micro-Opioid receptor (MOR) desensitization and endocytosis have been implicated in tolerance and dependence to opioids. The efficiency of each process is known to be agonist dependent; however, it is not known what determines the relative efficiency of various agonists at either process. In the present study, homologous MOR desensitization in locus ceruleus (LC) neurons and MOR internalization in HEK293 cells were examined using a series of agonists. The results show that the rank order of this series of agonists was different when comparing the magnitude of hyperpolarization and the ability to cause desensitization in LC neurons. Endocytosis of MOR was also examined in HEK293 cells using the same agonists. The relative ability to cause endocytosis in HEK293 cells correlated with the degree of desensitization in LC cells. This strong correlation suggests that the two processes are closely linked. The results also suggest that agonist efficacy is not necessarily a predictor of the ability to cause MOR desensitization or endocytosis. Identification and characterization of the biophysical properties of agonists that favor desensitization and internalization of receptors will lead to a better understanding of opioid signaling.
Chronic morphine treatment produces behavioral and cellular opioid tolerance that has been proposed to be caused by attenuated -opioid receptor (MOR) recovery from desensitization (resensitization). The process of MOR resensitization is thought to require arrestin-2 (arr-2)-dependent trafficking of desensitized receptors to endosomal compartments, followed by recycling of resensitized receptors back to the plasma membrane. However, there is little direct evidence for this, particularly in native neurons. This study used whole-cell patch-clamp recording in locus ceruleus (LC) neurons from wild-type (w.t.) and arr-2 knock-out (k.o.) mice to examine whether arr-2/dynamin-dependent trafficking is required for MOR resensitization in neurons from opioid-naive and morphine-treated mice. Surprisingly, recovery of MOR from acute desensitization in LC neurons does not require arr-2-or dynamin-dependent trafficking. To the contrary, MOR resensitization was accelerated by disruption of either arr-2 or dynamin function. Chronic morphine treatment caused cellular MOR tolerance and concurrently impaired MOR resensitization in neurons from w.t. mice, as expected from previous studies, but neither occurred in neurons from arr-2 k.o. mice. Moreover, the impairment of MOR resensitization caused by chronic morphine was reversed in w.t. neurons when G-protein-coupled receptor kinase-2 (GRK2) or dynamin function was disrupted. Together, these results establish that arr-2/dynamin-dependent receptor regulation is not required for MOR resensitization in LC neurons. Furthermore, chronic morphine treatment modifies GRK2-arr-2-dynamin-dependent MOR trafficking to impair receptor resensitization, thereby contributing to opioid tolerance in LC neurons by reducing the number of functional receptors on the surface membrane.
Agonists acting on m-opioid receptors (MOR) are very effective analgesics but cause tolerance during long-term or repeated exposure. Intensive efforts have been made to find novel opioid agonists that are efficacious analgesics but can elude the signalling events that cause tolerance. m-Opioid agonists differentially couple to downstream signalling mechanisms. Some agonists, such as enkephalins, ,N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO), methadone and sufentanyl are efficacious at mediating G-protein and effector coupling, as well as triggering MOR regulatory events that include MOR phosphorylation, b-arrestin binding, receptor endocytosis and recycling. By contrast, morphine and closely related alkaloids can mediate efficacious MOR-effector coupling but poorly trigger receptor regulation. Several models have been proposed to relate differential MOR regulation by different opioids with their propensity to cause tolerance. Most are based on dogma that b-arrestin-2 (barr-2) binding causes MOR desensitization and/or that MOR endocytosis and recycling are required for receptor resensitization. This review will examine some of these notions in light of recent evidence establishing that MOR dephosphorylation and resensitization do not require endocytosis. Recent evidence from opioid-treated animals also suggests that impaired MOR-effector coupling is driven, at least in part, by enhanced desensitization, as well as impaired resensitization that appears to be barr-2 dependent. Better understanding of how chronic exposure to opioids alters receptor regulatory mechanisms may facilitate the development of effective analgesics that produce limited tolerance. LINKED ARTICLESThis IntroductionOpioids are potent and effective analgesics. It is well established that nearly all clinically used opioids mediate their analgesic effects by activating the m-opioid receptor (MOR; Kieffer and Gaveriaux-Ruff, 2002). However, long-term use of m-opioid agonists produces adverse effects that include the development of tolerance and addiction, limiting their clinical utility (Williams et al., 2001;Christie, 2008;Morgan and Christie, 2011). Qualitatively, all MOR agonists produce tolerance in vivo although there are differences in the extent of tolerance (Morgan and Christie, 2011), suggesting that opioid analgesics resistant to tolerance could be developed. Recent promising approaches to limit tolerance have been extensively reviewed and include simultaneous activation of more than one opioid receptor type (e.g. MOR and DOR receptors), selective targeting of heteromultimers or opioids that differentially activate distinct intracellular signalling cascades, possibly involving differential activation of Ga subtypes (Pineyro and Archer-Lahlou, 2007), and particularly differential G-protein activation versus endocytosis (e.g. Martini and Whistler, 2007;Christie, 2008;Koch and Hollt, 2008;Berger and Whistler, 2010;von Zastrow, 2010). BJPBritish Journal of Pharmacology DOI:10.1111DOI:10. /j.1476DOI:10. -5381.2011 1704 British Journal of Pharmac...
Sustained stimulation of G-protein coupled receptors (GPCRs) leads to rapid loss of receptor function (acute desensitization). For manyGPCRs including the -opioid receptor (MOR), an accepted mechanism for acute desensitization is through G-protein coupled receptor kinase (GRKs) mediated phosphorylation of the receptor, which facilitates the binding of -arrestins (arrs) to the receptor and then promotes endocytosis. However, the mechanism(s) that mediate acute desensitization have not yet been well defined in native neurons. This study used whole-cell patch clamp recording of G-protein coupled inward-rectifying potassium (GIRK) currents to assay MOR function and identify mechanisms of acute MOR desensitization in locus ceruleus (LC) neurons. The rate and extent of MOR desensitization were unaffected by arr-2 knock-out. Disruption of GRK2 function via inhibitory peptide introduced directly into neurons also failed to affect desensitization in wild type or arr-2 knock-outs. Inhibition of ERK1/2 activation alone had little effect on acute desensitization. However, when both GRK2-arr-2 and ERK1/2 functions were disrupted simultaneously, desensitization of MOR was nearly abolished. Together, these results suggest that acute desensitization of MOR in native LC neurons is determined by at least two molecular pathways, one involving GRK2 and arr-2, and a parallel pathway mediated by activated ERK1/2.
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