Agonist-induced mu-opioid receptor (MOPr) internalization has long been suggested to contribute directly to functional receptor desensitization and opioid tolerance. In contrast, recent evidence ...suggests that opioid receptor internalization could in fact reduce opioid tolerance in vivo, but the mechanisms that are responsible for the internalization-mediated protection against opioid tolerance are controversely discussed. One prevailing hypothesis is, that receptor internalization leads to decreased receptor signaling and therefore to reduced associated compensatory changes in downstream signaling systems that are involved in the development of opioid tolerance. However, numerous studies have demonstrated that desensitized and internalized mu-opioid receptors are rapidly recycled to the cell surface in a reactivated state, thus counteracting receptor desensitization and opioid tolerance. Further studies revealed agonist-selective differences in the ability to induce opioid receptor internalization. Recently it has been demonstrated that the endocytotic efficacies of opioids are negatively correlated to the induced opioid tolerance. Thus, clearer understanding of the role of opioid receptor trafficking in the regulation of opioid tolerance and dependence will help in the treatment of patients suffering from chronic pain or drug dependence.
BACKGROUND AND PURPOSE Morphine activates the µ‐opioid receptor without causing its rapid endocytosis. In contrast, full agonists such as d‐Ala2‐MePhe4‐Gly‐olenkephalin (DAMGO) or etonitazene ...stimulate a rapid and profound internalization. However, the detailed molecular events underlying the differential regulation of receptor trafficking by distinct opioid agonists remain incompletely understood.
EXPERIMENTAL APPROACH Here, we have generated phosphosite‐specific antibodies for the carboxyl‐terminal residues serine 363 (Ser363), threonine 370 (Thr370) and serine 375 (Ser375), which enabled us to selectively detect either the Ser363‐, Thr370‐ or Ser375‐phosphorylated form of the receptor.
KEY RESULTS We showed that agonist‐induced phosphorylation occurs at Thr370 and Ser375, whereas Ser363 is constitutively phosphorylated in the absence of agonist. We further demonstated that DAMGO and etonitazene stimulated the phosphorylation of both Thr370 and Ser375. In contrast, morphine promoted the phosphorylation of Ser375, but failed to stimulate Thr370 phosphorylation. In the presence of DAMGO, Ser375 phosphorylation occurred at a faster rate than phosphorylation of Thr370, indicating that Ser375 is the primary site of agonist‐dependent phosphorylation. Activation of PKC by phorbol 12‐myristate 13‐acetate increased receptor phosphorylation only on Thr370, but not on Ser375, indicating that Thr370 can also undergo heterologous PKC‐mediated phosphorylation. We also showed that µ receptor dephosphorylation can occur within minutes at or near the plasma membrane, and that agonist removal is a major prerequisite for Thr370 and Ser375 dephosphorylation.
CONCLUSIONS AND IMPLICATIONS Together, we showed for the first time that distinct agonists stimulate site‐specific patterns of phosphorylation, which are intimately related to their ability to elicit µ‐opioid receptor sequestration.
LINKED ARTICLE This article is commented on by Kelly, pp. 294–297 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476‐5381.2011.01387.x
A re-balance of postsynaptic serotonin (5-HT) receptor signalling, with an increase in 5-HT1A and a decrease in 5-HT2A signalling, is a final common pathway multiple antidepressants share. Given that ...the 5-HT1A/2A agonist lysergic acid diethylamide (LSD), when repeatedly applied, selectively downregulates 5-HT2A, but not 5-HT1A receptors, one might expect LSD to similarly re-balance the postsynaptic 5-HT signalling. Challenging this idea, we use an animal model of depression specifically responding to repeated antidepressant treatment (olfactory bulbectomy), and test the antidepressant-like properties of repeated LSD treatment (0.13 mg/kg/d, 11 d). In line with former findings, we observe that bulbectomised rats show marked deficits in active avoidance learning. These deficits, similarly as we earlier noted with imipramine, are largely reversed by repeated LSD administration. Additionally, bulbectomised rats exhibit distinct anomalies of monoamine receptor signalling in hippocampus and/or frontal cortex; from these, only the hippocampal decrease in 5-HT2 related 35S-GTP-gamma-S binding is normalised by LSD. Importantly, the sham-operated rats do not profit from LSD, and exhibit reduced hippocampal 5-HT2 signalling. As behavioural deficits after bulbectomy respond to agents classified as antidepressants only, we conclude that the effect of LSD in this model can be considered antidepressant-like, and discuss it in terms of a re-balance of hippocampal 5-HT2/5-HT1A signalling.
Abstract Opioids are irreplaceable for the treatment of severe pain. However, opioid-induced immunomodulation affects therapies. Here we report that treatment of human T lymphocytes with the opioids ...fentanyl, methadone, loperamide and beta-endorphin resulted in a strong induction of the cytokine interleukin-4. In contrast, morphine and buprenorphine induced markedly and significantly lower levels of interleukin-4 mRNA and protein. These findings suggest agonist-biased μ opioid receptor signaling in T cells. In the future, better knowledge about agonist-specific immunomodulatory effects of opioids offers the possibility to select drugs for a therapy with more favorable and/or less detrimental side effects in immune cells.
Abstract Expression patterns of the second SDF-1 receptor RDC1/CXCR7 were examined after focal ischemia in rats using in situ hybridization. CXCR7 mRNA was identified in the ventricle walls as well ...as neuronal, astroglial, and vascular cells. After ischemia, intact cortical regions showed a rapid, 4 days-lasting increase in neuronal CXCR7 expression. In the ischemic tissue CXCR7 expression was scarce and associated with blood vessels. Between days 2 and 10 after ischemia-onset, SDF-1 expression increased strongly in the peri-infarct and infarct region, which was accompanied by the appearance of numerous CXCR4-expressing but not CXCR7-expressing cells. These patterns suggest that SDF-1 may influence vascular, astroglial, and neuronal functions via CXCR7 and mediate cell recruitment to ischemic brain areas via CXCR4.
The aim of this study was to characterize inhibitory mechanisms on T cell receptor signaling mediated by the cannabinoid receptors CB1 and CB2. Both receptors are coupled to Gi/o proteins, which are ...associated with inhibition of cyclic AMP formation. In human primary and Jurkat T lymphocytes, activation of CB1 by R(+)-methanandamide, CB2 by JWH015, and both by Δ9-tetrahydrocannabinol induced a short decrease in cyclic AMP lasting less than 1 h. However, this decrease was followed by a massive (up to 10-fold) and sustained (at least up to 48 h) increase in cyclic AMP. Mediated by the cyclic AMP-activated protein kinase A and C-terminal Src kinase, the cannabinoids induced a stable phosphorylation of the inhibitory Tyr-505 of the leukocyte-specific protein tyrosine kinase (Lck). By thus arresting Lck in its inhibited form, the cannabinoids prevented the dephosphorylation of Lck at Tyr-505 in response to T cell receptor activation, which is necessary for the subsequent initiation of T cell receptor signaling. In this way the cannabinoids inhibited the T cell receptor-triggered signaling, i.e. the activation of the ζ-chain-associated protein kinase of 70 kDa, the linker for activation of T cells, MAPK, the induction of interleukin-2, and T cell proliferation. All of the effects of the cannabinoids were blocked by the CB1 and CB2 antagonists AM281 and AM630. These findings help to better understand the immunosuppressive effects of cannabinoids and explain the beneficial effects of these drugs in the treatment of T cell-mediated autoimmune disorders like multiple sclerosis.
•LSD and DOB induce a ketanserin-sensitive increase in shaking behaviour.•LSD and DOB induced shaking behaviour is undermined by tolerance development.•Tolerance to DOB correlates with reduced ...frontocortical 5-HT2A binding sites.•Tolerance to LSD does not correlate with frontocortical 5-HT2A binding sites.•Tolerance to LSD correlates with reduced frontocortical glutamate binding sites.
Serotonergic hallucinogens, such as lysergic acid diethylamide (LSD) and dimethoxy-bromoamphetamine (DOB), provoke stereotype-like shaking behaviour in rodents, which is hypothesised to engage frontocortical glutamate receptor activation secondary to serotonin2A (5-HT2A) related glutamate release. Challenging this hypothesis, we here investigate whether tolerance to LSD and DOB correlates with frontocortical adaptations of 5-HT2A and/or overall-glutamate binding sites. LSD and DOB (0.025 and 0.25mg/kg, i.p.) induce a ketanserin-sensitive (0.5mg/kg, i.p., 30-min pretreatment) increase in shaking behaviour (including head twitches and wet dog shakes), which with repeated application (7× in 4ds) is undermined by tolerance. Tolerance to DOB, as indexed by DOB-sensitive 3Hspiroperidol and DOB induced 35SGTP-gamma-S binding, is accompanied by a frontocortical decrease in 5-HT2A binding sites and 5-HT2 signalling, respectively; glutamate-sensitive 3Hglutamate binding sites, in contrast, remain unchanged. As to LSD, 5-HT2 signalling and 5-HT2A binding, respectively, are not or only marginally affected, yet 3Hglutamate binding is significantly decreased. Correlation analysis interrelates tolerance to DOB to the reduced 5-HT2A (r=.80) as well as the unchanged 3Hglutamate binding sites (r=.84); tolerance to LSD, as opposed, shares variance with the reduction in 3Hglutamate binding sites only (r=.86). Given that DOB and LSD both induce tolerance, one correlating with 5-HT2A, the other with glutamate receptor adaptations, it might be inferred that tolerance can arise at either level. That is, if a hallucinogen (like LSD in our study) fails to induce 5-HT2A (down-)regulation, glutamate receptors (activated postsynaptic to 5-HT2A related glutamate release) might instead adapt and thus prevent further overstimulation of the cortex.
Opioids are widely used for the treatment of severe pain. However, it is also known that opioids, in particular morphine, cause immunosuppression. Therefore, their use may complicate treatment of ...persons with an already impaired immune system, e.g., patients suffering from cancer or AIDS. We investigated the mechanisms of opioid-induced immunosuppression in primary human T lymphocytes and the human T cell line Jurkat. We demonstrated that morphine and the endogenous opioid beta-endorphin inhibited the transcription of IL-2 in activated human T lymphocytes as well as the activation of the transcription factors AP-1, NFAT, and NF-kappaB, which transactivate IL-2. In addition, the TCR-induced calcium flux and MAPK activation were inhibited by the opioids, as well as proximal signaling events, such as the phosphorylation of the linker for activation of T cells and Zap70. A more detailed characterization of the mechanism revealed that incubation of T cells with the opioids caused a marked increase in cAMP. This in turn activated protein kinase A, which augmented the kinase activity of C-terminal Src kinase bound to phosphoprotein associated with glycosphingolipid-enrich microdomains, resulting in a further enhancement of the tonic inhibition of the leukocyte-specific protein tyrosine kinase Lck, thereby blocking the initiation of TCR signaling. These effects were mediated by mu opioid receptors. Our findings contribute to the understanding of immunosuppressive side effects of morphine. Since beta-endorphin is expressed and secreted by immune effector cells, including T cells, and up-regulated in these cells by various stimuli, our data also suggest an inhibitory role for beta-endorphin in the physiological regulation of T cell activation.
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