aldosterone to extracellular Ca(2+)-free solution. In contrast, the Ca2+ response to aldosterone, in the presence of 2 mM Ca2+ in the external bathing solution, was not decreased after intracellular Ca2+ stores were emptied by thapsigargin. Other mineralocorticoid hormones increased [Ca2+]i, whereas the glucocorticoid hydrocortisone failed to increase [Ca2+]i. These results demonstrate the existence of a mineralocorticoid-specific Ca(2+)-signaling pathway in human colonic T84 (crypt) epithelial cells.