Rapid Nongenomic Effects Of Aldosterone Research Articles

Role of Epithelial Sodium Channel in the Realization of Homeostatic Effects of Aldosterone on the Volume of Principal Cells of Cortical Collecting Ducts in Rats after Hypoosmotic Stress

The mechanism of aldosterone effects (10 nM) on the volume of the principal cells of cortical collecting ducts after hypoosmotic stress (280/140 mOsm/kg) was studied using fluorescence microscopy. The experiments with intracellular fluorescent dye calcein showed that aldosterone significantly decreases the amplitude and rate of increase in principal cell volume during hypoosmotic stress. Epithelial sodium channel blocker amiloride (10(-5)M) significantly attenuated the effects of aldosterone on the amplitude and rate of changes in cell volume. The obtained data attest to the contribution of epithelial sodium channel to the realization of rapid non-genomic effects of aldosterone on the amplitude and rate of changes in volume of the principal cells of cortical collecting ducts in rat kidney after hypoosmotic stress.

Extrarenal effects of aldosterone

The renal distal tubule has been considered for a long time as the main cellular target of aldosterone, where the hormone enhances sodium reabsorption and potassium secretion. However, other cell types in nonepithelial tissues, such as the heart, the vessels, adipose tissue, and macrophages, are now also recognized as targets for aldosterone. The functions that aldosterone exerts in these nonclassical target tissues are still a matter of debate. This review will highlight the recent findings on the extrarenal effects of aldosterone. Numerous studies showed that aldosterone exerts profibrotic and proinflammatory effects, but one or more cofactors such as salt, angiotensin II, and oxidative stress are required. Moreover, inflammation and macrophage infiltration are a prerequisite to aldosterone-induced cardiac fibrosis. This underlines a key role for aldosterone and the mineralocorticoid receptor in macrophages. Inflammatory effects of aldosterone in vascular smooth muscle cells involve trafficking to lipid rafts/caveolae through receptor tyrosine kinases. Finally, a growing body of evidence indicates a prominent role of aldosterone/mineralocorticoid receptor in the metabolic syndrome, in insulin resistance, and in adipocyte biology. The idiom from Socrates, 'the more we learn, the less we know', can be applied to aldosterone with its different facets and its pleiotropic effects. There is clear evidence for rapid nongenomic effects of aldosterone, mineralocorticoid receptor-dependent and mineralocorticoid receptor-independent signaling, in the heart, the vessels, and other nonepithelial tissues, leading to inflammation, fibrosis, and progression of cardiovascular diseases including hypertension and metabolic syndrome.

Effect of aldosterone on the regulation of the volume of principal cells of rat cortical collecting duct epithelium in early postnatal development

A rapid nongenomic regulatory effect of aldosterone on the volume of principal cells in the cortical segment of cortical collecting ducts (CCDs) of rat kidney was studied using the fluorescent dye calcein under hypoosmotic shock conditions (280‐140 mOsm/kg). In the presence of aldosterone (10 nM), the amplitude of cell volume growth significantly decreased ( p < 0.05) and the cell volume recovery time increased twice both in adult and ten-day-old animals. Age differences were detected when the time required for the cell volume recovery under hypoosmotic conditions was determined: in the adult animals, the cell volume recovered much more rapidly (by an order of magnitude) than in the ten-day-old animals both in the control and in the presence of aldosterone ( 0.09 ± 0.02 vs 2.8 ± 0.3 and 0.21 ± 0.03 vs 4.4 ± 0.5 , respectively, p < 0.001). This was the first study to demonstrate the involvement of aldosterone in the regulation of amplitude and dynamics of changes in the volume of CCD principal cells in early postnatal ontogeny. One of the key aspects of development of the osmoregulatory function of the kidney is the development of the molecular mechanisms of regulation of sodium transport in nephron epithelial cells by aldosterone. It is known that, in early ontogeny, kidneys of altricial animals including rats are insensitive to the antinatriuretic effect of aldosterone. Regulation of sodium transport in postnatal ontogeny of the rat is detected starting from the tenth day of life, when the transition from milk feeding to independent feeding is ended. The molecular mechanisms underlying the development of hormonal sensitivity of target cells of an immature kidney have been studied insufficiently well. It is known that the main proteins involved in the transmembrane sodium transport in the principal cells of the CCDs of rat nephron are the proteins of the apical epithelial of the sodium channel (ENaC) and the basolateral sodium pump ( Na + ,a + -ATPase) [6, 7]. The intracellular sodium concentration ([ ]) in principal cells is primarily determined by the dynamic equilibrium between the sodium influx through ENaC and its efflux form the cell into the intersticium against the concentration gradient by ATP-driven Na + ,a + -ATPase. Earlier, we showed that the expression of the genes encoding these proteins is suppressed and the intracellular sodium concentration in CCD principal cells is decreased in kidneys of tenday-old animals [1, 4]. At the same time, we discovered that aldosterone had a rapid nongenomic effect expressed in maintaining an increased [ ] in CCD principal cells at a low (14 mM) sodium concentration in the ambient medium both in adult and ten-day-old rats [4]. These data led us to assume that, along with the well-known mineralocorticoid effect, aldosterone is also involved in the regulation of the volume of CCD cells in rat nephron in hypoosmotic medium and that this mechanism functions even in the preweaning period. However, the rapid nongenomic effects of aldosterone on the regulation of the volume of CCD principal cells have been studied insufficiently comprehensively. In view of above, the goal of this study was to investigate the rapid nongenomic effect of aldosterone at a physiological concentration on the volume of CCD principal cells in kidneys of ten-day-old and adult rats under hypoosmotic shock conditions. Experiments were performed with CCDs of ten- and sixty-day-old Wistar rats, which were obtained by microdissection. The changes in the cell volume were assessed by the changes in the fluorescence intensity of the intracellular dye calcein and expressed in relative units. Fluorescence intensity was measured in the cells located at the open end of a CCD fragment; the basolateral and apical surfaces of these cells are in contact with the ambient solution. Continuous fluorescence recording during cell volume changes in response to osmotic shock was performed in our modification [11]. Renal tubule fragments contained in 100 µ l of suspension in aOa culture medium were loaded on a polylysinecoated slide and loaded with the fluorescent dye calcein

Effect of aldosterone on kinetics of intracellular sodium in cortical portion of collecting ducts in rat kidney

We studied the effect of protein kinase C inhibitor RO-31-8220 (10(-7) M) on rapid nongenomic effect of aldosterone in cells of isolated segment of distal region of collecting duct in rat kidney. Experiments with fluorescent dye Na-Green showed that the inhibitor abolished the modulating effect of aldosterone (10 nM) on intracellular sodium concentration at external sodium concentration of 14 mM. Aldosterone decreased by half the initial rate of the changes in internal sodium concentration in both 10-day and mature rats (p<0.05). Similarly to sodium channel blocker amiloride (10(-5) M), RO-31-8220 abolished rapid nongenomic effect of aldosterone on the rate of the changes in internal sodium concentration.

Rapid non-genomic effects of aldosterone on the renal vasculature

There is increasing evidence for the importance of rapid non-genomic effects of aldosterone on the human vasculature including renal vessels. Arima and collegues by examining isolated perfused afferent and efferent arterioles from rabbit kidneys found a vasoconstriction in both. In another study the same group showed that endothelium-derived nitric oxide (NO) modulates the vasoconstrictor response to aldosterone in rabbit preglomerular afferent arterioles. Disrupting the endothelium as well as blockade of endothelial NO synthase (eNOS) augmented aldosterone-induced vasoconstriction in this study. Uhrenholt et al. found no effect of aldosterone alone to afferent arterioles but a suppression of depolarisation-induced vasoconstriction. After the blockade of eNOS the aldosterone effect was completely suppressed. In a clinical study in healthy male volunteers injection of aldosterone had no statistically significant effects. Co-infusion of the eNOS inhibitor N(G)-monomethyl- l-arginine ( l-NMMA) changed the effect of aldosterone on renal hemodynamics. Aldosterone in co-infusion with l-NMMA decreased renal plasma flow (RPF) much stronger than l-NMMA alone. Infusion of l-NMMA alone increased GFR whereas aldosterone/ l-NMMA lowered GFR slightly. Aldosterone co-infused with l-NMMA strongly increased renal vascular resistance (RVR). The increase was on top of the smaller increase that was induced by l-NMMA infusion. These data indicate that aldosterone acts via rapid non-genomic effects in vivo in humans at the renal vasculature. Antagonizing the endothelial nitric oxide synthase unmasks these effects. Therefore, rapid non-genomic aldosterone effects increase renal vascular resistance and thereby may mediate arterial hypertension if endothelial dysfunction is present.

Aldosterone and the Cardiovascular System: Genomic and Nongenomic Effects

There is clear evidence for rapid nongenomic effects of aldosterone in the cardiovascular system in addition to its well characterized effects of unidirectional transepithelial sodium transport. Many of these effects are mediated by the classical mineralocorticoid receptors, although others may be exerted independently. Given that mineralocorticoid receptors are largely constitutively occupied but not activated by physiological glucocorticoids, effects of aldosterone administered in vitro or in vivo may or may not equate with true physiological mineralocorticoid roles. In many systems (e.g. blood pressure regulation and cardiac fibrosis), the time course of effects is such that it is not possible, and perhaps not important, to distinguish between rapid nongenomic and classical genomic effects in the context of homeostatic physiology.

Rapid Nongenomic Effects of Aldosterone on the Renal Vasculature in Humans

There is increasing evidence for the importance of rapid nongenomic effects of aldosterone on the human vasculature. In vitro animal experiments in renal arterioles also suggest the presence of such effects on the renal vasculature. We conducted a clinical study to explore these effects in vivo in humans. Thirteen healthy male volunteers were examined. Aldosterone (500 microg) or placebo was injected intravenously with or without coinfusion of N(G) monomethyl-L-arginine (L-NMMA) in a randomized, double-blinded 4-fold crossover design. Renal plasma flow and glomerular filtration rate were measured by constant infusion clearance technique using inulin and para-aminohippuric acid. Injection of aldosterone without concomitant infusion of L-NMMA changed the renal plasma flow and glomerular filtration rate not statistically significant compared with placebo. Coinfusion of L-NMMA unmasked the effect of aldosterone: aldosterone with L-NMMA decreased the glomerular filtration rate slightly (-1.4+/-6.2 mL/min), whereas infusion of L-NMMA alone increased the glomerular filtration rate (8.3+/-9.8 mL/min; P=0.004). L-NMMA alone decreased renal plasma flow by 58.2+/-97.5 mL/min, and aldosterone with L-NMMA decreased renal plasma flow by 190.0+/-213.7 mL/min (P=0.074). Accordingly, Aldosterone with L-NMMA increased renal vascular resistance much more than L-NMMA alone (1588+/-237 versus 614+/-240 dynxsxcm(-5); P=0.014). These data indicate that aldosterone acts via rapid nongenomic effects in vivo in humans at the renal vasculature. Antagonizing the endothelial NO synthase unmasks these effects. Therefore, rapid nongenomic aldosterone effects increase renal vascular resistance and thereby mediate arterial hypertension if endothelial dysfunction is present.

Nongenomic Renal Effects of Aldosterone

There is increasing evidence for a dependency on aldosterone in the development of hypertension.1 How aldosterone influences the physiology that determines blood pressure or for that matter promotes the pathophysiology of high blood pressure turns out to be less than straightforward. There is the genomic pathway that begins with the binding of aldosterone to the classical mineralocorticoid receptor (MR) and ends with sodium reabsorption at the distal nephron. But aldosterone can convey nongenomic influences as well, actions that have received less attention, possibly in part because they seem counterintuitive to our genomic view of biology. Adding to the complexity of aldosterone’s actions is the fact that there are also nonepithelial (nondistal nephron) targets of aldosterone, such as the heart, the vasculature, the kidney, and other sites. In this issue of Hypertension , Schmidt et al2 describe a rapid (thus nongenomic) effect of administered aldosterone to increase resistance in renal vasculature (a nonepithelial target) in healthy human subjects. The findings follow on the heels of a rather large body of work on the genomic actions of aldosterone. Aldosterone’s nongenomic effects were first recognized more than a decade before an aldosterone-inducible gene was identified in the late 1990s.3 A nongenomic action characteristically occurs almost immediately, usually within several minutes,4 before sufficient time has elapsed for gene …

A rapid nongenomic effect of aldosterone on intracellular sodium concentration in the distal nephron segment of the rat

A rapid nongenomic effect of aldosterone on intracellular sodium concentration in the distal nephron segment of the rat

Nongenomic Cardiovascular Actions of Aldosterone: A Receptor for All Seasons?

Untilrecently,aldosteroneeffectshavebeendistinguished as acute nongenomic vs. more persistent genomic actions. Nongenomic actions are characterized by rapid onset and cannot be blocked by spironolactone nor mimicked by cortisol. On this basis, these rapid nongenomic effects of aldosterone were attributed to a membrane receptor, distinct from the classical mineralocorticoid receptor (MR), although such a receptor has not been characterized to date . Recently, however, Mano et al. (1) reported specific binding in plasma membranes of cardiac myocytes, with maximal levels of only 10.3 0.4 fmol/mg protein, and no correlation with functional data. The paper by Michea et al. (2) in the current issue of Endocrinology provides strong additional evidence that both the nongenomic and genomic actions of aldosterone, at least incardiovasculartissues,aremediatedviathesame,classical MR. The new mineralocorticoid antagonist, eplerenone, which binds specifically to the cytosolic receptor (3–6), blocks the rapid aldosterone effect on intracellular Ca 2 and pH as well as aldosterone-induced vasoconstriction. From other recent studies, there is a growing body of evidence that rapid nongenomic effects of aldosterone on vascular smooth muscle cells (7, 8) and cardiomyocytes (9, 10) are similarly mediated via classical MRs. A previous study from the Marusic laboratory (7) showed that human vascular smooth muscle cells express 11-HSD1 and 11-HSD2, and nanomolar concentrations of aldosterone acutely (5 min) increased intracellular pH in these cells. This effect was mediated by acute activation of the Na/H exchanger, as it was blocked by the amiloride derivative ethylisoprolpylamiloride but not spironolactone; in contrast, the water soluble, open E-ring MR antagonist RU28318 blocked the aldosterone-induced increase in pH. In keeping with other studies on the rapid effects of aldosterone, cortisol alone was without effect. When, however, the cortisol-metabolizing, reduced nicotinamide adenine dinucleotide-generating enzyme 11-HSD2 is blocked by addition of carbenoxolone, the glucocorticoid becomes active, mimicking the aldosterone action on intracellular pH.

Aldosterone Rapidly Activates Na+/H+ Exchange in M-1 Cortical Collecting Duct Cells via a PKC-MAPK Pathway

Background: In this study, the mechanism of the rapid non-genomic effect of aldosterone on Na⁺/H⁺ exchanger (NHE)-mediated intracellular pH (pH_i) recovery from an acid load in murine M-1 cortical collecting duct cells was assessed. Methods: Spectrofluorescence microscopy and Western blot analysis was carried out and NH₄Cl was used to induce the acid load. Results: Aldosterone (10 nM) induced a rapid (<5 min) concentration-dependent increase in pH_i recovery in M-1 cells, an effect mimicked by its precursor deoxycorticosterone (1 nM). This response was unaffected by the mineralocorticoid receptor (MR) antagonist spironolactone (10 µM) but was significantly reduced by the NHE antagonists 5′-(N-ethyl- N-isopropyl)amiloride (EIPA) (20 µM) and cariporide (1 µM). The PKC inhibitor chelerythrine chloride (1 µM) significantly attenuated the aldosterone-induced increase in NHE1 activity. HBDDE (80 µM), a PKC_α inhibitor, inhibited the rapid aldosterone effect whereas rottlerin (15 µM), a PKC_δ antagonist, did not. The glucocorticoid receptor agonists hydrocortisone (1 µM) and dexamethasone (100 nM) decreased NHE activity, whereas the synthetic mineralocorticoid fludrocortisone (1 nM) had no significant effect. MAPK inhibition using PD98059 (25 µM) significantly attenuated the rapid aldosterone effect; Western blot analysis showed that aldosterone activation of ERK 1/2 was unaffected by pretreatment with spironolactone but was inhibited following chelerythrine chloride. Conclusion: Aldosterone causes a rapid non-genomic increase in NHE1 activity in M-1 cells via a PKC_α/MAPK pathway independent of the classical MR.

Eplerenone Blocks Nongenomic Effects of Aldosterone on the Na+/H+ Exchanger, Intracellular Ca2+ Levels, and Vasoconstriction in Mesenteric Resistance Vessels

There is increasing evidence for rapid nongenomic effects of aldosterone. Aldosterone has been demonstrated to alter intracellular pH and calcium in isolated cells. However, few studies have correlated these effects with aldosterone-mediated physiological responses. Therefore, we studied rapid effects of aldosterone on vascular reactivity, intracellular Ca2+, and pH in resistance vessels. Furthermore, we explored whether the new antimineralocorticoid drug eplerenone could effectively block nongenomic aldosterone-mediated effects. The vasoconstrictor action of aldosterone was examined directly by determining the diameter of small resistance mesenteric vessels (160-200 microm resting diameter), simultaneously with intracellular pH or Ca2+. Aldosterone (10 nm) caused a rapid constriction of resistance vessels (8.1% +/- 1.0% reduction in the diameter below control conditions, P < 0.05). Aldosterone potentiated phenylephrine-mediated constriction in small and large mesenteric vessels. Aldosterone induced a rapid increase of intracellular Ca2+ and cellular alkalinization. Vasoconstrictor action of aldosterone and nongenomic effects on the sodium-proton exchanger (NHE1) activity or intracellular Ca2+ responses was abolished by eplerenone. The vasoconstrictor response of aldosterone was related to phosphatidylinositol 3-kinase (PI3-K): the hormone decreased protein kinase B phosphorylation; pharmacological inhibition of PI3-K (10 microm LY294002 or 1 microm wortmannin) increased arterial contractility. Inhibitors of ERK 1/2 phosphorylation (15 microm PD98059) had no effect on aldosterone-mediated vasoconstriction. Inhibition of protein kinase C with 1 microm bi-sindolylmaleimide I and/or inhibition of NHE1 with 100 microm amiloride abolished aldosterone vasoconstrictor action of resistance mesenteric arteries. We conclude that aldosterone-mediated increase in vascular tone is related to a nongenomic mechanism that involves protein kinase C, PI3-K, and NHE1 activity. Eplerenone is an effective blocker of nongenomic effects of aldosterone in vascular tissue.

Rapid non-genomic effects of aldosterone on rodent vascular function.

The main role of aldosterone is to maintain body sodium homeostasis by promoting salt reabsorption in the collecting ducts of the kidney. In the cardiovascular system, aldosterone may be harmful in a number of disease states by inducing fibrosis and vascular dysfunction. The present review describes novel results from several laboratories, which show that aldosterone also has beneficial effects in the cardiovascular system by stimulating the production of nitric oxide (NO) from the endothelium. The effect of aldosterone is seen within minutes, and is not inhibited by blockers of gene transcription, thus pointing to a non-genomic mechanism. Furthermore, this potentially beneficial effect is observed at low physiological concentrations of aldosterone (0.1-10 pm). The effect is mediated by the classical mineralocorticoid receptor, and it involves heat shock protein 90, phosphatidylinositol (PI)-3 kinase, protein kinase B, endothelial nitric oxide synthase, and liberation of NO. It is proposed that in healthy individuals with a functioning NO system, the detrimental effects of aldosterone on cardiovascular function are balanced by activation of the potentially beneficial effect of NO. However, in situations with endothelial dysfunction, such as congestive heart failure and hypertension, the negative effects of aldosterone are unopposed and inhibition of aldosterone is warranted.

Letters to the Editor

To the Editor: Deducing the physiological role of aldosterone from studies using systemic infusions of hormone is difficult because of the interdependent nature of many of the often counter-regulatory organ responses that are evoked. It is because of such general limitations that venous occlusion plethysmography (VOP), combined with intra-arterial drug infusion, has been used extensively to study (regional) human vascular physiology in vivo. Using this technique, Schmidt et al1 report rapid nongenomic effects of aldosterone …

Rapid, nongenomic effects of aldosterone in the heart mediated by epsilon protein kinase C.

Aldosterone elevates Na+/K+/2Cl- cotransporter activity in rabbit cardiomyocytes within 15 min, an effect blocked by K-canrenoate and thus putatively mineralocorticoid receptor mediated. Increased cotransporter activity raises intracellular [Na+] sufficient to produce a secondary increase in Na+-K+ pump activity; when this increase in intracellular [Na+] is prevented, a rapid effect of aldosterone to lower pump activity is seen. Addition of transcription inhibitor actinomycin D did not change basal or aldosterone-induced lowered pump activity, indicating a direct, nongenomic action of aldosterone. We examined a possible role for protein kinase C (PKC) in the rapid nongenomic effects of aldosterone. Single ventricular myocytes and pipette solutions containing 10 mm intracellular [Na+] were used in patch clamp studies to measure Na+-K+ pump activity. Aldosterone lowered pump current, an effect abolished by epsilon PKC (epsilonPKC) inhibition but neither alphaPKC nor scrambled epsilonPKC; addition of epsilonPKC activator peptide mimicked the rapid aldosterone effect. In rabbits chronically infused with aldosterone, the lowered pump current in cardiomyocytes was acutely (< or =15 min) restored by epsilonPKC inhibition. These studies show that rapid effects of aldosterone on Na+-K+ pump activity are nongenomic and specifically epsilonPKC mediated; in addition, such effects may be prolonged (7 d) and long-lived ( approximately 4 h isolated cardiomyocyte preparation time). The rapid, prolonged, long-lived effects can be rapidly (< or =15 min) reversed by epsilonPKC blockade, suggesting a hitherto unrecognized complexity of aldosterone action in the heart and perhaps by extension other tissues.

Aldosterone induces contraction of the resistance arteries in man

Very rapid nongenomic effects of aldosterone in vitro have been described in recent years and in vivo evidence has been reported as well. In the present study, we investigated the rapid effect of aldosterone on resistance arteries in vivo in man. We performed a randomized, placebo-controlled, double-blind crossover study on ten healthy male volunteers. Forearm blood flow (FBF) was measured using venous occlusion plethysmography in both forearms. FBF was reduced by administration of aldosterone 2.5 pmol/min at min 4 (from 4.45±0.03 to 3.3±0.25 ml/100 ml tissue) and reached its nadir at min 12 (from 4.45±0.03 to 1.6±0.08 ml/100 ml tissue, P<0.001). Our study documents a direct nongenomic effect of aldosterone on the resistance arteries in vivo in man. The rapid vasoconstrictive effect of aldosterone at physiological concentrations opens the way to investigations on the vascular role of this steroid in several disorders, such as hypertension, characterized by elevated peripheral vascular resistance.

Non-genomic actions of aldosterone: role in hypertension.

There is universal acceptance of the existence of rapid, non-genomic effects of aldosterone, although their physiological relevance and potential importance in hypertension are not yet clear. What has emerged over the year under review is that at least some of such rapid non-genomic effects of aldosterone may be mediated by the activation of the classical intracellular mineralocorticoid receptor, rather than a putative membrane receptor. The post-receptor mechanisms of rapid aldosterone action appear variously to involve protein kinase C, calcium, cyclic adenosine 3', 5'-monophosphate and inositol 1, 4, 5-triphosphate, with downstream effects on a variety of ion pumps and channels.

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.