Agonist-induced formation of opioid receptor-G protein-coupled receptor kinase (GRK)-G beta gamma complex on membrane is required for GRK2 function in vivo

G protein-coupled receptor kinases (GRKs) catalyze agonist-induced receptor phosphorylation on the membrane and initiate receptor desensitization. Previous in vitro studies have shown that the binding of GRK to membrane-associated G beta gamma subunits plays an important role in translocation of GRK2 from the cytoplasm to the plasma membrane. The current study investigated the role of the interaction of GRK2 with the activated delta-opioid receptor (DOR) and G beta gamma subunits in the membrane translocation and function of GRK2 using intact human embryonic kidney 293 cells. Our results showed that agonist treatment induced GRK2 binding to DOR, GRK2 translocation to the plasma membrane, and DOR phosphorylation in cells expressing the wild-type DOR but not the mutant DOR lacking the carboxyl terminus, which contains all three GRK2 phosphorylation sites. DORs with the GRK2 phosphorylation sites modified (M3) or with the acidic residues flanking phosphorylation sites mutated (E355Q/D364N) failed to be phosphorylated in response to agonist stimulation. Agonist-induced GRK2 membrane translocation and GRK-receptor association were observed in cells expressing M3 but not E355Q/D364N. Moreover, over-expression of G beta gamma subunits promoted GRK2 binding to DOR, whereas over-expression of transducin alpha or the carboxyl terminus of GRK2 blocked binding. Further study demonstrated that agonist stimulation induced the formation of a complex containing DOR, GRK2, and G beta gamma subunits in the cell and that agonist-stimulated formation of this complex is essential for the stable localization of GRK2 on the membrane and for its catalytic activity in vivo.     

Expression of delta opioid receptors by splenocytes from SEB-treated mice and effects on phosphorylation of MAP kinase

Delta opioid receptors (DORs) are known to modulate multiple T-cell responses. However, little is known about the expression of these receptors. These studies evaluated the expression of DOR mRNA and protein after a single in vivo exposure to staphylococcal enterotoxin B (SEB). SEB (20 microg, ip) significantly enhanced splenocyte DOR mRNA expression 8 and 24 h after injection. SEB also increased the fractions of the total splenocyte (5 to 20%) and T-cell (8 to 50%) populations expressing DOR protein. In saline-treated animals, DOR relative fluorescence intensity per cell was 11.1 +/- 0.62 units (mean +/- SEM), increasing to 16.1 +/- 1.7 after exposure to SEB. DOR fluorescence intensity significantly increased to 33.5 +/- 2.0 units in a subpopulation of T-cells. Thus, SEB significantly increased DOR expression in vivo, affecting both mRNA and protein levels primarily within the T-cell population. To determine whether T-cell DORs modulate the activity of extracellular-regulated kinases (ERKs), the phosphorylation of ERKs 1 and 2 was studied using splenocytes from SEB-treated mice. At concentrations from 10(-8) to 10(-6) M, [d-Ala(2)-d-Leu(5)]-enkephalin, a selective DOR agonist, significantly inhibited anti-CD3-epsilon-induced phosphorylation of the ERKs. Therefore, the DORs expressed by activated T-cells are capable of attenuating T-cell activation that depends on ERK phosphorylation.     

Activation of delta opioid receptors induces receptor insertion and neuropeptide secretion

Here we describe a novel mechanism for plasma membrane insertion of the delta opioid receptor (DOR). In small dorsal root ganglion neurons, only low levels of DORs are present on the cell surface, in contrast to high levels of intracellular DORs mainly associated with vesicles containing calcitonin gene-related peptide (CGRP). Activation of surface DORs caused Ca(2+) release from IP(3)-sensitive stores and Ca(2+) entry, resulting in a slow and long-lasting exocytosis, DOR insertion, and CGRP release. In contrast, membrane depolarization or activation of vanilloid and P2Y(1) receptors induced a rapid DOR insertion. Thus, DOR activation induces a Ca(2+)-dependent insertion of DORs that is coupled to a release of excitatory neuropeptides, suggesting that treatment of inflammatory pain should include blockade of DORs.     

Mitochondrial ERK plays a key role in delta-opioid receptor neuroprotection against acute mitochondrial dysfunction

It is well established that stimulating delta-opioid receptor (DOR) with its specific agonists elicits neuroprotection against hypoxia/ischemia. Mitochondrial dysfunction plays a key role in hypoxic neuronal injury, but the effects of DOR activation on mitochondrial dysfunction in neurons are poorly elucidated. In this investigation, we studied the effects of [d-Ala2, d-Leu5] enkephalin (DADLE), a potent DOR agonist, on acute mitochondrial dysfunction and ensuing cell damage induced by sodium azide in primary rat cortical neuronal cultures, and explored possible mechanisms underlying. Here, we show that DADLE reverses NaN₃-induced acute mitochondrial dysfunction by selectively activating DOR, mainly including mitochondrial membrane depolarization, mitochondrial Ca²⁺ overload and reactive oxygen species generation. DOR stimulation also inhibits cytochrome c release and caspase-3 activation, and attenuates neuronal death caused by acute NaN₃ insults. Furthermore, DOR activation with DADLE protects neurons from acute NaN₃ insults mainly through PKC-ERK pathway, and mitochondrial ERK activation is especially required for DOR neuroprotection against acute mitochondrial dysfunction.     

Particulate Matter Facilitates C6 Glioma Cells Activation and the Release of Inflammatory Factors Through MAPK and JAK2/STAT3 Pathways

It has been widely accepted that astrocytes, play a role in regulating almost every physiological system. In the present study, we investigated the role of particulate matter (PM) in regulating activation of astrocytes. The glial cell strain C6 was cloned from a rat glioma which was induced by N-nitrosomethylurea. The C6 cells were plated at a density of 5 × 10⁶ cells/10 cm diameter dish and incubated with different concentrations (0, 12, 25, 50, 100, 200, and 400 μg/mL) of PM for 24 h and different time (0, 1, 3, 6, 8,12, and 24 h) with 100 μg/mL at 37 °C. The study revealed that PM stimulated the expression of inducible nitric oxide synthase (iNOS) as well as the production of IL-1β in a dose- and time-dependent manner. Furthermore, activation of JAK2/STAT3 and p38/JNK/ERK MAPKs was found in astrocytes following PM treatment. Blockage of JAK and p38/JNK/ERK MAPKs with their specific inhibitors, AG490, SB202190, SP600125 and U0126 significantly reduced PM-induced iNOS expression and IL-1β production. In addition, it was demonstrated that inhibition of p38, JNK and JAK prevented STAT3 tyrosine phosphorylation induced by PM, while blocking ERK did not. MAPKs (p38 and JNK) could regulate tyrosine STAT3 phosphorylation, which suggested that the JAK2/STAT3 pathway might be the downstream of p38/JNK MAPK pathways.     

A novel role for activating transcription factor-2 in 15(S)-hydroxyeicosatetraenoic acid-induced angiogenesis

To investigate the mechanisms underlying 15(S)-HETE-induced angiogenesis, we have studied the role of the small GTPase, Rac1. We find that 15(S)-HETE activated Rac1 in human retinal microvascular endothelial cells (HRMVEC) in a time-dependent manner. Blockade of Rac1 by adenovirus-mediated expression of its dominant negative mutant suppressed HRMVEC migration as well as tube formation and Matrigel plug angiogenesis. 15(S)-HETE stimulated Src in HRMVEC in a time-dependent manner and blockade of its activation inhibited 15(S)-HETE-induced Rac1 stimulation in HRMVEC and the migration and tube formation of these cells as well as Matrigel plug angiogenesis. 15(S)-HETE stimulated JNK1 in Src-Rac1-dependent manner in HRMVEC and adenovirus-mediated expression of its dominant negative mutant suppressed the migration and tube formation of these cells and Matrigel plug angiogenesis. 15(S)-HETE activated ATF-2 in HRMVEC in Src-Rac1-JNK1-dependent manner and interference with its activation via adenovirus-mediated expression of its dominant negative mutant abrogated migration and tube formation of HRMVEC and Matrigel plug angiogenesis. In addition, 15(S)-HETE-induced MEK1 stimulation was found to be dependent on Src-Rac1 activation. Blockade of MEK1 activation inhibited 15(S)-HETE-induced JNK1 activity and ATF-2 phosphorylation. Together, these findings show that 15(S)-HETE activates ATF-2 via the Src-Rac1-MEK1-JNK1 signaling axis in HRMVEC leading to their angiogenic differentiation.     

Interaction with vesicle luminal protachykinin regulates surface expression of delta-opioid receptors and opioid analgesia

Opioid and tachykinin systems are involved in modulation of pain transmission in the spinal cord. Regulation of surface opioid receptors on nociceptive afferents is critical for opioid analgesia. Plasma-membrane insertion of delta-opioid receptors (DORs) is induced by stimulus-triggered exocytosis of DOR-containing large dense-core vesicles (LDCVs), but how DORs become sorted into the regulated secretory pathway is unknown. Here we report that direct interaction between protachykinin and DOR is responsible for sorting of DORs into LDCVs, allowing stimulus-induced surface insertion of DORs and DOR-mediated spinal analgesia. This interaction is mediated by the substance P domain of protachykinin and the third luminal domain of DOR. Furthermore, deletion of the preprotachykinin A gene reduced stimulus-induced surface insertion of DORs and abolished DOR-mediated spinal analgesia and morphine tolerance. Thus, protachykinin is essential for modulation of the sensitivity of nociceptive afferents to opioids, and the opioid and tachykinin systems are directly linked by protachykinin/DOR interaction.     

Microtubule inhibitors elicit differential effects on MAP kinase (JNK, ERK, and p38) signaling pathways in human KB-3 carcinoma cells

Microtubule inhibitors are widely used in cancer chemotherapy, but the signaling mechanisms that link microtubule disarray to destructive or protective cellular responses are poorly understood. Because members of the mitogen-activated protein kinase (MAPK) family have been implicated in regulation of cell survival and cell death, we examined the extent and kinetics of activation of JNK, ERK, and p38 MAPKs in response to treatment of KB-3 carcinoma cells with several microtubule inhibitors. All four agents tested (vinblastine, vincristine, Taxol, and colchicine) caused significant (6- to 13-fold) activation of JNK, concomitant inactivation of ERK, and a reduction in basal p38 MAPK activity. JNK activation and ERK inactivation occurred prior to caspase 3 activation. The microtubule inhibitors also induced phosphorylation of Raf-1 kinase. SEK-1, upstream of JNK, was also activated and phosphorylated in response to the microtubule inhibitors, and sustained phosphorylation of three endogenous JNK substrates (c-Jun, ATF-2, and JunD) was observed. By comparison, the antitumor agent doxorubicin induced activation of JNK and p38 but had no effect on ERK activity or Raf-1. These data demonstrate that microtubule inhibitors elicit distinct and specific effects on MAPK-mediated signaling pathways and suggest in particular that coordinate and reciprocal alterations in JNK and ERK activities are important facets of the cellular response to microtubule disruption.