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CELLULAR AND MOLECULAR

Endogenous Regulator of G Protein Signaling Proteins Reduce µ-Opioid Receptor Desensitization and Down-Regulation and Adenylyl Cyclase Tolerance in C6 Cells

Pharmacology Department, University of Michigan Medical Center, Ann Arbor, Michigan

Received July 21, 2004; accepted September 21, 2004.

	Abstract

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Chronic exposure of cells to µ-opioid agonists leads to tolerance which can be measured by a reduced ability to activate signaling pathways in the cell. Cell signaling through inhibitory G proteins is negatively regulated by RGS (regulator of G protein signaling) proteins. Here we examine the hypothesis that the GTPase accelerating activity of RGS proteins, by altering the lifetime of G and G, plays a role in the development of cellular tolerance to µ-opioids. C6 glioma cells were stably transfected with µ-opioid receptor and pertussis toxin (PTX)-insensitive G_o that was either sensitive or insensitive to endogenous RGS proteins. Cells were treated with PTX to uncouple endogenous G proteins followed by exposure to the µ-opioid agonists [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAMGO) or morphine. Receptor desensitization as measured by agonist-stimulated [³⁵S]GTPS binding and receptor down-regulation as measured by [³H]diprenorphine binding were increased in cells expressing RGS-insensitive G_o. Exposure to high concentrations of morphine or the peptidic µ-opioid agonist DAMGO led to a tolerance to inhibit adenylyl cyclase activity in both cell types with a rapid (30 min) and a slower component. Using a submaximal concentration of DAMGO to induce a reduced level of tolerance, a shift in the concentration-effect curve for DAMGO to inhibit adenylyl cyclase activity was seen in the cells expressing RGS-insensitive G_o, but not in the cells expressing RGS-sensitive G_o, which can be partly explained by an increased supersensitization of the adenylyl cyclase response. The results show that RGS proteins endogenously expressed in C6 cells reduce agonist-induced µ-opioid receptor desensitization, down-regulation, and sensitivity to tolerance to inhibit adenylyl cyclase activity.

The µ-opioid receptor is a member of the G protein-coupled receptor (GPCR) family that couples to heterotrimeric, pertussis toxin (PTX)-sensitive proteins of the G_i/o family. Agonist activation of the receptor leads to exchange of GDP bound to the G subunit in the resting state for GTP and dissociation of the G-GTP and G subunits which function as signaling molecules. One function of G subunits is to recruit GPCR kinases (GRK) to the plasma membrane. These kinases phosphorylate the receptor and allow the subsequent binding of -arrestin which prevents activation of G protein giving desensitization (Kupnick and Benovic, 1998). The binding of -arrestin plays an important role in opioid tolerance in vivo (Bohn et al., 2000, 2002). Active G-GTP and G signaling molecules are returned to their inactive state by the intrinsic GTPase activity of G, causing G-bound GTP to be hydrolyzed to GDP with subsequent reassociation of the G-GDP and G subunits. This process is accelerated by RGS (regulator of G protein signaling) proteins, which bind to the G subunit and speed up hydrolysis of G-bound GTP. RGS proteins function as negative regulators of GPCR signaling (for review see Hollinger and Hepler, 2002). RGS proteins comprise a family of more than 30 members. All have a conserved RGS domain that is responsible for the GTPase accelerating properties (GAP) of the proteins. However, there is some degree of selectivity for G subunits, and the many members of the family have a wide variety of other protein-protein interacting domains which provide for selectivity and allow binding to different intracellular proteins to provide a scaffolding role (Hollinger and Hepler, 2002; Neubig and Siderovski, 2002).

RGS proteins have been shown to decrease specific aspects of µ-opioid signaling in vitro (Chuang et al., 1998; Potenza et al., 1999; Clark et al., 2003; Gold et al., 2003). In vivo, inhibition of RGS9 levels in mice using antisense oligonucleotides leads to an increase in the potency and duration of morphine antinociception with delayed development of antinociceptive tolerance (Garzón et al., 2001). Similarly, RGS9 knockout mice show enhanced responses to morphine with delayed antinociceptive tolerance (Zachariou et al., 2003). Following chronic morphine treatment of rats, RGS4 protein levels are increased in the locus coeruleus (Gold et al., 2003), and RGS9 levels are increased on acute morphine treatment in the mouse striatum, spinal cord, and periaqueductal gray but decreased on chronic treatment (Zachariou et al., 2003). Consequently, RGS proteins and more specifically RGS9 appear to be important in the acute and chronic pharmacology of opioids.

In this study, we examined the hypothesis that by shortening the lifetime of G-GTP and G RGS proteins function to reduce µ-opioid receptor-mediated responses and so regulate opioid tolerance. This hypothesis was tested by determining agonist-induced changes in µ-opioid receptor desensitization and down-regulation and reduction in the ability of µ-opioid agonists to inhibit adenylyl cyclase activity. For the studies, we used rat C6 glioma cells expressing a µ-opioid receptor (C6µ) and G_o that is either sensitive or insensitive to the GAP activity of all RGS proteins (Clark et al., 2003). G_o is rendered RGS-insensitive (RGSi) by a G184S mutation at the interface where it binds RGS protein (Lan et al., 1998; Clark et al., 2003). G_o is also made insensitive to PTX (PTXi) by mutation of the sensitive cysteine in the C-terminal tail (C351G; Milligan, 1988). When expressed in C6µ cells, the effects of the RGS-insensitive mutation on cell signaling can be studied following treatment of the cells with PTX to inactivate endogenous G subunits.

The results show that agonist-induced µ-opioid receptor desensitization and down-regulation is increased in cells expressing RGS-insensitive G_o compared with cells expressing RGS-sensitive G_o. There is also a loss of µ-agonist potency to inhibit adenylyl cyclase activity along with a concomitant increase in supersensitization of adenylyl cyclase. This implies that at the cellular level endogenous RGS protein action works to prevent agonist-induced desensitization and down-regulation of the µ-opioid receptor and so contributes to the maintenance of signaling on chronic agonist exposure.

	Materials and Methods

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Materials. [³⁵S]GTPS and [³H]diprenorphine were from PerkinElmer Life and Analytical Sciences (Boston, MA), and cAMP radioimmunoassay kits were from Diagnostic Products (Los Angeles, CA). Morphine sulfate was obtained through the Narcotic Drug and Opioid Peptide Basic Research Center at the University of Michigan (Ann Arbor, MI). PTX was from List Biological Laboratories Inc. (Campbell, CA). DAMGO, IBMX, forskolin, and all other biochemicals were from Sigma-Aldrich (St. Louis, MO). Tissue culture media, LipofectAMINE Plus reagent, Geneticin, Zeocin, fetal bovine serum, and trypsin were from Invitrogen (Carlsbad, CA). Mouse, PTXi G_o (C351G), and RGS and pertussis toxin-insensitive G_o (RGS/PTXi; G184S/C351G) DNAs were a kind gift from Steve Ikeda (National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD) (Jeong and Ikeda, 2000).

Expression of PTXi or RGS/PTXi G_o in C6µ Cells and Cell Culture. PTXi or RGS/PTXi G_o DNA was inserted into Zeocin resistance vector pcDNA3.1Zeo^- then transfected into C6 glioma cells stably expressing rat µ-opioid receptor (C6µ) (Lee et al., 1999) using LipofectAMINE Plus reagent as described previously (Clark et al., 2003). Cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum under 5% CO₂ in the presence of 0.25 mg/ml Geneticin (to maintain expression of the µ-opioid receptor in a Geneticin-resistant plasmid) and 0.4 mg/ml Zeocin. Clones were typically subcultured at a ratio of 1:20 to 1:30 with partial replacement of the media on day 4 and again on day 6 before sub-culturing or harvesting at day 7.

Receptor Number and Down-Regulation. C6µ cells expressing PTXi G_o or RGS/PTXi G_o grown as above were treated with PTX (100 ng/ml) 2 days before harvesting followed by the addition of 1 µM DAMGO 18 h before harvesting. After collection, cells were homogenized and membranes prepared as described in Clark et al. (2003) with a 15-min incubation at 37°C between the two centrifugation steps. Membranes (10–20 µg) were incubated in 50 mM Tris-HCl, pH 7.4, with a supermaximal concentration (4 nM) of the opioid antagonist [³H]diprenorphine with or without 50 µM naloxone to define nonspecific binding in a total volume of 0.2 ml for 60 min at 25°C. Samples were filtered through glass fiber filters (no. 32; Schleicher & Schuell, Keene, NH), mounted in a Brandel cell harvester (Brandel Inc., Gaithersburg, MD), and rinsed three times with ice-cold 50 mM Tris-HCl, pH 7.4. Radioactivity retained on each filter was counted by liquid scintillation counting in a 4-ml EcoLume scintillation mixture (ICN Pharmaceuticals Biochemicals Division, Aurora, OH). Data from three experiments, each carried out in triplicate, were expressed as mean ± standard error of the mean and differences analyzed by student's t test.

Stimulation of [³⁵S]GTPS Binding. Membranes (14–20 µg) were incubated for 60 min in a shaking water bath at 25°C in 20 mM Tris-HCl, pH 7.4 containing 5 mM MgCl₂, 100 mM NaCl, 0.1 mM dithiothreitol, 30 µM GDP, 0.1 nM [³⁵S]GTPS, and 0.01 to 10 µM DAMGO or distilled water as described by Traynor and Nahorski (1995). Samples were filtered through no. 32 glass fiber filters (Schleicher & Schuell), mounted in a Brandel cell harvester, and rinsed three times with an ice-cold solution containing 50 mM Tris-HCl, pH 7.4, 5 mM MgCl₂, and 100 mM NaCl. Each filter was removed and counted by liquid scintillation counting as above.

Inhibition of cAMP Accumulation. Cells were plated to 80 to 90% confluency in 24-well plates 2 days before the assay and treated overnight with 100 ng/ml PTX. Varying concentrations of DAMGO or morphine were added without removing the PTX for varying times from 0.5 to 24 h before the start of the assay. To assay for opioid inhibition of cAMP accumulation, media were replaced with serum-free media containing 30 µM forskolin, 1 mM IBMX, and varying concentrations of DAMGO, morphine, or vehicle (distilled water) as control for 5 min (for single, high concentrations of agonist) or 15 min (for concentration-response curves) at 37°C. The reaction was stopped by replacing the media with ice-cold 3% perchloric acid. Accumulated cAMP was measured by radioimmunoassay as previously described (Clark et al., 2003). Inhibition of cAMP formation was determined as a percentage of forskolin-stimulated cAMP accumulation in the absence of opioid agonist.

Data Analysis. For [³⁵S]GTPS and cAMP accumulation assays, concentration-response data were fitted to sigmoidal dose response curves using GraphPad Prism (GraphPad Software Inc., San Diego, CA) to determine EC₅₀ and maximal levels. Data from at least three separate experiments, each carried out in duplicate, were presented as mean ± standard error of the mean. Significance differences were determined by Student's t test.

	Results

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C6µ cells expressing similar levels of PTX-insensitive G_o without (PTXi) or with (RGS/PTXi) the mutation for RGS insensitivity (Clark et al., 2003) were treated overnight with PTX to inactivate endogenously expressed G proteins in all experiments.

µ-Opioid Receptor Desensitization. The potency (EC₅₀) and maximal effect (E_max) of DAMGO, a full µ-opioid agonist, to cause stimulation of [³⁵S]GTPS binding were similar in membranes from cells expressing G_o with only the PTXi mutation (EC₅₀ = 330 ± 40 nM, E_max = 333 ± 45% of basal) or with the RGS/PTXi mutation (EC₅₀ = 440 ± 40 nM, E_max = 368 ± 44% of basal). The potency of DAMGO is lower than in wild-type C6µ cells but is consistent with our previous findings (Clark et al., 2003). Pretreatment of cells with 1 µM DAMGO for varying times before membrane preparation reduced the maximal DAMGO stimulation of [³⁵S]GTPS binding in a time-dependent manner but without any consistent change in the EC₅₀ for DAMGO (Table 1). The reduction in maximal [³⁵S]GTPS stimulation was evident within 30 min and was significantly greater in cells expressing G_o that was insensitive to the action of RGS proteins (RGS/PTXi; Table 1).

View this table: [in this window] [in a new window]   TABLE 1 Stimulation of [35S]GTPS binding by DAMGO in membranes from C6µ cells expressing PTX-insensitive and RGS-sensitive (PTXi) or PTX-insensitive and RGS-insensitive (RGS/PTXi) Go following pretreatment with 1 µM DAMGO PTX-treated C6µ cells expressing Go that was PTXi or RGS/PTXi were treated for varying times with 1 µM DAMGO. Cells were washed, membranes prepared, and DAMGO stimulation of [35S]GTPS binding was measured as described under Materials and Methods. Shown are the combined data from four to five assays each measured in duplicate. Emax is given as the percentage of stimulation compared with cells that did not receive DAMGO pretreatment (time 0).

µ-Opioid Receptor Down-Regulation. µ-Opioid receptor number, determined with a single saturating concentration of the nonselective antagonist [³H]diprenorphine, was the same (6.4 pmol/mg protein; Table 2) in membranes from PTXi G_o-expressing cells and cells expressing RGS/PTXi G_o. The µ-opioid receptor expression level did vary when cells were grown in different batches of fetal bovine serum, but the effects were the same across clones (data not shown). After a 60-min treatment of the cells with 1 µM DAMGO, there was a similar small decrease in µ-opioid receptor number in membranes prepared from cells expressing PTXi G_o or RGS/PTXi G_o (Table 2). After overnight treatment with 1 µM DAMGO, receptor number was further reduced; however, the reduction in receptor level was significantly greater in cells expressing RGS-insensitive G_o.

View this table: [in this window] [in a new window]   TABLE 2 µ -Opioid receptor numbers (Bmax) in membranes from C6µ cells expressing PTX-insensitive and RGS-sensitive (PTXi) or PTX-insensitive and RGS-insensitive (RGS/PTXi) Go following pretreatment with 1 µM DAMGO PTX-treated C6µ cells expressing Go that was PTXi or RGS/PTXi were treated for varying times with 1 µM DAMGO. Cells were washed, membranes prepared, and µ- opioid receptor numbers determined with 4 nM [3H]diprenorphine as described under Materials and Methods. Shown are the combined data from three assays each measured in triplicate.

Tolerance of the Adenylyl Cyclase Response. The effect of the observed agonist-induced µ-opioid receptor desensitization and down-regulation on tolerance, measured as a reduction in µ-opioid inhibition of adenylyl cyclase, was examined. Cells were treated with varying concentrations of DAMGO or morphine, and the ability of a challenge concentration (10 µM) of DAMGO or morphine to inhibit forskolin-stimulated adenylyl cyclase was determined. This high challenge concentration was used to ensure that maximal inhibition of adenylyl cyclase was obtained, since cells expressing G_o that is only PTXi or both RGS/PTXi differ by more than 10-fold in their sensitivity to acute DAMGO (Clark et al., 2003). The challenge with DAMGO or morphine was added without washout of the pretreatment drug to avoid the overshoot in forskolin-stimulated cAMP levels due to adenylyl cyclase supersensitization caused by treatment agonist that would be triggered by agonist removal (Watts, 2002; Clark et al., 2004). Following pretreatment with high (1 µM) concentrations of DAMGO or morphine for 18 h, the ability of 10 µM DAMGO or 10 µM morphine to cause inhibition of forskolin-stimulated adenylyl cyclase was completely lost in cells expressing RGS/PTXi or PTXi G_o (Fig. 1) indicating tolerance. The pretreatment concentration of DAMGO or morphine necessary to cause a 50% reduction in the adenylyl cyclase inhibition by an acute challenge with 10 µM DAMGO or 10 µM morphine, respectively, was not significantly different between cells expressing G_o that was RGS-sensitive or RGS-insensitive (Fig. 1).

View larger version (31K): [in this window] [in a new window]   Fig. 1. Reduction in acute 10 µM DAMGO (A) or 10 µM morphine (B) inhibition of forskolin-stimulated cAMP accumulation following pretreatment with varying concentrations of DAMGO or morphine. PTX-treated C6µ cells expressing PTXi Go or RGS/PTXi Go were treated for 18 h with varying concentrations of DAMGO (A) or morphine (B). The media were replaced with serum-free media containing 1 mM IBMX, 30 µM forskolin, and 10 µM DAMGO (A) or 10 µM morphine (B) to start the assay, which was allowed to proceed for 5 min at 37°C. Data are expressed as a fraction of the cAMP accumulated in vehicle (distilled water)-treated cells. Shown are the combined data from three assays each performed in duplicate. The pretreatment concentration of DAMGO (EC50: PTXi = 274 ± 17 nM; RGS/PTXi = 287 ± 58 nM, p = 0.84) or morphine (EC50: PTXi = 192 ± 41 nM; RGS/PTXi = 213 ± 55 nM, p = 0.78) necessary to cause a 50% reduction in the adenylyl cyclase inhibition by an acute challenge with 10 µM DAMGO or 10 µM morphine, respectively, was not significantly different (Student's t test) between PTXi or RGS/PTXi Go-expressing cells.

Since RGS proteins accelerate the GTPase activity of G,it can be hypothesized that RGS proteins should affect the rate of tolerance development rather than the degree of tolerance achieved. After 30 min of treatment with 1 µM DAMGO, there was a reduction in the ability of subsequently added 10 µM DAMGO to inhibit forskolin-stimulated adenylyl cyclase (Fig. 2). This reduction was similar (p = 0.13) in cells expressing PTXi G_o (reduced by 0.26 ± 0.09 pmol/µg protein) and RGS/PTXi G_o (reduced by 0.22 ± 0.07 pmol/µg protein). After 24 h of pretreatment with 1 µM DAMGO, there was a greater loss of the effectiveness of acute 10 µM DAMGO but no significant difference (p = 0.22) between cells expressing PTXi G_o (reduced by 0.52 ± 0.13 pmol/µg protein) or RGS/PTXi G_o (reduced by 0.89 ± 0.11 pmol/µg protein).

View larger version (18K): [in this window] [in a new window]   Fig. 2. Acute inhibition of forskolin-stimulated cAMP accumulation following varying lengths of pretreatment with 1 µM DAMGO. PTX-treated C6µ cells expressing PTXi Go or RGS/PTXi Go were treated with DAMGO for the times indicated. The media were replaced with serum-free media containing 1 mM IBMX, 30 µM forskolin, and 10 µM DAMGO to start the assay. cAMP accumulation was determined after 5 min at 37°C. Data are expressed as a fraction of the cAMP accumulated in vehicle (distilled water)-treated cells. Shown are the combined data from three assays each measured in duplicate. DAMGO-inhibited cAMP levels after preincubation for 0.5 to 24 h were significantly different from the response in cells that were not pretreated (time 0, p 0.05).

It is possible that differential responses between the PTXi and RGS/PTXi G_o-expressing cells are present at lower pretreatment concentrations but masked by the supramaximal concentrations (10 µM) of the agonists employed to examine tolerance. Cells were pretreated with 100 nM DAMGO, followed by two washes. This pretreatment caused no change in the concentration of subsequently added acute DAMGO required to reach half-maximal inhibition of cAMP accumulation in the cells expressing PTXi G_o (EC₅₀ = 321 ± 79 nM untreated, EC₅₀ = 353 ± 42 nM treated). In contrast, the EC₅₀ was increased 7.6-fold (EC₅₀ = 27.5 ± 11.1 nM untreated, EC₅₀ = 208 ± 32 nM treated, p 0.05) in the RGS/PTXi G_o-expressing cells (Fig. 3A). When plotted as concentrations (picomole) cAMP, it can be seen that there was a small increase in cAMP levels following washout of pretreatment agonist in the PTXi G_o-expressing cells (16.7 ± 4.3%), but this response was four times greater in the RGS/PTXi G_o-expressing cells (76.5 ± 2.8%). This represents the supersensitization of adenylyl cyclase which occurs as the result of chronic opioid drug exposure and is revealed by washout of the agonist (Watts, 2002; Clark et al., 2004). Despite this supersensitization, DAMGO inhibited cAMP accumulation to the same level in the treated or untreated PTXi G_o-expressing cells (58.2 ± 10.8% and 47.9 ± 14.7%, respectively) or RGS/PTXi G_o-expressing cells (84.2 ± 2.2% and 84.0 ± 2.4%, respectively).

View larger version (21K): [in this window] [in a new window]   Fig. 3. DAMGO inhibition of cAMP accumulation following overnight pretreatment with 100 nM DAMGO. PTX-treated C6µ cells expressing PTXi Go or RGS/PTXi Go treated with PTX overnight followed by 18 h of treatment with 100 nM DAMGO. Cells were rinsed twice with serum-free media at 37°C, and the assay was started immediately by adding serum-free media containing 1 mM IBMX, 30 µM forskolin, and appropriate concentrations of DAMGO or vehicle. cAMP accumulation was measured after 15 min. Data are presented as percentage of cAMP accumulation in the presence of forskolin only (A) or as picomoles of cAMP accumulated per micrograms of protein (B). Shown are the combined data from three assays each performed in duplicate.

Increased activity of adenylyl cyclase due to increased supersensitization could contribute to the reduction in potency of DAMGO in the RGS/PTXi G_o-expressing cells. To attempt to mimic this, DAMGO inhibition of cAMP accumulation was measured in the presence of increasing concentrations of forskolin (3, 10, or 30 µM) to provide increased levels of adenylyl cyclase stimulation. The EC₅₀ values for DAMGO inhibition increased with the degree of forskolin-stimulated adenylyl cyclase in cells expressing PTXi G_o, but this was not significant (p = 0.11). In contrast in cells expressing RGS/PTXi G_o, the EC₅₀ values for DAMGO inhibition were 2-fold higher (p 0.01) at the highest concentration of forskolin (Table 3). This demonstrates that at least part of the loss of effect of DAMGO in the RGS/PTXi G_o-expressing cells is due to supersensitization of adenylyl cyclase.

View this table: [in this window] [in a new window]   TABLE 3 Effect of forskolin concentration on cAMP accumulation and the potency of DAMGO to inhibit cAMP accumulation in C6µ cells expressing PTX-insensitive and RGS-sensitive (PTXi) or PTX-insensitive and RGS-insensitive (RGS/PTXi) Go C6µ cells expressing PTXi or RGS/PTXi Go grown in 24-well plates were treated with PTX overnight. Media were replaced with serum-free media containing 1 mM IBMX, 3, 10, or 30 µM forskolin, and 0.001 to 10 µM DAMGO. After incubation for 15 min at 37° C, cAMP accumulation was measured as described under Materials and Methods. Shown are the combined data from three assays each measured in duplicate.

	Discussion

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The aim of this study was to define a role for endogenous RGS proteins in cellular tolerance to µ-opioid agonists identified by a reduction in ability to inhibit forskolin-stimulated adenylyl cyclase. Agonist-induced receptor desensitization and receptor down-regulation in C6µ cells expressing RGS-sensitive or insensitive G_o resulted in a reduction in the ability of morphine and DAMGO to inhibit adenylyl cyclase activity. There was a greater degree of desensitization and down-regulation and a higher sensitivity to tolerance in RGS-insensitive G_o-expressing cells compared with cells expressing RGS-sensitive G_o. This indicates that RGS proteins in this cell model serve to protect against µ-opioid mediated tolerance. In this study, G_o was the only functional G_i/o protein confirming the ability of this G protein to mediate chronic effects of µ-opioid agonist exposure in the absence of other functional adenylyl cyclase inhibitory G proteins.

The ability of DAMGO to stimulate [³⁵S]GTPS was the same in cells expressing RGS-sensitive or -insensitive G_o, but the potency to inhibit adenylyl cyclase activity was 10-fold lower in the RGS-sensitive G_o-expressing cells confirming a role for RGS proteins in inhibiting signaling downstream of G protein (Clark et al., 2003). After overnight treatment with increasing concentrations of DAMGO or morphine, there was a concentration-dependent loss in the ability of the µ-agonists to inhibit adenylyl cyclase activity. Despite the 10-fold higher potency of µ-agonists to acutely inhibit adenylyl cyclase activity in the RGS-insensitive G_o-expressing cells, no difference in the potency of morphine or DAMGO to induce tolerance was observed between RGS-insensitive and -sensitive G_o-expressing cells using a high challenge concentration of agonist to detect tolerance. However, in cells pretreated overnight with 100 nM DAMGO to induce a mild adenylyl cyclase tolerance, there was a 10-fold shift in DAMGO potency to inhibit adenylyl cyclase activity in the RGS-insensitive G_o-expressing cells but no change in the RGS-sensitive G_o-expressing cells_. This suggests that cells expressing RGS-insensitive G_o are more susceptible to tolerance development and therefore endogenous RGS proteins in the C6µ cells are acting to inhibit tolerance, although tolerance does still develop.

The development of adenylyl cyclase tolerance to µ-opioid agonists had a fast component apparent within 30 min and a slow component which was not apparent until 6 h of agonist exposure in both the RGS-sensitive and -insensitive G_o-expressing cells. This fast component is likely due to desensitization of receptor activation of G proteins which was evident after 30 min of treatment with 1 µM DAMGO. The degree of desensitization was greater in the RGS-insensitive G_o-expressing cells (Fig. 2). C6 cells express GRK2, GRK5, and arrestin (Gray et al., 2001) so this is probably the result of increased recruitment of a GRK leading to increased receptor phosphorylation and -arrestin binding. This could occur due to the lengthened lifetime of free G as a result of the inability of endogenous RGS proteins to accelerate the hydrolysis of G-bound GTP in the presence of the RGS-insensitive G_o mutant. Additionally, activation of the mitogen-activated protein kinase cascade, which is negatively regulated by RGS protein action in C6µ cells (Clark et al., 2003), has also been implicated in the rapid phosphorylation and desensitization of the µ-opioid receptor (Polakiewicz et al., 1998; Schmidt et al., 2000).

Receptor down-regulation was not apparent until more than 1 h after pretreatment with µ-opioid agonist and was also seen to a greater degree in cells expressing RGS-insensitive G_o. Down-regulation could be involved in the slower developing tolerance; however, there is little literature support for this idea. Gomes et al. (2002) showed that PTX-treatment can eliminate tolerance without affecting receptor down-regulation. There are also ligand-specific differences in the ability of µ-opioids to cause receptor down-regulation that do not correlate with differences in tolerance (Patel et al., 2002). In addition, µ-opioid antinociceptive tolerance in mice is reduced by treatment with G_i2 antisense oligodeoxynucleotides, although µ-opioid receptor down-regulation is not affected (Yoburn et al., 2003). These and other studies (Chakrabarti et al., 1997; Yabaluri and Medzihradsky, 1997; Pak et al., 1999) provide evidence for separate mechanisms of receptor down-regulation, namely a G protein-dependent and -independent mechanism (Pak et al., 1999) which may show some redundancy. The increase in receptor down-regulation in the RGS-insensitive G_o-expressing C6µ cells implies at least the presence of a G protein-dependent component.

Although increased levels of µ-opioid receptor desensitization and down-regulation in C6µ cells contribute to adenylyl cyclase tolerance, there is also an increased adenylyl cyclase supersensitization in cells expressing RGS-insensitive G_o (Clark et al., 2004; present results) which could be a factor in adenylyl cyclase tolerance. Use of increasing concentrations of forskolin to raise the level of adenylyl cyclase activation in naive cells expressing RGS-insensitive G_o resulted in a reduced potency of DAMGO suggesting that the supersensitized state does contribute to the observed tolerance. Moreover, supersensitization of the adenylyl cyclase response is apparent by 30 min in G_o-expressing C6µ cells (Clark et al., 2004) and therefore could, along with desensitization, contribute to the fast development of tolerance. Finally, it is also possible that in the RGS-sensitive G_o-expressing cells there is RGS-mediated inhibition of pathways that contribute to the slower developing tolerance, in particular, those leading to changes in gene transcription (Chao and Nestler, 2004) that may be promoted in the C6µ cells expressing RGS-insensitive G_o. For example, activation of the mitogen-activated protein kinase pathway by µ-opioids is negatively regulated by RGS proteins (Clark et al., 2003) and could be an important pathway for transducing signals to the nucleus resulting in changes in gene expression that contribute to tolerance (Ma et al., 2001; Eitan et al., 2003).

The suggestion that RGS proteins protect against tolerance at adenylyl cyclase appears to disagree with data on antinociception in the mouse and in vitro data in several systems. Wild-type mice show decreased morphine antinociception with more rapid antinociceptive tolerance than their RGS9 littermate knockouts (Zachariou et al., 2003). These authors suggest that tolerance is caused by the morphine-mediated increase in RGS9 levels leading to an increased negative regulation of G protein signaling. Similarly, treatment of mice with RGS9 antisense oligodeoxynucleotides (Garzón et al., 2001) increases the potency and duration of opioid antinociception, eliminates acute tolerance, and delays longer-term tolerance. Knockdown of other members of the R7 family of RGS proteins which contain dishevelled, egl-10 and pleckstrin (DEP) and G protein gamma subunit-like (CGI) domains (RGS6, RGS7, and RGS11; Hollinger and Hepler, 2002) also prolongs the duration of µ-opioid antinociception and reduces acute antinociceptive tolerance (Garzón et al., 2003). However, C6 glioma cells express RGS2, 3, 8, 10, 12, and 14 but not members of the R7 family (Snow et al., 2002). Thus, it is possible that the proposed role of RGS proteins in promoting tolerance is not simply due to the GAP activity of proteins but rather requires domains specific to members of the R7 family (Sánchez-Blázquez et al., 2003). Other studies suggest that RGS4 can modulate µ-opioid receptor signaling (Gold et al., 2003). mRNA for RGS4 is slightly up-regulated by acute morphine (Bishop et al., 2002), and RGS4 immunoreactivity is increased by chronic morphine (Gold et al., 2003). However, again C6 cells do not express RGS4 (Ni et al., 1999).

In vitro, RGS proteins serve to limit µ-opioid signaling (Potenza et al., 1999; Clark et al., 2003) and to potentiate acute desensitization of the effector response thus promoting tolerance probably by limiting the supply of G-GTP and G (Chuang et al., 1998). Of course by acting as negative regulators of G protein signaling, RGS proteins will also reduce activation of those processes that contribute to tolerance. Thus, the overall effect of endogenous RGS proteins in vivo will depend on a balance of effects. A reduction in signaling to effector molecules that promote agonist responses will support tolerance; a reduction in receptor desensitization, receptor down-regulation, and adenylyl cyclase supersensitization will inhibit tolerance. This in turn will depend on the types of RGS proteins expressed and their selectivity for particular receptor G protein and G protein-effector molecule combinations. Certainly it would seem possible that RGS action will switch off agonist signaling to effectors that are very sensitive to the action of RGS proteins, such as G protein gated inwardly rectifier K⁺ channels (Chuang et al., 1998) and adenylyl cyclase (Clark et al., 2003) but protect signaling to pathways that are not so responsive, for example, signaling to intracellular calcium (Clark et al., 2003).

In summary, endogenously expressed RGS proteins in C6µ glioma cells decrease the chronic opioid effects of desensitization and receptor down-regulation. This results in a reduced sensitivity to tolerance as measured by opioid-agonist induced inhibition of adenylyl cyclase. The decreased sensitivity to tolerance afforded by endogenous RGS proteins could in part be due to an inhibition of adenylyl cyclase supsersensitization. The findings are opposite to experiments in vivo where knockdown or knockout of members of the R7 family of RGS proteins inhibits tolerance to the antinociceptive effects of morphine (Garzón et al., 2001; Sánchez-Blázquez et al., 2003; Zachariou et al., 2003). Taken together, these findings show that endogenous RGS proteins act as a modulators rather than play a critical role in controlling the various signaling processes that contribute to tolerance such that the degree of tolerance observed will depend on the system, the output measure, and the complement of RGS proteins expressed.

	Acknowledgements

 
We thank Dr. Huda Akil for the µ-opioid receptor and Dr. Stephen Ikeda for the RGS- and PTX-insensitive G_o mutants.

	Footnotes

 
This study was supported by Grant DA04087 (to J.R.T.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.104.074641.

ABBREVIATIONS: GPCR, G protein-coupled receptor; PTX, pertussis toxin; GRK, G protein-coupled receptor kinase; RGS, regulator of G protein signaling; GAP, GTPase-accelerating protein; RGSi, RGS-insensitive; PTXi, PTX-insensitive; DAMGO, [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin; IBMX, 3-isobutyl-1-methylxanthine.

Address correspondence to: Dr. John R. Traynor, Department of Pharmacology, University of Michigan Medical School, 1301 Medical Science Research Building III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0632. E-mail: jtraynor{at}umich.edu

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