Carmine J. Coscia

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.

Differential postnatal development of mu and delta opiate receptors

We found a differential postnatal development of mu and delta opiate receptors. Mu receptors labelled with low concentrations of [3H]naloxone appeared to develop earlier than did delta receptors labelled with [3H]D-Ala2-D-Leu5-enkephalin (0.5 nM). Competition binding studies also revealed a delayed appearance of delta receptors (day 12 postnatal).

Hydroxylation of geraniol and nerol by a monooxygenase from Vinca rosea

Abstract A microsomal mixed function oxidase isolated from V. rosea seedlings was shown to catalyze the hydroxylation of the monoterpene alcohols, geraniol and nerol, to their corresponding 10-hydroxy derivatives. Hydroxylase activity was dependent upon NADPH and oxygen and was associated with the 100,000 X g pellet which exhibited a characteristic reduced P-450-CO binding spectra. Light reversible inhibition by CO as well as differential sensitivity to other inhibitors established the hydroxylase as a cytochrome P-450 type. Cis-trans isomerase activity was not observed in this preparation. Both geraniol and nerol were shown to be hydroxylated almost exclusively at the C-10 methyl group.

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.

The prenatal developmental profile of expression of opioid peptides and receptors in the mouse brain

Although the postnatal development of opioid systems of mammalian brain has been well studied, little is known about the ontogeny of and relationship between embryonic (E) opioid peptides and their receptors. Moreover, a simultaneous assessment of levels of the 3 classes of opioid peptides and their putative receptors during embryonal development has not been made. To this end, the ontogeny of opioid peptides and receptors in mouse brain were examined during the period E11.5 to postnatal day 1 (P1). Met-enkephalin, dynorphin and beta-endorphin immunoreactivity were detected before their putative opioid receptors. beta-Endorphin can be discerned as early as E11.5, whereas mu binding was first observed at E12.5. Although dynorphin and Met-enkephalin were measurable at the same time as beta-endorphin, kappa-receptors were not detected until E14.5 and delta sites were not found at all prenatally. Differences in immunoreactivity levels of the 3 peptides occur with dynorphin being lower than Met-enkephalin and beta-endorphin, consistent with a low Bmax for kappa binding. Expression of the 3 opioid peptides as well as mu and kappa opioid receptors rapidly increase in parallel from E14.5 to E18.5. Interestingly, levels of beta-endorphin diminish by P1, the stage at which a sharp rise of mu receptors occurs. In a comparative study of the binding of beta-endorphin 1-31, its truncated form (1-27) and their N-acetyl derivatives to E14.5 brain membranes, beta-endorphin 1-31 exhibited the highest affinity.

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.

Localization of enzymes and alkaloidal metabolites in Papaver latex

In continuing studies on the metabolic activity of Papaver somniferum, latex has been examined for its enzyme and alkaloidal metabolite content. After an initial centrifugation of latex at 1000g, the pellet which contained a heterogeneous population of dense organelles was further resolved on sucrose gradients. Of the enzymes monitored, acid phosphatase and L-3,4-dihydroxyphenylalanine decarboxylase were found to be in the latex 1000g supernatant, whereas catecholase (polyphenolase) was localized in two distinct organelles within the 1000g sediment. The lighter organelles, sedimenting at 30% sucrose, contained a soluble enzyme which was readily released on organelle plasmolysis, whereas the catecholase found within the heavier organelles, sedimenting at 55-60% sucrose, was membrane bound and showed significant activity only in the presence of Triton X-100. These latter organelles also contained the alkaloids, including morphine and thebaine, and were observed to readily accumulate [14CH3]morphine. The alkaloid precursor, dopamine, was localized in the same dense vesicle fraction as the alkaloids. The rate of uptake of [7-14C]dopamine into these fractions at room temperature, however, was markedly lower than that of morphine. Electron microscopic examination of the organelles of various densities revealed that they possessed different morphology. The results are consistent with the concept that both the 1000g and supernatant fractions of the latex are required for alkaloid biosynthesis and that a subpopulation of dense organelles found in the 1000g sediment have at least a function as a storage compartment for both alkaloids and their catecholamine precursor.

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.