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CELLULAR AND MOLECULAR

Endogenous Regulator of G Protein Signaling Proteins Suppress Go-Dependent, µ-Opioid Agonist-Mediated Adenylyl Cyclase Supersensitization

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

Received December 22, 2003; accepted March 9, 2004.

	Abstract

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Chronic µ-opioid agonist treatment leads to dependence with withdrawal on removal of agonist. At the cellular level withdrawal is accompanied by a supersensitization of adenylyl cyclase, an effect that requires inhibitory G proteins. Inhibitory G protein action is modulated by regulator of G protein signaling (RGS) proteins that act as GTPase activating proteins and reduce the lifetime of G-GTP. In this article, we use C6 glioma cells expressing the rat µ-opioid receptor (C6µ) to examine the hypothesis that Go alone can mediate µ-opioid agonist induced adenylyl cyclase supersensitivity and that endogenous RGS proteins serve to limit the extent of this supersensitization. C6µ cells were stably transfected with pertussis toxin (PTX)-insensitive Go 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 or morphine. Supersensitization was observed in cells expressing wild-type G, but this was lost on PTX treatment. In cells expressing PTX-insensitive Go supersensitization was recovered, confirming that Go alone can support supersensitization. In cells expressing the RGS-insensitive mutant Go, there was a greater degree of supersensitization and the concentration of µ-agonist needed to achieve half-maximal supersensitization was reduced by 10-fold. The amount of supersensitization seen did not directly relate to the degree of acute inhibition of adenylyl cyclase. These results demonstrate a role for Go in adenylyl cyclase supersensitization after µ-agonist exposure and show that this action is modulated by endogenous RGS proteins.

Adenylyl cyclase supersensitivity is observed at several receptor systems that, like the µ-opioid receptor, are G protein-coupled receptors that activate inhibitory pertussis toxin (PTX)-sensitive Gi/o proteins and modulate intracellular pathways via GTP bound G subunit or the G subunit dimers. For the µ-receptor, these pathways include opening of G protein-gated inwardly rectifying K⁺ channels (GIRK), stimulation of mitogen-activated protein kinase (MAPK) phosphorylation, inhibition of adenylyl cyclase, closing of voltage-sensitive Ca²⁺ channels, and release of Ca²⁺ from intracellular stores. G proteins are, in turn, modulated by regulator of G protein signaling (RGS) proteins, which act as GTPase activating proteins (GAPs) to increase the rate of GTP hydrolysis by the G subunit and decrease the lifetime of the active G-GTP and free G subunits, thus limiting signaling to downstream effectors. For example, RGS proteins have been shown to decrease specific aspects of µ-opioid signaling in vitro (Potenza et al., 1999; Clark et al., 2003; Gold et al., 2003); RGS9 antisense oligonucleotide treatment of mice leads to an increase in the antinociceptive potency of morphine and a decrease in the level of acute, but not long-term tolerance (Garzón et al., 2001); and RGS9 knockout mice show enhanced responses to morphine (Zachariou et al., 2003). In HEK cells expressing GIRK channels and µ-opioid receptors, RGS4 expression accelerates both µ-agonist activation and deactivation of GIRK (Chuang et al., 1998).

Adenylyl cyclase supersensitization is thought to involve an enhanced interaction between Gs and specific isoforms of adenylyl cyclase (for review, see Watts, 2002), although how this arises after occupation of receptors that stimulate inhibitory G proteins is unknown. Functional inhibitory G proteins are a requirement for supersensitization to develop (Sharma et al., 1975), but the relative importance of different G subunits in the development of supersensitization is not clear. For example Go, but not Gi₁, Gi₂, or Gi₃, is essential for supersensitization after agonist activation of D_2L receptors expressed in NS20Y cells (Watts et al., 1998). Similarly, after chronic µ-agonist opioid treatment of HEK293 cells, supersensitization cannot be supported by Gz (Tso and Wong, 2000a), Gi₂ (Tso and Wong, 2000b), Gi₁ or Gi₃ (Tso and Wong, 2001) when expressed alone. The authors suggest that simultaneous activation of multiple G proteins may be necessary (Tso and Wong, 2001), although a role for Go was not investigated. In COS-7 cells that express Gz, Gi₂, and Gi₃, but not Go, Ammer and Christ (2002) have shown that supersensitization is observed but suggested that the ability of different G subunits to induce supersensitization is dependent on the type of adenylyl cyclase expressed. Additional factors controlling adenylyl cyclase activity, including levels of adenylyl cyclase-stimulating agents such as Ca²⁺/calmodulin, or levels or phosphorylation state of certain adenylyl cyclase isoforms, as well as changes in levels and phosphorylation state of cAMP response element-binding protein have all been implicated in the development of adenylyl cyclase supersensitization (for review, see Law et al., 2000).

The PTX-sensitive G protein Go can be rendered insensitive to PTX by a C351G mutation (Milligan, 1988; Jeong and Ikeda, 2000) and insensitive to the GAP activity of RGS proteins by a G184S mutation (Lan et al., 1998). We have expressed Go containing the PTX-insensitive mutation (Go-PTXi) or the PTX-insensitive and the RGS-insensitive mutation (Go-RGS/PTXi) in C6 glioma cells stably expressing the µ-opioid receptor (C6µ) and have shown that endogenously expressed RGS proteins decrease µ-opioid signaling to adenylyl cyclase and extracellular signal-regulated kinase (ERK) (Clark et al., 2003). Here, we use these same cells, in the presence of PTX treatment to ADP-ribosylate endogenous Gi/o proteins, to test the hypothesis that Go alone can support µ-agonist induced adenylyl cyclase supersensitization, and that endogenous RGS proteins modulate the development and/or expression of this event by decreasing the lifetime of active Go-GTP.

	Materials and Methods

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Materials. [³H]Diprenorphine was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). cAMP radioimmunoassay kits were purchased from Diagnostic Products (Los Angeles, CA). Tissue culture media, LipofectAMINE Plus Reagent, Geneticin, Zeocin, fetal bovine serum, and trypsin were purchased from Invitrogen (Carlsbad, CA). PTX was purchased from List Biological Laboratories Inc. (Campbell, CA). Morphine sulfate was obtained through the Narcotic Drug and Opioid Peptide Basic Research Center at the University of Michigan (Ann Arbor, MI). Trizma base, IBMX, forskolin, DAMGO, and other biochemicals were purchased from Sigma-Aldrich (St. Louis, MO). PTX-insensitive (C351G) GoA DNA and RGS and PTX-insensitive (G184S and C351G) GoA DNA were obtained from Steve Ikeda (Guthrie Research Institute, Sayre, PA).

Expression of PTXi or RGS/PTXi Go in C6µ Cells and Cell Culture. Mouse Go-PTXi or Go-RGS/PTXi DNA was inserted into Zeocin resistance vector pcDNA3.1zeo^- and 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). Clones were chosen based on expression levels of Go determined by Western blot (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 subculturing or harvesting at day 7.

Measurement of Adenylyl Cyclase Activity. This was determined as the accumulation of cAMP. Cells were plated to 80 to 90% confluence 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, the media were replaced with serum-free media containing 30 µM forskolin, 1 mM IBMX, and varying concentrations of DAMGO or morphine for 15 min at 37°C. To assay for adenylyl cyclase supersensitization, the media were replaced with serum-free media containing 30 µM forskolin, 1 mM IBMX, and 10 µM naloxone for 5 min at 37°C. The reactions were stopped by replacing the media with ice-cold 3% perchloric acid. After at least 30 min at 4°C, 0.4 ml was removed from each sample, neutralized with 0.08 ml of 2.5 M KHCO₃, vortexed, and centrifuged at 15,000g for 1 min. Accumulated cAMP was measured by radioimmunoassay in a 10-µl aliquot of the supernatant from each sample. Inhibition of cAMP formation by agonists was determined as a percentage of forskolin-stimulated cAMP accumulation in the absence of opioid agonist. Supersensitization was determined as the percentage of increase in forskolin-stimulated cAMP accumulation compared with cells that had not been exposed to chronic agonist. Dose-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, are presented as mean ± standard error of the mean. Data were compared using two-way analysis of variance or Student's t test as appropriate and differences considered significant if p 0.05.

Receptor Number. Membranes from C6µ cells expressing either Go-PTXi or Go-RGS/PTXi were prepared as described in Clark et al. (2003). The affinity of the opioid antagonist [³H]diprenorphine for µ-receptors in C6µ cells is 0.13 nM (Lee et al., 1999). Consequently, B_max was defined by the level of binding of a single, supermaximal concentration (30 times the K_D) of [³H]diprenorphine to ensure binding to all µ-receptors, as follows. Membranes (10–20 µg) were incubated in 50 mM Tris-HCl, pH 7.4, with a supermaximal concentration (4 nM) of [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 mounted in a cell harvester (Brandel Inc., Gaithersburg, MD) and rinsed three times with ice-cold 50 mM Tris-HCl, pH 7.4. Radioactivity retained was counted by liquid scintillation counting. Data from three experiments, each carried out in triplicate, are expressed as mean ± standard error of the mean.

	Results

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Acute Inhibition of cAMP. Stably transfected C6µ cells that expressed the same level of Go as either Go-RGS/PTXi or Go-PTXi determined by Western blot (Clark et al., 2003) were used throughout this study, unless stated, to allow direct comparison of the role of the RGS-insensitive mutation. The chosen clones also expressed the same number of µ-opioid receptors (6.4 ± 0.1 and 6.4 ± 0.4 pmol/mg protein, respectively). This is considerably higher than levels of µ-opioid receptors in cells endogenously expressing these receptors. Nonetheless, the C6µ cells respond in a conventional way to µ-agonists; for example, efficacy differences between agonists are maintained (Emmerson et al., 1996) and the µ-receptors only couple to Gi/o proteins (Emmerson et al., 1996). In agreement with this, overnight treatment with 100 ng/ml PTX eliminated opioid-agonist-mediated inhibition of cAMP accumulation in C6µ cells not expressing PTX-resistant Go (Clark et al., 2003). In most experiments described, cells were treated overnight with 100 ng/ml PTX to eliminate endogenous Gi/o activity.

Although a difference in the degree of forskolin (30 µM)-stimulated cAMP accumulation in C6µ cells was observed between cells expressing the Go that was either PTXi or RGS/PTXi [F (1,8) = 10.6; p = 0.01], the opposite effect was seen in a second set of clones [F (1,8) = 11.3; p = 0.01], showing no consistent effect of the RGS-insensitive mutation (Table 1). Inclusion of 10 µM naloxone during the assay had no effect on forskolin-stimulated cAMP accumulation in naive cells [F(1,8) = 0.06; p = 0.81] and was used as control in all subsequent studies. There was a significant difference in the ability of maximal concentrations of DAMGO (10 µM) and morphine (10 µM) to inhibit forskolin-stimulated cAMP accumulation dependent upon cell type [Go-RGS/PTXi cells or Go-PTXi cells; F(1.8) = 92.6; p < 0.0001] when both clones expressed a similar level of Go (Table 1, clones 1) and a significant difference between morphine and DAMGO [F(1,8 = 13.02; p = 0.0069], independent of cell type. In a second set of clones, the level of Go-PTXi expressed was higher than the level of RGS/PTXi-Go, but the level of adenylyl cyclase inhibition was similar. These findings confirm the role of the RGS-insensitive mutation in promoting signaling (Clark et al., 2003).

View this table: [in this window] [in a new window]   TABLE 1 cAMP accumulation in GoPTXi- and GoRGS/PTXi-expressing C6µ cells during 5-min incubation with 30 µM forskolin in the absence or presence of 10 µM naloxone, and the degree of inhibition by 10 µM DAMGO or 10 µM morphine

Adenylyl Cyclase Supersensitization. This was measured as an increase in 30 µM forskolin-stimulated cAMP accumulation upon addition of 10 µM naloxone after opioid agonist treatment. In C6µ wild-type cells, there was a 142 ± 40% increase in cAMP accumulation after 18-h exposure to 1 µM DAMGO, followed by naloxone challenge. This increase in cAMP accumulation was completely eliminated when the C6µ cells were treated overnight with PTX (data not shown). To examine a role for Go alone in adenylyl cyclase supersensitization, cells were treated with PTX to ADP-ribosylate endogenous G proteins leaving only the transfected PTXi Go proteins functional. Under these conditions, PTX-treated C6µ cells expressing Go-PTXi showed supersensitization after 18-h treatment with DAMGO (31 ± 8% overshoot) or morphine (28 ± 9% overshoot). The maximal degree of supersensitization was significantly increased (p < 0.01) in C6µ cells expressing Go-RGS/PTXi (81 ± 7% DAMGO, 76 ± 4% morphine; Fig. 1). The concentration of DAMGO or morphine required to cause 50% of maximum supersensitization was also 10- to 14-fold lower in cells expressing Go-RGS/PTXi (DAMGO, EC₅₀ of 5.5 ± 2.7 nM; morphine, EC₅₀ of 11.1 ± 1.6 nM) than cells expressing Go-PTXi (DAMGO, EC₅₀ of 55 ± 33 nM; morphine, EC₅₀ of 155 ± 32 nM; Fig. 1, A and B). These findings were confirmed in a second set of clones where supersensitization was again observed in PTX-treated C6µ cells when Go-PTXi was expressed, with the maximal supersensitization higher (p < 0.01) and the EC₅₀ lower (p < 0.01) in Go-RGS/PTXi (94 ± 18%; EC₅₀ of 19 ± 2 nM) than in the Go-PTXi (59 ± 6%; EC₅₀ of 42 ± 5 nM) expressing cells, after overnight treatment with DAMGO.

View larger version (17K): [in this window] [in a new window]   Fig. 1. Forskolin-stimulated cAMP accumulation in PTX-treated C6µ cells expressing Go-PTXi or Go-RGS/PTXi is increased by 18-h exposure to varying concentrations of DAMGO (A) or morphine (B), followed by acute treatment with naloxone (10 µM). PTX-treated cells were exposed to agonists for 18 h, and medium was then replaced with serum-free medium containing 1 mM IBMX, 30 µM forskolin, and 10 µM naloxone to start the assay, which was allowed to proceed for 5 min at 37°C. Data are expressed as percentage of overshoot, which represents the increase of the cAMP accumulated in treated cells compared with naive cells. Shown are the combined data from four assays, each measured in duplicate. The maximum overshoot in the Go-RGS/PTXi-expressing cells was significantly greater (p 0.001) than in cells expressing Go-PTXi.

The addition of naloxone was not necessary to observe the increase in cAMP. Thus, treatment of cells for 18 h with 100 nM DAMGO and then removing the agonist and washing the cells once gave an overshoot in cAMP levels of 18 ± 2.8% (n = 3) in the Go-PTXi-expressing cells and 76.5 ± 2.8% (n = 3) in the Go-RGS/PTXi expressing cells, similar to values in the presence of naloxone.

It could be suggested that the degree of supersensitization observed after chronic exposure to a specific agonist concentration is dependent upon the level of acute inhibition of adenylyl cyclase caused by that same concentration of agonist. To examine this, Go-PTXi cells were treated with 300 nM DAMGO and Go-RGS/PTXi cells with 10 nM DAMGO for 18 h before naloxone (10 µM) challenge. These doses of DAMGO gave comparable acute inhibition of cAMP accumulation (26.7 ± 4.0 and 21.8 ± 5.2%, n = 4, in Go-PTXi and Go-RGS/PTXi cells, respectively; Fig. 2A). Under these conditions, there was a significantly (p = 0.02) higher cAMP overshoot in Go-RGS/PTXi-expressing cells (50.8 ± 5.8%, n = 4) compared with Go-PTXi-expressing cells (25 ± 6.7%, n = 4; Fig. 2).

View larger version (11K): [in this window] [in a new window]   Fig. 2. Concentrations of DAMGO that cause similar acute inhibition of adenylyl cyclase (A) cause a greater degree of cyclic AMP supersensitization in Go-RGS/PTXi-expressing cells than in Go-PTXi expressing cells (B). A, PTX-treated C6µ cells were exposed to DAMGO (10 nM for Go-RGS/PTXi-expressing cells or 300 nM for Go-PTXi expressing cells) for 15 min in the presence of 1 mM IBMX and 30 µM forskolin, and cAMP accumulation was determined as described under Materials and Methods. B, PTX-treated cells were exposed for 18 h to 10 nM DAMGO (Go-RGS/PTXi) or 300 nM DAMGO (Go-PTXi). Medium was then replaced with serum-free medium containing 1 mM IBMX, 30 µM forskolin, and 10 µM naloxone to start the assay, which was allowed to proceed for 5 min at 37°C. Data are expressed as percentage of overshoot, which represents the increase in cAMP accumulated in treated compared with naive cells. Shown are the combined data from four assays, each measured in duplicate. *, p 0.05.

To determine whether there was a difference in the rate of onset of supersensitization C6µ cells expressing Go-RGS/PTXi and Go-PTXi were treated with 1 µM DAMGO for varying lengths of time. Supersensitization showed a rapid onset and reached maximal effect after a 30-min treatment with agonist in Go-RGS/PTXi-expressing cells and did not increase significantly with 24-h exposure. Similarly, supersensitization reached maximal effect in the Go-PTXi-expressing cells after 1 h (Fig. 3A). If the concentration of DAMGO was reduced 100-fold (to 10 nM) then it can be seen with Go-RGS/PTXi cells that there was a slowing in the development of supersensitization in that peak effect was not reached until 3 h of exposure to the agonist (Fig. 3B), although the degree of overshoot was similar. When comparing equieffective doses of DAMGO to cause acute inhibition of adenylyl cyclase (300 nM in Go-PTXi cells, 10 nM in Go-RGS/PTXi cells), the time course for the development of supersensitivity was similar.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Naloxone-precipitated increase in forskolin-stimulated cAMP accumulation after varying preincubation times with DAMGO. A, PTX-treated C6µ cells expressing Go-PTXi or Go-RGS/PTXi were treated with 1 µM DAMGO for different lengths of time. B, PTX-treated C6µ cells expressing Go-PTXi were treated with 300 nM DAMGO and cells expressing Go-RGS/PTXi with 10 nM DAMGO for different lengths of time. Media were replaced with serum-free media containing 1 mM IBMX, 30 µM forskolin, and 10 µM naloxone to start the assay, which was allowed to proceed for 5 min at 37°C. Data are expressed as percentage of overshoot, which represents the increase in cAMP accumulated in treated compared with naive cells. Shown are the combined data from three assays, each measured in duplicate.

To examine whether supersensitization was caused by an increase in the potency or maximal response to forskolin, dose-response curves were generated (Fig. 4A). There was no difference in the maximal response or EC₅₀ for forskolin between untreated Go-RGS/PTXi (2.3 ± 0.3 pmol cAMP/µg protein, 7.9 ± 0.8 µM) and Go-PTXi (2.3 ± 0.3 pmol cAMP/µg protein, 6.4 ± 0.6 µM)-expressing C6µ cells. After DAMGO (1 µM, 18 h) pretreatment, followed by 10 µM naloxone challenge, the maximal forskolin response increased slightly, but nonsignificantly, in cells expressing either Go-RGS/PTXi or Go-PTXi. The EC₅₀ for forskolin was decreased by the DAMGO pretreatment in Go-RGS/PTXi (3.1 ± 0.9 µM, representing a 2.5-fold shift, p < 0.05) but was unaltered in Go-PTXi (4.9 ± 1.4 µM, representing a 1.3-fold shift, p = 0.4)-expressing cells. In C6µ cells expressing endogenous G subunits without PTX treatment, exposure overnight to 1 µM DAMGO led to a 7.5-fold decrease in the EC₅₀ for forskolin from 22.6 ± 6.1 to 3.0 ± 0.5 µM, with no significant change in the maximal effect (Fig. 4B).

View larger version (18K): [in this window] [in a new window]   Fig. 4. A, stimulation of cAMP accumulation by increasing concentrations of forskolin in PTX-treated C6µ cells expressing Go-PTXi or Go-RGS/PTXi after 18-h treatment with or without 1 µM DAMGO, followed by naloxone (10 µM). B, stimulation of cAMP accumulation by increasing concentration of forskolin in C6µ cells expressing endogenous G proteins, not treated with PTX, after 18-h treatment without or with 1 µM DAMGO, followed by naloxone (10 µM). Cells were incubated with 1 µM DAMGO for 18 h, and media were replaced with serum-free media containing 1 mM IBMX, varying concentrations of forskolin, and 10 µM naloxone to start the assay, which was allowed to proceed for 5 min at 37°C. Data are expressed as picomoles of cAMP per microgram of protein. Shown are the combined data from three assays, each measured in duplicate.

₂-Adrenergic receptors are endogenously expressed in C6 cells. It is pertinent, therefore, to ask whether adenylyl cyclase supersensitization after chronic opioid can be seen if the enzyme is stimulated by isoproterenol acting at Gs coupled ₂-adrenergic receptors. Isoproterenol stimulated adenylyl cyclase activity to the same degree in naive C6µ cells expressing Go-RGS/PTXi (3.2 ± 0.2 pmol cAMP/µg protein, EC₅₀ of 4.5 ± 0.2 nM) or Go-PTXi (3.2 ± 0.2 pmol cAMP/µg protein, EC₅₀ of 2.4 ± 0.9 nM). After DAMGO (1 µM, 18 h) pretreatment and naloxone (10 µM) challenge, the maximal isoproterenol response did not significantly change in cells expressing Go-RGS/PTXi (21 ± 8%) or Go-PTXi (8 ± 2%). The isoprotenenol EC₅₀ value was not changed by the DAMGO treatment in either Go-RGS/PTXi- (3.9 ± 0.5 nM) or Go-PTXi (2.2 ± 0.8 nM)-expressing cells. In contrast, in non-PTX-treated C6µ cells expressing endogenous G subunits, exposure overnight to 1 µM DAMGO led to a significant increase in the maximal effect for isoproterenol with no change in the EC₅₀ value (Fig. 5B)

View larger version (17K): [in this window] [in a new window]   Fig. 5. A, stimulation by isoproterenol of cAMP accumulation in PTX-treated C6µ cells expressing Go-PTXi or Go-RGS/PTXi after 18-h treatment with or without 1 µM DAMGO, followed by naloxone (10 µM). B, stimulation by isoproterenol of cAMP accumulation in C6µ cells expressing endogenous G proteins not treated with PTX after 18-h treatment without or with 1 µM DAMGO, followed by naloxone (10 µM). Cells were incubated with 1 µM DAMGO for 18 h, medium was replaced with serum-free media containing 1 mM IBMX, 30 µM forskolin, and 10 µM naloxone to start the assay, which was allowed to proceed for 5 min at 37°C. Data are expressed as picomoles of cAMP per microgram of protein. Shown are the combined data from three assays, each measured in duplicate. The maximum response in the DAMGO-treated cells was significantly greater (p = 0.02) than in naive cells.

	Discussion

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The effect of endogenous RGS proteins on receptor-mediated signaling has been studied in various systems by expressing RGS-insensitive or -sensitive Go (Jeong and Ikeda, 2000; Boutet-Robinet et al., 2003; Clark et al., 2003). Insensitivity of Go to endogenous RGS proteins increases receptor signaling to some, but not all, pathways that have been studied. In this work we have expressed RGS-insensitive and -sensitive Go in C6 glioma cells expressing the rat µ-opioid receptor. This allowed us to test two hypotheses, namely, that coupling of the µ-opioid receptor to Go can provide for adenylyl supersensitization and that RGS proteins decrease the degree of supersensitization because of their effect in shortening the lifetime of the active signaling molecule Go-GTP and its G counterpart. The results show the presence of active Go alone allows for the development of µ-opioid agonist-mediated adenylyl cyclase supersensitization and that this effect is greatly enhanced when Go is insensitive to RGS protein action. This suggests that in cells expressing wild-type Go, endogenous RGS proteins are important in limiting adenylyl cyclase supersensitization. Because supersensitization is a cellular correlate of withdrawal, the findings support a model in which endogenous RGS protein action decreases the expression of withdrawal symptoms after chronic exposure to opioid drug.

C6µ cells after overnight treatment with DAMGO or morphine showed a marked supersensitization of adenylyl cyclase on challenge with naloxone or removal of agonist; an effect that was lost on PTX pretreatment. Our finding that transfected PTX-insensitive Go can restore adenylyl cyclase supersensitivity agrees with data from Watts et al. (1998) using D_2L receptors expressed in NS20Y or HEK293 cells that Go is able to support adenylyl cyclase supersensitization. However, our results disagree with suggestions that multiple G subunits are needed to observe µ-opioid induced supersensitization in HEK293 cells (Tso and Wong, 2000a,b), although in those studies a role for Go was not examined. The supersensitization observed with both mutant forms of Go alone was much reduced compared with C6µ wild-type cells that express mainly Gi₂. Similarly, in NS20Y cells Go only gives approximately 50% of the supersensitization seen in the wild-type cells (Watts et al., 1998). This relatively poor ability of Go to mediate supersensitization may relate to the expression level of Go, the weak ability of Go to couple to adenylyl cyclase (McKenzie and Milligan, 1990; Moon et al., 2001; Clark et al., 2003), or the fact that Go is not endogenous to C6 cells (Charpentier et al., 1993), and so the correct machinery is not in place, including isoforms of adenylyl cyclase. This last point is particularly pertinent because Ammer and Christ (2002) have shown that the ability of G subunits to mediate adenylyl cyclase supersensitivity is isoform-specific.

There was a much higher level of supersensitization in C6µ cells expressing Go-RGS/PTXi, indicating that endogenous RGS proteins reduce the magnitude of supersensitization. In addition, the EC₅₀ values of DAMGO and morphine to produce supersensitization were 10-fold lower in cells expressing Go-RGS/PTXi than in cells expressing GoPTXi, indicating endogenous RGS proteins also reduced the potency of the agonists to produce supersensitization. It can be hypothesized that RGS proteins reduce supersensitization simply by reducing the degree of agonist inhibition of adenylyl cyclase, by removing G-GTP through GAP activity. On the other hand, there does not seem to be a direct correlation between the acute inhibition of adenylyl cyclase and the degree of supersensitization. In the Go-PTXi-expressing cells, morphine treatment induced the same level of supersensitization as DAMGO, even though it was less efficacious acutely than DAMGO. Moreover, the level of supersensitization in the Go-PTXi-expressing cells never approached the level of supersensitization in the Go-RGS/PTXi-expressing cells at any concentration of agonist. Finally, when concentrations of chronic DAMGO were used that gave the same degree of acute inhibition of adenylyl cyclase in the Go-PTXi- and Go-RGS/PTXi-expressing cells, the supersensitization was significantly greater in the cells expressing Go-RGS/PTXi. Certainly, in HEK293 cells expressing different Gi subunits, inhibition of adenylyl cyclase can be obtained without supersensitization, confirming the two events are separate (Tso and Wong, 2001).

The onset of supersensitization after treatment with a supramaximal concentration of DAMGO was rapid both in the Go-RGS/PTXi and the Go-PTXi cells, reaching maximal effect after 30 min and 1 h, respectively. Using lower concentrations of DAMGO that caused equivalent acute inhibition of adenylyl cyclase (20%), the time to maximal supersensitization was longer, occurring in approximately 3 h in both Go-PTXi- and Go-RGS/PTXi-expressing cells. As with other systems (for review, see Watts, 2002), this rapid time course shows protein expression is unlikely to be responsible for supersensitization. One proposed mechanism behind supersensitization is an enhanced interaction between Gs and adenylyl cyclase (Watts and Neve, 1996; Ammer and Schulz, 1998). Indeed, supersensitization is lost in cells expressing Gs-insensitive mutants of adenylyl cyclase type V (Watts et al., 2001). In the current study, opioid agonist pretreatment increased the potency of forskolin to stimulate adenylyl cyclase. Because forskolin is believed to act synergistically with Gs, this is consistent with a role for Gs in supersensitization (Watts and Neve, 1996). This increased interaction of adenylyl cyclase with Gs would be predicted to increase the maximal effect of adenylyl cyclase stimulated through a Gs-coupled receptor. Using isoproterenol to stimulate endogenous ₂ adrenoreceptors in C6µ cells, no significant increase in maximal effect or shift in the potency of isoproterenol was observed after exposure to µ-agonist. This was an unexpected finding that may be due to the use and/or expression level of Go, which, as discussed above, does not support adenylyl cyclase inhibition or supersensitization as well as endogenous G proteins. Certainly, a robust effect on the isoproterenol maximal response was seen in the C6µ cells expressing wild-type G proteins.

The lifetime of Go-GTP, which is decreased by the GAP activity of RGS proteins, is likely the important parameter in mediating supersensitization. The GAP activity of RGS proteins not only decreases the accumulation of G-GTP but also the level of free G dimers. This is an important consideration since G has been implicated in opioid supersensitization (Avidor-Reiss et al., 1996; Rubenzik et al., 2001). G dimers activate some adenylyl cyclase isoforms (AC II and AC IV), although AC type VI predominately expressed in C6 cells (Debernardi et al., 1993) is neither stimulated or inhibited by G (Sunahara et al., 1996), suggesting a direct action of G on adenylyl cyclase is unlikely. However, an indirect action of G through signaling pathways with possible feedback regulatory functions, such as ERK/MAPK is possible. In support of this, Raf-1, a protein kinase in the MAPK signal transduction cascade, has been shown to have a role in -opioid mediated supersensitization in human -opioid receptor/Chinese hamster ovary cells by use of a selective inhibitor of Raf-1 (Varga et al., 2002). A role for ERK is an interesting possibility because we recently showed that ERK activation by µ-agonists is increased in C6µ cells expressing RGS-insensitive Go (Clark et al., 2003). Finally, RGS proteins are multifunctional (for review, see Zhong and Neubig, 2001), and a direct interaction between RGS proteins and adenylyl cyclase or other proteins having a role in supersensitization cannot be ruled out given the multiple binding domains on many RGS proteins. On the other hand, the effects of RGS proteins were revealed in this study using a G184S mutation on Go that prevents the binding of RGS, so any non-GAP RGS effects must presumably involve the Go G184S site. This could include antagonism of effectors (Hepler et al., 1997; Tesmer et al., 1997; Zhong and Neubig, 2001) that may be important in the development or expression of supersensitization. Alternatively, regions outside the RGS box might be responsible for recruiting proteins involved in supersensitization to the vicinity of active Go. For example, D-AKAP2, which contains an RGS domain, may function to scaffold G protein mediated cAMP signaling (Huang et al., 1997; Siderovski et al., 1999). Given the structural variety of RGS proteins (Zhong and Neubig, 2001), further studies to identify which RGS protein(s) modulate the development of supersensitization would be of great interest.

Overall, our results support the view that Go alone is capable of supporting cyclic AMP supersensitization mediated through µ-opioid receptors coupled to inhibitory G proteins and that this supersensitization is modulated by endogenous RGS proteins expressed in C6µ cells. Recently, Zachariou et al. (2003) have suggested that RGS9 is essential for µ-opioid activity. Using RGS9 knockout mice, they demonstrated a 10-fold increase in the potency of morphine to induce reward and an increased antinociceptive potency with reduced tolerance. The mice also showed enhanced morphine withdrawal after chronic treatment, indicating a greater degree of physical dependence. However, C6 cells do not express RGS9 or the other members of the RGS9 subfamily (RGS 6, 7, and 11; Snow et al., 2002) that contain similar structural domains and motifs (Hollinger and Hepler, 2002), so there is either some redundancy of RGS activity or RGS protein action in altering µ-agonist responses is cell-specific. However, together these findings suggest that the severity of dependence and withdrawal is greater in systems expressing reduced RGS activity; therefore, pharmacological interventions that increase RGS action could be useful in reducing symptoms of opioid dependence and withdrawal.

	Acknowledgements

 
We thank Dr. Huda Akil for the µ-opioid receptor DNA and Dr. Stephen Ikeda for the Go mutant DNA.

	Footnotes

 
This study was supported by Grants DA04087 (to J.R.T.) and GM39561 (to R.R.N.). Portions of these data have been published in abstract form in FASEB J. 17:A218, abstract 145.6.

DOI: 10.1124/jpet.103.064824.

ABBREVIATIONS: PTX, pertussis toxin; GIRK, G protein-gated inwardly rectifying K⁺ channel; MAPK, mitogen-activated protein kinase; RGS, regulator of G protein signaling; GAP, GTPase-activating protein; HEK, human embryonic kidney; ERK, extracellular signal-regulated kinase; IBMX, 3-isobutyl-1-methylxanthine; DAMGO, [D-Ala²,MePhe⁴,Gly⁵-ol]enkephalin; PTXi, pertussis toxin-insensitive.

Address correspondence to: Dr. John R. Traynor, Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, MI 48109-0632. E-mail: jtraynor{at}umich.edu

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