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(Hypertension. 1995;25:117-123.)
© 1995 American Heart Association, Inc.

Articles

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

Michael Christ; Kathrin Douwes; Christoph Eisen; Günther Bechtner; Karl Theisen; Martin Wehling

From the Medizinische Klinik, Klinikum Innenstadt, University of Munich (FRG).

	Abstract

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Abstract 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 µmol/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.

Key Words: ion transport • aldosterone • inositol 1,4,5-trisphosphate • carrier proteins • steroids • muscle, smooth, vascular • sodium channels

	Introduction

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The Na⁺-H⁺ exchanger of the cell membrane is a key membrane transport system for the control of intracellular pH and Na⁺ in vascular smooth muscle cells (VSMCs).¹ Although rapid activation of the exchanger by mitogens such as epidermal growth factor, platelet-derived growth factor, and phorbol esters² points to its possible involvement in cell growth, its activation by vasoconstrictors such as angiotensin II (Ang II) or by -thrombin suggests its regulatory significance for various other functions.^{3 4} In addition, the glucocorticoid hydrocortisone stimulates the Na⁺-H⁺ antiport of VSMCs after a latency of at least 4 hours,⁵ presumably by the classic genomic pathway of steroid action. It also induces a phenotypic change of the VSMC and inhibits proliferation of cultured rat aortic VSMCs, if proliferation is induced by 10% fetal calf serum (FCS).⁵ These actions are likely to involve binding of steroids to classic intracellular receptors and modification of transcription, translation, and protein synthesis and are characterized by a latency of more than 2 hours.

Besides these classic, genomic mechanisms, there is increasing evidence for rapid nongenomic steroid actions, including neural effects after local application of steroids and fast effects of steroids on the -aminobutyric acid A receptor, on luteinizing hormone–releasing peptide secretion, on dopamine release, on oocyte maturation, and on the acrosome reaction in spermatozoa (for review, see Reference 6⁶ ). Recently, a rapid stimulation of the Na⁺-H⁺ antiport in human mononuclear leukocytes (HMLs) by aldosterone⁷ has been shown, characterized by pharmacological and kinetic properties identical with those of radioactive binding of ¹²⁵I-labeled aldosterone to plasma membranes from HMLs.⁸

In the present study, we investigated rapid steroid effects on the Na⁺-H⁺ antiporter and inositol 1,4,5-trisphosphate (IP₃) levels in rat VSMCs. As a hypothesis, a rapid stimulation of the Na⁺-H⁺ antiporter in these cells by aldosterone would suggest that not only known agonists such as Ang II or platelet-derived growth factor but also aldosterone may be involved in circulatory regulation through similar mechanisms, including vasoconstriction or cell growth.

	Methods

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Materials
Steroids, actinomycin D, cycloheximide, bumetanide, and trypan blue were obtained from Sigma Chemical Co. Canrenone was from Societá Prodotti Antibiotici, and ethylisopropylamiloride (EIPA) was kindly provided by Hoechst AG. Neomycin, U-73122, and U-73343 were from Biomol. ²²Na⁺ was obtained from New England Nuclear. Minimum essential medium, Waymouth's 752/1 medium (WM-752), Ham's F-12 nutrient mixture (H-F12), antibiotics, FCS, and trypsin-EDTA were from GIBCO BRL GmbH; elastase and Ang II were from Boehringer Mannheim GmbH; collagenase (CLS I) was from Worthington Biochemical (distributor, Pansystems); and soybean trypsin inhibitor was from Serva Feinbiochemica GmbH. The monoclonal mouse antibody for the -actin isoform of smooth muscle was purchased from Progen Biotechnik. Ouabain, Tris, HEPES, bovine serum albumin, and other reagents (analytical grade) were from E Merck. The IP₃ assay kit (TRK 1000) was purchased from Amersham Buchler.

Isolation and Primary Culture of Rat VSMCs
VSMCs were prepared from enzymatically dissociated rat thoracic aortas by an adaptation of the method described by Gunther et al⁹ for rat mesenteric arteries. All procedures were carried out under aseptic conditions. Male Sprague-Dawley rats (220 to 250 g) on standard laboratory chow and tap water ad libitum were killed by cervical dislocation after CO₂ anesthesia. After sternotomy, the thoracic aorta was excised and placed in ice-cold minimum essential medium with standard amounts of antibiotics and 25 mmol/L HEPES (pH 7.4). After removal of fat, adventitia, and venous structures by blunt dissection in a Petri dish, the vessel was cut longitudinally. The intima was gently removed with a scalpel. With the use of two fine forceps, the medial layer was stripped off and placed in a centrifuge tube containing 5 mL enzyme dissociation mixture (1 mg/mL collagenase [169 U/mg], 0.25 mg/mL elastase [105 U/mg], and 0.375 mg/mL soybean trypsin inhibitor [76 U/mg]). After incubation at 37°C for 75 to 90 minutes in a shaker bath, the suspension was gently triturated in a 12-gauge stainless steel needle and the reaction terminated with 20% FCS. From an aliquot of the digestion suspension, the number of cells was determined in a hemocytometer, and the enzymes were removed from the supernatant after centrifugation (120g, 10 minutes). After resuspension of the pellet in culture medium (50% WM-752, 50% H-F12) supplemented with 10% heat-inactivated FCS and standard amounts of antibiotics according to Dartsch et al,¹⁰ cells were seeded in plastic tissue culture flasks (Costar Corp) at a density of approximately 10⁴ cells per centimeter squared and incubated at 37°C in a humidified atmosphere of 6% CO₂/94% air. Cell viability determined by trypan blue exclusion was approximately 80%, and plating efficiency of primary cultures ranged from 35% to 60%. After 24 hours, the cultures were washed once with WM-752/H-F12 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 (Zeiss). Confluent monolayers of primary cultures typically formed within 10 to 12 days.

VSMCs were harvested by incubation with trypsin-EDTA (0.05% trypsin, 0.02% EDTA [wt/vol]) for 4 to 5 minutes and passaged at a density of 10⁴ cells per centimeter squared in 75-cm² culture flasks; plating efficiency ranged from 94% to 98%. For influx experiments, cells between passages 4 and 10 were plated into 35-mm six-well culture dishes (Greiner Labortechnik) and refed every other day. VSMCs showed a typical hill-and-valley configuration and stained positive with a specific antibody against the -actin isoform of smooth muscle.

Measurement of ²²Na⁺ Influx
²²Na⁺ influx in VSMCs was measured by a method adapted from Vallega et al.⁴ Cells were refed with WM-752/H-F12 medium, 10% FCS, and standard amounts of antibiotics until 48 hours before experiment. In experiments labeled "10% FCS," 10% FCS was continued until cells were used. In experiments with growth-arrested cells labeled as "0.4% FCS," FCS was reduced to 0.4% for 48 hours before the experiments. In experiments labeled as "0% FCS," FCS was kept at 0.4% from 24 to 48 hours before the experiment and then completely removed. For influx experiments, the dishes were washed three times with an HCO₃^--free and Na⁺-free Tris-buffered salt solution containing (mmol/L) choline chloride 130, KCl 5, CaCl₂ 2, MgCl₂ 1, glucose 10, and Tris-HEPES 20 (pH 7.0) at 37°C and preincubated with this buffer for 25 minutes. Subsequent incubation of VSMCs with steroids was in the same buffer for 4 minutes in the presence of 1 mmol/L ouabain and 0.1 mmol/L bumetanide. Stock solutions of steroids at a concentration of 10 mmol/L were prepared in ethanol, and maximal ethanol concentration in experiments was 0.01%. Incubation with vehicle alone had no influence on Na⁺ influx of VSMCs. The preincubation buffer was then replaced by medium containing 1 µCi/mL ²²Na⁺ and (mmol/L) ouabain 1, bumetanide 0.1, NaCl 100, choline chloride 30, KCl 5, CaCl₂ 2, MgCl₂ 1, glucose 10, and Tris-HEPES 20 (pH 7.4) to which 60 µmol/L EIPA and/or steroids were added as indicated. The EIPA concentration was based on previous studies indicating an inhibition constant (K_i) of 39 nmol/L for the inhibition of the Na⁺-H⁺ antiport in VSMCs by EIPA.¹¹ After 3 minutes, ²²Na⁺ influx was terminated by rapid aspiration of the medium followed by six rapid washes with ice-cold 100 mmol/L MgCl₂ to remove extracellular radioactivity. The dishes then were treated with 1.5 mL of 0.2% sodium dodecyl sulfate and 0.1 mmol/L NaOH to solubilize VSMCs and collect intracellular radioactivity. Radioactivity was counted in a gamma counter (Hewlett-Packard) at an efficiency of approximately 80%. The total protein content of every set of dishes was determined by the Peterson modification of the Lowry procedure¹² in quadruplicate.

Measurement of IP₃ in VSMCs
Levels of IP₃ in HMLs were measured by a radioreceptor assay kit according to Sato et al.¹³ Briefly, VSMCs were trypsinized for 1 to 2 minutes, washed in HEPES-buffered WM-752/H-F12 medium (20 mmol/L, pH 7.4), and resuspended at a concentration of 11x10⁶ to 12x10⁶ cells per milliliter. After preincubation of the cells at 37°C, addition of aldosterone or Ang II started the stimulation of IP₃. The reaction was stopped with 50 µL ice-cold 20% perchloric acid, and the mixture was kept on ice for 20 minutes. After centrifugation, the supernatant was titrated to pH 7.5 with 1.5 mmol/L KOH and 60 mmol/L HEPES in siliconized tubes according to the method of Palmer et al.¹⁴ After centrifugation, IP₃ levels of the supernatants were measured with a radioreceptor assay kit.

Statistical Analysis
The two-sided Mann-Whitney U test for unpaired data was used, with results presented as mean±SEM. Values of P<.05 were considered significant.

	Results

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Rapid Aldosterone Effects on EIPA-Sensitive ²²Na⁺ Influx
To minimize the effects of internal Na⁺ on the activity of the Na⁺-H⁺ antiport, rat VSMCs were preincubated in an isosmotic Na⁺/HCO₃^--free buffer at pH 7.0. Under these conditions, internal Na⁺ decreases and the cells are acidified to a resting pH of approximately 6.8. On readdition of Na⁺, there is a rapid influx of Na⁺ that is linear for the first minute and then slightly decreases⁴ over the next 5 minutes.

The short-term effect of aldosterone on sodium influx was tested for different FCS concentrations in the various media used over the 48 hours before the experiment (Fig 1). With 10% FCS in the growing medium, basal Na⁺ influx of VSMCs was 26.2±0.9 nmol/mg protein per minute and decreased to 9.2±0.6 nmol/mg protein per minute with 60 µmol/L EIPA in the medium. After addition of 1 nmol/L aldosterone to the medium for 4 minutes, values for Na⁺ influx were 26.7±1.1 nmol/mg protein per minute and 9.9±0.4 nmol/mg with 60 µmol/L EIPA, not significantly different from Na⁺ influx obtained without aldosterone (Fig 1, top). In growth-arrested VSMCs (48 hours in medium containing 0.4% FCS; Fig 1, middle), basal Na⁺ influx increased (P<.05) to 34.2±0.9 nmol/mg protein per minute, and addition of aldosterone 4 minutes before measurement did not significantly alter Na⁺ influx (34.7±0.84 nmol/mg protein per minute). After incubation with 60 µmol/L EIPA, Na⁺ influx was also not different (9.7±0.9 and 10.3±0.8 nmol/mg protein per minute without and with 1 nmol/L aldosterone, respectively).

View larger version (19K): [in this window] [in a new window]   Figure 1. Bar graphs show stimulation of Na+ influx in rat vascular smooth muscle cells by aldosterone. Na+ influx was measured after incubation of cells in 10% fetal calf serum (FCS, top), after growth arrest for 48 hours with 0.4% FCS (middle), and after growth arrest with 0.4% FCS from 24 to 48 hours before experiment and subsequent incubation without FCS until cells were used (bottom). Cells were incubated in incubation buffer alone (INC) or with 1 nmol/L aldosterone (ALDO) ±60 µmol/L ethylisopropylamiloride (EIPA) (ALDO and ALDO-EIPA, respectively) for 4 minutes before application of Na+ influx measuring medium. EIPA-sensitive Na+ influxes were calculated by subtraction (INC-EIPA and ALDO-EIPA).*P<.05, incubation buffer vs aldosterone. Mean±SEM, n=9.

Finally, VSMCs were growth arrested with 0.4% FCS from 24 to 48 hours before experiments and then incubated without FCS until cells were used (Fig 1, bottom). VSMCs remain attached to the tissue plastic dishes and do not alter their morphological appearance under these conditions. A constant number of cells per well was demonstrated for 24 hours of incubation without FCS. The percentage of vital cells remained constant under these conditions, as shown by trypan blue exclusion (not shown).

Basal Na⁺ influx decreased significantly (P<.05) to 22.1±1.8 nmol/mg protein per minute compared with values after incubation for 48 hours with 0.4% FCS. Aldosterone (1 nmol/L) significantly increased Na⁺ influx to 28.6±1.5 nmol/mg protein per minute when added to the preincubation medium 4 minutes before the addition of the radiotracer. Basal Na⁺ influx after incubation with EIPA with and without aldosterone was 9.8±1.3 and 9.7±1.1 nmol/mg protein per minute, respectively. Thus, aldosterone stimulates EIPA-sensitive Na⁺ influx, which is significantly (P<.05) increased from 12.3±1.6 to 18.9±1.3 nmol/mg protein per minute, whereas aldosterone does not influence EIPA-insensitive Na⁺ influx. The effect of aldosterone was almost maximal after 5 minutes, with a small further increase after 15 and 30 minutes (not shown). To investigate a possible involvement of phospholipase C in this rapid effect of aldosterone, we preincubated VSMCs with the specific inhibitors of phospholipase C, neomycin (300 µmol/L) or U-73122 (10 µmol/L), for 25 minutes. Aldosterone (10 nmol/L) was not able to stimulate the sodium-proton antiporter under these conditions, whereas preincubation with U-73343 (10 µmol/L), an inactive congener of U-73122, did not inhibit aldosterone-induced stimulation of EIPA-sensitive sodium influx (Table). Control values of sodium influx in the presence of these inhibitors alone were not different from the baseline values without inhibitors.

View this table: [in this window] [in a new window]   Table 1. Effects of Phospholipase C Inhibition on Aldosterone-Induced Increases of EIPA-Sensitive Na+ Influx and Intracellular IP3 Levels in Rat VSMCs

EIPA-sensitive Na⁺ influx was stimulated by 1 nmol/L aldosterone despite the presence of the inhibitors of transcription and protein synthesis, actinomycin D (5 µg/mL) and cycloheximide (10 µg/mL). Na⁺ influx stimulation was 89.8±3.9% (actinomycin D) and 104.9±6.8% (cycloheximide) of influx stimulation with aldosterone alone (three experiments in triplicate).

Dose-Response Studies for Aldosterone and Other Steroids
Increasing aldosterone concentrations resulted in a dose-related stimulation of EIPA-sensitive Na⁺ influx when VSMCs were growth arrested with 0.4% FCS from 24 to 48 hours before experiments and then incubated without FCS until cells were used (Fig 2). The half-maximal effect (EC₅₀) of aldosterone occurs at a concentration between 0.1 and 0.5 nmol/L; fludrocortisone and deoxycorticosterone were also active at concentrations between 0.1 and 100 nmol/L, with EC₅₀ at approximately 0.5 nmol/L (Fig 3). Hydrocortisone (Fig 3) did not stimulate EIPA-sensitive Na⁺ influx in rat VSMCs up to concentrations of 1 µmol/L. EIPA-insensitive Na⁺ influx was not significantly affected by any of the steroids tested, with a mean value for all measurements of 9.8±1.2 nmol/mg protein per minute.

View larger version (20K): [in this window] [in a new window]   Figure 2. Bar graph shows dose-response curve for aldosterone effects on the Na+-H+ antiport (ethylisopropylamiloride [EIPA]–sensitive Na+ influx) in vascular smooth muscle cells after incubation for 4 minutes. Incubation conditions were the same as in Fig 1 (bottom). Mean±SEM, n=8.

View larger version (19K): [in this window] [in a new window]   Figure 3. Line graph shows dose-response curves for fludrocortisone (FLUDRO), deoxycorticosterone (DOCA), and hydrocortisone (HYDRO) effects on ethylisopropylamiloride (EIPA)–sensitive Na+ influx (percent of control) in vascular smooth muscle cells after incubation for 4 minutes. Incubation conditions were the same as in Fig 1 (bottom). Mean±SEM, n=6.

Incubation of VSMCs with 1 nmol/L aldosterone plus 0.1 or 1 µmol/L canrenone did not result in values different from those after incubation with aldosterone alone, and 0.1 and 1 µmol/L canrenone alone were inactive in terms of EIPA-sensitive Na⁺ influx (Fig 4).

View larger version (19K): [in this window] [in a new window]   Figure 4. Bar graph shows effect of canrenone on rapid aldosterone action in vascular smooth muscle cells. Cells were incubated for 4 minutes in incubation buffer alone (INC), 1 nmol/L aldosterone (ALDO), 1 nmol/L aldosterone plus 0.1 µmol/L canrenone (ALDO+0.1 C) or 1 µmol/L canrenone (ALDO+1 C), and 0.1 µmol/L (0.1 C) or 1 µmol/L (1 C) canrenone alone. Ethylisopropylamiloride (EIPA)–sensitive Na+ influx was measured and calculated under the same incubation conditions as in Fig 1 (bottom). Mean±SEM, n=4.

Effects of Aldosterone on Intracellular IP₃ Levels
The basal intracellular IP₃ content of VSMCs was 3.41±1.03 pmol per 10⁶ cells (n=6). During incubation of the cells in WM-752/H-F12 medium containing 0.01% ethanol (maximal ethanol concentration), a stable baseline of intracellular IP₃ levels was obtained. Aldosterone (10 nmol/L) significantly increased (P<.05, Fig 5) intracellular IP₃ levels to a maximum of 6.07±2.02 pmol per 10⁶ cells (166.7±9.7%) after 30 seconds, whereas 100 nmol/L Ang II increased intracellular IP₃ to a maximum of 7.32±1.93 pmol per 10⁶ cells (197.9±21.9%) after 15 seconds (Fig 5). Fig 6 shows the dose-response curve for the effect of aldosterone on intracellular IP₃ levels. Aldosterone was active at concentrations higher than 0.1 nmol/L, and maximal IP₃ levels were reached at concentrations of 10 nmol/L. EC₅₀ of aldosterone was seen at approximately 0.5 to 1 nmol/L. Aldosterone increased intracellular IP₃ levels despite inhibition of the Na⁺-H⁺ exchanger by 60 µmol/L EIPA (data not shown). Hydrocortisone did not stimulate IP₃ generation at concentrations up to 1 µmol/L (Fig 6).

View larger version (21K): [in this window] [in a new window]   Figure 5. Line graph shows time course of inositol 1,4,5-trisphosphate stimulation in vascular smooth muscle cells by aldosterone and angiotensin II. Cells were incubated in HEPES-buffered Waymouth's 752/1 medium and Ham's F-12 medium plus 0.01% ethanol (maximum vehicle concentration during steroid stimulation) alone (control), with 10 nmol/L aldosterone, or with 100 nmol/L angiotensin II. Levels of inositol 1,4,5-trisphosphate were measured by a specific radioreceptor assay kit (see "Methods"). Mean±SEM; six experiments were run in triplicate (*P<.05 vs control).

View larger version (21K): [in this window] [in a new window]   Figure 6. Line graph shows dose-response curve for inositol 1,4,5-trisphosphate stimulation by aldosterone and hydrocortisone in vascular smooth muscle cells after incubation for 30 seconds. Six (aldosterone) and three (hydrocortisone) experiments were run in triplicate (mean±SEM).

Preincubation of cells with neomycin (300 µmol/L) or U-73122 (10 µmol/L) but not with U-73343 (10 µmol/L) abolished aldosterone effects on intracellular IP₃ levels (Table). After preincubation of the cells with these inhibitors alone, intracellular IP₃ levels were not different from the baseline values without inhibitors.

	Discussion

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The aim of the present study was to explore the possibility of rapid, nongenomic stimulation of the Na⁺-H⁺ exchanger by aldosterone in rat VSMCs, an effect that has already been shown in HMLs.⁷

The main findings of the study are as follows: (1) A significant stimulation of the Na⁺-H⁺ exchanger by aldosterone as determined by EIPA-sensitive ²²Na uptake was seen after 4 minutes when VSMCs were incubated in serum-free medium for 24 hours before the experiments. The apparent EC₅₀ was between 0.1 and 0.5 nmol/L. (2) Fludrocortisone and deoxycorticosterone were active at concentrations similar to that of aldosterone, whereas hydrocortisone was ineffective at concentrations up to 1 µmol/L. (3) The classic inhibitor of mineralocorticoid action, canrenone, did not block aldosterone effects at concentrations up to 1000-fold higher than that of aldosterone. (4) Aldosterone significantly stimulated IP₃ generation within 30 seconds at a slightly higher EC₅₀ value, whereas hydrocortisone was ineffective at concentrations up to 1 µmol/L. (5) Specific inhibitors of phospholipase C (neomycin and U-73122) inhibit aldosterone-induced stimulation of both EIPA-sensitive Na⁺ influx and intracellular IP₃ levels, suggesting an involvement of phospholipase C in rapid aldosterone effects. U-73343, an inactive congener of U-73122, did not block these effects.

Basal Na⁺ influx values of proliferating VSMCs (cells grown in 10% FCS) are in good agreement with Vallega et al⁴ and Berk et al.⁵ The EIPA-sensitive Na⁺ influx is stimulated to approximately 150% in growth-arrested VSMCs⁴ (0.4% FCS), although the intracellular pH is more alkaline in proliferating than in growth-arrested VSMCs.¹⁵ The exact mechanisms responsible for this long-term activation of Na⁺-H⁺ antiport in growth-arrested cells are unknown, but downregulation of protein kinase C as seen after in vitro addition of serum or phorbol esters seems to play a key role. Thus, an increased Na⁺-H⁺ antiport activity appears to be a characteristic feature of the growth-arrested phenotype of VSMCs.¹⁵

Aldosterone stimulates the Na⁺-H⁺ antiport in VSMCs within a few minutes if incubation with FCS is discontinued 24 hours before experiments, whereas there is no effect in cells fed with 10% or 0.4% FCS before the experiment. Basal EIPA-sensitive Na⁺ influx in these "starving" VSMCs is lower than in growth-arrested cells, perhaps reflecting incomplete removal of serum factors in the latter group. The activation of the Na⁺-H⁺ antiport by growth factors contained in FCS occurs via intracellular signaling mechanisms involving diacylglycerol and protein phosphorylation,¹⁶ and incubation for 24 hours in serum-free medium removes these growth factors from the cells and reduces basal protein phosphorylation levels,¹⁷ thought to be associated with a lower basal activity of the Na⁺-H⁺ antiport. Thus, aldosterone appears to stimulate the antiport only if basal activity is low.

The physiological relevance of the restriction of the aldosterone effect to starved VSMCs is still unclear. In vivo, a protection of VSMCs from growth factors and steroids by the endothelial barrier either by specific transport mechanisms or by nonspecific lipophilic separation could play a role. Another hypothesis would imply that in vitro, for technical and statistical reasons, the effect of aldosterone on the Na⁺-H⁺ antiport is visible only if increased to its maximum by creating a low basal level of the Na⁺-H⁺ antiport activity. In vivo, even smaller changes might be physiologically relevant for the "fine tuning" of VSMC activity in connection with other cardiovascular hormones.

Long-term effects of mineralocorticoids on electrolyte balance are commonly understood as results of their renal effects. These are thought to be mainly located at the distal tubule involving genomic mechanisms and both transcriptional and translational processes. Given the polarity of the membrane distribution of Na⁺,K⁺-ATPase, transcellular transport of sodium and potassium is typical of these cells.¹⁸ Besides these renal actions of mineralocorticoids, there is increasing evidence for extrarenal corticosteroid action, eg, on VSMCs. For instance, deoxycorticosterone acetate induces changes in vascular reactivity to vasoconstrictors, which initiate development of systemic vascular resistance in unilateral nephrectomized pigs,¹⁹ suggesting the existence of direct actions of mineralocorticoids in VSMCs. Hydrocortisone induces morphological changes in rat VSMCs, decreases the proliferative response of VSMCs to 10% FCS, and stimulates the Na⁺-H⁺ antiport within 12 hours.⁵ Actinomycin D and cycloheximide inhibit these responses, indicating the involvement of the classic genomic pathway of steroid action. These effects have been explained by an increase of vasoconstrictor-induced IP₃ generation as second messenger.¹³ Nongenomic, fast effects were not observed by Berk et al,⁵ presumably because of the FCS present in the preincubation period and hydrocortisone concentrations of "only" 2 µmol/L; 1 µmol/L hydrocortisone has been shown in the present study to be insufficient for a rapid activation of the Na⁺-H⁺ antiport.

In addition to these genomic, late-onset actions, there is increasing evidence for rapid nongenomic effects of steroids in various tissues. In renal cells, aldosterone induces an intracellular alkalinization within 20 minutes by stimulation of the Na⁺-H⁺ antiport.²⁰ Extrarenal, rapid actions of mineralocorticoids have been reported by Moura and Worcel,²¹ who demonstrated a late Na,K-ATPase–dependent and early ouabain-independent efflux of ²²Na⁺artery VSMCs after aldosterone injection. Late, ouabain-inhibitable effects were blocked by actinomycin D, implying a genomic aldosterone action, whereas ouabain- and actinomycin D–independent ²²Na⁺ efflux was stimulated as early as 15 minutes after aldosterone application, indicating a nongenomic effect.

Successful identifications of high-affinity steroid binding sites in preparations of pituitary membranes,²² liver membranes,^{23 24} neuronal membranes,²⁵ and recently HMLs⁷ support the hypothesis of nongenomic steroid action involving plasma membrane receptors different from the classic intracellular steroid receptors. In addition, functional studies have shown rapid, nongenomic steroid effects in other cells, eg, neurons,²⁶ and effects on neuronal luteinizing hormone–releasing hormone release²⁷ and on the acrosome reaction in spermatozoa.²⁸ Dufy et al²⁹ observed a rapidly increased Ca²⁺-dependent spiking activity in pituitary cells after estradiol application. Progesterone-induced rapid Ca²⁺ mobilization appears to be involved in meiotic maturation of the oocyte.³⁰

In HMLs, changes of intracellular sodium, potassium, and calcium and accompanying shifts of water and volume 1 hour after aldosterone application were found^{31 32 33} and appeared to depend on a primary activation of the sodium-proton antiport within 1 to 2 minutes.⁷ The time interval between aldosterone application and stimulation of the Na⁺-H⁺ antiport in HMLs is too short to be compatible with a genomic response. Actinomycin D, an inhibitor of transcriptional processes, was not able to block this effect. Thus, the rapid action of aldosterone was assumed to result from a direct interaction with specific membrane receptors for aldosterone. Subsequently, such receptors could be shown by radioactive binding studies to plasma membrane preparations from HMLs.^{8 34} Binding characteristics, including a high aldosterone selectivity over hydrocortisone and canrenone, were distinctly different from that of the classic type I receptor, which does not separate aldosterone from hydrocortisone.³⁵ In addition, a rapid stimulation of intracellular IP₃ by aldosterone could be demonstrated in HMLs, exposing pharmacological properties similar to those of Na⁺-H⁺ antiporter stimulation and membrane binding.³⁶

The data on aldosterone effects in VSMCs presented here share major similarities with the receptor-effector mechanisms for nongenomic aldosterone action in HMLs. This includes the rapid onset of stimulation of the EIPA-dependent Na⁺ influx, an effector selectivity for aldosterone that has an at least 1000-fold higher activity than hydrocortisone and canrenone, and the ineffectiveness of the inhibitors of transcription and protein synthesis, actinomycin D and cycloheximide. A rapid stimulation of IP₃ production by aldosterone could be demonstrated in VSMCs, with effects being similar to those observed earlier in HMLs.³⁶ As in HMLs, a selectivity for aldosterone versus cortisol was found as a "landmark" characteristic. The EC₅₀ for the effect of aldosterone on IP₃ production was slightly higher than for the effect on sodium-proton exchange (0.5 to 1 versus 0.1 to 0.5 nmol/L), an insignificant difference that could reflect the deteriorating effect of trypsinization in the case of IP₃ determination. This effect of aldosterone on IP₃ production and the inhibition of aldosterone-induced stimulation of Na⁺ influx and intracellular IP₃ levels by neomycin and U-73122 may indicate a possible involvement of phospholipase C and the phosphoinositide pathway in the intracellular signaling for rapid aldosterone effects. These findings are in agreement with the data of Steiner et al,³⁷ who previously showed steroid-induced breakdown of phosphoinositides in rat VSMCs.

With regard to the functional consequences of aldosterone-induced stimulation of IP₃ levels, effects of aldosterone on free intracellular calcium have been studied in single VSMCs. Effects on calcium are seen that are almost immediate, reach a plateau after 3 to 5 minutes only, and are characterized by high specificity for mineralocorticoids versus glucocorticoids (unpublished data, 1994). Aldosterone is an agonist with an estimated apparent EC₅₀ of 0.1 nmol/L, whereas progesterone, cortisol, corticosterone, and estradiol have much lower potency (EC₅₀ of approximately 0.5 to 5 µmol/L). The effect of aldosterone is blocked by neomycin and short-term treatment with phorbol esters but is augmented by staurosporine, indicating an involvement of phospholipase C and protein kinase C. The calcium effect appears biphasic, with the release of intracellular calcium as shown by the inhibitory effect of thapsigargin, followed by the influx of extracellular calcium. These data and those presented in the present study are in perfect agreement and support the assumption that intracellular signaling of rapid aldosterone effects involves both IP₃ and calcium.

It is obvious that membrane receptors for aldosterone, but not the classic type I mineralocorticoid receptors, are ideal candidates for the transmission of rapid aldosterone effects (for review, see Reference 38³⁸ ). In addition, the EC₅₀ of aldosterone effects in HMLs and VSMCs of approximately 0.1 to 0.5 nmol/L is close to the physiological concentration of free aldosterone in human (approximately 0.1 nmol/L³⁹ ) and rat (approximately 0.2 nmol/L⁴⁰ ) plasma, thus pointing to a possible physiological cardiovascular relevance of the effector mechanism in VSMCs.

The findings of the present study represent further evidence for the existence of a novel rapid pathway for nongenomic aldosterone action. Immediate postural responses of aldosterone plasma levels would be without effect in a slow-reacting, genomic effector system. However, they could be effective for the peripheral regulation of circulation through an alternative pathway of mineralocorticoid action involving the VSMCs, the main peripheral cardiovascular effector cell.

	Acknowledgments

 
The study was supported by the "Wilhelm-Sander-Stiftung" (88.015.2) and the "Deutsche Forschungsgemeinschaft" (We 1184/4-2Sc4/9-4). We thank Janet Aktas and Katrin Sippel for expert technical assistance.

	Footnotes

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

Received January 31, 1994; first decision March 2, 1994; accepted October 3, 1994.

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