Martin Wehling

Rapid Effects of Aldosterone on Sodium Transport in Vascular Smooth Muscle Cells

Increasing evidence has accumulated for rapid nongenomic steroid actions in various cell systems and, more recently, for rapid aldosterone effects on the Na(+)-H+ antiport in human mononuclear leukocytes. The aim of the present study was to demonstrate a rapid, nongenomic aldosterone action in rat vascular smooth muscle cells as a key effector cell in cardiovascular regulation. Basal 22Na+ influx in quiescent vascular smooth muscle cells was 22.1 +/- 1.9 nmol/mg protein per minute (mean +/- SEM, n = 9). Aldosterone (1 nmol/L) stimulated influx to 28.6 +/- 1.5 nmol/mg protein per minute after 4 minutes (n = 9, P < .05), with a half-maximal effect between 0.1 and 0.5 nmol/L; the effects were inhibited by ethylisopropylamiloride, the specific inhibitor of the Na(+)-H+ exchanger, demonstrating the involvement of this transport system in rapid effects of aldosterone. Hydrocortisone (1 mumol/L) was ineffective, and fludrocortisone and deoxycorticosterone increased influx with half-maximal effects at approximately 0.5 nmol/L. Canrenone, a classic antagonist of aldosterone action, did not inhibit stimulation by aldosterone at a 1000-fold excess concentration. Aldosterone significantly stimulated intracellular inositol 1,4,5-trisphosphate levels (P < .05) after 30 seconds; the inhibitors of phospholipase C, neomycin and U-73122, inhibited aldosterone-stimulated Na+ influx and increase of intracellular inositol 1,4,5-trisphosphate. The rapid stimulation of sodium transport in vascular smooth muscle cells and the pharmacological characteristics of this effect are clearly incompatible with the classic, genomic pathway of steroid action and represent further evidence for nongenomic effects of aldosterone.

Nongenomic actions of steroid hormones

The genomic theory of steroid action has dominated research in the field over the past 4 decades. Despite early observations on rapid steroid effects being clearly incompatible with this theory, only recently has immediate steroid action been more widely recognized and is summarized here, with the emphasis on aldosterone. Further evidence for nongenomic steroid effects is also gathered rapidly for various groups of other steroids, such as neurosteroids, vitamin D(3), and estrogens.

Non-classical receptors for aldosterone in plasma membranes from pig kidneys.

Rapid, nongenomic in vitro effects of aldosterone on intracellular electrolytes, cell volume and the sodium-proton antiporter have been found in human mononuclear leukocytes (HML), as have related membrane receptors. In the present study, binding of 125I-labeled aldosterone to plasma membrane preparations from pig kidneys was studied, since nongenomic in vitro effects of aldosterone have also been described in cultured kidney cells. In this preparation, binding of aldosterone shares important features with both functional and binding data in HML. These include a very low apparent Ki of approximately 0.1 nM for aldosterone, a high turnover rate and binding selectivity for aldosterone and fludrocortisone. Desoxycorticosterone acetate and corticosterone show intermediate affinity, with apparent Ki values of approximately 1 and 100 nM, with hydrocortisone even less active. Thus binding of aldosterone to kidney plasma membranes is compatible with the major features of its nongenomic renal effects.

Looking beyond the dogma of genomic steroid action: insights and facts of the 1990s

The genomic theory of steroid action has been the unquestioned dogma for the explanation of steroid effects over the past four decades. Despite early observations on rapid steroid effects being clearly incompatible with this theory, only recently has nongenomic steroid action been more widely recognized and led to a critical reappraisal of unsolved questions about this dogma. Evidence for nongenomic steroid effects is now coming from all fields of steroid research, and mechanisms of agonist action are being studied with regard to the membrane receptors and second messengers involved. A prominent example of a receptor/effector cascade for nongenomic steroid effects has been described for rapid aldosterone effects in various cell types, including lymphocytes and vascular smooth muscle cells. Rapid in vitro effects of aldosterone on the sodium proton antiport have been found in human lymphocytes, cultured vascular smooth muscle, and endothelial cells involving nonclassical membrane receptors with a high affinity for aldosterone, but not for cortisol, and phosphoinositide turnover. Another important second messenger, [Ca2+]i, is consistently increased by aldosterone within 1–2 min. In vascular smooth muscle cells, calcium is released from perinuclear stores while in endothelial cells a predominant increase of subplasmalemmal calcium is seen. Effects are half-maximal at physiological concentrations of free aldosterone (0.1 nM), while cortisol is inactive up to 0.1 μM; the classical mineralocorticoid antagonist canrenone is ineffective in blocking the action of aldosterone. The data show that intracellular signaling for nongenomic aldosterone effects also involves calcium, but pathways of cell activation may vary between different cell types. Further evidence for nongenomic steroid effects is encountered presently for various groups of steroids such as neurosteroids, mineralocorticoids, vitamin D3, and sex hormones. Future research will have to target the cloning of the first membrane receptor for steroids and evaluate the clinical relevance of these rapid steroid effects.

Characterization of high affinity progesterone-binding membrane proteins by an anti-peptide antiserum

A chemically synthesized 15-mer oligopeptide derived from the N terminus of high affinity progesterone-binding membrane site(s) from porcine liver was used to generate site-specific antibodies. Western blotting experiments confirmed the specificity of the anti-peptide serum obtained. In further investigations this antiserum was used for the identification of the native progesterone-binding membrane protein complex that represents an oligomer with an apparent molecular mass of about 200 kDa. In temperature-induced Triton X-114 phase separation experiments combined with Western-blotting, the progesterone-binding site was identified as an hydrophobic (integral) membrane protein. In addition, in Western blotting analyses the antiserum reacted with the progesterone-binding or related proteins in membrane fractions from a wide array of different tissues in various species.

Aldosterone influences free intracellular calcium in human mononuclear leukocytes in vitro

Mineralocorticoid receptors have been detected in human mononuclear leukocytes (HML) and a physiological effector mechanism was demonstrated subsequently by which aldosterone is able to prevent the loss of intracellular sodium, potassium and cell water during incubation in an aldosterone-free medium. In the present paper, free intracellular calcium, [Ca2+]i, was measured in HML from normal subjects by Quin-2 and Fura-2 fluorescence after incubation for 1 h at 37 degrees C in RPMI-1640 medium. In fresh HML, [Ca2+]i was 54 +/- 15 nM (Fura-2, mean +/- SD, n = 26). After incubation without aldosterone, [Ca2+]i in HML was 118 +/- 27 nM (Quin-2, n = 11) and 50 +/- 13 nM (Fura-2). After incubation with 1.4 (Fura-2) or 2.8 nM (Quin-2) aldosterone, [Ca2+]i was 139 +/- 38 nM (Quin-2, P less than 0.05 compared with value after incubation without aldosterone) and 57 +/- 11 nM (Fura-2, P less than 0.00001). The Kd-value for dose-response curve was 0.4 nM. The effect of aldosterone was antagonized by N-ethyl-isopropylamiloride, but not by canrenoate, canrenone, cycloheximide and actinomycin D. It was absent in a sodium-free buffer. Corticosterone and hydrocortisone were active as agonists. These results show that aldosterone exerts an effect on the [Ca2+]i in HML in vitro which could be involved in hemodynamic responses to mineralocorticoids if also present in cardiovascular tissues.

Are high-affinity progesterone binding site(s) from porcine liver microsomes members of the σ receptor family?

Membrane progesterone binding sites have been purified recently from pig liver. Since progesterone is considered as an endogenous sigma (sigma) receptor ligand, these sites were characterized pharmacologically by ligands selective for sigma receptor and dopamine receptor binding sites, and by other drugs from distinct pharmacological classes. Binding studies using the radioligand [3H]progesterone were done in crude membrane preparations and solubilized fractions to determine half-maximal inhibitory concentration (IC50) values, from which inhibitory constants (Ki values) were calculated. Radioligand binding was inhibited by the sigma receptor ligands haloperidol, carbetapentane citrate, 1,3-Di(2-tolyl)guanidine (DTG), R(-)-N-(3-phenyl-1-propyl)-1-phenyl-2 aminopropane HCl (R(-)-PPAAP HCl), or sigma receptor antagonists like (+)-3-(3-hydroxyphenyl)-N-propylpiperidine HCl (R(+)-PPP HCl) and cis-9-[3-(3,5-dimethyl-1-piperazinyl)propyl]-9H-carbazole dihydrochloride (rimcazole 2HCl). The hierarchy of inhibitory action was not fully compatible with either sigma receptor class I (moderate affinity of pentazocine, diphenylhydantoin (phenytoin) insensitivity) or II sites (high affinity of carbetapentane). The data thus suggest that progesterone binding sites in porcine liver membranes are related to the sigma receptor binding site superfamily, but may represent a particular species with progesterone specificity.

Aldosterone-specific membrane receptors and rapid non-genomic actions of mineralocorticoids

Functional studies in extrarenal, non-epithelial cells such as smooth muscle cells and more recently circulating human lymphocytes have provided increasing evidence that aldosterone produces not only classical genomic effects, but also rapid, non-genomic effects on transmembrane electrolyte movements. These involve activation of the sodium/proton exchanger of the cell membrane at very low, physiological concentrations of aldosterone with an acute onset within 1-2 min. A second messenger cascade involved is the inositol 1,4,5-trisphosphate/calcium pathway which responds over the same rapid time course. Such changes clearly cannot be explained by genomic mechanisms, which are responsible for later effects than the membrane related rapid responses. The mechanisms underlying these rapid effects of aldosterone on electrolytes have been extensively studied in human lymphocytes, which thus may represent valuable tools in the delineation of the receptor-effector mechanisms involved. The unique characteristics of this new pathway for steroid action include its rapid time course, 10,000-fold selectivity for aldosterone over cortisol and the ineffectiveness of spironolactones, classical mineralocorticoid antagonists, as antagonists of the response.

High affinity aldosterone binding to plasma membrane rich fractions from mononuclear leukocytes: Is there a membrane receptor for mineralocorticoids?

In vitro effects of aldosterone on the intracellular concentrations of sodium, potassium and calcium, cell volume and the sodium-proton-antiport have been described in intact human mononuclear leukocytes (HML). In the present paper, the binding of a [125I]-labeled aldosterone derivative to plasma membrane rich fractions of HML was studied. High affinity binding of the radioligand with an apparent Kd of approximately 0.1 nM was found. Aldosterone displaced the tracer at a similar Kd. Both canrenone and cortisol were inactive as ligands up to concentrations of 0.1 microM. The findings are the first to demonstrate membrane binding sites with a high affinity for aldosterone, but not for cortisol. These data are perfectly compatible with major properties of steroidal effects on the sodium-proton-antiport in HML and thus very likely represent membrane receptors for aldosterone.

Photoaffinity labeling of plasma membrane receptors for aldosterone from human mononuclear leukocytes

Non-genomic effects of aldosterone on the sodium-proton-antiport have been shown in human mononuclear leukocytes which could be related to a new aldosterone membrane receptor. In the present paper plasma membranes from human mononuclear leukocytes were covalently photolabeled with a [125I]-aldosterone derivative. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed significant aldosterone binding at a molecular weight of approximately 50000 Dalton which was absent with 1 microM cold aldosterone, but not cortisol in the binding media. The presence of the sulfhydryl agent dithiothreitol did not affect results suggesting the absence of disulfide bridges in the steroid binding domain of the receptor. These data are the first to define the molecular weight of the membrane receptor for aldosterone.