Search

J. Neurochem. (2010) 112, 1431–1441. As embryonic stem cell-derived neural progenitors (NPs) have the potential to be used in cell replacement therapy, an understanding of the signaling mechanisms that regulate their terminal differentiation is imperative. In previous studies, we discovered the presence of functional μ opioid receptors (MOR) and κ opioid receptors (KOR) in mouse embryonic stem cells and NPs. Here, MOR and KOR immunoreactivity was detected in NP-derived oligodendrocytes during three stages of their maturation in vitro. Moreover, we examined the modulation of retinoic acid-induced NP differentiation to astrocytes and neurons by μ, [D-ala2, mephe4, gly-ol5] enkephalin, or κ, U69, 593, opioids. Both opioid agonists inhibited NP-derived neurogenesis and astrogenesis via their corresponding receptors as determined by immunocytochemistry. By administering selective inhibitors, we found that opioid inhibition of NP-derived astrogenesis was driven via extracellular-signal regulated kinase (ERK), while the p38 mitogen-activated protein kinase pathway was implicated in opioid attenuation of neurogenesis. In addition, μ and κ opioids stimulated oligodendrogenesis from NP-derived NG2+ oligodendrocyte progenitors via both ERK and p38 signaling pathways. Accordingly, both opioids induced ERK phosphorylation in NG2+ cells. These results indicate that small molecules, such as MOR and KOR agonists may play a modulatory role in NP terminal differentiation. [ABSTRACT FROM AUTHOR]

Although μ, κ, and δ 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 μ opioids rapidly (min) activate ERK and their slower (h) actions to inhibit epidermal growth factor (EGF)-induced ERK-mediated astrocyte proliferation. The μ-opioid agonists ([d-ala2, mephe4, gly-ol5] enkephalin and morphine) promoted the phosphorylation of ERK/MAP kinase within 5 min via Gi/o protein, calmodulin (CaM), and β-arrestin2-dependent signaling pathways in immortalized and primary astrocytes. This was based on the attenuation of the μ-opioid activation of ERK by pertussis toxin (PTX), the CaM antagonist, W-7, and siRNA silencing of β-arrestin2. All three pathways were shown to activate ERK via an EGF receptor transactivation-mediated mechanism. This was disclosed by abolishment of μ-opioid-induced ERK phosphorylation with the EGF receptor-specific tyrosine phosphorylation inhibitor, AG1478, and μ-opioid-induced reduction of EGF receptor tyrosine phosphorylation by PTX, and β-arrestin2 targeting siRNA in the present studies and formerly by CaM antisense. Long-term (h) treatment of primary astrocytes with [d-ala2,mephe4,gly-ol5] enkephalin or morphine, attenuated EGF-induced ERK phosphorylation and proliferation (as measured by 5′-bromo-2′-deoxy-uridine labeling). PTX and β-arrestin2 siRNA but not W-7 reversed the μ-opioid inhibition. Unexpectedly, β-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 μ-opioid signaling pathways to ERK and their cellular responses. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

NAD(P)H:quinone oxidoreductase (DT-diaphorase) was detected in 100 000 × g supernatant fractions of extracts of a wide variety of higher plants. Smaller amounts were also found in microsomes and chloroplast fractions. The enzyme was partially purified from soluble extracts of several plants and the quinone reductase from Catharanthus roseus was enriched 25-fold. Plant quinone reductases have molecular weights in the range of 38 000 - 53 000 as determined by gel filtration. The plant enzyme is far less sensitive to dicoumarol than its mammalian counterpart and it is inhibited by superoxide dismutaste. Quinone reductase is capable of reducing simple p-benzoquinone and naphthoquinone including vitamins K3 and K1. These results indicate that, although the plant enzyme exhibits a similar substrate specificity, it is distinguishable from mammalian DT-diaphorase particularly with respect to its mechanism of reduction. [ABSTRACT FROM AUTHOR]