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Aldosterone Increases T-Type Calcium Currents in Human Adrenocarcinoma (H295R) Cells by Inducing Channel Expression

Division of Endocrinology and Diabetology, (O.L., M.F.R.) Department of Internal Medicine, Laboratory of Clinical Chemistry (A.C., M.F.R.), Department of Pathology, University Hospital, Geneva 14, Switzerland

Address all correspondence and requests for reprints to: Dr. Michel F. Rossier, Division of Endocrinology and Diabetology, University Hospital, 24 rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland. E-mail: rossier{at}cmu.unige.ch

	Abstract

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In adrenal glomerulosa cells, low-threshold voltage-activated (T-type) calcium channels are known to play a crucial role in coupling physiological variations of extracellular potassium to aldosterone biosynthesis. On the other hand, aldosterone itself has been recently shown to regulate Ca²⁺ currents in its target cells. In the present study, we have investigated the effect of aldosterone on Ca²⁺ channels of the steroidogenic human adrenocarcinoma cell line, using both electrophysiological and molecular techniques. Cell incubation with aldosterone (1 µM) for 24 h increased by 39% the density of T-type calcium currents, as assessed with the patch clamp technique. This effect of aldosterone was not related to a modification of T channel activation and inactivation properties. In contrast, L-type calcium currents remained unaffected by aldosterone treatment. The mineralocorticoid receptor antagonist, spironolactone, blunted the aldosterone-induced increase in T-type calcium current. By RT-PCR, we detected in human adrenocarcinoma cells the presence of mRNA coding for the ₁ subunits of three different calcium channels: the ₁H isoform of T channels and the ₁C and ₁D isoforms of the L channels. The presence of mRNA coding for the mineralocorticoid receptor was also found in these cells. Aldosterone treatment induced a 36% increase of mRNA coding for ₁H, as assessed by real-time PCR. This aldosterone-evoked stimulation of mRNA expression was maximal at 24–48 h and reversed by spironolactone, suggesting a receptor-mediated genomic effect of aldosterone. Pregnenolone production in response to KCl stimulation was increased after aldosterone treatment, in parallel to T channel expression, confirming the essential role of these channels in the steroidogenic response to potassium. Taken together, these data indicate that, in human adrenocarcinoma cells, aldosterone increases, through an autocrine pathway, the expression of T-type calcium channels and therefore modifies the ability of these cells to respond to steroidogenic agonists.

	Introduction

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VOLTAGE-DEPENDENT CALCIUM CHANNELS control the rapid entry of Ca²⁺ ions into a wide variety of cell types and are therefore involved in both electrical and cellular signaling. Early electrophysiological studies have identified two major classes of Ca²⁺ channels, namely the high voltage- and the low voltage-activated channels (1, 2) with the latter being also identified as T-type Ca²⁺ channels (3). T-type Ca²⁺ channels were originally defined by their activation at low membrane potential, their fast kinetics of inactivation, and their small unitary conductance (4, 5).

These channels have been identified in a large variety of neurons, and it has become obvious that significant functional diversity exists in the gating behavior of T-type channels, particularly in their inactivation kinetics, voltage dependence of steady-state inactivation, and pharmacology (6). The recent identification of several novel genes encoding a subset of homologous Ca²⁺ channel ₁ subunits, i.e. the ₁G (7, 8), the ₁H (9, 10) and the ₁I isoforms (11), has revealed that diversity of T-type voltage-dependent calcium channels is primarily related to the expression of distinct ₁ subunits. Indeed, the expression of the ₁G and ₁H isoforms produces Ca²⁺ currents with the typical signature of T-type channels but with specific features, such as a different sensitivity to block by Ni²⁺, which discriminates between ₁G and ₁H currents (12).

Angiotensin II (AngII) and potassium ion (K⁺) are major regulators of calcium influx into adrenal glomerulosa cells, a crucial step in the stimulation of aldosterone production. Both stimuli are able to maintain a sustained influx of Ca²⁺ into these cells. AngII induces a biphasic response of the cytosolic free Ca²⁺ concentration ([Ca²⁺]_c). An initial transient rise owing to inositol 1,4,5-trisphosphate-induced release of Ca²⁺ from the intracellular stores is followed by a sustained plateau phase, resulting from the activation of the capacitative influx triggered by the depletion of intracellular Ca²⁺ pools (13, 14). AngII also activates voltage-operated Ca²⁺ channels of both T- and L-types by inducing cell depolarization through inhibition of K⁺ channels. Calcium influx through these channels also contributes to the sustained Ca²⁺ entry triggered by AngII (15, 16). In bovine and human glomerulosa cells, the presence of both high-threshold, long-lasting (L-type) and low-threshold, transient (T-type) voltage-operated Ca²⁺ channels has been demonstrated (17, 18).

Recently a clear dissociation between L- and T-type channel functions in these cells has been established. Indeed, Ca²⁺ entering through each channel appears to have distinct functions and destinations (19). L-type channels appeared to be the major mediators of the large [Ca²⁺]_c variations observed in response to low, physiological concentrations of extracellular K⁺, whereas the cytosolic Ca²⁺ signal resulting from T channel activation was barely detectable. However, inhibition of L-type channels by selective pharmacological drugs did not markedly affect steroidogenesis, as demonstrated by Barrett et al. (20) and by our own laboratory (19). In contrast, in these and other studies, T-type channel activity has been shown to be more closely related to aldosterone production. For example, T channel inhibition with the relatively specific alkaloid tetrandrine (21) or with mibefradil (22) strongly reduced aldosterone production.

Although the stimulation of aldosterone production by AngII and K⁺ and the involvement of calcium channels in this process have been extensively documented (14, 23, 24), much less is known about the modulation of calcium channels by aldosterone in aldosterone-producing cells. The goals of the present study were therefore to investigate the effect of aldosterone on calcium channels in steroidogenic cells and to determine whether aldosterone exerts an autocrine feedback control on its own production. For this purpose, we used the human adrenocarcinoma (H295R) cell line (25) for two main reasons: (1) the human origin of these cells allowed us to design PCR experiments directly from sequences of the various channel isoforms currently available in databases, and (2) in preliminary studies, these cells appeared to express T channels at a higher level than freshly prepared bovine glomerulosa cells maintained in primary culture. On one hand, we used the patch-clamp technique to characterize the properties of calcium channels present in H295R cells and to evaluate the effect of aldosterone on their T-type calcium channel activity. On the other hand, by a molecular approach using real-time PCR, we examined the expression of calcium channels under aldosterone treatment.

	Materials and Methods

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Reagents and test substances
Aldosterone, spironolactone, ACTH, tetrodotoxin, sodium ATP, sodium GTP were purchased from Sigma (St. Louis, MO) and AngII from Bachem AG (Bubendorf, Switzerland). Cs₄-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid was obtained from Molecular Probes, Inc. (Eugene, OR). SYBR Green kits for RNA amplification on Light Cycler were purchased from Roche Diagnostics (Rotkreuz, Switzerland) and the EZ-rTth system for conventional PCR from PE-Biosystems (Rotkreuz, Switzerland). [³H]pregnenolone was obtained from NEN Life Science Products (Geneva, Switzerland), antipregnenolone antiserum from Biogenesis Ltd. (Poole, UK) and WIN 19758 from Farillon (London, UK). IGF-I was a generous gift from Dr. Jean-Paul Thissen, Université Catholique de Louvain, Belgium.

H295R cell culture
H295R cells have been obtained from Dr. W. E. Rainey (University of Texas, Dallas). Cells were grown in Dulbecco’s modified Eagle’s and Ham’s F12 media 1:1 (vol/vol) supplemented with 15 mM HEPES. Complete DMEM-F12 was prepared by adding 1% ITS Plus (insulin, transferrin, selenium; Collaborative Biomedical Product, Bedford, MA), 120 UI/ml penicillin and 120 µg/ml streptomycin (Life Technologies, Inc., Gaithersburg, MD), 0.5 µg/ml Fungizone, 6 IU/ml nystatin, 40 µg/ml Garamycin and 2% Ultroser SF (BioSepra SA, Villeneuve-la-Garenne, France).

In the present study, H295R adrenocortical cells were grown in 150-cm² flasks and incubated at 37 C in a humidified atmosphere containing air/carbon dioxide (95%/5%, vol/vol). The medium was changed every 3 d and cells were subcultured after detaching them with 0.25% trypsin every 7 d.

Before experiments, cells were plated into culture Petri dishes or on glass coverslips. Cells were then maintained overnight in complete DMEM-F12 but without ITS and Ultroser before starting aldosterone and/or spironolactone 24-h treatment. For longer treatment periods, fresh ITS- and serum-containing medium was added on the first day and maintained for 3 d. Aldosterone (1 µM) or spironolactone (20 µM) were added at different times during the treatment. Serum was removed 6 h before experimental stimulation (pregnenolone production) or cell harvesting for RNA extraction, but steroids were maintained during this period.

Patch-clamp experiments
The activity of voltage-operated Ca²⁺ channels in single H295R cells was recorded under voltage clamp in the whole-cell configuration of the patch clamp technique, as previously described (26). The bath solution contained 117 mM tetraethylammonium chloride, 20 mM BaCl₂, 0.5 mM MgCl₂, 5 mM D-glucose, 32 mM sucrose, and 200 nM tetrodotoxin and was buffered to pH 7.5 with 10 mM HEPES (CsOH). The patch-pipette (3–6 megohm; Clark 150T, Reading, UK) contained 85 mM CsCl, 10 mM tetrabutylammonium chloride, 6 mM MgCl₂, 5 mM sodium ATP, 0.04 mM sodium GTP, 0.9 mM CaCl₂, and 11 mM Cs₄-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; pH was adjusted to 7.2 with 20 mM HEPES (CsOH). The reference electrode was placed in a KCl solution linked to the bath with an Agar bridge; the resulting liquid junction potential was smaller than 2 mV and has been neglected. The cell was voltage clamped (Axopatch 1D, Axon Instruments Inc., Foster City, CA) at a holding potential of -90mV and depolarized as indicated. Cells were relatively homogenous in size with a membrane capacitance of 6.88 ± 1.57 picofarads (mean ± SD, n = 34). The Ba²⁺ currents were filtered at 1–2 kHz and sampled at 5 kHz using a TL-1–125 interface (Axon Instruments). The leak was subtracted automatically by a P/4 protocol (pclamp6, Axon Instruments Inc.).

RNA extraction
Total RNA isolation from 1 million H295R cells in culture was performed using the RNAgents Total RNA isolation system kit (Promega Corp., Madison, WI), as indicated in the manufacturer’s instructions. Dry RNA pellets were dissolved in nuclease-free water and stored frozen at a concentration of 50 ng/µl.

Messenger RNA extraction was carried out using the Dynabeads mRNA direct kit (DynAl A.S., Oslo, Norway) from 2 million to 4 million homogenized cells. Finally, mRNA was stored frozen in 20 µl 10 mM Tris-HCl.

RT-PCR procedure
Conventional RT-PCR was performed using the "one step, one enzyme" EZ-rTth system from PE-Biosystems following manufacturer’s instructions except for the enzyme amount and cycling conditions. Briefly, first-strand cDNA was generated by loading 3 µl of extracted mRNA in the master mix (50 µl) containing 0.5 µM specific primers for calcium channels (Table 1) and other reagents, as specified by the manufacturer. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts were reverse transcribed and analyzed in parallel to evaluate mRNA integrity. Reverse transcription (RT) was achieved by incubation at 61 C for 25 min after a 5 min-step at 50 C. A 3-min denaturation step at 94 C then preceded 45 PCR cycles according to the following protocol: denaturation at 94 C (30 sec), annealing at 54 C (30 sec), and elongation at 72 C (1 min). Amplified fragments were then resolved on a 2% agarose gel.

Quantification by real-time RT-PCR on LightCycler
A one-step conventional RT-PCR protocol has been adapted for the LightCycler (Roche Diagnostics AG). Individual glass capillaries were filled with a solution containing 9 µl RT-PCR mix and 2 µl total RNA template (50 ng/µl). The reaction mix was composed of primer oligonucleotides (0.5 µM), MgCl₂ (5 mM), LightCycler RT-PCR enzyme mix and LightCycler RT-PCR reaction mix/SYBR Green I, itself containing reaction buffer, dNTP, and SYBR Green I dye at concentrations optimized by the manufacturer. The RT of the RNA template occurred during 5 min at 54 C and was followed by a 2-min denaturation of cDNA at 95 C. The amplification of target cDNA was then performed for 35–50 cycles according to the following steps: denaturation at 95 C (1 sec), annealing at 54 C (5 sec), and elongation at 72 C (7 sec). After each elongation step, the temperature was raised to 83 C to measure SYBR Green fluorescence at a temperature preventing a contribution of primer dimers. At the end of the PCR, a melting curve analysis was performed by gradually increasing temperature from 63 C to 95 C (0.1 C/sec). Moreover, at the end of some experiments, RT-PCR products were removed from capillaries and analyzed by gel electrophoresis to confirm the presence and assess the purity of the amplicons of interest.

After PCR was completed, the SYBR Green fluorescent signal was analyzed and converted into a relative number of copies of target molecules. For this purpose, the results of a series of standards prepared by successive dilutions and plotted against the logarithm of the concentration were used to estimate the relative amount of specific mRNA initially present in the various samples. Each sample was analyzed in quadruplicate.

Pregnenolone measurement
Pregnenolone production was determined by direct RIA in the medium of cells incubated in a Krebs-Ringer buffer containing either 3 mM (basal) or 12 mM KCl (stimulated). For this purpose, H295R cells were cultured for 3 d in 24-well plates (500,000 cells/well) in the presence of aldosterone (1 µM) or spironolactone (20 µM) for various periods. At the time of stimulation, cells were washed with a Krebs-Ringer buffer (136 mM NaCl, 5 mM NaHCO₃, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 1.8 mM KCl, 1.2 mM CaCl₂, 5.5 mM D-glucose, buffered to pH 7.4 with 20 mM HEPES) and incubated for 90 min in the same buffer, supplemented or not with 9 mM KCl (12 mM final concentration). To prevent the conversion of pregnenolone to progesterone, WIN 19758 (5 µM) was present in each well throughout the stimulation. At the end of the incubation period, medium was removed for pregnenolone assay, as previously described (27), and adherent cells were washed and detached in NaOH (0.5 M) to determine protein content by the Coomassie blue method (Bio-Rad Laboratories, Inc., Reinach, Switzerland). The polyclonal antibody used for measuring pregnenolone was highly specific with very low cross-reactivity to spironolactone (0.0016%) and to aldosterone (undetectable).

Statistics
If not otherwise indicated, data are expressed as mean ± SE in the text and in the graphs. The statistical significance of the changes induced by aldosterone or other treatments was analyzed by unpaired t tests.

	Results

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Voltage-activated calcium currents in H295R cells
Calcium channels in the H295R cell line, were analyzed with the patch-clamp technique in the whole-cell configuration, with barium as a charge carrier. Step depolarization from a holding potential of -90 mV to various potentials revealed the presence of rapidly activating inward currents (Fig. 1A) that partially inactivated within 100–150 msec to stabilize at a sustained plateau. The analysis of the amplitude of the peak current as a function of voltage (Fig. 1B) indicated that it was maximal at -5 mV and reversed above 60 mV, with a bell-shape characteristic of voltage-operated calcium currents. The presence of both T- and L-type channels in these cells, suggested by the kinetics of the currents, was then confirmed by the consecutive activation of both low threshold (T-type) and high threshold (L-type) currents during a ramp depolarization from -110 to +40 mV (Fig. 1C).

View larger version (18K): [in this window] [in a new window]   Figure 1. Voltage-gated calcium currents in H295R cells. Currents were measured with the patch-clamp technique in the whole-cell configuration, as described in Materials and Methods, and barium was used as charge carrier. A, Inward currents were evoked by step depolarization (100 msec duration) of increasing amplitude from a holding potential of -90 mV up to +70 mV with 20-mV increments. Leak currents were automatically subtracted with a P/N protocol (pClamp 6.0). B, Current-voltage relationship of the peak current shown in A. C, Low- and high-threshold inward currents were subsequently elicited by a unique ramp depolarization from -110 up to +40 mV for a period of 2 sec. D, Inward current was activated by a step depolarization from -90 to 0 mV and maintained for 600 msec. The decay of the current was best approximated by a biexponential function with two time constants, of 25.6 and 160 msec. The amplitude of the L current was measured between 450 and 500 msec after depolarization (when most of T channels are inactivated) and amounted to 46.4 pA in this particular cell. E, Slowly deactivating (T-type) current was elicited upon repolarization of the cell to -65 mV after a short period (20 msec) of channel activation at +20 mV. The time constant of the slowly decaying tail current in this cell was 3.4 msec. The amplitude of the maximal current, occurring at the time of cell repolarization, was determined by extrapolating the tail current fitted to a single exponential and amounted to 875 pA. Cell capacitance was 8.8 pF. These currents are representative of currents recorded in more than 80 individual H295R cells.

The tail current through T channels was also determined in the same cell, by measuring slowly deactivating Ca²⁺ currents ( = 3.5 msec) evoked upon repolarization of the cell to -65 mV after a 20-ms depolarization period at +20 mV (Fig. 1E), as previously described (26). Currents were recorded during T channel deactivation, after L channels had almost completely closed (within the first 3 msec after cell repolarization). Then they were fitted to single exponential functions and the values of T currents present at the time of cell repolarization were determined by extrapolation. The density (amplitude per capacitance unit) of tail currents owing to T channels in H295R cells was relatively high (24 ± 3 pA/pF at -65 mV), compared with that generally measured under the same conditions in bovine adrenal glomerulosa cells (10 pA/pF) or in newborn rat ventricular cardiomyocytes (1 pA/pF) in primary culture.

Analysis of voltage-dependent activation and steady-state inactivation of the tail currents in this particular H295R cell showed that half of the channels are activated at -26 mV and inactivated at -49 mV (not shown). These values are close to those previously determined for T channels in bovine glomerulosa cells (28).

Effect of aldosterone treatment on T- and L-type calcium currents
We observed a significant increase in T-type current amplitude on treatment of the cells for 24 h with 1 µM aldosterone, an effect completely reversed in the presence of the MR antagonist spironolactone (Fig. 2). This increase of the current was not owing to variations in cell size after treatment, as assessed by the measurement of the cell capacitance, that amounted to 6.8 ± 0.4 pF for control cells (n = 11), 6.6 ± 0.5 pF for aldosterone-treated cells (n = 12), and 7.3 ± 0.4 pF for aldosterone and spironolactone-treated cells (n = 9). Moreover, data were normalized to the membrane capacitance and current densities rather than current amplitudes were analyzed.

View larger version (29K): [in this window] [in a new window]   Figure 2. Effect of aldosterone treatment on the density of T- and L-type currents. H295R cells were treated or not for 24 h with aldosterone alone (1 µM) or with aldosterone and spironolactone (20 µM) before being voltage clamped as described in Fig. 1. No serum was present in culture medium during the treatment. The amplitude of T-type and L-type currents (in pA) was determined using the voltage protocols indicated in Fig. 1, E and D, respectively, and then normalized for the cell capacitance (in pF). The resulting current densities obtained for each treatment were then compared (mean ± SEM, n = 9–12 cells from seven independent preparations). Ctrl, Control (untreated) cells; ns, not significantly different. *, Significantly different from control (P < 0.05).

In contrast, L-type calcium current density was unaffected by aldosterone (Fig. 2, right panel), but spironolactone induced a 36% increase of this current, possibly revealing a chronic inhibition of L channels by endogenous aldosterone. The modulation by aldosterone of T and L currents in opposite directions is an additional argument in favor of a specific action of the hormone on the corresponding channels, not simply related to a change of cell size or biophysical properties of the plasma membrane.

Various agonists had been previously investigated in our laboratory for their capacity to modulate calcium currents in these cells. We have found that treatment for 24–72 h with ACTH (100 nM), AngII (100 nM), or IGF-I (500 ng/ml) generally resulted in the inhibition of both T and L current densities (unpublished data). In this regard, ACTH was particularly efficient, reducing T current density by 43% and L current density by 79% (P < 0.05, n = 11). This inhibition could appear quite paradoxical for agonists of aldosterone secretion like AngII and ACTH, but other signaling pathways, in addition to calcium entry through voltage-operated calcium channels, are known to be also involved upon glomerulosa cell stimulation by these hormones (14).

Lack of effect of aldosterone on T channel activation and inactivation properties
To determine whether the apparent increase in T current density induced by aldosterone was owing to a change in the sensitivity of the channel for the membrane potential, tail currents were analyzed to establish T channel activation and steady-state inactivation curves for control and aldosterone-treated cells (Fig. 3). It appeared that the curves obtained with each group are very similar and that channel properties were unaffected by aldosterone treatment. The mean V_1/2 readings for T-channel activation were not significantly different for control (-27.2 ± 6.3 mV) and aldosterone-treated cells (-25.5 ± 1.2 mV). Similarly, V_1/2 readings for channel steady-state inactivation were very close in both cell groups (-54.1 ± 4.0 mV vs. -54.9 ± 1.0 mV for control and treated cells, respectively).

View larger version (16K): [in this window] [in a new window]   Figure 3. Lack of effect of aldosterone on T channel activation and inactivation. H295R cells were treated or not with aldosterone (1 µM) for 24 h and individually analyzed with the patch-clamp technique to determine the voltage-dependent activation and steady-state inactivation properties of their T channels. For this purpose, tail (slowly deactivating, T-type) currents were recorded at -65 mV after 20-msec activation at various potentials or after steady-state inactivation for 10 sec at various holding potential followed by a 20-msec stimulation at +20 mV, as described in detail elsewhere (26 ). The amplitude of the maximal tail current, occurring upon repolarization to -65 mV, was determined and plotted as a function of test potential to be fitted to Boltzman’s equation. The parameters of the equation were then determined and the currents were normalized before being averaged. Activation (, •) and inactivation (, ) curves were established for control (, ) and aldosterone-treated cells (•, ). Data are the mean ± SEM from 11 control and 12 aldosterone-treated cells obtained from seven independent preparations. Voltages of half activation and steady-state inactivation (V1/2) were not significantly different in each group.

View larger version (48K): [in this window] [in a new window]   Figure 4. Identification of the voltage-operated calcium channels expressed in H295R cells. The mRNA was extracted from untreated H295R cells as described in Materials and Methods and then reverse transcribed and amplified by RT-PCR, using sets of primers targeting a region of the 1 subunits (second large intracellular loop, LII-III) that is specific for each of the three isoforms previously characterized for T channels (1H, 1G, and 1I) and for the four L-type channel isoforms (1C, 1D, 1S, and 1F). An additional RT-PCR was performed with primers recognizing a fragment of the GAPDH mRNA. Amplified fragments were then resolved by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. The 1 isoforms tested are indicated at the bottom of the gel and the size of the molecular weigh markers (in base pairs) is shown at the level of the corresponding band. The identity of the four positive bands (1H, 1C, 1D, and GAPDH) has been confirmed by sequencing of the corresponding DNA products.

Real-time PCR of the 1H channel

A typical experiment, showing the evolution of the fluorescent signal as a function of cycle number, is shown in Fig. 5A. Although no fluorescence signal was detected, even after 50 cycles, in negative controls devoid of target mRNA (H₂O), in the other samples, the amount of mRNA present before the amplification dictated the number of cycles required to reach a point in which the fluorescent signal was first recorded as statistically significant above background. Indeed, in a series of successive dilutions of total RNA extracted from untreated control cells, performed to obtain 500, 100, or 20 ng RNA per reaction sample, the most concentrated sample (1) arrived first in the apparent exponential phase of the PCR amplification. A relative standard curve could be then established after setting a user-defined signal threshold (noise band) and analyzing the distribution of the crossing points between the fluorescence curves and this threshold line. A linear standard curve was automatically generated, the slope of this curve reflecting the efficiency of the PCR amplification (not shown). The relative concentration of specific mRNA in unknown samples was extrapolated, using the LightCycler software (v.3) of the instrument.

View larger version (28K): [in this window] [in a new window]   Figure 5. Effect of aldosterone treatment on T channel expression. Total RNA extracted from control or treated H295R cells was quantified by spectrophotometry and its relative content in mRNA coding for 1H was determined by real-time RT-PCR on a LightCycler instrument (Roche Diagnostics), monitoring the amplification reaction with the fluorescent dye SYBR Green I, as described in Materials and Methods. A, Evolution of fluorescence during the PCR process. Standard (S) samples (1 2 3 ) were obtained by successive dilution of RNA extracted from control cells; experimental samples (4 5 6 ) are RNA from cells treated as described in Fig. 2. B, Temperature dependence of SYBR Green I fluorescence determined at the end of the PCR and revealing the phase transition ("melting") of the double-stranded DNA product. Negative derivative of the melting curve is shown for an easier visualization of the transition temperature(s). C, 1H mRNA was quantified by analyzing traces similar to those shown in panel A and obtained with RNA extracted from cells treated or not with aldosterone alone (Aldo, 1 µM) or with aldosterone and spironolactone (Spiro, 20 µM). Each measurement was performed in quadruplicate. Data were then normalized to the amount of mRNA coding for GAPDH present in the same cell extracts and determined with the same technique. Results were expressed as a percentage of the amount of mRNA found in control cells in each experiment (n = 7). Aldosterone treatment did not significantly affect GAPDH mRNA expression. *, Significantly different from control (P < 0.05), ns, not significant.

Because the SYBR Green I dye detects the presence of any double-stranded DNA, the specificity of the PCR products was systematically assessed by analyzing a DNA melting curve obtained at the end of each amplification protocol by slowly increasing the temperature from 63 to 95 C and continuously monitoring the fluorescence. The resulting DNA melting curve (Fig. 5B) shows that all samples (1, 2, 3, 4, 5, 6) display a similar phase transition at approximately 91 C, reflecting a rapid separation of DNA strands at this temperature and therefore the disappearance of dye fluorescence. The negative first derivative of the fluorescence was plotted to better visualize the phase transition. In contrast, the negative control sample (H₂O) showed a much more spread transition occurring at lower temperatures and reflecting the presence of unspecific DNA products, probably primer-dimers, that generally appear in PCR experiments performed with samples devoid of target DNA. This result clearly demonstrates the necessity of recording the fluorescent signal above 82 C (instead of 72 C) during the amplification process to prevent the interference of unspecific products in the quantification of ₁H mRNA.

In preliminary experiments, we have also controlled, by gel electrophoresis, that the PCR products obtained on the LightCycler were composed of a single band of the expected size (data not shown).

Effect of aldosterone on ₁H channel expression
The analysis of mRNA coding for the ₁H channel by real-time PCR revealed that it was significantly increased (by 36 ± 16%) in aldosterone-treated cells and that the effect of the steroid was completely abolished by the presence of spironolactone (Fig. 5C). To minimize errors owing to variations occurring during RNA extraction and quantification, the results were normalized to the amount of mRNA coding in the same sample for GAPDH, a housekeeping gene, the expression of which was unaffected by aldosterone treatment. Analysis of GAPDH was also useful as quality control for the RNA extraction procedure.

Spironolactone reversed the action of aldosterone on both calcium currents and calcium channel expression, suggesting the involvement of the MR. We therefore verified that this receptor is really expressed in H295R cells by RT-PCR performed with specific primers (see Materials and Methods) on cell mRNA. After resolution of the PCR products by gel electrophoresis (Fig. 6), a band at the expected size appeared only in sample in which an RT had been performed before the PCR (RT+), excluding the amplification of a DNA contaminant of the mRNA preparation. The presence of mRNA coding for the MR in H295R cells is therefore in agreement with a genomic action of aldosterone in these cells.

View larger version (82K): [in this window] [in a new window]   Figure 6. Expression of the MR in H295R cells. RT-PCR was performed on mRNA extracted from H295R cells using primers specifically targeting the MR in the presence (RT+) or in the absence (RT-) of reverse transcriptase. Amplified fragments were then resolved by gel electrophoresis and visualized by ethidium bromide staining. The size of the molecular weigh markers is shown at the level of the corresponding band. The identity of the main product has been confirmed by DNA sequencing.

View larger version (24K): [in this window] [in a new window]   Figure 7. Kinetics of aldosterone action on T channel expression and steroidogenesis. H295R cells were incubated in the presence of aldosterone (1 µM, /) or spironolactone (20 µM, /•) for various periods of time before determining 1H mRNA levels by real time RT-PCR (A) or basal (open symbols) and KCl-stimulated (closed symbols) pregnenolone formation (B), as described in Materials and Methods. Channel mRNA was normalized to GAPDH mRNA and expressed as a percentage of the amount found in control (untreated) cells, and steroid production was normalized per milligram cell protein and expressed as fold increase of the basal (unstimulated) and control (no treatment) values that averaged 18.2 ± 5.5 pmol/mg protein per 90 min. Data are the mean values from three independent experiments yielding similar results.

Surprisingly, treatment of H295R cells with spironolactone for 6 h or more markedly increased both basal and KCl-stimulated pregnenolone synthesis (Fig. 7B). Basal pregnenolone levels after 24-h spironolactone treatment were 10.5 ± 0.4-fold those of control cells. A concomitant increase in corticosterone formation was also observed in independent experiments in which the inhibitor of pregnenolone conversion was omitted (data not shown). This effect of spironolactone suggests that endogenously produced aldosterone exerts a strong chronic negative feedback on early steps of steroidogenesis, probably independently of its positive action on T channel expression.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we have shown that aldosterone positively regulates T-type calcium channels in H295R cells, that this modulation is made by a stimulation of the expression of the ₁H channel isoform, and that it is accompanied by a parallel increase in the steroidogenic response of the cells to a challenge with high potassium.

Calcium channels are classically modulated by hormones and neurotransmitters through protein kinases and phosphatases or through G proteins (16, 29, 30). For example, in bovine adrenal glomerulosa cells, AngII has been previously shown to affect T-type channel activity either positively through a G protein-dependent pathway (31), possibly involving the calmodulin-dependent kinase II (32), or negatively through the activation of PKC (26). In each case, the hormone exerted its action on T channels through a leftward or rightward shift of the channel activation curve, a modification probably reflecting the presence of specific phosphorylated residues on the channel itself or on a closely associated protein. In contrast, no change in current properties was observed in H295R cells after aldosterone treatment (Fig. 3).

A control of calcium fluxes through the genomic expression of specific channels therefore appeared as an alternative mechanism by which hormones like aldosterone could exert their effect on their target cells, although probably with a much longer delay. Indeed, although the action of AngII on glomerulosa cell T channels was already observed a few minutes after cell stimulation, a 24-h treatment of H295R cells with aldosterone was required (Fig. 7). This delay of the response as well as the inhibition of aldosterone effect on T channels by spironolactone, an antagonist of the MR, are both in agreement with a genomic action of the steroid. This hypothesis has been strongly reinforced in the present study by the demonstration that the MR is effectively expressed in H295R cells (Fig. 6). Although the presence of MR has been demonstrated in several tissues and organs, including renal collecting duct, distal colon, lung, myocardium, aorta, bone, adipose tissue, lymphocytes, and brain, to our knowledge, this is the first demonstration of the presence of an MR in steroidogenic cells, themselves producing aldosterone. This finding is reinforced by the report of Mazzocchi et al. (33) showing that human adrenal cortex also expresses 11ß hydroxysteroid dehydrogenase type 2, an enzyme that confers specificity for aldosterone to its receptor in mineralocorticoid target tissues.

Aldosterone has been recently shown to increase, in a spironolactone-sensitive manner, calcium currents in rat cardiomyocytes (34), a response that was not observed before 6-h stimulation, but completely developed after 24 h. Prevention of aldosterone effect in cardiomyocytes by actinomycin or cycloheximide was an additional argument in favor of a genomic action of the hormone. Although calcium currents have not been systematically resolved in the latter study, it was assumed that L channels were the main targets of aldosterone in cardiomyocytes because T channels are normally poorly expressed in adult rat heart.

In contrast, in H295R cells, T-type current amplitudes are much higher and discrimination from L currents is made possible because of their slow kinetics of deactivation. Analysis of aldosterone effect on each type of calcium current in these cells clearly demonstrated a specific increase of T-type current density, and L-type currents remained largely unaffected by aldosterone treatment (Fig. 2). In the presence of spironolactone, T current density was reduced slightly below the control levels, suggesting that the basal production of endogenous aldosterone by H295R cells is sufficient to exert a partial stimulation of T channel expression. Interestingly, the MR antagonist increased L current density by approximately 36%, although this effect was not statistically significant. It is nevertheless tempting to propose that endogenously produced aldosterone simultaneously stimulates T channel expression and maintains L channels at their lowest levels. These observations have to be considered in relation to the previous demonstration that T- and L-type channels fulfill distinct functions, at least in bovine adrenal glomerulosa cells (19, 20). Aldosterone appears, therefore, less able to regulate the quantity of calcium entry into the cell than to control the quality of the calcium signal. The physiological relevance of this differential regulation, in terms of plasma membrane electrophysiological properties or local activation of other types of ionic channels, remains however to be evaluated.

A more direct demonstration of the effect of aldosterone on channel expression consisted in measuring the amount of mRNA coding for specific channels (in fact, their ₁ subunit), but this approach previously required the determination of what channel isoforms are actually expressed in H295R cells. By conventional RT-PCR, we found that among the three possible ₁ isoforms coding for T channels, only ₁H was detectable in H295R cells. This finding was supported by the electrophysiological and pharmacological properties of the currents recorded in the same cells. Indeed, the rapid inactivation of the transient current ( = 25 msec at 0 mV) excluded a contribution to this current of ₁I that inactivates (and activates) much more slowly (35) and that is almost exclusively expressed in the nervous system (36). Moreover, the observation that the slowly deactivating current was inhibited by micromolar concentrations of Ni²⁺ (IC₅₀ = 45 µM, not shown) was in favor of the presence of ₁H rather than ₁G that presents an IC₅₀ around 250 µM (12). Recently, ₁H has been found to be the only isoform of T channels expressed in rat and bovine adrenal glomerulosa cells (37).

Although only ₁H was effectively detected in H295R cells under our experimental conditions, we have been recently able to induce the expression of ₁G in the same cells by modifying the composition of the culture medium. The functional consequences of ₁G expression in term of steroidogenesis are currently under investigation in our laboratory.

A precise quantitative analysis of ₁H mRNA by real time RT-PCR allowed us then to show that aldosterone induced an increase in the amount of channel transcripts that is in the same proportion as the increase in current density. Because the electrophysiological properties of the channel were apparently not affected by aldosterone, we propose that variations in current density observed upon aldosterone treatment are mainly explained by a genomic action of the steroid on channel expression and that the amount of mRNA probably faithfully reflects the amount of channels functionally inserted in plasma membrane, although a definitive conclusion on this matter would require single channel analysis.

In contrast to T channels, less direct correlation was obtained between L-type currents and the expression of L channel isoforms. Firstly, two distinct isoforms coding for L-type channels have been detected in H295R cells, with ₁C being apparently more abundant than ₁D (Fig. 4), but the contribution of each isoform to the current change induced by spironolactone is unknown. Secondly, less amounts of ₁C and ₁D were present in H295R cells, compared with ₁H, making much more difficult an accurate assessment of their levels by real-time RT-PCR. The appearance of primer dimers was a limitation in the precision of the method (a 40% increase corresponding to only a fraction of PCR cycle) and made results much less reproducible. Thirdly, contrary to T channels, L channels are built of several ancillary subunits that participate to channel insertion in plasma membrane (38). It therefore appears that L current density in H295R cells is less likely to be closely related to ₁C or ₁D expression alone.

Finally, we investigated whether ₁H channel induction by aldosterone was responsible for an increased ability of H295R cells to produce steroids. Production of pregnenolone, an early intermediate in the steroidogenic pathway, appeared augmented in response to KCl after 24–48 h of treatment with aldosterone, a modification that paralleled T channel expression. However, the marked increase in basal steroidogenesis observed upon treatment with spironolactone strongly suggests that aldosterone also exerts a sustained negative feedback on this pathway, probably independently of calcium channels, because the latter are mostly closed at 3 mM KCl. A direct action of aldosterone on early steps of steroidogenesis, by inhibiting P450 enzyme or StAR expression, and/or their activity, is a possibility that merits further investigation.

Opposite effects of aldosterone on steroidogenesis—an activation through T channel expression and an inhibition through a distinct mechanism—is rather confusing but certainly not the first example of an hormone simultaneously activating the brakes and the accelerator in a signaling pathway. For instance, in bovine glomerulosa cells, AngII is known to stimulate T-type channels through cell depolarization and to inhibit the same channels through a PKC-dependent pathway (26).

In conclusion, in the adrenocarcinoma H295R cells, aldosterone appears to induce, through an autocrine genomic action involving its own receptor, a qualitative change in calcium influx, favoring calcium flow through T channels in detriment of calcium entry through L channels. The physiological significance of this modulation is still unclear because the specific function(s) of each calcium channel isoform in this particular cell type is not yet completely elucidated. Nevertheless, concerning steroidogenesis, aldosterone appears to exert a complex regulation involving a moderate positive action related to T channel expression, which contrasts with a stronger inhibition of basal steroid production, through still undefined effectors.

	Acknowledgments

 
We are particularly grateful to Dr. N. Lalevée for helpful discussions and to Ms. M. Jayo and G. Dorenter for their excellent technical assistance.

	Footnotes

 
This work was supported by Swiss National Science Foundation Grant 32-58948.99 and by a grant from the Jubiläumsstiftung der Schweizerischen Lebensversicherungs und Rentenanstalt für Volksgesundheit und medizinische Forschung.

Abbreviations: AngII, Angiotensin II; [Ca²⁺]_c, cytosolic free Ca²⁺ concentration; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H295R, human adrenocarcinoma; K⁺, potassium ion; RT, reverse transcription.

Received February 5, 2001.

Accepted for publication June 26, 2001.

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