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Endocrinology Vol. 143, No. 3 747-754
Copyright © 2002 by The Endocrine Society

INTRACELLULAR SIGNAL SYSTEMS

Evidence for a Direct Negative Coupling between Dopamine-D2 Receptors and PLC by Heterotrimeric Gi1/2 Proteins in Rat Anterior Pituitary Cell Membranes

R. Rasolonjanahary, C. Gerard, M. N. Dufour, V. Homburger, A. Enjalbert and G. Guillon

Unite Mixté de Recherche 6544 Centre National de la Recherche Scientifique (R.R., C.G., A.E.), Institut Fédératif Jean Roche, Faculté de Médecine Nord, 13916 Marseille Cedex 20; Unité Propre de Recherche 9023 Centre National de la Recherche Scientifique (M.N.D., V.H.) and U 469 Institut National de la Santé et de la Recherche Médicale (G.G.), Centre National de la Recherche Scientifique-Institut National de la Santé et de la Recherche Médicale de Pharmacologie-Endocrinologie, 34094 Montpellier Cedex, France

Address all correspondence and requests for reprints to: R. Rasolonjanahary, Interactions Cellulaires Neuroendocriniennes-Unité Mixte de Recherche UMR 6544 Centre National de la Recherche, Faculté de Médecine Nord, Bd Pierre Dramard, 13916 Marseille Cedex 20, France. E-mail: . rasolo.r{at}jean-roche.univ-mrs.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dopamine (DA) is known to inhibit basal and hormone TRH- or angiotensin II (AngII)-stimulated PRL secretion and inositol phosphate accumulation in rat pituitary cells in primary culture. This inhibition persists when cells are incubated in a calcium-free medium (a condition in which DA could not inhibit PLC activities by blocking calcium influx) and is abolished by a Pertussis toxin treatment. These data suggest that DA receptor could be negatively coupled to PLC by a direct mechanism involving a Pertussis toxin-sensitive G protein. To demonstrate this hypothesis, we measured PLC activities on crude plasma membranes obtained from rat pituitary cells in primary culture grown in the presence of tritiated myo-inositol. We showed that 1) DA and quinpirole or RU24926 (specific D2 agonists) inhibited both basal and TRH- or AngII-stimulated membrane PLC activities. 2) Such inhibitions were completely prevented by sulpiride (specific D2 antagonist). 3) Heterotrimeric Gi1/2 proteins coupled the DA receptors to PLC because DA inhibitions were completely reversed by preincubation either with Pertussis toxin or with a specific G{alpha}i1/{alpha}i2 antibody. Such data are in favor of the existence of a direct negative coupling between DA-D2 receptor and PLC on a native physiological plasma membrane model.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PRL SECRETION by lactotroph cells is under the stimulatory and inhibitory control of hypothalamic factors. Substances such as angiotensin II (AngII), TRH, or VIP stimulate and other like dopamine (DA) or somatostatin inhibit this secretion (1, 2, 3, 4).

DA is considered as the major potent PRL inhibiting factor for PRL secretion in vivo as in vitro (1, 2, 4). Activation of dopamine-D2 like (DA-D2) receptors by DA reduces both basal and hormone-stimulated PRL secretions induced by various peptides such as TRH and AngII. This inhibition appears as a complex mechanism because DA-D2 receptors are known to regulate multiple transduction pathways (5, 6, 7, 8, 9, 10, 11, 12). Yet, as PRL secretion is mainly stimulated by an increase in intracellular concentration of cAMP, inositol phosphates (InsPs) and/or calcium, the inhibitory effects of DA were mainly studied on the accumulation of these second messengers. Thus, in rat pituitary cells in primary culture, DA inhibits VIP-stimulated PRL secretion via an inhibition of intracellular cAMP accumulation. This inhibitory effect is triggered by a direct negative coupling between the DA-D2 receptor subtype and adenylyl cyclase via a Pertussis toxin (PTX)-sensitive G protein. In the same biological model, many groups including ours have shown that DA also inhibits TRH- and AngII-stimulated PRL secretion via a decrease of intracellular InsPs accumulation (13, 14). At least two distinct PTX-sensitive mechanisms are involved (13). The first one corresponds to an inhibition of voltage-dependent ionic channels. DA selectively blocks the activity of an L-type calcium channel via DA-D2 receptor (13, 14). As a consequence, both basal and hormone-stimulated calcium influxes are inhibited and the intracellular calcium concentration drops, leading to a parallel decrease of intracellular InsPs concentration because the activity of PLC is highly calcium sensitive (15). The second, fast and Ca2+ independent, might involve a direct inhibition of PLC by DA-D2 receptors (13). Such an hypothesis has been also suggested in many studies where agents such as adenosine, calcitriol, carbachol, histamine, or quisqualate have been reported to inhibit InsP accumulation (16, 17, 18). Moreover, further evidences for a direct inhibition of PLC activity was also reported by Litosch (19, 20). On various acellular systems, these studies demonstrated that GTP{gamma}S at low concentrations could inhibit the activity of this enzyme via a PTX-sensitive G protein. The concept of a direct negative coupling between some classes of G protein-coupled receptors (GPCRs) and the adenylyl cyclase via a PTX-sensitive G protein is now well established (21). This kind of negative coupling between other classes of GPCRs and PLC is also strongly suspected (16, 17, 18). Yet, to our knowledge, no clear experimental demonstration has been provided.

To validate this new transduction pathway and to verify its physiological relevance, we decided to study the effect of specific D2-dopaminergic agonists on the PLC activity present in a crude plasma membrane preparation derived from rat pituitary cells in primary culture. This experimental model allows to eliminate the eventual cross-talk with the other cytoplasmic components and/or the second messengers implicated in the DA-D2 receptor activation. In this study, we presented for the first time the evidence in favor of a direct negative coupling between DA-D2 receptors and PLC via heterotrimeric Gi1/2 proteins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult female Sprague Dawley rats (175–200 g) were obtained from Iffa Credo (L’Arbresle, France) and housed in a controlled environment with food and water available ad libitum.

Animal manipulations were performed according to the recommendations of the French Ethical Committee and under the supervision of authorized investigators.

Pharmacological agents
They were obtained from the following sources: Dopamine HCl from Calbiochem (La Jolla, CA); (+/-)quinpirole HCl, (-)-sulpiride, (+)-SCH-23390 HCl, (+)-SKF-38393 HCl and PTX from Research Biochemicals International (Natick, MA); RU24926 from Roussel Uclaf (Romainville, France); TRH and AngII from Neosystem (Strasbourg, France); myo-[3H]inositol (myo-[2-3H]inositol) from NEN Life Science Products (Boston, MA); and all other compounds were purchased from Sigma (St. Louis, MO).

Cell culture
The rat pituitary cells were prepared by enzymatic dispersion as previously described by Hopkins and Farquhar (22) and adapted to the anterior pituitary by Enjalbert et al. (5). Briefly, the anterior pituitaries were rapidly removed and dissected in sterile conditions after rat decapitation. They were rinsed, cut into small pieces in DMEM-0.3% BSA, pH 7.3 (medium A) and incubated in DMEM-0.5% trypsin (from bovine pancreas) for 15 min at 37 C. Deoxyribonuclease, 2 µg/ml, was then added to the medium for 2 min. After enzymatic digestion, the medium was removed and anterior pituitaries were incubated in DMEM containing a trypsin inhibitor, type I-S from Soybean, 1 mg/ml, for 5 min at 37 C. The medium was then discarded and pituitaries were incubated in Ca2+ and Mg2+-free medium containing 2 mM EDTA for 5 min at 37 C, followed by a 15-min incubation in the same medium containing only 1 mM EDTA. Both the incubations were run under very gentle shaking. The cells were rinsed with Ca2+ and Mg2+-free medium and dispersed mechanically in the same medium. After centrifugation at 500 x g for 10 min and resuspension in medium A, the cells were counted and plated at a density of 15 x 106 cells per dish culture. Cells were maintained 3 d at 37 C in DMEM supplemented with: 10% FCS (pretreated overnight at 4 C with dextran 0.1% and norit A charcoal 1%), 2 mM glutamine, 0.05 mg/ml penicillin and streptomycin in water-saturated atmosphere of 5% CO2.

Myo-[3H]inositol prelabeled membrane preparation
Forty-eight hours before membrane preparation, myo-[3H]inositol, 3 µCi/ml (20 Ci/mmol) was added in the culture medium. Myo-[3H]inositol prelabeled membranes used for PLC activity measurement were prepared according to previous report (23) and adapted to anterior pituitary cells. Briefly, the day of the experiment, the radioactive medium was discarded and the cells were preincubated for 45 min at 37 C in DMEM without serum and myo-[3H]inositol. Cells were then washed three times with cold Ca2+ and Mg2+-free PBS, scrapped, and homogenized in Elvehjem glass/glass potter homogenizer in a cold medium containing: 10 mM Tris-HCl buffer, pH 7.4, 10 mM LiCl, 1 mM EGTA, 0.1 mM ATP, and 0.1 mM phenylmethylsulfonyl fluoride. The cellular homogenate was centrifuged for 3 min at 500 x g at 4 C. The supernatant was recentrifuged for 15 min at 15,000 x g at 4 C and the resulting pellet resuspended in the same Tris-HCl buffer. Rat pituitary cell membrane preparations were either used immediately or preincubated 4 h at 4 C with the antibody studied (0.1 mg purified IgG/ml of membrane suspension). Proteins content was determined using the Bradford assay, with BSA as standard.

Determination of InsP production
Myo-[3H]inositol prelabeled membranes freshly prepared or preincubated with IgG were incubated for various times at 37 C in the presence of 25 µl of vehicle or substance(s) to be tested. The final incubation medium (150 µl) contained: Tris-HCl 10 mM, pH 7.4, 10 mM LiCl, 1 mM EGTA, 5 mM MgCl2, and CaCl2 and GTP at indicated concentrations. The incubation was initiated by the addition of 75 µl of membranes (30–50 µg proteins). The reaction was stopped by addition of 150 µl PCA 10% and 50 µl BSA (20 mg/ml). [3H]-Prelabeled InsPs which accumulated were separated by ion exchange chromatography on Dowex AG 1 x 8 (200–400 Mesh, formate form) as previously described (23). [3H]Inositol 1-phosphates were first eluted and discarded because we previously shown on membrane preparations from WRK1 cells (23) or from rat pituitary cells (data not shown) that InsP1 accumulation was not increased by hormonal stimulation. Then [3H]inositol 1,4-bisphosphates and [3H]inositol 1,4,5-trisphosphates (InsPs) were collected and counted by liquid scintillation spectroscopy.

Antisera
Polyclonal antibodies raised against the C-terminal part of {alpha}i1/2 or {alpha}q/11 were generated in New Zealand rabbits. IgGs obtained from sera were affinity-purified on {alpha}i1/2 or {alpha}q/11 peptide-linked gels as previously described (10, 24, 25). Purified anti-{alpha}i1/2 antibodies recognized specifically both G{alpha}i1 and G{alpha}i2 proteins but did not cross-react with other G{alpha}-subunits (10). Anti-{alpha}q/11 antibodies were shown to be specific to this class of G{alpha} protein (25).

Data analysis
Results were obtained from at least three independent experiments, each performed in quadruplicate. Either representative experiments or means of individual experiments were illustrated. Data were submitted to unpaired t test or ANOVA. The significant differences shown were calculated with absolute dpm values. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of membrane PLC activities from rat pituitary cells in primary culture
Because hormonal stimulations of membrane PLC activities are generally weak and less than in intact cells, preliminary experiments were run to optimize the basal and hormone-induced PLC activities measured on rat pituitary cell membrane preparations. For this purpose, optimal free Ca2+ and GTP concentrations were determined in the absence or presence of TRH an hormone known to stimulate InsPs accumulation in entire pituitary cells (13).

As illustrated in Fig. 1AGo, GTP stimulated in a dose-dependent manner basal InsPs production. In the absence of exogenous GTP, TRH (1 µM) had no significant effect. The hormonal stimulation of InsPs production increased with the GTP level and became maximal (30% stimulation, P < 0.01), in the presence of 0.1 mM GTP. Similarly, the free calcium concentration represented a crucial parameter. As shown in Fig. 1BGo, the basal PLC activity increased dose dependently with free calcium concentration. Maximal activity (5.7-fold stimulation, P < 0.001) was observed for 1 µM free calcium. In the presence of 1 µM TRH, a similar dose-response curve was obtained but slightly shifted for lower calcium concentrations. TRH stimulation was found maximum (53% stimulation, P < 0.001) in the presence of 0.2 µM free calcium, a concentration in the range of intracellular free calcium concentration measured in intact pituitary cells (26). We thus decided to measure hormonal PLC activation in the presence of 0.1 mM GTP and 0.2 µM free calcium. Under these conditions, as shown in Fig. 2Go, AngII and TRH increased InsPs production in a dose-dependent manner with an EC50 = 1.2 ± 0.4 and 4.6 ± 1.8 nM, respectively (n = 3). Maximal stimulations (36 ± 3 and 39 ± 4%, for AngII and TRH, respectively, n = 24) were reproducible and statistically significant (P < 0.01).



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  		Figure 1. Influence of GTP and calcium on basal and TRH-stimulated membrane PLC activities. Myo-[3H]inositol-prelabeled cell membranes, prepared as described in Materials and Methods, were incubated 2 min at 37 C without (control) or with 1 µM TRH in the absence (basal) or presence of increasing amounts of GTP and 0.1 µM free calcium concentration (A) or with increasing doses of free calcium and 0.1 mM GTP (B). Accumulated [3H]InsPs were measured and expressed as dpm per mg of membrane proteins. Results are the mean ± SEM of quadruplicate determinations from one representative experiment of three (panel A) to four (panel B). *, P < 0.05; **, P < 0.01, TRH effects vs. corresponding control values.

 


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  		Figure 2. Effects of TRH and AngII on membrane PLC activities. Myo-[3H]inositol-prelabeled cell membranes were incubated 2 min at 37 C in a medium containing 0.1 mM GTP and 0.2 µM free calcium in the presence of increasing amounts of TRH or AngII. Accumulated [3H]InsPs were measured and expressed as percentage of basal value (100% = 4,145 ± 150 dpm per mg of membrane proteins). Results represent the mean ± SEM of quadruplicate determinations from one representative experiment of four. **, P < 0.01; ***, P < 0.001, TRH or °, P < 0.05; °°, P < 0.01 AngII effects vs. basal values.

 
To further study the nature of the G protein involved in the coupling between TRH receptor and PLC, we preincubated myo-inositol-prelabeled rat pituitary membranes with or without affinity-purified anti-{alpha}q/{alpha}11 antibodies. As illustrated on Fig. 3Go, this pretreatment completely prevented the TRH stimulation of PLC activity. Such results demonstrate the involvement of heterotrimeric Gq/11 protein in the coupling between TRH receptors and PLC as previously described on intact cells (27). Moreover, they indicate that during the course of rat pituitary membrane preparation no abnormal coupling between GPCRs and G proteins occurred.



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  		Figure 3. Involvement of Gq/11 in the TRH stimulation of membrane PLC activities. Myo-[3H]inositol-prelabeled cell membranes were preincubated 4 h at 4 C either with nonspecific rabbit IgG (control), or with purified anti-{alpha}q/{alpha}11 antibodies. (IgG final concentration was 0.1 mg/ml). Membranes were further incubated 2 min at 37 C as described in Fig. 2Go without (basal, B) or with increasing amounts or TRH. Accumulated [3H]InsPs were measured and expressed as percentage of basal control (100% = 4,145 ± 150 dpm per mg of membrane proteins). Values are the mean ± SEM of quadruplicate from one representative experiment of five. **, P < 0.01; ***, P < 0.001, TRH effects vs. basal values.

 
Effects of dopamine-D2 agonists or antagonists on membrane PLC activities
DA inhibited the basal PLC activity in a dose-dependent manner with an IC50 = 0.27 µM (Fig. 4AGo and Table 1Go). For DA concentrations higher than 0.1 µM, this effect was partial but significant (20%, P < 0.001). In the same conditions, RU 24926 and quinpirole, two well known specific D2 agonists mimicked the DA action (Fig. 4, B and CGo). Whatever the dopaminergic agonist used, the same maximal inhibition of basal PLC activity was observed with a similar efficiency (Table 1Go).



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  		Figure 4. Inhibition of basal and TRH-stimulated membrane PLC activities by DA and DA-D2 agonists. Myo-[3H]inositol-prelabeled cell membranes were incubated as described in Fig. 2Go with 1 µM TRH or with vehicle (control) in the presence of increasing amounts of DA (panel A), RU24926 (panel B), quinpirole (panel C), or without DA or DA agonist (basal condition, B). Accumulated [3H]InsPs were measured and expressed as percentage of basal control value (100% = 3,210 ± 190 dpm per mg of membrane proteins). Results represent the mean ± SEM of quadruplicate determinations from one representative experiment of 8. *, P < 0.05; **, P < 0.01; ***, P < 0.001, DA, RU24926, or quinpirole effects vs. TRH alone and °, P < 0.05; °°, P < 0.01; °°°, P < 0.001, DA, RU24926, or quinpirole effects vs. control basal values.

 

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  		Table 1. Dopaminergic inhibition of basal and TRH-stimulated membrane PLC activities

 
Dopamine, RU 24926, or quinpirole also inhibited dose dependently the TRH-stimulated InsPs production, with an IC50 not statistically different from those observed for basal PLC activity (Fig. 4Go and Table 1Go). Maximal dopaminergic inhibitions of TRH-stimulated PLC activity were partial (21–25%) but statistically significant whatever the dopamine agonist used (Fig. 4Go and Table 1Go). Similar data were also obtained when PLC was stimulated by AngII (data not shown).

To determine the nature of the DA receptor subtype involved in these inhibitory processes, we performed pharmacological studies. As illustrated on Fig. 5AGo, 1 µM sulpiride (a specific D2 antagonist), 1 µM SCH-23390 (a specific D1 antagonist), or 1 µM SKF-38393 (a specific D1 agonist) did not significantly modify the basal PLC activity. In contrast, the PLC inhibitions induced either by DA or specific D2 agonists were selectively blocked by preincubation with sulpiride but not by SCH-23390 or SKF-38393. These data strongly suggest that the basal PLC inhibition is mediated by D2 dopaminergic receptors.



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  		Figure 5. Pharmacological profiles of dopaminergic inhibition of membrane PLC activities. A, Myo-[3H]inositol-prelabeled cell membranes were preincubated, as described in Fig. 2Go, 15 sec at 37 C with vehicle (control), 1 µM sulpiride (SULP), 1 µM SCH-23390 (SCH), or 1 µM SKF-38393 (SKF). Then 10 µM DA, 10 µM quinpirole (QUIN), 10 µM RU 24926 (RU), or vehicle (basal) was added in the incubation medium and the reaction allowed to proceed for 2 additional minutes. B, Myo-[3H]inositol prelabeled membranes were preincubated 15 sec at 37 C with vehicle (control), 1 µM SULP, with or without 10 µM DA. Then either 1 µM TRH, 1 µM AngII, or vehicle (basal) was added in the incubation medium and the reaction allowed to proceed for 2 additional minutes. Accumulated [3H]InsPs were measured in each condition and expressed as percentage of control basal value (100% = 4,960 ± 210 dpm per mg of membrane proteins). Results represent the mean ± SEM of three distinct experiments each performed in quadruplicate. **, P < 0.01; ***, P < 0.001.

 
To verify whether the same DA-D2 receptors were involved in the inhibition of neuropeptide-stimulated PLC activities, we tested the effect of sulpiride on the dopaminergic inhibition of TRH- or AngII-induced InsPs production. As shown in Fig. 5BGo, sulpiride did not modify the basal or hormone-stimulated PLC activities but was able to fully reverse the dopaminergic inhibition of TRH- or AngII-stimulated InsPs production.

Involvement of G proteins in the dopaminergic inhibitions of membrane PLC activities
Because on intact pituitary cells, the dopaminergic inhibitions of InsP accumulation were PTX sensitive (7, 13), we further tested the effect of this toxin on membrane PLC activities. As illustrated in Fig. 6AGo, a PTX pretreatment did not alter the stimulatory effect of TRH or AngII on InsPs production. By contrast, it almost completely reversed the dopaminergic inhibition of basal and TRH- or AngII-stimulated InsPs production. These results suggest the involvement of Gi and/or Go proteins.



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  		Figure 6. Involvement of G proteins in the dopaminergic inhibition of membrane PLC activities. A, Rat pituitary cells were infected without (control) or with PTX (0.1 µg/ml) for the last 18 h of culture. Myo-[3H]inositol-prelabeled cell membranes were preincubated 15 sec at 37 C without or with 10 µM DA. Then either 1 µM TRH, 1 µM AngII, or vehicle (basal) was added in the incubation medium and the reaction allowed to proceed for 2 additional minutes. Accumulated [3H]InsPs were measured in each condition and expressed as percentage of corresponding basal control value (100% = 3,430 ± 150 and 2,560 ± 130 dpm per mg protein for membrane preparations deriving from control and PTX-pretreated cells, respectively). Values are the mean ± SEM of three to five distinct experiments each performed in quadruplicate. *, P < 0.05. B and C, Myo-[3H]inositol-prelabeled cell membranes were incubated 4 h at 4 C either with rabbit IgG (B) or with anti-{alpha}i1/2 antibody (C). Membranes were then incubated as described in panel A. Accumulated [3H]InsPs were measured and expressed as % of corresponding basal control value (100% = 3,120 ± 150 and 3,270 ± 180 dpm per mg protein from membranes preincubated with IgG and specific antibody, respectively). Values represent the mean ± SEM of quadruplicate determinations from one representative experiment of four. **, P < 0.01; ***, P < 0.001.

 
To verify this assumption, before PLC assays, we incubated rat pituitary membranes either with a nonspecific IgG (Fig. 6BGo) or with an affinity-purified anti-{alpha}i1/2 antibodies (Fig. 6CGo). While nonspecific IgG did not alter the inhibitory effects of DA on basal, TRH-, and AngII-stimulated PLC activities, pretreatment with specific G{alpha}i1/2 antibodies completely reversed these inhibitory effects of DA.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
As previously shown in rat anterior pituitary cells in primary culture, DA-D2 receptors inhibited InsP accumulation via multiple molecular mechanisms (13, 14). The first one to be described involved the inhibition of voltage-dependent calcium channels (13, 14). In the present study, we characterize a more direct negative coupling between DA-D2 receptors and PLC via a heterotrimeric Gi1/2 protein.

To demonstrate a direct coupling between GPCRs, heterotrimeric G proteins, and PLC, we decided to work on plasma membrane preparations obtained from rat pituitary cells in primary culture, grown in the presence of myo-[3H]inositol rather than on membranes labeled by addition of exogenous [3H]phosphatidyl-inositol 4,5 bisphosphate. This latter technique, known to give a better signal for measurement of GTP{gamma}S-stimulated PLC activity (28), needs the presence of deoxycholate in the assay medium. This ionic detergent allows the incorporation of labeled phospholipids into plasma membrane preparation but also uncouples the hormonal receptors from the G proteins and thus prevents the measurement of hormone-stimulated PLC activity (data not shown). Moreover, by working on membrane preparations, we eliminated some potential cross-talk with cytosolic proteins (PKC, PKA, calmodulin kinases...) and/or second messengers (cAMP, calcium...) known to potentially modify InsP accumulation. Such an experimental approach has been successfully used to demonstrate either a direct positive and negative coupling between some GPCRs and adenylyl cyclase (21) or a direct positive coupling with PLC (23, 29). We also studied hormonal PLC activity modulation on a native physiological plasma membrane model. This preparation represents a convenient system to study interactions between DA-D2 and AngII or TRH receptors since the lactotroph cells (60% of cells in our primary culture) express both type of receptors (30, 31, 32). As the DA-D2 receptor isoform triggering the inhibition of InsPs accumulation is not defined, we did not use established PRL secreting pituitary cell lines such as GH4C1 or MMQ because these cells express no or only one DA-D2 receptor isoform (33, 34). We also did not use transfected cells because sometimes some abnormal couplings occurred. Thus, Vallar et al. (35) described that DA-D2 receptors transfected in different cell lines lead to differential couplings to PLC activities. Surprisingly, stimulation of InsPs accumulation was observed in transfected Ltk-fibroblasts.

First, we optimized the PLC assay to get a maximal hormonal stimulation of inositol bis and trisphosphate production. As previously observed on other membrane systems, both GTP and Ca2+ concentrations represent critical parameters (23, 36). Both basal and hormone-stimulated PLC activities increased with GTP and calcium. Maximal TRH and AngII stimulations were observed in the presence of 0.1 mM GTP and 0.2 µM free calcium. Under these conditions, we showed on membrane preparations that TRH and AngII stimulated InsPs production with an EC50 similar to those found in intact cells (13). Moreover, these values are similar to the affinities of TRH and AngII found for their specific membrane receptors (37, 38). This suggests that the membrane preparations used exhibited a functional hormonal receptor/PLC coupling. Its efficiency was, however, reduced when compared with that measured in intact cells. This could be explained by: 1) a lack of cytosolic components that regulate GPCRs signaling like RGS/GAIP (39, 40, 41); 2) a disorganization of the cellular cytoskeleton because some G proteins, namely {alpha}q/{alpha}11 that couple TRH and AngII receptors to PLC isoforms, are known to be associated with actin or tubulin networks (25, 42, 43). In addition, this disorganization could also implicate other factors such as gelsolin, which has been recently described to play a pivotal role in cytoskeletal remodeling and PLC signaling (44). This reduction seems to be a general phenomenon because it has been previously observed in different membrane preparations for the effects of various hormonal receptors coupled to PLC but also to adenylyl cyclase (23, 36, 45). Despite this weak hormonal stimulation, the membrane PLC assay we developed is reliable. InsPs production induced by AngII or TRH is statistically significant, very reproducible, and obtained for physiological hormone concentrations. In addition, using specific anti-{alpha}q/{alpha}11 antibodies, we confirmed that heterotrimeric Gq/11 proteins couple the TRH receptors to PLC as previously described on intact cells (27). All together, these data validate the use of this experimental model for studying direct couplings between GPCRs and PLC.

Then, we described on membrane preparations a dose-dependent inhibition of basal and hormone-stimulated PLC activities by DA. Pharmacological data strongly suggest the involvement of DA-D2 receptor subtype because: 1) RU 24926 and quinpirole, two well known specific D2 agonists, mimicked the DA inhibitory effects. 2) SKF-38393, a specific D1 agonist, did not modify both the basal and hormone-stimulated PLC activities. 3) Sulpiride, a DA-D2-specific antagonist, but not SCH-23390, a DA-D1-specific antagonist, completely suppressed the inhibitory effects of DA on PLC activities. Compared with entire pituitary cells, the maximal DA inhibition obtained on acellular preparations was reduced by 2-fold (13). This reduced efficiency might be due to 1) the loss of cytosolic components potentially involved in these inhibitory coupling mechanisms. For instance, spinophilin (46) and ABP-280 proteins (47), which interact with the third cytoplasmic loop of D2 receptors, may play a role as scaffolding/adapter protein in organizing the D2 receptor signaling; 2) the suppression of the indirect negative coupling between DA-D2 receptors and PLC via calcium voltage-dependent channels as previously described (13, 14). Thus, on intact lactotroph cells, two distinct mechanisms involving the DA-D2 receptor negatively regulate the PLC activity: a direct one observed on membrane preparation (this study) and an indirect one involving calcium voltage-dependent channels (13, 14). These fine tunings of PLC activity could contribute to the major regulation of PRL secretion exerted by DA under physiological conditions.

As PTX pretreatment completely abolished the dopaminergic inhibition of basal and hormone-stimulated PLC activities, we hypothesized that heterotrimeric Gi/o proteins triggered the negative coupling between DA-D2 receptors and PLC. Previous studies performed on rat pituitary tumor cell lines have demonstrated that G{alpha}i2 protein preferentially coupled the DA-D2 receptors to adenylyl cyclase (48, 49). In addition, the Gi1/2 proteins are expressed in the rat pituitary tissue (50, 51). Thus, we further tested the influence of specific anti-{alpha}i1/2 antibodies on the dopaminergic inhibition of basal and TRH- or AngII-induced PLC activities. Data presented in this study favored a role for Gi1/2. Further experiments are now in progress to specify the G protein subunit involved in this inhibitory process because: 1) the antibody used could not discriminate between G{alpha}i1 and G{alpha}i2 and 2) the Gß{gamma} dimers have been shown to directly bind and regulate numerous effectors including PLC (for review, see Ref. 52). Similarly, the DA-D2 receptor isoform involved in this negative coupling remains to be characterized because the rat anterior pituitary tissue contains mRNA encoding for both the short and long isoforms of DA-D2 receptors (for review, see Ref. 2). The nature of the PLC subtype involved in these inhibitory processes also remains to be clarified. PLCß isoforms are probably involved since they have been shown to be modulated by GPRCs (53). In fact, TRH and AngII receptors stimulate PLCß isoforms through coupling to G proteins belonging to the Gq subfamily (27, 53). Yet, we cannot exclude the involvement of other PLC subtypes because it had been described that the AT1 AngII receptor can also be coupled to PLC{gamma} isoform (for review, see Ref. 54).

In conclusion, our study demonstrates that DA-D2 receptors could inhibit PLC activity via a mechanism that involves heterotrimeric Gi1/2 proteins. Such a mechanism indicates that PLC, like adenylyl cyclase, could be directly regulated either in a positive or negative way by seven-transmembrane domain receptors through distinct heterotrimeric G proteins.


   Acknowledgments
 
We wish to thank Dr. S. Drouva and Dr. J. Bockaert for advice and criticisms during the preparation of this manuscript. We also thank Mrs. M. Passama for drawing the illustrations and Mrs. M. Challier for correcting the manuscript.


   Footnotes
 
Abbreviations: AngII, Angiotensin II; DA, dopamine; DA-D2, dopamine-D2 like; GPCR, G protein-coupled receptors; InsP, inositol phosphate; PTX, Pertussis toxin.

Received August 10, 2001.

Accepted for publication November 15, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lamberts SWJ, MacLeod RM 1990 Regulation of prolactin secretion at the level of the lactotrophs. Physiol Rev 70:279–317[Free Full Text]
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