BACKGROUND: Opioid analgesics remain widely used for pain treatment despite the related serious side effects. Some of those, such as opioid tolerance and opioid-induced hyperalgesia may be at least partially due to modulation of opioid receptors (OR) function at nociceptive synapses in the spinal cord dorsal horn. It was suggested that increased release of different chemokines under pathological conditions may play a role in this process. The goal of this study was to investigate the crosstalk between the μOR, transient receptor potential vanilloid 1 (TRPV1) receptor and C-C motif ligand 2 (CCL2) chemokine and the involvement of spinal microglia in the modulation of opioid analgesia. METHODS: Patch-clamp recordings of miniature excitatory postsynaptic currents (mEPSCs) and dorsal root evoked currents (eEPSC) in spinal cord slices superficial dorsal horn neurons were used to evaluate the effect of μOR agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), CCL2, TRPV1 antagonist SB366791 and minocycline. Paw withdrawal test to thermal stimuli was combined with intrathecal (i.t.) delivery of CCL2 and DAMGO to investigate the modulation in vivo. RESULTS: Application of DAMGO induced a rapid decrease of mEPSC frequency and eEPSC amplitude, followed by a delayed increase of the eESPC amplitude, which was prevented by SB366791. Chemokine CCL2 treatment significantly diminished all the DAMGO-induced changes. Minocycline treatment prevented the CCL2 effects on the DAMGO-induced eEPSC depression, while mEPSC changes were unaffected. In behavioral experiments, i.t. injection of CCL2 completely blocked DAMGO-induced thermal hypoalgesia and intraperitoneal pre-treatment with minocycline prevented the CCL2 effect. CONCLUSIONS: Our results indicate that opioid-induced inhibition of the excitatory synaptic transmission could be severely attenuated by increased CCL2 levels most likely through a microglia activation-dependent mechanism. Delayed potentiation of neurotransmission after μOR activation is dependent on TRPV1 receptors activation. Targeting CCL2 and its receptors and TRPV1 receptors in combination with opioid therapy could significantly improve the analgesic properties of opioids, especially during pathological states.

• MeSH
• Anilides pharmacology   MeSH
• Chemokine CCL2 pharmacology   MeSH
• Cinnamates pharmacology   MeSH
• Enkephalin, Ala(2)-MePhe(4)-Gly(5)- pharmacology   MeSH
• Excitatory Postsynaptic Potentials drug effects   MeSH
• Rats MeSH
• Spinal Cord drug effects   MeSH
• Miniature Postsynaptic Potentials drug effects   MeSH
• Synaptic Transmission drug effects   MeSH
• Neurons drug effects   MeSH
• Nociception drug effects   MeSH
• Analgesics, Opioid pharmacology   MeSH
• Rats, Wistar MeSH
• Spinal Cord Dorsal Horn drug effects   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

The objectives of this study were to investigate the role of endogenous opioids in the mediation of stress-induced cardiomyopathy (SIC), and to evaluate which opioid receptors regulate heart resistance to immobilization stress. Wistar rats were subjected to 24 h immobilization stress. Stress-induced heart injury was assessed by 99mTc-pyrophosphate accumulation in the heart. The opioid receptor (OR) antagonists (naltrexone, NxMB - naltrexone methyl bromide, MR 2266, ICI 174.864) and agonists (DALDA, DAMGO, DSLET, U-50,488) were administered intraperitoneally prior to immobilization and 12 h after the start of stress. In addition, the selective micro OR agonists PL017 and DAMGO were administered intracerebroventricularly prior to stress. Finally pretreatment with guanethidine was used. Naltrexone did not alter the cardiac 99mTc-PP accumulation in stressed rats. NxMB aggravated stress-induced cardiomyopathy (P=0.005) (SIC). The selective micro OR agonist DALDA, which does not cross the blood-brain barrier, completely prevented (P=0.006) SIC. The micro OR agonist DAMGO exhibited weaker effect than DALDA. The selective delta ligand (DSLET) and kappa OR ligand (U-50,488) did not alter stress-induced 99mTc-pyrophosphate accumulation in the heart. Intracerebroventricular administration of the micro OR agonists aggravated SIC. Pretreatment with guanethidine abolished this effect (P=0.01). Guanethidine alone exhibited cardioprotective properties. A stimulation of central micro OR promotes an appearance of SIC. In contrast, stimulation of peripheral micro OR contributes to an increase in cardiac tolerance to stress.

• MeSH
• Enkephalin, Ala(2)-MePhe(4)-Gly(5)- pharmacology   therapeutic use   MeSH
• Immobilization adverse effects   psychology   MeSH
• Cardiotonic Agents pharmacology   therapeutic use   MeSH
• Rats MeSH
• Myocardium metabolism   MeSH
• Naltrexone pharmacology   MeSH
• Narcotic Antagonists pharmacology   MeSH
• Heart Diseases chemically induced   metabolism   prevention & control   MeSH
• Analgesics, Opioid pharmacology   therapeutic use   MeSH
• Rats, Wistar MeSH
• Stress, Psychological metabolism   psychology   MeSH
• Receptors, Opioid, mu agonists   metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

There are some indications that biased μ-opioid ligands may diversely affect μ-opioid receptor (MOR) properties. Here, we used confocal fluorescence recovery after photobleaching (FRAP) to study the regulation by different MOR agonists of receptor movement within the plasma membrane of HEK293 cells stably expressing a functional yellow fluorescent protein (YFP)-tagged μ-opioid receptor (MOR-YFP). We found that the lateral mobility of MOR-YFP was increased by (D-Ala(2),N-MePhe(4),Gly(5)-ol)-enkephalin (DAMGO) and to a lesser extent also by morphine but decreased by endomorphin-2. Interestingly, cholesterol depletion strongly enhanced the ability of morphine to elevate receptor mobility but significantly reduced or even eliminated the effect of DAMGO and endomorphin-2, respectively. Moreover, the ability of DAMGO and endomorphin-2 to influence MOR-YFP movement was diminished by pertussis toxin treatment. The results obtained by agonist-stimulated [(35)S]GTPγS binding assays indicated that DAMGO exhibited higher efficacy than morphine and endomorphin-2 did and that the efficacy of DAMGO, contrary to the latter agonists, was enhanced by cholesterol depletion. Overall, our study provides clear evidence that biased MOR agonists diversely affect receptor mobility in plasma membranes as well as MOR/G protein coupling and that the regulatory effect of different ligands depends on the membrane cholesterol content. These findings help to delineate the fundamental properties of MOR regarding their interaction with biased MOR ligands and cognate G proteins.

• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Cell Membrane drug effects   metabolism   MeSH
• Cholesterol deficiency   MeSH
• Enkephalin, Ala(2)-MePhe(4)-Gly(5)- metabolism   pharmacology   MeSH
• Fluorescence Recovery After Photobleaching MeSH
• Guanosine 5'-O-(3-Thiotriphosphate) metabolism   MeSH
• HEK293 Cells MeSH
• Microscopy, Confocal MeSH
• Humans MeSH
• Ligands MeSH
• Luminescent Proteins genetics   metabolism   MeSH
• Morphine metabolism   pharmacology   MeSH
• Narcotic Antagonists pharmacology   MeSH
• Oligopeptides metabolism   pharmacology   MeSH
• Pertussis Toxin pharmacology   MeSH
• GTP-Binding Protein alpha Subunits, Gi-Go metabolism   MeSH
• Receptors, Opioid, mu agonists   genetics   metabolism   MeSH
• Recombinant Fusion Proteins metabolism   MeSH
• Transfection MeSH
• Protein Transport MeSH
• Dose-Response Relationship, Drug MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH

BACKGROUND: The functional activity of trimeric guanine-nucleotide-binding proteins (G-proteins) represents an essential step in linking and regulation of the opioid receptor (mu-, delta- and kappa-OR)-initiated signaling pathways. Theoretical basis and/or molecular mechanism(s) of opioid tolerance and addiction proceeding in the central nervous system were not studied in the forebrain cortex of mammals with respect to quantitative analysis of opioid-stimulated trimeric G-protein activity. MATERIAL/METHODS: G-protein activity was measured in PercollR-purified plasma membranes (PM) isolated from the frontal brain cortex of control and morphine-treated rats by both high-affinity [32P]GTPase and [35S]GTPgammaS binding assays. Exposition to morphine was performed by intra-muscular application of this drug. Control animals were injected with sterile PBS. RESULTS: Both mu-OR (DAMGO)- and delta-OR (DADLE)-responses were clearly desensitized in PM isolated from morphine-treated rats; kappa-OR (U-69593)- and baclofen (GABAB-R)-stimulated [35S]GTPgammaS binding was unchanged, indicating the specificity of the morphine effect. Under such conditions, the amount of G-protein alpha subunits was unchanged. The order of efficacy DADLE>DAMGO>U-69593 was the same in control and morphine-treated PM. Behavioral tests indicated that morphine-treated animals were fully drug-dependent and developed tolerance to subsequent drug addition. CONCLUSIONS: Prolonged exposure of rats to high doses of morphine results in decrease of the over-all output of OR-stimulated G-protein activity in the forebrain cortex but does not decrease the amount of these regulatory proteins. These data support the view that the mechanism of the long-term adaptation to high doses of morphine is primarily based on desensitization of OR-response preferentially oriented to mu-OR and delta-OR.

• MeSH
• Substance Withdrawal Syndrome physiopathology   MeSH
• Baclofen pharmacology   MeSH
• Cell Membrane drug effects   enzymology   MeSH
• Time Factors MeSH
• Behavior, Animal drug effects   MeSH
• Enkephalin, Ala(2)-MePhe(4)-Gly(5)- pharmacology   MeSH
• Adaptation, Physiological drug effects   MeSH
• Guanosine 5'-O-(3-Thiotriphosphate) metabolism   MeSH
• Guanosine Diphosphate metabolism   MeSH
• Rats MeSH
• Morphine administration & dosage   pharmacology   MeSH
• Cerebral Cortex drug effects   metabolism   MeSH
• Rats, Wistar MeSH
• GTP-Binding Protein alpha Subunits metabolism   MeSH
• Receptors, Opioid, delta agonists   metabolism   MeSH
• Receptors, Opioid, mu agonists   metabolism   MeSH
• Drug Tolerance MeSH
• Dose-Response Relationship, Drug MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH