Heterodimerization of opioid receptor-like 1 and mu-opioid receptors impairs the potency of micro receptor agonist

Nociceptin activation of ORL1 (opioid receptor-like 1 receptor) has been shown to antagonize mu receptor-mediated analgesia at the supraspinal level. ORL1 and mu-opioid receptor (muR) are co-expressed in several subpopulations of CNS neurons involved in regulating pain transmission. The amino acid sequence of ORL1 also shares a high degree of homology with that of mu receptor. Thus, it is hypothesized that ORL1 and muR interact to form the heterodimer and that ORL1/muR heterodimerization may be one molecular basis for ORL1-mediated antiopioid effects in the brain. To test this hypothesis, myc-tagged ORL1 and HA-tagged muR are co-expressed in human embryonic kidney (HEK) 293 cells. Co-immunoprecipitation experiments demonstrate that ORL1 dimerizes with muR and that intracellular C-terminal tails of ORL1 and muR are required for the formation of ORL1/muR heterodimer. Second messenger assays further indicate that formation of ORL1/muR heterodimer selectively induces cross-desensitization of muR and impairs the potency by which [D-Ala(2),N-methyl-Phe(4),Gly-ol(5)]enkephalin (DAMGO) inhibits adenylate cyclase and stimulates p42/p44 mitogen-activated protein kinase (MAPK) phosphorylation. These results provide the evidence that ORL1/muR heterodimerization and the resulting impairment of mu receptor-activated signaling pathways may contribute to ORL1-mediated antiopioid effects in the brain.     

Cyclic biphalin analogues with a novel linker lead to potent agonist activities at mu,delta, and kappa opioid receptors

In an effort to improve biphalin’s potency and efficacy at the µ-(MOR) and δ-opioid receptors (DOR), a series of cyclic biphalin analogues 1–5 with a cystamine or piperazine linker at the C-terminus were designed and synthesized by solution phase synthesis using Boc-chemistry. Interestingly, all of the analogues showed balanced opioid agonist activities at all opioid receptor subtypes due to enhanced κ-opioid receptor (KOR) activity. Our results indicate that C-terminal flexible linkers play an important role in KOR activity compared to that of the other cyclic biphalin analogues with a hydrazine linker. Among them, analogue 5 is a potent (Ki?=?0.27, 0.46, and 0.87?nM; EC₅₀?=?3.47, 1.45, and 13.5?nM at MOR, DOR, and KOR, respectively) opioid agonist with high efficacy. Based on the high potency and efficacy at the three opioid receptor subtypes, the ligand is expected to have a potential synergistic effect on relieving pain and further studies including in vivo tests are worthwhile.     

Heterodimerization of ORL1 and Opioid Receptors and Its Consequences for N-type Calcium Channel Regulation

We have investigated the heterodimerization of ORL1 receptors and classical members of the opioid receptor family. All three classes of opioid receptors could be co-immunoprecipitated with ORL1 receptors from both transfected tsA-201 cell lysate and rat dorsal root ganglia lysate, suggesting that these receptors can form heterodimers. Consistent with this hypothesis, in cells expressing either one of the opioid receptors together with ORL1, prolonged ORL1 receptor activation via nociceptin application resulted in internalization of the opioid receptors. Conversely, μ-, δ-, and κ-opioid receptor activation with the appropriate ligands triggered the internalization of ORL1. The μ-opioid receptor/ORL1 receptor heterodimers were shown to associate with N-type calcium channels, with activation of μ-opioid receptors triggering N-type channel internalization, but only in the presence of ORL1. Furthermore, the formation of opioid receptor/ORL1 receptor heterodimers attenuated the ORL1 receptor-mediated inhibition of N-type channels, in part because of constitutive opioid receptor activity. Collectively, our data support the existence of heterodimers between ORL1 and classical opioid receptors, with profound implications for effectors such as N-type calcium channels.     

Modulation of Na, K-ATPase activity by prostaglandin E1 and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin

Adenylyl cyclase is activated by prostaglandin E and inhibited by mu-opioids. Since cAMP-related events influence the activity of the Na Pump and its biochemical correlate Na,K-ATPase in many systems, we tested the hypothesis that prostaglandin E1 and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), a mu-opioid agonist, have opposing actions on Na,K-ATPase activity. Studies were conducted with alamethicin-permeabilized SH-SY5Y human neuroblastoma cells. Prostaglandin E1 (1 microM) transiently inhibited Na,K-ATPase activity for 10-15 min. A direct activator of protein kinase A, 8-Br-cAMP (150 and 500 microM), also inhibited, but more rapidly and for a shorter duration. Both DAMGO (1 microM) and Rp-adenosine 3',5'-cyclic monophosphorothioate (500 microM), a protein kinase A-inhibitor, reversed the inhibitory effect of prostaglandin E1. DAMGO alone (1 microM) stimulated Na,K-ATPase activity up to nearly three-fold control activity. The stimulatory action of DAMGO was blocked by cyclosporine A (2 microM), an inhibitor of calcineurin, and was dependent on Ca2+ entry through nifedipine-sensitive Ca2+ channels. In the presence of 1 mM EGTA, DAMGO inhibited Na,K-ATPase activity. DAMGO-induced inhibition was blocked by the inositol 1,4,5-trisphosphate receptor antagonist xestospongin C (1 microM). Na,K-ATPase is poised to modulate neuronal excitability through its roles in maintaining the membrane potential and transmembrane ion gradients. The differential effects of prostaglandin E1 and opioids on Na,K-ATPase activity may be related to their actions in hyperalgesia.     

Methamphetamine-induced behavioral sensitization in mice: alterations in μ-opioid receptor

Summary We had previously demonstrated that opioid receptors contribute to the induction and expression of behavioral sensitization induced by repeated daily injection with 2.5 mg/kg of methamphetamine for 7 days. Using the same regimen, the present study investigated the alterations in μ-opioid receptor during the induction (on days 2, 5, and 8) and expression (on days 11 and 21) periods of behavioral sensitization. Radioligand binding revealed that the maximal binding of μ-opioid receptor was not changed on days 2 and 5, but down-regulated on day 8. After cessation of drug treatment, the maximal binding of μ-opioid receptor gradually and time-dependently returned to normal level on day 11 and up-regulated on day 21. In contrast, no changes in δ- and κ-opioid receptors were detectable on any given day examined. The potency of DAMGO for [³⁵S]-GTPγS coupling was enhanced on days 2, 5, 11, and 21. Moreover, 1 μM of naltrexone or β-chlornaltrexamine significantly suppressed the basal [³⁵S]-GTPγS coupling on days 2, 11, and 21. These findings indicate enhanced responsiveness and elevated constitutive activity of μ-opioid receptor. In summary, our data clearly demonstrate that alterations in μ-opioid receptor are involved in and may contribute to the sensitization to locomotor stimulating effect of methamphetamine.     

Changes in the Sensitivity of the Brain Cortex to the Action of Convulsants within the Remote Period after Electrostimulation-Induced Kindling in Rats

In acute experiments on rats, we measured the intensity of epileptiform events after applications of agonists of the receptors to excitatory amino acids, NMDA (10.0 mg/ml) and kainic acid (2.0 mg/ml), to the cortex. The rats were preliminarily subjected to a kindling procedure using electrical stimulations (ES) of the ventrobasal amygdala. Within the remote post-kindling period (three weeks after the last ES session), the sensitivity to the above drugs increased: the latency of the first EEG spike potential decreased, the amplitude of epileptic discharges within the period of the highest activity increased, and the epileptic nidi existed for a longer period. Applications of NMDA and kainic acid after preliminary intraventricular injections of an agonist of the -opiate receptors, DAMGO (0.5 g), on the one hand, evoked more intensive epileptogenic effects in kindling rats, and, on the other hand, did not change the above-mentioned indices of epileptogenesis in the control animals. The effects of applications of other convulsants (30 mg/ml strychnine or 10,000 IU/ml benzylpenicillin) showed no changes under the influence of DAMGO.     

G protein-coupled receptor kinase 2 mediates mu-opioid receptor desensitization in GABAergic neurons of the nucleus raphe magnus

Nucleus raphe magnus (NRM) sends the projection to spinal dorsal horn and inhibits nociceptive transmission. Analgesic effect produced by mu-opioid receptor agonists including morphine partially results from activating the NRM-spinal cord pathway. It is generally believed that mu-opioid receptor agonists disinhibit spinally projecting neurons of the NRM and produce analgesia by hyperpolarizing GABAergic interneurons. In the present study, whole-cell patch-clamp recordings combined with single-cell RT-PCR analysis were used to test the hypothesis that DAMGO ([D-Ala(2),N-methyl-Phe(4),Gly-ol(5)]enkephalin), a specific mu-opioid receptor agonist, selectively hyperpolarizes NRM neurons expressing mRNA of glutamate decarboxylase (GAD(67)). Homologous desensitization of mu-opioid receptors in NRM neurons could result in the development of morphine-induced tolerance. G protein-coupled receptor kinase (GRK) is believed to mediate mu-opioid receptor desensitization in vivo. Therefore, we also investigated the involvement of GRK in mediating homologous desensitization of DAMAMGO-induced electrophysiological effects on NRM neurons by using two experimental strategies. First, single-cell RT-PCR assay was used to study the expression of GRK2 and GRK3 mRNAs in individual DAMGO-responsive NRM neurons. Whole-cell recording was also performed with an internal solution containing the synthetic peptide, which corresponds to G(betagamma)-binding domain of GRK and inhibits G(betagamma) activation of GRK. Our results suggest that DAMGO selectively hyperpolarizes NRM GABAergic neurons by opening inwardly rectifying K(+) channels and that GRK2 mediates short-term homologous desensitization of mu-opioid receptors in NRM GABAergic neurons.     

Diffusion of the mu opioid receptor at the surface of human neuroblastoma SH-SY5Y cells is restricted to permeable domains

Previous single-molecule studies have shown a long-term diffusion superimposed to a short-term confinement of the human mu opioid (hMOP) receptors at the surface of heterologous cells. However, additional ensemble average measurements are required to reach a complete understanding of the undergoing process. Here, we analyse, by fluorescence recovery after photobleaching measurements, the lateral diffusion of fully functional T7-EGFP-hMOP receptors in neuroblastoma SH-SY5Y cells naturally expressing a low level of the wild-type receptor. Experiments carried out at variable observation radii demonstrate the restriction of the receptors diffusion to sub-micrometer sized domains. Furthermore, consistently with the long-term single-molecule data, the domains are found permeable.     

Filamin A Mutant Lacking Actin-Binding Domain Restores Mu Opioid Receptor Regulation in Melanoma Cells

We have previously reported that the protein filamin A (FLA) binds to the carboxyl tail of the mu opioid receptor (MOPr). Using human melanoma cells, which do not express filamin A, we showed that receptor down-regulation, functional desensitization and trafficking are deficient in the absence of FLA (Onoprishvili et al. Mol Pharmacol 64:1092–1100, 2003). Since FLA has a binding domain for actin and is a member of the family of actin cytoskeleton proteins, it is usually assumed that FLA functions via the actin cytoskeleton. We decided to test this hypothesis by preparing cDNA coding for mutant FLA lacking the actin binding domain (FLA-ABD) and expressing FLA-ABD in the human melanoma cell line M2 (M2-ABD cell line). We report here that this mutant is capable of restoring almost as well as full length FLA the down-regulation of the human MOPr. It is similarly very effective in restoring functional desensitization of MOPr, as assessed by the decrease in G-protein activation after chronic exposure of M2-ABD cells to the mu agonist DAMGO. We also found that A7 cells, expressing wild type FLA, exhibit rapid activation of the MAP kinases, ERK 1 and 2, by DAMGO, as shown by a rise in the level of phospho-ERK 1 and 2. This is followed by rapid dephosphorylation (inactivation), which reaches basal level between 30 and 60 min after DAMGO treatment. M2 cells show normal activation of ERK 1 and 2 in the presence of DAMGO, but very slow inactivation. The rapid rate of MAPK inactivation is partially restored by FLA-ABD. We conclude that some functions of FLA do not act via the actin cytoskeleton. It is likely that other functions, not studied here, may require functional binding of the MOPr-FLA complex to actin. Special issue article in honor of Dr. Ji-Sheng Han.