2؉ mobilization, but it was blocked upon phospholipase C inhibition. These results suggest a novel mechanism wherein, upon DAMGO binding, CaM is released from the receptor and activates phospholipase C. Subsequently, phospholipase C generates diacylglycerides that activate PKC⑀. In contrast, U69,593 appears to act via phosphoinositide 3-kinase, PKC, and Ca 2؉ mobilization. These signaling components were implicated based on studies with specific inhibitors and a dominant negative mutant of PKC. 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 and opioids.

Phosphorylation of the MAPK isoform ERK by G protein-coupled receptors involves multiple signaling pathways. One of these pathways entails growth factor receptor transactivation followed by ERK activation. This study demonstrates that a similar signaling pathway is used by the -opioid receptor (

We investigated the postnatal ontogeny of opioid receptors in rat brain under assay conditions which, when combined with computerized analysis, effectively reflect the developmental profile of high affinity binding to mu, delta, and kappa subpopulations. Concentrations of mu sites were assessed with the selective ligand 3H-[D-ala2,mePhe4,gly-ol5]enkephalin (DAGO). The other two sites were analyzed in binding assays with less selective radioligands but in the presence of specific unlabeled ligands which suppress cross-reactivity. We utilized 3H-[D-ala2,D-leu5]enkephalin (DADL) in the presence of 10 nM DAGO to label delta sites and 3H-ethylketocyclazocine (EKC) in the presence of 100 nM DADL + 100 nM [D-ala2,mePhe4,Met(0)ol5]enkephalin to detect kappa receptors. After birth, the density (femtomoles per milligram of wet weight) of mu sites declined for several days and then rose sharply over the next 2 weeks, increasing 2-fold by adulthood. Delta (delta) sites appeared in the second week postnatal and increased more than 8-fold in the next 2 weeks. Levels of kappa receptors were relatively low at birth and increased slowly (2-fold, overall). Computerized analyses of binding data revealed that DAGO and DADL were binding to single populations of sites throughout the postnatal period. DAGO and EKC affinities did not fluctuate in this period, whereas DADL affinities were low for the first week and then rose to adult levels. In summary, mu, kappa, and delta receptors exhibit differential postnatal developmental profiles. The former two are present at birth, whereas the latter appears in the second week. The postnatal increase for all three sites appear to be preceded by the previously demonstrated emergence of opioid peptides.

GTP binding regulatory protein (G protein)‐coupled receptors can activate MAPK pathways via G protein‐dependent and ‐independent mechanisms. However, the physiological outcomes correlated with the cellular signaling events are not as well characterized. In this study, we examine the involvement of G protein and β‐arrestin 2 pathways in kappa opioid receptor‐induced, extracellular signal‐regulated kinase 1/2 (ERK1/2)‐mediated proliferation of both immortalized and primary astrocyte cultures. As different agonists induce different cellular signaling pathways, we tested the prototypic kappa agonist, U69593 as well as the structurally distinct, non‐nitrogenous agonist, C(2)‐methoxymethyl salvinorin B (MOM‐Sal‐B). In immortalized astrocytes, U69593, activated ERK1/2 by a rapid (min) initial stimulation that was sustained over 2 h and increased proliferation. Sequestration of activated Gβγ subunits attenuated U69593 stimulation of ERK1/2 and suppressed proliferation in these cells. Furthermore, small interfering RNA silencing of β‐arrestin 2 diminished sustained ERK activation induced by U69593. In contrast, MOM‐Sal‐B induced only the early phase of ERK1/2 phosphorylation and did not affect proliferation of immortalized astrocytes. In primary astrocytes, U69593 produced the same effects as seen in immortalized astrocytes. MOM‐Sal‐B elicited sustained ERK1/2 activation which was correlated with increased primary astrocyte proliferation. Proliferative actions of both agonists were abolished by either inhibition of ERK1/2, Gβγ subunits or β‐arrestin 2, suggesting that both G protein‐dependent and ‐independent ERK pathways are required for this outcome.

Although it is well-established that G protein-coupled receptor signaling systems can network with those of tyrosine kinase receptors by several mechanisms, the point(s) of convergence of the two pathways remains largely undelineated, particularly for opioids. Here we demonstrate that opioid agonists modulate the activity of the extracellular signal-regulated protein kinase (ERK) in African green monkey kidney COS-7 cells transiently cotransfected with mu-, delta-, or kappa-opioid receptors and ERK1- or ERK2-containing plasmids. Recombinant proteins in transfected cells were characterized by binding assay or immunoblotting. On treatment with corresponding mu- ([D-Ala2,Me-Phe4,Gly-ol5]enkephalin)-, delta- ([D-Pen2,D-Pen5]enkephalin)-, or kappa- (U69593)-selective opioid agonists, a dose-dependent, rapid stimulation of ERK1 and ERK2 activity was observed. This activation was inhibited by specific antagonists, suggesting the involvement of opioid receptors. Pretreatment of cells with pertussis toxin abolished ERK1 and ERK2 activation by agonists. Cotransfection of cells with dominant negative mutant N17-Ras or with a betagamma scavenger, CD8- beta-adrenergic receptor kinase-C, suppressed opioid stimulation of ERK1 and ERK2. When epidermal growth factor was used to activate ERK1, chronic (>2-h) opioid agonist treatment resulted in attenuation of the stimulation by the growth factor. This inhibition was blocked by the corresponding antagonists and CD8- beta-adrenergic receptor kinase-C cotransfection. These results suggest a mechanism involving Ras and betagamma subunits of Gi/o proteins in opioid agonist activation of ERK1 and ERK2, as well as opioid modulation of epidermal growth factor-induced ERK activity.