Mariana M. Belcheva

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

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.

Diversity of G Protein-Coupled Receptor Signaling Pathways to ERK/MAP Kinase

One of the most intriguing examples of cross talk between signaling systems is the interrelationship between G protein-coupled receptor and growth factor receptor pathways leading to activation of the ERK/MAP kinase phosphorylation cascade. This review focuses on the mechanism of this cross talk, denoting primarily signaling components known to occur in the G protein-coupled receptor branch of the MAP kinase pathways in neural cells. Recent evidence is presented on the existence of a plethora of pathways, due to the multiplicity of G protein-coupled receptors, their differential interaction with heterotrimeric G protein isoforms, various effectors and second messengers. In light of this rich diversity, the review will discuss different points of convergence of G protein-coupled receptor and growth factor receptor pathways that may feature a requirement for growth factor receptor transactivation, receptor internalization and scaffolds to assemble receptor, adaptor and anchoring proteins into multiprotein complexes.

Mitogenic Signaling via Endogenous κ-Opioid Receptors in C6 Glioma Cells: Evidence for the Involvement of Protein Kinase C and the Mitogen-Activated Protein Kinase Signaling Cascade

Abstract: As reports on G protein‐coupled receptor signal transduction mechanisms continue to emphasize potential differences in signaling due to relative receptor levels and cell type specificities, the need to study endogenously expressed receptors in appropriate model systems becomes increasingly important. Here we examine signal transduction mechanisms mediated by endogenous κ‐opioid receptors in C6 glioma cells, an astrocytic model system. We find that the κ‐opioid receptor‐selective agonist U69,593 stimulates phospholipase C activity, extracellular signal‐regulated kinase 1/2 phosphorylation, PYK2 phosphorylation, and DNA synthesis. U69,593‐stimulated extracellular signal‐regulated kinase 1/2 phosphorylation is shown to be upstream of DNA synthesis as inhibition of signaling components such as pertussis toxin‐sensitive G proteins, L‐type Ca2+ channels, phospholipase C, intracellular Ca2+ release, protein kinase C, and mitogen‐activated protein or extracellular signal‐regulated kinase kinase blocks both of these downstream events. In addition, by overexpressing dominant‐negative or sequestering mutants, we provide evidence that extracellular signal‐regulated kinase 1/2 phosphorylation is Ras‐dependent and transduced by Gβγ subunits. In summary, we have delineated major features of the mechanism of the mitogenic action of an agonist of the endogenous κ‐opioid receptor in C6 glioma cells.

μ- and κ-Opioids Induce the Differentiation of Embryonic Stem Cells to Neural Progenitors

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 μ-opioid receptor (MOR-1) and κ-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-Ala2,MePhe4,Gly-ol5]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-Ala2,MePhe4,Gly-ol5]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.

The Fibroblast Growth Factor Receptor Is at the Site of Convergence between μ-Opioid Receptor and Growth Factor Signaling Pathways in Rat C6 Glioma Cells

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 μ-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 antagonistd-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 Gβγ 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 κ-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.

Evidence for the implication of phosphoinositol signal transduction in μ-opioid inhibition of DNA synthesis

Abstract: An opioid receptor agonist, [D‐Ala2, Me‐Phe4, Glyol5]enkephalin (DAMGE), decreased [3H]thymidine incorporation into DNA of fetal rat brain cell aggregates. This action proved to depend on the dose of this enkephalin analog and the interval the aggregates were maintained in culture. The opioid antagonist naltrexone and the μ‐specific antagonist cyclic D‐Phe‐Cys‐Tyr‐D‐Trp‐Orn‐Thr‐Pen‐Thr amide (CTOP) reversed the DAMGE effect, arguing for a receptor‐mediated mechanism. The μ‐opioid nature of this receptor was further established by inhibiting DNA synthesis with the highly μ‐selective agonist morphiceptin and blocking its action with CTOP. Several other opioids, pertussis toxin, and LiCl also diminished DNA synthesis, whereas cholera toxin elicited a modest increase. Naltrexone completely reversed the inhibition elicited by the combination of DAMGE and low doses of LiCl but not by that of high levels of LiCl alone. The enkephalin analog also reduced basal [3H]inositol trisphosphate and glutamate stimulated [3H]inositol monophosphate and [3H]inositol bisphosphate accumulation in the aggregates. These DAMGE effects were reversed by naltrexone and were temporally correlated with the inhibition of DNA synthesis. A selective protein kinase C inhibitor, chelerythrine, also in hibited thymidine incorporation dose‐dependently. The effect of DAMGE was not additive in the presence of chelerythrine but appeared to be consistent with their actions being mediated via a common signaling pathway. These results suggest the involvement of the phosphoinositol signal transduction system in the modulation of thymidine incorporation into DNA by DAMGE.

KAPPA OPIOIDS PROMOTE THE PROLIFERATION OF ASTROCYTES VIA Gβγ AND β-ARRESTIN 2 DEPENDENT MAPK- MEDIATED PATHWAYS

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.

K-Opioid Agonist Modulation of [3H]Thymidine Incorporation into DNA: Evidence for the Involvement of Pertussis Toxin-Sensitive G Protein-Coupled Phosphoinositide Turnover

Abstract: A body of evidence has indicated that μ‐opioid agonists can inhibit DNA synthesis in developing brain. We now report that K‐selective opioid agonists (U69593 and U50488) modulate [3H]thymidine incorporation into DNA in fetal rat brain cell aggregates in a dose‐ and developmental stage‐dependent manner. K agonists decreased thymidine incorporation by 35% in cultures grown for 7 days, and this process was reversed by the K‐selective antagonist, norbinaltorphimine, whereas in 21‐day brain cell aggregates a 3,5‐fold increase was evident. Cell labeling by [3H]thymidine was also inhibited by the K‐opioid agonist as shown by autoradiography. In addition, U69593 reduced basal rates of phosphoinositide formation in 7‐day cultures and elevated it in 21‐day cultures. Control levels were restored by norbin‐altorphimine. Pertussis toxin blocked U69593‐mediated inhibition of DNA synthesis. The action of K agonists on thymidine incorporation in the presence of chelerythrine, a protein kinase C (PKC) inhibitor, or in combination with LiCl, a noncompetitive inhibitor of inositol phosphatase, was attenuated in both 7‐ and 21‐day cultures. These results suggest that K agonists may inhibit DNA synthesis via the phosphoinositide system with a pertussis toxin‐sensitive G protein as transducer. In mixed glial cell aggregates, U50488 increased thymidine incorporation into DNA 3.1‐fold, and this stimulation was reversed by the opioid antagonist naltrexone.

μ-Opioid Agonist Inhibition of κ-Opioid Receptor-Stimulated Extracellular Signal-Regulated Kinase Phosphorylation Is Dynamin-Dependent in C6 Glioma Cells

Abstract: In previous studies we found that μ‐opioids, acting via μ‐opioid receptors, inhibit endothelin‐stimulated C6 glioma cell growth. In the preceding article we show that the κ‐selective opioid agonist U69,593 acts as a mitogen with a potency similar to that of endothelin in the same astrocytic model system. Here we report that C6 cell treatment with μ‐opioid agonists for 1 h results in the inhibition of κ‐opioid mitogenic signaling. The μ‐selective agonist endomorphin‐1 attenuates κ‐opioid‐stimulated DNA synthesis, phosphoinositide turnover, and extracellular signal‐regulated kinase phosphorylation. To investigate the role of receptor endocytosis in signaling, we have examined the effects of dynamin‐1 and its GTPase‐defective, dominant suppressor mutant (K44A) on opioid modulation of extracellular signal‐regulated kinase phosphorylation in C6 cells. Overexpression of dynamin K44A in C6 cells does not affect κ‐opioid phosphorylation of extracellular signal‐regulated kinase. However, it does block the inhibitory action on κ‐opioid signaling mediated by the κ‐opioid receptor. Our results are consistent with a growing body of evidence of the opposing actions of μ‐ and κ‐opioids and provide new insight into the role of opioid receptor trafficking in signaling.

Opioid receptor density changes in Alzheimer amygdala and putamen

Since opioids can influence the release of acetylcholine, substance P and a number of other neurotransmitters that have been implicated in the pathogenesis of Alzheimers disease (AD), it is of interest to assess opioid receptor levels in AD. We have examined mu, delta and kappa opioid receptor binding parameters, binding sensitivity to a GTP analog and distribution in amygdala, frontal cortex and putamen of AD brain. Control brains were matched according to age, sex, post-mortem interval and storage time. Kd values and GTP analog binding sensitivity did not differ in AD and control brains. Bmax values for mu ([3H]DAMGE) sites also appeared unaffected by in vitro binding assays. In contrast, kappa ([3H]U69593) and delta ([3H]DSLET) opioid receptor levels, were significantly changed. In AD amygdala kappa Bmax values increased from control levels of 123 +/- 12 to 168 +/- 13 fmol/mg protein, whereas densities of kappa and delta sites were decreased from 94 +/- 8 to 48 +/- 8 and 102 +/- 3.6 to 69 +/- 8.5 fmol/mg protein, respectively, in putamen. Autoradiography revealed corresponding differences in the distribution of kappa opioid receptors. The findings indicate that the kappa binding site, which is quantitatively the major opioid receptor class in human brain, undergoes marked changes in AD amygdala and putamen.