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Opioid receptor signaling via EGE receptor (EGFR) transacti- vation and ERK/MAPK phosphorylation initiates diverse cellu- lar responses that are cell type-dependent. In astrocytes, multiple μ opioid receptor-mediated mechanisms of ERK activation exist that are temporally distinctive and feature different out- comes. Upon discovering that chronic opiate treatment of rats down-regulates thrombospondin 1 (TSP1) expression in the nucleus accumbens and cortex, we investigated the mechanism of action of this modulation in astrocytes. TSP1 is synthesized in astrocytes and is released into the extracellular matrix where it is known to play a role in synapse formation and neurite out- growth. Acute morphine (hours) reduced TSP1 levels in astrocytes. Chronic (days) opioids repressed TSP1 gene expression and reduced its protein levels by g.μ opioid receptor and ERK-dependent mechanisms in astrocytes. Morphine also depleted TSP1 levels stimulated by TGFβ1 and abolished ERK activation induced by this factor. Chronic morphine treatment of astrocyte-neuron co-cultures reduced neurite outgrowth and synapse formation. Therefore, inhibitory actions of morphine were detected after both acute and chronic treatments. An acute mechanism of morphine signaling to ERK that entails depletion of TSP1 levels was suggested by inhibition of morphine activa- tion of ERK by a function-blocking TSP1 antibody. This raises the novel possibility that acute morphine uses TSP1 as a source of EGF-like ligands to activate EGFR. Chronic morphine inhibi- tion of TSP1 is reminiscent of the negative effect of μ opioids on EGFR-induced astrocyte proliferation via a phospho-ERK feedback inhibition mechanism. Both of these variations of classical EGFR transactivation may enable opiates to diminish neurite outgrowth and synapse formation. [ABSTRACT FROM AUTHOR]

In addition to its use for heroin addiction pharmacotherapy in general, buprenorphine has advantages in treating maternal heroin abuse. To examine the gestational effects of buprenorphine on opioid receptor signaling, the [35S]-GTPγS in situ binding induced by the μ agonist [D-Ala2,MePhe4,Gly5-ol] enkephalin (DAMGO) or the nociceptin/orphanin FQ (N/OFQ) agonist was measured in mesolimbic structures of pup brains from pregnant rats administered with buprenorphine±naloxone, naloxone, or methadone by osmotic minipump. Drug- and gender-based changes in DAMGO- and N/OFQ-induced GTPγS binding were discovered in mesolimbic regions of dam, P2, and P7 brains. Buprenorphine and/or methadone gestational treatment attenuated DAMGO-induced GTPγS binding in some dam and male P2 mesolimbic regions. Methadone diminished DAMGO-induced GTPγS binding in almost all monitored brain regions of the dam but had few effects on their N/OFQ-induced GTPγS binding. Naloxone used in combination with buprenorphine blocked the inhibition by buprenorphine alone on DAMGO-induced GTPγS binding. In contrast to its inhibitory effects on DAMGO-induced GTPγS binding, buprenorphine stimulated N/OFQ-induced GTPγS binding in male P2 nucleus accumbens and lateral septum. Brain region-dependent gender differences in DAMGO-induced GTPγS binding were seen in P2 pups, and males showed greater sensitivity to buprenorphine and methadone than females. Our findings on μ opioid receptor (MOR) GTP-binding regulatory protein (G protein) coupling and its gender dependency are consistent with our earlier studies on μ receptor binding adaptation induced by buprenorphine in dams and neonatal rats after in utero treatment regimens, and they extend the gestational effects of this opiate to μ and N/OFQ receptor functionality. [Copyright &y& Elsevier]

Chronic opioids induce synaptic plasticity, a major neuronal adaptation. Astrocyte activation in synaptogenesis may play a critical role in opioid tolerance, withdrawal, and dependence. Thrombospondins 1 and 2 (TSP1/2) are astrocyte-secreted matricellular glycoproteins that promote neurite outgrowth as well as dendritic spine and synapse formation, all of which are inhibited by chronic μ opioids. In prior studies, we discovered that the mechanism of TSP1 regulation by μ opioids in astrocytes involves crosstalk between three different classes of receptors, μ opioid receptor, EGFR and TGFβR. Moreover, TGFβ1 stimulated TSP1 expression via EGFR and ERK/MAPK activation, indicating that EGFR is a signaling hub for opioid and TGFβ1 actions. Using various selective antagonists, and inhibitors, here we compared the mechanisms of chronic opioid regulation of TSP1/2 isoform expression in vivo and in immortalized rat cortical astrocytes. TSP1/2 release from astrocytes was also monitored. Acute and chronic μ opioids, morphine, and the prototypic μ ligand, DAMGO, modulated TSP2 protein levels. TSP2 but not TSP1 protein content was up-regulated by acute (3 h) morphine or DAMGO by an ERK/MAPK dependent mechanism. Paradoxically, TSP2 protein levels were altered neither by TGFβ1 nor by astrocytic neurotrophic factors, EGF, CNTF, and BMP4. TSP1/2 immunofluorescence was increased in astrocytes subjected to scratch-wounding, suggesting TSPs may be useful markers for the "reactive" state of these cells and potentially for different types of injury. Previously, we determined that chronic morphine attenuated both neurite outgrowth and synapse formation in cocultures of primary astrocytes and neurons under similar temporal conditions that μ opioids reduced TSP1 protein levels in astrocytes. Here we found that, after the same 8 day treatment, morphine or DAMGO diminished TSP2 protein levels in astrocytes. Therefore, μ opioids may deter synaptogenesis via both TSP1/2 isoforms, but by distinct mechanisms.

Although micro, kappa, and delta opioids activate extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase, the mechanisms involved in their signaling pathways and the cellular responses that ensue differ. Here we focused on the mechanisms by which micro opioids rapidly (min) activate ERK and their slower (h) actions to inhibit epidermal growth factor (EGF)-induced ERK-mediated astrocyte proliferation. The micro-opioid agonists ([d-ala(2), mephe(4), gly-ol(5)] enkephalin and morphine) promoted the phosphorylation of ERK/MAP kinase within 5 min via G(i/o) protein, calmodulin (CaM), and beta-arrestin2-dependent signaling pathways in immortalized and primary astrocytes. This was based on the attenuation of the micro-opioid activation of ERK by pertussis toxin (PTX), the CaM antagonist, W-7, and siRNA silencing of beta-arrestin2. All three pathways were shown to activate ERK via an EGF receptor transactivation-mediated mechanism. This was disclosed by abolishment of micro-opioid-induced ERK phosphorylation with the EGF receptor-specific tyrosine phosphorylation inhibitor, AG1478, and micro-opioid-induced reduction of EGF receptor tyrosine phosphorylation by PTX, and beta-arrestin2 targeting siRNA in the present studies and formerly by CaM antisense. Long-term (h) treatment of primary astrocytes with [d-ala(2),mephe(4),gly-ol(5)] enkephalin or morphine, attenuated EGF-induced ERK phosphorylation and proliferation (as measured by 5'-bromo-2'-deoxy-uridine labeling). PTX and beta-arrestin2 siRNA but not W-7 reversed the micro-opioid inhibition. Unexpectedly, beta-arrestin-2 siRNA diminished both EGF-induced ERK activation and primary astrocyte proliferation suggesting that this adaptor protein plays a novel role in EGF signaling as well as in the opioid receptor phase of this pathway. The results lend insight into the integration of the different micro-opioid signaling pathways to ERK and their cellular responses.

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 beta-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 Gbetagamma subunits attenuated U69593 stimulation of ERK1/2 and suppressed proliferation in these cells. Furthermore, small interfering RNA silencing of beta-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, Gbetagamma subunits or beta-arrestin 2, suggesting that both G protein-dependent and -independent ERK pathways are required for this outcome.

Growth factors, hormones, and neurotransmitters have been implicated in the regulation of stem cell fate. Since various neural precursors express functional neurotransmitter receptors, which include G protein-coupled receptors, it is anticipated that they are involved in cell fate decisions. We detected mu-opioid receptor (MOR-1) and kappa-opioid receptor (KOR-1) expression and immunoreactivity in embryonic stem (ES) cells and in retinoic acid-induced ES cell-derived, nestin-positive, neural progenitors. Moreover, these G protein-coupled receptors are functional, since [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin, a MOR-selective agonist, and U69,593, a KOR-selective agonist, induce a sustained activation of extracellular signal-regulated kinase (ERK) signaling throughout a 24-h treatment period in undifferentiated, self-renewing ES cells. Both opioids promote limited proliferation of undifferentiated ES cells via the ERK/MAP kinase signaling pathway. Importantly, biochemical and immunofluorescence data suggest that [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin and U69,593 divert ES cells from self-renewal and coax the cells to differentiate. In retinoic acid-differentiated ES cells, opioid-induced signaling features a biphasic ERK activation profile and an opioid-induced, ERK-independent inhibition of proliferation in these neural progenitors. Collectively, the data suggest that opioids may have opposite effects on ES cell self-renewal and ES cell differentiation and that ERK activation is only required by the latter. Finally, opioid modulation of ERK activity may play an important role in ES cell fate decisions by directing the cells to specific lineages.

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.

Astroglia are a principal target of long-term mu antiproliferative actions. The mitogen-activated protein (MAP) kinase known as extracellular signal-regulated kinase (ERK), is a key mediator of cell proliferation. In studies on the mechanism of short- and long-term mu opioid regulation of the ERK signaling pathway, we show that the mu opioid agonist [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), acting via the endogenous mu opioid receptor (MOR), induced sequential epidermal growth factor receptor (EGF) receptor (EGFR) Tyr phosphorylation, Ser phosphorylation, and down-regulation in immortalized rat cortical astrocytes. The short-term action of DAMGO resulted in the stimulation of ERK phosphorylation. 4(3-Chlorophenylamino)-6,7-dimethoxyquinazoline (Tyrphostin AG1478), a selective inhibitor of EGFR Tyr kinase activity, blocked EGFR and ERK activation by short-term DAMGO administration, implicating EGFR transactivation in its stimulation of ERK activity. Inhibitors of matrix metalloproteinases attenuated MOR-mediated ERK phosphorylation, suggesting that shedding of EGF-like ligands from the plasma membrane may be involved in the EGFR transactivation process. Prolonged DAMGO exposure induced EGFR internalization/down-regulation, did not activate ERK, and inhibited exogenous EGF-stimulated ERK phosphorylation. MOR-mediated EGFR down-regulation seems to be MAP kinase-dependent, because it was inhibited by the ERK kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene (U0126), and tyrphostin AG1478. The kappa opioid agonist (5alpha,7alpha,8beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl) benzeneacetamide (U69,593) induced Tyr and Ser phosphorylation of EGFR and activation of ERK. However, long-term application of U69,593 neither down-regulated EGFR nor inhibited EGF-induced ERK activation. Instead, it engendered a sustained activation of ERK. Collectively, our data suggest that long-term application of DAMGO initiates heterologous down-regulation of EGFR via a mechanism involving ERK in astrocytes.

Mitogenic signaling of G protein-coupled receptors (GPCRs) can proceed via sequential epidermal growth factor receptor (EGFR) transactivation and extracellular signal-regulated kinase (ERK) phosphorylation. Although the mu-opioid receptor (MOR) mediates stimulation of ERK via EGFR transactivation in human embryonic kidney 293 cells, the mechanism of acute MOR signaling to ERK has not been characterized in rat C6 glioma cells that seem to contain little EGFR. Herein, we describe experiments that implicate fibroblast growth factor (FGF) receptor (FGFR) transactivation in the convergence of MOR and growth factor signaling pathways in C6 cells. MOR agonists, endomorphin-1 and morphine, induced a rapid (3-min) increase of ERK phosphorylation that was abolished by MOR antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2. By using selective inhibitors and overexpression of dominant negative mutants, data were obtained to suggest that MOR signaling to ERK is transduced by Gbetagamma and entails Ca2+- and protein kinase C-mediated steps, whereas the FGFR branch of the pathway is Ras-dependent. An intermediary role of FGFR1 transactivation was suggested by MOR- but not kappa-opioid receptor (KOR)-induced FGFR1 tyrosine phosphorylation. A dominant negative mutant of FGFR1 attenuated MOR- but not KOR-induced ERK phosphorylation. Thus, a novel transactivation mechanism entailing secreted endogenous FGF may link the GPCR and growth factor pathways involved in MOR activation of ERK in C6 cells.