Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

Investigations into cellular mechanisms underlying chronic opioid action revealed significant changes in the sensitivity of transmembrane AC signaling (for review see Johnson and Fleming, 1989; Childers, 1991). In general, prolonged exposure to an opioid produces two adaptational phenomena: desensitization of opioid receptor function (tolerance) and sensitization of AC (dependence) (Wüster et al., 1985). Opioid receptor desensitization has been investigated in great detail and represents a complex mechanism composed of multiple regulatory steps, such as phosphorylation (Pei et al., 1995), internalization (Law et al., 1984) and down-regulation (Law et al., 1983) of opioid receptors. There is also evidence for the involvement of additional post-receptor events in opioid tolerance, such as uncoupling of inhibitory signal transmission at the level of inhibitory G proteins (Vachon et al., 1987) and direct alteration of AC activity (Avidor-Reiss et al., 1995).

Sensitization of AC has been observed in various cell systems after chronic activation of opioid receptors, such as neuroblastoma × glioma NG108-15 hybrid cells (Sharma et al., 1975; Traber et al., 1975), human neuroblastoma SH-SY5Y cells (Yu et al., 1990; Ammer and Schulz, 1993a) and primary cultures of rat striatal neurons (Van Vliet et al., 1991), as well as in stably opioid receptor-transfected Chinese hamster ovary (Law et al., 1994; Avidor-Reiss et al., 1995) and Neuro_2A cells (Chakrabarti et al., 1995). At the cellular level, sensitization of AC is made manifest by a transient increase in enzyme activity after termination of chronic inhibitory opioid receptor action. This supersensitive AC response, also termed "cAMP overshoot" or "rebound phenomenon," represents a cellular correlate of opioid withdrawal and thus defines the state of opioid dependence (Sharma et al., 1975; Collier, 1984; Thomas and Hoffman, 1987). In an attempt to explore the underlying regulatory mechanism, we recently proposed an active counter-regulation at the level of excitatory AC-coupled signal transduction pathways as revealed by opioid-dependent NG108-15 (Ammer and Schulz, 1993b; 1995) and SH-SY5Y (Ammer and Schulz, 1996) cells. In these cells, up-regulation of stimulatory AC signaling is associated with a significant enhancement in PGE₁ receptor/G_s coupling, establishing a new equilibrium in the stimulatory control of AC. We initiated the present study in order to examine the underlying biochemical mechanisms more closely, using a cellular model that features the expression of a well-characterized endogenous stimulatory receptor system.

The human mammary epidermoid carcinoma A431 cell line, which carries a large number of functionally active beta-2 ARs (Delavier-Klutchko, 1984; Lohse, 1992), has been stably transfected with the recently cloned rat MOR cDNA (Chen et al., 1993) to allow investigation of chronic opioid-regulated beta-2 AR activity. The present report describes that long-term exposure of clonal A431/µ13 cells to morphine (10 µM; 2 d) induced cellular correlates of both opioid tolerance and opioid dependence. With respect to dependence, we found that sensitization of AC is characterized by an increased efficacybut not an increased potencyof beta-2 AR-stimulated AC. Up-regulation of stimulatory AC signaling is mediated by an increase in the steady-state levels of beta-2 ARs, which suggests that stimulatory receptor systems play an important role in the cellular mechanisms associated with opioid dependence.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Chemicals. The following drugs and chemicals were used in this study: [³H]diprenorphine (37 Ci/mmol) (DuPont/NEN, Dreieich, Germany), [¹²⁵I]-CYP (2200 Ci/mmol) and [³H]DAGO (Amersham Buchler, Braunschweig, Germany), [¹²⁵I]cAMP tracer (2000 Ci/mmol) (ICN Biomedicals, Meckenheim, Germany), anti-cAMP antiserum (BioMakor, Rehovot, Israel). The following ligands were used: R()-isoproterenol bitartrate, naloxone hydrochloride, S()-propranolol hydrochloride, ICI 118,551 (Research Biochemicals International, Köln, Germany) and DAGO (Bachem, Heidelberg, Germany). Forskolin, PTX, IBMX, and Ro 20-1724 were from Calbiochem (Bad Soden, Germany). Cell culture reagents and geneticin (G418) were purchased from Gibco/BRL (Karlsruhe, Germany). All other reagents were of analytical grade and were obtained from Sigma (Deisenhofen, Germany) or Merck (Darmstadt, Germany).

Cell culture, stable expression of the rat MOR in A431 cells and chronic opioid treatment. Human epidermoid A431 carcinoma cells were cultured in DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 IU/ml penicillin and 100 mg/ml streptomycin in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. A431 cells were transfected (DOTAP; Boehringer, Mannheim) with the expression vector pRc/CMV containing a full-length cDNA of the rat MOR (Chen et al., 1993). Cell colonies were isolated after selection with neomycin (G418, 800 µg/ml), and MOR expression was assessed by [³H]diprenorphine binding. The cell clone chosen for further analysis (A431/µ13) was maintained in growth medium supplemented with 200 µg/ml G418 and was routinely subcultured every 4 to 5 days after trypsination (0.05% trypsin, 0.02% EDTA) in the ratio . At 50% confluency, cells were subjected to chronic opioid treatment (morphine 10 µM; 2 d) unless otherwise indicated. In each experiment, untreated cells of the same passage, which were kept for 2 days in the absence of the opiate, served as controls. In some cases, chronically morphine-treated cells were coincubated with naloxone (10 µM) or PTX (16 ng/ml).

Membrane preparation. All steps were performed at 4°C. Cells were harvested and washed twice with PBS (10 min; 300 × g). They were resuspended in 10 volumes of homogenization buffer (5 mM Tris-HCl, pH 7.4, supplemented with 1 mM EDTA, 1 mM DTT, 1 mM benzamidine) and lysed using a Polytron (Brinkman Instruments) for 10 s (setting 6). The homogenate was centrifuged for 10 min at 1000 × g, the supernatant was recovered, and membranes were pelleted for 30 min at 18,000 × g. The pellet was washed once with 10 ml of the above buffer, resuspended in the appropriate assay buffers and immediately used in binding experiments. For use in AC assays, the membranes were resuspended in homogenization buffer at a concentration of 10 mg/ml and stored in aliquots at 80°C until used. Protein was determined by the method of Peterson (1983) using bovine serum albumin as standard.

Binding experiments. Total MOR levels were measured by saturation binding experiments using [³H]diprenorphine (0.01-2.5 nM) as tracer. Incubations (200 µl total volume) were for 30 min at 30°C in 50 mM Tris-HCl buffer (pH 7.4) containing 100 µg of membrane protein. Nonspecific binding was defined in parallel tubes with 10 µM naloxone. The high-affinity fraction of MOR was evaluated by homologous [³H]DAGO (2 nM) displacement experiments using a range of unlabeled competing DAGO (0.1 nM-10 µM). Reactions (90 min; 4°C) contained 50 µg of membrane protein in 50 mM Tris-HCl buffer, pH 7.4, supplemented with 5 mM MgCl₂. All binding reactions were terminated by rapid vacuum filtration through Whatman GF/B glass-fiber filters that had been pretreated with 0.05% polyethylenimine. The filters were extracted overnight in scintillation fluid before radioactivity was counted at 60% efficiency (Beckman LS 1801). All reactions were done in duplicate.

cAMP accumulation assay. A431/µ13 cells were seeded onto 24-well plates at a density of 10⁴ cells/well and allowed to grow overnight. Cells were kept for 2 more days either in the absence (control) or in the presence of 10 µM morphine. Thereafter, media were removed, cell layers were washed 2 times with prewarmed DMEM and subsequently incubated for 30 min at 37°C with DMEM containing 0.2 mM IBMX. In case of chronically opioid-treated cells, morphine was left in place at the concentration used for pretreatment. cAMP accumulation (10 min; 37°C) was determined after replacement of the medium by 250 µl of assay medium (DMEM and 0.2 mM IBMX, containing the appropriate receptor ligands as indicated). The reactions were stopped with 750 µl of ice-cold 0.05 N HCl, and intracellular cAMP was extracted for 30 min on ice. The amount of cAMP in the supernatant fraction was determined by radioimmunoassay according to Frandsen and Krishna (1977) after dilution of the samples (1/4-) in 50 mM sodium acetate (pH 6.0). Data are expressed in pmol cAMP/10⁶ cells/10 min.

AC assay. AC activity in A431/µ13 cell membranes was determined at 32°C for 10 min essentially as described by Vachon et al. (1987). Each reaction tube (100-µl volume) contained 10 µg of membrane protein in a buffer consisting of 40 mM Tris-HCl, pH 7.4, 0.2 mM EDTA, 0.2 mM DTT, 100 mM NaCl, 10 mM MgCl₂, 0.5 mM ATP, 5 µg/ml phosphocreatine, 5 IU/ml creatine phosphokinase, 10 µM GTP and 30 µM Ro 20-1724. Stimulation of AC was induced by ()-isoproterenol (1 nM-10 µM), whereas morphine (10 µM) was used to assess MOR-mediated inhibition of effector activity. The reactions were terminated with 500 µl of ice-cold 0.01 N HCl, and the amount of cAMP generated was measured as described above.

Western blot analysis of G_s. Determination of the relative abundance of G_s in membranes of A431/µ13 cells was performed as described (Ammer and Schulz, 1995). Briefly, 10 µg of membrane proteins were electrophoresed over 10% (vl/vl) polyacrylamide gels and subsequently transferred onto poly(vinylidene difluoride) membranes (Immobilon P; Millipore). The blots were probed with a subtype-specific anti-G_s antibody (S ) followed by a goat anti-rabbit alkaline phosphatase-conjugated antibody as secondary reagent. Immuncomplexes were visualized using 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium as the substrate. The intensity of the respective G_s bands was quantitated by video densitometry using the Herolab E.A.S.Y. RH-3 system.

Beta-2 AC/G_s coupling. The number of beta-2 AR-activated G_s molecules was evaluated in situ by the use of a C-terminal anti-G_s antibody (S ) that binds to a putative receptor recognition site on G_s and thus blocks receptor/G protein interaction (Simonds et al., 1989). In this assay, the amount of S antibodies required for half-maximal attenuation of ()-isoproterenol-stimulated AC provides an estimate of the relative beta-2 AR/G_s stoichiometry (Ammer and Schulz, 1996). Briefly, membranes (5 µg/tube) were incubated with various amounts of protein A-purified S antibodies diluted in 50 mM Tris-HCl buffer, pH 7.4, containing 150 mM NaCl, 10 mM MgCl₂ and 10 mg/ml preimmune rabbit IgG and were incubated for 2 h at 4°C before ()-isoproterenol (1 µM)-stimulated AC was determined. In case of morphine-treated membranes, the opiate (morphine; 10 µM) was included into the reaction mixtures during both antibody pretreatment and AC assay.

Data analysis. Radioligand binding data were analyzed according to the method of Scatchard (1949), which yielded estimates for maximal binding capacity (B_max) and ligand affinity (K_d). ED₅₀ values for ()-isoproterenol-stimulated AC and IC₅₀ values for S antibody-mediated inhibition of AC were determined by nonlinear least-squares regression curve fitting, using SigmaPlot (Jandell Scientific) software. Statistical differences were determined with Student's t test.

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

Stable expression of the rat MOR in A431 cells. A431 cells were transfected to express stably the rat MOR. One of the clones isolated (A431/µ13) was chosen for further studies because it expresses a physiologic amount of MOR (B_max = 302.9 ± 46 fmol/mg membrane protein, K_d = 1.3 ± 0.6 nM; n = 6). Nontransfected A431/µ13 cells showed no specific [³H]diprenorphine binding (fig. 1).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Binding parameters of stably MOR-expressing A431/µ13 cells. Scatchard analysis of [3H]diprenorphine binding in membranes prepared from clonal A431/µ13 cells. Data are from six experiments.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Functional coupling of stably expressed MOR to AC. Opioid inhibition of basal (A) or ()-isoproterenol (1 µM)-stimulated (B) cAMP accumulation was investigated in 431/µ13 cells. Cells were incubated either in the absence or in the presence of morphine (10 µM) alone or together with naloxone (10 µM). Cellular cAMP levels are expressed in picomoles per 10 min per 106 cells. The data shown are mean values ± S.D. of n = 6 experiments each performed in duplicate. *** Significantly different at P < .001 as compared with control cells.

Chronic opioid regulation of AC. To determine whether prolonged MOR activation in A431/µ13 cells produces cellular correlates of opioid tolerance and dependence, the cells were incubated for 2 days in the presence of 10 µM morphine. Chronic opioid treatment resulted in an almost complete loss of the inhibitory effect of morphine on ()-isoproterenol (1 µM)-stimulated cAMP accumulation (38.9 ± 5 vs. 9.4 ± 4% inhibition; mean values ± S.D.; n = 5; P < .001), even at a 10 times higher concentration of morphine (100 µM) than was originally used for pretreatment. This decreased responsiveness seems to be mediated by homologous desensitization of MOR function, because oxotremorine M (10 µM), which acts at the muscarinic cholinergic receptor, retains its full inhibitory effect (46.7 ± 6 vs. 48.2 ± 4% inhibition; mean values ± S.D.; n = 3) (fig. 3). Apart from opioid receptor desensitization, chronic exposure of the cells to morphine (10 µM; 2 d) also up-regulated the AC effector system, as demonstrated by an approximately 30% to 40% increase in both basal (0.85 ± 0.2 vs. 1.18 ± 0.1 pmol cAMP; mean values ± S.D.; P < .05; n = 6 and n = 4 independent experiments, respectively) and ()-isoproterenol (1 µM)-stimulated cAMP generation (148.6 ± 6.7 vs. 201.3 ± 8.5 pmol cAMP; mean values ± S.D.; n = 6; P < .001). Most interestingly, these increased cAMP levels occur despite the presence of the inhibitory opioid given chronically (fig. 4A).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Desensitization of MOR from AC inhibition following chronic morphine pretreatment. A431/µ13 cells were grown for 2 days either in the absence (naive) or in the presence (chronic morphine) of morphine (10 µM) before the acute inhibitory effects of morphine () and oxotremorine M (10 µM, ) on ()-isoproterenol (1 µM)-stimulated cAMP accumulation were determined. The morphine concentrations used were 10 µM for naive and 100 µM for opioid tolerant cells, respectively. Attenuation of intracellular cAMP generation is expressed in percent of 1 µM ()-isoproterenol-stimulated cAMP levels, which were set 100%. All values represent the mean ± S.D. of five (morphine) and three (oxotremorine M) individual experiments. *** Significantly different at P < .001 as compared with isoproterenol alone.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effect of chronic morphine treatment on beta-2 AR-stimulated AC. A) Increased capacity of ()-isoproterenol (1 µM) to stimulate intracellular cAMP generation in chronically morphine-pretreated (10 µM; 2 d) A431/µ13 cells. Morphine-dependent cells were examined in the presence of the opioid given chronically. The data shown are mean values ± S.D. of six (control) and four (morphine exposed cells) experiments, each carried out in duplicate determination. *** Significantly different at P < .001 from controls. B) Dose-response relationship of ()-isoproterenol-stimulated AC regulation in membranes derived from control () or chronically morphine-pretreated () A431/µ13 cells. Beta-2 AR-regulated AC activity was determined as described in "Materials and Methods." AC activity is expressed in picomoles of cAMP formed per milligram of membrane protein per minute. The data shown are mean values ± S.D. of three independent experiments. Vertical bars indicate calculated ED50 values.

Characterization of chronic opioid-induced up-regulation of AC. We first determined whether up-regulation of AC is associated with an increase in the sensitivity of beta-2 AR-stimulated AC activity. For this purpose, ()-isoproterenol dose-response curves were established in particulate membrane preparations. Chronic morphine treatment (10 µM; 2 d) led to an approximately 28% increase in the maximum capacity of beta-2 AR-stimulated AC, whereas no statistically significant change in the ED₅₀ value for ()-isoproterenol was observed (ED₅₀ = 79.6 ± 21 and 62.5 ± 12 nM for control and chronically morphine-exposed cells, respectively; mean values ± S.D., n = 3) (fig. 4B). Thus chronic opioid-induced up-regulation of AC in A431/µ13 cells is characterized by increased functional capacity rather than increased sensitivity of beta-2 AR signaling.

View this table: [in this window] [in a new window]   TABLE 1 Blockade of chronic opioid-induced increase of AC activity in A431/µ13 cells by PTX and naloxone A431/µ13 cells were kept either in the absence (no pretreatment) or in the presence of various ligands and substances at the concentrations and times indicated. Subsequently, basal or ()-isoproterenol (1 µM)-stimulated cAMP levels were determined. All data are mean values ± S.D. from four experiments. Percentage change in intracellular cAMP accumulation after chronic morphine pretreatment as compared with untreated controls is given in parentheses.

Regulation of stimulatory beta-2 adrenoceptors by chronic morphine treatment. To investigate more closely the cellular adaptations causing the increase in stimulatory AC signaling, we examined regulation of beta-2 ARs. [¹²⁵I]-CYP saturation binding experiments in naive A431/µ13 cells revealed a B_max value of 78.5 ± 4.6 fmol/mg membrane protein with a K_d value of 22.4 ± 3.2 nM (mean values ± S.D.; n = 6). In response to chronic morphine exposure (10 µM; 2 d) beta-2 AR levels increased by some 40% (B_max = 109.4 ± 16.6 fmol/mg membrane protein) without any change in drug affinity (K_d = 22.9 ± 2.8 nM; mean values ± S.D.; n = 4). A representative Scatchard plot is given in figure 5. Chronic morphine treatment had no effect on the concentrations of postreceptor components of the stimulatory branch of AC. As shown in Figure 6, immunoblot analysis of membrane proteins from A431/µ13 cells revealed the presence of two G_s isoforms that had relative molecular weights of 45 and 48 kDa, respectively, the smaller form being more prominent. Moreover, chronic exposure of the cells to morphine (10 µM; 2 d) failed to affect the abundance of G_s as verified by video densitometry of the bands (100 vs. 96.2 ± 7.3% for the 45-kDa form; 100 vs. 99.8 ± 2.3% for the 48-kDa form; mean values ± S.D.; n = 3). The overall capacity of AC was determined in the presence of 5.6 mM Mn⁺⁺ and 100 µM forskolin, which directly activate the catalytic moiety of the effector enzyme (May et al., 1985). Again, no apparent change in total AC activity could be detected after chronic morphine treatment of the cells (385.1 ± 27.7 vs. 382.5 ± 24.3 pmol of cAMP/min/mg membrane protein for control and opioid-dependent cells, respectively; mean values ± S.D.; n = 3).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Chronic morphine regulation of stimulatory beta-2 AR number. Scatchard analysis of [125]I-CYP binding data determined with membranes from control () or chronically morphine-pretreated ()-A431/µ13 cells. The experiment shown is representative for six (naive) and four (morphine-pretreated cells) separate experiments, all yielding similar results.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Chronic morphine treatment fails to affect the steady-state levels of Gs. A431/µ13 cells were incubated either in the absence or in the presence of morphine (10 µM; 2 d). Cell membranes (10 µg/lane) were resolved by 10% (w/v) sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotted using the S1/3 antibody at a dilution of 1/5000. Subsequently, the blot was incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG as secondary antibody. Shown is a representative blot. Arrows indicate the 45- and 48-kDa isoforms of Gs.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Functional coupling efficiency between beta-2 ARs and Gs. The functional beta-2 AR/Gs stoichiometry was investigated in situ by application of a C-terminal anti-Gs antibody that blocks receptor-mediated activation of Gs and thus of AC activity. Membranes from control () or chronically morphine (10 µM; 2 d)-pretreated () A431/µ13 cells were incubated on ice for 2 h with increasing amounts of protein A-purified S antibodies diluted in a total of 10 mg/ml preimmune IgG as described in "Materials and Methods." Thereafter, beta-2 AR-stimulated AC activity was determined at 32°C for 10 min. Isoproterenol (1 µM)-stimulated enzyme activity obtained in the absence of specific antibody was set as 100% for each control and morphine-treated cells separately. In the experiment shown, maximal ()-isoproterenol (1 µM)-stimulated adenylate cyclase activity was 45.3 ± 2.6 and 57.9 ± 3.1 pmol of cAMP/min/mg membrane protein (mean value ± variation of triplicate determination) for control and chronically morphine-exposed cells, respectively. IC50 values were 10.5 (control) and 8.4 (chronic morphine) µg S antibodies/5 µg membrane protein. The data shown are the mean values of a representative experiment that was repeated three times with similar results.

View this table: [in this window] [in a new window]   TABLE 2 Reversal of chronic morphine-induced up-regulation of AC by an inverse beta-2 AR agonist Basal cAMP accumulation was determined in control and chronically morphine-pretreated A431/µ13 cells either in the absence or in the presence of ICI 118,551 or ICI 118,551 together with ()-propranolol. Percentage reduction of basal AC activity by ICI 118,551 activity is given in brackets. All data are mean values ± S.D. from four or six experiments.

Opioid withdrawal failed to produce a supersensitive AC response. Opioid dependence is usually accompanied by the generation of a cAMP overshoot upon removal of the inhibitory drug given chronically. In morphine-dependent A431/µ13 cells, however, termination of prolonged MOR activation by naloxone (100 µM) fails to elicit a further increase in AC activity (fig. 8A). Removal of morphine by extensive washing of the cells also failed to induce a withdrawal response [()-isoproterenol (1 µM) plus morphine (10 µM), 181.8 ± 9.3; after washout of morphine, 204.9 ± 20.1 pmol cAMP/10⁶ cells/10 min; n = 3]. Because it is assumed that a minimum number of functionally G protein-coupled opioid receptors is required to trigger changes in intracellular signaling processes enhancing AC activity (Law et al., 1994), the number of functionally active, high-affinity MORs was determined. Homologous displacement experiments in A431/µ13 cells using the mu agonist [³H]DAGO (2 nM) as the tracer revealed the presence of only 40.2 ± 8.2 fmol of high-affinity binding sites/mg membrane protein (K_d = 2.7 ± 1.1 nM; n = 4). This value is only some 13% of that obtained by [³H]diprenorphine binding. In addition, we observed no effect of chronic morphine treatment on the proportion of high-affinity [³H]DAGO binding (B_max = 40.3 ± 9.4 fmol/mg membrane protein; K_d = 3.1 + 1.2 nM; n = 4), which indicates that chronic morphine-induced desensitization of MOR function in A431/µ13 cells is not accompanied by MOR down-regulation. Because of the low number of G protein-coupled MOR in A431/µ13 cells, precipitation of a cAMP overshoot was investigated in a high-MOR-expressing cell clone (A431/µ2) that carries 1.02 ± 0.1 pmol of [³H]DAGO binding sites. Although chronic exposure of the cells to morphine (10 µM; 2 d) had no effect on high-affinity [³H]DAGO binding (B_max = 1.03 ± 0.1 pmol receptors/mg protein; K_d = 1.7 ± 0.3 nM), again withdrawal of the opioid failed to increase intracellular cAMP accumulation further in A431/µ2 cells. This suggests that MOR-expressing A431 cells per se are not capable of producing a supersensitive AC response (fig. 8B).

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Sensitization of AC in stably MOR-expressing A431 cells is not associated with the production of a cAMP overshoot phenomenon upon naloxone-precipitated withdrawal. The effect of opioid withdrawal on intracellular cAMP accumulation was investigated in two different A431 cell clones that expressed different amounts of functionally G protein-coupled opioid receptors. A431/µ13 (40.2 fmol; panel A) and (B) A431/µ2 cells (1.02 pmol high-affinity receptors/mg membrane protein; panel B) were kept in the absence or in the presence of morphine (10 µM) for 2 days before ()-isoproterenol (1 µM)-stimulated cAMP generation was determined. Morphine-dependent cells were measured either in the presence of morphine (10 µM) or after naloxone (100 µM)-precipitated withdrawal. cAMP levels are expressed in picomoles produced in 10 min per 106 cells. Each data point represents the mean value ± S.D. from three separate experiments performed in duplicate.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

The aim of the present study was to investigate the function of stimulatory receptor systems in opioid-dependent cells. For this purpose, recently cloned rat MOR (Chen et al., 1993) was stably transfected into human mammary epidermoid carcinoma A431 cells, which feature the expression of an endogenous, and hence functional competent, stimulatory beta-2 AR system (Delavier-Klutchko et al., 1984). Chronic morphine treatment (10 µM; 2 d) of clonal A431/µ13 cells (B_max = 302.9 fmol/mg protein; K_d = 1.3 nM) significantly increased the capacity of stimulatory AC signaling, a phenomenon found to be associated with a 40% increase in the number of excitatory beta-2 ARs. These results provide evidence for a compensatory adaptation within the stimulatory branch of AC in response to chronic inhibitory opioid treatment, a mechanism previously postulated by Collier (1980) on the basis of theoretical considerations.

Sensitization of AC is thought to represent a cellular correlate of opioid dependence and has been observed in a variety of neuronal cell lines (Sharma et al., 1975; Yu et al., 1990), as well as in distinct areas of the CNS (Nestler, 1992), after chronic morphine treatment. In most tissues, this chronic opioid effect is characterized by a transient increase in AC activity upon withdrawal of the opioid. An interesting finding of the present study is that chronic morphine treatment of A431/µ13 cells increases AC activity during the course of chronic inhibitory opioid treatment, whereas induction of opioid withdrawal does not further amplify AC activity. The failure of A431/µ13 cells to exhibit a cAMP overshoot upon opioid withdrawal does not necessarily imply the absence of a dependent state, because a withdrawal sign represents only one criterion by which drug dependence can be recognized (Collier, 1984). For example, both primary cultures of rat striatal neurons (Van Vliet et al., 1991) and the rat locus ceruleus (Duman et al., 1988) have been judged opioid-dependent because they display an increased AC response toward excitatory stimuli. Similarly, chronic etorphine treatment also failed to elicit a cAMP overshoot in neuroblastoma N18TG2 cells under conditions where a high degree of tolerance was established (Law et al., 1982). Thus up-regulation of AC as observed in A431/µ13 cells during the course of chronic opioid treatment is suggested to represent a homeostatic principle associated with opioid dependence.

Chronic opioid-induced sensitization of AC in A431/µ13 cells is not associated with an increased potency of beta-2 AR-stimulated AC activity. This finding confirms previous data from opioid-dependent neuronal cell lines that partially exhibit an increased capacity of stimulatory AC signaling (Yu et al., 1990; Van Vliet et al., 1991; Ammer and Schulz, 1993a; Avidor-Reiss et al., 1995), whereas no change (Traber et al., 1975) or even a decrease (Ammer and Schulz, 1996) in the sensitivity of AC toward excitatory drugs was observed. Thus, on a cellular level, opioid dependence is characterized by an increased capacity, rather than an elevated sensitivity, of stimulatory AC signal transduction pathways.

Sensitization of AC in A431/µ13 cells appears to be mediated by up-regulation of stimulatory beta-2 ARs. This interpretation is based on two different observations: First, the increase in basal cAMP accumulation could be completely reversed by the "inverse beta-2 AR agonist" ICI 118,551 (Samama et al., 1994). Second, determination of the relative beta-2 AR/G_s stoichiometry by application of a C-terminal anti-G_s antibody (Simonds et al., 1989; Ammer and Schulz, 1996) revealed an increase in the number of functional beta-2 AR/G_s complexes during the state of opioid dependence. The latter finding suggests that the number of stimulatory beta-2 ARs is directly related to the magnitude of AC activation. Consequently, the increase in beta-2 AR number observed in response to chronic morphine treatment is likely to contribute to up-regulation of AC in A431/µ13 cells. According to receptor theory, variation of receptor number should also affect the potency of AC activation (Whaley et al., 1994). However, in the present study, we failed to demonstrate a leftward shift in the ED₅₀ value of ()-isoproterenol-stimulated AC. This discrepancy might be due to the fact that in A431/µ13 cells, the beta-2 AR is limiting within the stimulatory flow cascade, so an increase in receptor number preferentially results in the elevation of AC capacity. Although there is currently no information about the stoichiometry of stimulatory signal transduction components in this cell system, the functional capacity of both G_s and AC seems to be far in excess of that of excitatory beta-2 ARs, because maximal forskolin-stimulated cAMP levels are much higher than those obtained with ()-isoproterenol (unpublished observation).

The failure of naloxone to precipitate a cAMP overshoot phenomenon in opioid-dependent A431/µ13 cells clearly demonstrates that inactivation of the opioid receptor per se is insufficient to elicit a supersensitive AC response. It follows that simple elimination of persistent AC inhibition, as proposed by Griffin et al. (1985), is unlikely to account for the withdrawal sign. Thus manifestation of a cAMP overshoot requires additional biochemical events on the postreceptor level that result in amplification of AC activity. Though we can only speculate at this time about the nature of such a mechanism, our recent work demonstrated an increased functional coupling efficiency between PGE₁ receptors and G_s during the state of opioid dependence (Ammer and Schulz, 1995; 1996). Because receptor/G protein coupling represents a critical step in the amplification of receptor-generated signals (Gilman, 1987), alteration in the activity state of G_s may contribute to sensitization of AC. In this context, the present finding of an unaltered beta-2 AR/G_s interaction in chronically morphine-treated A431/µ13 cells might explain the absence of a cAMP overshoot in these cells.

It has been reported that the induction of a cAMP withdrawal response requires the presence of a minimum number of functional active opioid receptors (Law et al., 1994). Thus the failure of naloxone to precipitate AC supersensitivity in A431/µ13 cells could be due to a loss of high-affinity opioid receptors during the course of chronic morphine treatment. Reduction of opioid receptor number would not necessarily exclude the establishment of dependence, because the agonist concentrations required to induce sensitization of AC have been reported to be much lower than those that produce receptor desensitization (Chakrabarti et al., 1995). Although chronic morphine treatment had no effect on overall MOR number in A431/µ13 cells, the capacity of high-affinity receptors was surprisingly low (~40 fmol/mg membrane protein). We therefore examined a second cell clone (A431/µ2) that expresses much higher levels of G protein-coupled µ receptors (~1.0 pmol/mg membrane protein). However, as in A431/µ13 cells, opioid withdrawal failed to elicit a cAMP overshoot in these cells. In addition, chronic morphine treatment had no effect on high-affinity [³H]DAGO binding in A431/µ2 cells, which indicates that uncoupling of the opioid receptors is unlikely to account for the lack of naloxone to precipitate a cAMP withdrawal response. This notion supports the general theory that desensitization of opioid receptor function and sensitization of AC represent two separate cellular adaptation processes (Wüster et al., 1985).

In conclusion, the present study shows that long-term exposure of stably MOR-transfected human mammary epidermoid carcinoma A431 cells to morphine counter-regulates the stimulatory beta-2 AR system, resulting in an overall enhanced AC activity. These results directly demonstrate a critical role of excitatory receptor systems in the cellular mechanisms underlying opioid dependence. Moreover, they may provide a mechanistic background for the use of beta adrenergic antagonists in the control of aversive effects during opioid withdrawal (Harris and Aston-Jones, 1993; Funada et al., 1994).