Role of extracellular signal-regulated kinases in opioid-induced adenylyl cyclase superactivation in human embryonic kidney 293 cells.

The mu-opioid receptor stimulates the activity of extracellular signal-regulated protein kinase 1/2 (ERK1/2) in recombinant human embryonic kidney (HEK) 293 cells but this stimulatory response is abolished by prolonged opioid treatment. Chronic opioid treatment of the same cells has also been shown to induce adenylyl cyclase (AC) superactivation. This study examined the role of ERK1/2 activity in opioid-induced AC superactivation. Acute opioid treatment of HEK 293 cells expressing mu-opioid receptors resulted in the activation of ERK1/2, and this response was abolished in the presence of U0126, a MEK1/2 inhibitor. Despite a complete blockade of ERK1/2 phosphorylation, U0126 did not affect opioid-induced AC superactivation, indicating that ERK1/2 activity was not required for opioid-induced AC superactivation in HEK 293 cells.

Opioid-induced adenylyl cyclase supersensitization in human embryonic kidney 293 cells requires pertussis toxin-sensitive G proteins other than G(i1) and G(i3).

Chronic activation of opioid receptors in cultured mammalian cells is known to induce adenylyl cyclase (AC) supersensitization via the pertussis toxin-sensitive G(i/o) proteins. To examine the role of G(i1) and G(i3) in opioid-induced AC supersensitization, pertussis toxin-resistant mutants of Galpha(i1) and Galpha(i3) (Galpha(i1)CG and Galpha(i3)CG) were stably co-expressed with different opioid receptors (mu, delta or kappa) in human embryonic kidney (HEK 293) cells. Although the opioid receptors were capable of inhibiting AC via Galpha(i1)CG and Galpha(i3)CG in pertussis toxin-treated cells, AC supersensitization induced by chronic opioid treatment remained sensitive to pertussis toxin. Our results demonstrated that despite their ability to interact with opioid receptors, the pertussis toxin-sensitive G(i1) and G(i3) proteins on their own are incapable of supporting opioid-induced AC supersensitization.

Deciphering the role of Gi2 in opioid-induced adenylyl cyclase supersensitization.

Prolonged opioid treatment of HEK 293 cells expressing opioid receptors are known to induce adenylyl cyclase supersensitization, a process that requires pertussis toxin (PTX)-sensitive G(i/o) proteins. Here, the role of Gi2 in adenylyl cyclase supersensitization was investigated. A PTX-insensitive G alpha(i2)/z chimera was stably co-expressed with mu-, kappa- or delta-opioid receptors in HEK 293 cells. Functional coupling of G alpha(i2)/z to the opioid receptors was demonstrated by opioid-induced inhibition of adenylyl cyclase and stimulation of ERK1/2 phosphorylation in PTX-treated cells. Chronic opioid treatment of each cell line led to adenylyl cyclase supersensitization but this response was blocked by PTX. Our results demonstrated that although PTX-sensitive G proteins are obligatory for opioid-induced adenylyl cyclase supersensitization, Gi2 alone was insufficient to mediate this response.

G(z) can mediate the acute actions of mu- and kappa-opioids but is not involved in opioid-induced adenylyl cyclase supersensitization.

The three subtypes of opioid receptors (delta, micro, and kappa) are known to regulate multiple effectors through either pertussis toxin-sensitive or -insensitive G proteins. In opioid-induced inhibition of adenylyl cyclase, both G(i) and G(z) proteins can serve as the signal transducer. Our previous study showed that opioid-induced adenylyl cyclase supersensitization in human embryonic kidney (HEK) 293 cells expressing the delta-opioid receptor requires G(i) but not G(z) proteins. Herein, we studied the ability of mu- and kappa-opioid receptors to regulate the activities of adenylyl cyclase through G(z). In HEK 293 cells coexpressing G(z) with the mu- or kappa-opioid receptors, opioid agonists induced inhibition of adenylyl cyclase in a pertussis toxin-insensitive manner. However, adenylyl cyclase supersensitization induced by chronic opioid treatments remained sensitive to pertussis toxin. We also showed that the responsiveness of cAMP-dependent response element-binding proteins to forskolin was not altered after prolonged opioid treatment but was higher in cells coexpressing G(z). Although the mu- and kappa-opioid receptors mediated acute activation of extracellular signal-regulated protein kinase 1/2 via both G(i) and G(z), these responses were abolished by chronic opioid treatment. These studies showed that G(z) could mediate acute actions of mu- and kappa-opioids but G(z) alone was insufficient to mediate adenylyl cyclase supersensitization induced by the chronic activation of opioid receptors.

Regulation of adenylyl cyclase, ERK1/2, and CREB by Gz following acute and chronic activation of the delta-opioid receptor.

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 Gz (a member of the Gi subfamily), which inhibits adenylyl cyclase and stimulates mitogen-activated protein kinases (MAPKs). Here, we established stable human embryonic kidney 293 cell lines expressing delta-opioid receptors with or without Gz to examine the role of Gz in opioid receptor-regulated signaling systems. Each cell line was acutely or chronically treated with [D-Pen2,D-Pen5]enkephalin (DPDPE), a delta-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 signal-regulated protein kinases (ERKs) 1 and 2. In cells coexpressing Gz, DPDPE inhibited forskolin-stimulated cAMP accumulation in a PTX-insensitive manner, but Gz could not replace Gi 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 Gi and Gz mediated DPDPE-induced activation of ERK1/2, but these responses were abolished by chronic opioid treatment. Collectively, our results show that although Gz mediated opioid-induced inhibition of adenylyl cyclase and activation of ERK1/2, Gz alone was insufficient to mediate opioid-induced adenylyl cyclase supersensitization.