Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

In pregnant rat myometrium, ₂-adrenoceptor (AR) signaling pathways differentially modulate ₂-AR-mediated regulation of adenylyl cyclase (AC) at midpregnancy and at term (Mhaouty et al., 1995). At midterm, ₂-AR activation potentiates adenylyl cyclase activity stimulated by ₂-AR, thus enhancing uterine relaxation in response to catecholamines. This augmentation of AC activity induced by ₂-AR probably involves the type II family isoform of AC and is caused by the input of G released from Gi (Gi₂ and/or Gi₃) that synergizes with Gs to further elevate cAMP levels (Mhaouty-Kodja et al., 1997). In contrast, at term, myometrial ₂-AR/Gi signaling pathways reduce the ₂-AR-induced-cAMP generation to allow intracellular Ca²⁺ increase and cell contraction. This switch in the stimulatory versus inhibitory input to ₂-AR-dependent cAMP generation that occurs between mid- and late pregnancy may be influenced by changes in the expression of AC isoforms, G proteins, and/or ₂-AR subtypes expression.

When comparing mid- and late pregnancy, no substantial modification in the amounts of specific types of AC transcripts and no alteration in the basal activity of the AC system (Mhaouty-Kodja et al., 1997) could be found. In particular, the expression of transcripts encoding for members of AC type II family, which are involved in this potentiation process, persisted throughout the time course of pregnancy up to parturition (Mhaouty-Kodja et al., 1997). Conversely, as shown by pharmacological data (Bouet-Alard et al., 1997) and Northern blot analysis (Mhaouty et al., 1995), the two ₂-AR subtypes expressed in rat myometrium, _2A- and _2B-AR, were differentially expressed at midpregnancy and term (midpregnancy _2A/D-AR _2B-AR; term _2B  2A/D-AR). Also, significant changes in the Gi₂/Gi₃ ratio could be detected by immunoblot analysis (Tanfin et al., 1991; Cohen-Tannoudji et al., 1995). Altogether, these data suggested that the switch of regulation mediated by ₂-adrenoceptors toward ₂-dependent cAMP production could result from a specific signaling of ₂-AR subtypes toward AC II activity and/or alteration in receptor coupling to G proteins.

As an initial approach, we used DDT1-MF2 (hamster vas deferens smooth muscle cell) cotransfectants stably expressing _2A/D-AR (RG20) or _2B-AR (₂C2) and AC type II isoform and studied the regulation induced by each ₂-AR subtypes on ₂-stimulated AC II activity.

We report, herein, agonist and receptor specific regulation of ACII that involves selective coupling to Gi₂ versus Gi₃. Our results also shed light upon molecular mechanism by which clonidine acts as a partial agonist through _2B-AR.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

[³H]cAMP (30 Ci/mmol), [³²P]ATP (30 Ci/mmol), [³H]rauwolscine (81 Ci/mmol), [-³²P]GTP (3000 Ci/mmol), and [¹²⁵I]cAMP radioimmunoassay kit were purchased from NEN Life Sciences Products (Les Ulis, France). ARC-239 bichloride (2[2-[4(O-methoxy-phen) piperazine-1-y]4,4dimethyl-1,3,2H-4H] isoquinolinedione) was a gift from Karl Thomae (Biberach, Germany). 4-Azidoanilide was obtained from Fluka Biochemicals (Saint Quentin Fallavier, France). 1-Ethyl-3-[3-(dimethylamino)propyl] carbodiimide HCl and Dowex 50W-X4 (100-200 mesh, hydrogen form) were from Bio-Rad (Ivry sur Seine, France). PEI-cellulose plates and aluminum oxide (90 active neutral) were purchased from Merck (Nogent sur Marne, France). Cell culture supplies were obtained from Life Technologies (Cergy-Pontoise, France). Pansorbin cells were supplied from Calbiochem (Meudon, France). GF/C glass-fiber filters were from Millipore (Saint Quentin en Yvelines, France). All remaining drugs were from Sigma-Aldrich (Saint Quentin Fallavier, France).

Cell Culture and Transfection. DDT1-MF2 cells were grown and stably transfected with human _2B (₂C2) or rat _2A/D (RG20) cDNA as described previously (Duzic and Lanier, 1992). Resistant clones were tested for their ₂-AR binding capacity using the selective tritiated antagonist [³H]rauwolscine as described under binding studies. Clones expressing a receptor density between 1 and 1.5 pmol/mg of membrane proteins, were further cotransfected with the ACII cDNA expression vector and the drug resistance cassette pHyg according to the transfection strategy described by Gorman (1986). Transfected cells were selected by their resistance to hygromycin B (750 mg/ml). Each clone was then analyzed for AC II expression by Northern blot using a ³²P-labeled cDNA AC II probe [rat full-length (4 kilobases)] as described previously (Marjamaki et al., 1997) and tested for enzyme activity.

Partially Purified Membrane Preparation. Membranes were prepared by hypotonic lysis in ice-cold lysis buffer (5 mM Tris-HCl, pH 7.5, 5 mM EDTA, 5 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, and 10 mg/ml pepstatin A) and collected by centrifugation (17,000g for 15 min at 4°C). Membrane pellet was resuspended in 50 mM HEPES, pH 8.0, for adenylyl cyclase assay or 50 mM Tris, pH 7.5, 5 mM MgCl₂, 0.6 mM EDTA for binding studies. Protein concentration was determined according to the method of Schacterle and Pollack (1973) using bovine serum albumin as standard.

Binding Studies. For saturation experiments, membranes (30-40 µg) were incubated with the required concentrations of [³H]rauwolscine [1-50 nM] for 20 min at 25°C in a final volume of 100 µl. Nonspecific binding was determined in the presence of 10 µM phentolamine. Competition studies were performed in presence of increasing concentrations (10 pM-50 µM) of various competitors and 8 nM [³H]rauwolscine (a concentration near the K_D value). Bound radioligand was separated from the free by vacuum filtration over GF/C glass-fiber filters as described previously (Bouet-Alard et al., 1997). Radioactivity was counted by liquid scintillation in a 1214 Rack- spectrometer (LKB, Rockville, MD) with a counting efficiency of approximately 30%.

Adenylyl Cyclase Assay and Determination of Intracellular cAMP Accumulation. Adenylyl cyclase activity was measured as described previously (E. Duzic and S.M. Lanier, 1992) using 50 µg of crude membrane. For intracellular cAMP accumulation, cells were plated at a concentration of 5 × 10⁵ cells/well in six-well plates and incubated at 37°C for 24 h. One hour before starting the experiment, the medium was removed and replaced with 4 ml of serum-free DMEM containing 20 mM HEPES, pH 7.5, and 250 mM isobutyl-L-methylxanthine. Then, cells were incubated with drugs to be tested for 10 min at 37°C. The reaction was stopped by aspiration of the medium and cells were disrupted by the addition of 1 ml of 10% ice-cold trichloracetic acid per well. After recovering the cellular lysate by scrapping the wells, samples were centrifuged (10,000g, 15 min at 4°C). The supernatants were then extracted 3 times with diethyl ether (1v/4v) and cAMP contents were determined by a cAMP radioimmunoassay system obtained from NEN Life Sciences Products.

Immunoblot and Immunoprecipitation. For immunoblotting, 50 µg of membranes obtained from DDT1-MF2 cells or tissues were resolved on 10% SDS-PAGE and transfer to polyvinylidene difluoride-transfer membrane POLYSCREEN (NEN Research Products, DuPont de Nemours, France). After transfer, the blots were probed with anti-G protein subtype specific antibodies as described previously (Cohen-Tannoudji et al., 1995). Briefly, after blocking, the polyvinylidene difluoride blots were incubated with the primary antibody [AS/7 (anti-Gi₂/Gi₁) or EC/2 (anti-Gi₃/Go) or GC/2 (anti-Go)] for 1 h in a high-detergent 5% nonfat dry milk/Tris-buffered saline at room temperature at a dilution of 1:1000. After four successive washes, the blots were incubated for 1 h with HRP-conjugated secondary antibody in high-detergent 5% nonfat dry milk/Tris-buffered saline. After washing, bound antibodies were visualized using enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, les Ulis, France). For immunoprecipitation, antisera were raised against the C-terminal decapeptide (amino acids 345-354) of Gi₃ and against the C-terminal decapeptide (amino acids 345-354) that is shared by both Gi₁ and Gi₂ as described previously (Gettys et al., 1994). The antisera were characterized with respect to titer, specificity, and cross-reactivity using lysates from bacteria transformed with cDNA for each of the G proteins. Each antiserum was desalted and purified as described previously (Raymond et al., 1993; Gettys et al., 1994).

Photolabeling of Membrane G Proteins. The 4-azidoanilido-[-³²P]GTP ([-³²P]AA-GTP) was synthesized according to the method described by Offermanns et al., 1990 and 1991), except that [-³²P]AA-GTP was purified using a thin-layer chromatography and finally resuspended in water at a concentration of 4 × 10⁹ cpm/ml. Photoaffinity labeling of G proteins was performed as described by Offermanns et al. (1991). Briefly, cell membranes (50 µg) were preincubated with ₂-AR agonists and/or antagonists for 10 min at 30°C in 50 µl of assay buffer containing 30 mM HEPES, pH 7.5, 100 mM NaCl, 100 mM EDTA, 1 mM benzamidine, 5 mM MgCl_2, 50 mM leupeptin, and 3 µM GDP. Then, 10 µl of [-³²P]AA-GTP (0.4 × 10⁹ cpm/ml) diluted in distilled water was added to each sample and the labeling reaction was allowed to proceed for 10 min. The reaction was stopped by the addition of 100 µl of ice-cold assay buffer and immediate 4°C centrifugation at 12,000g for 10 min. All subsequent procedures were performed at 4°C. Supernatants containing free [-³²P]AA-GTP were removed. Membrane pellets were rapidly resuspended in 55 µl of assay buffer supplemented with 2 mM dithiothreitol and exposed, on ice, to UV light (254 nm, 15 W) in the dark for 4 min. Before immunoprecipitation, photolabeled membranes (50 µl) were then solubilized by incubation in presence of 0.25% SDS at 60°C for 5 min and addition of 50 µl of an immunoprecipitation buffer (0.5% SDS, 2% Nonidet P40, 1% cholate, 150 mM NaCl). Solubilized samples were first incubated 30 min at 4°C with prewashed Pansorbin cells (25 µl of a 10% solution in 50 mM sodium phosphate, pH 7.4) to further minimize the nonspecific antibody binding. After removing Pansorbin cells by centrifugation (700 g), each sample was divided in two aliquots and incubated in presence of anti-Gi₂ or -Gi₃ IgG (1:50) over night at 4°C under constant rotation. The immunocomplexes were collected by the addition of 25 µl of Pansorbin cell suspension and centrifugation at 700g. Then, pellets were washed two times in PBS and resuspended in 30 µl of 1.5% SDS and 30 µl of Laemmli buffer (Laemmli, 1970). Samples were boiled for 5 min before 10% SDS-PAGE analysis. After drying the gel, photolabeled proteins were visualized by autoradiography on Kodak X-Omat AR-5 films (Sigma-Aldrich). Incorporation of [-³²P]AA-GTP into immunoprecipitated G proteins  subunits was quantified by densitometric analysis of autoradiograms with an Imstar computer-assisted image analyzer. Results are expressed as -fold incorporation of [-³²P]AA-GTP into immunoprecipitated G protein  subunits compared with unstimulated control subunits.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Establishment of the Experimental System. DDT1-MF2 cells express useful common and distinct signaling entities in comparison with pregnant myometrium. Indeed, they display a similar density of ₂ adrenoceptors and Gs proteins (Hadcock et al., 1991; Vivat et al., 1992). They also express the same isoforms of pertussis toxin (PTX)-sensitive G proteins (Gi₂ and Gi₃) that exert a similar tonic inhibition of adenylyl cyclase activity in an agonist-independent manner (Tanfin et al., 1991; Cohen-Tannoudji et al., 1995). However, none of ₂-AR subtypes (Philippe et al., 1989; Duzic and Lanier, 1992) nor AC isoforms type II and IV could be detected (Marjamaki et al., 1997). Thus we established, in this cell line, an experimental system expressing _2A/D- or _2B-AR subtype in presence of AC II using stable gene transfection method to further assess their functional characterization.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Analysis of pharmacological properties and functionality of the transfected 2B-AR in DDT1-MF2 cells. A, membranes were prepared from DDT1-MF2 cells stably transfected with the 2B-AR cDNA. Competition studies were performed in the presence of 8 nM [3H]rauwolscine (a concentration near the KD value) and increasing concentrations (0.1 nM to 1 mM) of various competitors. Values represent the mean ± S.E. of three separate determinations performed in duplicate. The inset indicates pKi values. B, cells were incubated with 1 µM isoproterenol and increasing concentrations of clonidine (0.1 nM to 1 mM). cAMP accumulation was determined as described under Materials and Methods. The specificity of clonidine (1 µM) was evaluated in the presence of yohimbine (0.1 mM). Basal cAMP and isoproterenol (1 µM) stimulated-cAMP accumulation in presence of GTP (pmol/mg of protein) were, respectively: 55.6 ± 12.2 and 2499 ± 493. Data are expressed as the percentage of isoproterenol-stimulated cAMP production (control = 100%) and represent the mean ± S.E. of three independent experiments performed in duplicate.  and , clonidine effect alone and clonidine effect in presence of yohimbine, respectively. Arrow represents the first time point with significance (p < 0.05) versus isproterenol control.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Effect of GTPS on adenylyl cyclase activity in DDT1-MF2 cells stably transfected with cDNAs encoding 2B-AR (2B) alone and 2B-AR and adenylyl cyclase II (2B-AC II). Adenylyl cyclase activity was measured as described under Materials and Methods using 50 µg of membrane protein. Maximally stimulated enzyme activity was determined in the presence of 10 µM GTPS. Basal AC activity (pmol of cAMP/10 min/mg of protein): DDT1-MF2-2B, 73 ± 42; DDT1-MF2-2B-ACII, 136 ± 7. Values represent the mean ± S.E. of three independent determinations performed in duplicate.

Effect of 2-AR Activation on Cellular cAMP in DDT1-MF2 Cotransfectants. Whereas epinephrine stimulation potentiated the ₂-activated cAMP production in DDT1-MF2-_2B-ACII cotransfectants, clonidine decreased it (Fig. 3A). Indeed, epinephine enhanced ₂-stimulated cAMP production up to 52% ± 2 at 10 µM with an ED₅₀ value of 114 ± 33 nM. Conversely, clonidine produced a dose-dependent attenuation of cAMP accumulation over a concentration range of 1 nM to 0.1 mM. Maximal inhibition (43%) was obtained at 1 µM (*p < 0.05). Half-maximal inhibition (ED₅₀) occurred at 10 nM clonidine. With higher concentrations of clonidine, negative input persisted, although it was reduced. The inhibitory influence of the clonidine-activated _2B-AR did not seem to be caused by low receptor expression in _2B-ACII cells. Indeed, in these cells, B_max was 1.3 ± 1 pmol of receptor/mg of membrane protein, a receptor density equivalent to the one measured in _2A/D-ACII cells (1.2 ± 0.045 pmol/mg). Furthermore, we tested six _2B-ACII clones ranging in receptor density from 1 to 3 pmol/mg of protein; none produced significant potentiation of isoproterenol-stimulated cAMP accumulation using clonidine (data not shown). Conversely, they all reduced cAMP-₂-AR dependent generation. These data demonstrated that in DDT₁-MF₂ cells expressing type II AC isoform, the _2B-AR could translate into opposite response depending on the type of agonist used (epinephrine or clonidine).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effect of clonidine (clo) or epinephrine (epi) on isoproterenol-stimulated cAMP production in DDT1-MF2 -2B-AC II (A) or 2A/D-AC II (B) transfectants. Cells were incubated with isoproterenol (1 µM) and increasing concentrations of clonidine or epinephrine and cAMP concentrations were determined as described under Materials and Methods. Data are expressed as the percentage of isoproterenol-stimulated cAMP production (control = 100%). Results are the mean ± S.E. of three independent determinations performed in duplicate. The dose-response significance as well as dose-response curve differences were analyzed by one-way analysis of variance followed by Bonferroni's multiple range test.  and , effects of epinephrine and clonidine, respectively.  and , effects of agonists in presence of yohimbine. Arrows represent the first time point with significance (p < 0.05) versus isoproterenol control. Basal cAMP concentrations were 44 ± 5 pmol/mg of protein and 19 ± 3 in 2B-ACII cells and 2A/D-ACII cells, respectively. Isoproterenol (1 µM) -stimulated cAMP accumulations were 1959 ± 234 and 1894 ± 187 pmol/mg of protein in 2B-ACII and 2A/D-AC II cells, respectively.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effect of clonidine or epinephrine on isoproterenol-stimulated cAMP production in DDT1-MF2-2B-AC II or 2A/D-AC II cotransfectants after cell preincubation in presence or absence of PTX. Confluent plates of cells were pretreated with pertussis toxin (100 ng/ml) or vehicle for 18 h at 37°C in normal culture medium. Cells were further incubated with isoproterenol (1 µM) in the absence and presence of clonidine (1 µM) or epinephrine (1 µM) for 10 min at 37°C. Data are expressed as percentage inhibition or augmentation of isoproterenol-induced elevation of cAMP production and represent the mean ± S.E. of three to six separate experiments performed in duplicate. Values obtained in the presence and absence of pertussis toxin were compared using an unpaired Student's t test. *Statistically significant difference (p < 0.05).

Selective Recruitment of Gi Proteins by _2B- or _2A/D -AR in Response to Clonidine or Epinephrine. Within the family of PTX-sensitive G proteins, DDT₁-MF₂ cells and myometrium express Gi₂ and Gi₃ (Fig. 5) (Hadcock et al., 1991; Tanfin et al., 1991, Cohen-Tannoudji et al., 1995). Thus, agonist-specific adenylyl cyclase II response could be the consequence of a differential ₂-AR subtype specific recruitment of Gi₂ and/or Gi₃. So, we questioned whether the differences observed in the receptor coupling to AC for clonidine and epinephrine in the cell models reflected specific coupling to Gi₂ or Gi₃. This issue was addressed by incubation of membranes with the photoreactive GTP analog, 4-azido-anilido-[³²P]GTP ([-³²P]AA-GTP) in the presence of ligand, followed by cross-linking, solubilization, and selective immunoprecipitation of Gi₂ or Gi₃.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Identification of Gi and Go proteins in DDT1-MF2 cotransfectants cell membranes. Immunoblotting was performed with antibodies directed against Gi1 and Gi2 (AS/7), Gi3 and Go (EC/2), or Go (GC/2) as described under Materials and Methods. Br is used for rat brain, DDT1 for DDT1-MF2 cells, and Myo for rat pregnant myometrium. Molecular masses (kDa) of protein are given next to molecular size markers.

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Effect of clonidine (A) and epinephrine (B) on [-32P]AA-GTP incorporation into Gi2 and Gi3 proteins in membranes obtained from 2B-AC II transfectants. Cell membranes (50 µg) were incubated with [-32P]AA-GTP and increasing concentrations of clonidine (clo) or epinephrine (epi) as described under Materials and Methods. After solubilization, photolabeled aliquots (20 µg) were incubated with anti-Gi1/2 or anti-Gi3/o. Immunocomplexes were precipitated and analyzed on SDS-PAGE as described under Materials and Methods. Gels were submitted to autoradiography with intensifying screens for 5 to 7 days. The specificity of epinephrine (1 µM) was determined in the presence of 100 µM yohimbine (Yo). The autoradiograms were scanned with Imstar computer-assisted image analyzer. Results are expressed as the percentage of incorporation of [-32P]AA-GTP into immunoprecipitated G protein  subunits assuming unstimulated controls as 100%. The curves were fit by least-squares and the autoradiograms are representative of four to seven experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Effect of clonidine on [-32P]AA-GTP incorporation into Gi2 and Gi3 proteins in membranes obtained from 2A/D-AC II transfectants. Cell membranes (50 µg) were incubated with 10 nM or 1 µM clonidine (clo) and [-32P]AA-GTP as described under Materials and Methods. The specificity of clonidine (1 µM) was determined in presence of 100 µM yohimbine (yo). After solubilization, photolabeled aliquots (20 µg) were incubated with anti-Gi1/2 or anti-Gi3/o. Immunocomplexes were precipitated and analyzed on SDS-PAGE as described under Materials and Methods. Gels were submitted to autoradiography with intensifying screens for 5 to 7 days. The autoradiograms were scanned with Imstar computer-assisted image analyzer. Results are expressed as percent of incorporation of [-32P]AA-GTP into immunoprecipitated G proteins  subunits assuming unstimulated controls as 100%. The autoradiograms are representative of seven experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Activation of ₂-AR subtypes induces multiple cellular effects, including inhibition of adenylyl cyclase or, in some physiological models or cell systems, an increase of cAMP levels. The mechanisms responsible for inhibitory or stimulatory input on adenylyl cyclase activity/cAMP production greatly depend on their interaction with PTX-sensitive G inhibitory proteins and, additionally, also reflect the type of adenylyl cyclases expressed in various cells.

The present work was motivated by the observation that, in the midpregnancy and term myometrium, the cross talk between activated ₂- and ₂-ARs differently affect the degree of intracellular cAMP generation (Mhaouty et al., 1995) and, consequently, the relaxed or contractile state of the uterus (Do Khac et al., 1986). Molecular events that underlie these subtle changes in sensitivity of the smooth muscle to catecholamines may result from 1) the alteration of the _2A/D-/_2B-subtypes expression pattern (Bouet-Alard et al., 1997); 2) the drastic changes of Gi₂/Gi₃ protein ratio (Tanfin et al., 1991; Cohen-Tannoudji et al., 1995); and/or 3) the functional properties of the pregnant myometrium adenylyl cyclase population (Mhaouty-Kodja et al., 1997; Suzuki et al., 1997). As an initial approach, we investigated whether _2A/D- and _2B-AR subtypes could exert different regulatory roles on ₂-AR catalyzed cAMP production. To address this issue, DDT1-MF2 cells provided an interesting context, because they endogenously express some of the molecular entities (₂-AR, Gi₂, Gi₃, and G_s proteins) involved in myometrium ₂-/₂-AR cross talk but lack ₂-AR subtypes and AC type II isoform. Thus, we separately expressed _2B- or _2A/D-AR subtype in this cell line and, further on, cotransfected each clone with the adenylyl cyclase II isoform that potentiated cAMP production in response to ₂-AR agonists in pregnant myometrium. Selected clonal cell lines with comparable functional pools of ARs and adenylyl cyclase have provided useful test models for a careful examination on how ₂-AR differ in their ability to modulate adenylyl cyclase and to couple to endogenous Gi protein.

In this context, we found that, without any ACII expression, clonidine induced an inhibition of ₂-dependent cAMP production in both _2A- and _2B-AR transfectants (at 1 µM clonidine _2A/D- and _2B-AR transfectants: 34 ± 3% for and 62 ± 2% rspectively). This result is consistent with those reported by Duzic and Lanier (1992) in the same cell line, demonstrating that _2B- and _2A/D-AR activation similarly inhibits forskolin-induced increase in intracellular cAMP. When AC II was coexpressed in DDT1-MF2-_2A/D transfectants, epinephrine as well as clonidine were able to switch the inhibitory signal into a stimulatory input through PTX-sensitive G proteins. In DDT1-MF2-_2B-AC II cotransfectants, despite a similar functional pool of AC II and an equivalent density of receptor, clonidine was unable to trigger such a switch, in contrast with epinephrine. Direct measurement of G protein activation by photoaffinity labeling with [-³²P]AA-GTP followed by selective separation of individual G protein  subunits revealed that clonidine, acting on _2B-AR, mediated an exclusive coupling to Gi₂, whereas the full agonist, epinephrine, led to the recruitment of both PTX-sensitive G proteins, Gi₂ and Gi₃. Thus, using this photoaffinity probe, it seems clear that, in DDT1-MF2 cells overexpressing AC II, the ability of _2B-AR to switch from a negative to a positive input to Gs-stimulated cAMP production greatly depends on the recruitment of Gi_3.

Previous works have established that the potentiation of Gs-stimulated cAMP production is caused by the input of G released from Gi to AC II that synergizes with Gs to further elevate cAMP levels (Tang and Gilman, 1991; Federman et al., 1992). Nevertheless, this phenomenon can only occur from a threshold concentration of released Gi (Tang and Gilman, 1991). Thus, the persistent inhibitory effect observed when Gi₂ is activated alone could be explained by the following hypothesis: the total amount of Gi₂ released upon receptor activation would be insufficient to overcome inhibitory influences exerted by Gi₂ on endogenous AC. On the contrary, when both Gi proteins (Gi₂ and Gi₃) were recruited, the threshold concentration would be reached, thus allowing AC II potentiation. Here, we should note that the possibility of overcoming Gi₂ inhibition was probably reinforced by the low ability of Gi₃ to induce AC inhibition (Raymond et al., 1993; Gettys et al., 1994). This might also reflect the fact that Gi₂ dimers released upon _2B-AR activation poorly interact or activate AC II compared with Gi₃ . Although there is no direct evidence that Gi₂ and Gi₃ differ in their capacity to potentiate Gs-stimulated AC, such a hypothesis must be taken into account. Indeed, several studies report that the regulation of -sensitive effectors depends on the composition of the G dimers with which they are interacting (Müller et al., 1997; Bayewitch et al., 1998; Maier et al., 2000). Finally, because post-translational modifications are considered important criteria in determining the potency by which G complexes modulate effectors (Ford et al., 1998), differential modifications of Gi₂ and/or - versus Gi₃ and/or - might also contribute to the clonidine-specific effect.

The present work also brings some evidence as to whether changes in the ratio of Gi₂ to Gi₃ proteins in late pregnant myometrium may play a crucial role for the switch in the stimulatory versus inhibitory input to AC population from the ₂-AR/Gi protein signaling. The down-regulation of Gi₃ protein, together with Gi₂-increased expression (Cohen-Tannoudji et al., 1995), could prevent ACII potentiation, thus allowing the decrease of ₂-AR stimulated cAMP production at term. The inhibition of the synthesis of smooth muscle relaxation factor (cAMP) together with the increase of intracellular Ca²⁺ would promote myometrial contractions at term.

In summary, these data provide the molecular basis of clonidine partial agonist effect when acting through _2B-AR, because they reveal that this compound selectively uncouples the receptor from one of the normally targeted G proteins: Gi₃. From this finding, it can be predicted that, in the situation where clonidine behaves as the full agonist, the second messenger pathway would be exclusively regulated by Gi₂ in Gi₂/Gi₃ expressing cells. On the other hand, in systems in which clonidine acts as partial agonist or even as an antagonist, Gi₃ would play a determinant or exclusive role. Furthermore, they suggested that Gi₂ and Gi₃ have specific roles in modulating AC II effector through  and/or subunits. Finally, they shed light upon a possible molecular mechanism that might allow the versatility of signal routed through myometrial ₂-AR during pregnancy involved changes in Gi₂/Gi₃ ratio.