Regulation of Adenylyl Cyclase, ERK1/2, and CREB by G_z Following Acute and Chronic Activation of the δ‐Opioid Receptor

Abstract

Opioid tolerance and physical dependence in mammals can be rapidly induced by chronic exposure to opioid agonists. Recently, opioid receptors have been shown to interact with the pertussis toxin (PTX)-insensitive G z (a member of the G i subfamily), which inhibits adenylyl cyclase and stimulates mitogen-activated protein kinases (MAPKs). Here, we established stable human embryonic kidney 293 cell lines expressing ␦-opioid receptors with or without G z to examine the role of G z in opioid receptor-regulated signaling systems. Each cell line was acutely or chronically treated with [D-Pen 2 ,DPen 5 ]enkephalin (DPDPE), a ␦-selective agonist, in the absence or presence of PTX. Subsequently, the activities of adenylyl cyclase, cyclic AMP (cAMP)-dependent response element-binding proteins (CREBs), and MAPKs were measured by determining cAMP accumulation and phosphorylation of CREBs and the extracellular signalregulated protein kinases (ERKs) 1 and 2. In cells coexpressing G z , DPDPE inhibited forskolin-stimulated cAMP accumulation in a PTX-insensitive manner, but G z could not replace G i to mediate adenylyl cyclase supersensitization upon chronic opioid treatment. DPDPE-induced adenylyl cyclase supersensitization was not associated with an increase in the phosphorylation of CREBs. Both G i and G z mediated DPDPE-induced activation of ERK1/2, but these responses were abolished by chronic opioid treatment. Collectively, our results show that although G z mediated opioid-induced inhibition of adenylyl cyclase and activation of ERK1/2, G z alone was insufficient to mediate opioid-induced adenylyl cyclase supersensitization.

Molecular Pharmacology ofδ-Opioid Receptors

“…Failure of PKA inhibitors to modify acute morphine analgesia also supports the notion that cAMP effectors are minimally involved in the acute analgesic responses to opioids (Bernstein and Welch, 1999), as does the observation that phosphodiesterase inhibitors do not modify analgesia caused by intracerebroventricular or intrathecal administration of DPDPE (Suh et al, 1995). On the other hand, cAMP signaling has been consistently implicated in cellular adaptations that result from sustained DOPr stimulation and may contribute to analgesic tolerance (Tso et al, 2000;Varga, 2003;Zhang et al, 2006). These adaptations generally involve an increase in AC catalytic activity, although the mechanisms contributing to such an increase vary according to the type of AC involved (Avidor-Reiss et al, 1997;Varga, 2003).…”

“…In immortalized cell lines expressing either endogenous or recombinant DOPr, Gai2 has been frequently associated with cAMP inhibition, although additional subtypes are also involved in this response (Table 4). For example, DOPrs expressed in Chinese hamster ovary (CHO), COS cells (George et al, 2000;Fan et al, 2005), and HEK293 cells (Tsu et al, 1997;Tso et al, 2000) activate Gaz and modulate AC activity in a PTXinsensitive manner. In COS and CHO cells, AC inhibition by DOPr agonists was only evident when these receptors were coexpressed with MOPrs (George et al, 2000); however, in HEK cells, DOPrs expressed by themselves were able to both inhibit (Tsu et al, 1997;Ho and Wong, 2000;Tso et al, 2000) or stimulate cAMP production via Gaz (Tsu et al, 1997;Ho et al, 2001).…”

Expression of the Third Intracellular Loop of the δ-Opioid Receptor Inhibits Signaling by Opioid Receptors and Other G Protein-Coupled Receptors

“…It has been previously demonstrated that ␦-or -ORs expressed in HEK293 cells or Rat-1 fibroblasts stimulate ERK1/2 activity via PTX-sensitive or -insensitive G protein signaling mechanisms (Burt et al, 1996;Tso et al, 2000;Belcheva et al, 2002). To examine whether the ␦-i3L peptide can be also used as potential inhibitor of this type of GPCR signaling, ERK activation was measured in response to ␦-and -opioid agonist stimulation in serum-starved HEK293 cells expressing these receptors.…”

Differential chemokine activation of CC chemokine receptor 1‐regulated pathways: ligand selective activation of Gα 14‐coupled pathways

“…Cells were assayed for ERK and JNK activities as previously described [36,37]. Briefly, cells were challenged with chemokines and the cell contents were extracted, separated by SDS-PAGE, and transferred to nitrocellulose membrane before incubation with antisera against phospho-ERK1/2 or phospho-JNK and detection using the ECL kit.…”