Plasticity of Adenylyl Cyclase-Related Signaling Sequelae after Long-Term Morphine Treatment

Abstract

Adaptations to long-term morphine treatment resulting in tolerance are protective by counteracting the consequences of sustained opioid receptor activation. Consequently, the manifestation of specific adenylyl cyclase (AC)-related neurochemical sequelae of long-term morphine treatment should depend on the consequences of short-term -opioid receptor (MOR) activation. We tested this by comparing complementary chemical sequelae of long-term morphine treatment among cells in which short-term MOR activation inhibited instead of stimulated AC activity. Short-term activation of MOR in Chinese hamster ovary (CHO) cells stably transfected with MOR (MOR-CHO) inhibits AC activity. Long-term morphine treatment of these cells increased AC and G␤ phosphorylation, membrane protein kinase C␥ (PKC␥) translocation, and MOR G s association. All converge, shifting the consequences of short-term MOR activation from G␣ i /G␣ o inhibitory to AC stimulatory signaling. In contrast, overexpression of the G␤␥-stimulated AC isoform AC2 (which converted MOR-coupled inhibition to stimulation of AC) eliminated or reversed these adaptations to long-term morphine treatment; it negated the increase in G␤ phosphorylation and PKC␥ translocation while reversing the increase in AC phosphorylation and MOR G s association. These adaptations greatly attenuated MOR-coupled stimulation of AC activity. Altered overexpression of AC protein per se was not a confounding factor because MOR-CHO overexpressing AC1, which is inhibited by short-term MOR activation, manifested adaptations to long-term morphine treatment qualitatively identical with those of MOR-CHO. These results reveal that adaptations elicited by long-term morphine treatment depend on the effects of short-term MOR activation. This dynamic and pliable nature of tolerance mechanisms could represent a new paradigm for pharmacotherapeutics.All formulations of opioid tolerance are based on the tenet that it is adaptive. All identified neurochemical adaptations to long-term morphine enable cell survival by reinstating initial steady-state conditions. For example, up-regulation of the adenylyl cyclase (AC) system, believed to underlie the AC "superactivation" that is manifest upon short-term precipitated opioid withdrawal of long-term opioid-treated tissue (Chavkin and Goldstein, 1984;Chakrabarti et al., 1995;Cox and Crowder, 2004), would neutralize the persistent ongoing opioid receptor-coupled inhibition of AC. Opioid receptor uncoupling and its arrestin-mediated internalization that has been demonstrated in some preparations after persistent opioid exposure (Appleyard et al., 1999;Bohn et al., 2000) has been postulated to compensate for the sustained inhibitory action of drugs like morphine that are resistant to proteolytic degradation and thus persist in the extracellular milieu. Augmented AC stimulatory signaling via G␤␥ (Chakrabarti et al., 1998a(Chakrabarti et al., ,b, 2005bChakrabarti and Gintzler, 2003) and increased interaction of the -opioid receptor (MOR) with G s (Chakrabarti et ...

Pleiotropic Opioid Regulation of Spinal Endomorphin 2 Release and Its Adaptations to Opioid Withdrawal Are Sexually Dimorphic

“…We had reported previously that chronic morphine increases MOR G s coupling in cell lines as well as spinal cord (Chakrabarti et al, 2005a;Chakrabarti and Gintzler, 2007;Shy et al, 2008). The present results reaffirm those findings and put them into a physiological context, i.e., enhanced MOR G s signaling during withdrawal enables opioid facilitation of EM2 release, which in turn contributes to withdrawalcoping mechanisms.…”

Subcellular Localization of μ-Opioid Receptor Gs Signaling

“…6). Because increased MOR G s ␣ signaling is associated with the formation of opioid tolerance (Chakrabarti et al, 2005;Chakrabarti and Gintzler, 2007;Shy et al, 2008) and caveolae are generally considered to be important subcellular domains specifying the formation of signaling complexes, understanding the caveolae-associated substrates for MOR G s ␣ AC signaling could provide unexpected insights into critical subcellular determinants of tolerance and the related phenomenon of addiction.…”

“…1) In MOR-CHO membranes, sufentanil stimulated the translocation of G s ␣ into Triton-insoluble membrane compartments. 2) Sufentanil enhanced the coimmunoprecipitation (co-IP) of G s ␣ and adenylyl cyclase (AC) with caveolin-1 (a marker for caveolae) from the Triton-insoluble membrane fraction of spinal cord Pharmacological analyses (Xu et al, 1989;Shen and Crain, 1990;Gintzler and Xu, 1991;Cruciani et al, 1993;Wang and Gintzler, 1997;Szucs et al, 2004) and biochemical analyses (Chakrabarti et al, 2005;Chakrabarti and Gintzler, 2007;Shy et al, 2008) provide convergent evidence for -opioid receptor (MOR) signaling via G s , which has been long debated among opioid researchers. Our recent demonstrations that MOR is present in G s ␣ immunoprecipitate (IP), obtained from a variety of MOR-expressing cell lines and spinal cord, and the content of MOR in G s ␣ IP increases after chronic morphine exposure underscores the putative relevance of MOR G s ␣ signaling to acute and chronic opioid responsiveness.…”

The Ambiguities of Opioid Tolerance Mechanisms: Barriers to Pain Therapeutics or New Pain Therapeutic Possibilities: Fig. 1.

“…The complexity and multidimensionality of cellular mechanisms underlying opioid tolerance are underscored by the recent report that a subset of inter-related cell signaling adaptations to chronic morphine exposure does not represent a fixed set of adaptations; rather, these inter-related cell signaling adaptations are cell state-dependent (Shy et al, 2008).…”

“…The qualitative difference in the consequences of acute MOR activation (AC inhibition versus stimulation) has a profound effect on the manifestation of multiple, complementary AC-related adaptations to chronic morphine, many of which are diametrically opposite (Shy et al, 2008). It is striking that none of the AC/cAMP-related adaptations to chronic morphine observed in MOR-CHO and AC1-MOR-CHO (increased AC and G ␤ phosphorylation, membrane protein kinase C␥ translocation and MOR G s association (Chakrabarti et al, 1998b(Chakrabarti et al, , 2005aChakrabarti and Gintzler, 2003) is observed in AC2-MOR-CHO.…”