Inhibition of the T-Type Ca2+ Current by the Dopamine D1 Receptor in Rat Adrenal Glomerulosa Cells: Requirement of the Combined Action of the Gß{gamma} Protein Subunit and Cyclic Adenosine 3',5'-Monophosphate

Patrick Drolet, Lyne Bilodeau, Alzbeta Chorvatova, Liette Laflamme, Nicole Gallo-Payet and Marcel D. Payet

Department of Physiology and Biophysics (P.D., L.B., A.C., M.D.P.) and Department of Medicine Endocrine Service (P.D., L.L., N.G-P.), Faculty of Medicine, University of Sherbrooke Sherbrooke, Quebec, Canada J1H 5N4


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Modulation of ionic Ca2+ currents by dopamine (DA) could play a pivotal role in the control of steroid secretion by the rat adrenal glomerulosa cells. In the present study, we report that DA decreases the T-type Ca2+ current amplitude in these cells. The use of pharmacological agonists and antagonists reveals that this effect is mediated by activation of the D1-like receptors. Modulation by cAMP is complex inasmuch as preincubation of the cells with 8-Br-cAMP or the specific adenylyl cyclase inhibitor, 2',3'-dideoxyadenosine, have no effect per se, but prevent the DA-induced inhibition. The inhibitory effect of DA was abolished by addition of GDPßS to the pipette medium but not by pertussis toxin. If a cell is dialyzed with medium containing G{alpha}s-GDP, the inhibitory effect is reduced and cannot be recovered by the addition of GTP{gamma}S, indicating that the {alpha}s is not involved, but rather the ß{gamma}-subunit. Indeed, DA-induced inhibition was mimicked by Gß{gamma} in the pipette and 8-Br-cAMP in the bath. Similarly, Gß{gamma} release from the activation of the AT1 receptor of angiotensin II did affect the current amplitude only in the presence of 8-Br-cAMP in the bath. The mitogen-activated protein kinase cascade, which can be activated by receptors coupled to Gs, was not involved as shown by the lack of activation of p42mapk by DA and the absence of effect of the mitogen-activated protein kinase inhibitor, PD 098059, on the DA-induced inhibition. Because the binding of Gß{gamma}-subunits to various effectors involves the motif QXXER, we therefore tested the effect of the QEHA peptide on the inhibition of the T-type Ca2+ current induced by DA. The peptide, added to the medium pipette (200 µM), abolished the effect of DA. We conclude that the presence of the Gß{gamma} and an increase in cAMP concentration are both required to inhibit the T-type Ca2+ current in rat adrenal glomerulosa cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroid secretion by the glomerulosa cells of adrenal glands is modulated by a number of factors. The main secretagogues, ACTH, angotensin II (Ang II), and K+, have been widely studied in terms of steroid secretion, second messenger production, and ionic current levels (1, 2). Negative modulation of steroid secretion is now well established, and the two main factors were identified as the atrial natriuretic factor (3) and the neuromediator dopamine (4, 5, 6). Other factors such as somatostatin, neuropeptide Y, or interleukins have also been shown to inhibit glomerulosa cell secretion (for a review, see Ref.7). Moreover, the recent findings of the presence of chromaffin cells originating from the medulla in cortical layers (8, 9) strengthen the concept of a neurohormonal control of zona glomerulosa cell secretion, formerly proposed by McCarty et al. (10).

Both dopamine (DA) receptors, D1 and D2, have been described in rat and bovine glomerulosa cells (11, 12). Binding of DA or specific agonists to D1 receptors activates adenylyl cyclase through a Gs protein, which results mainly in an increase in cytosolic cAMP. In contrast, one of the best known effects of D2 receptors is the inhibition of adenylyl cyclase through the pertussis toxin (PTX)-sensitive Gi/Go family of G proteins (13). Modulation of ionic channels is also an important component of the physiological effects of DA. In rat neostriatal neurons, D1 receptors inhibit high voltage-activated (HVA) currents (N- and P-types) through the activation of a Ser/Thr phosphatase (PP1-type) (14) and depress the amplitude of the Na+ current through a direct cAMP-protein kinase A (PKA)-dependent phosphorylation of the channels (15). The role of D2 receptors in the regulation of K+ and Ca2+ currents has been reported in many cell types. The signaling pathway was described in rat pituitary cells, where Go{alpha} and Gi3{alpha} proteins are involved (16).

Little is known about the modulation of ionic currents by DA in adrenal glomerulosa cells. A recent report showed that the T-type Ca2+ current is blocked by activation of the D2 receptor (17). In the present study, we also report that DA inhibits the T-type Ca2+ current in glomerulosa cells. However, using agonists and antagonists of DA receptors, we demonstrate that the type of DA receptor involved is the D1 receptor. We also show that the DA-induced inhibition is mediated by the Gs-coupling G protein, through its ß{gamma}- subunit. Because current inhibition was prevented by the QEHA peptide, we propose that the ß{gamma}-subunit must bind to a specific site on the T-channel. Moreover, at odds with previous works on {gamma}-subunit-modulated ionic channels, we demonstrate that the inhibitory effect of DA also requires cAMP-dependent phosphorylation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effect of Dopamine on the T-Type Ca2+ Current
The patch-clamp experiments were done in whole cell configuration with external and internal solutions adjusted to block outward currents. In these conditions, application of a depolarizing pulse from a holding potential of -80 mV to -20 mV elicited a transient inward current with voltage-dependent and kinetic properties identical to that formerly described by our group for the T-type Ca2+ current of adrenal glomerulosa cells (18). Figure 1AGo shows the current traces obtained in control conditions and after addition of 0.3 mM DA to the bath. DA caused the current amplitude to decrease rapidly, reaching a plateau after 70 sec, with a level of inhibition of 33.0 ± 1.8% (n = 75). The current-voltage relationships (I/V curve) are illustrated in Fig. 1BGo. The current threshold was close to -60 mV, the peak current voltage near -30 mV, and the zero current voltage around +50 mV. DA reduced the current amplitude at each potential without any change in the voltage threshold or peak current voltage. Figure 1CGo illustrates the dose-dependent blockage effect of DA. A cumulative dose-response curve was constructed with five DA concentrations (0.1 to 0.5 mM). Current traces are presented in the inset. Percent inhibition of the current amplitude was plotted as a function of DA concentration (Fig. 1DGo); 50% inhibition (IC50) was obtained with 0.25 mM DA. Total inhibition was never observed, even with DA concentrations as high as 1 mM. Partial recovery of the current amplitude was obtained after washing (data not shown).



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Figure 1. Inhibition of the T-Type Ca2+ Current by DA in Rat Adrenal Glomerulosa Cells

A, Addition of 0.3 mM DA to the external solution rapidly decreases the current amplitude. Inset ({square}, control; {blacksquare}, DA) shows current traces generated by a depolarizing pulse from a holding potential of -80 mV to -20 mV. Scale: horizontal, 30 msec; vertical, 220 pA. B, Current-voltage relationships of the T-current in control conditions ({circ}) and with 0.3 mM DA ({square}). Current threshold near -60 mV; zero current voltage near +50 mV; peak current voltage near -30 mV. C, Cumulative dose-response curve of current inhibition by DA constructed with (1) 0.1 mM, (2) 0.2 mM, (3) 0.3 mM, (4) 0.4 mM, and (5) 0.5 mM DA. Inset shows current traces in control conditions (C) and for each DA concentration (1, 2, 3, 4, 5). Horizontal scale, 30 msec; vertical 200 pA. D, Relationship between percent inhibition of the current amplitude and DA concentration. The IC50 was found to be 0.25 mM.

 
Receptor Subtype Involved in the Blocking Effect of DA
Freshly isolated glomerulosa cells possess D1- and D2-subtypes of DA receptors whereas 3-day cultured cells exhibit only D1 subtypes (5, 6). The nature of the DA receptor subtype responsible for the blockage of the T-type current was determined using specific agonists and antagonists of the D1- and D2-like families (see Ref.19). The full D1 receptor agonist 6-chloro-APB HBr (Cl-APB) (SKF 82958), at a concentration of 5 µM, caused an average (n = 6) 44.2 ± 7.1% reduction of the T-type current amplitude (Fig. 2AGo). In addition, preincubation of the cells with the D1 antagonist SCH 23390 (5 µM) markedly reduced their response to DA (0.8 mM) (Fig. 2BGo). We also found that 5 or 10 µM (n = 5) SCH 23390 has by itself a blocking effect on the T-type current. In contrast, when the D2 antagonist spiperone (10 µM) was added to the bath before DA (0.3 mM), the current amplitude was still reduced with similar time course and potency as observed in the absence of the antagonist (Fig. 2CGo, n = 5). These results suggest that the inhibitory effect of DA we have recorded on the T-type Ca2+ current is produced through the activation of the D1 receptor subtype.



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Figure 2. Pharmacology of the DA Effect

A, Inhibition of the T-type Ca2+ current by the D1-like receptor agonist 6-chloro-ABP HBr (5 µM). All current recording was done as in Fig. 1Go: inset ({square}, control; {blacksquare}, Cl-APB HBr); scale: horizontal, 15 msec; vertical, 100 pA. B, Addition of the D1 antagonist SCH 23390 (5 µM) before 0.3 mM DA prevents current inhibition. Inset ({square}, control; {circ}, SCH 23390; {blacksquare}, DA). Scale: horizontal, 15 msec; vertical, 150 pA. C, The D2 antagonist spiperone (10 µM) applied before DA (0.3 mM) does not interfere with the inhibition of the T-type current. Inset: {square}, control + spiperone; {blacksquare}, DA. Scale: horizontal, 15 msec; vertical, 145 pA.

 
Second Messengers Involved in the DA Inhibition
It has been previously shown that DA, through the D1 receptor type, activates adenylyl cyclase, which leads to an increase in cAMP concentration (5, 6). Figure 3AGo confirms and extends these observations. The maximun increase in cAMP production measured with DA was 11.6-fold, with a concentration of 0.3 mM (n = 3), compared with a 7.2-fold increase with the D1 agonist Cl-APB (1 µM) (n = 3). The use of specific D1 and D2 antagonists indicates that the increase in cAMP production is linked only to D1 receptor activation, since SCH 23390 blocked the DA stimulation, while sulpiride was ineffective.



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Figure 3. Implication of cAMP in DA-Induced Inhibition of the T-Type Ca2+ Current

A, DA increases cAMP production in cultured glomerulosa cells through activation of the D1 receptor. Cells were incubated in the absence (C) or in the presence of 0.3 mM DA (DA), 1 µM Cl-APB (APB), DA plus 1 µM SCH 23390 (DA + SCH), or DA plus 1 µM sulpiride (DA + Sul). B, Addition of 1 mM 8-Br-cAMP to the bath has no effect on the current amplitude but blocks DA-mediated current inhibition. Inset: {square}, control; {blacksquare}, DA, 0.3 mM; {circ}, DA, 0.6 mM. Scale: horizontal, 15 msec; vertical, 140 pA. C, Inhibition of the adenylyl cyclase by DDA (100 µM) has no effect on current amplitude but abrogates DA-induced current inhibition; Inset, {square}, control; {blacksquare}, DA. Scale: horizontal, 15 msec; vertical, 110 pA.

 
Because activation of the D1 receptor by DA increases the cAMP level, we asked whether T-type current inhibition could be induced by cAMP. Figure 3BGo shows that addition of 1 mM 8-Br-cAMP to the bath did not affect the current amplitude, which indicates that cAMP does not modulate directly the T-current in adrenal glomerulosa cells. However, pretreatment of the cells with 8-Br-cAMP drastically decreased DA potency (9.1 ± 1.9% inhibition; n = 7). Moreover, when the cells were preincubated 5 min in the presence of the specific adenylyl cyclase inhibitor 2',3'-dideoxyadenosine (DDA, 100 µM) (20, 21, 22), DDA had no effect on the T-type Ca2+ current, but prevented current inhibition by DA (0.3 mM) (5.2 ± 3.6%, n = 5) (Fig. 3CGo). Thus, although cAMP does not directly affect the channel activity, adenylyl cyclase activation is part of the D1-receptor-signaling pathway. Activation of the cAMP-dependent PKA, known to phosphorylate L-type Ca2+ channels in numerous cell types, seems to be involved in the regulation of the T-type Ca2+ channel, because preincubation of the cells with the PKA inhibitor H-89 (10 µM) reduced the impact of DA on the current amplitude (16.4 ± 3.1% inhibition; n = 11); the IC50 of the dose-effect curve was 8.1 µM (data not shown). It has been reported that D1 receptor can signal through the phospholipase C pathway (23, 24, 25). However, application of Ang II (100 nM) had no effect on the T-type Ca2+ current and did not modulate the DA-induced inhibition of the current (data not shown).

Several ionic channels are modulated by the p21ras protein (26) or by the Ras/Raf transduction cascade (27). This possibility was tested in glomerulosa cells. The T-type Ca2+ current was not affected by an antibody raised against the p21ras protein added to the pipette medium (35.2 ± 1.2% inhibition; n = 4; data not shown), or when the Ras/Raf cascade was blocked at a more distal stage by preincubation of the cells with the MAP kinase inhibitor PD 098059 (1 h, 30 µM) (Fig. 4AGo; 30.3 ± 5.7% inhibition; n = 7). Furthermore, MAP kinase activity was assessed by in-gel kinase analysis after cell stimulation by Ang II or DA. As shown in Fig. 4BGo, treatment of glomerulosa cells with Ang II for 5 min activates the p42mapk (ERK2) MAP kinase (see also Ref.28), while DA treatment was ineffective. A PP1-type phosphatase, activated by PKA-dependent phosphorylation, was shown to be involved in the D1-like receptor-induced inhibition of HVA Ca2+ currents (14). This possibility was also ruled out, because okadaic acid (1 mM), a blocker of Ser/Thr phosphatase, did not impair DA-induced current inhibition (38.8 ± 3.2%; n = 8; data not shown).



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Figure 4. Lack of Involvement of the MAP Kinase in DA-Induced Inhibition of T-Type Ca2+ Current

A, The specific inhibitor PD 098059 (30 µM, 60 min) does not impair the DA effect; Inset, {square}, control; {blacksquare}, DA. Scale: horizontal, 15 msec; vertical, 75 pA. B, MAP kinase activity in glomerulosa cells. Cells were incubated for 5 min in the absence (lane 1) or in the presence of 100 nM Ang II (lane 2) or 0.3 mM DA (lane 3). Samples from whole-cell extracts were subjected to in-gel analysis with substrate MBP copolymerized into the gel. p42mapk (ERK2) activity is shown from the autoradiogram, as well as other higher molecular mass kinases, after enzyme phosphorylation of MBP.

 
Involvement of a G Protein in the Blocking Effect of DA
It is well established that the D1-receptor subtype is linked to its effector through a Gs protein (19, 29). Addition of GDPßS (2 mM) to the pipette medium completely abolished current inhibition of 0.3 mM DA (0.16 ± 3.8%; n = 4) (Fig. 5AGo), which confirms the involvement of a G protein in the signaling pathway between the D1 receptor and the T-type Ca2+ channel. To identify the type of G protein, the cells were incubated 18 h with 100 ng/ml PTX. In glomerulosa cells, this PTX concentration was shown to inactivate the Gi protein through ADP ribosylation (30). Figure 5BGo shows that DA was still able to reduce the T-type current amplitude (35. 6 ± 7.2% inhibition; n = 5). Similar results were found when an antibody raised against the {alpha}-subunit of the Gi protein was added to the pipette medium (data not shown, n = 4). The absence of effect of Ang II, as mentionned earlier, confirms that neither Gi nor Gq coupling proteins were involved. However, if Ang II (100 nM) and 8-Br-cAMP (1 mM) were added together, a slow inhibition took place, reaching 25.3 ± 3.9% (Fig. 5CGo; n = 4).



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Figure 5. A G Protein Is Involved in the DA-Induced Inhibition of the T-Type Ca2+ Current

A, GDPßS (2 mM) added to the medium pipette prevents current inhibition by 0.3 mM DA. Inset: {square}, control; {blacksquare}, DA. Scale: horizontal, 30 msec; vertical, 50 pA. B, Cells preincubated with pertussis toxin (PTX, 100 ng/ml, 18 h) still respond to DA (0.3 mM) by a decrease in T-type current amplitude. Inset: {square}, control of PTX-treated cells; {blacksquare}, DA. Scale: horizontal, 30 msec; vertical, 110 pA. C, Ang II (100 nM) and 8-Br-cAMP (1 mM) added simultaneously to the bath induce a gradual decrease in the current amplitude. Inset: {square}, control; {blacksquare}, Ang II + 8-Br-cAMP. Scale: horizontal, 15 msec; vertical, 130 pA.

 
The acetylcholine-activated inwardly rectifying K+ channel (IKAch) was shown to be modulated by the Gß{gamma}-subunit of the G protein by Clapham’s group (31, 32). This modulation would result from a direct binding of the Gß{gamma}-subunit to the IKAch channel (33). Here, when Gß{gamma} concentration was reduced by cell dialysis with G{alpha}s-GDP (300 nM), placed in the pipette medium, the inhibitory effect of DA was considerably decreased (Fig. 6AGo, 16.9 ± 2.3% inhibition; n = 4). When GTP{gamma}S (1 µM) was added to the G{alpha}s-GDP medium pipette, DA did not recover its inhibitory effect (10.2 ± 3.2% inhibition; n = 4; data not shown), suggesting that the G{alpha}-subunit was not involved in DA-induced inhibition of the T-type Ca2+ current. In contrast, although {gamma}-subunits (100 nM), added to the pipette medium, did not affect the current under control conditions, DA inhibition was impaired (Fig. 6BGo; 14.8 ± 3.9% inhibition; n = 6). Denaturated Gß{gamma} was inactive (data not shown). We previously showed that DA-induced inhibition of the T current was considerably reduced by cAMP (Fig. 3BGo). However, when 8-Br-cAMP (1 mM) was added to the bath medium and Gß{gamma}-subunit to the pipette medium, current inhibition increased slowly (Fig. 6CGo) to reach a level similar to the one measured with DA (Fig. 6DGo; 31.2 ± 4.9% inhibition; n = 4).



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Figure 6. Involvement of the Gß{gamma}-Subunit in the DA-Dependent Inhibition of the T-Type Ca2+ Current

A, Dialysis of the cell with a medium containing G{alpha}s-GDP (300 nM) suppressed the DA effect. Inset: {square}, control; {blacksquare}, DA. Scale: horizontal, 15 ms; vertical, 110 pA. B, Bovine brain Gß{gamma}-subunit added to the medium pipette (100 nM) also prevented DA-induced inhibition. Note that Gß{gamma} per se has no inhibitory effect. Inset: {square}, control; {blacksquare}, DA. Scale: horizontal, 15 msec; vertical, 165 pA. C, With a pipette medium containing Gß{gamma}-subunits (100 nM), addition of 8-Br-cAMP (1 mM) to the bath induced a slow decrease in current amplitude; Inset: {square}, control; {blacksquare}, DA. Scale: horizontal, 15 msec; vertical, 200 pA;. D, Histogram showing percent inhibition of current amplitude by DA (0.3 mM), 8-Br-cAMP (1 mM), Gß{gamma}-subunit (100 nM), and Gß{gamma}-subunit plus 8-Br-cAMP. The presence of Gß{gamma}- subunits potentiates the effect of 8-Br-cAMP on the T-type current, reaching a percent inhibition equivalent to that obtained with DA. **, P < 0.01, *, P < 0.05, difference compared with control conditions.

 
Binding of Gß{gamma}-subunits to various effectors involves the motif QXXER (34). The effects of Gß{gamma} have been reported to be specifically blocked by a peptide containing the sequence corresponding to residues 956 to 982 of adenylyl cyclase 2, the peptide called QEHA (34). We therefore tested the effect of the QEHA peptide on the DA-induced inhibition of the T-type Ca2+ current. The peptide, added to the medium pipette (200 µM), suppressed the effect of DA (Fig. 7AGo; 10.3 ± 3.7% inhibition; n = 4); QEHA peptide blunted the DA inhibition in a dose-dependent manner (Fig. 7BGo).



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Figure 7. Effect of the QEHA Peptide on DA-Induced Inhibition of the T-Type Ca2+ Current

A, The synthetic QEHA peptide, added to the pipette medium (200 µM), prevented current inhibition by 0.3 mM DA. Inset: {square}, control; {blacksquare}, DA. Scale: horizontal, 15 msec; vertical, 65 pA. B, Effect of various concentrations of the QEHA peptide on DA (0.3 mM) induced inhibition of the T-type current. Numbers of experiments are 4, 9, 6, and 4, for 1, 10, 100, and 200 µM QEHA, respectively. C, Amino acids sequences of four different proteins that possess the QXXER motif (ßARK-1, ß-adrenergic receptor kinase-1; AC2, adenylyl cyclase type 2; GIRK-1, G protein-coupled inward rectifier K+ channel; rbE-II, {alpha}1 subunit of the cloned T-type channel).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this paper, we report that dopamine inhibits the T-type Ca2+ current in glomerulosa cells from rat adrenal glands. We first demonstrate, by using a pharmacological approach, that this effect is mediated by a D1-like receptor type. Our second finding is that the ß{gamma}-subunit of the Gs-coupling protein is involved in the inhibition of the current. However, our data clearly demonstrated that cAMP plays a pivotal role in the inhibition of the current.

Dopamine Receptors in Adrenal Glomerulosa Cells
Several subtypes of DA receptors have been described and classified in two families: the D1-like (D1 and D5) and the D2-like (D2, D3, D4) receptors (for reviews, see Refs. 19 and 29). This classification is based mainly on the modulation of adenylyl cyclase. The D1-like receptors stimulate adenylyl cyclase through a Gs protein (35), whereas the D2-like receptors inhibit the enzyme activity through a Gi protein (36). D1-receptor activation was also associated with stimulation of phosphoinositide hydrolysis (23, 24, 25) and Ca2+ mobilization (37, 38) whereas D2 receptors were reported to stimulate ATP-mediated arachidonic acid release (39).

Conflicting results were reported concerning the role of DA in the control of steroid secretion from isolated glomerulosa cells (40, 41). They were shown to originate from experimental conditions. Freshly isolated cells express DA receptors from both D1- and D2-subclasses, while 3-day cultured cells express only the D1 subclass (5, 6). In this study, all experiments were performed on glomerulosa cells after 1 or 2 days of culture, when the majority of DA receptors are expected to belong to the D1-like subclass. Expression of the D1-like receptors was confirmed by an increase in cAMP production measured after the addition of DA or the D1 agonist Cl-APB (see Fig. 3AGo). Moreover, preincubation of the cells with the D1 antagonist SCH 23390 prevented DA-mediated increase in cAMP production. The fact that cells preincubated with the D2 antagonist sulpiride did not respond to DA by a faster rate of cAMP production indicates that the DA receptors expressed were mainly of the D1 type.

DA Receptors Modulate Ionic Currents
In the majority of the cells we studied (95%), DA reduced the current amplitude of the T-type Ca2+ channel in a dose-dependent manner, whereas transient inhibition were occasionally observed (data not shown) as previously reported for K+ current enhancement by PACAP 38 (42). Several experiments were designed to confirm that DA inhibition of the T-type current was mediated through the activation of a D1 receptor type. First, the inhibitory potency of DA was not modified by the presence of spiperone, a specific D2 antagonist (Fig. 2CGo). Second, the D1 agonist Cl-APB was also able to reduce the T-type Ca2+ current amplitude. Finally, the DA-inhibitory effect was greatly attenuated when cells were pretreated 5 min with the D1 antagonist SCH 23390. As shown in Fig. 2BGo, this D1 antagonist SCH 23390 does have an inhibitory effect per se on the Ca2+ channel activity. Such an effect was recently reported on calcium currents in rat retinal ganglion cells (43).

Most cases of ionic current modulation by DA reported in the literature were believed to be due to the activation of D2-like receptors. D2 receptor activation was reported to modulate Ca2+ currents. It was found that LVA and/or HVA Ca2+ currents are inhibited by DA or D2-agonists (17, 44, 45) and that this inhibition is suppressed by PTX (44). DA also induces an hyperpolarization of the cell membrane, which was attributed to an activation of K+ channels (44, 46, 47, 48). This hyperpolarization is believed to play a key role in the inhibition of cell secretion by DA. PTX treatment of the cells blocks both the DA-induced membrane hyperpolarization and activation of the K+ channels, indicating that a Gi/o protein is involved (44, 47, 48). Modulation of K+ and Ca2+ currents by D2 type receptors occurs through different G protein coupling: Gi3{alpha} for IK and IA channels, and Go{alpha} for the T- and L-types of Ca2+ channels (16).

Modulation of ionic currents by D1 receptor activation has been less frequently reported. However, cAMP-dependent PKA phosphorylation was always involved. For instance, in rat striatal neurons, D1 receptor activation reduces the amplitude of the fast Na+ current. This effect is mediated by a GTP-binding protein that induces an increase in cAMP level, which then activates the cAMP-dependent PKA (15). Phosphorylation of the Na+ channel by PKA results in a reduction in current amplitude (49). D1 receptor activation also inhibits HVA Ca2+ currents (N- and P-types) of neostriatal neurons (14). In this case, the cAMP-dependent PKA activates a protein phosphatase (PP1), which dephosphorylates the Ca2+ channels. In some neurons, D1 receptor activation leads to an increase in the L-type Ca2+ current (14). An increase in the cytosolic Ca2+ concentration in rat pituitary GH4C1 cells transfected with the human D1 receptor was also explained by an L-type channel activation (38).

Coupling between the D1 Receptor and the T-Type Ca2+ Channel
Our results show that the T-type current inhibition by DA is mediated by the cAMP-dependent PKA. The PKA inhibitor H-89 (50, 51) blocks the DA-inhibitory effect. However, the fact that 8-Br-cAMP did not inhibit the T current provides strong evidence that in addition to a PKA-dependent phosphorylation, a parallel signal must be delivered. The involvement of a PKA/phosphatase cascade, as described for the inhibition of the HVA Ca2+ channels in neurons (14), was ruled out using a Ser/Thr phosphatase inhibitor, okadaic acid. Some reports suggest that phospholipase C activation could also be part of the D1 receptor-signaling pathway (23, 24), through a Gq-coupling protein (25). However, previous results from our group showed that DA was not able to increase inositol phosphate production in rat glomerulosa cells (6), which, together with the lack of effect of Ang II application on the T-type Ca2+ current, ruled out a possible involvement of the phospholipase C pathway.

A G protein coupling is part of the D1 receptor-inhibitory process. Blockage of G protein activity with GDPßS completely prevented current inhibition by DA. Pretreatment of the cells with PTX or the use of an antibody raised against the {alpha}i-subunit did not impair the inhibitory action of DA. These results, along with those showing that Ang II application has no effect on the T current amplitude (data not shown), indicate that the inhibition of the T current we recorded is not transduced by the coupling protein Gi and thus strengthen our findings that D2-like receptors are not involved.

Whether G protein modulation of ionic channels is mediated by the G{alpha}- or Gß{gamma}-subunit is a matter of debate (52, 53, 54). Here, the fact that Gß{gamma}-subunits from bovine brain, added to the internal solution, did not have any effect on the T-type currents (Fig. 6BGo) raised several possibilities. The effectiveness of the Gß{gamma}-subunits we used in our experiments could be questioned. However, DA-induced inhibition of the T current was greatly reduced by Gß{gamma} dialysis of the cell whereas temperature-inactivated {gamma} was ineffective. On the basis of the existence of several {gamma}- combinations (53), the bovine brain Gß{gamma}-subunit may not efficiently interfere with the inhibitory pathway of the T-type Ca2+ channels. However, it has been recently shown that various Gß{gamma}-forms, including bovine brain Gß{gamma}, activate the IKAch channel (32) by direct binding (33). Consequently, this absence of effect did not exclude the participation of {gamma} in the DA-signaling pathway, but raises the possibility of a second factor that should act together with Gß{gamma} to inhibit the T-type Ca 2+ current.

The involvement of the Gß{gamma}-subunit in the inhibition of the T-type Ca2+ current by the D1 receptor was further assessed by decreasing the Gß{gamma} concentration in the cell using specific Gß{gamma}-subunit scavengers. First, we added the {alpha}-subunit of the Gs protein under its {alpha}-GDP form to the pipette medium. Under these conditions, DA could no longer inhibit the T-type current. Second, it has been shown that a 27-amino acid peptide, the QEHA peptide, was able to block the Gß{gamma}-induced regulation of adenylyl cyclase (AC), phospholipase C-ß3, the ß-adrenergic receptor kinase (ß-ARK), and the G protein-coupled inward rectifer K + channel (GIRK-1) (34). The inhibitory effect of DA was also abrogated by the peptide placed in the pipette medium. Moreover, the IC50 was found near 100 µM, a concentration similar to that reported by Chen et al. (34) for various effectors of the Gß{gamma}-subunit. These results suggest that binding of Gß{gamma} on a site that could be located on the T channel is involved in current inhibition, as demonstrated for the IKAch channel (32, 33). Because several effectors of the Gß{gamma}-subunit (AC2, AC4, ß-ARK-1 and 2, and GIRK-1) display a common amino acid sequence (see Ref.34), we looked for this sequence in the T-type channel. A Ca2+ channel has been cloned from rat brain cells (55). When expressed in oocytes, the {alpha}1-subunit (rbE-II) shows voltage-dependent and pharmacological properties similar to the LVA Ca2+ channel (T-type) found in neuronal cells (56) or glomerulosa cells (18). A unique consensus sequence (QQIER) was found at position 325 (Fig. 7CGo). Cloned Ca2+ channels can be classified into two subfamilies: the dihydropyridine (DHP)-insensitive (T-, N-, P-, and Q-type) and the DHP-sensitive (L-type) families. The two-Ca2+ channel subfamilies display the QXXER motif, which may confer the modulating properties of the Gß{gamma}-subunit. Indeed, it has been recently reported that N-type (57) and P/Q-type (58) Ca2+ channels are modulated by various forms of the Gß{gamma}-subunit. However, evidence has not yet been obtained in the case of the DHP-sensitive channel.

As shown in Fig. 3BGo, superfusion of the cells with 8-Br-cAMP (1 mM) completely abolished the inhibition of the T current induced by DA. Nevertheless, in order to trigger the DA effect, it appears that cAMP is required at a level higher than the basal level. Indeed, we show that DA inhibition of the Ca2+ current will not occur if the adenylyl cyclase is blocked by the DDA (100 µM) (Fig. 3CGo). Requirement of cAMP for the development of the T-type inhibition was further strengthened by results showing that bovine {gamma}-subunit, placed in the medium pipette or disengaged from Gq and/or Gi by Ang II binding on the AT1 receptor, only reduces Ca2+ current in the presence of 8-Br-cAMP.

This kind of double regulation by a Gs-coupled membrane receptor has been recently reported. It has been shown that the ß-adrenergic receptor is coupled to a Gs protein that activates adenylyl cyclase and potently activates the MAP kinase cascade through {gamma} and that application of 8-Br-cAMP inhibits this effect in a PKA-dependent way (59). Similarly, the pituitary adenylyl cyclase-activating polypeptide (PACAP 38) increases the amplitude of the K+ current both by the Gß{gamma}-Ras/Raf and the Gs-adenylyl cyclase cAMP pathways; nevertheless, this effect is blocked by preincubation with cAMP (42). Using a specific inhibitor of the MAP kinase cascade, the PD 098059 (60), and the MAP kinase assay (70), we ruled out the MAP kinase pathway in favor of a direct interaction of the Gß{gamma}-subunit with the T channel.

Conclusion
Our data support the fact that the T-type Ca2+ channel is inhibited upon binding of the ß{gamma}-subunit of the G-coupling protein. Various forms of Gß{gamma}-subunits could be involved, since they can originate from the Gs protein (D1-like receptor), the Gq and/or Gi protein(s) (AT1 receptor of Ang II), or from bovine brain (ß1{gamma}2). Since the first work of Clapham and co-workers (31), showing that the Gß{gamma}-subunit was able to activate the IKAch channel, numerous effectors of Gß{gamma}-subunits have been described (34, 42, 54, 57, 58). One important observation is that several Gß{gamma} combinations are effective, which implies that the same effector can be modulated by different G protein-coupled receptors. Pathways specificity for an effector can be obtained in different ways: binding of G{alpha} or Gß{gamma} alone or a synergistic or antagonistic binding of G{alpha} and Gß{gamma} (53). Yet another factor could be the relative proximity between the effectors and the receptors releasing {gamma}-subunits (62). Our results show that, for the T-type channel, cAMP is a strong modulator of the Gß{gamma} effect. The question can be raised whether the T channel and/or the ß{gamma}-subunit should be phosphorylated by PKA. The {alpha}1 (rbE-II)-subunit displays the concensus sequence RRXS for a putative phosphorylation by PKA. However, experimental evidence for PKA-dependent phosphorylation of the T-type channel has not yet been provided. On the other hand, PKC-dependent phosphorylation of the ß{gamma}-subunit has been reported (63, 64). As stated by Neer (62), the role of ß{gamma}-subunit phosphorylation, if any, is not known. In any case, PKA is not able to phosphorylate the ß{gamma}-subunit (64) and, more important, unphosphorylated forms of ß{gamma}-subunit are active on several effectors (31, 32, 33, 34). In the framework of our results we can postulate that the ß{gamma}-subunits bind to the T channel and that activation of PKA, by an increase in cAMP, phosphorylates the channel. The sequence of the events and the mechanism involved are not yet resolved. This could constitute an example in which cAMP acts as a gating factor (62). Indeed, we show that the Ang II-signaling pathway, which was shown to increase T-type current amplitude (65, 66, 67), could be modulated by a cAMP-dependent process in such a way that T-type current is blocked. This raises the possibility of interactions between hormones and neurotransmitters coupled to G protein receptors in the control of aldosterone secretion by adrenal glomerulosa cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chemicals
The chemicals used in the present study were obtained from the following sources: [3H]adenine (24 Ci/mmol) from Amersham (Oakville, Ontario, Canada); ATP, cAMP, 8-Br-cAMP, glutathione, DNAse; DDA from Sigma (St. Louis, MO); pertussis toxin from List Biological Laboratories (Campbell, CA); GTP{gamma}S and GDPßS from Boeringher (Indianapolis, IN); the {alpha}-subunit of the Gs coupling protein and the bovine brain Gß{gamma}-subunit from Calbiochem (San Diego, CA); H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonanide) from Seikagaku America (St. Petersburg, FL); angiotensin II from Bachem (Marina Delphen, CA); dopamine, 6-chloro-APB Hbr (SKF 82958), SCH 23390, and spiperone from RBI (Natich, MA); collagenase, MEM (Eagle medium) and OPTI-MEM from GIBCO (Burlington, Ontario, Canada). The specific inhibitor of the MAP kinase cascade, PD 098059, was a gift from Dr. D. T. Dudley (Parke-Davis Pharmaceuticals Research Division, Warner-Lambett Co, Ann Harbor, MI 48105). The QEHA peptide was synthesized by "Service de Séquence de Peptides de l’Est du Québec" (Le Centre Hospitalier de l’Université Laval, Qué, Canada). The peptide was purified by HPLC (> 90%) and its identity verified by mass spectrometry. All other chemicals were of A-grade purity.

Preparation of Glomerulosa Cells
The zonae glomerulosa were obtained from adrenal glands of female Long Evans rats weighting 200–250 g. Rats were killed according to a protocol approved by the Local Ethics Animal Care Committee, and adrenals were isolated according to the method described in detail elsewhere (6). The successive steps of zona glomerulosa isolation and cell dissociation were performed in MEM Eagle medium (supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin). After a 20-min incubation at 37 C in collagenase (2 mg/ml, 4 capsules/ml) and DNAse (25 µg/ml), the cells were disrupted by gentle aspiration with a sterile 10 ml pipette, filtered, and centrifuged for 10 min at 100 x g. They were then resuspended in OPTI-MEM supplemented with 2% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin and plated in 35-mm tissue culture dishes at a density of approximately 5 x 104 cells per dish. The cells were cultured at 37 C in a humidified atmosphere of 95% air-5% CO2. The culture medium was changed every day, and the cells were used after 1 or 2 days of culture.

Solutions and Recording Conditions
Solutions
The physiological solutions used for the patch clamp experiments had the following compositions. The basic extracellular solution contained (mM): NaCl, 100; CaCl2, 10; tetraethylammonium, 35; MgCl2, 1; CsCl, 5.4; HEPES, 5; and glucose 2 g/liter at pH 7.4. The pipette solution contained (mM): CsCl, 126; NaCl, 18; CaCl2, 1; EGTA, 11; MgCl2, 2; HEPES, 5; ATP, 3; and GTP, 0.4 at pH 7.2. The external solution was supplemented with glutathione to avoid DA oxydation. Solutions containing hormones, antibodies, or drugs were freshly prepared before each experiment.

Electrophysiology
Experiments were performed at room temperature and in the dark. The Petri dish (1 ml volume solution) was mounted on the stage of an inverted microscope, and the cells were observed at a magnification of 300x. Ionic currents were recorded using the whole-cell configuration of the patch-clamp method (68). Patch electrodes with a resistance of 3 to 5 megohms (M{Omega}) were pulled from Pyrex Glass capillaries (Corning 7740, Corning Glass Works, Corning, NY). Ionic currents were recorded with an axopatch 1B (Axon Instruments, Burlingame, CA), whereas pulse stimulation and data acquisition were performed with an A/D interface DAS 16F (Metrabyte Taunton, MA) and an IBM-compatible computer under the control of a custom-built program. Linear leak and capacitative currents were subtracted. Currents were filtered at 2 kHz and sampled at 5 kHz. Analysis was performed with a custom-made program. Each figure is representative of several experiments conducted on the number of cells (n) indicated in the text.

Cyclic AMP Determination
Intracellular cAMP production was determined by measuring the conversion of [3H]ATP into [3H]cAMP, as described previously (6). Briefly, cultured cells were incubated at 37 C in the same MEM Eagle culture medium containing 2 µCi/ml [3H]adenine. After 1 h, the cells were washed and then incubated in Hanks buffer saline glucose containing 1 mM isobutyl methylxanthine for 15 min at 37 C. Hormones or drugs were then added to the medium for an additional 15-min incubation period at 37 C. The reaction was stopped by aspiration of the medium and addition of 1 ml TCA 5%. The cells were scraped with a rubber policeman and transfered to 100 µl of cold 5 mM ATP-cAMP. Cell membranes were pelleted at 5,000 x g for 15 min, and the supernatant was sequentially chromatographed on Dowex and alumina columns, according to the method of Salomon (69), allowing the separation of [3H]ATP from [3H]cAMP. Cyclic AMP accumulation was calculated from the equation: % conversion = ([3H]cAMP/[3H]cAMP + [3H]ATP) x 100.

Analysis of MAP Kinase Activity
Cells are washed and stimulated for 5 min at 37 C with or without Ang II (100 nM) or DA (0.3 mM). Cells are then washed and solubilized in 300 ml of cold lysis buffer (50 mM HEPES, pH 7.8, 1% Triton X-100, 2.5 mM EDTA, 100 mM sodium fluoride, 10 mM sodium PPi, 2 mM sodium orthovanadate, 0.5 U aprotinin, 1 mM phenylmethylsulfonylfluoride, 1 mM benzamidin) and centrifuged at 10,000 x g for 15 min at 4 C. Equal amounts of protein (10 µg) were electrophoresed on a 12% SDS-polyacrylamide gel containing 0.5 mg/ml myelin basic protein (MBP, Sigma, Mississauga, Ontario, Canada). Kinase assays in MBP-containing gel were performed at room temperature as described by Holt et al. (70). After further denaturation and renaturation of the proteins, the kinase reaction was initiated by placing the gel in fresh kinase buffer containing 30 µM [{gamma}32P]ATP (9 µCi/ml) and incubated for 1 h at room temperature with gentle agitation. The gel was washed extensively with repeated changes of wash buffer (5% trichloroacetic acid, 1% sodium PPi), dried, and subjected to autoradiography.


    ACKNOWLEDGMENTS
 
The authors would like to thank Dr. Gilles Dupuis for critical discussions and Ms. Denise Girardin for typing the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Marcel D. Payet, Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, 3001–12th Avenue N., Sherbrooke, Quebec Canada J1H 5N4.

Nicole Gallo-Payet is a recipient of a Scholarship from "Le Fonds de La Recherche en Santé du Québec." This work was supported by grants (MT-10998 and MA-6813) from the Medical Research Council of Canada and the "Fondation des Maladies du Coeur du Québec" to Marcel Daniel Payet and Nicole Gallo-Payet.

Received for publication October 18, 1996. Revision received January 13, 1997. Accepted for publication January 17, 1997.


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