Introduction

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Opioid receptors belong to the family of seven-transmembrane domain G protein-coupled receptors (for review see: Reisine and Bell, 1993). Their acute activation leads to the inhibition of AC activity, an effect that is mediated by pertussis toxin-sensitive G proteins of the G_i/G_o class (Childers, 1991). Chronic exposure to an opioid, however, produces multiple adaptational changes within the stimulatory branch of AC resulting in an increased capacity of stimulatory AC signaling during the state of dependence (Ammer and Schulz, 1993; 1995; 1997). Up-regulation of stimulatory AC signaling usually is masked as long as the inhibitory opioid is present but manifests in a significant enhancement of cAMP production after removal of the agonist (Sharma et al., 1975). This regulatory phenomenon, generally referred to as "cAMP overshoot" or "AC supersensitivity," represents a cellular correlate for opioid withdrawal and frequently has been used to define the state of dependence (Sharma et al., 1975; Collier, 1984). AC supersensitivity had been detected originally in chronically morphine-treated neuronal cell lines, such as neuroblastoma x glioma (NG108-15) hybrid cells (Sharma et al., 1975; Law et al., 1984) and human neuroblastoma SH-SY5Y cells (Yu et al., 1990). Heterologous expression of the recently cloned opioid receptor cDNA (delta, kappa, mu) revealed that AC supersensitivity can be reconstituted with all three opioid receptor types (Law et al., 1994; Avidor-Reiss et al., 1995a, 1995b). Moreover, AC supersensitivity apparently represents a more common means of cellular adaptation toward chronic inhibitory drug action, because several other inhibitory receptors, such as alpha₂ adrenergic, muscarinic cholinergic and somatostatin receptors also induce this phenomenon (Thomas and Hoffman, 1987). Although the role of AC supersensitivity in the development of drug dependence is well recognized (Nestler et al., 1993), the biochemical signal mediating the increase in AC activity is still unknown.

The activity of opioid-regulated AC is under the control of stimulatory receptor systems (Collier, 1984). Signal transduction from stimulatory receptors to AC involves the heterotrimeric stimulatory G protein (G_s) which, upon activation, dissociates into its GTP-bound G_s and G subunits (Gilman, 1987). Activated G_s subsequently binds to AC, thereby stimulating catalytic activity. Recent molecular cloning has permitted identification of at least nine distinct AC isoforms which show several common and disparate features in the regulation of effector activity (for review see: Sunahara et al., 1996). Whereas all AC isoforms can be stimulated by both activated G_s and forskolin, several additional regulatory factors exist which may positively or negatively modulate catalytic activity in a subtype-specific manner. For instance, direct G_i-mediated inhibition of enzymatic activity has been shown for AC types I, V and VI (Wong et al., 1991; Sunahara et al., 1996), whereas G subunits may either attenuate AC type I activity (Tang and Gilman, 1991) or synergistically activate G_s-stimulated AC types II and IV (Tang and Gilman, 1991; Federman et al., 1992). More complex and indirect modes of regulation have been shown for Ca⁺⁺-dependent calmodulin, Ca⁺⁺ and phosphorylation by protein kinases A and C (Choi et al., 1992; Premont et al., 1992; Kawabe et al., 1994).

Because of the molecular diversity and regulatory complexity, investigation into the regulatory mechanism underlying AC supersensitivity is highly complicated. In a first step to decipher the stimulatory signal, the present study was initiated to determine the role of the stimulatory G protein in mediating the enhancement of AC activity in opioid-withdrawn cells. As a model system we used chronically morphine treated NG108-15 hybrid cells, which carry high levels of inhibitory delta opioid receptors (Hamprecht et al., 1985). Although morphine acts as a partial agonist on delta opioid receptors in this cell line (Vachon et al., 1987), this opiate proved particularly advantageous in inducing cellular dependence, most likely because it fails to desensitize delta opioid receptor signaling (Law et al., 1984; Keith et al., 1996). Our results demonstrate that the enhanced AC activity in morphine withdrawn NG108-15 hybrid cells is not a function of an increased stimulation by G_s, but involves an additional regulatory event that requires coincident stimulation of AC by receptor-activated G_s.

	Experimental Procedures

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Materials. Reagents were purchased from the following sources: [³H]forskolin (31 Ci/mmol) from NEN DuPont (Dreieich, Germany); [¹²⁵I]cAMP tracer (2000 Ci/mmol) from Amersham International (Braunschweig, Germany); forskolin, Ro 20-1724, and CTX from Calbiochem (Bad Soden, Germany); DSLET from Bachem (Heidelberg, Germany); rabbit anti-cAMP antibody from BioMakor (Rehovot, Israel). Tissue culture reagents were obtained from PAN Systems (Aidenbach, Germany) and Gibco/BRL (Eggenstein, Germany). PGE₁ and all standard laboratory reagents were from Sigma (Deisenhofen, Germany).

Cell culture, chronic opioid treatment. Neuroblastoma x glioma (NG108-15) hybrid cells (Hamprecht et al., 1985) were grown as monolayers in Dulbecco's modified Eagle's medium, containing 5% heat-inactivated fetal calf serum, 100 µM hypoxanthine, 1 µM aminopterin and 16 µM thymidine, in a humidified atmosphere of 5% CO₂ and 95% air at 37°C. Subconfluent monolayers were split in the ratio of 1:5, grown overnight and subjected to chronic inhibitory opioid treatment for another 3 days with the addition of 10 µM morphine. This treatment regimen has been shown previously to produce high degrees of cellular dependence in NG108-15 hybrid cells (Ammer and Schulz, 1995). Untreated cultures of individual passages served as controls.

[³H]Forskolin binding. [³H]Forskolin binding in whole NG108-15 hybrid cells was determined essentially as described (Alouisi et al., 1991; Kim et al., 1995). Cells from two 75 cm² culture flasks were harvested, washed three times (200 × g; 10 min) with ice-cold 20 mM HEPES-buffered Dulbecco's modified Eagle's medium (pH 7.4), and resuspended in the same buffer at a density of 2.5 × 10⁶ cells/ml. Cells were equilibrated on ice for 30 min either in the absence (control) or presence of 10 µM morphine (opioid-dependent cells). Binding reactions were at 4°C for 60 min in a total volume of 500 µl HEPES-buffered Dulbecco's modified Eagle's medium containing 5 × 10⁵ cells, 10 nM [³H]forskolin and various concentrations of PGE₁ to stimulate formation of G_s/AC complexes. Nonspecific binding was defined with 10 µM unlabeled forskolin. In some experiments, binding of [³H]forskolin was displaced with increasing concentrations of unlabeled forskolin. Chronically morphine-treated cells were assayed in the presence of either morphine (10 µM) for investigation of the state of dependence or of naloxone (100 µM) for analysis of the state of opioid withdrawal. Binding reactions were terminated by rapid filtration over Whatman GF/C filters, followed by four washes with 5 ml each of ice-cold 50 mM Tris HCl buffer, pH 7.4, containing 10 mM MgCl₂. Cell associated radioactivity was determined by scintillation counting of the filters.

Adenylyl cyclase assay. AC activity was determined in a particulate membrane preparation according to Vachon et al. (1987). Cells were collected, washed three times (200 × g; 10 min) with phosphate-buffered saline (pH 7.4) and membranes were prepared in 5 mM Tris-HCl buffer (pH 7.4), containing 1 mM DTT and 1 mM EGTA. Membranes were resuspended in the above buffer at a concentration of 10 mg/ml and stored in aliquots at 70°C until use. AC activity was measured in 40 mM Tris-HCl buffer (pH 7.4), containing 0.2 mM EGTA, 0.2 mM DTT, 100 mM NaCl, 10 mM MgCl₂, 0.5 mM ATP, 5 µM phosphocreatine, 5 units/ml creatine kinase, 10 µM GTP and 30 µM Ro 20-1724. Reactions (100 µl total volume) were started by the addition of 10 µg of membrane protein, maintained for 10 min at 32°C and stopped with 500 µl of ice-cold 10 mM HCl. Membranes derived from opioid-dependent cells were first equilibrated at 4°C for 30 min with 10 µM morphine before AC activity was determined either in the presence of morphine (state of dependence) or after addition of 100 µM naloxone to uncover effector supersensitivity (precipitated withdrawal). The high concentration of naloxone (100 µM) used to induce opioid withdrawal did not produce nonspecific effects on AC activity in membranes from control cells (not shown) and was essential in stably reproducing the phenomenon of AC supersensitivity (Lee et al., 1988; Law et al., 1994; Ammer and Schulz, 1995). Stimulation of effector activity was achieved with either PGE₁ or forskolin at the concentrations indicated. The amount of cAMP generated was quantitated by radioimmunoassay after dilution of the samples as described (Ammer and Schulz, 1995).

Modification of G_s activity. In some experiments the functional integrity of G_s was altered before determination of AC activity. Selective inactivation of G_s function was achieved by transient exposure of the membranes to low pH (Childers and LaRiviere, 1984). Membranes were recovered by centrifugation (10,000 × g; 15 min), resuspended in 50 mM sodium acetate (pH 4.5) and incubated for 20 min at 4°C. Controls were kept in the presence of 50 mM Tris-HCl (pH 7.4) buffer. Reactions were stopped by dilution with 10 volumes of ice-cold NMT buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl₂; pH 7.4) and membranes were collected as above. Subsequently, membranes were resuspended in NMT buffer (0.5 mg/ml) and equilibrated for another 30 min at 4°C either without (control) or with 10 µM morphine (dependence) before determination of AC activity.

Data analysis. EC₅₀, E_max and IC₅₀ values were determined by nonlinear least-squares regression curve fitting, with SigmaPlot (Jandell Scientific, Erkrath, Germany) software. [³H]Forskolin binding parameters were calculated from homologous displacement curves according to the method of DeBlasi et al. (1989). Statistical differences were determined by one-way ANOVA or Student's two-tailed t test where appropriate.

	Results

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Comparison of PGE₁ receptor-stimulated AC activation and G_s/AC interaction. To determine whether the expression of AC supersensitivity in opioid-withdrawn NG108-15 hybrid cells is a function of enhanced activation of the catalytic component of AC by G_s, we compared the effects of PGE₁ on the stimulation of AC activity with those on the promotion of high-affinity [³H]forskolin binding, which provides a direct measure for the binding of activated G_s to AC (Alouisi et al., 1991; Kim et al., 1995). In membranes from naive cells, activation of PGE₁ receptors dose-dependently stimulated effector activity with an EC₅₀ value of 36.5 ± 6 nM (mean ± S.D.; n = 4). Chronic morphine treatment (10 µM; 3 days) per se had no effect on both the potency (EC₅₀ = 39.8 ± 3 nM; mean ± S.D.; n = 4) as well as the maximum capacity of PGE₁ to activate AC as long as the assays were performed in the presence of the inhibitory opioid used for pretreatment (E_max = 285.1 ± 12 vs. 288.7 ± 32 pmol/min/mg protein for control and opioid-dependent cells, respectively; means ± S.D.; n = 4). However, precipitation of opioid withdrawal by the addition of naloxone (100 µM) resulted in an approximately 40% increase in the capacity of PGE₁ receptor-stimulated AC activity (E_max = 401.3 ± 20 pmol/min/mg protein; mean ± S.D.; n = 4; P < .001), without any change in its EC₅₀ value (40.5 ± 9 nM; mean value ± S.D.; n = 4). Thus, AC supersensitivity is characterized by an increased capacity rather than a change in the sensitivity of PGE₁ receptor signaling.

The activated, GTP-bound form of G_s represents the principle stimulator of all membrane-bound AC isoforms currently known (Sunahara et al., 1996). High-affinity [³H]forskolin binding experiments were performed to evaluate if AC supersensitivity after opioid withdrawal is caused by an enhanced stimulation of AC by G_s. Although high-affinity [³H]forskolin binding to intact NG108-15 hybrid cells was only barely detectable in the absence of a stimulatory ligand, PGE₁ receptor-mediated activation of G_s resulted in the formation of G_s/AC complexes and, thus, in a strong increase in high-affinity [³H]forskolin binding. However, as shown in figure 1B, the dose-response curves obtained for naive, opioid-dependent and opioid-withdrawn cells were almost superimposable, yielding identical values for EC₅₀ (10.9 ± 3, 8.4 ± 0.2, and 7.7 ± 2 nM; means ± S.D.; n = 4) and E_max (76.7 ± 8, 73.6 ± 5, and 79.8 ± 6 fmol [³H]forskolin binding sites/10⁶ cells; means ± S.D.; n = 4). These results demonstrate that the enhancement of AC activity after opioid withdrawal is not associated with changes in the functional interaction between G_s and AC. Thus, the expression of AC supersensitivity is not a function of an enhanced stimulation of AC by activated G_s but involves an additional regulatory event.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Comparison of PGE1 receptor-stimulated AC activity with high-affinity [3H]forskolin binding in NG108-15 cells. NG108-15 hybrid cells were grown either in the absence (control; ) or presence of morphine (10 µM; 3 days) to induce dependence. A: Cells were harvested and the dose-response relationship of PGE1-stimulated AC activity was determined in a particulate membrane preparation. Membranes from opioid-dependent cells were assayed either in the presence of morphine () or naloxone () to investigate the states of dependence and opioid withdrawal, respectively. Enzymatic activity is expressed in picomoles of cAMP formed per min per mg of membrane protein. The data shown are mean values ± S.D. of n = 4 experiments. B: Binding of Gs to AC was assessed by high-affinity [3H]forskolin binding to intact cells as described under "Experimental Procedures." Binding of [3H]forskolin (10 nM) was simulated dose dependently by the addition of increasing concentrations of PGE1 (0.1 nM 10 µM). Opioid-dependent cells were measured either in the presence of morphine () or after naloxone-precipitated withdrawal (). The number of Gs/AC complexes formed is given in femtomoles of [3H]forskolin bound per 106 cells. The data presented are mean values ± S.D. from n = 4 experiments. Vertical bars indicate calculated EC50 values.

Effect of opioid withdrawal on the affinity of [³H]forskolin binding. Although both chronic morphine treatment and opioid withdrawal lack any effect on the maximum number of PGE₁ receptor stimulated G_s/AC complexes, binding of an additional component to AC possibly could alter the conformation of the binding site for [³H]forskolin and, hence, affect its affinity. Therefore, homologous displacement curves of PGE₁ (10 µM)-stimulated high-affinity [³H]forskolin binding were constructed. As shown in figure 2, there is no difference in the inhibition curves, regardless of whether naive, chronically morphine- or opioid-withdrawn cells were measured. Calculation of the apparent affinity constants by the method of DeBlasi et al. (1989) resulted in almost identical K_d values for forskolin (20.1 ± 14, 19.0 ± 16, and 19.7 ± 23 nM for naive, opioid-dependent and opioid-withdrawn cells, respectively; means ± S.D.; n = 4).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Homologous displacement of high-affinity [3H]forskolin binding in naive, opioid-dependent and opioid-withdrawn NG108-15 hybrid cells. The affinity of forskolin for the Gs/AC complex was determined by homologous displacement of PGE1 (10 µM)-stimulated high-affinity [3H]forskolin (10 nM) binding with increasing concentrations of competing unlabeled forskolin. Either native (), chronically morphine (10 µM; 3 days) -treated (), or opioid-withdrawn cells () were measured. Specific high-affinity [3H]forskolin binding obtained in the presence of 10 µM forskolin was set at 100%. Each data point represents the mean value of n = 4 experiments. Standard deviation was routinely less than 7% of the mean. Binding parameters were calculated according to the formalism of DeBlasi et al (1989). Dotted lines indicate half-maximal inhibition of [3H]forskolin binding, which is almost identical in control, opioid-dependent and opioid-withdrawn cells, respectively.

Regulation of [³H]forskolin binding by acute delta opioid receptor activation. The failure of opioid withdrawal to affect the affinity of [³H]forskolin binding raises the question of whether binding of an additional regulator of AC activity must necessarily alter the conformation of the G_s/AC complex, which represents the high-affinity binding site for forskolin (Alouisi et al., 1991). For this, the effect of acute delta opioid receptor activation on high-affinity [³H]forskolin binding was determined. In NG108-15 hybrid cells, inhibition of AC activity is mediated via the inhibitory G protein -subunit G_i2 (McKenzie and Milligan, 1990). Acute inhibition of AC activity, neither with morphine (10 µM) nor the full delta receptor agonist DSLET (1 µM), had any effect on PGE₁ receptor-stimulated [³H]forskolin binding parameters (table 1), which indicates that binding of G_i2 to AC does not interfere with the formation and conformation of G_s/AC complexes, confirming previous data (Kim et al., 1995).

Requirement of activated G_s for AC supersensitivity. The finding that the generation of AC supersensitivity in opioid-withdrawn NG108-15 hybrid cells is not a function of enhanced G_s activity raises the question of whether G_s is involved at all in the regulatory mechanism leading to the increase in AC activity. To investigate this issue we first tested the effect of different concentrations of forskolin on the expression of AC supersensitivity. The diterpene forskolin directly binds to and stimulates AC activity by mechanisms which depend on the concentration used. In the nanomolar range, forskolin potentiates receptor-stimulated AC by increasing the affinity of G_s to AC; whereas, high micromolar concentrations directly activate the effector molecule without the requirement of G_s for its full action (Sutkowski et al., 1996). Precipitation of opioid withdrawal by naloxone (100 µM) only resulted in AC supersensitivity when AC was stimulated with 100 nM but not 10 µM forskolin (table 2). The finding that opioid withdrawal failed to induce AC supersensitivity in the presence of 10 µM forskolin apparently was not caused by maximal activation of the effector molecule, because higher forskolin concentrations further enhanced AC activity (not shown). These results suggest that the expression of AC supersensitivity requires costimulation of AC by G_s. This issue was investigated further in membranes depleted of G_s function after transient exposure to acidic conditions (Childers and LaRiviere, 1984). Inactivation of G_s largely decreased PGE₁ receptor-mediated stimulation of AC by more than 95% in membranes from both naive and opioid-dependent cells. Conversely, low pH treatment increased the efficacy of morphine to acutely inhibit AC activity in membranes from naive cells (38.4 ± 6 vs. 29.4 ± 4% inhibition; means ± S.D.; n = 4), whereas no effect on delta opioid receptor desensitization was observed in membranes from chronically morphine treated cells, as assessed in the presence of 100 µM morphine (10.9 ± 5 vs. 11.3 ± 7% inhibition; means ± S.D.; n = 4). Thus, there are still functional delta opioid receptors present in low pH pretreated membranes derived from opioid-dependent cells that should be able to trigger AC supersensitivity after withdrawal. However, the addition of naloxone (100 µM) to G_s-depleted membranes failed to induce a cellular withdrawal response, which suggests that stimulation of AC activity by G_s represents an essential requirement for the expression of AC supersensitivity (fig. 3).

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Inhibition of AC supersensitivity by low pH pretreatment. Membranes prepared from naive or opioid-dependent NG108-15 cells were exposed transiently to pH 4.5 before determination of PGE1 (10 µM)-stimulated AC activity. Inhibition of AC activity in naive NG108-15 hybrid cell membranes was assessed with 10 µM morphine. The ability of naloxone (100 µM) to precipitate AC supersensitivity was tested in membranes from opioid-dependent cells which were equilibrated for 30 min in the presence of 10 µM of morphine to avoid spontaneous withdrawal. Enzymatic activity is expressed in picomoles of cAMP generated per mg of membrane protein per min. The data shown are mean values ± S.D. of n = 4 experiments each performed in triplicate. ***; statistically different from controls at P < .001 as determined by ANOVA.

AC supersensitivity requires intact PGE₁ receptor/G_s signaling. We further investigated whether either the presence of activated G_s per se or the functional intact stimulatory signaling during the state of opioid withdrawal is required for the expression of AC supersensitivity. For this, stimulatory signal transduction in membranes from opioid-dependent NG108-15 cells was modulated by two different approaches. First, short-term CTX treatment (10 µg/ml; 30 min) was used to uncouple G_s from its associated stimulatory receptors by constitutive activation of G_s. CTX treatment of the membranes increased basal AC activity by about 3-fold and almost completely abolished further stimulation via PGE₁ receptors, whereas no effect on acute delta opioid receptor-mediated inhibition of AC was observed. Receptor-independent activation of AC by constitutively activated G_s, however, prevented the expression of AC supersensitivity in opioid-withdrawn cell membranes (fig. 4). This result indicates that not only the presence of activated G_s alone but also an intact stimulatory control of AC is necessary to elicit AC supersensitivity. The latter conclusion was confirmed by experiments in which receptor-mediated activation of G_s was blocked in situ by the use of a C-terminal anti-G_s antibody (Ammer and Schulz, 1997). Blockade of PGE₁ receptor/G_s interaction by preincubation of NG108-15 hybrid cell membranes with a maximal effective concentration of S1/3 antibody (30 µg IgG/5 membranes) resulted in an approximately 90% attenuation of PGE₁ (0.1 µM)-stimulated AC activity. Anti-G_s antibody treatment apparently selectively interfered with stimulatory signal transduction, because acute inhibition of PGE₁ (0.1 µM)-stimulated AC [by morphine (10 µM)] remained unaffected (28.2 ± 3 vs. 21.1 ± 5 pmol cAMP/min/mg of membrane protein; means ± S.D.; n = 3). In contrast, disruption of PGE₁ receptor/G_s coupling in membranes from opioid-dependent cells completely abolished the generation of AC supersensitivity after naloxone (100 µM)-precipitated withdrawal (fig. 5). Preincubation of the membranes with control IgG (30 µg/5 µg) failed to affect both receptor mediated stimulation of AC and the induction of AC supersensitivity. Therefore, the expression of AC supersensitivity in opioid-withdrawn NG108-15 hybrid cells requires functional, intact stimulatory signal transduction pathways.

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Constitutive activation of Gs by CTX-catalyzed ADP-ribosylation inhibits AC supersensitivity after opioid withdrawal. Membranes from naive and chronically morphine-treated NG108-15 cells were subjected to short-term CTX treatment to constitutively activate Gs by a receptor-independent mechanism. Subsequently, the ability of morphine (10 µM) to inhibit and of naloxone (100 µM) to increase AC activity was determined in naive and opioid-dependent cells, respectively. Membranes from opioid-dependent cells were kept in the presence of morphine (10 µM) throughout the incubation periods. Enzymatic activity is expressed in picomoles of cAMP generated per mg of membrane protein per min. The data shown are mean values ± S.D. of n = 3 experiments each performed in triplicate. ***; statistically different from controls at P < .001 as determined by ANOVA.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Blockade of PGE1 receptor/Gs interaction abolishes the ability of naloxone to precipitate AC supersensitivity in opioid-dependent NG108-15 cells. Membranes (5 µg) from opioid-dependent NG108-15 cells were either incubated together with 30 µg of a protein A-purified C-terminal anti-Gs antibody or an identical concentration of control IgG to block PGE1 receptor/Gs interaction as described under "Experimental Procedures." All steps were performed in the presence of morphine (10 µM) to avoid spontaneous withdrawal. After antibody treatment, the ability of naloxone (100 µM) to precipitate AC supersensitivity was determined in the presence of 0.1 nM PGE1. Enzymatic activity is expressed in picomoles of cAMP generated per mg of membrane protein per min. The data shown are mean values ± S.D. of n = 3 experiments each performed in triplicate. ***; statistically different at P < .001 according to Student's t test.

	Discussion

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The results of the present study reveal that the enhancement of AC activity (AC supersensitivity) in opioid-withdrawn NG108-15 hybrid cells is not a function of an increased activation by G_s, the G protein -subunit mediating stimulation of enzyme activity. Nevertheless, the stimulatory G protein was critical in the generation of cellular withdrawal, because both G_s-independent activation of AC with high concentrations of forskolin as well as inactivation of G_s function by low pH pretreatment abolished the expression of AC supersensitivity. In addition, interruption of stimulatory receptor/AC signaling after constitutive activation of G_s by CTX-catalyzed ADP-ribosylation or by application of a C-terminal anti-G_s antibody further demonstrated that the regulatory mechanism leading to the enhancement of AC activity after opioid withdrawal requires functional, intact stimulatory AC signaling pathways.

The phenomenon of AC supersensitivity originally had been described in chronically morphine-treated NG108-15 hybrid cells after opioid withdrawal (Sharma et al., 1975) and subsequently was used to define the state of opioid dependence in several neuronal cell lines and tissues (Collier, 1984; Childers, 1991; Nestler et al., 1993). The enhancement of AC after drug withdrawal clearly represents a more general adaptive phenomenon toward chronic treatment with inhibitory drugs because prolonged activation of alpha₂ adrenergic, muscarinic cholinergic or somatostatin receptors was found to induce a similar cellular response (for review see: Thomas and Hoffman, 1987). Thus, the main criterion for the choice of a cell system to investigate the biochemical mechanisms underlying AC supersensitivity would be the development of a high degree of cellular dependence; whereas the nature of the inhibitory receptor system used to persistently inhibit AC is probably of secondary significance. Although the recent cloning of a mu opioid receptor cDNA (Chen et al., 1993), the opioid binding site associated with classical withdrawal in vivo (Nestler et al., 1993), has permitted the establishment of cell lines stably expressing high levels of a single population of mu opioid receptors (Avidor-Reiss et al., 1995b; Ammer and Schulz, 1997), the present study was performed with chronically morphine treated NG108-15 hybrid cells because this cell system is well characterized and has a long history in the investigation of cellular aspects of opioid dependence and withdrawal (Sharma et al., 1975; Collier, 1984; Childers, 1991). NG108-15 hybrid cells carry ample amounts of endogenous delta opioid receptors (Hamprecht et al., 1985) which are particularly advantageous in inducing cellular correlates of opioid dependence because morphine, unlike more selective delta agonists, fails to desensitize and down-regulate the delta opioid receptor (Law et al., 1984; Keith et al., 1996).

Regulation of membrane-bound AC is highly complex and largely depends on the type of AC present in a particular tissue (Sunahara et al., 1996). Whereas all AC isoforms are stimulated by the GTP-bound form of G_s and the diterpene forskolin (Sunahara et al., 1996), certain cyclases are also the target of regulation by G_i subunits (Wong et al., 1991), G subunits (Tang and Gilman, 1991; Federman et al., 1992), Ca⁺⁺/calmodulin (Choi et al., 1992) and protein kinases A and C (Premont et al., 1992; Kawabe et al., 1994). Thus, because of this regulatory complexity, the overall level of AC activity measured in a certain tissue reflects the sum of multiple stimulatory and inhibitory inputs. To gain insight into the regulatory mechanism underlying the phenomenon of AC supersensitivity, we first determined whether the enhancement of AC activity is caused by an increased stimulation by G_s, the principle activator of AC (Gilman, 1987), or by secondary regulation of G_s-stimulated AC activity. Discrimination between the effects of G_s on AC activity from other stimulatory signals was achieved by comparing PGE₁ receptor-mediated stimulation of AC activity with the promotion of high-affinity [³H]forskolin binding, which provides a direct measure for the physical interaction of activated G_s and AC (Alouisi et al., 1991). Although there is currently little information about the expression pattern of individual AC isoforms in NG108-15 cells (these cells contain AC type VI but not AC type I and II, others have not been searched for (MacEwan et al., 1996). The property of both forskolin and activated G_s to bind to all AC isoforms (Sutkowski et al., 1996) overcomes this limitation and renders high-affinity [³H]forskolin binding a reliable measure for the number of G_s/AC complexes formed, regardless of the cellular expression profile of different AC isoforms (Kim et al., 1995). With this approach we could demonstrate clearly that neither the states of opioid dependence nor withdrawal are associated with changes in the maximum number of G_s/AC complexes formed and the affinity of [³H]forskolin binding. These findings confirm that chronic morphine treatment per se has no effect on the steady-state levels of G_s (Ammer and Schulz, 1995) and is not likely to affect overall AC abundance, because changes in the expression levels of AC should be detected by this method (MacEwan et al., 1996). Moreover, the findings also indicate that the enhancement of AC activity after opioid withdrawal is not caused by an increased stimulation of AC by activated G_s. Thus, the phenomenon of AC supersensitivity seems to represent a regulatory phenomenon that is mediated by a stimulatory signal different of activated G_s. This notion is supported by our previous work in which we demonstrated that opioid withdrawal is not associated with an increased activation of G_s molecules by PGE₁ receptors (Ammer and Schulz, 1995). In addition, the observation that the expression of AC supersensitivity is confined specifically to only some (AC types I, V, VI, VIII) but not all AC isoforms and that tissue specific differences apparently exist in the ability of AC type I to exhibit AC supersensitivity (Thomas and Hoffman, 1996; Avidor-Reiss et al., 1997) further argues against a direct G_s effect as the underlying mechanism.

In addition to stimulation by G_s, there are several other potential regulatory mechanisms that could account for the enhancement of AC activity after opioid withdrawal, such as direct activation of AC type I by Ca⁺⁺-dependent calmodulin (Choi et al., 1992) and stimulation of AC types II and IV by G subunits released from inhibitory G proteins (Tang and Gilman, 1991; Federman et al., 1992). Both components act only at defined AC isoforms and their effects are highly synergistic or conditional to the presence of activated G_s (Sunahara et al., 1996). Although there is currently no information about the presence of one or more susceptible AC isoforms in NG108-15 hybrid cells, the results presented herein strongly suggest that the regulatory mechanism underlying AC supersensitivity must involve an additional component that requires costimulation of AC by activated G_s to exert its effect. This notion is based on the observation that opioid withdrawal is able to elicit AC supersensitivity only in the presence of nanomolar concentrations of forskolin, which act to potentiate receptor-mediated stimulation of AC (Sutkowski et al., 1996). Direct activation of catalytic activity by high micromolar forskolin concentrations prevents the development of a cellular withdrawal sign. In addition, transient exposure of membranes from opioid-dependent cells to low pH also was found to abolish the expression of AC supersensitivity. This effect is most likely caused by selective inactivation of G_s function (Childers and LaRiviere, 1984), which results in a dramatic decrease of G_s-mediated stimulation of AC (Ammer and Schulz, 1993). Both findings indicate that the expression of AC supersensitivity depends on the presence of functional active G_s. Such a cooperatively acting mechanism would explain the phenomenon that the percentage increase in AC activity after opioid withdrawal is identical regardless of whether AC activity is measured under basal or stimulated conditions (Yu et al., 1990; Ammer and Schulz, 1997; Thomas and Hoffman, 1996).

The involvement of a synergistically acting component in the regulatory mechanism underlying AC supersensitivity does not conflict with the finding that opioid withdrawal fails to affect the K_d value of high-affinity [³H]forskolin binding. Because the complex between activated G_s and AC constitutes the high-affinity binding site for forskolin (Alouisi et al., 1991; Sutkowski et al., 1996), a change in the high-affinity binding state would be observed only when the binding of an additional regulator of AC alters the conformation of the G_s/AC complex. In this respect, Taussig et al. (1994) demonstrated that G_i and G_s subunits bind to different domains at the effector molecule. To investigate whether the interaction of G_i subunits with AC complex would alter the high-affinity binding state for forskolin, the effect of acute delta opioid receptor mediated inhibition of AC on high-affinity [³H]forskolin binding was tested. Our results demonstrate that inhibition of AC activity, which in naive NG108-15 hybrid cells is transduced via the inhibitory G protein G_i2 (McKenzie and Milligan, 1990), lacks any effect on both the maximum capacity and the affinity of PGE₁ receptor-stimulated high-affinity [³H]forskolin binding. This finding suggests that the interaction of G_i subunits with AC does not influence both the formation and conformation of G_s/AC complexes.

The present study further revealed that the expression of AC supersensitivity not only requires the presence of activated G_s per se but requires functional intact stimulatory receptor signaling. This notion is based on the observation that receptor-independent activation of AC by CTX treatment blocks the ability of naloxone to precipitate AC supersensitivity. In contrast to receptor-mediated activation of G_s, CTX-catalyzed ADP-ribosylation of G_s results in constitutive activation of G_s by inhibiting its intrinsic GTPase activity (Cassel and Selinger, 1977). Consequently, CTX treatment leads to the uncoupling of stimulatory receptors from subsequent signal transduction pathways as indicated by the failure of PGE₁ to further stimulate effector activity. From this we concluded that intact stimulatory receptor/G_s interaction may represent a critical step in the expression of AC supersensitivity. To test this notion experiments were performed in which the interaction between PGE₁ receptors and G_s was blocked with a C-terminal anti-G_s antibody (Ammer and Schulz, 1997). Abrogation of stimulatory AC signaling completely abolished the expression of AC supersensitivity, which demonstrates that the enhancement of AC activity requires acute receptor mediated activation and dissociation of G_s into G_s and G subunits. Because AC supersensitivity apparently does not reflect a direct G_s effect, this finding may suggest that G subunits, acutely released from receptor-activated G_s, are critical in the regulatory mechanism underlying AC supersensitivity. In this context, Thomas and Hoffman (1996) and Avidor-Reiss et al. (1996) recently have proposed that G subunits contribute to the development of AC supersensitivity by a yet unidentified indirect mechanism, because heterologous expression of G scavengers was found to prevent the induction of a cellular withdrawal sign.

In conclusion, the present study demonstrates that the stimulatory G protein is involved in the regulatory mechanisms underlying AC supersensitivity. Although the enhancement of AC activity during the state of opioid withdrawal is not a direct function of G_s, it was found to require stimulation of AC by receptor-activated G_s. These results indicate the involvement of an additional stimulatory regulator of AC in the development of AC supersensitivity.