Opioid Receptor Function Is Regulated by Post-endocytic Peptide Processing

Most neuroendocrine peptides are generated in the secretory compartment by proteolysis of the precursors at classical cleavage sites consisting of basic residues by well studied endopeptidases belonging to the subtilisin superfamily. In contrast, a subset of bioactive peptides is generated by processing at non-classical cleavage sites that do not contain basic residues. Neither the peptidases responsible for non-classical cleavages nor the compartment involved in such processing has been well established. Members of the endothelin-converting enzyme (ECE) family are considered good candidate enzymes because they exhibit functional properties that are consistent with such a role. In this study we have explored a role for ECE2 in endocytic processing of δ opioid peptides and its effect on modulating δ opioid receptor function by using selective inhibitors of ECE2 that we had identified previously by homology modeling and virtual screening of a library of small molecules. We found that agonist treatment led to intracellular co-localization of ECE2 with δ opioid receptors. Furthermore, selective inhibitors of ECE2 and reagents that increase the pH of the acidic compartment impaired receptor recycling by protecting the endocytosed peptide from degradation. This, in turn, led to a substantial decrease in surface receptor signaling. Finally, we showed that treatment of primary neurons with the ECE2 inhibitor during recycling led to increased intracellular co-localization of the receptors and ECE2, which in turn led to decreased receptor recycling and signaling by the surface receptors. Together, these results support a role for differential modulation of opioid receptor signaling by post-endocytic processing of peptide agonists by ECE2.     

Evidence for the involvement of descending pain-inhibitory mechanisms in the attenuation of cancer pain by carvacrol aided through a docking study

Aims

The present study evaluated the carvacrol (CARV) effect on hyperalgesia and nociception induced by sarcoma 180 (S180) in mice.

Main methods

Carvacrol treatment (12.5–50 mg/kg s.c.) once daily for 15 days was started 24 h after injection of the sarcoma cells in the hind paw (s.c.). Mice were evaluated for mechanical sensitivity (von Frey), spontaneous and palpation-induced nociception, limb use and tumor growth on alternate days. CARV effects on the central nervous system were evaluated through immunofluorescence for Fos protein. Molecular docking studies also were performed to evaluate intermolecular interactions of the carvacrol and muscimol, as ligands of interleukin-10 and GABA_A receptors.

Key findings

CARV was able to significantly reduce mechanical hyperalgesia and spontaneous and palpation-induced nociception, improve use paw, decrease the number of positively marked neurons in lumbar spinal cord and activate periaqueductal gray, nucleus raphe magnus and locus coeruleus. CARV also caused significant decreased tumor growth. Docking studies showed favorable interaction overlay of the CARV with IL-10 and GABA_A.

Significance

Together, these results demonstrated that CARV may be an interesting option for the development of new analgesic drugs for the management of cancer pain.     

Morphine alleviates early stages of apoptosis in cultured CEM x174 cells infected with simian immunodeficiency virus

The reduction in apoptosis caused by short-term exposure of CEM x174 cells infected with SIVmac239 to morphine was investigated. Eeffects of morphine on the viability of normal and infected CEM x174 cells were determined by MTS assay. Apoptosis induced by SIVmac239 and the effects of morphine were analyzed by flow cytometry. cAMP levels, PKA activity, and the resulting histone H3 phosphorylation levels were measured. The results show a pronounced decrease in numbers of infected SIVmac239 cells compared to controls. Morphine elevated cell viability in the infected groups. Annexin V binding assays showed that 1 microM l(-1) morphine increased the percentage of viable cells and decreased apoptotic cells. Morphine also downregulated cAMP and PKA activity in both groups, but more markedly in the infected group. Histone H3 phosphorylation was elevated after virus infection and decreased in the presence of morphine. The results indicate that the cAMP-PKA signal transduction cascade is involved in morphine regulation of early SIVmac239-induced apoptosis.     

巴氯芬对慢性吗啡依赖后条件化位置偏好和戒断症状的抑制

小鼠连续7天腹腔注射吗啡(40mg/kg)建立条件化位置偏好模型,连续皮下递增注射吗啡(25、50、75、100、125、150mg/kg),成瘾后腹腔注射纳络酮(6mg/kg)诱导戒断症状(跳跃行为)建立戒断模型。腹腔注射GABAB受体激动剂巴氯芬(2mg/kg)可以有效地抑制吗啡诱导的条件化位置偏好和减轻纳络酮诱导的戒断症状,结果表明GABA系统参与动物成瘾后渴求和戒断过程,激动GABAB受体可以在一定程度上抑制成瘾的心理和生理戒断症状。     

Effects of prenatal cocaine, morphine, or both on postnatal opioid (mu) receptor development

We studied the effects of prenatal cocaine and morphine given separately and in combination on the (1) postnatal brain mu-opioid receptor development and (2) interaction of dopamine with mu receptors. Pregnant rats received single daily intraperitoneal (I.P.) injections of saline, cocaine (20 mg/kg), morphine (2 mg/kg), or the combination of both drugs from day 13 to day 20 of gestation. Postnatal days (P) 1, 7, 14, and 28, whole brains were analyzed for opioid receptor binding and mu mRNA. Prenatal cocaine administered by itself had no significant effect on the ontogeny of brain mu receptors on all the days studied when compared to controls. The morphine-treated group showed a significant increase in mu receptor binding on P1 and P7. Exposure to both cocaine and morphine showed a significant increase in mu receptor density on P1 and P7. In addition, there was also a significant increase in MOR mRNA in both the morphine alone and combination groups. Pretreatment with dopamine D2 receptor antagonist (sulpiride, 20 mg/kg) prior to drug administration showed decreased mu receptor binding on P1 and P7. These results suggest that prenatal exposure to morphine or a combination of cocaine and morphine significantly increases mu receptor density. By P14, mu-opioid receptor binding was no longer different than the control. This may suggest that the effect on receptor may be short-lived and that other key intracellular events may be activated to mediate the long-term effects. Also, the data show that dopaminergic mechanisms are (or opioid-dopamine interaction is) involved in the effects of morphine alone or morphine in combination with cocaine on mu receptor regulation.     

Protein kinase C inhibitor chelerythrine attenuates the morphine-induced excitatory amino acid release and reduction of the antinociceptive effect of morphine in rats injected intrathecally with pertussis toxin

Neuropathic pain syndromes respond poorly to opioid treatment. In our previous studies, we found that intrathecal (i.t.) injection of pertussis toxin (PTX) produces thermal hyperalgesia, which is poorly responsive to morphine and is accompanied by an increase in cerebrospinal fluid (CSF) levels of excitatory amino acids (EAAs) and protein kinase C (PKC) activation. In the present study, rats were implanted with an i.t. catheter for drug injection and a microdialysis probe for CSF dialysate collection. On the fourth day after injection of PTX (2 microg, i.t.), there was a significant reduction in the antinociceptive effect of morphine (10 microg, i.t.) which was accompanied by an increase in levels of EAAs. Pretreatment with the PKC inhibitor, chelerythrine (25 microg, i.t.) one hour before morphine injection markedly inhibited both effects. These results suggest that, in PTX-treated rats, PKC plays an important role in inhibiting the morphine-induced spinal EAA release, which might be related to the reduced antinociceptive effect of morphine.     

Up-regulated L-type high voltage-gated calcium channels cause increase in diazepam binding inhibitor induced by sustained morphine exposure in mouse cerebrocortical neurons

Mechanisms of increase in diazepam binding inhibitor (DBI) mRNA expression in mouse cerebrocortical neurons after sustained morphine exposure were investigated. Increases in DBI and its mRNA expressions induced by sustained morphine (0.3 μM) exposure for 3 days were completely abolished by naloxone and nifedipine, but not by ω-agatoxin VIA and ω-conotoxin GIVA. Increase in [³H]diltiazem binding to the particulate fractions from the morphine-treated neurons was due to increased B_max value with no changes in K_d value. Western blot analysis on l-type high voltage-gated calcium channel (HVCC) subunits revealed the increased expressions of α1C, α1D, and α2/δ1 subunits and decreased of β4 subunit expression, whereas expression of N- and P/Q-type HVCC subunits was not changed. These results indicate that morphine-induced increase in DBI mRNA expression is mediated via increased Ca²⁺ entry through up-regulated l-type HVCCs.     

Modeling the onset of drug dependence: a consideration of the requirement for protein synthesis

It has been proposed, with some supporting evidence, that development of opiate tolerance and dependence requires protein synthesis. However, a quantitative, biologically based model within which to analyse and support the data has been lacking. Utilizing such a framework or model, we recently compared the time course of onset of opiate dependence in laboratory animals, with the mathematical time course of general changes in protein levels. Not only did the time course of onset of dependence parallel the time course of increasing levels of a protein, but also the half-life of the putative protein required by the model was very similar to those of many brain proteins. In this study, we have more extensively tested the model by producing and examining a much more detailed and surprisingly complex time course of the onset of dependence. Applying the protein synthesis time course model to the data suggested the presence of two distinct components of dependence, an early transient component and a later long-lasting component. These components appear to correspond to acute and chronic dependence, respectively. The protein synthesis hypothesis more readily applies to the chronic dependence portion. Because consideration of the model can generate components that correspond to accepted and well-known components of dependence, both the utility of the model as well as the hypothesis that opiate dependence at least partially requires protein synthesis are supported. It is also possible that individual components of the withdrawal syndrome have individual and unique rate limiting mechanisms. In any case, time course analysis may be helpful in revealing underlying mechanisms of change.     

Science in drug control: the alkaloid content of afghan opium

Opium samples from Afghanistan were analyzed by HPLC for their content of morphine and three further alkaloids (codeine, thebaine, and papaverine). To our knowledge, this is the largest set of authentic opium samples analyzed in one study until now. The purpose was to assess possible correlations between samples and selected external factors, such as region of origin within Afghanistan, year of harvest, or intra-batch variation. In the investigated samples, a trend towards higher morphine concentrations in opium from the North-Eastern parts of Afghanistan was observed in the period from 2003 to 2005. More than 75% of the samples contained above 10% of morphine, the overall average was 14.4%.     

Brain peptide reverses effect of morphine on human lymphocytes

E-rosette formation by human lymphocytes incubated with sheep red blood cells (sRBC) is inhibited by morphine. We studied the ability of the opiate antagonists naloxone and Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH₂) to block this action. Active E-rosette formation by lymphocytes incubated with morphine was reduced from the control of 35.7±1.7% to 23.7±1.5% (p<0.001). Similarly, total E-rosette formation was reduced by morphine from the control of 65.8±1.3% to 53.2±2.9% (p<0.001). These effects were blocked by co-incubation of the lymphocytes with either Tyr-MIF-1 or naloxone (p<0.05). Tyr-MIF-1 was active (p<0.05) at concentrations as dilute as 10⁻¹³M. These results indicate that the neuropeptide Tyr-MIF-1 exerts an antiopiate effect at the human T-lymphocyte.