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.

Tolerance and dependence result from long-term exposure to opioids, and there is growing evidence linking acute receptor desensitization to these more long-term processes. Receptor desensitization encompasses a series of events leading to the loss of receptor function and internalization. This study examines the onset and recovery from desensitization in locus ceruleus neurons recorded in brain slices taken from animals that have been chronically treated with morphine. After chronic morphine treatment, desensitization was altered as follows. First, the rate of desensitization was increased. Second, recovery from desensitization was always incomplete, even after a brief (1-2 min) exposure to agonist. This contrasts with experiments in controls in which recovery from desensitization, after a brief exposure to agonist, was complete within 25 min. Finally, morphine-6-␤-D-glucuronide, a metabolite of morphine that was ineffective at causing desensitization in controls, induced significant desensitization in slices from morphine-treated animals. When brain slices from controls were treated with inhibitors of PKC or monensin, agents known to compromise G-protein-coupled receptor resensitization, desensitization was increased, and recovery was significantly reduced. These results indicate that receptor resensitization maintains signaling during periods of intense and sustained stimulation. After chronic morphine treatment, desensitization is potentiated, and receptor resensitization is compromised.

Morphine has been widely accepted as the opioid agonist that sustains signaling because it does not cause receptor desensitization or internalization. This notion has led to the hypothesis that long-term morphine treatment initiates downstream adaptations that underlie tolerance and dependence. This study uses whole-cell recordings from neurons in the locus ceruleus to measure the potassium current induced by morphine. The results show that morphine does cause short-term desensitization. The desensitization induced by morphine was slower and smaller then that induced by [MET] 5 -enkephalin (ME). After a brief application of a saturating concentration of ME, the current induced by morphine was smaller, and desensitization was not observed. In tissue taken from morphinetreated animals, the peak current induced by morphine was the same as in untreated animals, but morphine-induced desensitization was facilitated. The results suggest that morphine, like other agonists, can initiate receptor desensitization to decrease signaling.Morphine is one of a very few opioids that cause little or no desensitization, internalization, and recycling of -opioid receptors (MOR) (Sternini et al

Objective-Because previous preclinical and clinical studies have implicated the endogenous opioid system in major depression and in the neurochemical action of antidepressants, the authors examined how DNA variation in the μ-opioid receptor gene may influence population variation in response to citalopram treatment. Method-A total of 1,953 individuals from the Sequenced Treatment Alternatives to RelieveDepression (STAR*D) study were treated with citalopram and genotyped for 53 single nucleotide polymorphisms (SNPs) in a 100-kb region of the OPRM1 gene. The sample consisted of NonHispanic Caucasians, Hispanic Caucasians, and African Americans. Population stratification was corrected using 119 ancestry informative markers and principal components analysis. Markers were tested for association with phenotypes for general and specific citalopram response as well as remission.Results-Association between one SNP and specific citalopram response was observed. After Bonferroni correction, the strongest finding was the association between the rs540825 SNP and specific response. The rs540825 polymorphism is a nonsynonymous SNP in the final exon of the μ-opioid receptor-1X isoform of the OPRM1 gene, resulting in a histidine to glutamine change in the intra-cellular domain of the receptor. When Hispanic and Non-Hispanic Caucasians were analyzed separately, similar results in the population-corrected analyses were detected.Conclusions-These results suggest that rates of response to antidepressants and consequent remission from major depressive disorder are influenced by variation in the μ-opioid receptor gene as a result of either an effect on placebo response or true pharmacologic response.Antidepressant response rates in the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study averaged <50% for treatment with citalopram (level 1 treatment condition), with only approximately 30% of subjects remitting from major depressive disorder (1).Address correspondence and reprint requests to Dr. Hamilton, University of California, San Francisco, Department of Psychiatry, 401 Parnassus Ave., San Francisco, steveh@lppi.ucsf.edu. Previously presented at the Annual Meeting of the National Institute of Mental Health New Clinical Drug Evaluation Unit, June 11-14, 2007, Boca Raton, Fla.; and the Annual Meeting of Pharmacogenetics in Psychiatry, April 13-14, 2007, New York. Preliminary analyses were also presented in Psychiatric Times (April 15, 2008, vol. 25, no. 5).ClinicalTrials.gov registry number, NCT00021528. NIH Public Access Author ManuscriptAm J Psychiatry. Author manuscript; available in PMC 2010 June 15. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptInterindividual differences, such as genetic distinction, may contribute to this variation in response to antidepressant treatment, and two genes have already been identified in two studies (2,3). In the present study, we add to the findings of these investigations and show that the μ-opioid receptor gene is associated with antidepr...

Prolonged agonist stimulation of the -opioid receptor (MOR) initiates receptor regulatory events that rapidly attenuate receptor-mediated signaling (homologous desensitization). Emerging evidence suggests that persistent MOR stimulation can also reduce responsiveness of effectors to other G-protein-coupled receptors, termed heterologous desensitization. However, the mechanisms by which heterologous desensitization is triggered by MOR stimulation are unclear. This study used whole-cell patch-clamp recordings of ligand activated G-protein-activated inwardly rectifying potassium channel currents in mouse brain slices containing locus ceruleus (LC) neurons to determine the effects of prolonged stimulation of MOR on ␣ 2 -adrenoceptor (␣ 2 -AR) function. The results show distinct and sequential development of homologous and heterologous desensitization during persistent stimulation of MOR in LC neurons with Met 5 -enkephalin (ME). ME stimulation of MOR promoted rapid homologous desensitization that reached a steady state after 5 min and partially recovered over 30 min. Longer stimulation of MOR (10 min) induced heterologous desensitization of ␣ 2 -AR function that exhibited slower recovery than homologous desensitization. Heterologous (but not homologous) desensitization required ␤-arrestin-2 (␤arr-2) because it was nearly abolished in ␤arr-2-knockout (ko) mice. Heterologous (but not homologous) desensitization was also prevented by inhibition of ERK1/2 and c-Src signaling in wild-type (wt) mouse LC neurons. Heterologous desensitization may be physiologically relevant during exposure to high doses of opioids because ␣ 2 -AR-mediated slow inhibitory postsynaptic currents were depressed in wt but not ␤arr-2 ko LC neurons after prolonged exposure to opioids. Together, these findings demonstrate a novel mechanism by which ␤arr-2 can regulate postsynaptic responsiveness to neurotransmitter release.