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• W2023669293 abstract "Acute μ and κ opioids activate the ERK/MAPK phosphorylation cascade that represents an integral part of the signaling pathway of growth factors in astrocytes. By this cross-talk, opioids may impact neural development and plasticity among other basic neurobiological processes in vivo. The μ agonist, [d-ala2,mephe4,glyol5]enkephalin (DAMGO), induces a transient stimulation of ERK phosphorylation, whereas κ agonist, U69,593, engenders sustained ERK activation. Here we demonstrate that acute U69,593 and DAMGO stimulate ERK phosphorylation by utilization of different secondary messengers and protein kinase C (PKC) isoforms upstream of the growth factor pathway. Immortalized astrocytes transfected with either antisense calmodulin (CaM), a mutant μ opioid receptor that binds CaM poorly or a dominant negative mutant of PKCϵ were used as a model system to study μ signaling. Evidence was gained to implicate CaM and PKCϵ in DAMGO stimulation of ERK. DAMGO activation of PKCϵ and/or ERK was insensitive to selective inhibitors of Ca2+ mobilization, but it was blocked upon phospholipase C inhibition. These results suggest a novel mechanism wherein, upon DAMGO binding, CaM is released from the μ receptor and activates phospholipase C. Subsequently, phospholipase C generates diacylglycerides that activate PKCϵ. In contrast, U69,593 appears to act via phosphoinositide 3-kinase, PKCζ, and Ca2+ mobilization. These signaling components were implicated based on studies with specific inhibitors and a dominant negative mutant of PKCζ. Collectively, our findings on acute opioid effects suggest that differences in their mechanism of signaling may contribute to the distinct outcomes on ERK modulation induced by chronic μ and κ opioids. Acute μ and κ opioids activate the ERK/MAPK phosphorylation cascade that represents an integral part of the signaling pathway of growth factors in astrocytes. By this cross-talk, opioids may impact neural development and plasticity among other basic neurobiological processes in vivo. The μ agonist, [d-ala2,mephe4,glyol5]enkephalin (DAMGO), induces a transient stimulation of ERK phosphorylation, whereas κ agonist, U69,593, engenders sustained ERK activation. Here we demonstrate that acute U69,593 and DAMGO stimulate ERK phosphorylation by utilization of different secondary messengers and protein kinase C (PKC) isoforms upstream of the growth factor pathway. Immortalized astrocytes transfected with either antisense calmodulin (CaM), a mutant μ opioid receptor that binds CaM poorly or a dominant negative mutant of PKCϵ were used as a model system to study μ signaling. Evidence was gained to implicate CaM and PKCϵ in DAMGO stimulation of ERK. DAMGO activation of PKCϵ and/or ERK was insensitive to selective inhibitors of Ca2+ mobilization, but it was blocked upon phospholipase C inhibition. These results suggest a novel mechanism wherein, upon DAMGO binding, CaM is released from the μ receptor and activates phospholipase C. Subsequently, phospholipase C generates diacylglycerides that activate PKCϵ. In contrast, U69,593 appears to act via phosphoinositide 3-kinase, PKCζ, and Ca2+ mobilization. These signaling components were implicated based on studies with specific inhibitors and a dominant negative mutant of PKCζ. Collectively, our findings on acute opioid effects suggest that differences in their mechanism of signaling may contribute to the distinct outcomes on ERK modulation induced by chronic μ and κ opioids. Although both μ and κ opioids stimulate the MAPK 1The abbreviations used are: MAPK, mitogen activated protein kinase; Ab, antibody; ADAM, a disintegrin and metalloprotease; CaM, calmodulin; DAG, diacylglycerol; DAMGO, [d -ala2,mephe4,glyol5]enkephalin; dnPKC, dominant negative PKC; EGF, epidermal growth factor; EGFR, EGF receptor; ERK, extracellular signal-regulated kinase; GPCR, G protein-coupled receptor; KOR, κ opioid receptor; MMP, matrix metalloprotease; MOR, μ opioid receptor; PI3K, phosphoinositide-3 kinase; PLC, phospholipase C; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate. phosphorylation cascade via growth factor receptor transactivation, their effect on the extracellular signal-regulated kinase (ERK) varies in duration in immortalized astrocytes (1Belcheva M.M. Tan Y. Heaton V.M. Clark A.L. Coscia C.J. Mol. Pharmacol. 2003; 64: 1391-1401Crossref PubMed Scopus (52) Google Scholar). The μ agonist, [d-ala2,mephe4,gly-ol5]enkephalin (DAMGO), induces a transient activation of ERK that dissipates within 30 min, whereas that of the κ ligand U69,593 persists for several hours. In addition, chronic μ opioids inhibit growth factor-induced ERK activation, whereas chronic U69,593 does not. Examples of differential signaling mechanisms by subtypes of G protein-coupled receptors (GPCRs) are replete in the literature and are consistent with their possession of distinct functions (e.g. see Refs. 2Hawes B.E. Fried S. Yao X.R. Weig B. Graziano M.P. J. Neurochem. 1998; 71: 1024-1033Crossref PubMed Scopus (62) Google Scholar and 3Olivares-Reyes J.A. Jayadev S. Hunyady L. Catt K.J. Smith R.D. Mol. Pharmacol. 2000; 58: 1156-1161Crossref PubMed Scopus (12) Google Scholar). In studies of another astrocytic model system, rat C6 glioma cells, these differences in chronic μ and κ opioid regulation of ERK activity correlated well with their actions on mitogenesis, consistent with other evidence indicating that the ERK member of the MAPK family is implicated in cell proliferation (4Bohn L.M. Belcheva M.M. Coscia C.J. J. Neurochem. 2000; 74: 574-581Crossref PubMed Scopus (45) Google Scholar). The mechanisms that occur in the GPCR branch of the heterologous pathway by which μ and κ opioids signal to ERK/MAPK have not been studied in detail in comparison with the better understood steps downstream in the growth factor phase. Since opioid signaling to ERK may underlie basic mechanisms related to neuroplasticity as well as to cell proliferation, the early phase of this signaling pathway was investigated. PKC is an integral component of most GPCR signaling pathways to ERK/MAPK (5Belcheva M.M. Coscia C.J. Neurosignals. 2002; 11: 34-44Crossref PubMed Scopus (88) Google Scholar). Accordingly, PKC was found to be an early signaling component in the opioid pathway to ERK (6Fukuda K. Kato S. Morikawa H. Shoda T. Mori K. J. Neurochem. 1996; 67: 1309-1316Crossref PubMed Scopus (178) Google Scholar, 7Belcheva M.M. Szucs M. Wang D.X. Sadee W. Coscia C.J. J. Biol. Chem. 2001; 276: 33847-33853Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 8Bohn L.M. Belcheva M.M. Coscia C.J. J. Neurochem. 2000; 74: 564-573Crossref PubMed Scopus (79) Google Scholar). Nevertheless, little is known about the actual isoforms involved in this pathway. GPCR-mediated ERK activation via EGF receptor transactivation (9Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1329) Google Scholar) is one of a number of cell- and GPCR-specific variations of this type of cross-talk that were detected (5Belcheva M.M. Coscia C.J. Neurosignals. 2002; 11: 34-44Crossref PubMed Scopus (88) Google Scholar, 10Shah B.H. Catt K.J. Trends Neurosci. 2004; 27: 48-53Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). In earlier studies, PKC was found to play a role in directly activating Raf-1 and thereby circumventing involvement of the receptor tyrosine kinases (11Kolch W. Heidecker G. Kochs G. Hummel R. Vahidi H. Mischak H. Finkenzeller G. Marme D. Rapp U.R. Nature. 1993; 364: 249-252Crossref PubMed Scopus (1161) Google Scholar). Recently, PKC was also implicated in the activation of the MMP/ADAMs that are responsible for the release of extracellular membrane-bound EGF-like ligands, which trigger EGF receptor transactivation (12Pierce K.L. Tohgo A. Ahn S. Field M.E. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 2001; 276: 23155-23160Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Although the phosphoinositide kinases (PI3Ks) are found in many growth factor signaling pathways, the γ isoform can also mediate GPCR signaling to ERK upstream of the growth factor pathway in some cells (13Lopez-Ilasaca M. Crespo P. Pellici P.G. Gutkind J.S. Wetzker R. Science. 1997; 275: 394-397Crossref PubMed Scopus (629) Google Scholar, 14Hawes B.E. Luttrell L.M. Vanbiesen T. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 12133-12136Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 15Duckworth B.C. Cantley L.C. J. Biol. Chem. 1997; 272: 27665-27670Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). The Gβγ subunit of heterotrimeric GTP binding proteins activates PI3Kγ, which then acts on the 3′-OH position of phosphatidyl myo-inositol lipids to release different phosphorylated lipid products as secondary messengers. The initial evidence for its implication in the GPCR phase of the ERK signaling pathway arose from PI3K enzyme activity and co-immunoprecipitation studies in cultured Swiss 3T3 cells and human myeloid-derived cells (16Stephens L. Eguinoa A. Corey S. Jackson T. Hawkins P.T. EMBO J. 1993; 12: 2265-2273Crossref PubMed Scopus (137) Google Scholar, 17Kumagai N. Morii N. Fujisawa K. Nemoto Y. Narumiya S. J. Biol. Chem. 1993; 268: 24535-24538Abstract Full Text PDF PubMed Google Scholar). Subsequently, selective PI3K inhibitors (LY294002 and wortmannin) have been used to implicate this signaling component. Opioid stimulation of ERK is wortmannin-sensitive in COS-7 but not in C6 glioma cells, wherein MOR is overexpressed (18Belcheva M.M. Haas P.D. Tan Y. Heaton V.M. Coscia C.J. J. Pharmacol. Exp. Ther. 2002; 303: 909-918Crossref PubMed Scopus (71) Google Scholar). In prior investigations with HEK293 cells, we obtained evidence to suggest that the μ opioid pathway leading to EGF receptor and ERK activation featured the Ca2+-binding protein, CaM, as a secondary messenger and PKC as an intermediate (7Belcheva M.M. Szucs M. Wang D.X. Sadee W. Coscia C.J. J. Biol. Chem. 2001; 276: 33847-33853Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). This work was prompted by the discovery of the ability of CaM to bind to GPCRs such as MOR, dopamine, vasopressin, and the metabotropic glutamate receptor (19Bofill-Cardona E. Kudlacek O. Yang Q. Ahorn H. Freissmuth M. Nanoff C. J. Biol. Chem. 2000; 275: 32672-32680Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 20Wang D.X. Sadee W. Quillan J.M. J. Biol. Chem. 1999; 274: 22081-22088Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 21Minakami R. Jinnai N. Sugiyama H. J. Biol. Chem. 1997; 272: 20291-20298Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 22Nickols H.H. Shah V.N. Chazin W.J. Limbird L.E. J. Biol. Chem. 2004; 279: 46969-46980Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 23Nakajima Y. Yamamoto T. Nakayama T. Nakanishi S. J. Biol. Chem. 1999; 274: 27573-27577Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In the case of the metabotropic glutamate receptor subtypes 5 and 7, interaction with CaM was Ca2+-dependent, but MOR binding to CaM was shown to be at least partially Ca2+-independent (20Wang D.X. Sadee W. Quillan J.M. J. Biol. Chem. 1999; 274: 22081-22088Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Direct evidence that CaM binding to MOR initiates signaling to ERK was obtained by using cells stably transfected with wild type human MOR or with a mutant MOR (K273A) that was shown to bind CaM poorly and coupled more efficiently to G protein in the original studies by Sadee and co-workers (7Belcheva M.M. Szucs M. Wang D.X. Sadee W. Coscia C.J. J. Biol. Chem. 2001; 276: 33847-33853Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 20Wang D.X. Sadee W. Quillan J.M. J. Biol. Chem. 1999; 274: 22081-22088Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Here the roles of PKC isoforms, PLC, PI3K, and CaM in μ and κ opioid activation of ERK were assessed in astrocytes. Reagents—Chemicals were purchased from Sigma with the following exceptions: DAMGO-trifluoroacetate was obtained from Multiple Peptide Systems (San Diego, CA); U69,593 was from NIDA Drug Supply (Research Triangle, NC); EGF (human, recombinant) was from Invitrogen; Dulbecco's modified Eagle's medium and fetal bovine serum were from ATCC (Manassas, VA); phorbol 12-myristate 13-acetate (PMA), CaM inhibitors N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide and fluphenazine, PLC inhibitor U73122, PI3K inhibitors LY294002 and wortmannin, tyrphostin AG1478, and protein kinase C inhibitors bisindolylmaleimide I (GFX) and Gö6983 were from Calbiochem; Protein G plus A-agarose suspension was purchased from Oncogene Research Products (Cambridge, MA). The following antibodies (Abs) were purchased: anti-phospho-ERK Ab from Cell Signaling Technology (Beverly, MA); CaM and EGFR (sheep polyclonal or mouse monoclonal) Abs from Upstate (Charlottesville, VA); and ERK, PKC isoform, and actin Abs from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Cell Cultures—Rat cortical astrocytes (CTX TNA2, ATCC) were established from cultures of primary type 1 astrocytes from 1-day-old rat brain frontal cortex. The cultures were originally transfected with a DNA construct containing the oncogenic early region of SV40 under the transcriptional control of the human glial acidic fibrillary protein promoter (24Radany E.H. Brenner M. Besnard F. Bigornia V. Bishop J.M. Deschepper C.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6467-6471Crossref PubMed Scopus (79) Google Scholar). The astrocytes have the phenotypic characteristics of type 1. The cell line was maintained in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum at 37 °C in a humidified atmosphere of 95% air, 5% CO2 for up to 20–35 passages. For all experiments in which endogenous MOR or KOR were activated, early passage (passages 3–10) cells were grown in 6-well plates at least overnight to adhere well to the plate surface. Later passage (passages 10–35) cells were used for MOR/KOR transfection experiments. Optimal starvation of cultures was achieved in Dulbecco's modified Eagle's medium devoid of serum for 24 h. In all assays, agonists or inhibitors were delivered in serum-free media. Transient and Stable Transfections—Cells were transiently transfected with pcDNA3 (for mock transfections), rat or human MOR, human K273A-MOR, rat KOR (in pcDNA3 or pCMV-neo expression vectors), or dnPKCϵ or dnPKCζ. The aforementioned cDNAs of MORs, KOR, dnPKCϵ, and dnPKCζ were kind gifts from Drs. W. Sadee, H. Akil, P. Blumberg, and S. Gutkind, respectively). Transfections were conducted using 1–2 μg of cDNA and 3–6 μl of FuGENE 6 following the manufacturer's instructions. After 24–48 h of incubation, transfection medium was replaced with serum-free medium for an additional 24 h. The efficiency of transfection was determined to be 9 ± 1% (n = 6) by in situ identification of cells expressing β-galactosidase. Negative controls included untransfected and/or mock-transfected cells. Since the ATCC immortalized rat cortical astrocytes under investigation are transfectants selected with G418, we used zeocin (400 μg/ml) as a selection marker to prepare rat wild type MOR and/or antisense CaM stable transfections. Six clones were generated with stably transfected wild type MOR, and their MOR binding and DAMGO activation of ERK were assessed. The stable transfectants show a direct correlation between DAMGO binding to MOR and the extent of DAMGO-induced ERK phosphorylation. Stably transfected MOR clones activate ERK to a greater extent and have higher MOR binding levels than cells with endogenous MOR. DAMGO activation of ERK assays was performed only with clones containing 100–200 fmol/mg protein, a MOR concentration routinely found in rat forebrain. They gave on average 5-fold stimulation of ERK phosphorylation, whereas that of transiently transfected or untransfected cells was about 3-fold higher than basal levels in these studies. Antisense CaM stable transfectants were also generated, and 11 clones displayed CaM levels ranging from 20 to 90% lower than control immortalized astrocytes as determined by Western blotting with a CaM Ab (Upstate). Immunoblotting was performed following the manufacturer's instructions. Cell Membrane Preparation—Cells were harvested and homogenized by gentle disruption in a “cell cracker” as described (25Belcheva M. Barg J. Rowinski J. Clark W.G. Gloeckner C.A. Ho A. Gao X.M. Chuang D.M. Coscia C. J. Neurosci. 1993; 13: 104-114Crossref PubMed Google Scholar). A membrane fraction (P20) was prepared from cell homogenates by sedimenting at 20,000 × g for 20 min. A mixture containing 10 μg/ml leupeptin, 2 μg/ml pepstatin A, 200 μg/ml bacitracin, and 1 mm phenylmethylsulfonyl fluoride was added to the 50 mm Tris buffer, pH 7.4, during the preparation of this membrane fraction. These P20 membrane preparations were used in MOR binding and phospho-PKC immunoblotting experiments. MOR Binding—P20 membranes (300–500 μg/ml) were incubated with 1–5 nm3H-labeled DAMGO (35 Ci/mmol) at room temperature for 1 h. Nonspecific binding was determined in the presence of 1–10 μm DAMGO. Reactions were terminated by the addition of cold Tris buffer to the tubes followed by rapid filtration over GF/B filters in a Brandel cell harvester (Brandel Inc., Gaithersburg, MD). Filters were washed twice with cold 50 mm Tris-HCl, pH 7.4, buffer and then counted. Total and specific binding were calculated in fmol of MOR/mg of protein for each MOR-transfected cell line. PKC Isoform Translocation and Activation—Membrane and cytosolic fractions were isolated from rat astrocytes using the method of Krotova et al. (26Krotova K.Y. Zharikov S.I. Block E.R. Am. J. Physiol. 2003; 284: L1037-L1044Crossref PubMed Scopus (50) Google Scholar) with some modifications. Briefly, cells treated with opioids or vehicle were washed several times with PBS and collected by scraping in the presence of 5 mm Tris-HCl buffer, pH 7.4, containing 5 mm EGTA, 2mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 50 μg/ml aprotinin, and 50 μg/ml leupeptin. Cell suspensions were sonicated and were spun at 1000 × g for 5 min, to remove unbroken cells, cell debris, and nuclear pellet and then centrifuged at 100,000 × g for 60 min at 4 °C. The supernatant was used as the cytosolic fraction, whereas the pellet was resuspended in the above Tris buffer and used as the membrane fraction. Levels of PKCϵ and PKCζ were measured by immunoblotting with the corresponding Abs (27Brandlin I. Eiseler T. Salowsky R. Johannes F.J. J. Biol. Chem. 2002; 277: 45451-45457Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) in both cytosolic and membrane fractions from rat astrocytes. PKCϵ and PKCζ activation was also measured in P20 membrane fractions from rat astrocytes by immunoblotting, applying phospho-PKCϵ (Ser729; Santa Cruz Biotechnology) and phospho-PKCζ (Thr410/403; Cell Signaling Technology, Beverly, MA) Abs and following the manufacturers' instructions. ERK Assay—ERK phosphorylation was measured by immunoblotting as described (7Belcheva M.M. Szucs M. Wang D.X. Sadee W. Coscia C.J. J. Biol. Chem. 2001; 276: 33847-33853Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Briefly, cells were treated first with different inhibitors and then with DAMGO or U69,593 as described in each figure legends. Cells were then washed with PBS and lysed with buffer containing 20 mm HEPES, 10 mm EGTA, 40 mm β-glycerophosphate, 2.5 mm MgCl2, 2 mm sodium vanadate, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, and 20 μg/ml leupeptin. Cell lysates were centrifuged at 14,000 × g for 20 min at 4 °C, and protein concentration of the supernatants was determined. Samples (10–20 μg of protein/lane) were separated by 10% SDS-PAGE. Proteins were blotted on Immobilon P™ polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). Nonspecific sites were blocked with 5% milk in Tris-buffered saline plus 0.2% Tween 20 (TBST). Blots were then washed three times with TBST and incubated with anti-phospho-ERK Ab, diluted 1:2000 in TBST for at least 15 h at 4 °C. After three washes with TBST, blots were incubated with 1:2000 diluted goat anti-mouse horseradish peroxidase-conjugated IgG (Sigma) for 1 h at room temperature. For assurance of equivalent total ERK protein per lane, representative blots were stripped (0.2 m glycine, pH 2.5, 60 min at room temperature) and exposed to ERK Ab, followed by goat anti-rabbit horseradish peroxidase-conjugated IgG. Bands were visualized using an ECL chemiluminescence detection system (Amersham Biosciences) and exposure to Classic Blue sensitive x-ray film (Molecular Technologies, St. Louis, MO). Band intensities were determined by densitometric analysis using a Kodak DC120 digital camera, Kodak ds 1D version 3.0.2 software (Scientific Imaging Systems, New Haven, CT), and NIH ImageJ, version 1.32j. EGFR Immunoprecipitation—The EGFR immunoprecipitation protocol followed a previously described procedure (7Belcheva M.M. Szucs M. Wang D.X. Sadee W. Coscia C.J. J. Biol. Chem. 2001; 276: 33847-33853Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Briefly, serum-starved cells were administered DAMGO or U69,593 (0.1 μm, 3–5 min). Cultures were lysed by using a modified radioimmune precipitation buffer containing 50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mm Na3VO4, 1 mm NaF. Cell lysates of 0.8–1.2 mg of protein (diluted to ∼1 μg/μl) were used. EGFR was immunoprecipitated by adding 5–10 μg of either a mouse monoclonal or sheep polyclonal anti-EGFR Ab (Upstate) to the lysates and incubating overnight at 4 °C. This step was followed by the addition of a 50-μl suspension of protein G plus A-Sepharose beads per sample and incubation for 3–4 h at 4 °C. The beads were washed three times with PBS, resuspended in SDS loading buffer, and boiled for 5 min before SDS-PAGE. Proteins were blotted on Immobilon P™ polyvinylidene difluoride membranes and tested with Tyr(P) Ab (Cell Signaling) and peroxidase-conjugated mouse secondary Ab. Bands were visualized using a chemiluminescence detection system as described above. Protein Assay and Statistical Analysis—Protein concentrations were determined by the Bradford method (28Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) with bovine serum albumin (1 mg/ml) as a standard. Statistical determinations were made by t test analysis (version 2.01; GraphPad Software, Inc.). Data are expressed as the mean ± S.E. μ and κ Opioid-induced ERK Phosphorylation Is Mediated by Different PKC Isoforms—Previously, both time and dose dependence plots for DAMGO and U69,593 were obtained for cortical type 1 immortalized astrocytes to optimize conditions for ERK activation by these opioids (Ref. 1Belcheva M.M. Tan Y. Heaton V.M. Clark A.L. Coscia C.J. Mol. Pharmacol. 2003; 64: 1391-1401Crossref PubMed Scopus (52) Google Scholar and data not shown). Selective μ (CTAP) and κ (nor-binaltorphimine) opioids inhibited DAMGO and U69,593, respectively. Although PKC mediates the activation of ERK by all opioid receptor subtypes in a number of cell lines, including C6 glioma cells (4Bohn L.M. Belcheva M.M. Coscia C.J. J. Neurochem. 2000; 74: 574-581Crossref PubMed Scopus (45) Google Scholar, 6Fukuda K. Kato S. Morikawa H. Shoda T. Mori K. J. Neurochem. 1996; 67: 1309-1316Crossref PubMed Scopus (178) Google Scholar, 7Belcheva M.M. Szucs M. Wang D.X. Sadee W. Coscia C.J. J. Biol. Chem. 2001; 276: 33847-33853Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), initial experiments with the cortical astrocytes revealed that the general PKC inhibitor, GFX, blocked μ opioid-induced ERK phosphorylation but not that by κ (Fig. 1A). To determine whether the opioids under investigation here use different PKC isoforms in this signaling pathway than those in previously studied cells, we assessed the effects of another PKC inhibitor, Gö6983, which complements the PKC isoform selectivity profile of GFX (Fig. 1, B and C). Gö6983 abolished U69,593-induced ERK phosphorylation without affecting that of three different μ opioid agonists, DAMGO, morphine, and endomorphin. Of the ∼11 known isozymes of PKC, GFX inhibits all of the conventional isoforms (α, β1, β2, γ) that are DAG- and Ca2+-dependent and several novel isoforms (δ, ϵ) that are DAG-dependent but do not require Ca2+ (29Chow J.Y.C. Uribe J.M. Barrett K.E. J. Biol. Chem. 2000; 275: 21169-21176Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Whereas Gö6983 is also selective for conventional PKC isoforms, it inhibits PKCδ and the atypical PKCζ that is insensitive to Ca2+ and DAG (29Chow J.Y.C. Uribe J.M. Barrett K.E. J. Biol. Chem. 2000; 275: 21169-21176Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). On the basis of the isoform selectivity profiles of the two PKC inhibitors, we tested DAMGO for its ability to activate PKCϵ. Both PKCϵ membrane translocation (Fig. 2A) and phosphorylation assays (Fig. 2B) showed that the μ opioid activated this PKC. Time course studies on DAMGO-induced PKCϵ phosphorylation suggested that it peaks at 3 min, and by 5 min it is reduced to basal levels (data not shown). Transfection of astrocytes with a dominant negative mutant of PKCϵ attenuated DAMGO-induced phosphorylation of both PKCϵ (Fig. 2B) and ERK (Fig. 2C). Alternately, U69,593 signaling to ERK was not affected by the expression of dnPKCϵ in astrocytes (11 ± 1.4 in control cells versus 12 ± 1.5 in dnPKCϵ-expressing cells, n = 4). The data implicate PKCϵ in μ opioid signaling to ERK. μ Opioid-induced PKCϵ and ERK Phosphorylation Is Mediated by PLC but Not PI3K—There are reports that PKCϵ can be activated by either DAG, which is normally generated by PLC hydrolysis of phosphatidylinositide (30Chen C.C. FEBS Lett. 1993; 332: 169-173Crossref PubMed Scopus (86) Google Scholar), or by PI3K (29Chow J.Y.C. Uribe J.M. Barrett K.E. J. Biol. Chem. 2000; 275: 21169-21176Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). To address this question, the phorbol ester PMA, which mimics DAG by binding at its activation site on some PKCs, was tested and found to stimulate PKCϵ phosphorylation to about the same extent as DAMGO (Fig. 3A). This issue was further examined by subjecting astrocytes to chronic PMA, which down-regulates PKCs that contain a DAG binding site. Accordingly, overnight treatment of astrocytes with PMA abolished DAMGO- but not U69,593-induced ERK phosphorylation (Fig. 3B). Evidence that PLC generates the DAG in question was obtained by demonstrating that a specific inhibitor of this enzyme, U73112, attenuated DAMGO-induced phosphorylation of both PKCϵ (Fig. 3A) and ERK (Fig. 3C). Moreover, selective inhibitors of PI3K, wortmannin and LY294002, had no effect on μ opioid-induced ERK phosphorylation (Fig. 3C). As a member of the novel PKC isoform family, PKCϵ should be Ca2+-independent. Accordingly, we measured the intermediacy of Ca2+ mobilization and discovered that μ activation of ERK was not blocked by nifedipine, an L-type Ca2+ channel inhibitor, or by dantrolene, an inhibitor of microsomal Ca2+ release (Fig. 3D). CaM Is a Secondary Messenger in μ Opioid-induced ERK Phosphorylation—How is PLC activated to generate DAG? There is evidence to suggest that CaM can activate PLCβ (31McCullar J.S. Larsen S.A. Millimaki R.A. Filtz T.M. J. Biol. Chem. 2003; 278: 33708-33713Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). In prior studies using HEK293 cells, we discovered that the μ opioid pathway leading to growth factor transactivation featured CaM as a second messenger and PKC as an intermediate (7Belcheva M.M. Szucs M. Wang D.X. Sadee W. Coscia C.J. J. Biol. Chem. 2001; 276: 33847-33853Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Thus, we took three different approaches to implicate CaM in astrocytes. CaM antagonists, N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide and fluphenazine, were shown to attenuate DAMGO stimulation of ERK phosphorylation (data not shown). However, these antagonists are not completely selective. A second approach entailed the use of astrocytes stably transfected with CaM antisense cDNA. For the antisense experiments shown here a clone was adopted that displayed 55% less CaM than basal levels (n = 3; see inset in Fig. 4A). Astrocytes transfected with CaM antisense and transiently transfected with MOR were treated with DAMGO or, in some cases, morphine and were found to be incapable of stimulating ERK activity (Fig. 4A). However, CaM antisense clones transiently transfected with KOR retained their ability to mediate κ opioid activation of ERK (Fig. 4B). Astrocytes of similar passage number were also transiently transfected with MOR or KOR and used as controls in these experiments. In some cells, CaM has been implicated in the modulation of growth factor-induced ERK activation. This can occur either by a direct interaction with Ras-Raf (32Farnsworth C.L. Freshney N.W. Rosen L.B. Ghosh A. Greenberg M.E. Feig L.A. Nature. 1995; 376: 524-527Crossref PubMed Scopus (393) Google Scholar) or EGFR itself (33San Jose E. Benguria A. Geller P. Villalobo A. J. Biol. Chem. 1992; 267: 15237-15245Abstract Full Text PDF PubMed Google Scholar). Although CaM kinases have been implicated in activation of MAPKs (34Enslen H. Tokumitsu H. Stork P.J.S. Davis R.J. Soderling T.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10803-10808Crossref PubMed Scopus (261) Google Scholar), some have also been shown to phosphorylate EGFR and reduce its response to EGF in CHO cells (35Countaway J.L. Nairn A.C. Davis R.J. J. Biol. Chem. 1992; 267: 1129-1140Abs" @default.
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• W2023669293 date "2005-07-01" @default.
• W2023669293 modified "2023-10-16" @default.
• W2023669293 title "μ and κ Opioid Receptors Activate ERK/MAPK via Different Protein Kinase C Isoforms and Secondary Messengers in Astrocytes" @default.
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