Introduction

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Opioid receptors belong to the superfamily of receptors that regulates GTP-binding proteins (G proteins). The ability of a receptor to regulate different classes of G proteins causing the productive activation of multiple G protein signaling pathways has been convincingly documented (Kenakin, 1995). Much work has been performed to characterize transduction regulated by opioid receptors, not only in in vitro but also in in vivo systems, e.g., the production of antinociception. The i.c.v. injection of pertussis toxin into rodents demonstrated the regulation of G_i/G_o families by mu/delta opioid receptors in the mediation of analgesia (Parenti et al., 1986; Sánchez-Blázquez and Garzón, 1988, 1991; Lutfy et al., 1991). To gain information about the transduction system implicated in analgesia mediated by mu and delta opioid receptors, the authors' group pioneered in vivo studies with antibodies to G subunits. Possible neural processes facilitating the access of IgGs to G proteins have been discussed (Garzón, 1995). Supraspinal antinociception produced by agonists of delta receptors was reduced in mice receiving antibodies to G_i2 and G_i3 subunits by i.c.v. injection (Sánchez-Blázquez and Garzón, 1993; Sánchez-Blázquez et al., 1993). However, the analgesic profile of mu opioid receptor agonists appeared greatly diminished after injecting antibodies to pertussis toxin-sensitive G_i2 and pertussis toxin-insensitive G_x/z subunits (Sánchez-Blázquez et al., 1993, 1995). The assignment of G proteins to mu and delta opioid receptors has been substantiated by an alternative approach; the in vivo administration of antisense oligodeoxynucleotides complementary to portions of mRNAs expressing different subtypes of G subunits (Raffa el al., 1994; Rossi et al., 1995; Sánchez-Blázquez et al., 1995; Tang et al., 1995; Standifer et al., 1996).

Of potential interest was the finding that after reducing the availability of a single class of G subunits, agonist-antagonist properties of opioids were revealed (Garzón et al., 1994; Sánchez-Blázquez and Garzón, 1988). Because the various classes of G subunits exhibit structural differences in their C-terminal peptide sequences, it is feasible that agonist-bound receptors should display diverse efficacy in the activation of the subtypes of G proteins (Roerig et al., 1992; Chabre et al., 1994; Law et al., 1994; Lui et al., 1994; Offermanns et al., 1994). To obtain further insight into receptor-mediated effects, it is important to determine the classes of G-proteins regulated and their order of activation. The efficacy of activation of all, or some, of the G proteins regulated by a given receptor should shed some light on the agonist-antagonist properties of the ligands.

A number of laboratories have described a regulated association of G protein subunits with cytoskeletal proteins (see references in Neubig, 1994). In immunocytochemical studies, G proteins are often viewed as clusters distributed along the plasma membrane (Lewis et al., 1991). The finding that Triton X-100 solubilization of cell membranes provides insoluble complexes enriched in different types of proteins, receptors and GTP-binding proteins included, suggests that this constrained distribution corresponds to that of noncoated pits or caveolae-sites enriched in signal transducing elements (Neubig, 1994; Sargiacomo et al., 1993). In agreement with these findings, heterologous aggregates of proteins containing G-like immunoreactivity have been isolated after mild solubilization of cell membranes (Coulter and Rodbell, 1992; Jahangeer and Rodbell, 1993). Further, disaggregation was achieved using guanine nucleotides or extensive solubilization of the membranes (Nakamura and Rodbell, 1990). In the present investigation the characteristics of the GTP-dependent opioid-induced activation of different G proteins in the cell membrane were investigated using the rocket immunoelectrophoretic technique described by Laurell (1966).

An investigation was made of the activation of G_i2 and G_x/z subunits by agonists binding mu and/or delta opioid receptors in membranes from mouse PAG. This neural structure plays a major role in mediating the supraspinal analgesic effect of opioids when given by i.c.v. injection (Yaksh et al., 1976; Jensen and Yaksh, 1986). The results of this study indicate that mu opioid receptors couple with the G_i2 and G_x/z types, whereas delta opioid receptors prefer G_i2 over the G_x/z type. The observation that opioids displayed dissimilar "efficacy" at mu/delta opioid receptors in the activation of these G_i2/G_x/z transducer proteins is of potential interest.

	Methods

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Membrane Preparation

Experimental tissue was provided by albino male mice CD-1 (Charles River, Barcelona) weighing 22 to 25 g. P₂ fractions from PAG were prepared as previously described by Sánchez-Blázquez and Garzón (1989). Membranes were diluted in Tris.HCl buffer, pH 7.7, to a final protein concentration of 2 µg µl¹.

Antisera, SDS-PAGE and Immunoblotting

Anti-G sera were raised in New Zealand White rabbits (Biocentre, Barcelona, Spain). The corresponding synthetic peptides were conjugated to bovine thyroglobulin (Sigma no. T-1001, Sigma Chemical Co., St. Louis, MO) by means of (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride (Sigma no. E-6383), or m-maleimidobenzoyl-N-hydroxysuccinimide ester (Sigma no. M-2786). The antigenic sequences used were: G_i2 internal fragment [115-125: EEQGMLPEDLS] S/1, and the G_x/z internal fragment [111-125: C-TGPAESKGEITPELL] W/1 of the cDNA-predicted sequence of these proteins (Jones and Reed, 1987; Matsuoka et al., 1990). The antisera (S/1 anti-G_i2, W/1 anti-G_x/z) immunoreacted with proteins of 39 to 41 KDa in neural structures of mouse CNS (Sánchez-Blázquez et al., 1993).

Membranes from mouse PAG were solubilized in a buffer containing 50 mM Tris.HCl, 5% SDS, 10% glycerol, 5% 2-mercaptoethanol, pH 6.8, and boiled for 5 min at 100°C. Approximately 40 µg of protein were loaded on each lane of a 8 cm × 11 cm × 0.15 cm gel slab [7-18% acrylamide/1.9% bis-acrylamide (w/v)] (Hoefer Vertical Slab Unit SE 280). A 20 mA constant current was applied (ISCO Power Supply 595). Proteins were transferred (Mini Trans-Blot Electrophoretic Transfer Cell, BioRad) to polyvinylidene difluoride microporous (0.2 µm) membranes (BioRad) using Towing buffer (25 mM Tris.HCl, 192 mM glycine, 0.04% SDS and 20% methanol, pH 8.3) applying 70V (from 200 to 300 mA) for 120 min.

Unoccupied protein binding sites were blocked with non-fat dry milk (BioRad, no. M7439C) in tris-buffered saline (50 mM Tris.HCl, 500 mM NaCl, pH 7.7) for 1 hr at 37°C. Primary antiserum (1:3000 dilution) in TTBS, was added and incubation allowed overnight (Hoefer, Deca-Probe Incubation Manifold PR 150). After removing the antiserum the blot was washed with TTBS. Secondary antiserum [goat anti-rabbit IgG (FC) alkaline phosphatase conjugate (Promega, no. S373B)] in TTBS was added at 1:3000 dilution and left for 3 hr. The secondary antiserum was removed and the membrane washed with TTBS. Western Blue (Promega no. S384B) was used as a substrate (nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate in dimethylformamide) (fig. 1). Positive specificity of W/1 antiserum was determined by labeling recombinant G_x/z subunits (generously provided by Drs. T. Fields/P. J. Casey), but not G_i/G_o subunits (Calbiochem). The S/1 antibodies labeled the recombinant G_i2 subunits, but not G_i1, G_i3, G_o or G_x/z subunits (fig. 1). An identical pattern of selectivity was obtained when recombinant G subunits were studied in agarose gels containing either the S/1 or W/1 antibodies (fig. 1).

View larger version (52K): [in this window] [in a new window]   Fig. 1.   Reactivity of peptide antisera with recombinant G subunits and immunoblots of mouse PAG. Approximately 7 µg of Escherichia coli lysates containing recombinant G subunits, or 40 µg of protein from mouse PAG membranes were resolved by SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride microporous (0.2 µm) membranes. Immunodetection of S/1 and W/1 reactive material was performed as described in "Methods." Bottom right, E. coli lysates containing recombinant G subunits were electrophoresed in agarose plates loaded with anti-G sera. Anti-Gi2 sera: 1, 3 µg Gx/z; 2, 3 µg Go; 3, 3 µg Gi1; 4, 1 µg Gi2; 5, 3 µg Gi2; 6, 3 µg Gi3. Anti-Gx/z sera: 7, 1 µg Gx/z; 8, 3 µg Gx/z; 9, 3 µg Go; 10, 3 µg Gi1; 11, 3 µg Gi2; 12, 3 µg Gi3.

Incubation of P₂ Membranes with GTPS and Opioids

Concentrations of GTPS ranging from 1 fM to 300 µM were incubated with 40 to 80 µg protein from mouse PAG in 30 to 50 µl (25 mM Tris.HCl, pH 7.7) for 60 min at 25°C. The nucleotides GDP, GDPS and ATPS were used at 100 µM. The opioid agonists, morphine, -endorphin-(1-31), DAMGO, DPDPE, [D-Ala²]deltorphin II and the opioid antagonists, naloxone and ICI-174864, were incubated in this buffer supplemented with 1 pM GTPS. The interaction between opioids was allowed by adding a fixed concentration of the first ligand 10 min before a range of concentrations of the second ligand, followed by incubation for 50 min. The samples, membranes plus incubation medium, were SDS-solubilized (boiled for 3 min at 100°C) and the final ratio being 2 µg SDS µg¹ membrane protein. For immunoelectrophoresis, 2 to 4 µg protein were transferred to each sample well in volumes of 4 to 6 µl.

In a series of assays, the membranes were incubated for 60 min at 25°C with 1 µM morphine in the presence of 100 pM guanosine 5[-thio]triphosphate [³⁵S] (New England Nuclear, Boston, MA, NEG-030H, 1244 Ci/mmol). Samples were centrifuged at 20,000 × g for 20 min. The precipitate and the supernatant were SDS-solubilized and immunoelectrophoresis was performed in agarose plates loaded with anti G_i2 antiserum (fig. 1). After this the dried plates were exposed to X-OMAT film (Kodak). Films were developed in Kodak LX-24 developer and Kodak AL-4 fixer.

ADP-Ribosylation of Mouse PAG Membranes Catalyzed by Pertussis and Cholera Toxin

The method described by Wong et al., (1988) was followed with minor modifications. Pertussis toxin, 25 µg ml¹, was preactivated in 50 mM dithiothreitol for 1 hr at 25°C. Cholera toxin, 100 µg ml¹, was preactivated in 20 mM dithiothreitol for 1 hr at 25°C. PAG membranes were incubated in 100 mM Tris.HCl, pH 7.5 (final volume of 100 µl), 1 mM NAD, 1 mM EDTA, 1 mM ATP, 100 µM GTP, 10 mM thymidine, 75 µg protein of membranes, in the presence or absence of 2.5 µg preactivated pertussis toxin or 10 µg cholera toxin. After 60 min of incubation at 25°C the membranes were pelleted by centrifugation at 20,000 × g for 20 min and resuspended in the appropriate medium for incubation with or without GTPS.

Immunoelectrophoresis

Sample preparation. After GTP-dependent activation of G proteins a significant fraction of G subunits is released into the supernatant (McArdle et al., 1988; fig. 2, bottom right). Therefore, after incubating the guanine nucleotides and/or opioids, the membranes plus the supernatant were studied. Mouse PAG membranes, 80 to 100 µg protein in 50 µl of 25 mM Tris.HCl, pH 8.6, were SDS-solubilized using a ratio of 2 µg SDS per µg protein and centrifuged at 11,000 × g for 10 min. After precipitation with 0.15% deoxycholic acid (w/v) and 72% trichloroacetic acid (w/v), protein content of the solubilized samples was ascertained by the method of Lowry et al. (1951).

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Effect of GTP/GTPS on G protein -like immunoreactivity from mouse PAG membranes. A, Mouse PAG membranes were incubated with 100 µM GTPS, solubilized with 2 or 8 µg SDS µg1 protein and immunoelectrophoresed in agarose plates containing anti-Gi2 serum. Control samples were incubated without GTPS. B, Time-course for the GTP/GTPS-induced increases of G-related immunosignals. The guanine nucleotides were incubated at 10 µM with the PAG membranes. The samples were SDS-solubilized (2 µg µg1 protein) at different intervals and aliquots of 4 µg were electrophoresed in agarose gels containing anti-Gi2 serum. The immunoreactivity is expressed relative to that found in the absence of GTP/GTPS incubation (percentage of increment). C and D, A range of GTPS concentrations were incubated with the membranes for 60 min. After solubilizing the samples (2 µg SDS µg1 protein), 4 to 6 µg membrane protein were studied for immunoreactivity in agarose gels containing anti-Gi2 (C) or anti-Gx/z serum (D) Values are the mean ± S.E.M. from three to four independent determinations. Bottom left: Actual appearance of the immunoprecipitates. From 1 to 5 µg SDS-solubilized (2 µg µg1 protein) PAG membranes were electrophoresed in an Gx/z serum-agarose gel. Incubated in the absence (left) or presence (right) of 100 µM GTPS. Bottom right, Autoradiographic image of agarose plates loaded with antibodies to Gi2 subunits. Samples were incubated with (wells 1 and 2) or without 1 µm morphine (well 3) in the presence of 100 pM guanosine 5[-thio]triphosphate [35S]. Radioactivity associated with Gi2-like immunoreactivity detected in: 1, the supernatant; 2, the fraction of membranes; 3, supernatant plus membranes.

Immunoelectrophoresis. The method used was that described by Laurell (1966) with minor modifications (Dunbar, 1987). This nonisotopic procedure is based on the differential migration of the antigen and immunoglobulins in an appropriate buffer system. The electrophoretic migration of IgGs is very low at pH 8.6 whereas the G subunits with isoelectric point values in the range 5.4 to 6.1 (Goldsmith et al., 1988; Spicher et al., 1992) migrate rapidly in the electric field. As the antigen moves to the anode the content of IgGs inside the migrating track becomes reduced and the insoluble antigen-IgG complexes construct the sides of the rocket-shaped immunoprecipitate. When no antigen is available the immunoprecipitate ends in a sharp peak. The G subunits in complexes with other proteins or included in the aggregates, are bound very poorly by specific antibodies. The accessibility of the IgGs is enhanced after promoting the release of G subunits by either aggressive solubilizations, high concentrations of guanine nucleotides or agonist-liganded receptors (Nakamura and Rodbell, 1990). The final effect would be as if the amount of antigen had increased, thus producing greater immunosignals; the stoichiometric ratio G/IgG was reduced.

Agarose gel preparation. A solution of 1.5% (w/v) agarose (FMC no. 50002, LE) was prepared in electrode buffer: 20 mM Tris.HCl, 25 mM sodium barbital, 5 mM barbital, 0.85 mM calcium lactate, 0.03% (w/v) sodium azide (pH 8.6). Agarose was dissolved by constant stirring in the heated buffer (90°C, microwave oven). When cooled to 45°C, immune serum was added at 1:100 dilution. Once the solution became clear and thickened, 12 ml of the molten agarose were poured over the whole surface of leveled 85 × 100 mm plastic plates (GelBond FMC no. 53734). The casted gels were first cooled to room temperature and then placed in a humidity chamber at 4°C for at least 2 hr before use. Samples were applied to the gel in 2.5-mm round wells of approximately 7 µl capacity cut with a gel punch connected to a vacuum source. The gel minus the well area was covered with a plastic film and immunoelectrophoresis performed in a humid atmosphere on a cooling plate (LKB 2117 Multiphor II Electrophoresis System) connected to a thermostatic circulator bath (Haake G/3D) set at 10°C. Samples containing the antigen were loaded into the punched holes on the cathode side. A constant current of 7 mA per agarose gel plate was applied for 18 hr (Isco Power Supply 452). Antigen molecules migrated toward the anode and the insoluble immunocomplexes formed the rocket-shaped precipitate. A relationship between the height of the "rocket" and the amount of antigen applied was obtained (Laurell, 1966; Dunbar, 1987).

Staining and quantification. The agarose gel was separated from the plastic support. To remove the unreacted proteins the gel was placed between sheets of chromatography paper (Whatman 3 mm CHR) soaked in 100 mM NaCl and pressed (1 kg) for 2 × 30 min. The gel was then immersed and gently agitated in this saline solution for 20 min. After a second pressing for 30 min, the gel was dried under a warm air stream before staining. Immunocomplexes, containing the G-like immunoreactivity plus the specific IgGs, were stained with Coomassie Brilliant Blue R-250 (BioRad). Silver staining of the "rockets" was also performed with reagents suitable for agarose gels (Silver Stain Plus, BioRad), although increases in the signal were accompanied by higher backgrounds. The length of the immunoprecipitatesdistance from the center of the well to the peak of the rocketwas assessed by optical densitometry (Isco Gel Scanner 1312/UA-5).

Drugs

Human -endorphin-(1-31), DAMGO, DPDPE and [D-Ala²]deltorphin II were purchased from Peninsula Laboratories Europe (Merseyside, England). ICI-174864 was from CRB (Cambridge, England). Morphine sulfate came from Merck (Darmstadt, Germany), naloxone hydrochloride, GTPS, GDP, GDPS and ATPS from Sigma. Pertussis and cholera toxin were obtained from LIST Biological Labs (Campbell, CA). Synthetic peptides: EEQGMLPEDLS (Peninsula), and C-TGPAESKGEITPELL (Bio-Synthesis, Madrid, Spain). SDS biotechnology grade came from Amresco (Solon, OH). Recombinant G_i and G_o subunits were purchased from Calbiochem (San Diego, CA).

Statistics

The data are expressed as the mean ± S.E.M. of at least three independent experiments. Statistical significance was assessed using the Student's paired t test. P < .05 were considered to be significant. Calculations were performed using the SigmaStat computer program (Jandel Scientific Software, Erkrath, Germany).

	Results

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Detection of G subunits by immunoelectrophoresis. Mouse PAG membranes solubilized with SDS in the absence of thiol-reducing agents were loaded into agarose gel plates containing anti G_i2 or anti G_x/z serum at 1:100 dilution (fig. 2A). The length of the immunoprecipitates "rockets" increased linearly with the amount of protein undergoing electrophoresis. The curves correlating protein concentrations to immunosignals did not pass through the origin and showed a threshold for immunodetection. The concentration of the negatively charged detergent SDS in the solubilizing buffer influenced the size of the immunosignals. Increases in the ratio SDS/protein extended the thresholds and parallel curves could be observed (fig. 2A). Samples solubilized with ratios SDS/protein less than 0.5 hardly entered the agarose gel and the stained proteins were detected in the sample wells (not shown). For the purpose of the study, a constant ratio of 2 µg SDS µg¹ protein was selected. Immunodetection was achieved at low micrograms of membrane protein (1-6 µg) indicating that G subunits are detected in the nanogram range. The method is highly reproducible, the S.E.M. of the computed immunoprecipitates is less than ± 2% and the accuracy of the measurements increases with the length of the rockets (Laurell, 1966). In assays where differences are expressed as a percentage of the control, the threshold "blank" was subtracted from the length of the rockets.

GTPS produces a concentration-dependent increase of G-like immunoreactivity. Incubation of samples with GTP/GTPS produced an enhancement of the G-related immunosignals (fig. 2). The increases in immunoreactivity observed after raising the amount of SDS in the solubilizing buffer masked the effect of 100 µM GTPS (fig. 2A, bottom left). Samples incubated with GTPS and solubilized with 2 µg SDS µg¹ protein gave steeper curves than those produced by the corresponding controls. However, the threshold was practically maintained (more antigen being available for the IgGs) (fig. 2A). The time-course for the enhancing effect of GTP and GTPS on G_i2-like immunoreactivity is shown (fig. 2B). A concentration of 10 µM GTP/GTPS, was incubated with mouse PAG membranes at several intervals before stopping the reaction (SDS-solubilization). The GTPS raised the G_i2-related immunoreactivity over the basal values (obtained in the absence of the nucleotide). The pattern showed a fast rise during the first 15 min followed by a steady plateau that extended beyond 120 min a result in agreement with that reported by Milligan and Unson (1989) who used membranes of rat glioma C6 BU1 cells and Gpp(NH)p. A concentration of 10 µM GTPS promoted a steady increase of G_x/z-related immunoreactivity after 40 to 50 min of incubation (not shown). An interval of 60 min was therefore selected to carry out further assays with GTPS. The effect of GTP showed a biphasic pattern on G-like immunoreactivity: an initial rise lasting up to 30 min followed by a pronounced decrease that practically returned to the basal levels. The metabolism suffered by GTP was responsible for the reversible activation of the G subunits. At the end of the incubation period the fraction of activated G subunits depends on the concentration of GTP that remains. Considerable loss of sensitivity and experimental variation follows.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Specificity of the G-related immunosignals detected in immunoelectrophoresis. A, PAG membranes were incubated in the absence, and also in the presence of 100 µM GDP/GDPS for 45 min at 25°C. GDP was removed before incubation for an additional 60 min with 100 µM GTPS. Samples were also ADP-ribosylated with pertussis and cholera toxin as described in "Methods" before being incubated with or without 100 µM GTPS. The solubilized samples (2 µg SDS µg1 protein) were electrophoresed in agarose gels containing anti-Gi2 serum. * Significantly different to the corresponding group incubated in the absence of GTPS (open column). Significantly different to the control group when incubated in the absence of GTPS (open column), P < .05, Student's t test. B, Influence of 5% mercaptoethanol on GTPS-mediated enhancement of Gi2-like immunoreactivity. The membranes were incubated in the absence and presence of 100/300 µM GTPS and solubilized with and without 5% mercaptoethanol in the solubilization buffer (2 µg SDS µg1 protein). * Significantly different to the group solubilized without mercaptoethanol (open column). , Significantly different to the Control group incubated in the absence of GTPS and solubilized without mercaptoethanol (open column), P < .05, Student's t test. The empty boxes drawn for panels A and B indicate the threshold for immunodetection (no rocket is formed below this migration distance). C, Influence of GTPS and mercaptoethanol on delta receptor-related immunoreactivity. The membranes were incubated in the presence and absence of 100 µM GTPS and SDS-solubilized with and without 5% mercaptoethanol before electrophoresing the samples in agarose gels containing anti-delta opioid receptor serum.

Specificity of the GTPS effect on G-like immunoreactivity. Incubation of PAG membranes with 100 µM ATPS, a nonrelevant nucleotide in G protein activation, did not increase G immunoreactivity. GDP/GDPS, after stabilizing the associated conformation of the G proteins (Wong et al., 1985; McArdle et al., 1988), reduced the basal immunosignals associated with G_i2 subunits in mouse PAG (fig. 3A). After removing 100 µM GDP from the incubation media, 100 µM GTPS produced a significant increase of G-like immunoreactivity. In the presence of 100 µM GDPS, GTPS failed to increase this signal (fig. 3A). Pertussis toxin, which produces the ADP-ribosylation of trimeric G_i/G_o proteins (Wong et al., 1985; Milligan, 1987) and inhibits the receptor-activated GTPase activity of G subunits (Van Dop et al., 1984; Aktories et al., 1993), reduced the basal immunosignals and also prevented GTPS (100 µM) from increasing G_i2-like immunoreactivity (fig. 3A). Cholera toxin did not suppress the enhancing activity of the nucleotide on G_i2-like immunoreactivity of PAG (fig. 3A). After adding 2-mercaptoethanol to the SDS-solubilizing buffer, increases of G-like immunoreactivity could be observed (fig. 3B). This effect was maximal for 2% v/v of the reagent (data not shown). In this experimental protocol, GTPS failed to enhance the resulting immunosignals (fig. 3B). In agarose plates containing anti-delta opioid receptor serum (Garzón et al., 1994), incubation of PAG membranes with 100 µM GTPS did not modify immunoreactivity. However, the thiol-reducing agent significantly expanded the magnitude of the immunosignals (fig. 3C).

Opioid-induced GTPS-dependent activation of G transducer proteins. The opioid agonists, morphine, -endorphin-(1-31), DAMGO, DPDPE, [D-Ala²]deltorphin II, or the antagonists, naloxone and ICI-174864, when incubated in 25 mM Tris.HCl, pH. 7.5, in the absence of GTPS, did not modify G-related immunosignals. Concentrations of GTPS in the range 1 fM to 1 µM were subeffective or produced a limited increase of the G_i2-like immunoreactivity from PAG membranes (figs. 2C and 4A). Samples preincubated with combinations of 1 µM morphine [or -endorphin-(1-31)] plus GTPS ranging from 10 fM to 1 µM, significantly increased G_i2-related immunoreactivity (fig. 4A). In the presence of GTPS of less than 10 fM, the opioids did not alter the immunoprecipitates. Incubation of opioid agonists with 1 pM GTPS (subeffective concentration), provided concentration-related increases of G-related immunosignals (figs. 4 and 5). Thus, GTPS was required by opioid agonists to augment G-related immunosignals. The enhancement could be observed even in the presence of nonactivating concentrations of the nucleotide. This attribute preserved most of the trimeric G proteins for the study of agonist-mediated increases of G-like immunoreactivity. Therefore, the correlation of agonist concentration (gradual occupation of receptors) with the fractional activation of G transducer proteins was feasible.

View larger version (46K): [in this window] [in a new window]   Fig. 4.   Effect of opioids binding mu receptors in G-related immunoreactivity of mouse PAG membranes. A, The membranes were incubated with 1 µM morphine or -endorphin-(1-31) in the presence or absence of increasing concentrations of GTPS. * Shows a significant difference with respect to the corresponding Gi2-like immunoreactivity obtained in the absence of GTPS, P < .05, Student's t test. A wide range of morphine, -endorphin-(1-31) and DAMGO concentrations were incubated with the membranes in the presence of 1 pM GTPS. Solubilized samples (2 µg SDS µg1 protein) were studied for immunoreactivity in agarose gels containing anti-Gi2 (B) or anti-Gx/z serum (C). Immunoreactivity is expressed relative to the value obtained in the presence of 1 pM GTPS and the absence of opioid agonists (percentage increment). D, Antagonism of naloxone on morphine-evoked increase of Gi2-like immunoreactivity. The opioid antagonist was added 10 min before morphine to mouse PAG membranes in the presence of 1 pM GTPS. Incubation was continued for 50 min at 25°C. Samples were processed for immunoelectrophoresis in agarose gels containing anti-Gi2 serum. Values are the mean ± S.E.M. of three to four independent determinations. Bottom, Morphine-evoked increase Gi2-like immunosignals. Mouse PAG membranes were incubated with the opioid in the presence of 1 pM GTPS. From left to right: 1, no opioid (basal); 2 to 9, 30 pM, 100 pM, 1 nM, 3 nM, 10 nM, 100 nM and 10 µM morphine.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Effect of opioids binding delta receptors in G-related immunoreactivity of mouse PAG membranes. A wide range of DPDPE and [D-Ala2]deltorphin II concentrations were incubated with the membranes in presence of 1 pM GTPS. The opioid DPDPE at 1 nM was added 10 min before [D-Ala2]deltorphin II. ICI-174864 antagonist at delta opioid receptors was added also 10 min before DPDPE and [D-Ala2]deltorphin II. Solubilized samples (2 µg SDS µg1 protein) were studied for immunoreactivity in agarose gels containing anti-Gi2 (A and B) or anti-Gx/z serum (C). Experimental details as in figure 4.

View this table: [in this window] [in a new window]   TABLE 1 Potency of opioids to increase G-like immunoreactivity in mouse PAG membranes

	Discussion

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Our results provide a molecular basis for the explanation of the agonist-antagonist properties of opioids in promoting analgesia (Sánchez-Blázquez and Garzón, 1988; Garzón et al., 1994). It was observed that opioids displayed dissimilar "efficacy" at mu/delta opioid receptor-G_i2/G_x/z complexes because antagonist activities were revealed. Several agonists promoted a substantial activation of G_i2 and G_x/z transducer proteins at concentrations lower than those required to bind 50% of available receptors (K_D) (Sánchez-Blázquez and Garzón, 1989). The levels of agonists required to activate a substantial quantity of G proteins in membranes from mouse PAG may be very low. In intact tissues these effects might be achieved even at much lower agonist concentrations. This could be explained by receptor reserve. However, the existence of receptors with a very high affinity for opioids, e.g., the µ1 (Pasternak et al., 1980; Lutz et al., 1984) must also be considered.

The involvement of opioid receptors in the observed opioid-evoked activation of G proteins was demonstrated. Naloxone, a competitive antagonist of opioid receptors reduced the enhancing activity of opioids on G-like immunoreactivity in a concentration-dependent fashion. ICI174864, antagonist of delta opioid receptors, displays negative intrinsic activity or inverse agonist activity on basal GTPase activity of, and binding of [³⁵S]GTPS to, G subunits (Costa and Herz, 1989; Georgoussi and Zioudrou, 1993; Mullaney et al., 1996). The concentration of GTPS selected did not activate (bind) G subunits. Therefore, ICI174864 exhibited no reducing effect on basal G-related immunosignals. Interestingly, this antagonist exhibited high potency to reduce the activating effect of [D-Ala²]deltorphin II and DPDPE on G-like immunoreactivity. DPDPE showed even a greater potency to impair the effect of [D-Ala²]deltorphin II on G_x/z-like immunosignals. The antagonism of DPDPE on [D-Ala²]deltorphin II-evoked effects has already been documented for analgesia (Vanderah et al., 1994). Thus, it seems that ICI174864 and DPDPE, after binding to delta opioid receptors, reduce the affinity of certain classes of G proteins toward GTP/GTPS nucleotides. As a result of this inverse agonist activity, the efficacy of agonists in the activation of delta receptor-coupled G proteins is impaired. Further studies are in progress to explore this possibility.

It is established that G receptors display selectivity toward certain classes of G-transducer proteins, e.g., muscarinic acetylcholine receptors (Offermanns et al., 1994), somatostatin receptors (Law et al., 1994) or dopamine receptors (Lui et al., 1994). With respect to opioid receptors, evidence supporting the existence of a similar phenomenon is accumulating. The mu, delta and kappa opioid receptors exhibit differences in the classes of G proteins they regulate in the production of antinociception: mu opioid receptors regulate G_i2 and G_x/z, delta opioid receptors regulate G_i2 and G_i3 types and kappa₃-opioid receptors regulate G_i1 and G_i3 (Sánchez-Blázquez and Garzón, 1993; Sánchez-Blázquez et al., 1993, 1995; Raffa et al., 1994; Standifer et al., 1996). The results of this study show that mu opioid receptors in PAG couple with the G_i2 and G_x/z types, whereas delta opioid receptors prefer G_i2 over G_x/z proteins.

The i.c.v. injection of pertussis toxin into mice reduces the efficacy of opioids in invoking supraspinal analgesia (Sánchez-Blázquez and Garzón, 1988). The antinociception elicited by the preferential ligands of mu opioid receptors, DAMGO and FK 33824, of mu/delta receptors, [D-Ala²,Met⁵]enkephalinamide and [D-Ala²,D-Leu⁵]enkephalin, and of delta receptors, [D-Ala²]deltorphin II and DPDPE, appeared largely reduced in these mice. Most notably, the activities of morphine, -casomorphin (1-4) amide and human -endorphin were much more resistant to the bacterial toxin (Sánchez-Blázquez and Garzón, 1988, 1991). The results of our study and those of previous reports, confirm the involvement of pertussis toxin-resistant G proteins in the analgesic effects of certain opioids when acting at the supraspinal level (Sánchez-Blázquez et al., 1993; Garzón et al., 1994, 1995; Sánchez-Blázquez et al., 1995).

In pertussis toxin-treated mice, opioids for which activity was greatly reduced by this toxin behaved as antagonists of morphine-evoked analgesia (Sánchez-Blázquez and Garzón, 1988). Antagonism was also observed in mice that had received i.c.v. injection of IgGs to G_i2 or G_x/z subunits (Garzón et al., 1994). It is well documented that morphine is less effective than other opioids that also bind mu receptors, e.g., DAMGO. This has been observed for the rat in the locus coeruleus (Christie et al., 1987) and vas deferens (Huidobro et al., 1980), and after chronic morphine treatment in the guinea pig ileum (Porreca and Burks, 1983). This evidence shows that opioids, after binding to their receptors, exhibit different efficacy in the activation of the different classes of G proteins. Variations in the relative abundance of these G proteins might be responsible for different pharmacological profiles and hence be interpreted in terms of diversity of receptors.

It is well established that pertussis toxin disrupts the interaction of receptors with G proteins as well as the direct activation by mastoparan of G-related GTPase activity (Weingarten et al., 1990). In membrane preparations containing ADP-ribosylated G_i2 proteins GTPS failed to increase G_i2-like immunoreactivity. However, even though GTPS might bind pertussis toxin-acted G subunits (Winslow et al., 1986; Yi et al., 1991) our results, and those of other investigations (Sunyer et al., 1989), suggest that G subunits do not dissociate from the trimer. By fastening free G subunits to the trimeric G protein, pertussis toxin reduced the G_i2-like basal immunoreactivity. Obviously this effect is not observed for the pertussis toxin-insensitive G_x/z subunits. The stabilizing activity of GDP/GDPS on the trimeric G-proteins also promoted reductions of G_i2-related basal immunosignals.

In summary, the use of this sensitive, nonisotopic and quantifiable method allowed the detection of opioid-activated G proteins of mouse PAG membranes. Opioids showed different potency and efficacy in the activation of G_i2 and G_x/z subunits. For opioids acting on the same receptor, the rank order of potency in activating G subunits depended on the class of G protein analyzed. The concept of intrinsic activity of ligands might therefore be expanded from the receptor molecule to the different receptor-G protein complexes.