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J Biol Chem, Vol. 274, Issue 28, 19943-19948, July 9, 1999

Angiotensin II Negatively Modulates L-type Calcium Channels through a Pertussis Toxin-sensitive G Protein in Adrenal Glomerulosa Cells^*

Andrés D. Maturana^§, Andrés J. Casal, Nicolas Demaurex^¶, Michel B. Vallotton, Alessandro M. Capponi, and Michel F. Rossier^**

From the  Division of Endocrinology and Diabetology, Department of Internal Medicine, the  Laboratory of Clinical Chemistry, Department of Pathology, the ^¶ Department of Physiology, and the ^§ Fondation pour Recherches Médicales, University Hospital, 24 rue Micheli-du-Crest,CH-1211 Geneva 14, Switzerland

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In bovine adrenal glomerulosa cells, angiotensin II and extracellular K⁺ stimulate aldosterone secretion in a calcium-dependent manner. In these cells, physiological concentrations of extracellular potassium activate both T-type (low threshold) and L-type (high threshold) voltage-operated calcium channels. Paradoxically, the cytosolic calcium response to 9 mM K⁺ is inhibited by angiotensin II. Because K⁺-induced calcium changes observed in the cytosol are almost exclusively due to L-type channel activity, we therefore studied the mechanisms of L-type channel regulation by angiotensin II. Using the patch-clamp method in its perforated patch configuration, we observed a marked inhibition (by 63%) of L-type barium currents in response to angiotensin II. This effect of the hormone was completely prevented by losartan, a specific antagonist of the AT₁ receptor subtype. Moreover, this inhibition was strongly reduced when the cells were previously treated for 1 night with pertussis toxin. An effect of pertussis toxin was also observed on the modulation by angiotensin II of the K⁺ (9 mM)-induced cytosolic calcium response in fura-2-loaded cells, as well as on the angiotensin II-induced aldosterone secretion, at both low (3 mM) and high (9 mM) K⁺ concentrations. Finally, the expression of both G_o and G_i proteins in bovine glomerulosa cells was detected by immunoblotting. Altogether, these results strongly suggest that in bovine glomerulosa cells, a pertussis toxin-sensitive G protein is involved in the inhibition of L-type channel activity induced by angiotensin II.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Angiotensin II (AngII)¹ and potassium ion (K⁺) are the 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 due 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 (1, 2). Angiotensin II also activates voltage-operated Ca²⁺ channels of both T- and L-types by inducing cell depolarization through inhibition of K⁺ channels. Ca²⁺ influx through these channels also contributes to the sustained Ca²⁺ entry triggered by AngII (1). Extracellular K⁺, by depolarizing the cells, directly activates the voltage-operated Ca²⁺ channels, leading to a sustained Ca²⁺ entry into the cell (3, 4). Paradoxically, Ca²⁺ entry elicited by K⁺ is markedly inhibited upon addition of AngII (5, 6). Although this phenomenon was observed some years ago both in rat and bovine glomerulosa cells, the mechanism of this AngII effect on cytosolic Ca²⁺ homeostasis has never been elucidated.

In bovine 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 (7). Because of their low threshold of activation, T-type channels have been first thought to be the major mediators of the Ca²⁺ response to physiological increases of extracellular K⁺ (3, 8). Various laboratories have subsequently investigated the modulation of T-type channels by AngII, but contradictory results have been published. Lu et al. (9) have observed an increase of T channel activity induced by AngII and mediated by a G_i protein. In fact, they have shown that AngII shifts the activation curve of T channels to more negative voltage values, increasing the size of the permissive window of voltage of the channel and therefore allowing more Ca²⁺ to enter the cell in a steady state manner. In contrast, in our laboratory, we found that AngII shifts the activation curve of T channels to more positive voltages, thus reducing the steady-state current through these channels (10). In this study, Rossier et al. (10) demonstrated that AngII exerts a negative modulation on T channels and that this modulation is mediated by protein kinase C (PKC). In summary, both positive and negative effects of AngII on T channel activity have been observed in electrophysiological experiments performed under similar conditions, but no correlation with [Ca²⁺]_c has been ever obtained.

Recently, a clear dissociation between L- and T-type channel functions has been established. Indeed, Ca²⁺ entering through each channel appears to have distinct functions and destinations (11). Selective inhibition of L-type channels does not markedly affect steroidogenesis, as demonstrated by Barrett et al. (12) with the spider toxin -agatoxin-IIIA, as well as in our own laboratory, using low concentrations of dihydropyridines (13). 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 divalent ion nickel or the relatively specific alkaloid tetrandrine strongly reduced aldosterone production (14). Moreover, L-type channels appear to be the major mediators of the large [Ca²⁺]_c variations observed with fluorescent probes in response to extracellular K⁺, whereas at low, physiological concentrations of the agonist, the cytosolic Ca²⁺ signal resulting from T channel activation is barely detectable (15).

In order to understand the mechanism of the inhibition by AngII of the K⁺-induced cytosolic Ca²⁺ response, we therefore have investigated the modulation of the activity of L-type channels by the hormone, using both patch-clamp and microfluorometry methods.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Percoll was obtained from Amersham Pharmacia Biotech. Amphotericin B, nifedipine, nicardipine, phenylmethylsulfonyl fluoride, Triton X-100, aprotinin, leupeptin, 2-mercaptoethanol, pertussis toxin, and tetrodotoxin were purchased from Sigma; fura-2 acetoxymethyl ester was from Molecular Probes (Eugene, OR); Tween-20 was from Merck (Geneva, Switzerland); glycerol and bromphenol blue were from Fluka (Buchs, Switzerland); and Cell-Tak was from BioReba (Basel, Switzerland). Losartan (DuP753) was a generous gift from Dr. R. D. Smith, DuPont Merck Pharmaceutical Co. Rabbit polyclonal antibodies selectively raised against rat G_i and G_o proteins were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Adrenal Glomerulosa Cell Isolation and Culture-- Bovine adrenal glands were obtained from a local slaughterhouse, and adrenal glomerulosa cells were prepared by enzymatic dispersion, purified on a Percoll density gradient, and, for the patch-clamp and microfluorometry experiments, maintained in culture for 2-4 days on Cell-Tak-coated glass coverslips, as described previously in detail (14). Otherwise, cells were used freshly prepared, after two washes in Krebs-Ringer buffer (137 mM NaCl, 1.8 mM KCl, 1.2 mM KH₂PO₄, 1.25 mM MgSO₄, 5 mM NaHCO₃, 1.2 mM CaCl₂, 5.5 mM D-glucose, 20 mM Hepes, pH 7.4) for [Ca²⁺]_c measurements in cell populations.

Patch-clamp Measurements-- The activity of voltage-operated Ca²⁺ channels in single bovine adrenal glomerulosa cells was recorded under voltage-clamp in the perforated patch configuration of the patch-clamp method, as described previously (13, 16). 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, United Kingdom) contained 130 mM CsCl, 5 mM MgCl₂, and 1 mM CaCl₂, and the pH was adjusted to 7.2 with 20 mM Hepes (CsOH). The pipette solution also contained 0.24 mg/ml amphotericin B, but the tip of the pipette was filled with ionophore-free solution to allow the formation of the seal. The access resistance was reduced within approximately 10 min after seal formation. The reference electrode was placed in a KCl solution linked to the bath with an Agar bridge, reducing the liquid junction potential to negligible values. The cell was voltage-clamped (Axopatch 1D, Axon Instruments, Foster City, CA) at holding potential of 90 mV and depolarized as indicated. The 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).

Cytosolic Free Calcium Measurements-- Cytosolic [Ca²⁺] was determined in freshly isolated bovine glomerulosa cell populations loaded with the fluorescent probe fura-2. After isolation, cells were preincubated at 37 °C for approximately 1 h in Krebs-Ringer buffer. Cells were then washed, resuspended at 10⁷ cells/ml, and incubated in the dark in the same buffer for 45 min in the presence of 2 µM fura-2 acetoxymethyl ester. Dye excess was then washed away and the cells were kept in the dark at room temperature. Samples of 2 × 10⁶ cells were sedimented just before use and resuspended in 2 ml of Krebs-Ringer buffer in a cuvette placed in a thermostatted room at 37 °C. Fura-2 fluorescence was recorded with a Jasco CAF-110 fluorescence spectrometer (Hachioji City, Japan), and [Ca²⁺]_c was determined as described by Grynkiewicz et al. (17).

Cytosolic [Ca²⁺] was also determined in single cultured cells, plated on glass coverslips coated with Cell-Tak. Cells were incubated in a Krebs-Ringer buffer for 45 min at room temperature in the presence of 1 µM fura-2 acetoxymethyl ester. Loaded cells were then washed with the Krebs-Ringer buffer. Fura-2 fluorescence was monitored on an inverted Zeiss Axiovert S100TV microscope coupled to a PTI D104 photometer. The light source came from a Xenon XBO 75-W lamp. The fluorescent signal (excitation 340/380 nm, emission 510 nm) was recorded using the Felix 1.1 software on an AST 386/100 MHz computer.

Determination of Aldosterone Production-- Measurement of aldosterone production from cultured glomerulosa cells was performed as described elsewhere (1). Glomerulosa cells, after 2 days in culture, were incubated overnight in the presence or absence of pertussis toxin (500 ng/ml). The next day, the medium was removed, and cells were incubated for 1 h at 37 °C in multiwell plates containing a modified Krebs-Ringer medium and various concentrations of AngII. At the end of the incubation period, the aldosterone content of the medium was determined by direct radioimmunoassay, using a commercially available kit (Diagnostic Systems Laboratories, Webster, TX).

Extraction and SDS-PAGE Separation of Proteins-- Bovine glomerulosa cells in primary culture, as well as cultured GH₄ cells, were homogenized in lysis buffer (containing 10 mM sodium phosphate, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 1 µg/ml leupeptin) at 4 °C. Extracted proteins were quantified using a protein microassay (Bio-Rad). SDS-PAGE was performed according to the method of Laemmli (18). Extracts of total cellular protein (16 µg/lane) were solubilized in sample buffer (60 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, 10% (v/v) glycerol, and 0.01% (v/v) bromphenol blue) and loaded onto a SDS/polyacrylamide (10%) gel (MiniProtean II system, Bio-Rad). Electrophoresis was performed at 130 V for 1 h.

Blotting and Immunodetection of G Proteins-- SDS-PAGE-resolved proteins were electrophoretically transferred onto a nitrocellulose membrane (Macherey-Nagel, Düen, Germany). The membrane was then incubated in blocking buffer (phosphate-buffered saline containing 0.4% Tween-20 and 5% nonfat dried milk) overnight at 4 °C and then incubated for 1 h in phosphate-buffered saline containing 0.4% Tween-20 with rabbit polyclonal antibodies raised against the  subunits of the G_o and G_i proteins. The membrane was washed with the same buffer and then incubated for 1 h with horseradish peroxidase-labeled goat anti-rabbit IgG (CovalAb, Oullins, France). The membrane was then washed three times for 10 min with phosphate-buffered saline containing 0.4% Tween-20, and the antigen-antibody complex was revealed by enhanced chemiluminescence using a Western blot detection kit and Hyper-ECL film (Amersham Pharmacia Biotech).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Inhibition by Angiotensin II of the Cytosolic Calcium Response Induced by Potassium-- As previously observed in bovine glomerulosa cells (5) and confirmed by others in rat cells (6), 10 nM AngII markedly reduced the sustained elevation of [Ca²⁺]_c elicited upon addition of 9 mM extracellular K⁺ (Fig. 1A). Because challenge with AngII is normally accompanied by Ca²⁺ release from inositol ,4,5-trisphosphate-sensitive intracellular Ca²⁺ pools and by development of a capacitative Ca²⁺ influx, thapsigargin, an inhibitor of the microsomal Ca²⁺/Mg²⁺-ATPases, was added at the beginning of the experiment in order to empty Ca²⁺ stores and to allow an accurate determination of the inhibition of the Ca²⁺ signal by the hormone. The amount of this inhibition was estimated to be 63 ± 15% from 12 independent experiments. The addition of 2 µM nifedipine, a dihydropyridine antagonist, at the end of the experiment achieved to reduce [Ca²⁺]_c down to the level corresponding to 100% inhibition of the voltage-operated Ca²⁺ channels.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Inhibition of the potassium-induced cytosolic calcium response by angiotensin II. A, fura-2-loaded cells were sequentially exposed to 200 nM thapsigargin (Tg), 9 mM KCl, 10 nM AngII, and 2 µM nifedipine. Cytosolic Ca2+ values were calculated as described under "Experimental Procedures." Dotted lines represent maximal and minimal [Ca2+]c values measured before and after complete inhibition of voltage-operated calcium channels, respectively. B, same experiment as in A, but the addition of nifedipine and AngII was inverted. Traces are representative of 12 experiments yielding similar results.

Two mechanisms could be responsible for the decrease of [Ca²⁺]_c induced by AngII: 1) an inhibition of the calcium influx through voltage-operated calcium channels, or 2) an activation of the pumping of calcium out of the cytosol through plasma membrane Ca²⁺/Mg²⁺-ATPases. In order to discriminate between these possibilities, we completely blocked the Ca²⁺ channels opened by K⁺ with 2 µM nifedipine, before the addition of AngII (Fig. 1B). As expected, an almost complete inhibition of the sustained [Ca²⁺]_c response to K⁺ was observed upon addition of nifedipine, and AngII exerted only a very small effect on the residual [Ca²⁺]_c level thereafter, thus excluding a strong activation of the Ca²⁺ pumps by the hormone and therefore privileging an action of AngII on the dihydropyridine-sensitive Ca²⁺ channels.

Inhibition of L-type Currents by AngII-- Single bovine glomerulosa cells were voltage-clamped in the permeabilized patch configuration of the patch clamp technique and both T- and L-type Ca²⁺ channels were activated upon a step depolarization from 90 to 0 mV. In order to discriminate between T and L currents, we maintained the depolarized state at 0 mV for 600 ms (Fig. 2, inset). L current was measured after 500 ms, when most T channels are inactivated, as expected according to their fast inactivation characteristics (time constant,  = 20 ms at 0 mV) and when T current amplitude is therefore negligible.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Inhibition of L-type currents by AngII in bovine glomerulosa cells. Ba2+ currents were recorded from a single voltage-clamped glomerulosa cell maintained in the perforated patch configuration of the patch clamp technique, as described in detail under "Experimental Procedures." Currents were elicited every 30 s by a step depolarization from 90 to 0 mV for 600 ms and L-type current amplitudes were measured after 500 ms, when T-type current contribution is negligible. Inset, example of currents from the same cell and elicited before (control) and after the addition of AngII (100 nM) and nifedipine (1 µM). Data are representative of 14 independently tested cells.

A time-course study of L-type current amplitude (Fig. 2) confirmed its relative stability in the perforated patch configuration. Addition of 100 nM AngII induced a marked inhibition of the current in less than 1 min. Addition of a dihydropyridine antagonist, such as nifedipine or nicardipine, at the end of the experiment blocked the residual current and pharmacologically confirmed the identity of this current. The mean inhibition induced by AngII has been estimated to be 63 ± 11% (n = 14) of the maximal current, a value closely related to the inhibition of the [Ca²⁺]_c signal.

In contrast, the transient current observed during the first 50 ms depolarization and reflecting T channel activity remained unaffected by AngII or nifedipine treatment at 0 mV. Moreover, it is noteworthy that the action of AngII on L current was only rarely observed in the whole cell configuration and that the cells needed to be maintained metabolically intact (in the perforated patch configuration) for AngII to exert its inhibition on L channels.

Washing out the hormone (Fig. 3) allowed recovery of 30 ± 3% of L channel activity (n = 3), whereas addition of losartan (10 µM), a specific AT₁ receptor antagonist, a few minutes after the hormone reversed only 19 ± 5% of the AngII effect (n = 4). This lack of complete reversibility could be attributed to AngII receptor sequestration or endocytosis, as previously suggested by Ambroz and Catt (19).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Reversibility and voltage dependence of L current inhibition. Ba2+ currents were recorded from a single voltage-clamped glomerulosa cell as described in the legend of Fig. 2, and the inhibition induced by AngII was reversed by washing out the hormone from the medium. A, example of trace recordings obtained before (1) or after (2) the addition of 100 nM AngII and after washing out the hormone (3). B, the current-voltage relationship of the L-type Ba2+ current was established under each condition by varying the amplitude of the step depolarization. C, time course of L-current variations induced by AngII or during wash out. Numbers indicate the times at which current-voltage curves were determined and correspond to numbers in A and B.

Fig. 3B also shows the current-voltage relationship of the L-type current before () and after () the addition of 100 nM AngII, as well as after the wash-out of the hormone (). The inhibition of the current exerted by the hormone was observed at any voltage, and AngII did not change the shape of the current-voltage curve; threshold, peak, and reversal potentials of the Ba²⁺ current were not affected by AngII (n = 6).

Losartan Prevents the Inhibition of L-type Current by AngII-- We have previously shown that the presence of losartan (DuP753), prevents the action of AngII on the [Ca²⁺]_c signal induced by extracellular K⁺ (10). Similarly, losartan prevented AngII action on L-type channels. Indeed, as illustrated in Fig. 4, losartan (10 µM) had no effect by itself on the amplitude of L-type currents elicited by a depolarization to 0 mV but completely prevented the AngII-induced inhibition of this current (n = 4). The current remained sensitive to dihydropyridines and was almost completely abolished upon addition of 2 µM nicardipine.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Inhibition by losartan of the AngII effect on the L-current. The amplitude of L current elicited before and after the addition of AngII (100 nM) and nicardipine (2 µM) was determined as described in the legend of Fig. 2, but in the presence of losartan (10 µM). This result is representative of four independent experiments.

To further characterize the molecular mechanism through which AngII exerts its inhibition on L currents, we tested whether protein kinase C (PKC), which is coupled to the AT₁ receptor through the formation of diacylglycerol, is involved in the modulation of L-type channels. We have previously shown that in glomerulosa cells, T-type channels are negatively regulated by PKC and that aldosterone production is concomitantly reduced (10, 20); however, we have also demonstrated that activation of PKC with the phorbol ester phorbol 12-myristate 13-acetate is unable to mimic AngII action on the [Ca²⁺]_c signal induced by K⁺ (13). In agreement with the latter observation, phorbol 12-myristate 13-acetate had no effect on L-type currents and did not prevent the inhibition induced by AngII in 4 individually tested cells (data not shown).

The inhibition of L-type Current by AngII Is Sensitive to Pertussis Toxin Treatment-- In numerous cells, it has been shown that L-type calcium channels can be modulated by pertussis toxin-sensitive G proteins, such as G_i or G_o (21). We therefore treated bovine glomerulosa cells overnight with 500 ng/ml pertussis toxin (Ptx) before recording L-type currents. Under these conditions, we observed a marked reduction of the inhibition of L-type Ca²⁺ channels in response to AngII. Fig. 5 shows a cell in which AngII effect on L-type current was completely abolished by Ptx treatment, although the sensitivity to dihydropyridines remained unchanged. On average, the inhibition of the current elicited by AngII, which amounted to 63% in control cells, was reduced to only 21 ± 17% after treatment with Ptx (n = 16). The effect of Ptx was statistically significant according to an unpaired Student's t test with a p value < 0.0001. 

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Pertussis toxin treatment prevents AngII action on L current. Glomerulosa cells were treated overnight with 500 ng/ml pertussis toxin before being tested as described in the legend of Fig. 2. Inset, examples of currents recorded from the same cell and elicited before (control) and after the addition of AngII (100 nM) and nicardipine (2 µM). Data are representative of 16 independently tested cells.

Effect of Pertussis Toxin Treatment on AngII Action on the Cytosolic Ca²⁺ Signal and on Aldosterone Secretion-- In order to determine whether the effect of Ptx on the AngII-induced inhibition observed on Ba²⁺ currents is also exerted on [Ca²⁺]_c homeostasis, we performed Ca²⁺ microfluorometry experiments in single fura-2-loaded glomerulosa cells. Fig. 6 compares the [Ca²⁺]_c fluctuations obtained in a control cell (A) and in a cell treated overnight with Ptx (500 ng/ml) (B). Cells were successively exposed to thapsigargin, K⁺, and AngII, as described in Fig. 1A. After establishment of an elevated plateau of [Ca²⁺]_c at approximately 400 nM with K⁺, the addition of AngII led to a dramatic reduction of the [Ca²⁺]_c signal in the control cell (Fig. 6A), but had a much less pronounced effect in the Ptx-treated cell (Fig. 6B). The responses obtained in control individual cells (n = 3) were similar to those obtained in control cell populations (Fig. 1A); AngII-induced inhibition of the [Ca²⁺]_c signal amounted on average to 60 ± 5%. In contrast, in the Ptx-treated single cells, the inhibition of the K⁺-induced [Ca²⁺]_c elevation by AngII was reduced to only 19 ± 8% in the five tested cells.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Effect of pertussis toxin on the [Ca2+]c response to AngII in single adrenal glomerulosa cells. Cultured cells were incubated overnight in the absence (A) or in the presence (B) of 500 ng/ml Ptx before to be loaded with the fluorescent calcium probe fura-2. Variations of cytosolic calcium in single glomerulosa cells were recorded (as described under "Experimental Procedures") upon addition of thapsigargin (200 nM), KCl (9 mM), and AngII (100 nM).

In a separate series of experiments, we have assessed the effect of Ptx treatment on the [Ca²⁺]_c response to AngII at a low concentration of K⁺ (3 mM). Under these conditions, the [Ca²⁺]_c rise induced by AngII, measured during the plateau phase 3 min after hormone addition, was slightly more pronounced in Ptx-treated cells (93 ± 16 nM, n = 10) as compared with control cells (69 ± 11 nM, n = 12, data not shown), probably reflecting a lack of L channel inhibition in Ptx-treated cells.

Because treatment with Ptx maintains [Ca²⁺]_c at markedly higher levels upon stimulation with AngII, we have investigated the effect of Ptx on the steroidogenic response. As illustrated in Fig. 7, pretreatment with the toxin reduced by approximately 20% the aldosterone secretion evoked by AngII, without significantly changing the EC₅₀ value for the hormone. Similar results were obtained when aldosterone was stimulated with increasing concentrations of AngII in the presence of 9 mM instead of 3 mM K⁺ (not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Effect of pertussis toxin on the steroidogenic response to AngII. Cultured cells were incubated overnight in the presence or in the absence (control) of 500 ng/ml Ptx, before being exposed for 1 h at 37 °C to increasing concentrations of AngII. Potassium concentration in the medium amounted to 3 mM. Aldosterone production was determined in the medium by direct radioimmunoassay as described under "Experimental Procedures" and is expressed per mg of cell protein. Aldosterone secretion was significantly reduced in Ptx-treated as compared with control cells at AngII concentrations above 1 nM (p < 0.05, according to a paired Student's t test). Data are the mean values from four independent experiments performed in duplicate and were fitted to four parameter logistic functions; half-maximal stimulation was obtained at 1.5 and 3.0 nM AngII for control and Ptx-treated cells, respectively.

Identification of G Protein Isoforms Expressed in Bovine Glomerulosa Cells-- Because both G_o and G_i proteins are known to be sensitive to Ptx, we have investigated which isoform is expressed in bovine adrenal glomerulosa cells. The presence of G_o and G_i in cellular extracts was analyzed by SDS-PAGE and immunoblotting with specific antibodies raised against the  subunit of each isoform. Results were compared with those obtained with the GH₄C₁ rat pituitary cell line, in which the expression of these isoforms has been well characterized (22).

As expected, both G_o and G_i isoforms were detected in GH₄C₁ cells as strong bands at approximately 40 kDa (Fig. 8). Bands of similar intensity were also observed in proteins extracts of bovine adrenal glomerulosa cells, strongly suggesting that both G_i and G_o proteins are constitutively expressed in these cells.

View larger version (53K): [in this window] [in a new window]   Fig. 8.   Identification of G protein subtypes expressed in bovine adrenal glomerulosa cells by immunoblotting analysis. Proteins from cultured bovine adrenal glomerulosa (BAG) cells and from GH4C1 cells were extracted and separated by SDS-PAGE, as described under "Experimental Procedures." Proteins (16 µg/lane) were then transferred onto a cellulose membrane to be analyzed for the presence of specific G proteins with rat polyclonal antibodies raised against the  subunits of the Go and Gi proteins. Arrows indicate the position of markers with relative molecular masses of 29 and 45 kDa. Similar results were obtained in four independent preparations.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The aim of the present study was to characterize the molecular mechanism by which AngII exerts its inhibitory action on the cytosolic Ca²⁺ response to K⁺ (Fig. 1A), a phenomenon already observed several years ago in adrenal glomerulosa cells (5). Because this effect disappeared after voltage-operated calcium channel inhibition with dihydropyridines (Fig. 1B), it is deduced that the reduction of [Ca²⁺]_c evoked by AngII reflects the hormonal modulation of these channels rather than an activation of Ca²⁺ extrusion from the cytosol. In previous work (10), we have studied the modulation by AngII of the low threshold T-type calcium channels, which appeared, at this time, to be the main effectors of Ca²⁺ entry under very small depolarizations resulting from physiological K⁺ increases. More recently, we have focused our attention on the high threshold L-type channels for two reasons: 1) the inhibition of the steady-state current flowing through T-type channels with phorbol 12-myristate 13-acetate is not accompanied by a reduction of the [Ca²⁺]_c levels; and 2) the [Ca²⁺]_c response to low concentrations of extracellular K⁺ is mainly due to L-type channels, the contribution of T channels to the signal being negligible (13).

The main finding of the present work is that AngII clearly modulates negatively L channels in bovine adrenal glomerulosa cells, to the same extent as it reduces [Ca²⁺]_c. This inhibition does not result from a shift of the sensitivity of the channel to voltage and is observed at any membrane potential; however, further investigation will be required, particularly at the single channel level, in order to determine whether total current inhibition is due to a decrease of channel open probability, to a change of channel conductance or to a modulation of channel insertion within the plasma membrane. In any case, these results strongly suggest that the inhibition of L-type channels by AngII is responsible for the decreased [Ca²⁺]_c.

The AT₁ receptor subtype has been shown to be the major AngII receptor expressed in adrenal glomerulosa cells and to mediate most of the hormone actions in these cells (23). The AngII-induced inhibition of L-type Ca²⁺ channels appears to be also mediated by the AT₁ receptor. Indeed, the presence of the AT₁-selective antagonist losartan completely abolished AngII action (Fig. 4). This result is in agreement with the previous observation that losartan also prevents the action of AngII on the sustained [Ca²⁺]_c response to K⁺ (10).

Various cellular mechanisms of L-type Ca²⁺ channel modulation have been extensively investigated (21). From these studies, two major pathways have been highlighted: these involve either GTP binding proteins or channel phosphorylation by specific kinases. Whereas a role for PKC or tyrosine kinases, enzymes known to be linked to AT₁ receptor activation, could be recently excluded (24), we show here (Fig. 5) that the modulation of the L-type current by AngII is much reduced after pertussis toxin treatment. This finding strongly suggests that the transduction of the AngII signal from the AT₁ receptor to the L-type channels involves a Ptx-sensitive G protein of the G_i/G_o family.

A modulation of L channels by AngII through G proteins has been previously observed in other cell types, but with contrasting results. For example, Hescheler et al. (25) have demonstrated that AngII, through a G_i protein, stimulates L-type current in the murine adrenocortical cell line Y1. More recently, an inhibition by AngII of L-type currents has been reported in rabbit sinoatrial node cells (26) as well as in guinea pig cardiomyocytes (27), and these negative modulations have been proposed to be mediated by G_i through an inhibition of the cAMP-dependent pathway. Moreover, in each of these studies, pretreatment of the cells with Ptx abolished AngII action on L currents. It is, however, noteworthy that in contrast to what we observed in glomerulosa cells, in the above studies, AngII affected L currents only after they had been enhanced by -adrenergic stimulation, the hormone being inefficient on basal currents.

As expected, in bovine adrenal glomerulosa cells, Ptx treatment also markedly reduced the AngII-induced inhibition of the sustained [Ca²⁺]_c response to K⁺ (Fig. 6) and slightly increased the [Ca²⁺]_c response to AngII itself (not shown). This sensitivity of the [Ca²⁺]_c signal to Ptx confirms the previously described close relationship existing between [Ca²⁺]_c and L-type channel activity (13) and strongly reinforces the hypothesis that L channels are principally responsible for the variations of [Ca²⁺]_c observed upon stimulation of glomerulosa cells with physiological concentrations of extracellular K⁺.

Surprisingly, despite the fact that Ptx treatment maintained higher levels of [Ca²⁺]_c upon stimulation with AngII, aldosterone production was significantly reduced (Fig. 7). This result suggests that either the negative feedback exerted by AngII on the [Ca²⁺]_c signal induced by K⁺, or another factor controlled by a Ptx-sensitive G protein, is required for an optimal steroidogenic response. Relevant to this point is the suggestion by Barrett et al. (12) that Ca²⁺ influx through L-type channels could exert a negative action on the steroidogenic function of bovine glomerulosa cells.

Nevertheless, because pertussis toxin also negatively affects aldosterone production at relatively low [Ca²⁺]_c (expected to occur at basal extracellular K⁺ and concentrations of AngII as low as 0.1 nM; see Fig. 7), a bell-shaped dependence of steroidogenesis upon calcium is probably not the only explanation, and some still undetermined steroidogenic factor, controlled by a pertussis toxin-sensitive G protein, could also be involved in this phenomenon.

In rat glomerulosa cells, it has been shown that Ptx treatment helps AngII to maintain an optimal aldosterone secretion at supramaximal concentrations of the hormone but has no effect at lower AngII concentrations (28). This specific effect of Ptx in rat cells is presumably due to prevention of adenylyl cyclase inhibition by AngII through a G_i protein, although contradictory results in this species have been published by others (29, 30). In bovine glomerulosa cells, Barrett and Isales (31) have previously observed a 20% inhibition of the steroidogenic response to 10 nM AngII after treatment with Ptx, a result that is in complete agreement with our own observation (Fig. 7).

Both G_i and G_o proteins appear to be expressed in bovine adrenal glomerulosa cells (Fig. 8), but pertussis toxin does not allow to discriminate between the respective functions of each of these proteins. Nevertheless, in other cell types, G_o proteins have been generally shown to negatively modulate L-type channels, whereas in contrast, G_i protein activation is often associated with a direct (cAMP-independent) increase of the activity of these channels (21, 25, 32). Furthermore, we and others have recently shown that in bovine glomerulosa cells, AngII does not inhibit but rather potentiates cAMP production (33, 34), suggesting a poor coupling between the AT₁ receptor and some G_i protein in these cells. These two facts would therefore indirectly favor the involvement of a G_o protein in the negative modulation of L-type channels by AngII in bovine glomerulosa cells.

In conclusion, the negative modulation of [Ca²⁺]_c homeostasis exerted by AngII in bovine adrenal glomerulosa cells reflects the inhibition of L-type Ca²⁺ channels by this hormone. Moreover, the inhibition of these channels involves a Ptx-sensitive G protein, presumably of the G_o family, linked to the AT₁ receptor subtype (Fig. 9). It therefore appears that the same hormone, through the same receptor, is able to inhibit different types of Ca²⁺ channels in the cell through distinct mechanisms: T channel activity is negatively modulated through PKC activation, whereas L channels are partially closed via a Ptx-sensitive G protein. This differential control by AngII of Ca²⁺ entry occurring through various channels should probably represent an advantage for the cell in view of the growing evidence that different types of Ca²⁺ channels exert distinct functions in glomerulosa cells.

View larger version (13K): [in this window] [in a new window]   Fig. 9.   Model describing the modulation of voltage-operated calcium channels by AngII in bovine adrenal glomerulosa cells. The abbreviations used are as follows: AT1, angiotensin II receptor subtype 1; DAG, diacylglycerol; G, GTP-binding protein; L and T, voltage-operated calcium channels of types L and T; PLC, phosphoinositide-specific phospholipase C.

	ACKNOWLEDGEMENTS

We are particularly grateful to G. Dorenter and W. Dimeck for excellent technical assistance.

	FOOTNOTES

^* This work was supported by Swiss National Science Foundation Grants 32-49297.96, 31-42178.94, and 31-52779.97.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed. Tel.: 41-22-3729320; Fax: 41-22-3729329; E-mail: rossier@cmu.unige.ch.

	ABBREVIATIONS

The abbreviations used are: AngII, angiotensin II; PAGE, polyacrylamide gel electrophoresis; Ptx, pertussis toxin; PKC, protein kinase C.

	REFERENCES

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