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, 280 (30), 27662-9

Mu and Kappa Opioid Receptors Activate ERK/MAPK via Different Protein Kinase C Isoforms and Secondary Messengers in Astrocytes

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Mu and Kappa Opioid Receptors Activate ERK/MAPK via Different Protein Kinase C Isoforms and Secondary Messengers in Astrocytes

Mariana M Belcheva et al. J Biol Chem.

Abstract

Acute mu and kappa 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 mu agonist, [D-ala2,mephe4,glyol5]enkephalin (DAMGO), induces a transient stimulation of ERK phosphorylation, whereas kappa 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 mu opioid receptor that binds CaM poorly or a dominant negative mutant of PKCepsilon were used as a model system to study mu signaling. Evidence was gained to implicate CaM and PKCepsilon in DAMGO stimulation of ERK. DAMGO activation of PKCepsilon 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 mu receptor and activates phospholipase C. Subsequently, phospholipase C generates diacylglycerides that activate PKCepsilon. In contrast, U69,593 appears to act via phosphoinositide 3-kinase, PKCzeta, and Ca2+ mobilization. These signaling components were implicated based on studies with specific inhibitors and a dominant negative mutant of PKCzeta. 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 mu and kappa opioids.

Figures

F<sc>IG</sc>. 1
FIG. 1
Differential effects of PKC inhibitors, GFX and Gö6983, on μ and κ opioid agonist stimulation of ERK phosphorylation as determined by immunoblotting assays. A, GFX abolishes DAMGO but not U69,593 stimulation of ERK phosphorylation. Astrocytes were pretreated with GFX (0.1 μM, 30 min) before adding DAMGO (0.1 μM, 5-10 min) or U69,593 (0.1 μM, 10 min). Endogenous MOR activity was measured in DAMGO-treated cells, whereas transiently transfected KOR astrocytes were treated with U69,593. A representative immunoblot is shown. n = 4-7 experiments. *, significantly greater than controls (p < 0.05). #, significantly less than DAMGO alone (p < 0.05). B, Gö6983 did not alter μ opioid agonist stimulation of ERK phosphorylation. MOR stably transfected astrocytes were pretreated with Gö6983 (0.1 μM, 30 min) before treatment with DAMGO (0.1 μM), endomorphin (0.1-1 μM), or morphine (0.5 μM) for 5 min. A representative immunoblot is shown. n = 3-6 experiments. *, significantly greater than controls (p < 0.05). **, significantly greater than controls (p < 0.01). C, Gö6983 abolished U69,593 stimulation of ERK phosphorylation. Astrocytes transiently transfected with KOR were pretreated with Gö6983 (0.1 μM, 30 min) before treatment with U69,593 (0.1 μM,10 min). A representative immunoblot is shown. n = 6-12 experiments. *, significantly greater than controls (p < 0.05). #, significantly less than U69,593 alone (p < 0.05).
F<sc>IG</sc>. 2
FIG. 2
DAMGO activation of PKCε as determined by immunoblotting assays. A, DAMGO induced translocation of PKCε into the membrane fraction of astrocytes. Cells stably transfected with MOR were treated with DAMGO (0.1 μM) for 1, 3, or 10 min before sonication and preparation of membrane and cytosolic fractions by differential centrifugation. Representative immunoblots using a PKCε Ab and an actin Ab (loading control) are shown. n = 2-3 experiments. *, significantly greater than controls (p < 0.05.). B, DAMGO-induced PKCε phosphorylation was abolished by dnPKCε. Astrocytes stably transfected with MOR were transiently transfected with either an empty vector (pcDNA3) or dnPKCε and were treated with DAMGO (0.1 μM, 3 min). A representative immunoblot is shown. n = 3-4 experiments. *, significantly greater than controls (p < 0.05.). #, significantly less than DAMGO alone (p < 0.05). C, DAMGO induction of ERK phosphorylation was attenuated by dnPKCε. Astrocytes stably transfected with MOR were transiently transfected with either an empty vector (pcDNA3) or dnPKCε and were treated with DAMGO (0.1 μM, 3 min). A representative immunoblot is shown. n = 3 experiments. **, significantly greater than controls (p < 0.01). #, significantly less than DAMGO alone (p < 0.05).
F<sc>IG</sc>. 3
FIG. 3
Differential effects of PLC inhibitor (U73122), phorbol ester (PMA), PI3K inhibitors (wortmannin and LY294002), or Ca2+ mobilization inhibitors (dantrolene and nifedipine) on DAMGO stimulation of PKCε and/or ERK phosphorylation as determined by immunoblotting assays. A, DAMGO induced PKCε phosphorylation by a mechanism attenuated by U73122, a selective inhibitor of PLC. Astrocytes stably transfected with MOR were pretreated with 10 μM U73122 for 30 min before treatment with 0.1 μM DAMGO for 3 min. In some experiments, cells were treated with PMA (1 μM, 3 min) as a positive control for stimulation of PKCε phosphorylation. n = 4-5 experiments. **, significantly greater than controls (p < 0.01). #, significantly less than DAMGO alone (p < 0.05). B, effect of chronic PMA on U69,593 or DAMGO stimulation of ERK phosphorylation. Astrocytes transiently transfected with KOR or stably transfected with MOR were pretreated with PMA (1 μM) overnight and treated with 0.1 μM U69,593 for 10 min or 0.1 μM DAMGO for 5 min, respectively. n = 4-5 experiments. *, significantly greater than controls (p < 0.05). **, significantly greater than controls (p < 0.01). #, significantly less than DAMGO alone (p < 0.05). C, differential effects of U73122 (U73), wortmannin (WM), and LY294002 (LY) on DAMGO stimulation of ERK phosphorylation. Astrocytes stably transfected with MOR were pretreated with U73122 (10 μM), wortmannin (1 μM), or LY294002 (10 μM) for 30 min before treatment with 0.1 μM DAMGO for 5 min. n = 3-4 experiments. *, significantly greater than controls (p < 0.05). #, significantly less than DAMGO alone (p < 0.05). D, DAMGO induction of ERK phosphorylation was unaffected by an inhibitor of microsomal Ca2+ release (dantrolene) or L-type Ca2+ channels (nifedipine). Astrocytes were pretreated for 30 min with 1 μM dantrolene (Dan) or 1 μM nifedipine (Nif) before treatment with 0.1 μM DAMGO for 5 min, respectively. A representative immunoblot is shown. n = 5-10 experiments. *, significantly greater than controls, p < 0.05.
F<sc>IG</sc>. 4
FIG. 4
Evidence implicating CaM in μ but not κ opioid agonist stimulation of EGFR and/or ERK phosphorylation as determined by immunoblotting assays. A, CaM antisense abolished DAMGO-induced ERK phosphorylation. Astrocytes were stably transfected with antisense CaM cDNA and shown to have reduced CaM levels by immunoblotting. Control cells and antisense CaM (anti-CaM) clones were transiently transfected with MOR cDNA and treated with DAMGO (0.1 μM, 5 min) or PMA (10 μM, 5 min) before measuring ERK phosphorylation. Representative immunoblot is shown. n = 4-9 experiments. *, significantly greater than controls (p < 0.05). #, significantly less than agonist alone (p < 0.05). Inset, representative immunoblot showing levels of CaM in equivalent amounts of protein from lysates of control and antisense CaM cells. B, CaM antisense did not alter U69,593 stimulation of ERK phosphorylation. Control cells and antisense CaM-transfected clones were transiently transfected with KOR cDNA. Cells were treated with U69,593 (0.1 μM, 10 min) before measuring ERK phosphorylation. A representative immunoblot is shown. n = 4-7 experiments. *, significantly greater than controls (p < 0.05). C, CaM antisense abolished DAMGO- and morphine-induced EGFR phosphorylation. Control cells and antisense CaM-transfected clones were transiently transfected with MOR cDNA. Cells were treated with DAMGO or morphine (0.1 μM, 1 min) before measuring EGFR phosphorylation by immunoprecipitation with EGFR Ab and immunoblotting with phospho-Tyr Ab. n = 4-9 experiments. *, significantly greater than controls (p < 0.05). #, significantly less than agonist alone (p < 0.05). D, DAMGO modulation of ERK phosphorylation in wild type MOR and mutant K273A MOR-transfected rat astrocytes. Cells were transiently transfected with either wild type human MOR or mutant human K273A MOR cDNA. Cultures were treated with DAMGO (0.1 μM, 5 min) before immunoblotting. Representative immunoblot is shown. n = 8 experiments. *, significantly greater than controls (p < 0.05). #, significantly less than wild type MOR (p < 0.05).
F<sc>IG</sc>. 5
FIG. 5
Evidence implicating PI3K, PKCζ and Ca2+ mobilization in U69,593 stimulation of ERK phosphorylation as determined by immunoblotting assays. A, U69,593 induced PKCζ phosphorylation by a mechanism blocked by the PI3K inhibitor, LY294002. Astrocytes transiently transfected with KOR were pretreated with LY294002 (10 μM) for 30 min before treatment with 0.1 μM U69,593 for 5 min. *, significantly greater than controls (p < 0.05). #, significantly less than U69,593 alone (p < 0.05). B, astrocytes transfected with dnPKCζ attenuated U69,593 stimulation of ERK phosphorylation. Cells transiently transfected with KOR cDNA were cotransfected with either wild type (wt) or dnPKCζ. Astrocytes were treated with U69,593 (0.1 μM, 10 min), and ERK phosphorylation was measured. n = 5-6 experiments. **, significantly greater than controls (p < 0.01). #, significantly less than U69,593 alone, p < 0.05. C, U69,593 stimulation of ERK phosphorylation was abolished by LY294002. Cells transiently transfected with KOR were exposed to LY294002 (10 μM) for 30 min before treatment with 0.1 μM U69,593 for 10 min. n = 5-6 experiments. **, significantly greater than controls (p < 0.01). #, significantly less than U69,593 alone (p < 0.05). D, U69,593 stimulation of ERK phosphorylation was abolished by an inhibitor of microsomal Ca2+ release (dantrolene) or of L-type Ca2+ channels (nifedipine). Astrocytes were pretreated for 30 min with 1 μM dantrolene (Dan) or 1 μM nifedipine (Nif) before treatment with U69,593 (0.1 μM) for 10 min. A representative immunoblot is shown. n = 5-10 experiments. *, significantly greater than controls (p < 0.01). #, significantly less than U69,593 alone (p < 0.05).

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