Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Opioids are members of the large superfamily of G protein-coupled receptors that traverse the plasma membrane seven times and activate intracellular G proteins (Raynor et al., 1994). -Opioid receptors often couple to multiple effectors within the same cell and/or tissue. In neuroblastoma cells -opioid receptors regulate adenylyl cyclase activity (Prather et al., 1994c), Ca²⁺ channels (Hescheler et al., 1987), and phospholipase C (Jin et al., 1992). However, in other tissues the coupling of -opioid receptors to intracellular effectors appears to be more restricted. For example, in rat dorsal root ganglion neurons, -opioid receptors are able to inhibit adenylyl cyclase activity (Makman et al., 1988), but fail to couple to Ca²⁺ channels (Moises et al., 1994).

We have studied the binding and functional properties of cloned µ- and -opioid receptors expressed either alone or in combination in GH₃ cells (Piros et al., 1995, 1996a). Consistent with the previous studies in dorsal root ganglion neurons, we found that -opioid receptors expressed alone in GH₃DOR cells bound opioid ligands and that their activation led to the inhibition of adenylyl cyclase without having an effect on Ca²⁺ channel activity (Piros et al., 1996a). In contrast, µ-opioid receptors in GH₃MOR cells coupled to both adenylyl cyclase and L-type Ca²⁺ channels (Piros et al., 1995). Interestingly, activation of either - or µ-opioid receptors in GH₃ cells that contained both receptor subtypes (GH₃MORDOR cells) also resulted in inhibition of both adenylyl cyclase and L-type Ca²⁺ channel activity.

The dissimilar signaling of -opioid receptors expressed in these clones could be explained by the observation that GH₃DOR cells contained a relatively low density of -opioid receptors (0.55 pmol/mg of protein) compared with the greater number of -opioid receptors expressed in GH₃MORDOR cells (2.45 pmol/mg of protein). If confirmed, this would indicate that different thresholds of -opioid receptor density might be required for coupling to different effectors in the same cell. This is supported by studies in which increasing the density of transfected G protein-coupled receptors enhances both agonist potency and efficacy for regulation of several intracellular effectors. This has been investigated most extensively for ₂-adrenergic stimulation of adenylyl cyclase (Whaley et al., 1994), but also has been observed for opioid inhibition of adenylyl cyclase (Hirst et al., 1997). Additionally, in CHO cells transfected with low densities of M₂-muscarinic receptors, carbachol produces only inhibition of adenylyl cyclase. However, when M₂-receptor density is increased, activation of phospholipase C is also observed (Ashkenazi et al., 1987). Lastly, overexpression of ₂-adrenergic receptors in CHO cells results in the ability of the receptor to couple not only to G_i but also to G_s, a G protein with which it does not normally couple (Eason et al., 1992). Consequently, it is possible that -opioid receptors couple to a unique blend of intracellular effectors in neurons, depending upon the stoichiometric ratio of receptors, G proteins, and effectors present within specific neurons.

Because -opioid receptors are able to couple to Ca²⁺ channels only in GH₃ cells that contain both µ- and -opioid receptors, it is also possible that these receptors interact to allow the activation of different G proteins to allow signaling to Ca²⁺ channels. Indeed, recent studies have demonstrated that the binding and signaling of some G protein-coupled receptors, including opioid receptors can be influenced by the formation of heterodimers (Hebert et al., 1996; Jordan and Devi, 1999). In the case of GABA_B receptors, coupling to K⁺ channels is not observed unless the GABA_BR1 and GABA_BR2 subunits are expressed together (White et al., 1998). Furthermore, the coexpression of - and -opioid receptors, both of which function individually, produces a heterodimer combination with different binding and functional properties seen when either of the individual receptors is expressed alone (Jordan and Devi, 1999). The changes in receptor signaling that are seen upon expression of receptors either alone, or in combination with another receptor subtype, are presumably caused by the differential activation of G proteins. Hence, the formation of µ/-opioid receptor dimers may enable coupling to Ca²⁺ channels through the activation of specific G proteins not activated when -opioid receptors are expressed in GH₃ cells alone.

Therefore, the purpose of the present study was to determine whether the differential coupling of -opioid receptors to these two effectors results from differences in receptor density and/or the activation of specific G proteins. Using the -opioid receptor alkylating agent SUPERFIT, reduction of -opioid receptors in GH₃MORDOR cells to a density similar to that of -opioid receptors in the GH₃DOR cells resulted in abolishment of coupling to Ca²⁺ channels, but not to adenylyl cyclase. Furthermore, although significantly greater amounts of all G proteins were activated by -opioid receptors in GH₃MORDOR cells, -opioid receptor activation in GH₃DOR cells resulted in coupling to the identical pattern of G proteins as that in GH₃MORDOR cells. These findings suggest that a threshold density of -opioid receptors is required to activate critical amounts of individual G proteins needed to produce coupling to certain effectors and that -opioid receptors couple more efficiently to adenylyl cyclase than to L-type Ca²⁺ channels.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. [-³²P]GTP (3000 Ci/mmol) and antisera (EC2 and GC2) were purchased from NEN Life Science Products (Boston, MA). SUPERFIT was a generous gift from Dr. Kenner C. Rice (National Institutes of Health, Bethesda, MD). [³H]Adenine (27 Ci/mmol), [³H]diprenorphine (36 Ci/mmol), [-³²P]ATP (17 Ci/mmol), and ECL reagents were purchased from Amersham Life Science Products (Arlington Heights, IL). [³H]DPDPE (18 Ci/mmol) was provided by the National Institute on Drug Abuse (Bethesda, MD). DPDPE, CTOP, and somatostatin were obtained from Peninsula Laboratories (Belmont, CA). All tissue culture reagents, including geneticin (G418) and hygromycin-B, were purchased from Life Technologies (Gaithersburg, MD). All other reagents were purchased from Sigma (St. Louis, MO).

Cell Culture. GH₃ cells (CCL 82.1), obtained from the American Type Culture Collection (Rockville, MD), were maintained in DMEM with 10% (v/v) fetal calf serum, 0.05 I.U./ml penicillin and were incubated in a humidified atmosphere of 10% CO₂, 90% O₂ at 37°C. To prevent cells from adhering to one another, 30% conditioned media were included in all incubation media. Cells were harvested once each week by detachment with 0.1% phosphate-buffered saline supplemented with 0.4% EDTA (PBS/EDTA) and reseeded at 20% of their original density. The incubation medium was changed every 2 to 3 days. All experiments were conducted with cells maintained between passages 4 and 10.

Transfection. Stable cell lines that expressed only - (GH₃DOR) or both µ- and - (GH₃MORDOR) opioid receptors were generated for this study. The transfection and subsequent characterization of GH₃ cells to obtain the GH₃MORDOR cell lines (Piros et al., 1996a) has been reported previously. To produce the GH₃DOR cell line, GH₃ cells (5 × 10⁶ in 0.5 ml of phosphate-buffered saline, pH 7.4) were stably transfected by electroporation (500 µF, 250 V) in the presence of 10 µg of pCDNA1neo plasmids, which contained the cDNA encoding for the -opioid receptor (DOR-1). The DOR-1 construct was subcloned into the XhoI site of the pCDNA1neo plasmid (Invitrogen, San Diego, CA) and consisted of a 1.8-kb cDNA representing the coding region and a 700-base pair 3'-noncoding region of the mouse -opioid receptor. GH₃ cells that stably incorporated these plasmids were selected by picking colonies that survived culturing in the presence of 1 mg/ml Geneticin (G418). Confirmation of -opioid receptor expression was determined by performing competition for [³H]diprenorphine (2 nM) binding by DPDPE (1 µM) as described below. The clone that expressed the highest level of -opioid receptor binding was selected for future studies and was designated as the GH₃DOR clone.

Receptor Binding. Membranes used in binding assays were prepared from GH₃ cells by centrifugation of homogenates minus nuclei at 100,000g for 60 min as described previously (Prather et al., 1994a). All opioid receptor binding was performed using 250 µg of membrane protein. Binding incubation was performed in 50 mM Tris-HCl, pH 7.6, with 10 mM MgCl₂, at room temperature for 90 min, as described previously (Prather et al., 1994b). For saturation binding studies using GH₃DOR membranes, concentrations of [³H]diprenorphine from 0.1 to 10 nM were used. For saturation binding studies using GH₃MORDOR membranes, concentrations of 0.05 to 20 nM [³H]DPDPE were used. In all experiments, nonspecific binding was determined in the presence of nonradioactive DPDPE (1 µM). Data obtained were subjected to Scatchard analysis, and estimates of affinity (K_d) and receptor density (B_max) were obtained using the LIGAND computer program. In competition binding experiments, the ability of increasing concentrations (0.01 nM-100 µM) of DPDPE to compete for the binding of [³H]DPDPE (10 nM) or [³H]diprenorphine (2 nM) was assessed. The concentration of opioid receptor ligands to produce a 50% reduction in radioligand binding (IC₅₀) was determined by the computer program Sigmaplot (Jandel Scientific, San Rafael, CA) and then converted to a measure of receptor affinity (K_i).

Measurement of Opioid-Mediated Inhibition of Adenylyl Cyclase Activity. The conversion of the [³H]adenine-labeled ATP pools to cyclic AMP was used as a measure of opioid ligand effect on cyclic AMP levels as described previously (Prather et al., 1994a). Briefly, measurements were made with GH₃ cells seeded into 17-mm (24-well) plates (4 × 10⁶ cells/plate), which resulted in 100% confluence when cultured for 4 days. The incubation medium was changed 24 h before the assay. On the day of the assay, media were removed and replaced with incubation mixture (warmed to 37°C) (DMEM containing 0.09% NaCl, 500 µM 3-isobutyl-1-methylxanthine, and 2 µCi/well [³H]adenine) for 1 h. At the time of the assay, plates were placed in an ice-water bath for 5 min. The incubation mixture was then removed and replaced with ice-cold assay mixture [Krebs-Ringer HEPES buffer (110 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 25 mM glucose, 55 mM sucrose, and 10 mM HEPES, at pH 7.4) containing 500 µM 3-isobutyl-1-methylxanthine, 10 µM forskolin] and the opioid ligand to be tested. The plates were then incubated at 37°C for 15 min and placed back in the ice-water bath for 5 min. After termination of incubations with 50 µl of 3.3 N perchloric acid and subsequent addition of [³²P]cyclic AMP as an internal standard, radioactive cyclic AMP was separated from other ³H-labeled nucleotides by a double-column chromatographic method. Seven milliliters of scintillation fluid was then added and samples were immediately counted in a Beckman LS2800 scintillation counter.

Electrophysiological Recordings. Single cells were voltage clamped, and voltage-activated Ca²⁺ channel activity was recorded from whole GH₃ cell clones using a List EPC-7 patch-clamp amplifier as described previously (Piros et al., 1995, 1996a). Before recording, culture dishes containing cells were superfused (flow rate, 2 ml/min) with a solution containing 140 mM NaCl, 2.8 mM KCl, 2 mM MgCl₂, 1 mM CaCl₂, and 10 mM HEPES (adjusted to pH 7.2 with NaOH). After a high-resistance seal was established and access to the interior of the cell was obtained, the external solution was replaced by a solution containing 125 mM NaCl, 5.4 mM CsCl, 10.8 mM BaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 0.5 µM tetrodotoxin (adjusted to pH 7.2 with NaOH). The recording electrode contained 120 mM CsCl, 10 mM EGTA, 1 mM MgCl₂, 3 mM magnesium-ATP, and 10 mM HEPES (adjusted to pH 7.2 with CsOH). Ba²⁺ currents were activated by step depolarizations of membrane potential from a holding potential of 80 mV for 100 ms. Capacitance and series resistance compensations were achieved using the patch-clamp amplifier. Residual artifacts and leakage currents were nulled using a P/4 subtraction. Patch electrodes were manufactured from thin-walled, borosilicate glass pipettes (World Precision Instruments, Sarasota, FL) using a Narishige (PP-83) electrode puller. Whole-cell currents, monitored using the EPC-7 amplifier, were low-pass filtered with an eight-pole Bessel filter (Frequency Devices, Haverhill, MA) at 1 kHz, digitized (Labmaster DMA interface; Axon Instruments, Foster City, CA) at a frequency of 5 kHz, and stored on an IBM PC hard disk. Data were acquired and analyzed using pCLAMP software (Axon Instruments). Drugs (prepared daily from frozen stock solutions) were applied to the bath, and all recordings were performed at room temperature (20-22°C).

Photoaffinity Labeling of G Subunits with [-³²P]AA-GTP. The method for synthesis and purification of [-³²P]AA-GTP can be found in Prather et al. (1994a). The photoaffinity labeling of G subunits with [-³²P]AA-GTP has also been recently reported (Prather et al., 1994a,b, 1995; Chakrabarti et al., 1995). Plasma membranes (50 µg/assay) were incubated in the presence or absence of agonist for 6 min at 30°C in 100 µl of buffer I (50 mM HEPES, pH 7.4, 0.1 mM EDTA, 10 mM MgCl₂, 30 mM NaCl, 50 µM GDP). After agonist incubation, [-³²P]AA-GTP (1 µCi/assay) was added, and samples were incubated for an additional 6 min at 30°C. The reaction was terminated by placing samples on ice. Membranes were then collected by centrifugation at 12,000g for 10 min and resuspended in 100 µl of buffer II (50 mM HEPES, pH 7.4, 0.1 mM EDTA, 10 mM MgCl₂, 30 mM NaCl, 2 mM dithiothreitol). Resuspended pellets (droplets) were then irradiated at 4°C with 240 mJ from an ultraviolet lamp (254 nM, 150 W) at a distance of 15 cm. Samples were centrifuged as before, resuspended in sample buffer, and separated by SDS-PAGE (see below). After electrophoresis, [-³²P]AA-GTP-labeled G subunits were visualized autoradiographically by a Molecular Dynamics Inc. PhosphorImager 445 SI (Sunnyvale, CA) and quantified by densitometry using the NIH Image software program (version 1.56). To determine the amount of radioactivity incorporated by individual G proteins, the area of each band was traced, multiplied by its mean density, and the femtomoles of radioactivity determined by comparison with the density produced by a range of ³²P standards using linear regression.

SDS-PAGE and Immunoblotting. To identify G subunits, membranes were separated on 20-cm separating gels containing 10% acrylamide and 6 M urea (Prather et al., 1994a,b, 1995; Chakrabarti et al., 1995). Before separation, samples were resuspended in 80 µl of electrophoresis loading buffer (0.065 M Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol), and heated at 90°C for 2 min. The ECL method of immunoblotting was used (Amersham Life Science Products). Gels were transferred to Hybond-ECL nitrocellulose membranes and incubated overnight at 4°C with 10% milk in blotting buffer (TBS-0.1%) (25 mM Tris-HCl, pH 7.6, 0.09% NaCl, 0.1% Tween 20). Blots were then washed three times (5 min each) with TBS-0.1% and incubated with primary antibodies (1:1000-2000) for 1 h at room temperature while shaking. The primary antibodies were then removed and blots were washed as described previously. Secondary antibody (donkey anti-rabbit or anti-mouse immunoglobin horseradish peroxidase, 1:5000) was then added and incubated for 30 min, with shaking. The secondary antibody was removed and blots were washed with 3 × 5-min washes with TBS-0.3%, followed by 3 × 5-min washes with TBS-0.1%. Blots were then incubated for 1 min with equal volumes of ECL detection reagents 1 and 2, wrapped in Saran plastic wrap, and exposed to Hybond-ECL X-ray film for periods varying between 30 s and 10 min.

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Data Analysis. Unless otherwise stated, data reported represent the mean ± standard error of at least three separate experiments that were each performed in triplicate. Data obtained from full dose-response curves using selective opioid agonists were subjected to sigmoidal curve fitting. The minimum and maximum plateau values for the amount of G subunits activated (expressed in fmol/mg of protein) and the amount of agonist required to produce 50% of maximal activation (i.e., ED₅₀) were determined from the best-fit curves. The maximum amount of G subunits activated was defined as the difference between the minimum and maximum plateau values. Percentage increase in G protein activation was defined as the maximal femtomoles per milligram of protein of [-³²P]AA-GTP incorporated in the presence of agonist, divided by basal incorporation, times 100%. Statistical significance of the data was determined by ANOVA followed by comparison using either the nonpaired two-tailed Student's t test or Tukey's method.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

-Opioid Receptor Binding and Regulation of Adenylyl Cyclase and Calcium Channels in Transfected GH₃DOR and GH₃MORDOR Cells. GH₃ cells were stably transfected by electroporation with plasmids containing cDNAs encoding for opioid receptors to obtain clones that expressed only - (GH₃DOR) or both - and µ-opioid receptors (GH₃MORDOR) (Piros et al., 1996a). The density of each receptor (B_max) and the affinity of the -opioid receptor-selective ligand DPDPE for the expressed receptors were determined in both clones by saturation and competition binding, respectively. The results of these characterizations are presented in Table 1. The density of -opioid receptors in the GH₃MORDOR clone was almost 5-fold greater than that observed in the GH₃DOR clone (P < .01). Additionally, the affinity of DPDPE for -opioid receptors in both clones reported here is similar to those reported for mammalian brain membranes (Goldstein and Naidu, 1989).

View this table: [in this window] [in a new window]   TABLE 1 -Opioid receptor binding and regulation of adenylyl cyclase and calcium channel activity in transfected GH3DOR and GH3MORDOR cells Receptor density (Bmax) values were determined from Scatchard analysis of saturation binding of [3H]diprenorphine (GH3DOR) or [3H]DPDPE (GH3MORDOR). Affinity (Ki) values were determined from competition binding experiments using the same radiolabeled ligands with increasing concentrations of DPDPE (0.01 nM to 10 µM) as described under Experimental Procedures. IC50 and maximal inhibition (Imax) values for -opioid receptor inhibition of adenylyl cyclase activity and Ca2+ currents by DPDPE were determined as described under Experimental Procedures. For receptor binding and adenylyl cyclase experiments, data represent the mean ± S.E.M. of a minimum of three separate experiments each performed in triplicate. For electrophysiological experiments, data represent the mean ± S.E.M. of measurements obtained from a minimum of four cells.

Effect of the -Opioid Receptor Alkylating Agent SUPERFIT on Opioid Receptor Binding, Inhibition of Adenylyl Cyclase Activity, and Ba²⁺ Currents in GH₃MORDOR Cells. Because GH₃MORDOR cells expressed 5-fold more  receptors than GH₃DOR cells, we examined whether the differential coupling of -opioid receptors to Ca²⁺ channels in these clones was the result of differences in receptor density. To accomplish this, the alkylating agent SUPERFIT (Zhu et al., 1996) was used to selectively and irreversibly block -opioid receptors in GH₃MORDOR cells and subsequently examine opioid receptor binding and the ability of the -opioid agonist DPDPE to inhibit adenylyl cyclase activity and the amplitude of Ba²⁺ currents (Fig. 1). Pretreatment of GH₃MORDOR cells with increasing concentrations of SUPERFIT (37°C for 45 min) followed by extensive washing reduced binding of the -opioid agonist [³H]DPDPE in a concentration-dependent manner with an IC₅₀ value of 6.1 nM (Fig. 1, top). This was similar to the IC₅₀ of 7.1 nM observed in CHO cells expressing -opioid receptors alone and suggests that the presence of the µ-opioid receptors does not influence the affinity of SUPERFIT (Zhu et al., 1996). Furthermore, the binding of the µ-opioid agonist [³H]DAMGO was not significantly altered when cells were pretreated with a 100 nM SUPERFIT, suggesting that the -opioid selective antagonist does not interact with µ-opioid binding sites (data not shown). These data argue against a confounding influence of a significant level of heterodimers (under Discussion). To confirm that the reduction in -opioid binding resulted from a decrease in receptor number and not receptor affinity, Scatchard analysis of [³H]DPDPE saturation binding was performed (Table 2). Pretreatment of GH₃MORDOR cells with SUPERFIT resulted in a dose-dependent (IC₅₀ = 4.9 nM) reduction in -opioid receptor B_max, without a significant reduction in K_d. These results are consistent with the previous observation of Zhu et al. (1996) that SUPERFIT was a highly selective irreversible -opioid receptor ligand and further demonstrated that we could selectively reduce the density of available -opioid receptors in GH₃MORDOR cells.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Effect of SUPERFIT on -opioid receptor binding, inhibition of adenylyl cyclase activity, and Ba2+ currents in GH3MORDOR cells. A, GH3MORDOR cells were pretreated with increasing concentrations of SUPERFIT (0.1-100 nM) for 45 min at 37°C, followed by extensive washing. The percentage of specific [3H]DPDPE binding remaining relative to control cells (i.e., those not treated with SUPERFIT) was calculated for each SUPERFIT concentration as described under Experimental Procedures. Each point represents the mean ± S.E. of three separate experiments. B, GH3MORDOR cells were cultured in 24-well plates (adenylyl cyclase assays; ) or in 35-mm dishes (electrophysiological experiments; ). Cells were incubated with SUPERFIT (0.6-600 nM) for 45 min at 37°C and subsequently washed extensively with warmed DMEM. The remainder of the assay was conducted as described under Experimental Procedures. For both adenylyl cyclase assays and electrophysiological experiments, the percentage of maximal inhibition was calculated by dividing the amount of inhibition produced by DPDPE (10 nM) in cells pretreated with each SUPERFIT concentration, by the inhibition produced by DPDPE in control cells (i.e., those not treated with SUPERFIT). For the adenylyl cyclase assays, each point represents the mean ± S.E. of three separate experiments. For the electrophysiological experiments, each point represents the mean ± S.E. for measurements obtained from a minimum of four cells. The best sigmoidal curve fit for all of these data was calculated using the computer program Sigmaplot. a, statistically different from the inhibition obtained after 600 nM SUPERFIT pretreatment. b, statistically different from the inhibition of adenylyl cyclase activity obtained after pretreatment with a corresponding concentration of SUPERFIT.

View this table: [in this window] [in a new window]   TABLE 2 -Opioid receptor density in membranes prepared from GH3MORDOR cells pretreated with increasing concentrations of the -alkylating agent SUPERFIT GH3MORDOR cells were incubated with the indicated concentration of SUPERFIT for 45 min, washed extensively, and membranes were prepared. Receptor density (Bmax) and affinity (Kd) values were determined from Scatchard analysis of saturation binding utilizing [3H]DPDPE as described under Experimental Procedures. The Kd and Bmax values represent estimates for each parameter ±S.E.M. determined by the computer program LIGAND from a single experiment performed in triplicate.

Effect of - (SUPERFIT) and µ- (-FNA) Opioid Receptor Alkylating Agents on µ- and -Opioid Receptor Inhibition of Adenylyl Cyclase Activity and Ba²⁺ Currents in Transfected GH₃ Cells. We performed additional experiments to determine whether the -opioid alkylating agent SUPERFIT influenced the ability of µ-opioid receptors to couple to adenylyl cyclase (Fig. 2) and/or Ca²⁺ channels (Fig. 3) in GH₃MORDOR cells. For ease of comparison, the data presented in Fig. 1 for the effect of 12 and 60 nM SUPERFIT on inhibition of adenylyl cyclase activity and Ba²⁺ currents produced by DPDPE (10 nM) are also presented in Figs. 2A and 3A (left). Although both 12 and 60 nM SUPERFIT significantly reduced the maximal inhibition of adenylyl cyclase activity produced by DPDPE, SUPERFIT (12 nM) had no effect on the ability of the µ-opioid agonist DAMGO to reduce intracellular cAMP levels (Fig. 2A). However, the highest concentration of SUPERFIT tested (60 nM) did diminish the amplitude of the DAMGO (1 µM)-evoked inhibition of cAMP accumulation by 69%. Although 60 nM SUPERFIT also reduced the maximal inhibition of adenylyl cyclase activity by DAMGO in GH₃MOR cells by 14%, this effect was not statistically significant. SUPERFIT (12 nM) also significantly reduced the ability of DPDPE to inhibit Ba²⁺ currents in GH₃MORDOR cells; after pretreatment with the higher concentration of SUPERFIT (60 nM) the effect of DPDPE on Ba²⁺ currents was all but abolished (Fig. 3A). Both concentrations of SUPERFIT also reduced the ability of the µ-opioid agonist DAMGO to inhibit Ba²⁺ currents by 45 and 66%. The agent had no effect on Ca²⁺ channel function as evidenced by the unaltered amplitude of Ba²⁺ currents recorded from cells pretreated by 12 or 60 nM SUPERFIT (Fig. 3C). Therefore, these data suggest that at in cells that express both receptor subtypes SUPERFIT can decrease the function of µ- as well as -receptors.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Effects of SUPERFIT and -FNA on the coupling of µ- and -receptors to adenylyl cyclase in GH3 MORDOR and GH3MOR cells. A, GH3MORDOR cells were cultured in 24-well plates and incubated with SUPERFIT (0, 12, or 60 nM) for 45 min at 37°C. After extensive washing with warmed DMEM, cells were exposed to an assay mix containing either DPDPE (left) or DAMGO (right) for 15 min (described under Experimental Procedures). The percentage of maximal inhibition of adenylyl cyclase activity was calculated by dividing the amount of inhibition produced by agonists in cells pretreated with each SUPERFIT concentration by the inhibition produced by agonists in control cells (i.e., those not treated with SUPERFIT). B, same experimental design as presented in A was used except that GH3MORDOR cells were pretreated for 45 min with the µ-opioid receptor alkylating agent -FNA (0, 10, or 20 nM). C, GH3MOR cells were incubated for 45 min with either SUPERFIT (0, 12, or 60 nM) or -FNA (0, 10, or 20 nM), extensively washed, and the inhibition of adenylyl cyclase produced by DAMGO was measured. The values presented are the mean ± S.E. determined from four separate experiments. Statistical significance of the data was determined by a nonpaired two-tailed Student's t test. *, statistically different from cells receiving no pretreatment, P < .05.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Effects of SUPERFIT and -FNA on the coupling of µ- and -receptors to Ca2+ channels in GH3MORDOR cells. A, GH3MORDOR cells were cultured in 30-mm dishes and incubated with SUPERFIT (0, 12, or 60 nM) for 45 min at 37°C. After extensive washing with warmed DMEM, cells were exposed to either DPDPE (left) or DAMGO (right) for 15 min (described under Experimental Procedures). The patch-clamp technique was used to measure the percentage of maximal inhibition of Ba2+ currents. This was calculated by dividing the amount of inhibition produced by agonists in cells pretreated with each SUPERFIT concentration by the inhibition produced by agonists in control cells (i.e., those not treated with SUPERFIT). The values presented are the mean ± S.E. determined from a minimum of four cells. B, same experimental design as presented in A was used except that GH3MORDOR cells were pretreated for 45 min with the µ-opioid receptor alkylating agent -FNA (0 or 10 nM). C, neither SUPERFIT (12 and 60 nM) nor -FNA (10 nM) had any effect on the amplitude of Ba2+ currents evoked by depolarizing GH3MORDOR cells from 80 to 0 mV. The values presented are the mean ± S.E. Statistical significance of the data was determined by a nonpaired two-tailed Student's t test. *, statistically different from cells receiving no pretreatment, P < .05.

GH₃ Cells Express Four Pertussis Toxin-Sensitive G Subunits. In addition to effects of receptor density, the differential coupling of opioid receptors to L-type Ca²⁺ channels in GH₃ cells might also be explained by activation of a distinct pattern of G proteins by -opioid receptors in the two clones. Because opioid receptor-mediated inhibition of adenylyl cyclase activity was blocked by pretreatment of cells with pertussis toxin, which ADP-ribosylates only G_i/G_o-type G proteins), we determined the identity of these G subunits in GH₃ cells. Membranes (50 µg/sample) were incubated with the photoaffinity label [³²P]AA-GTP, and labeled proteins were separated by urea/SDS-PAGE. After transfer to nitrocellulose membranes, blots were subjected to autoradiography, followed by immunoblotting with selective antibodies for individual G subunits (Fig. 4). In the absence of opioid, [³²P]AA-GTP was incorporated into three detectable bands (Fig. 4, A-D, lane 1). When membranes prepared from GH₃MORDOR cells were incubated with [³²P]AA-GTP in the presence of a -opioid agonist (Fig. 5), a fourth band incorporating the photoaffinity label was found that migrated above the first heavily labeled band. Consequently, these four bands were designated as proteins 1 to 4, from highest to lowest molecular weight.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Photoaffinity labeling and Western analysis of Gs in GH3 membranes using specific antisera. Membranes (50 µg) from GH3 cells were incubated with the photoaffinity label [32P]AA-GTP and subsequently separated by urea/SDS-PAGE. Proteins were then transferred onto nitrocellulose membrane and subjected to autoradiography followed by Western analysis of the same immunoblot with antibodies selective for individual G subunits. A-D, depicted in lanes 1 and 2 are the autoradiogram (AR) and the subsequent immunoblot of that autoradiogram, respectively. Specific G subunit antisera used were 978 (A, Gi1), EC2 (B, Gi3), GC2 (C, Go1 and Go2), or LEP4 (D, Gi1 and Gi2). Antibody-protein complexes were visualized using ECL and goat anti-rabbit conjugated with horseradish peroxidase as secondary antibodies.

View larger version (41K): [in this window] [in a new window]   Fig. 5.   Concentration-dependent [32P]AA-GTP labeling of individual pertussis toxin-sensitive G protein -subunits by the -opioid agonist DPDPE in GH3 MORDOR membranes. Top, autoradiogram of G subunits photoaffinity labeled with [32P]AA-GTP (1 µCi) in the presence of increasing concentrations (0.3-1000 nM) of the -opioid agonist DPDPE in GH3 MORDOR membranes (50 µg), separated by urea/SDS-PAGE. Bottom, to determine the amount of radioactivity incorporated by individual G subunits, the area of each band was traced, multiplied by its mean density, and the femtomoles of radioactivity determined by comparison with the density produced by a range of 32P standards using linear regression. The amount of each G subunit activated in femtomoles per milligram protein is plotted against the corresponding DPDPE concentrations. The values presented for each concentration represent the mean ± S.E. determined in five separate experiments.

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i3

o1

i2

o2

i3

o1,

i2

o2

Stimulation of a Higher Density of -Opioid Receptors in GH₃MORDOR Cells by DPDPE Results in a Greater Amount, but Not a Different Pattern of G Protein Activation Than in GH₃DOR Cells. Pretreatment of cells with pertussis toxin blocked regulation of adenylyl cyclase activity in both cell lines. Therefore, the activation of pertussis toxin-sensitive G_i/o subunits was examined in GH₃ membranes. One approach to measure the activation of G subunits by receptors is to use agonist-stimulated incorporation of [³²P]AA-GTP into G subunits, followed by separation using urea/SDS-PAGE and subsequent autoradiography (Prather et al., 1994a,b, 1995; Chakrabarti et al., 1995). The results of a typical photoaffinity-labeling experiment using GH₃MORDOR membranes are presented in Fig. 5. The -opioid receptor agonist DPDPE produced dose-related increases in the incorporation of [³²P]AA-GTP into all four previously identified G subunits. As was described previously for inhibition of adenylyl cyclase activity in GH₃MORDOR cells, to eliminate any nonselective stimulation of G protein labeling by higher DPDPE concentrations acting at µ-opioid receptors in GH₃ MORDOR membranes, all photoaffinity experiments using GH₃ MORDOR (but not GH₃DOR) cells were conducted in the presence of 300 nM CTOP.

o1

i2

o2

i3

o1

i3

o1

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o2

View larger version (61K): [in this window] [in a new window]   Fig. 6.   Maximum amount of G subunits activated by -opioid receptors in GH3 clones. Full dose-response curves (0.01-1000 nM) using the -opioid agonist DPDPE to induce photoaffinity labeling of G subunits with [32P]AA-GTP (1 µCi) were performed using 50 µg of membranes prepared from the indicated GH3 clones. Photolabeled G subunits were subsequently separated by urea/SDS-PAGE, exposed for autoradiography, and quantitated by densitometry. To determine the amount of radioactivity incorporated by individual G proteins, the area of each band was traced, multiplied by its mean density, and the femtomoles of radioactivity determined by comparison with the density produced by a range of 32P standards using linear regression. Minimum and maximum plateau values (expressed in fmol/mg of protein) were determined by curve fitting of the sigmoidal dose-response curves. The maximum amount of G subunits activated was defined as the difference between the minimum and maximum plateau values. Data reported represent the mean ± S.E. of at least three to five separate experiments. Statistical significance of the data was determined by ANOVA followed by comparisons using Tukey's method. a, statistically different from Gi3. b, statistically different from Go1. c, statistically different from Gi2. d, statistically different from Go2.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

In some central nervous system neurons and several cellular models, -opioid receptors are able to regulate the activity of multiple intracellular effectors. For example, -opioids inhibit both Ca²⁺ currents (Stefani et al., 1994) and adenylyl cyclase activities (Childers et al., 1992) in striatal neurons. In addition, in neuroblastoma cells -opioid receptors regulate adenylyl cyclase activity (Prather et al., 1994c), Ca²⁺ channels (Hescheler et al., 1987), and phospholipase C (Jin et al., 1992). Interestingly, in some other cells the coupling of -opioid receptors to intracellular effectors appears to be more restricted. In rat dorsal root ganglion neurons, µ- and - but not -opioid receptors are able to couple to Ca²⁺ channels (Moises et al., 1994) despite the fact that all three receptor subtypes are located on these cells (Ji et al., 1995). -Opioid receptors are also unable to regulate Ca²⁺ currents in several other central nervous system regions, including the nucleus tractus solitarius (Rhim and Miller, 1994), the basal forebrain (Soldo and Moises, 1997), and the neurohypophysis (Rusin et al., 1997). One possible mechanism that could underlie the coupling specificity of -opioid receptors to distinct effectors in the same cell might be the requirement for activation of different densities of receptors to regulate individual intracellular effectors.

In support of this hypothesis, we observed that in GH₃ cells stably transfected with a relatively low -opioid receptor density of 0.55 pmol/mg of protein (GH₃DOR), activation of -opioid receptors produced inhibition of adenylyl cyclase activity but was unable to alter current through Ca²⁺ channels. The density of -opioid receptors in GH₃DOR cells is similar to that reported for the neuroblastoma cell line NG108-15 (0.52 pmol/mg of protein) and slightly higher than that demonstrated in the striatum (0.29 pmol/mg of protein) (Law et al., 1983; Sim et al., 1996). In contrast to our observation with GH₃DOR cells, activation of -opioid receptors in a clone that contained a high density of -opioid receptors (2.45 pmol/mg of protein) coexpressed with µ-opioid receptors (GH₃MORDOR), resulted in not only the expected inhibition of adenylyl cyclase activity but also produced inhibition of L-type Ca²⁺ currents. The coupling of opioid receptors to L-type Ca²⁺ channels is rarely seen in neuronal preparations and it is possible that the high levels of receptor expression achieved in our recombinant system enable this signal transduction pathway to function (Piros et al., 1996b). It is also possible that in those instances in which such coupling is seen in cells with native receptors that this could be due to elevated receptor expression. Alternatively, coupling of opioid receptors to L-type Ca²⁺ channels may be enabled by an interaction with a specific G protein not present in all neurons. Because GH₃MORDOR cells expressed 5-fold more -opioid receptors than GH₃DOR cells, in the present study we examined whether the differential coupling of -opioid receptors to Ca²⁺ channels in these clones was the result of differences in receptor density. Selective and irreversible blockade of -opioid receptors using the alkylating agent SUPERFIT (Zhu et al., 1996) resulted in a concentration-dependent reduction in the ability of DPDPE to maximally inhibit both adenylyl cyclase and Ba²⁺ currents. The inhibition of Ba²⁺ currents by DPDPE was barely detectable when cells were pretreated with a concentration of SUPERFIT (12 nM) that resulted in approximately an 80% reduction in available -opioid receptors (0.5 pmol/mg), a level similar to the density of -opioid receptors expressed in GH₃DOR cells (0.55 pmol/mg). In contrast, more than 60% of the maximal inhibition of adenylyl cyclase activity by DPDPE was retained even when SUPERFIT pretreatment reduced specific binding by 99%. These results suggest that the inability of -opioid receptors to couple to L-type Ca²⁺ channels in GH₃DOR cells results from an insufficient number of available receptors and that -opioid receptors couple more efficiently to adenylyl cyclase than they do to L-type Ca²⁺ channels. These results might also help explain a general observation that -opioid receptors, when present, are able to regulate adenylyl cyclase activity in most brain regions and cell lines studied, but in many of these same tissues fail to couple to Ca²⁺ channels.

Overexpression of several different G protein-coupled receptors results in coupling to G proteins and effectors that would not be regulated at lower receptor densities (Ashkenazi et al., 1987; Eason et al., 1992). Hence, the differential coupling of -opioid receptors to L-type Ca²⁺ channels in GH₃ cells might be explained by higher densities of -opioid receptors producing activation of different (or additional) G protein(s) relative to those activated by lower densities of -opioid receptors. Therefore, we compared the pattern of maximal G protein activation produced by -opioid receptors in GH₃DOR and GH₃MORDOR cells. Dose-dependent activation of opioid receptors by agonists should produce a concomitant increase in the incorporation of [-³²P]AA-GTP into the G proteins with which they interact. Data obtained from full dose-response curves using selective agonists revealed that, stimulation of -opioid receptors in both GH₃ clones resulted in coupling to an identical, specific pattern of G proteins, the greatest activation of G_o1, followed by G_i2 = G_o2 > G_i3. Interestingly, the selective coupling of -opioid receptors to specific G proteins was different than observed previously when opioid receptors were transfected into CHO cells in which activation of µ-, -, or -opioid receptors resulted in nonselective, simultaneous coupling to multiple G proteins (Prather et al., 1994b, 1995; Chakrabarti et al., 1995). Although others have noted differential coupling of -opioid receptors to G proteins, the pattern of selectivity observed in the present study is different. Laugwitz et al. (1993) reported that - and µ-opioid receptors in SH-SY5Y cells preferentially activated G_i1 and G_i3, respectively, whereas both receptor subtypes interacted with G_i2 and G_o. The present findings suggest that increasing densities of -opioid receptors does not change the selectively of activation of different G subunits.

Although the pattern of G protein activation produced by -opioid receptors was not different in the two GH₃ clones, the maximal amount of all G proteins activated was significantly greater in GH₃MORDOR cells that expressed 5-fold more receptors. We have observed a similar direct relationship between - and µ-opioid receptor density and amount of G protein activated in other cells lines (Prather et al., 1994b; Chakrabarti et al., 1995). Based on these observations, it is tempting to speculate that stimulation of -opioid receptors in GH₃MORDOR, but not GH₃DOR cells resulted in activation of a critical threshold of specific G proteins required for Ca²⁺ channel inhibition. Several studies have attempted to identify the distinct G proteins responsible for coupling opioid receptors to Ca²⁺ channels. For example, intracellular application of G_o subunits was more effective than application of G_i in reconstitution of the -opioid receptor-mediated inhibition of Ca²⁺ currents in NG108-15 cells treated with pertussis toxin (Hescheler et al., 1987). Furthermore, a mutant G_oA subunit resistant to pertussis toxin, was able to rescue -opioid receptor-mediated inhibition of Ca²⁺ channels in pertussis toxin-treated NG108-15 cells (Taussig et al., 1992). Intracellular dialysis of dorsal root ganglion sensory neurons with antibodies selective for G_o, but not G_i, blocked both µ- (Moises et al., 1994) and - (Wiley et al., 1997) opioid receptor inhibition of Ca²⁺ currents. Similarly, -opioid inhibition of Ca²⁺ channels in NG108-15 cells is attenuated by preinjection of antibodies selective for G_o, but not G_i (McFadzean et al., 1989). Collectively, this evidence suggests that opioid receptors couple to Ca²⁺ channels through interaction with G proteins associated with the G_o-class of subunits (either G and/or G subunits). In any case, the results from the present study suggest that activation of higher densities of -opioid receptors does not produce promiscuous coupling to additional G proteins, resulting in the inhibition of Ca²⁺ channels in GH₃ cells. Alternatively, it seems more likely that a greater number of -opioid receptors are required to produce activation of increased amounts of specific G proteins that enable coupling to Ca²⁺ channels.

Recent studies have demonstrated that the coexpression of fully functional - and -opioid receptors produces a heterodimer combination with different binding and functional properties seen when either of the individual receptors are expressed alone (Jordan and Devi, 1999). Therefore, it is also possible that the presence of the µ-opioid receptor and/or the formation of µ/-opioid receptor dimers may enable coupling to Ca²⁺ channels in GH₃MORDOR cells. To examine this possibility, we conducted additional experiments with -FNA at concentrations (10 and 20 nM) suitable to all but abolish µ-opioid receptor activation by DAMGO (Chen et al., 1996). This action had no significant effect on the ability of -opioid receptors to couple to either adenylyl cyclase or Ca²⁺ channels in GH₃MORDOR cells. This suggests that the µ-opioid receptor is not influencing the function of -opioid receptors in this cell line.

We also performed additional experiments with SUPERFIT (12 and 60 nM) to determine whether this relatively selective -opioid receptor alkylating agent influenced the ability of µ-opioid receptors to couple to adenylyl cyclase and/or Ca²⁺ channels. Interestingly, we found that both concentrations of SUPERFIT diminished coupling of µ-opioid receptors to Ca²⁺ channels in GH₃MORDOR cells. SUPERFIT had no direct effect on Ca²⁺ channel function as evidenced by the unaltered amplitude of Ba²⁺ currents recorded from cells pretreated with 12 and 60 nM. Additionally, although 12 nM SUPERFIT had no effect on the ability of µ-receptors to couple to adenylyl cyclase, the highest concentration of SUPERFIT (60 nM) tested did diminish the amplitude of the DAMGO (100 nM)-evoked inhibition of cAMP accumulation. Because the same concentrations of SUPERFIT produced a greater effect in GH₃MORDOR cells than in GH₃ MOR cells, it appears that SUPERFIT at high concentrations can decrease the function of µ- as well as -receptors in cells transfected with both receptor subtypes. This suggests that µ-opioid receptor function may be influenced by coexpression with -opioid receptors and that specific concentrations of SUPERFIT may preferentially block such heterodimers. This interesting possibility will be tested in a subsequent study. However, as mentioned above blockade of µ-receptors by -FNA has no effect on the function of DPDPE in GH₃MORDOR cells and -receptors couple to the same G proteins regardless of whether the µ-receptor is present. These findings lead us to conclude that differences in the coupling between -receptors and Ca²⁺ channels seen in GH₃DOR and GH₃MORDOR cells are best explained by the higher level of -receptor expression in the latter. This is supported by the fact that SUPERFIT reduces -receptor coupling to Ca²⁺ channels in the GH₃ MORDOR cells at all concentrations tested.