Isolation and characterization of new exon 11-associated N-terminal splice variants of the human mu opioid receptor gene

Alternative splicing of the mu opioid receptor genes to create multiple mu receptor subtypes has been demonstrated in animals and humans. Previously, we identified a number of C-terminal variants in mice, rats and human, followed by several N-terminal variants associated with a new upstream exon in mice (exon 11). Behavioral studies in exon 11 knockout mice suggest an important role for the exon 11 variants in the analgesic actions of heroin and morphine-6β-glucuronide, but not morphine or methadone. We now have identified a homologous human exon 11 and three similar human exon 11-associated variants, suggesting conservation of exon 11 and its associated variants across species. hMOR-1i has an additional 93 amino acids at the tip of the N-terminus but is otherwise identical to hMOR-1. When expressed in Chinese hamster ovary cells, the additional 93 amino acids in hMOR-1i had little effect on opioid binding, but significantly altered agonist-induced G-protein activation. hMOR-1G1 and hMOR-1G2 predicted six transmembrane domain variants, similar to those seen in mice. The regional expression of these exon 11-associated variants, as determined by RT-PCR, varied markedly, implying region-specific alternative splicing. The presence of exon 11-associated variants in humans raises questions regarding their potential role in heroin and morphine-6β-glucuronide actions in people as they do in mice.     

Acute analgesic effect of loperamide as compared to morphine after intrathecal administration in rat

Loperamide, a mu opioid receptor agonist, which is commonly used as an antidiarrhoeal agent has been reported to possess analgesic activity after intrathecal administration. However, the exact analgesic profile, i.e., onset, duration and intensity of analgesia in relation to morphine is not fully known. In the present study, the acute analgesic effect of loperamide (5 microg) was compared with that of morphine (5 microg) and morphine + loperamide (5 microg of each) using the tail flick method after intrathecal administration. Naloxone (5 mg/kg) reversibility of the analgesic effect was also studied. The analgesic response of loperamide was significantly higher than morphine. Even after 22 hr, maximum possible effect was greater than 49%. Naloxone partially antagonized the analgesic effect of loperamide. This suggested that loperamide may be acting through blockade of Ca2+ channels besides activating mu opioid receptors. Loperamide may prove to be a better substitute for morphine as spinal analgesic.     

Antisense oligodeoxynucleotide targeting distinct exons of the cloned mu-opioid receptor distinguish between endomorphin-1 and morphine supraspinal antinociception in mice.

Antisense oligodeoxynucleotides (ODN) were used to investigate the supraspinal antinociceptive effects of endomorphin-1, an endogenous peptide whose analgesic profile suggests that it is a ligand at the mu-opioid receptor. To selectively restrict the expression of this receptor, five ODN targeting distinct exons of the gene sequence were injected subchronically by the intracerebroventricular route (i.c.v.) into mice. The antinociception induced by endomorphin-1 was greatly reduced in animals receiving the ODN directed to nucleotides 677-697, which code for a sequence located on the second extracellular loop of the mu receptor. ODN-mu(un), one of the two antisense ODN directed to exon 1, also impaired endomorphin-1 antinociception. ODN targeting exons 2 and 4 were totally inactive. In contrast, all five ODN blocked the antinociception induced by morphine and beta-casomorphin. The analgesic potency of endomorphin-1, morphine, and beta-casomorphin remained unaltered by administration of an ODN to nucleotides 29-46 of the murine delta-opioid receptor gene sequence of a random-sequence ODN. This suggest the existence of diverse molecular forms for the mu-opioid receptor that mediate the antinociceptive effects of endomorphin-1 and morphine/beta-casomorphin.     

Mu and delta receptors: their role in analgesia in the differential effects of opioid peptides on analgesia

Utilizing the mouse tail-flick assay, the rank order of analgesic potency for various opioids (i.c.v.) is beta h-endorphin greater than D-Ala2-D-Leu5-enkephalin greater than morphine greater than D-Ala2-met-enkephalinamide much greater than met-enkephalin much greater than leu-enkephalin. Assuming mu receptor mediation of analgesia, there is an affinity and analgesic potency (ie: D-Ala2-Leu5-enkephalin has 1/7 the affinity of morphine for the mu receptor but is 18X more potent as an analgesic). Additionally, sub-analgesic doses of various opioid peptides have opposite effects on analgesic responses. Leu-enkephalin, D-Ala2-D-Leu5-enkephalin or beta h-endorphin potentiate morphine or D-Ala2-met-enkephalinamide analgesia whereas met-enkephalin or D-Ala2-met-enkephalinamide antagonize opioid-induced analgesia. Using the enkephalins as the prototypic delta ligands (100 fold selective) and based on their effects on analgesia, we suggest that Leu-enkephalin-like peptides interact with the delta receptor as an "agonist" to facilitate and met-enkephalin-like peptides as an "antagonist" to attenuate analgesia. Given the biochemical evidence of a coupling between mu and delta receptors, we suggest that the mechanism of facilitation or attenuation of analgesia by the enkephalins is a direct in vivo consequence of this coupling. Further, the analgesic potencies of various opioid ligands can be better correlated to the combination of their simultaneous occupancy of mu and delta receptors.     

Single‐nucleotide polymorphism (A118G) in exon 1 of OPRM1 gene causes alteration in downstream signaling by mu‐opioid receptor and may contribute to the genetic risk for addiction

The opioid receptor mu1 (OPRM1) mediates the action of morphine. Although genetic background plays an important role in the susceptibility toward abuse of drugs as evident from familial, adoption and twin studies, association of specific single‐nucleotide polymorphisms of OPRM1 gene with narcotic addiction is to be established. Here, we demonstrate the involvement of A118G polymorphism of exon1 of human OPRM1 gene (hOPRM1), with heroin and alcohol addiction, in a population in eastern India. Statistical analysis exhibited a significant association of G allele with both heroin and alcohol addiction with a risk factor of P_trend < 0.05. The functional significance of G allele in A118G single‐nucleotide polymorphisms was evaluated by studying the regulation of protein kinase A (PKA), pCREB, and pERK1/2 by morphine in Neuro 2A cells, stably transfected with either wild type or A118G mutant hOPRM1. Unlike acute morphine treatment, both chronic morphine exposure and withdrawal precipitated by naloxone were differentially regulated by A118 and G118 receptor isoforms when both PKA and pERK1/2 activities were compared. Results suggest that the association of A118G polymorphism to heroin and alcohol addiction may be because of the altered regulation of PKA and pERK1/2 during opioid and alcohol exposures.     

Morphine tolerance in mice changes response of heroin from mu to delta opioid receptors

Heroin produced antinociception in the tail flick test through mu receptors in the brain of ICR and CD-1 mice, a response inhibited by 3-O-methylnaltrexone. Tolerance to morphine was produced by subcutaneous morphine pellet implantation. By the third day, the heroin response was produced through delta opioid receptors. The response was inhibited by simultaneous intracerebroventricular (i.c. v.) administration of naltrindole, a delta opioid receptor antagonist. More specifically, delta1 rather than delta2 receptors were involved because 7-benzylidenenaltrexone, a delta1 receptor antagonist, inhibited but naltriben, a delta2 antagonist, did not. Also, antinociception produced by i.c.v. heroin was inhibited by intrathecal administration of bicuculline and picrotoxin consistent with the concept that delta1 receptors in the brain mediated the antinociceptive response through descending neuronal pathways to the spinal cord to activate GABAA and GABAB receptors rather than spinal alpha2-adrenergic and serotonergic receptors activated originally by the mu agonist action in naive mice. The mu response of 6-monoacetylmorphine, a metabolite of heroin, was changed by morphine pellet implantation to a delta2 response (inhibited by naltriben but not 7-benzylidenenaltrexone). The agonist action of morphine in these morphine-tolerant mice remained mu. Thus, the opioid receptor selectivity of heroin and 6-monoacetylmorphine in the brain is changed by production of tolerance to morphine. Such a change explains how morphine tolerant mice are not cross-tolerant to heroin.     

Antinociceptive potency of a fluorinated cyclopeptide Dmt-c[D-Lys-Phe-p-CF3-Phe-Asp]NH2

Opioid peptides and opiate drugs such as morphine, mediate their analgesic effects, but also undesired side effects, mostly through activation of the mu opioid receptor. However, delta- and kappa-opioid receptors can also contribute to the analgesic effects of opioids. Recent findings showed that simultaneous activation of multiple opioid receptors may result in additional analgesia with fewer side effects. Here, we evaluated the pharmacological profile of our formerly developed mixed mu/kappa-opioid receptor ligands, Dmt-c[D-Lys-Phe-Phe-Asp]NH₂ (C-36) and Dmt-c[D-Lys-Phe-p-CF₃-Phe-Asp]NH₂ (F-81). The ability of these peptides to cross the blood–brain barrier was tested in the parallel artificial membrane permeability (PAMPA) assay. On the basis of the hot-plate test in mice after central and peripheral administration, analog F-81 was selected for the anti-nociceptive and anti-inflammatory activity assessment after peripheral administration.     

Analgesic (sub anesthetic) nitrous oxide interacts with the endogenous opioid system: a review of the evidence

The concept that anesthesia and analgesia are distinct states and therefore are possibly mediated by different mechanisms is stressed. Analgesic nitrous oxide is shown to act at specific rather than non specific central nervous system sites, as well as having a large number of actions similar to morphine the classical opioid. This includes the fact that specific opioid antagonists attenuate the effects of both morphine and analgesic nitrous oxide. Evidence is also provided showing that nitrous oxide may be a partial agonist and that it may interact with the endogenous opioid system by the release of endogenous opioids, and/or by direct action at the mu, delta, sigma and kappa receptors.     

Sex differences in opioid analgesia, hyperalgesia, tolerance and withdrawal: Central mechanisms of action and roles of gonadal hormones

This article reviews sex differences in opiate analgesic and related processes as part of a Special Issue in Hormones and Behavior. The research findings on sex differences are organized in the following manner: (a) systemic opioid analgesia across mu, delta and kappa opioid receptor subtypes and drug efficacy at their respective receptors, (b) effects of the activational and organizational roles of gonadal steroid hormones and estrus phase on systemic analgesic responses, (c) sex differences in spinal opioid analgesia, (d) sex differences in supraspinal opioid analgesia and gonadal hormone effects, (e) the contribution of genetic variance to analgesic sex differences, (f) sex differences in opioid-induced hyperalgesia, (g) sex differences in tolerance and withdrawal-dependence effects, and (h) implications for clinical therapies.     

家兔伏核—杏仁核神经通路在吗啡镇痛中的作用

用辐射热照射家兔鼻嘴侧部皮肤,测量其躲避反应潜伏期作为痛反应阈,简称痛阈。通过预先埋植的慢性套管向伏核或杏仁核内进行注射,结果表明:(1)在家兔的伏核内微量注射吗啡可产生镇痛作用,该作用可被杏仁核内注射纳洛酮所削弱,并有量效依从关系;在杏仁核内注射甲啡肽抗血清(ME AS)或β-內啡肽抗血清(β-EP AS)亦可削弱上述镇痛作用;(2)在杏仁核内微量注射吗啡可产生镇痛作用,此作用不能被伏核内注射纳洛酮所阻断;(3)在伏核内注射吗啡所产生的镇痛作用可被同一部位注射γ-氨基丁酸(GAEA)受体阻断剂氯甲基荷包牡丹碱所增强,被 GABA 受体激动剂异鹅羔胺所削弱。上述结果提示:在家兔脑内从伏核到杏仁核可能存在一条与镇痛有关的神经通路,伏核内的阿片样物质及杏仁核内的甲啡肽,β-内啡肽可能参与镇痛信息的传递,而伏核内的 GABA 可能有对抗吗啡镇痛的作用。     

Potentiation of opioid analgesia by cocaine: the role of spinal and supraspinal receptors.

These studies examined the effect of cocaine on the analgesia produced by systemically and centrally administered opioid agonists. Cocaine (50 mg/kg, s.c.) increased the analgesic potency of systemic, ICV and IT morphine; and the ICV and IT analgesic effects of the delta selective peptide, [D-Pen2,D-Pen5]enkephalin (DPDPE). Cocaine also increased the analgesic potency of the mu selective ligand [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAGO) administered ICV. However, cocaine did not alter the ED50 for IT DAGO. GC-MS studies indicated that brain cocaine concentration was approximately 3.0 micrograms/g wet weight 45 min following s.c. administration. These results suggest that cocaine-induced increases in opioid analgesic potency are mediated at brain mu and delta receptors and spinal mu receptors. Furthermore, there might be functional differences between spinal and supraspinal sites at which DAGO produces analgesia.     

The effect of opiates and opiate antagonists on heat latency response in the parasitic nematode Ascaris suum

The effects of the opiates morphine and morphine-6-glucuronide (M6G), the mu opioid receptor specific antagonist D-Phe-Cys-Tyr-D-Trp-Om-Thr-Pen-Thr-NH(2) (CTOP), and the general opiate antagonist naloxone on the latency of response to thermal stimulation were determined in the parasitic nematode Ascaris suum. Thermal detection and avoidance behaviors of the worms were evaluated with a tail flick analgesia meter using a modification of a technique employed for nociception experiments in rodents. Morphine and M6G were shown to have a dose dependent analgesic effect on A. suum's latency of response to heat with morphine being the most potent. The analgesic effect of morphine was reversed by naloxone but not CTOP. Neither naloxone nor CTOP was able to block the analgesia of M6G. CTOP but not naloxone had significant analgesic effects on its own. These findings are generally consistent with previous results on the effects of opiates and nitric oxide release from A. suum tissue. Apparently these nematodes possess opioid receptors that effect nociception.     

Bioconversion of codeine to semi-synthetic opiate derivatives by the cyanobacterium <Emphasis Type="Italic">Nostoc muscorum</Emphasis>

Very limited studies have been done to investigate the algal biotransformation of codeine to its opioid derivatives. On the other hand, microalgae have been recently introduced as potential tools for green synthesis of various organic compounds. In the present work, the capability of biotransformation of codeine by a locally isolate strain of cyanobacterium, Nostoc muscorum, was evaluated. Incubation of the whole cells of Nostoc muscorum with codeine (I) under continuous light photoregime of 60 μmol photons/m²s at 25°C for 5 days gave rise to four transformation products. The bioproducts were separated by gas chromatography and identified as 6-acetylcodeine (II), oxycodone (III), norcodeine (IV), morphine (V) and based on their mass spectra. Observed modifications included O-demethylation, N-demethylation, C6-acetylation, C14-hydroxilation, Δ⁷-reduction, and C6-oxidation. The ability of N. muscorum to convert codeine to oxycodone (III) represents an uncommon pattern of codeine metabolism in microorganisms that may be of industrial importance.     

Analgesia induced by intrathecal injection of dynorphin B in the rat

A dose-dependent analgesic effect of intrathecally injected dynorphin B was observed in rats using the tail flick as nociceptive test. Intrathecal injection of 20 nmol of dynorphin B increased the tail flick latency by 90 +/- 23%, an effect that lasted about 90 min. For the same degree of analgesia, dynorphin B was 50% more potent than morphine on a molar basis. The analgesic effect of this dose of dynorphin B was partially blocked by 10 mg/kg, but not by 1 mg/kg, of subcutaneous naloxone, showing a relative resistance to naloxone reversal as compared with morphine analgesia. The analgesia produced by dynorphin B was unchanged in morphine-tolerant rats but was significantly decreased in rats tolerant to ethylketazocine. These results suggest that dynorphin B produces its potent analgesic effect by activation of kappa rather than mu opioid receptors in the rat spinal cord.     

纳布啡在围手术期镇痛的研究进展

纳布啡是一种新型的菲族镇痛药,属于混合型阿片类受体激动/拮抗剂,可在脊髓水平激动κ受体发挥强效的镇痛效果,其镇痛作用起效迅速、药效持久、疗效确切;同时由于纳布啡独特的部分μ受体拮抗特性,使其与吗啡相比,在发挥镇痛作用的同时呼吸抑制轻、血流动力学平稳以及恶心呕吐、皮肤瘙痒、成瘾性等不良反应发生率更低,因此,纳布啡在围手术期镇痛和临床麻醉等多个领域有着广阔的应用前景。现结合纳布啡独特的药代动力学、药理学特点及作用机制,对纳布啡在围手术期镇痛的研究进展作一综述,以期为临床上合理、有效镇痛提供理论参考和实践依据。     

High-affinity naloxone binding to filamin a prevents mu opioid receptor-Gs coupling underlying opioid tolerance and dependence

Ultra-low-dose opioid antagonists enhance opioid analgesia and reduce analgesic tolerance and dependence by preventing a G protein coupling switch (Gi/o to Gs) by the mu opioid receptor (MOR), although the binding site of such ultra-low-dose opioid antagonists was previously unknown. Here we show that with approximately 200-fold higher affinity than for the mu opioid receptor, naloxone binds a pentapeptide segment of the scaffolding protein filamin A, known to interact with the mu opioid receptor, to disrupt its chronic opioid-induced Gs coupling. Naloxone binding to filamin A is demonstrated by the absence of [(3)H]-and FITC-naloxone binding in the melanoma M2 cell line that does not contain filamin or MOR, contrasting with strong [(3)H]naloxone binding to its filamin A-transfected subclone A7 or to immunopurified filamin A. Naloxone binding to A7 cells was displaced by naltrexone but not by morphine, indicating a target distinct from opioid receptors and perhaps unique to naloxone and its analogs. The intracellular location of this binding site was confirmed by FITC-NLX binding in intact A7 cells. Overlapping peptide fragments from c-terminal filamin A revealed filamin A(2561-2565) as the binding site, and an alanine scan of this pentapeptide revealed an essential mid-point lysine. Finally, in organotypic striatal slice cultures, peptide fragments containing filamin A(2561-2565) abolished the prevention by 10 pM naloxone of both the chronic morphine-induced mu opioid receptor-Gs coupling and the downstream cAMP excitatory signal. These results establish filamin A as the target for ultra-low-dose opioid antagonists previously shown to enhance opioid analgesia and to prevent opioid tolerance and dependence.     

Interactions of Opioid and Chemokine Receptors: Oligomerization of mu, kappa, and delta with CCR5 on Immune Cells

Activation of opioid receptors by morphine was previously shown to specifically induce the expression of chemokine receptor CCR5, promoting simian AIDS virus entry and replication in immune cells. The present study was undertaken to determine whether these two structurally and functionally distinct G-protein-coupled receptors are in close proximity and form an oligomeric complex in the cell membrane so that the activation of one triggers the activity of the other. Both human CEM ×174 and monkey lymphocytes were used in this study and gave similar results. Immunoprecipitation experiments showed that CCR5, but not CD4 nor Na⁺/H⁺ exchanger, coprecipitates with all three subtypes (mu, delta, and kappa) of opioid receptors. A single protein band immunoreactive with antibodies against both the CCR5 and the opioid receptors was identified after electrophoresis on nondenaturing polyacrylamide gels. Chemical crosslinking experiments using glutaraldehyde or BS³ indicate that these receptors are closely situated on the cell membrane with an intermolecular distance less than 11.4Å. Functional studies revealed that a combination treatment of cells with morphine, an agonist for mu, and MIP-1β, a ligand for CCR5, suppresses the inhibitory effect of MIP-1β and increases the stimulatory effect of morphine on CCR5 expression. These results suggest that oligomerization of chemokine receptor CCR5 and opioid receptors on the cell membrane of human or monkey lymphocytes may modulate receptor functions.     

Effects of stress and beta-funal trexamine pretreatment on morphine analgesia and opioid binding in rats

This study was essentially an in vivo protection experiment designed to test further the hypothesis that stress induces release of endogenous opioids which then act at opioid receptors. Rats that were either subjected to restraint stress for 1 hr or unstressed were injected ICV with either saline or 2.5 micrograms of beta-funaltrexamine (beta-FNA), an irreversible opioid antagonist that alkylates the mu-opioid receptor. Twenty-four hours later, subjects were tested unstressed for morphine analgesia (tail-flick assay) or were sacrificed and opioid binding in brain was determined. [3H]D-Ala2NMePhe4-Gly5(ol)enkephalin (DAGO) served as a specific ligand for mu- opioid receptors, and [3H]-bremazocine as a general ligand for all opioid receptors. Rats injected with saline while stressed were significantly less sensitive to the analgesic action of morphine 24 hr later than were their unstressed counterparts. Beta-FNA pretreatment attenuated morphine analgesia in an insurmountable manner. Animals pretreated with beta-FNA while stressed were significantly more sensitive to the analgesic effect of morphine than were animals that received beta-FNA while unstressed, consistent with the hypothesis that stress induces release of endogenous opioids that would protect opioid receptors from alkylation by beta-FNA. beta-FNA caused small and similar decreases in [3H]-DAGO binding in brain of both stressed and unstressed animals. Stressed rats injected with saline tended to have increased levels of [3H]DAGO and [3H]-bremazocine binding compared to the other groups. This outcome may be relevant to the tolerance to morphine analgesia caused by stress.