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J. Biol. Chem., Vol. 276, Issue 41, 37779-37786, October 12, 2001

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Constitutively Active µ-Opioid Receptors Inhibit Adenylyl Cyclase Activity in Intact Cells and Activate G-proteins Differently than the Agonist [D-Ala²,N-MePhe⁴,Gly-ol⁵]Enkephalin^*

Jing-Gen Liu, Michael B. Ruckle, and Paul L. Prather

From the Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205

Received for publication, July 1, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The most convincing evidence demonstrating constitutive activation of µ-opioid receptors is the observation that putative inverse agonists decrease basal G-protein activity in membrane preparations. However, it is not clear whether constitutively active receptors in isolated membranes have any physiological relevance in intact cells. GH₃ cells expressing µ-opioid receptors (GH₃MOR) exhibit higher basal G-protein activity and lower basal cAMP levels than wild-type GH₃ cells, indicative of constitutively active receptors. This study determined whether alkylation of µ-opioid receptors by the irreversible antagonist -funaltrexamine would decrease spontaneous receptor activity in intact cells, revealing constitutive activity. GH₃MOR cells were pretreated with increasing concentrations of -funaltrexamine followed by functional testing after removal of unbound drug. -Funaltrexamine pretreatment produced a concentration-dependent decrease in µ-opioid receptor binding with an IC₅₀ of 0.98 nM and an E_max of 77%. Similar concentrations of -funaltrexamine pretreatment produced a half-maximal reduction in basal [³⁵S]GTPS binding, a decrease in basal photolabeling of G-proteins with azidoanilido-[-³²P]GTP, and an increase in basal adenylyl cyclase activity in intact cells. Therefore, µ-opioid receptors are constitutively active in intact cells, producing stimulation of G-proteins and inhibition of adenylyl cyclase. Importantly, photolabeling of G-subunits with azidoanilido-[-³²P]GTP demonstrated that constitutively active µ-opioid receptors activate individual G-proteins differently than the agonist [D-Ala²,N-MePhe⁴,Gly-ol⁵]enkephalin.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The µ-opioid receptor is a member of the superfamily of G-protein-coupled receptors (GPCRs)¹ (1). Activation of µ-opioid receptors leads to the regulation of several intracellular effectors including the inhibition of adenylyl cyclase activity (2), the closing of voltage-gated Ca²⁺ channels (3), and the activation of inwardly rectifying K⁺ channels (4). All of these events are mediated by pertussis toxin (PTX)-sensitive G-proteins. Although the analgesic effect of clinically relevant opioids is mediated by the µ-opioid receptor, this receptor also appears to be critically involved in mediating the tolerance and dependence that occurs upon the chronic use of opioids (5). Although the development of opioid tolerance and dependence has been studied extensively, the exact mechanisms underlying these processes remain largely unknown.

Many GPCRs exhibit constitutive activity, producing spontaneous regulation of effectors in the absence of activation by agonists (6). Ligands that can reduce or abolish this spontaneous, agonist-independent activity are termed inverse agonists. Inverse agonists have been observed for several GPCRs including 5-HT_2c, ₂-adrenergic, ₂-adrenergic, D₂-dopaminergic, and retinoic acid receptors (7). Definitive evidence for constitutive activity of -opioid receptors has also been well established by use of the inverse agonist ICI-174,864 (8-12). In contrast, demonstration of constitutively active µ-opioid receptors has been less conclusive, perhaps because of the lack of a fully efficacious inverse agonist. The most convincing evidence for such spontaneous, agonist-independent activity to date is the ability of putative inverse agonists to decrease basal G-protein activity in membrane preparations containing relatively high densities of µ-opioid receptors (13, 14). However, it remains to be determined whether constitutive activation of µ-opioid receptors in isolated membrane preparations containing unusually high receptor densities has any physiological relevance in intact cells. Such evidence might be provided by employing a cellular model expressing physiological levels of µ-opioid receptors and demonstration of the effector(s) regulated by constitutively activated receptors and G-proteins.

Interestingly, it has also recently been reported that chronic treatment with morphine increases basal [³⁵S]GTPS binding in HEK 293 cells transfected with µ-opioid receptors (14). We further demonstrated that prolonged exposure of GH₃ cells expressing µ-opioid receptors (GH₃MOR) to either of the agonists morphine or [D-Ala²,N-MePhe⁴,Gly-ol⁵]enkephalin (DAMGO) converts the antagonists naloxone and -chlornaltrexamine into inverse agonists. This was indicated by production of a concentration-dependent inhibition of basal [³⁵S]GTPS binding by these ligands (15). Therefore, chronic opioid treatment may produce and/or enhance constitutive activation of µ-opioid receptors, and this process may play a role in the development of tolerance and/or dependence that occurs upon their prolonged use.

It is well established that certain ligands can bind irreversibly to (i.e. alkylate) µ-opioid receptors (16). Once alkylated, receptors are no longer able to bind agonist (or antagonist) and thus cannot be activated subsequently. Consequently, this technique has been used selectively to reduce the number of available µ-opioid receptors and monitor the effect on effector coupling (17). According to the two-state receptor model, to account for constitutive activity of certain GPCRs, it has been postulated that a high density of receptors would result in a greater degree of constitutive activity (18). This is due to a greater chance of encounter of a receptor in the active conformation with a G-protein. Therefore, it is possible that irreversible binding by specific ligands to constitutively active µ-opioid receptors might produce an inactive conformation of the receptor and thereby constrain spontaneous activity. If true, this technique might be utilized to demonstrate constitutive activity of GPCRs when no inverse agonists are available.

-Funaltrexamine (-FNA) is a well characterized antagonist that selectively and irreversibly binds to µ-opioid receptors (19, 20). GH₃MOR cells express a relatively moderate physiological density of µ-opioid receptors (0.39 pmol/mg) (3) and exhibit higher basal G-protein activity and lower basal cAMP levels that wild-type GH₃ cells. These observations suggest the potential presence of constitutively active µ-opioid receptors. However, our previous study failed to detect constitutive activity in opioid naive GH₃MOR cells after screening several ligands for potential inverse agonist activity (15). Therefore, the purpose of the present study was to determine whether alkylation of µ-opioid receptors by -FNA would reveal constitutive activity in intact GH₃MOR cells. This was accomplished by reducing available µ-opioid receptor number in a concentration-dependent manner using -FNA pretreatment. Following the removal of unbound -FNA by extensive washing, constitutive activity was assessed by correlating reductions in µ-opioid receptor density with basal G-protein activation and adenylyl cyclase activity. We report that constitutively active µ-opioid receptors inhibit adenylyl cyclase activity in intact cells and activate individual G-proteins differently than those stimulated by the agonist DAMGO. Furthermore, because GH₃MOR cells express physiologically relevant levels of µ-opioid receptors, it is likely that constitutive activation of µ-opioid receptors is not limited to artificially transfected systems, but rather it may serve important functions in the brain.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- GH₃ (CCL 82.1) or stably transfected GH₃MOR (3) cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum, 100 units/ml penicillin, 100 mg/ml streptomycin, and 2.5 mg/ml Geneticin (for GH₃MOR cells). Cells were incubated in a humidified atmosphere of 5% CO₂, 95% air at 37 °C.

-FNA Pretreatment-- For receptor and GTPS binding experiments, cells from separate T175 flasks were harvested and resuspended in 20 ml of Dulbecco's modified Eagle's medium containing various concentrations of -FNA (0.1-100 nM). Following incubation at 37 °C for 45 min in an orbital shaker (150 rpm), cells were washed three times with 50 ml of warmed phosphate-buffered saline to remove residual -FNA, and cell pellets were frozen at 80 °C. For adenylyl cyclase assays, cells were cultured in 24-well plates. On the day of the assay, the medium was replaced with a warmed incubation mixture containing [8-³H]adenine (26 Ci/mmol; Amersham Pharmacia Biotech) and different concentrations of -FNA for 45 min. The incubation mixture was removed, cells were washed three times with 1.5 ml of warmed Dulbecco's modified Eagle's medium to remove residual -FNA, and the assay was performed. In some cases, cells were cultured in the presence of 100 ng/ml PTX for 24 h.

Receptor Binding-- Membrane preparation and receptor binding experiments were conducted as described previously (15). Aliquots of 200 µg of membrane protein/sample were incubated with 1 nM [³H]diprenorphine (36 Ci/mmol; PerkinElmer Life Sciences) in 50 mM Tris-HCl, pH 7.4, with 10 mM MgCl₂ at room temperature for 90 min. Nonspecific binding was defined by the inclusion of 10 µM naloxone. The reaction was terminated by rapid filtration, and bound radioactivity was determined by liquid scintillation counting in a Packard Tri-Carb 2100TR liquid scintillation counter (Packard Instrument Co.).

[³⁵S]GTPS Binding-- [³⁵S]GTPS binding was performed as detailed elsewhere (15). Membranes (50 µg/sample) were incubated with [³⁵S]GTPS (0.1 nM) in a binding buffer composed of 20 mM HEPES, pH 7.4, 10 mM MgCl₂ 100 mM KCl or NaCl, and 10 µM GDP at 30 °C for 1 h. Nonspecific binding was determined in the presence of nonradioactive GTPS (10 µM). The reaction was terminated by rapid filtration, and bound radioactivity was determined by liquid scintillation counting.

Adenylyl Cyclase Activity in Intact Cells-- The effect of the absence or presence of opioids on the conversion of [³H]adenine-labeled ATP pools to cAMP in whole, intact cells was measured as described previously (21). Briefly, cells were seeded into 24-well plates and cultured until ~80% confluent. On the day of the assay, the medium was removed and replaced with a warmed (37 °C) incubation mixture containing [³H]adenine and -FNA for 45 min (for all ingredients see previous "-FNA Pretreatment"). After incubation and extensive washing to remove residual -FNA, the mixture was replaced with an ice-cold assay mixture (Krebs-Ringer HEPES buffer containing 500 µM 3-isobutyl-1-methylxanthine, and the absence or presence of the opioid ligand to be tested). Plates were then floated on a water bath at 37 °C for 15 min, and the reaction was terminated by the addition of 50 µl of 2.2 N HCl. cAMP was separated using Alumina column chromatography, and radioactivity was determined by liquid scintillation counting. Importantly, in all assays presented, the effect of treatments on basal adenylyl cyclase activity was examined (i.e. in the absence of any exogenous stimulator of adenylyl cyclase).

Photoaffinity Labeling of G-subunits with Azidoanilido-[-³² P]GTP (AA-[³²P]GTP)-- The photoaffinity labeling of G-subunits with AA-[³²P]GTP was performed as detailed elsewhere (21-25). Membranes (50 µg/sample) were incubated in the presence or absence of the drugs to be tested 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, 10 or 50 µM GDP). GDP (50 or 10 µM) was included in buffer I to reduce basal G-protein labeling to allow optimal measurement of agonist stimulated or constitutive activation, respectively. After incubation, AA-[³²P]GTP (1 µCi/sample) was added, and samples were incubated for an additional 6 min at 30 °C. The reaction was terminated by placing samples on ice, and membranes were then collected by centrifugation at 12,000 × g for 10 min. Following resuspension in 100 µl of buffer II (50 mM HEPES, pH 7.0, 0.1 mM EDTA, 10 mM MgCl₂, 30 mM NaCl, 2 mM dithiothreitol), samples were irradiated at 4 °C with 240 mJ from an ultraviolet lamp (254 nM, 150 w) at a distance of 15 cm. Membranes were collected by centrifugation as before, resuspended in electrophoresis loading buffer, and separated on SDS-polyacrylamide gels containing 10% acrylamide and 6 M urea. AA-[³²P]GTP labeled G-subunits were visualized autoradiographically by a Molecular Dynamics PhosphorImager 445 (Sunnyvale, CA) and quantified by densitometry using the NIH Image software program (version 1.56). To determine the amount of relative radioactivity incorporated by individual G-subunits (i.e. optical density units), the area of each band was traced and multiplied by its mean optical density. In instances where photoaffinity labeling of total G-proteins was determined, the optical density units for all individual G-subunits from a single sample were summed.

Data Analysis-- Unless otherwise stated, data reported represent the mean ± the standard error of at least three separate experiments that were each performed in triplicate. The ED₅₀ and E_max values were obtained from full dose-response curves, subjected to sigmoidal curve fitting. For statistical comparisons involving three or more groups, differences between means were determined by a one-way ANOVA followed by post hoc comparisons using Dunnett's or Tukey's tests. When only two groups were compared, differences between the means were determined by the nonpaired Student's t test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Effect of KCl and PTX on Basal [³⁵S]GTPS Binding in Wild-type GH₃ and GH₃MOR Cells-- The basal level of [³⁵S]GTPS binding was significantly higher (p < 0.05) in membranes prepared from GH₃MOR (50.3 fmol/mg) relative to wild-type GH₃ cells (30.3 fmol/mg) (Fig. 1). Replacement of Na⁺ with K⁺ ions in the assay buffer led to a further increase in basal [³⁵S]GTPS binding in both GH₃ and GH₃MOR cells lines to 42.8 and 74.6 fmol/mg, respectively. Pretreatment with 100 ng/ml PTX (24 h) abolished the enhanced basal [³⁵S]GTPS binding produced by KCl in both cell lines. These results indicated the presence of substantial spontaneous activity of receptors coupled to G_i/G_o proteins in GH₃MOR, relative to wild-type GH₃ cells. Because greater constitutive activity was observed in the presence of K⁺ ions, all subsequent [³⁵S]GTPS binding experiments included KCl instead of NaCl in the assay buffer.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   The effect of KCl and PTX on basal [35S]GTPS binding in wild-type GH3 (GH3WT) and GH3MOR cells. Basal [35S]GTPS binding was determined in GH3 wild type (left panel) or GH3MOR (right panel) membranes with 0.1 nM [35S]GTPS in a buffer containing either NaCl (100 mM) or KCl (100 mM). Nonspecific binding was defined by the inclusion of 10 µM GTPS. Values represent the mean ± S.E. of at least four independent experiments performed in triplicate. , significantly different from Basal/NaCl in GH3 wild type; p < 0.05 (one-way ANOVA plus Dunnett's post hoc test). #, significantly different from corresponding condition in GH3WT; p < 0.05 (Student's t test).*, significantly different from Basal/NaCl in GH3MOR, p < 0.05 (one-way ANOVA plus Dunnett's post hoc test).

The Effect of -FNA Pretreatment on Subsequent Opioid Receptor Binding and Agonist Activity in GH₃MOR Cells-- The ability of -FNA to alkylate µ-opioid receptors in GH₃MOR cells was examined first. Saturation binding with the opioid antagonist [³H]diprenorphine previously showed that GH₃MOR cells express a moderate density of µ-opioid receptors of 0.39 pmol/mg (3). Pretreatment of cells with increasing amounts of -FNA (0.1-100 nM; 37 °C for 45 min) followed by complete drug washout resulted in a concentration-dependent reduction of specific [³H]diprenorphine binding with an ED₅₀ of 0.98 nM and E_max of 77% (Fig. 2A; Table I). The ED₅₀ presented is similar to the affinity of -FNA for µ-opioid receptors (16). The observed decrease in binding reflected a loss of available functional µ-opioid receptors because pretreatment of cells with a maximal concentration of -FNA (100 nM) resulted in abolishment of the ability of the selective µ-opioid agonist DAMGO (1 µM) to stimulate [³⁵S]GTPS binding (64.2 versus 6.3% stimulation; p < 0.01) and to inhibit basal adenylyl cyclase activity (45.2 versus 4.5% inhibition; p < 0.01) (Fig. 2B). DAMGO (1 µM) produced no effect in either functional assay in wild-type GH₃ cells (data not shown). Pretreatment with 100 nM -FNA was used in all subsequent experiments to examine the effect of a maximal reduction in available µ-opioid receptors on several functional assays.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Effect of -FNA pretreatment on subsequent opioid receptor binding and agonist activity in GH3MOR cells. A, membranes prepared from extensively washed GH3MOR cells pretreated with increasing concentrations of -FNA were incubated with 1 nM [3H]diprenorphine in the presence or absence of 10 µM naloxone and expressed as a percent of the specific binding in non-pretreated GH3MOR cells. B, the ability of DAMGO to stimulate [35S]GTPS binding in membranes (left panel) or to inhibit basal adenylyl cyclase activity in intact cells (right panel) was evaluated following no treatment or after -FNA pretreatment (100 nM, 45 min). Values presented for both graphs represent the mean ± S.E. of three or four independent experiments conducted in triplicate. *, significantly different from corresponding no -FNA pretreatment condition; p < 0.01 (Student's t test).

View this table: [in this window] [in a new window]   Table I Comparison of the concentration of -FNA pretreatment required to reduce specific µ-opioid receptor binding by 50% with that needed to produce half-maximal effects on the basal activity in several functional assays

The Effect of -FNA Pretreatment on Subsequent Basal G-protein Activation and Adenylyl Cyclase Activity in Wild-type GH₃ and GH₃MOR Cells-- Similar to that observed in Fig. 1, basal [³⁵S]GTPS binding was significantly higher (p < 0.05) in GH₃MOR (66.6 fmol/mg) relative to wild-type GH₃ cells (46.9 fmol/mg) (Fig. 3B). To determine whether the elevated basal [³⁵S]GTPS binding activity in GH₃MOR cells could be correlated with the density of available µ-opioid receptors, cells were pretreated with -FNA (0.1-100 nM), and basal G-protein activation was measured in membranes prepared from cells following complete drug removal by extensive washing. This resulted in a concentration-dependent reduction in basal [³⁵S]GTPS binding with an E_max of 35.7% and an ED₅₀ of 1.59 nM (Fig. 3A; Table I). The decrease in G-protein activation produced by -FNA pretreatment paralleled the reduction in µ-opioid receptor binding, requiring a similar concentration to produce a half-maximal response. Pretreatment of GH₃MOR cells with a maximal concentration of -FNA (100 nM) reduced basal G-protein activation to a level (43.2 fmol/mg) not statistically different from that observed in GH₃ cells (Fig. 3B, right panel). The effect of -FNA on basal [³⁵S]GTPS binding in GH₃MOR cells was reversed by inclusion of the opioid antagonist naloxone (10 µM) during pretreatment. Importantly, exposure of wild-type GH₃ cells to 100 nM -FNA produced no effect on basal [³⁵S]GTPS binding (Fig. 3B, left panel).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Effect of -FNA pretreatment on subsequent basal [35S]GTPS binding in wild-type GH3 (GH3WT) and GH3MOR membranes. A, membranes prepared from extensively washed GH3MOR cells pretreated with increasing concentrations of -FNA were incubated with 0.1 nM [35S]GTPS in the presence or absence of 10 µM GTPS and expressed as a percent of the specific binding in membranes prepared from non-pretreated GH3MOR cells. B, basal [35S]GTPS binding was performed in membranes prepared from GH3 wild type (left panel) or GH3MOR (right panel) cells following no treatment, -FNA pretreatment (100 nM, 45 min), or pretreatment with -FNA plus naloxone (10 µM, GH3MOR cells only). Values presented for both graphs represent the mean ± S.E. of three to eight independent experiments conducted in triplicate. , significantly different from no -FNA pretreatment condition in GH3WT; p < 0.05 (Student's t test). *, significantly different from no -FNA pretreatment condition in GH3MOR; p < 0.05 (one-way ANOVA plus Dunnett's post hoc test).

Another approach to measure activation of G-proteins by GPCRs is to examine agonist-stimulated incorporation of AA-[³²P]GTP into G-subunits (21-25). An advantage of this technique is that the activation of individual G-subunits can be examined following separation by SDS-polyacrylamide gel electrophoresis. By comparison of the electrophoretic mobility of bands obtained by autoradiography with those detected by Western analysis of the same immunoblot, we have previously reported (21) and the autoradiograms presented in Figs. 4, 6, and 7 demonstrate that GH₃ cells express four pertussis toxin-sensitive G-proteins labeled by AA-[³²P]GTP. The identity of these G-subunits from higher to lower molecular weight were G_i3, G_o1, G_i2, and G_o2, respectively (21). When the activation of all G-subunits are considered together (i.e. total G-protein activation), basal photoaffinity labeling of G-proteins with AA-[³²P]GTP was significantly higher (p < 0.01) in GH₃MOR (8.35 OD units) relative to wild-type GH₃ cells (3.1 OD units) (Fig. 4B). Pretreatment with -FNA (0.1-100 nM) resulted in a concentration-dependent reduction in labeling of all G-proteins, resulting in a maximal inhibition of 46.5% and an ED₅₀ of 7.72 nM (Fig. 4A; Table I). The maximal decrease in labeling produced by 100 nM -FNA was significantly reversed (p < 0.01) by the addition of the opioid antagonist naloxone during pretreatment. As expected, these results are virtually identical to those presented for [³⁵S]GTPS binding.

View larger version (42K): [in this window] [in a new window]   Fig. 4.   Effect of -FNA pretreatment on subsequent basal photoaffinity labeling of G-proteins with AA-[32P]GTP in wild-type GH3 (GH3WT) and GH3MOR membranes. A, top, autoradiogram of individual G-subunits photoaffinity-labeled with AA-[32P]GTP (1 µCi) in membranes prepared from GH3MOR cells pretreated with increasing concentrations of -FNA. The optical density units for all individual G-subunits from a single sample were summed to determine the activation of total G-proteins, and data were expressed as a percent of labeling in non-pretreated GH3MOR cells. B, basal AA-[32P]GTP labeling in membranes prepared from GH3 wild type (left panel) or GH3MOR (right panel) cells following no treatment, -FNA pretreatment (100 nM, 45 min, GH3MOR cells only), or pretreatment with -FNA plus naloxone (10 µM, GH3MOR cells only). Values presented for both graphs represent the mean ± S.E. of four or five independent experiments. , significantly different from no -FNA pretreatment condition in GH3WT; p < 0.01 (Student's t test). *, significantly different from no -FNA pretreatment condition in GH3MOR; p < 0.01 (one-way ANOVA plus Dunnett's post hoc test).

Because acute activation of µ-opioid receptors by agonists results in an inhibition of adenylyl cyclase activity via G_i/G_o G-proteins (Fig. 2B), it would be expected that basal cAMP levels would be lower in intact cells containing constitutively active receptors. As predicted, basal intracellular cAMP levels were statistically reduced (p < 0.01) in GH₃MOR (6.1 fmol/mg/min) relative to GH₃ cells (11.9 fmol/mg/min) (Fig. 5B). Furthermore, -FNA pretreatment (0.1-100 nM) produced a concentration-related increase in basal cAMP (Fig. 5A) to a level (10.4 fmol/mg/min) that did not statistically differ from wild-type GH₃ cells (Fig. 5B). Concentrations of -FNA pretreatment that produced 50% alkylation of µ-opioid receptors (ED₅₀ = 0.98 nM) also resulted in half-maximal increases in cAMP levels (ED₅₀ = 2.2 nM) (Table I). Importantly, a maximal concentration of -FNA (100 nM) produced no effect on basal cAMP levels in GH₃ cells, and the effect in GH₃MOR cells could be reversed by inclusion of naloxone (10 µM) during pretreatment (Fig. 5B).

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Effect of -FNA pretreatment on subsequent basal adenylyl cyclase activity in wild-type GH3 (GH3WT) and GH3MOR cells. A, basal cAMP levels were measured in intact GH3MOR cells following pretreatment with increasing concentrations of -FNA and expressed as a percent of cAMP levels determined in non-pretreated GH3MOR cells. B, basal cAMP levels in intact GH3 wild type (left panel) or GH3MOR (right panel) cells following no treatment, -FNA pretreatment (100 nM, 45 min), or pretreatment with -FNA plus naloxone (10 µM, GH3MOR cells only). Values presented for both graphs represent the mean ± S.E. of three or five independent experiments conducted in triplicate. , significantly different from no -FNA pretreatment condition in GH3 wild type; p < 0.05 (Student's t test). *, significantly different from no -FNA pretreatment condition in GH3MOR; p < 0.05 (one-way ANOVA plus Dunnett's post hoc test).

When GH₃MOR cells were pretreated with a maximal concentration of -FNA (100 nM) for only a short duration (10 min), no effect on basal [³⁵S]GTPS binding or cAMP levels was observed (data not shown). Interestingly, acute addition of -FNA (100 nM) to the [³⁵S]GTPS binding buffer or to the cAMP assay mixture resulted in a slight stimulation (16.1 ± 1.5%) of [³⁵S]GTPS binding and had no effect on basal cAMP levels. Therefore, -FNA does not appear to act acutely as an inverse agonist but rather, possibly, as a partial agonist in these assays. To determine whether the observed effects of -FNA were due to potential partial agonist properties, GH₃MOR cells were pretreated with the full agonist DAMGO (1 and 10 µM) or the antagonist naloxone (10 µM) for 45 min. Following a complete washout of drugs, no effect on either [³⁵S]GTPS binding or basal adenylyl cyclase activity was observed (data not shown).

Comparison of G-proteins Activated by the µ-Opioid Agonist DAMGO Versus Those Stimulated by Constitutively Active µ-Opioid Receptors in GH₃MOR Cells-- The final experiments compared the individual G-subunits activated by the µ-opioid agonist DAMGO to those stimulated by constitutively active µ-opioid receptors. In GH₃MOR cells, DAMGO produced a concentration-dependent increase in the labeling of total G-proteins with a maximal effect of 333% and an ED₅₀ of 32.6 nM (Fig. 6). Activation by DAMGO (1 µM) was completely abolished by inclusion of 10 µM of the opioid antagonist naloxone (data not shown). A maximal concentration of DAMGO (1 µM) produced a distinct graded stimulation of individual G-subunits with the greatest amount of G_o2 activated (4.18 OD units), followed by G_i2 (3.0 OD units)  G_o1 (2.31 OD units) > G_i3 (0.89 OD units) (Fig. 7A, right panel). Interestingly, when the data are presented as percent of increase from control, DAMGO activated equal percentages of G_o2 (418%) and G_o1 (383%) but significantly less G_i2 (253%) and G_i3 (189%) (Fig. 7B, right panel).

View larger version (46K): [in this window] [in a new window]   Fig. 6.   Concentration-dependent photoaffinity labeling of total G-proteins by AA-[32P]GTP in response to the µ-opioid agonist DAMGO in GH3MOR membranes. Top, autoradiogram of individual G-subunits photoaffinity labeled by AA-[32P]GTP (1 µCi) in the presence of increasing concentrations (0.3-1000 nM) of DAMGO in membranes prepared from GH3MOR cells. Bottom, the optical density units for all individual G-subunits from a single sample were summed to determine the activation of total G-proteins, and data were expressed as a percent of labeling in the absence of agonist. The values represent mean ± S.E. from five independent experiments.

View larger version (54K): [in this window] [in a new window]   Fig. 7.   Comparison of the individual G-subunits activated by the µ-opioid agonist DAMGO versus those stimulated by constitutively active µ-opioid receptors in GH3MOR membranes. A, top, presented is the autoradiogram of individual G-subunits photoaffinity labeled with AA-[32P]GTP (1 µCi) in membranes prepared from GH3WT or GH3MOR cells under basal conditions (left panel) or photolabeling of individual G-subunits in the absence or presence of a maximal concentration (1 µM) of the agonist DAMGO in GH3MOR membranes (right panel). Bottom, G-protein stimulation by constitutively active µ-opioid receptors was defined as the difference in photolabeling of individual G-subunits (in optical density units) between GH3 wild type (GH3WT) and GH3MOR membranes (left panel). Agonist-induced stimulation of G-proteins was defined as the difference in photoaffinity labeling obtained in the absence and presence of DAMGO (1 µM) in GH3MOR membranes (right panel). B, data from panel A were expressed as a percent of control labeling (i.e. GH3 wild type for constitutive activation or photolabeling in the absence of DAMGO for agonist-induced activation). Values presented for both graphs represent the mean ± S.E. of four independent experiments. a, b, c, d, significantly different from the same value determined for Gi3 (a), Go1 (b), Gi2 (c), and Go2 (d); p < 0.01 (one-way ANOVA plus Tukey's post hoc test).

G-proteins stimulated by constitutively active receptors were determined by quantifying the increase in photoaffinity labeling of individual G-subunits in GH₃MOR cells relative to that observed in wild-type GH₃ cells. In contrast to the selective pattern of G-protein activation produced by DAMGO, putative constitutively active µ-opioid receptors activated all four G-subunits in a relatively nonselective manner (Fig. 7, A and B, left panels). For example, while slightly less G_i3 (0.68 OD units) was activated relative to other G-proteins, equivalent amounts of G_o1 (1.23 OD units), G_i2 (1.61 OD units), and G_o2 (1.76 OD units) were activated. Similarly, with the exception of G_o1 (124%), equal percentages of G_i3 (207%), G_i2 (220%), and G_o2 (176%) were activated. Importantly, Western analysis with selective antibodies revealed no differences in the expression levels of any G subunit between wild-type GH₃ and GH₃MOR cells (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Many GPCRs exhibit constitutive activity, producing spontaneous activation of G-proteins and regulation of effectors in the absence of agonists (6, 7, 26). In cells expressing -opioid receptors, the inverse agonist ICI-174,864 inhibits basal GTPase activity (8, 9), reduces basal [³⁵S]GTPS binding (12, 27), and increases adenylyl cyclase activity in whole cells (10, 11). This provides conclusive evidence that constitutively active -opioid receptors activate inhibitory G_i/G_o G-proteins and inhibit adenylyl cyclase activity in intact cells. Although several studies have failed to demonstrate constitutive activity of µ-opioid receptors in membrane preparations (12, 15), other recent reports show that -chlornaltrexamine inhibits basal [³⁵S]GTPS binding in membranes prepared from cells transfected with relatively high densities of µ-opioid receptors, indicative of inverse activity (13, 14). This suggests that µ-opioid receptors are constitutively active and stimulate G-proteins in membranes. However, it remains to be determined whether constitutive activation of µ-opioid receptors in isolated membrane preparations containing abnormally high receptor densities has any physiological relevance in intact cells. This is a crucial question given the recent evidence that chronic exposure to opioids enhances the apparent constitutive activity of µ-opioid receptors in membrane preparations (14, 15) and thus may play a role in the development of opioid tolerance and/or dependence (28). The present study utilized a novel approach to confirm that µ-opioid receptors are constitutively active in intact GH₃MOR cells expressing a physiological density of receptors. µ-Opioid receptors in these cells demonstrate spontaneous, agonist-independent stimulation of G-proteins and inhibition of adenylyl cyclase activity. Furthermore, constitutively active µ-opioid receptors activate G-proteins differently than those stimulated by the agonist DAMGO.

It was first shown that GH₃MOR exhibit higher basal G-protein activity and lower basal cAMP levels than GH₃ cells, suggesting the presence of constitutively active receptors. First, basal G-protein activity was significantly greater in GH₃MOR, relative to GH₃ membranes as measured by [³⁵S]GTPS binding or photoaffinity labeling of G-proteins with AA-[³²P]GTP. Second, under conditions known to increase constitutive activity of GPCRs (i.e. replacement of Na⁺ by K⁺ ions) (12, 27, 29), the amount of basal [³⁵S]GTPS binding in GH₃MOR and GH₃ membranes increased further. Third, PTX pretreatment of both cells lines significantly reduced basal [³⁵S]GTPS binding. Because PTX blocks receptor-G-protein coupling but not GTP binding (30), this confirmed the presence of constitutively active receptors stimulating predominately G_i/G_o G-proteins. Although the effects of K⁺ and PTX on basal G-protein activity were also observed in wild-type GH₃ membranes, the magnitude of response was significantly less than that observed in GH₃MOR membranes and most likely indicated the presence of constitutively active GPCRs other than µ-opioid receptors. Fourth, because acute activation of µ-opioid receptors by agonists results in an inhibition of adenylyl cyclase activity via G_i/G_o G-proteins (2), it would be expected that basal cAMP levels would be lower in intact cells containing constitutively active receptors. As predicted, it was also demonstrated that basal intracellular cAMP levels were statistically reduced in GH₃MOR relative to GH₃ cells.

To provide direct evidence that constitutively active µ-opioid receptors were responsible for the observed elevated basal G-protein activation and reduced basal cAMP levels in GH₃MOR cells, the selective and irreversible µ-opioid receptor alkylating agent -FNA (16, 17) was employed. Specifically, the hypothesis that irreversible binding by -FNA to µ-opioid receptors would produce an inactive conformation of the receptor and thereby constrain spontaneous activity was tested. GH₃MOR cells were pretreated with increasing concentrations of -FNA followed by functional testing after removal of unbound drug by extensive washing. -FNA pretreatment produced a concentration-dependent decrease in specific µ-opioid receptor binding. Importantly, the most definitive evidence supporting constitutive activation of µ-opioid receptors in this study was the observation that concentrations of -FNA pretreatment producing a 50% loss of available receptors also produced half-maximal reductions in two independent measures of basal G-protein activity and increases in basal adenylyl cyclase activity in intact cells (Table I). The observation that all effects of -FNA were reversed by co-pretreatment with the opioid antagonist naloxone indicated that the actions of -FNA were specifically due to action at µ-opioid receptors. Additionally, it is highly unlikely that -FNA produced the observed effects by alkylation of other, nonspecific cellular proteins. This is indicated by the finding that no effects of pretreatment with a maximal concentration of -FNA were observed in nontransfected GH₃ cells and that the ED₅₀ of -FNA in all assays was similar to its affinity for µ-opioid receptors (16). Collectively, the data presented strongly indicate that µ-opioid receptors are constitutively active in intact GH₃MOR cells, resulting in stimulation of G-proteins and inhibition of adenylyl cyclase activity.

Importantly, constitutive activation of GPCRs is most readily observed in transfected cells containing high levels of receptors (6). This is predicted by the two-state receptor model in which a high density of receptors would result in a greater degree of constitutive activity because of an increased chance of encounter of a receptor in the active conformation with a G-protein (18). In agreement with this prediction, the only two studies demonstrating constitutive activity of µ-opioid receptors in opioid naive cells employed transfected HEK 293 cells expressing a relatively high level of µ-opioid receptors (~4 pmol/mg) (13, 14). Because the density of µ-opioid receptors in most brain regions is more than 10-fold lower than these levels (31), it is possible that the constitutive activation reported in these studies could simply be because of an unusually high µ-opioid receptor density and thus have little physiological relevance. However, this hypothesis is not supported by findings presented in the present study. Constitutive activation of µ-opioid receptors was demonstrated in GH₃MOR cells that express a relatively moderate physiological density of µ-opioid receptors (i.e. 0.39 pmol/mg). This receptor density is similar to endogenous levels of µ-opioid receptors reported in several brain regions such as the striatum (i.e. 0.30 pmol/mg) (31). Because relatively low levels of constitutively active µ-opioid receptors were also shown to produce physiological relevant effects in intact cells, it is likely that constitutive activation of µ-opioid receptors is not limited to artificially transfected systems but rather may serve important functions in the brain.

Finally, it was demonstrated that constitutively active µ-opioid receptors in GH₃MOR cells activate individual G-proteins differently than the agonist DAMGO. For example, a maximal concentration of DAMGO (1 µM) produced a distinct graded stimulation of individual G-subunits, with the greatest selectivity for G_o2. In contrast, comparison of basal photolabeling of individual G-subunits between GH₃ and GH₃MOR cells revealed that constitutively active µ-opioid receptors couple to G-proteins rather nonselectively, producing similar amounts and percentages of activation. In contrast to these observations, another study provided evidence that constitutively active and morphine-stimulated µ-opioid receptors produced similar selective activation of G_i3 in membranes prepared from transfected HEK cells (13). It is possible that differences between studies might be attributed to the choice of agonist, the cell lines examined, the densities of transfected receptors, or the techniques employed.

Interestingly, it has been shown previously (21) that stimulation of -opioid receptors expressed in GH₃ cells (GH₃DOR) by the full agonist [D-Pen²D-Pen⁵]enkephalin (DPDPE) produces a distinct, yet significantly different pattern of individual G-subunit activation than that produced by DAMGO in GH₃MOR cells presented in the present study. For example, agonist stimulation of -opioid receptors produced the greatest activation of G_o1, followed by G_i2 = G_o2 > G_i3. However, agonist-stimulated µ-opioid receptors preferentially activate G_o2. These observations are significant because they suggest that closely related GPCRs, expressed in the same cell type can selectively couple to a unique pattern of individual G-subunits in response to activation by agonists.

In any case, the present study provides new evidence for the existence of multiple active conformations of µ-opioid receptors. For example, it is possible that constitutively active and agonist-stimulated receptors assume distinct active conformations resulting in different coupling to G-proteins. The suggestion of multiple active receptor conformations is inconsistent with the original two-state receptor model that has been proposed to account for constitutive activity in which receptors exist in an equilibrium between inactive (R) and only a single active (R*) state. Agonists stabilize the R* state, inverse agonists stabilize the R state, and antagonists have equal preferences for both states (6). However, experiments demonstrating that both agonists and inverse agonists are able to afford protection of ₂-adrenergic receptors from proteolysis (32) and thermal denaturation (33) indicate that this simple model of only two conformational states of the receptor may be inaccurate. Instead, these studies suggest that a receptor not bound by ligand is subject to denaturation and represents one conformational state. Additionally, binding of either agonists or inverse agonists produce presumably different conformational states, both of which, however, provide protection. Therefore, a model has been proposed in which at least three conformational states of the receptor exist: R, R*, and R^o. Agonists, stabilize the R* state, inverse agonists stabilize the R^o state, and antagonists stabilize all three states (34). Importantly, the findings presented here provide additional support for this new and intriguing model.

In conclusion, evidence was presented that µ-opioid receptors are constitutively active in intact cells expressing physiological levels of receptors. Furthermore, the spontaneous, agonist-independent activation of these receptors results in the physiological consequence of G-protein stimulation and adenylyl cyclase inhibition. These findings are significant because constitutive activation of µ-opioid receptors has been implicated in the development of tolerance and/or dependence following sustained opioid exposure. Additionally, it was shown that constitutively active and agonist-stimulated µ-opioid receptors activate G-proteins differently, indicative of the potential formation of multiple active receptor conformations. Finally, data were provided that suggest inactivation of constitutively active receptors by the irreversible binding of specific ligands might be utilized to demonstrate constitutive activity of GPCRs when no inverse agonists are available.

	Addendum

While this manuscript was under review, Wang et al. (35) demonstrated that a maximal concentration of several putative inverse µ-opioid agonists inhibited basal [³⁵S]GTPS binding and increased forskolin-stimulated cAMP levels in intact HEK cells transfected with a moderate density of µ-opioid receptors (1.048 pmol/mg).

	FOOTNOTES

^* This work was supported in part by National Institute on Drug Abuse Grant DA10936 (to P. L. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology, Mail Slot 611, College of Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR 72205. Tel.: 501-686-5512; Fax: 501-686-5521; E-mail: pratherpaull@uams.edu.

Published, JBC Papers in Press, August 10, 2001, DOI 10.1074/jbc.M106104200

	ABBREVIATIONS

The abbreviations used are: GPCR, G-protein-coupled receptor; PTX, pertussis toxin; GH₃MOR, GH₃ cells expressing µ-opioid receptors; DAMGO, [D-Ala²,N-MePhe⁴,Gly-ol⁵]enkephalin; -FNA, -funaltrexamine; ANOVA, analysis of variance; AA-[³²P]GTP, azidoanilido-[-³²P]GTP.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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