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NEUROPHARMACOLOGY

Converging Protein Kinase Pathways Mediate Adenylyl Cyclase Superactivation upon Chronic -Opioid Agonist Treatment

Departments of Pharmacology (E.V.V., M.K.R., D.S., M.S., V.G. W.R.R., H.I.Y.), Biochemistry and Psychiatry (H.I.Y.), Medicine (W.R.R.), and Chemistry (V.J.H.), and the Sarver Heart Center (E.V.V., W.R.R., H.I.Y.), The University of Arizona Health Sciences Center, Tucson, Arizona; and National Institute of Diabetes and Digestive and Kidney Diseases, Laboratory of Medicinal Chemistry (K.C.R.), National Institutes of Health, Bethesda, Maryland

Received January 27, 2003; accepted March 20, 2003.

	Abstract

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Adenylyl cyclase (AC) superactivation is thought to play an important role in opioid tolerance, dependence, and withdrawal. In the present study, we investigated the involvement of protein kinases in chronic -opioid agonist-mediated AC superactivation in Chinese hamster ovary (CHO) cells stably expressing the human -opioid receptor (hDOR/CHO). Maximal forskolin-stimulated cAMP formation in hDOR/CHO cells increased by 472 ± 91, 399 ± 2, and 433 ± 73% after chronic treatment with the -opioid agonists (+)-4-[(R)--((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxy-benzyl]-N,N-diethyl benzamide (SNC 80), [D-Pen²,D-Pen⁵]-enkephalin, and deltorphin II, respectively. Concurrently, chronic SNC 80 (1 µM, 4-h) treatment augmented ³²P incorporation into a 200-kDa protein immunoreactive with the ACV/VI antibody by 300 ± 60% in hDOR/CHO cell lysates. The calmodulin antagonist calmidazolium significantly attenuated chronic deltorphin II-mediated AC superactivation. Tyrosine kinase (genistein) and protein kinase C (chelerythrine) inhibitors individually had minimal effect on chronic -opioid agonist-mediated AC superactivation. Conversely, simultaneous treatment with both genistein and chelerythrine significantly attenuated AC superactivation. Because we showed previously that the Raf-1 inhibitor 3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one (GW5074) attenuates AC superactivation, we hypothesize that parallel calmidazolium-, chelerythrine-, and genistein-sensitive pathways converge at Raf-1 to mediate AC superactivation by phosphorylating AC VI in hDOR/CHO cells.

We have reported previously that in Chinese hamster ovary (CHO) cells stably expressing the human -opioid receptor (hDOR/CHO), chronic -opioid agonist treatment gives rise to AC superactivation (Malatynska et al., 1996). In addition, we also demonstrated that chronic -opioid agonist treatment of the hDOR/CHO cells augments ³²P incorporation into proteins immunoreactive with an AC V/VI-specific antibody (Varga et al., 1999). The identity of protein kinase(s) involved in chronic -opioid agonist-mediated phosphorylation in hDOR/CHO cells, and the role of the phosphorylation in AC superactivation, however, have not been investigated previously. In the present work, we studied the effect of protein kinase inhibitors on chronic -opioid agonist-mediated AC superactivation.

Recent data (Tan et al., 2001) indicate the important role of the protein kinase p74Raf-1 in phosphorylation of adenylyl cyclase VI in transfected human embryonic kidney 293 cells. Moreover, Raf-1-mediated phosphorylation led to the sensitization of AC VI to subsequent stimulation by forskolin or G_s. The investigators (Tan et al., 2001) have also demonstrated that a dominant negative Raf-1 construct (NRaf) attenuates both phosphorylation and sensitization of AC VI. Interestingly, we found previously that AC VI is the major adenylyl cyclase isoenzyme in CHO cells and that a selective inhibitor of Raf-1 (GW5074) significantly attenuates chronic deltorphin II treatment-mediated adenylyl cyclase superactivation in hDOR/CHO cells (Varga et al., 2002).

Raf-1 is the key protein kinase in the p42/44 mitogen-activated protein kinase signal transduction cascade. It has been suggested that -opioid receptors activate the p42/44 mitogen-activated protein kinase (MAPK) cascade using multiple, parallel signal transduction pathways in Chinese hamster ovary cells (Fukuda et al., 1996). In the present work, we used selective protein kinase inhibitors to demonstrate that similar, redundant signal transduction pathways are involved in the molecular mechanisms of AC superactivation in hDOR/CHO cells.

	Materials and Methods

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Chemicals. Calmidazolium was purchased from Calbiochem (San Diego, CA), genistein was from Tocris Cookson, Inc. (Ellisville, MO), and chelerythrine and 3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one (GW5074) were from Sigma-Aldrich (St. Louis, MO). (+)-4-[(R)--((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxy-benzyl]-N,N-diethyl benzamide (SNC 80) was synthesized at the National Institutes of Health (Bethesda, MD), in the laboratory of Kenner C. Rice. [D-Pen²-D-Pen⁵]-enkephalin (DPDPE) and deltorphin II ([D-Ala²]deltorphin II) were synthesized at the University of Arizona (Tucson, AZ), in the laboratory of Victor J Hruby. [³²P]Orthophosphate (3000 Ci/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA). All other compounds were obtained from commercial sources.

Construction of the hDOR/CHO Cell Line. The cDNA encoding the hDOR was identified and cloned in our laboratory (Knapp et al., 1994). CHO cells were transfected with the cloned hDOR inserted into the pREP10 mammalian expression vector to produce the hygromycin-resistant hDOR/CHO cell line (Malatynska et al., 1995). Radioligand binding experiments were used to characterize the cell line. The K_d value for [³H]naltrindole was 139 pM with a B_max value of 968 fmol/mg protein (Malatynska et al., 1996). The selective -opioid agonist SNC 80 inhibited forskolin-stimulated cAMP formation in hDOR/CHO cells with an EC₅₀ value of 1.3 ± 0.4 nM (n = 9) (E_max: 6 ± 2% of control) (Rubenzik et al., 2001).

Drug Pretreatment. hDOR/CHO cells were incubated (4–24 h, 37°C) in the presence or absence of SNC 80 (10 nM–1 µM) or deltorphin II (100 nM). The kinase inhibitors chelerythrine (5 µM), calmidazolium (0.5 µM), or genistein (100 µM) were added 30 to 60 min before starting the agonist treatment. The cells were washed in IMDM three times (15 min, 37°C) before forskolin-stimulated cAMP formation-, or ³²P incorporation into AC VI was determined.

Measurement of Forskolin-Stimulated cAMP Formation. After chronic drug treatment, the cells were washed three times (15 min each) with fresh IMDM. The IMDM was then aspirated and replaced with 5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich) in IMDM. Adenylyl cyclase was stimulated with 100 µM water-soluble forskolin (7-deacetyl-7-(O-N-methylpiperazino)--butyryl, diHCl) (Calbiochem). The cells were incubated in a humidified incubator at 37°C (5% CO₂), for 20 min. The reaction was terminated by replacing the medium with ice-cold Tris-EDTA buffer (50 mM Tris-HCl, 4 mM EDTA, pH 7.5). The cells were lysed by boiling (10 min) and centrifuged. Then 50 µl of the supernatant was incubated with 4 nM [³H]cAMP (PerkinElmer Life Sciences) and 30 µg/ml protein kinase A (Sigma-Aldrich). Serial dilutions of cAMP were run in parallel to obtain a cAMP standard curve. After a 2-h incubation at 4°C, activated charcoal (26 mg/ml) (NORIT, Amersfoort, The Netherlands) was added to adsorb free cAMP. The mixture was then centrifuged and 200 µl of the supernatant was counted in EcoLite (ICN Pharmaceuticals, Costa Mesa, CA) scintillation fluid.

Phosphorylation of Adenylyl Cyclase VI in the hDOR/CHO Cells after Chronic -Opioid Agonist (SNC 80) Treatment. A previously described (Varga et al., 1999) metabolic labeling/immunoprecipitation method was used to measure ³²P incorporation into the protein band immunoreactive with an ACV/VI-specific antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Briefly, hDOR/CHO cells were phosphate-starved and labeled with [³²P]orthophosphate. In experiments where specific protein kinase inhibitors were used, the inhibitors were added together with the [³²P]orthophosphate. After a 1-h preincubation, SNC 80 (10 nM–1 µM) was added and the incubation continued for 1–24 h. After the agonist treatment, the cells were thoroughly washed and homogenized in solubilization buffer (50 mM Tris, 250 mM sucrose, 1 mM EDTA, 5 mM MgCl₂, 1 mM dithiothreitol, 50 mM NaF, 10 mM Na-pyrophosphate, and 10 µl/ml protease inhibitor cocktail, 0.2 mM Na-orthovanadate, and 100 nM okadaic acid, pH 7.4). The lysate was centrifuged, precleared by incubating in the presence of 1 µg of preimmune rabbit IgG, and 10 µl of protein A-agarose for 1 h. After centrifugation, the precleared lysate was incubated with the ACV/VI-specific antibody and protein A-agarose beads for 4 h. After thorough washes in solubilization buffer, with detergent concentrations reduced to 0.075% Triton X-100, 0.05% Igepal CA-630 and 0.1% digitonin (4°C), the immuno-complexes, were eluted in glycine-Cl buffer (pH 2.3), neutralized, solubilized in Laemmli buffer, and resolved on 7.5% SDS-PAGE. The gel was silver-stained, dried, and subjected to autoradiography. The amounts of immunoprecipitated protein, and the extent of ³²P incorporation, were quantified by scanning densitometry of the silver stained gel and the autoradiography film, respectively.

Data Analysis. Radioligand binding data were analyzed by nonlinear regression, using the Prism (version 3.0) computer program. Data are represented as mean ± S.E.M., unless otherwise indicated. Standard statistical tests, such as one-way ANOVA followed by Dunnett's multiple comparison tests, were used to determine statistical differences between treatment groups (*p < 0.05, **p < 0.01, and ***p < 0.001).

	Results

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Effect of Chronic -Opioid Agonist Treatment on Forskolin-Stimulated cAMP Formation in hDOR/CHO Cells. We have demonstrated previously (Malatynska et al., 1996; Rubenzik et al., 2001) that chronic (4–24 h) SNC 80 (100–1000 nM) treatment of hDOR/CHO cells significantly increases forskolin-stimulated cAMP formation after the removal of the agonist. In the present work, we tested the ability of other, structurally different -opioid agonists to produce adenylyl cyclase superactivation upon chronic treatment in hDOR/CHO cells. As seen in Fig. 1, chronic (4-h) treatment of the hDOR/CHO cells with 100 nM SNC 80; DPDPE, or deltorphin II augmented forskolin (100 µM)-stimulated cAMP formation to 472 ± 91% (n = 10); 399 ± 2% (n = 4), and 433 ± 73% (n = 13) of the IMDM-treated control, respectively. Basal cAMP formation was not significantly different between IMDM-treated and agonist-treated hDOR/CHO cells [5.8 ± 4.4 (n = 24) and 6.3 ± 14 (n = 22) pmol/million cells, respectively, p > 0.5]. The selective -opioid antagonist naltrindole (1 µM) attenuated deltorphin-mediated adenylyl cyclase superactivation (forskolin-stimulated cAMP formation: 123 ± 2% of control, p > 0.5). Statistical significances (p < 0.001) were determined using one-way ANOVA followed by Dunnett's multiple comparison test.

View larger version (40K): [in this window] [in a new window]   Fig. 1. Maximal forskolin-stimulated cAMP formation in hDOR/CHO cells after chronic treatment with different -opioid agonists and/or naltrindole. Pretreatment (4 h) of hDOR/CHO cells with 100 nM SNC 80, DPDPE, or deltorphin II augmented forskolin (100 µM)-stimulated cAMP formation by 472 ± 91% (n = 10), 399 ± 2% (n = 4), and 433 ± 73% (n = 13) of IMDM-treated control, respectively. Maximal forskolin-stimulated cAMP formation after pretreatment of hDOR/CHO cells with deltorphin II (100 nM) in the presence of naltrindole (1 µM), on the other hand, was not significantly different from buffer-treated control cells (123 ± 2% of IMDM-treated control).

Phosphorylation of Adenylyl Cyclase VI in the hDOR/CHO Cells upon Chronic -Opioid Agonist (SNC 80) Treatment. It was shown previously that chronic opioid treatment-mediated AC superactivation is isoenzyme-specific (Avidor-Reiss et al., 1997). AC VI was one of the few AC isoenzymes that became sensitized to forskolin stimulation upon chronic opioid receptor stimulation (Avidor-Reiss et al., 1997). We have previously determined that AC VI is a major native isoenzyme in CHO cells (Varga et al., 1998). Thus, we hypothesized that AC VI is that major isoenzyme involved in forskolin-stimulated cAMP overshoot upon chronic -opioid agonist treatment in the hDOR/CHO cells.

Because the catalytic activity of adenylyl cyclase isoenzymes is frequently regulated by protein kinases (Ishikawa, 1998), we tested the effect of chronic -opioid agonist treatment on the phosphorylation state of AC VI in hDOR/CHO cells. A metabolic labeling/immunoprecipitation method, followed by SDS-PAGE, was used to measure ³²P incorporation into AC VI upon chronic SNC 80 treatment, as described previously (Varga et al., 1999). Two bands, with apparent molecular masses of about 130 and 200 kDa, were routinely obtained on both the silver-stained gel and the autoradiography film. Figure 2 shows a representative autoradiography film obtained after SNC 80 treatment of hDOR/CHO cells for 4 h with increasing doses of the drug. The 200-kDa band is presumably the glycosylated form of the 130-kDa protein, because only the 130-kDa band was apparent on the gel when after the immunoprecipitate was pretreated with N-aminoglycosidase F (data not shown). Immunoprecipitation was prevented by saturation of the antibody with a blocking peptide (Santa Cruz Biotechnology, Inc.). The optical densities of the bands in the silver-stained gel (protein content) and in the autoradiography film (³²P incorporation) were measured by scanning densitometry. Because a similar protein/phosphate ratio was measured for the 130- and the 200-kDa bands in initial experiments, we routinely used the 200-kDa band to quantitate phosphorylation. Data in Figs. 2 and 3 show the density ratios (³²P incorporation/protein stain) calculated as the percentage of the IMDM-treated control. Chronic (4-h) SNC 80 treatment-mediated phosphorylation of AC VI was at a maximal level (557% of untreated control, range 400–714, n = 2) at 100 nM SNC 80 concentrations (Fig. 2). Figure 3 shows a time course for SNC 80 (1 µM)-mediated ³²P incorporation into the 200-kDa band. Phosphorylation was maximal (303 ± 61% of control, p < 0.001, n = 7) after 4-h treatment. Phosphorylation of AC VI remained at similar levels upon longer (up to 24 h, 204 ± 29%, p < 0.01, n = 3) treatment.

View larger version (30K): [in this window] [in a new window]   Fig. 2. Dose-response of chronic (4 h) SNC 80-mediated phosphorylation of AC VI: hDOR/CHO cells were metabolically labeled with 200 µCi/ml [32P]orthophosphate and treated with increasing concentrations of SNC 80 for 4 h. Cell lysates were immunoprecipitated by an AC V/VI-specific antibody and protein A-agarose. The immunoprecipitated proteins were resolved on 7.5% SDS-PAGE. The gels were silver-stained and subjected to autoradiography. The figure shows a representative autoradiogram. The locations of the molecular weight standards on the corresponding silver-stained gel are indicated by arrows. Optical densities in the silver-stained gel (protein content) and in the autoradiography film (32P incorporation) were measured for the 200-kDa band using scanning densitometry. 32P incorporation was normalized for the amount of protein by calculating a ratio between the measured optical densities. The values underneath each SNC 80 dose show the range and the mean of the normalized 32P incorporation, calculated as percentage of IMDM-treated control (n = 2).

View larger version (15K): [in this window] [in a new window]   Fig. 3. Time course of SNC 80-mediated phosphorylation of AC VI: hDOR/CHO cells were metabolically labeled with 200 µCi/ml [32P]orthophosphate before treatment with SNC 80 (1 µM) for different time intervals. The cell lysates were immunoprecipitated, and the immunoreactive proteins were measured as described in the legend for Fig. 2. The density ratio (32P incorporation/protein) increased to 303 ± 61% of IMDM-treated control (n = 7) after 4 h SNC 80 pretreatment, and remained at a similar level upon longer (up to 24 h, 204 ± 29%, n = 3) treatment.

Effect of Protein Kinase Inhibitors on Chronic SNC 80-mediated AC Superactivation in hDOR/CHO Cells. In the next set of experiments, we investigated the effect of selective protein kinase inhibitors on SNC 80-mediated adenylyl cyclase superactivation. The calmodulin antagonist calmidazolium completely attenuated chronic SNC 80-mediated AC superactivation (Fig. 4). Forskolin-stimulated cAMP formation in calmidazolium (0.5 µM)-pretreated hDOR/CHO cells was similar after IMDM or chronic SNC 80 treatment (108 ± 3% of IMDM-treated control, n = 3). It should be noted, however, that there was a considerable difference in maximal forskolin-stimulated cAMP formation in the control IMDM- and calmidazolium-treated hDOR/CHO cells (669 ± 140 and 1821 ± 109 pmol/million cells, respectively, p < 0.001) in the absence of SNC 80. The protein kinase C (PKC) inhibitor chelerythrine, on the other hand, had only a minimal effect on cAMP overshoot in -opioid agonist-treated hDOR/CHO cells (Fig. 5). Forskolin-stimulated cAMP formation was 375 ± 87% (p > 0.1, n = 7).

View larger version (18K): [in this window] [in a new window]   Fig. 4. Effect of calmidazolium pretreatment on forskolin-stimulated cAMP formation in SNC 80-treated hDOR/CHO cells: forskolin-stimulated cAMP formation after chronic treatment with SNC 80 (100 nM, 4 h) was 180 ± 11% of IMDM-treated control hDOR/CHO cells at maximal (100 µM) forskolin concentration (, p < 0.05, n = 3). Conversely, after pretreatment (30 min) in the presence of 0.5 µM calmidazolium (CMZ), chronic SNC 80 treatment did not lead to a cAMP overshoot in hDOR/CHO cells. Forskolin-stimulated cAMP formation in calmidazolium-treated cells was 108 ± 3% of IMDM-treated control (p > 0.1, n = 3). The data are normalized as a percentage of maximal stimulation in IMDM-treated control cells in the absence or presence of calmidazolium, respectively. Raw values of maximal forskolin-stimulated cAMP formation in the absence of SNC 80 were 669 ± 140 and 1821 ± 109 pmol/million cells in IMDM- and calmidazolium-treated control hDOR/CHO cells, respectively (p < 0.001).

View larger version (18K): [in this window] [in a new window]   Fig. 5. Simultaneous inhibition of PKC- and Tyr-kinases attenuates chronic deltorphin II treatment-mediated cAMP overshoot: hDOR/CHO cells were pretreated (0.5–1 h) in the absence or presence of 5 µM chelerythrine or 100 µM genistein individually or with both inhibitors simultaneously. The treatment continued for a further4hinthe presence or absence of 100 nM deltorphin II. The cells were thoroughly washed and forskolin (100 µM)-stimulated cAMP formation was determined. Data are represented as percentage (mean ± S.E.M.) of maximal forskolin-stimulated cAMP formation in IMDM-treated control cells (n = 7). Chronic deltorphin II-mediated cAMP overshoot was 355 ± 65% of control in IMDM-treated cells. cAMP overshoot was not significantly different in hDOR/CHO cells pretreated with genistein (213 ± 34, p > 0.05) or chelerythrine (375 ± 87%, p > 0.1), individually. Conversely, simultaneous application of the two inhibitors led to a significant blockade of deltorphin II-mediated AC superactivation (cAMP overshoot: 143 ± 19% of deltorphin II-treated control, , p < 0.05) in hDOR/CHO cells. Basal cAMP formation was 4.3 ± 0.6, 4.6 ± 0.6, 7.0 ± 1.3, and 4.7 ± 0.5 pmol/million cells in untreated, genistein-, chelerythrine-, and genistein + chelerythrine-treated cells, respectively. Maximal forskolin-stimulated cAMP formation was 163 ± 21, 305 ± 37, 96 ± 70, and 217 ± 27 pmol/million cells in untreated, genistein-, chelerythrine-, and genistein + chelerythrine-treated cells, respectively.

Phosphorylation and sensitization of AC VI by the Raf-1 protein kinase has been recently demonstrated (Tan et al., 2001). Therefore, previously we have tested the effect of a selective Raf-1 inhibitor (GW5074) on chronic -opioid agonist-mediated AC superactivation in hDOR/CHO cells. We found that pretreatment (30 min, 37°C) of hDOR/CHO cells with GW5074 (10 µM) attenuated chronic deltorphin II-mediated AC superactivation by 40% (**p < 0.01) (Varga et al., 2002). Because the catalytic activity of Raf-1 is modulated by multiple independent mechanisms (Belcheva and Coscia, 2001), to achieve complete inhibition it is frequently necessary to inhibit multiple pathways simultaneously (Fukuda et al., 1996). Accordingly, chronic deltorphin II treatment-mediated cAMP overshoot (355 ± 65% of control) was not notably attenuated in hDOR/CHO cells pretreated individually with the PKC inhibitor chelerythrine (375 ± 87%, 91 ± 7% of control, p > 0.05) or the tyrosine kinase inhibitor genistein (213 ± 34, 78 ± 9% of control, p > 0.05). Simultaneous application of the two inhibitors, however, led to a significant blockade (cAMP overshoot: 143 ± 19% of deltorphin II-treated control, p < 0.05, n = 7) of AC superactivation in hDOR/CHO cells (Fig. 5). Statistical significances (*p < 0.05, **p < 0.01, and ***p < 0.001) were determined using one-way ANOVA followed by Dunnett's multiple comparison tests.

	Discussion

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Chronic stimulation of G_i/o protein-coupled receptors often leads to a compensatory increase in the response of adenylyl cyclase to stimulators (AC superactivation) after the removal of the inhibitory agonist. AC superactivation was first described for the opioid receptors in NG108-15 cells (Sharma et al., 1975). Although AC superactivation is thought to play an important role in opioid tolerance, dependence, and withdrawal, its molecular mechanism is still not completely understood (Williams et al., 2001). Down-regulation of the opioid receptors is probably not involved in AC superactivation because increased cAMP levels (cAMP overshoot) become evident after agonist withdrawal. Down-regulation of G_i/o proteins (vanVliet et al., 1991), subcellular translocation of G_i (Bayewitch et al., 2000) and G_s (Ammer and Schulz, 1997) proteins, attenuation of phosphodiesterase activity (Law and Loh, 1993), and switching from G_i/o-mediated signaling to G_s-mediated signaling (Crain and Shen, 1998) have been suggested as possible explanations for cAMP overshoot.

AC superactivation has been observed in brain areas involved in opiate addiction, but it can also be demonstrated in recombinant cell lines heterologously expressing opioid receptors. We (Malatynska et al., 1996), and others (Avidor-Reiss et al., 1995) demonstrated that chronic opioid treatment of CHO cells expressing -or µ-opioid receptors leads to AC superactivation. It has also been shown previously that adenylyl cyclase superactivation is isoenzyme-specific (Thomas and Hoffman, 1996; Avidor-Reiss et al., 1997, Cumbay and Watts, 2001). Using a reverse transcription-polymerase chain reaction method, we previously found that AC VI and AC VII are the major AC isoenzymes expressed in CHO cells (Varga et al., 1998). It was demonstrated previously that AC VI is one of the few AC isoenzymes that becomes sensitized to forskolin after chronic opioid receptor stimulation and subsequent withdrawal (Avidor-Reiss et al., 1997). Therefore, we hypothesized that the AC VI isoenzyme has an important role in augmentation of forskolin-stimulated cAMP formation by chronic -opioid agonist treatment in hDOR/CHO cells.

Compensatory feedback regulation of the concentration or/and catalytic activity of adenylyl cyclase is an attractive hypothesis to account for cAMP overshoot. Thus, it was shown previously that chronic opioid treatment increases the mRNA levels of AC isoenzymes in the nucleus accumbens (Nestler and Aghajanian, 1997) and AC VII in the ileum longitudinal muscle myenteric plexus (LMMP) (Rivera and Gintzler, 1998). The relatively rapid kinetics and cycloheximide insensitivity of AC superactivation in hDOR/CHO cells (Avidor-Reiss et al., 1995; Rubenzik, 2002), however, suggests that new protein synthesis is not the sole mechanism involved in this process. It was also demonstrated previously that G protein -subunits, released upon agonist-stimulation of the transiently transfected µ-opioid receptor, have a major role in AC superactivation in COS-7 cells (Avidor-Reiss et al., 1996). Previously, we also found that overexpression of a free -subunit scavenger (the -subunit of rod transducin, t1) completely attenuates SNC 80-mediated AC superactivation in hDOR/CHO cells (Rubenzik et al., 2001).

Because phosphorylation regulates the catalytic activity of adenylyl cyclase isoenzymes (Ishikawa, 1998), previously we tested whether chronic -opioid agonist treatment leads to phosphorylation of the AC VI isoenzyme in hDOR/CHO cells. We found that concurrent to AC superactivation, chronic SNC 80 treatment augments ³²P incorporation into a 130- and a 200-kDa protein band, immunoprecipitated by an AC V/VI-specific antibody from hDOR/CHO cells. Because only the 130-kDa band was apparent after aminoglycosidase F treatment of the hDOR/CHO cell extracts, the 200-kDa band presumably corresponds to the glycosylated form of the 130-kDa protein. Chronic SNC 80-mediated ³²P incorporation in the hDOR/CHO cells was attenuated by naltrindole, a selective opioid receptor antagonist (Varga et al., 1999) and was SNC 80 dose- and treatment time-dependent (this work). Importantly, the time course of the onset of SNC 80-mediated AC superactivation (Rubenzik, 2002) and AC phosphorylation (this work) are remarkably similar. Interestingly, Chakrabarti et al. (1998) simultaneously observed that chronic morphine treatment augments AC phosphorylation in LMMP preparations.

To test the involvement SNC 80-mediated phosphorylation in AC superactivation we examined the effect of protein kinase inhibitors on chronic -opioid agonist-mediated cAMP overshoot in hDOR/CHO cells. We found that the calmodulin antagonist calmidazolium completely attenuates chronic SNC 80-mediated AC superactivation, whereas the PKC inhibitor chelerythrine had only minimal effect. In preliminary experiments, we also tested the protein kinase inhibitor sensitivity of SNC 80-mediated phosphorylation of AC VI in hDOR/CHO cells. The protein kinase inhibitor H-89 and the calmodulin antagonist calmidazolium completely attenuated SNC 80-induced ³²P incorporation into the immunoreactive bands.

Chakrabarti et al. (1998) previously demonstrated that chronic morphine treatment-mediated phosphorylation of adenylyl cyclase in guinea pig LMMP preparation was attenuated by chelerythrine pretreatment, indicating the involvement of PKC in this process. Agonist-activated opioid receptors in recombinant CHO cells have been shown to stimulate IP₁ formation (Rubenzik et al., 2001), arachidonate release, and phosphatidylinositol-3 kinase (Fukuda et al., 1996). Either of these pathways may lead to the activation of PKC isoenzymes in hDOR/CHO cells. However, because we found that the PKC inhibitor chelerythrine has only a very moderate effect on AC superactivation, PKC-mediated phosphorylation of the adenylyl cyclase isoenzymes is unlikely to play a major role in chronic -opioid agonist-mediated AC superactivation in hDOR/CHO cells.

Recently, phosphorylation of AC VI by the Raf-1 protein kinase has been demonstrated (Tan et al., 2001). Interestingly, similar to chronic opioid treatment-mediated AC superactivation, Raf-1-mediated phosphorylation was shown to sensitize the AC VI to stimulators, such as forskolin and G_s. Therefore, we tested the involvement of Raf-1 in chronic -opioid agonist-mediated AC superactivation in hDOR/CHO cells. We found that pretreatment of hDOR/CHO cells with the selective Raf-1 inhibitor GW5074 significantly attenuates chronic -opioid agonist-mediated AC superactivation (Varga et al., 2002).

Raf-1 is a serine/threonine kinase that acts downstream of activated tyrosine kinases in the p42/44 MAPK signal transduction cascade (Belcheva and Coscia, 2002). The catalytic activity of Raf-1 is modulated by multiple independent mechanisms. Thus, in CHO cells only simultaneous inhibition of both tyrosine kinases and PKC attenuated DPDPE-mediated MAPK phosphorylation completely (Fukuda et al., 1996). Our present data demonstrate that similarly, inhibition of tyrosine kinases or PKC individually has only minimal effect on AC superactivation, whereas simultaneous pretreatment with both genistein and chelerythrine obliterates chronic -opioid agonist-mediated AC superactivation.

Previously, it was concluded that the mitogen-activated protein kinase pathway is not involved in AC superactivation, because neither overexpression of dominant negative Ras nor the phosphatidylinositol-3-kinase inhibitor wortmannin was able to attenuate AC superactivation in COS-7 cells cotransfected with the µ-opioid receptor and AC V (Avidor-Reiss et al., 1996). However, because the catalytic activity of Raf-1 is modulated by multiple mechanisms, blocking a single pathway may only shunt the signal to alternative, parallel pathways. Thus, simultaneous inhibition of both ras-dependent- and ras-independent pathways may be necessary to achieve complete inhibition of AC superactivation, as we indeed found in hDOR/CHO cells.

We also found that both AC superactivation and AC VI phosphorylation can be completely attenuated by pretreatment with calmidazolium. Calmidazolium is a calmodulin antagonist that competes with calmodulin-sensitive intracellular effectors in low micromolar concentrations (Gietzen et al., 1981). Interestingly, the involvement of a calmodulin-sensitive step, upstream of opioid-mediated transactivation of receptor- and nonreceptor tyrosine kinases and the small G protein Ras, was previously demonstrated in opioid receptor-mediated MAPK activation (Belcheva et al., 2001). In addition, recent data indicate that calmidazolium also inhibits Raf-1 directly, by antagonizing calmodulin binding to the enzyme (Egea et al., 2000). It should be noted, however, that maximal values for forskolin-stimulated cAMP formation were also different in IMDM- and calmidazolium-treated hDOR/CHO cells. Because we previously found that CHO cells do not express calmodulin- or calmodulin kinase-sensitive AC isoforms the reason for increased basal cAMP formation in calmidazolium-treated cells is presently not clear. Calmidazolium, however, is a nonselective calmodulin antagonist that interferes with a number of other intracellular enzymes, such as calmodulin-dependent protein phosphatases and phosphodiesterases. This may change the basal phosphorylation of every AC isoenzyme in CHO cells and also may affect cellular cAMP degradation rate, contributing to increased basal cAMP formation in calmidazolium-treated cells.

In summary, we have demonstrated that chronic SNC 80 treatment augments ³²P incorporation into two protein bands immunoreactive with the ACV/VI specific antibody. Both chronic SNC 80-mediated ³²P incorporation and AC superactivation are naltrindole-sensitive and exhibit similar SNC 80 dose and time dependence. Calmidazolium and a selective Raf-1 inhibitor (GW5074) significantly attenuated chronic -opioid agonist-mediated adenylyl cyclase superactivation in hDOR/CHO cells. Tyrosine kinase (genistein) and PKC (chelerythrine) inhibitors individually had a minimal effect. However, simultaneous treatment with both genistein and chelerythrine abolished AC superactivation. Based on our experimental data, we suggest that multiple redundant pathways contribute to -opioid receptor-mediated activation of Raf-1, that in turn leads to the phosphorylation and sensitization of AC VI in hDOR/CHO cells. Figure 6 shows a putative molecular model to interpret the role of chelerythrine-, genistein-, and calmidazolium-sensitive signal transduction pathways in Raf-1 activation and AC VI phosphorylation. Adenylyl cyclase superactivation is an important molecular mechanism contributing to the development of tolerance and dependence to chronic opioid treatment. Better understanding of this important cellular compensatory mechanism should ultimately lead to the development of longer acting analgesic drugs with fewer side effects.

View larger version (26K): [in this window] [in a new window]   Fig. 6. Putative molecular mechanism of -opioid agonist-mediated phosphorylation of adenylyl cyclase VI in hDOR/CHO cells. Stimulation of the hDOR by an opioid agonist (*) in CHO cells liberates G protein subunits. Free G protein subunits interact with multiple effectors, leading to the activation of PKC isoforms; recruitment of -arrestin (-arr) and the nonreceptor tyrosine kinase Src; and transactivation of tyrosine kinase receptors (RTK), such as the platelet-derived growth factor receptor. Activation of PKC and Src, on the other hand, results in Raf-1 activation through Ras-independent- or Ras-dependent pathways, respectively. Activated Raf-1 phosphorylates and sensitizes adenylyl cyclase VI (AC VI), leading to a cAMP overshoot. The putative molecular targets of the inhibitors used in the present work are indicated in the figure. Calmidazolium is a calmodulin antagonist, genistein is a tyrosine kinase inhibitor, whereas chelerythrine nonselectively inhibits PKC isoenzymes. GW5074 is a selective Raf-1 inhibitor. MEK, mitogen-activated protein kinase kinase.

	Acknowledgements

 
We thank Carol Haussler and Michelle Thacher for the maintenance of the hDOR/CHO cell culture.

	Footnotes

 
This work was supported in part by grants from the National Institutes of Health and the Arizona Disease Control Research Commission.

DOI: 10.1124/jpet.103.049643.

ABBREVIATIONS: AC, adenylyl cyclase; CHO, Chinese hamster ovary; hDOR, human -opioid receptor; GW5074, 3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one; MAPK, mitogen-activated protein kinase; SNC 80, (+)-4-[(R)--((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxy-benzyl]-N,N-diethyl benzamide; DPDPE, [D-Pen²-D-Pen⁵]-enkephalin; IMDM: Iscove's modified Dulbecco's medium; PAGE, polyacrylamide gel electrophoresis; ANOVA, analysis of variance; PKC, protein kinase C; LMMP, longitudinal muscle myenteric plexus; H-89, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinoline sulfonamide, 2HCl; IP1, inositol phosphate-1.

Address correspondence to: Dr. Henry I. Yamamura, Department of Pharmacology, College of Medicine, University of Arizona Health Sciences Center, Tucson, AZ 85724. E-mail: hiy{at}u.arizona.edu

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