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(Circulation Research. 1995;76:973-979.)
© 1995 American Heart Association, Inc.

Articles

Nongenomic Effects of Aldosterone on Intracellular Ca²⁺ in Vascular Smooth Muscle Cells

Martin Wehling, Craig B. Neylon, Meryl Fullerton, Alex Bobik, John W. Funder

From the Baker Medical Research Institute, Victoria, Australia.

	Abstract

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Abstract Genomic mechanisms of steroid action have been increasingly elucidated over the past four decades. In contrast, rapid steroid actions have been widely recognized only recently, and detailed analysis of the mechanisms involved are still lacking. The present article describes rapid effects of mineralocorticoid hormones on free intracellular calcium in vascular smooth muscle cells as determined by fura 2 spectrofluorometry in single cultured cells from rat aorta. These effects are almost immediate and reach a plateau after only 3 to 5 minutes and are characterized by high specificity for mineralocorticoids versus glucocorticoids. The potent mineralocorticoids aldosterone and fludrocortisone are agonists with estimated apparent EC₅₀ values of 0.1 to 0.5 nmol/L; deoxycorticosterone acetate is an agonist with an EC₅₀ of 5 nmol/L; and progesterone, cortisol, corticosterone, and estradiol have much lower potency (EC₅₀ values of 0.5 to 5 µmol/L). The effect of aldosterone is blocked by neomycin and short-term treatment with phorbol esters but augmented by staurosporine, indicating an involvement of phospholipase C and protein kinase C. The Ca²⁺ effect appears to involve the release of intracellular Ca²⁺, as shown by the inhibitory effect of thapsigargin; intriguingly, a relatively small maximum effect (40 nmol/L increase) is consistently seen. This mechanism operates at physiological subnanomolar aldosterone concentrations and appears to be a likely candidate for rapid fine tuning of cardiovascular responsivity. It may also contribute to known clinical features of mineralocorticoid action that are difficult to explain by the traditional genomic mechanism alone.

Key Words: free intracellular Ca²⁺ • aldosterone • nongenomic steroid action • vascular smooth muscle cells

	Introduction

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The genomic action of mineralocorticoids involves binding of aldosterone to nuclear type I receptors with subsequent specific protein synthesis. Classic intracellular mineralocorticoid receptors have been cloned^{1 2} and are characterized by equivalent high affinity for aldosterone, cortisol, and corticosterone and relatively high affinity for canrenone, the classic mineralocorticoid antagonist. More recently, however, rapid in vitro effects of aldosterone on intracellular electrolyte concentration and cell volume, on the activity of the Na⁺-proton exchanger, and on inositol trisphosphate (IP₃) production have been described variously in human mononuclear leukocytes (HMLs^{3 4 5 6 7} ), vascular smooth muscle cells (VSMCs⁸ ), and kidney cells.⁹ These effects are clearly incompatible with an action via classic intracellular mineralocorticoid receptors: their onset is too rapid to involve genomic events, and they are highly specific for aldosterone over cortisol and canrenone. These characteristic effects suggested the existence of distinct receptors, which have been described for aldosterone in HML cell membranes^{10 11} and are currently of increasing interest in terms of the action of a variety of other steroids (for review, see Reference 12¹² ). In the present article, the involvement of free intracellular Ca²⁺ as a second messenger in the rapid effects of aldosterone has been studied in VSMCs.

The rationale for the present study was derived from the rapid effects of aldosterone on IP₃, which might be expected to be accompanied by changes of [Ca²⁺]_i via the action of IP₃ on intracellular Ca²⁺ stores.¹³ The experiments have been performed in one of the cell types in which rapid effects of aldosterone on the Na⁺-proton exchanger and IP₃ have been found^{3 4 5 6 7 8} and include the pharmacological and physiological characterization of the rapid aldosterone effect at the subcellular level.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Thrombin, angiotensin II, EGTA, staurosporine, neomycin, phorbol myristate acetate (PMA), pertussis toxin, genistein, and steroids were obtained from Sigma Chemical Co, except for spironolactone, which was from CIBA-GEIGY. Thapsigargin was from LC Services; ionomycin, from Calbiochem; and fura 2-AM, from Molecular Probes.

The monoclonal mouse antibody for the -smooth muscle isoform of actin was purchased from Progen Biotechnik; collagenase and elastase, from Worthington Biochemical; medium 199, from ICN; and DMEM and fetal calf serum (FCS), from Commonwealth Serum Laboratory.

Isolation and Primary Culture of Rat VSMCs
VSMCs were prepared enzymatically from rat or, with minor modifications, rabbit thoracic aortas as described earlier.¹⁴ In brief, adult Wistar-Kyoto rats or New Zealand White rabbits, fed standard laboratory chow and tap water ad libitum, were killed by cervical dislocation. After sternotomy under aseptic conditions, the thoracic aorta was excised and placed in ice-cold DMEM containing 60 µg/mL penicillin and 10% FCS for the preparation of rat VSMCs and 5% FCS for the preparation of rabbit VSMCs. After removal of fat, adventitia, and venous structures by blunt dissection in a Petri dish, the vessel was cut longitudinally and placed in DMEM (rat VSMCs) or medium 199 (rabbit VSMCs) supplemented with collagenase (3 mg/mL). With two fine forceps, the medial layer was stripped off and placed in a centrifuge tube containing 5 mL of enzyme dissociation mixture (3 mg/mL collagenase, 1 mg/mL elastase). After incubation at 37°C for several hours in a shaker bath, the suspension was centrifuged (900 rpm, 3 minutes), and the reaction was terminated with 5% FCS. After resuspension of the pellet in DMEM supplemented with 10% FCS, cells were grown on coverslips. After 24 hours, the cultures were washed once to remove nonadherent cells and debris and fed with fresh medium. Medium was routinely exchanged at 2- to 3-day intervals under examination by an inverted phase-contrast microscope. Early passages (passages 3 to 6) were used 36 to 72 hours after seeding. VSMCs showed a typical hill-and-valley configuration and stained positive with a specific antibody against the smooth muscle isoform of myosin and against tropomyosin, both of which do not react with rat fibroblasts or endothelial cells.¹⁵ They also stained positively with a monoclonal mouse antibody for the -smooth muscle isoform of actin.

Measurement of [Ca²⁺]_i
Determination of [Ca²⁺]_i was performed in single rat or rabbit aortic VSMCs. Early passage^{3 4 5 6} VSMCs were grown on glass coverslips, washed three times with 2 mL physiological saline solution (PSS, containing [mmol/L] NaCl 135, KCl 5, CaCl₂ 1.8, MgCl₂ 0.8, HEPES 10, and glucose 5.5, pH 7.4) to remove serum and loaded with 2 µmol/L fura 2-AM from a 0.1% stock solution in dimethyl sulfoxide for 30 minutes at room temperature. At the end of the loading period, cells were washed with PSS buffer (three times, 2 mL) and kept at room temperature in the dark; cells were used within 1 hour. They were rinsed with PSS buffer immediately before use to remove any residual, possibly leaked, dye and placed in a thermostatically controlled ring chamber (37°C) holding 1 to 2 mL of incubation fluid. All drugs were added in volumes of 10 to 100 µL to a final incubation volume of 1 mL PSS buffer. Single-cell measurement of [Ca²⁺]_i was performed using a SPEX dual-wavelength 1681 fluorolog spectrometer attached to a Nikon Diaphot inverted fluorescence microscope with a fluor 40/1.30 oil immersion objective. In a few experiments (Figs 4a and 7), a dual-wavelength imaging system (Till Photonics GmbH) attached to an Aviovert 35 inverted fluorescence microscope (Zeiss) with a fluor 40/1.30 oil immersion objective was used in an analogous manner. Excitation wavelengths were 340 and 380 nm, and emitted light was collected via a dichroic mirror at 510 nm. Slit widths were set at 1.5 mm on both excitation monochromators and 2.5 mm on the emission side. A 450-W xenon arc lamp was used as light source. Integration time was 0.3 seconds at each wavelength, with a time increment of 1 second. Autofluorescence was determined in each experiment by the addition of 2 mmol/L MnCl₂ and 2 µmol/L ionomycin to quench intracellularly located dye. The autofluorescence level was subtracted from each reading before calculation of [Ca²⁺]_i and was typically 45% of the total signal at the end of the experiment. Leakage/bleaching of the dye was 25% within 20 minutes. Thus, relative autofluorescence at the beginning was only 35%. Within these limits, the ratio mode for the calculation of [Ca²⁺]_i resulted in a stable baseline (shift, <10 nmol/L for 20 minutes) in control experiments, with no dependence on absolute fluorescence signals.

View larger version (17K): [in this window] [in a new window]   Figure 4. a, The effect of 100 nmol/L aldosterone on [Ca2+]i in rat aortic vascular smooth muscle cells is shown for Na+-free medium (isoosmotically replaced by choline chloride directly before measurement). b, The effect of pretreatment with 1 µmol/L thapsigargin, which produces a large rise in [Ca2+]i, reflecting the emptying of intracellular Ca2+ stores, is depicted. Representative tracings of [Ca2+]i from four experiments are given.

View larger version (19K): [in this window] [in a new window]   Figure 7. Effect of pretreatment of rat aortic vascular smooth muscle cells by 100 nmol/L aldosterone (b) versus control without aldosterone (a) on the [Ca2+]i response to angiotensin II (10-13 to 10-9 mol/L). Representative tracings of [Ca2+]i from four experiments are given.

Addition of steroids (aldosterone, cortisol, and progesterone) at the maximal concentrations used in the present study did not change autofluorescence of cells without dye.

The system was calibrated by the method of Grynkiewicz et al,¹⁶ and the following equation was used for the calculation of [Ca²⁺]_i by dM3000 software (SPEX Industries Inc):

where R is the fluorescence ratio for the excitation wavelengths of 340 and 380 nm, R_min is the ratio (0.66) for fura 2 acid in solution at zero calcium (1 mmol/L EGTA), R_max is the ratio (31.2) at 1 mmol/L calcium, and S_f2 and S_b2 indicate fluorescence at 0 and 1 mmol/L Ca²⁺, respectively, for an excitation wavelength of 380 nm (11.66 in our system). All readings were checked for stability of baseline for 3 to 5 minutes after initial application of 100 µL PSS buffer alone. In all experiments, cell viability and responsiveness were confirmed by the final addition of 1 U/mL thrombin, which produced an instant, transient, several-fold increase of [Ca²⁺]_i, and by 1 µmol/L ionomycin, which produced a sustained increase.

At the times indicated, steroids were added, with final concentrations given as log moles per liter in all figures. Stock solutions were 1 mmol/L in ethanol. At a final steroid concentration of 10 µmol/L, which was used in a few experiments, the ethanol concentration was 1%, which produced a small effect of its own; 0.1% ethanol corresponding to a final steroid concentration of 1 µmol/L was without effect on [Ca²⁺]_i.

Neomycin, PMA, thapsigargin, and staurosporine were added from 1:1000 stock solutions in dimethyl sulfoxide at the times indicated.

Statistical Analysis
The two-sided t test for unpaired or paired data was used, with values of P<.05 being considered significant. Results are presented as mean±SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Rapid Aldosterone Effects on [Ca²⁺]_i in VSMCs
The experiments show rapid effects of aldosterone on [Ca²⁺]_i in cultured VSMCs from rats and rabbits (Figs 1 and 2). The onset of the rise in Ca²⁺ follows the addition of aldosterone by <1 minute, some of which probably reflects mixing time in the incubation chamber. A small but significant (P<.05, n=6) effect was seen at a concentration of 0.01 nmol/L, with half-maximal effects at 0.1 nmol/L and near-maximal effects at 1 to 10 nmol/L. The apparent EC₅₀ for aldosterone is thus 0.1 nmol/L. Data were pooled from experiments in which two to six cumulative concentrations of the steroids were added, depending on the stability of the preparation. There was no apparent influence of the range of concentrations tested on the resulting rise in [Ca²⁺]_i. For example, the mean increase of [Ca²⁺]_i at 0.1 nmol/L aldosterone was 22±6 versus 19±3 nmol/L for cells pretreated with two versus one lower concentration of aldosterone. No quantitative differences were seen between the cell systems studied; in some experiments, a small initial overshoot was seen (eg, see Figs 1c and 2a), preceding a slower rise, which reaches a plateau after 3 to 5 minutes. The maximum rise of [Ca²⁺]_i was only 40 nmol/L (or 40%), given a basal value for [Ca²⁺]_i of 100 nmol/L in these cells.

View larger version (17K): [in this window] [in a new window]   Figure 1. Determination of [Ca2+]i in single rat aortic vascular smooth muscle cells by fura 2 spectrofluorometry (for details see "Materials and Methods"). At the times indicated, steroids were added with final concentrations given as log moles per liter in all figures. Tracings are representative experiments from a total of nine experiments with aldosterone alone or from quadruplicate experiments in other studies. Because of the natural variability of basal [Ca2+]i in single-cell determinations and the overall modest extent of steroid effects on [Ca2+]i, ordinate scales have been adjusted to basal [Ca2+]i in all related figures.

View larger version (18K): [in this window] [in a new window]   Figure 2. Determination of [Ca2+]i in aortic vascular smooth muscle cells from rabbits, as described in Fig 1. The response of [Ca2+]i to aldosterone in physiological saline solution is shown in panel a; the response in Ca2+-free buffer to which 25 µmol/L EGTA was added is shown in panel b. Representative tracings from four or five experiments are shown.

Dose-Response Studies for Different Steroids: Effect of Spironolactone
The dose-response curves for the effects of various steroids on [Ca²⁺]_i in VSMCs are shown in Fig 3. The potent mineralocorticoid fludrocortisone is an agonist with an estimated apparent EC₅₀ of 0.5 nmol/L; deoxycorticosterone acetate (DOCA) is an agonist with an EC₅₀ of 5 nmol/L; and progesterone, cortisol, corticosterone, and estradiol have much lower potency (EC₅₀, 0.5 to 5 µmol/L), three or four orders of magnitude lower than that of aldosterone. For instance, the glucocorticoid cortisol is ineffective at doses up to 0.1 µmol/L (Fig 1b). Significant differences for steroid effects were found at the concentration next to or below its EC₅₀. No differences between rat and rabbit VSMCs were found in terms of their response to aldosterone, fludrocortisone, DOCA, and cortisol. The classic mineralocorticoid antagonist spironolactone at 10 µmol/L does not block the effect of 10 nmol/L (1000-fold lower concentration) aldosterone if given 5 minutes before aldosterone (Fig 1c). The same result was seen if rat VSMCs were pretreated with 10 µmol/L spironolactone for 30 minutes, in that 10 nmol/L aldosterone then elevated [Ca²⁺]_i from normal mean basal levels (115±13 versus 117±8 nmol/L in separate untreated control VSMCs) to levels seen in the absence of the antagonist. The mean increase of [Ca²⁺]_i by aldosterone in the absence of spironolactone was 41±7 compared with 39±6 nmol/L in the presence of spironolactone (n=5, P=NS).

View larger version (21K): [in this window] [in a new window]   Figure 3. Dose-response curves showing the effects of different steroids on [Ca2+]i in rat aortic vascular smooth muscle cells under conditions described in Fig 1. The increase of [Ca2+]i above baseline was read 200 to 250 seconds after the addition of steroid, by which time [Ca2+]i had reached a stable plateau (see Fig 1). This value (the difference between the baseline and plateau level of [Ca2+]i) was plotted even if an initial "overshoot," which is rarely seen, might have exceeded the plateau level. Lines are hand drawn and represent values for (from left to right) aldosterone, fludrocortisone, deoxycorticosterone acetate (DOCA), progesterone/corticosterone, and cortisol. Data are mean±SEM from nine experiments with aldosterone or four experiments in other studies. *P<.05, P<.01 vs baseline. For sake of clarity, all significances are shown for aldosterone, but for other steroids only the lowest concentration at which a significant effect was observed is indicated.

Effects of Aldosterone in Ca²⁺-Free Medium: Influence of Other Agents
To test the importance of extracellular Ca²⁺ for the intracellular Ca²⁺ signal induced by aldosterone, experiments were performed in Ca²⁺-free buffer. Under these conditions, basal [Ca²⁺]_i is slightly lower than in separate control experiments (83±12 nmol/L versus 116±9 nmol/L, P=NS); the initial aldosterone-induced increase of [Ca²⁺]_i is largely unaffected, although [Ca²⁺]_i quickly falls almost back to basal levels (Fig 2). The mean maximal response is 26±4 nmol/L (P<.001 versus baseline value, n=5), thus being only insignificantly lower than the plateau response in the presence of Ca²⁺ (41±7 nmol/L). The response can be reproduced by a higher aldosterone concentration (Fig 2). The type of Ca²⁺ channels involved remains unclear as the L-type Ca²⁺ channel blocker D-verapamil (10 µmol/L) does not affect the response to aldosterone (not shown). To assess a possible contribution of the Na⁺-Ca²⁺ exchanger and the Na⁺-proton antiport to the effect of aldosterone on [Ca²⁺]_i, experiments were performed in Na⁺-free medium (replaced by choline chloride) in which these ion transport systems were inactivated. A normal response of [Ca²⁺]_i to aldosterone (increase from 102±7 to 143±27 nmol/L, n=4) was seen (Fig 4a) in the absence of Na⁺. In the presence of the general Ca²⁺ channel blocker La³⁺, the initial rise of [Ca²⁺]_i is also visible but blunted (not shown).

The relevance of intracellular Ca²⁺ stores for the Ca²⁺ signal was studied by pretreatment of cells (Fig 4b) with 1 µmol/L thapsigargin for 15 minutes, which depletes intracellular Ca²⁺ stores by inhibition of Ca²⁺ reuptake into organelles such as endoplasmic reticulum. Thereby, thapsigargin produces a large rise in [Ca²⁺]_i, reflecting the emptying of intracellular Ca²⁺ stores, and subsequently blocks the response of [Ca²⁺]_i to aldosterone.

The involvement of protein kinase C in the effects of aldosterone on [Ca²⁺]_i was investigated indirectly by maneuvers that either stimulate or inhibit this enzyme. In short-term preincubations, PMA (0.1 µmol/L) stimulates protein kinase C; this blunts the [Ca²⁺]_i response from a maximum aldosterone (10 to 1000 nmol/L) effect of 41±7 to 13±6 nmol/L (n=4, P<.05).

In contrast, preincubation with PMA (0.1 µmol/L) for 20 hours (ie, conditions under which protein kinase C is partially downregulated) tends to augment the response to 53±8 nmol/L (P=NS, Fig 5a), whereas basal [Ca²⁺]_i was not affected by PMA.

View larger version (22K): [in this window] [in a new window]   Figure 5. Determination of [Ca2+]i in rat aortic vascular smooth muscle cells as described in Fig 1. The effects of pretreatment with phorbol myristate acetate (PMA) for 20 minutes or 20 hours (a) and with staurosporine (b) on the rise in [Ca2+]i induced by aldosterone are depicted. Representative tracings from experiments run in quadruplicate are shown, except for panel b, where the experiment with the smallest increase is chosen.

On the other hand, inhibition of protein kinase C by 0.1 µmol/L staurosporine increases the maximum intracellular Ca²⁺ response to aldosterone almost fourfold, to 168±32 nmol/L (n=4, P<.001, Fig 5b). Pretreatment with PMA and staurosporine tended to increase basal [Ca²⁺]_i, but differences were not significant (116±9 for separate control experiments with untreated VSMCs versus 142±30 nmol/L for pretreatment with PMA and 145±22 nmol/L for staurosporine pretreatment). However, it cannot be excluded that the absolute increase of [Ca²⁺]_i induced by aldosterone after short-term treatment with PMA is only smaller than in control experiments because basal [Ca²⁺]_i levels are slightly elevated under these conditions.

Pertussis toxin (1 µg/mL, pretreatment for 4 hours), an inhibitor of G-protein species, which play a key role in cellular signaling for various agonists, affected the response of [Ca²⁺]_i to aldosterone at borderline significance. The mean increase of [Ca²⁺]_i was 25±7 in the presence of pertussis toxin versus 41±7 nmol/L for control experiments (P<.06, n=6).

Because IP₃ has been reported to participate in rapid steroid effects,^{7 8} it was tested to determine whether the inhibition of phospholipase C (the enzyme generating IP₃) by neomycin (300 µmol/L) affects the response of intracellular Ca²⁺ to aldosterone; as expected, this compound completely blocks the effect of aldosterone (Fig 6a).

View larger version (17K): [in this window] [in a new window]   Figure 6. Effects of pretreatment of rat aortic vascular smooth muscle cells by 300 µmol/L neomycin for 15 minutes (a) or 100 µmol/L genistein for 30 minutes (b) before the addition of 100 nmol/L aldosterone. Representative tracings of [Ca2+]i from four experiments are given.

Platelet-derived growth factor (PDGF) has been shown to increase [Ca²⁺]_i in VSMCs through the activation of a tyrosine kinase,^{17 18 19 20} which is sensitive to inhibition by genistein. Similarly, this compound almost completely inhibits the action of aldosterone on [Ca²⁺]_i in VSMCs (Fig 6b). The maximum change of [Ca²⁺]_i was only 11±2 nmol/L in the presence of 100 or 200 µmol/L genistein (pretreatment for 30 minutes, n=14), which was significantly different from that found with 41±7 nmol/L in the presence of aldosterone alone (P<.001). Basal [Ca²⁺]_i in these experiments was insignificantly elevated from 124±9 (control) to 151±12 nmol/L.

To mimic an interaction with potential physiological relevance in vitro, the combined effects of aldosterone and angiotensin II on [Ca²⁺]_i were investigated in VSMCs. Pretreatment of VSMCs with 100 nmol/L aldosterone affected the threshold concentration for the Ca²⁺ response to angiotensin II. In Fig 7, it can be seen that 10^-13 mol/L angiotensin II raised [Ca²⁺]_i in the presence of aldosterone. However, even 10^-12 mol/L angiotensin II was ineffective without aldosterone. Angiotensin II at 10^-9 mol/L, added as a positive control, finally led to a marked increase of [Ca²⁺]_i in this experiment. Threshold increases of [Ca²⁺]_i of >25 nmol/L by angiotensin II were seen at a concentration of 10^-13 mol/L (six of seven experiments) in the presence of 100 nmol/L aldosterone. In the absence of aldosterone, 10^-13 mol/L angiotensin II did not significantly increase [Ca²⁺]_i in four of four experiments. Without aldosterone, threshold effects (increases of [Ca²⁺]_i of >25 nmol/L) were obtained at 10^-12 to 10^-11 mol/L angiotensin II.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The aim of the present study was to explore the possibility of rapid nongenomic effects of aldosterone on free intracellular Ca²⁺ in single cultured rat and rabbit VSMCs.

The main findings of the present study are as follows: (1) An immediate increase of [Ca²⁺]_i was seen in response to aldosterone, reaching a plateau after 3 to 5 minutes. The apparent EC₅₀ was 0.1 nmol/L. (2) Fludrocortisone was active at concentrations similar to those of aldosterone, whereas cortisol and other glucocorticoids were ineffective at concentrations up to 0.1 µmol/L. (3) The classic inhibitor of mineralocorticoid action, spironolactone, did not block aldosterone effects at concentrations up to 1000-fold higher than those of aldosterone.

The results reported here are perfectly in line with previous studies involving the rapid aldosterone effects both in HMLs and VSMCs. A significant stimulation of IP₃ production was found in these cells as early as 30 to 60 seconds after application of aldosterone, and rises in [Ca²⁺]_i were expected to occur in relation to the effect of aldosterone on this important intracellular second messenger. The time courses of the effects of aldosterone on IP₃ and [Ca²⁺]_i (as well as on the Na⁺-proton exchanger) in VSMCs were similar, as were the apparent EC₅₀ values of 0.1 nmol/L. Aside from this rapid time course, which is clearly incompatible with an involvement of the classical genomic pathway of steroid action, the specificity of the effects of aldosterone on [Ca²⁺]_i mirrors that shown by the other nongenomic mineralocorticoid effects studied to date and that shown for aldosterone membrane binding. This specificity includes the high affinity of the process for aldosterone, being 1000- to 10 000-fold more active than the glucocorticoids cortisol or corticosterone, and the insensitivity of the effect to 1000-fold excess concentrations of the classic mineralocorticoid antagonist spironolactone. These similarities of the effects of aldosterone at various subcellular levels suggest that we are looking at different elements of the same intracellular signaling process.

The maximum rise of [Ca²⁺]_i was 40 nmol/L, an effect that is small in comparison with that of other agonists such as angiotensin II or thrombin,²¹ which produce increases of several hundred nanomoles per liter. The latter agonists also induce a more immediate rise of [Ca²⁺]_i, which reaches its peak within a few seconds rather than within a few minutes as seen here. The effect of aldosterone on [Ca²⁺]_i in VSMCs is more reminiscent of the slower rise in [Ca²⁺]_i induced by PDGF, involving phosphorylation of phospholipase C through a receptor tyrosine kinase.^{17 18 19 20} This assumption is supported by the inhibition of the effect by the specific inhibitor of tyrosine kinases, genistein.

However, even if compared with the PDGF-induced increase of [Ca²⁺]_i, which is not as large as that induced by thrombin or angiotensin II, the effect of aldosterone on [Ca²⁺]_i is still much smaller. The effects of aldosterone shown here thus expose unique features and may represent the first example of a novel intracellular signaling cascade.

The modest effect of aldosterone may have been overlooked or misinterpreted as artifact in earlier studies. For example, Petzel et al²² have shown a small rapid increase (20 to 30 nmol/L) of [Ca²⁺]_i after the application of aldosterone to D6 cells from toad bladder. This rapid increase is followed by a considerably larger later spike in [Ca²⁺]_i, and the findings are discussed exclusively in terms of genomic actions.

The physiological implications of such a relatively small change may be considerable. For example, small continuous increases of intracellular electrolyte concentrations are the cellular correlate of the clinically relevant inotropic effect of ouabain, the Na⁺,K⁺-ATPase inhibitor, in that the effect of digitalis on the intracellular Na⁺ does not exceed 10% to 20% at maximum inotropic response of the myocardium to ouabain.²³ In guinea pig aortas,²⁴ a linear correlation between tension and [Ca²⁺]_i that would roughly predict a 50% increase of tension at a 50% to 60% increase of [Ca²⁺]_i has been described. Caffeine-induced contracture in pig intercostal muscle fibers was 20% of maximum at an increase of [Ca²⁺]_i from 80 to 150 nmol/L.²⁵ Thus, changes in [Ca²⁺]_i observed in the present study might be correlated with changes in contractile responses that are not large but are significant when considered in concert with other physiological stimulators of muscular contraction, such as angiotensin II. This assumption is further supported by the observation of a sensitization of VSMCs to the effects of angiotensin II on [Ca²⁺]_i by aldosterone as shown here.

Although no experimental data are yet available, the effector system described in the present study is a candidate mediator for the cardiovascular effects of rapid postural changes in aldosterone plasma levels.²⁶ A recent study has also demonstrated a nongenomic effect of aldosterone on baroreceptor discharge, similarly suggesting the cardiovascular relevance of the immediate effects of aldosterone.²⁷

Earlier reports of the nongenomic effects of progesterone on [Ca²⁺]_i in human sperm and Xenopus oocytes^{28 29} have described EC₅₀ values of 0.1 and 3 µmol/L; whether or not the effector mechanism involved was that described in the present study remains to be determined.

Preemptying intracellular Ca²⁺ stores by thapsigargin completely blocked the effect of aldosterone on [Ca²⁺]_i, indicating that release of Ca²⁺ from intracellular stores such as endoplasmic reticulum is essentially involved in this response. On the other hand, in Ca²⁺-free buffer the aldosterone-induced increase of [Ca²⁺]_i is still visible, but no plateau is maintained. These findings point to an additional involvement of extracellular Ca²⁺ in the effect of aldosterone on [Ca²⁺]_i.

D-Verapamil does not affect the response to aldosterone; these data are consistent with the observation of low levels of expression of verapamil-sensitive L-type Ca²⁺ channels in early-passage VSMCs as opposed to much higher levels of expression of T-type channels in these cells, for which no specific inhibitor is yet known.³⁰

The inhibitor of phospholipase C, neomycin, blocks the response to aldosterone, supporting earlier evidence for an involvement of the phosphoinositide breakdown in HMLs⁷ and VSMCs.⁸ The apparent modulation of aldosterone effects on [Ca²⁺]_i by PMA, which stimulates protein kinase C after short-term incubation, and by staurosporine, which inhibits protein kinase C, may suggest a participation of this enzyme in the signaling process. However, a baseline shift, a relatively simple explanation for the apparently blunted response after short-term PMA treatment, and the limited significance of perturbations in comparably small signals should be taken into account here, and a more excessive analysis of these data seems unwarranted.

Data on the effects of pertussis toxin and genistein point to a possible involvement of both G proteins and a tyrosine protein kinase function in the signaling cascade. Though still hypothetical at the moment, the existence of an intermediate tyrosine protein kinase in smooth muscle cells that is triggered through a special G protein has been postulated by Hollenberg.³¹ Our results are compatible with this hypothesis, although more detailed work on the nature of the initial steps in the intracellular signaling is necessary.

As shown for HMLs,⁵ aldosterone may affect cellular volume through effects on Na⁺-proton exchange, and effects on [Ca²⁺]_i could follow indirectly. An increase of [Ca²⁺]_i in hypoosmolar media resulting in cell swelling was demonstrated in bovine glomerulosa cells.³² However, we assume that the contribution of volume changes in the effects of aldosterone on [Ca²⁺]_i is minor, if relevant, since the inhibition of Na⁺-proton exchange in Na⁺-free medium, which should reduce volume shifts, did not block the Ca²⁺ response to aldosterone. However, an augmentation of the direct effects of aldosterone on [Ca²⁺]_i by volume shifts cannot be excluded.

In summary, the results presented are further evidence for a novel pathway of mineralocorticoid action, now characterized at various levels of the receptor-effector cascade: membrane receptors,^{10 11} phosphoinositides,^{7 8} and now Ca²⁺ and, indirectly, protein kinase C. The implications of, and possible physiological roles for, the "low ceiling" signaling process in VSMCs remain to be explored, as does the possibility of cross talk between the genomic and nongenomic effects of steroid, given the increasing evidence for second messenger–related modulation of transcriptional processes.^{33 34 35} Given the fact that this process is operating at low physiological concentrations of aldosterone (0.1 nmol/L in humans³⁶ ), mineralocorticoids could be effective in peripheral circulatory regulation through an alternative pathway of steroid action involving VSMCs as the main peripheral cardiovascular effector cells.

	Acknowledgments

 
This study was supported by the Wilhelm-Sander-Stiftung (88.015.2/3), the Deutsche Forschungsgemeinschaft (We 1184/4-2, We 1184/6-1, and Sc 9/4-2), and the National Heart and Medical Research Council of Australia. Dr Wehling is on sabbatical leave from the Medizinische Klinik, Klinikum Innenstadt, University of Munich, as a recipient of a Heisenberg scholarship from the Deutsche Forschungsgemeinschaft.

	Footnotes

 
Reprint requests to Martin Wehling, MD, Medizinische Klinik, Klinikum Innenstadt, University of Munich, Ziemssenstr 1, 80336 Munich, FRG.

Received June 7, 1994; accepted February 15, 1995.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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