Differential regulation of the cloned kappa and mu opioid receptors

To directly compare the regulation of the cloned kappa and mu opioid receptor, we expressed them in the same cells, the mouse anterior pituitary cell line AtT-20. The coupling of an endogenous somatostatin receptor to adenylyl cyclase and an inward rectifier K+ current has been well characterized in these cells, enabling us to do parallel studies comparing the regulation of both the kappa and the mu receptor to this somatostatin receptor. We show that the kappa receptor readily uncoupled from the K+ current and from adenylyl cyclase after a 1 h pretreatment with agonist, as indicated by the loss in the ability of the agonist to induce a functional response. The desensitization of the kappa receptor was homologous, as the ability of somatostatin to mediate inhibition of adenylyl cyclase or potentiation of the K+ current was not altered by kappa receptor desensitization. The mu receptor uncoupled from the K+ current but not adenylyl cyclase after a 1 h pretreatment with agonist. Somatostatin was no longer able to potentiate the K+ current after mu receptor desensitization, thus this desensitization was heterologous. Interestingly, pretreatment with a somatostatin agonist caused uncoupling of the mu receptor but not the kappa receptor from the K+ current. These results show that in the same cell line, after a 1 h pretreatment with agonist, the kappa receptor displays homologous regulation, whereas the mu receptor undergoes only a heterologous form of desensitization. mu receptor desensitization may lead to the alterations of diverse downstream events, whereas kappa receptor regulation apparently occurs at the level of the receptor itself. Broad alterations of non-opioid systems by the mu receptor could be relevant to the addictive properties of mu agonists. Comparison of kappa and mu receptor regulation may help define the properties of the mu receptor which are important in the development of addiction, tolerance, and withdrawal to opioid drugs. These are the first studies to directly compare the coupling of the kappa and mu receptors to two different effectors in the same mammalian expression system.     

Palmitoylation of the rat mu opioid receptor

We describe a structured and uniform resident experience in operative endoscopy and analyze the costs of implementing such a program at an urban academic medical center. The residency curriculum at Northwestern Memorial Hospital incorporates a five-part approach to endoscopy training: weekly endoscopy rounds, an annual animal laboratory for residents, an individual animal laboratory, supervision by skilled endoscopic surgeons, and a laparoscopic training facility. Thirty-two residents have completed the training over 4 years. The annual cost of the entire program is $34,500, which can be offset partially by vendor support. A comprehensive and continuous endoscopic training program is an important and affordable part of resident education.     

Ligand recognition in mu opioid receptor: experimentally based modeling of mu opioid receptor binding sites and their testing by ligand docking

For three-dimensional understanding of the mechanisms that control potency and selectivity of the ligand binding at the atomic level, we have analysed opioid receptor-ligand interaction based on the receptor's 3D model. As a first step, we have constructed molecular models for the multiple opioid receptor subtypes using bacteriorhodopsin as a template. The S-activated dihydromorphine derivatives should serve as powerful tools in mapping the three-dimensional structure of the mu opioid receptor, including the nature of the agonist-mediated conformational change that permits G protein-coupling to "second messenger' effector molecules, and in identifying specific ligand-binding contacts with the mu opioid receptor. The analyses of the interactions of some opioid ligands with the predicted ligand binding sites are consistent with the results of the affinity labeling experiments.     

Carboxyl-terminal splicing of the rat mu opioid receptor modulates agonist-mediated internalization and receptor resensitization

The rat mu opioid receptor is alternatively spliced into two isoforms (MOR1 and MOR1B) which differ in length and amino acid composition at the carboxyl terminus. When stably expressed in HEK 293 cells, both splice variants bind the mu receptor agonist [D-Ala2,N-Me-Phe4,-Gly-ol5]enkephalin (DAMGO) with similar affinity and exhibit functional coupling to adenylyl cyclase with similar efficiency. However, the shorter isoform, MOR1B, desensitized at a slower rate during prolonged DAMGO exposure (4 h) but resensitized at a faster rate than MOR1 during agonist withdrawal (20 min). Immunocytochemical analysis revealed that DAMGO-induced internalization of MOR1B proceeded much faster than that of MOR1 followed by rapid recycling of the receptor to the cell surface. In addition, the greater resistance of MOR1B to homologous desensitization compared with MOR1 as well as MOR1B resensitization was abolished when receptor reactivation/recycling was blocked with monensin, an inhibitor of endosomal acidification. It is concluded that the sequence at the cytoplasmic tail of MOR1B facilitates clathrin-coated vesicle-mediated endocytosis which, in turn, promotes accelerated receptor reactivation. Taken together, our findings suggest that carboxyl-terminal splicing of the rat mu opioid receptor modulates agonist-induced internalization and receptor resensitization.     

Effect of selective mu 1, mu 2 and delta 2 opioid receptor agonists on gastric functions in the rat

Because the role of mu and delta opioid receptors in modulating gastric functions remains uncertain, we studied whether intracerebroventricular (i.c.v.) and subcutaneous (s.c.) injections of new opioid peptides with high selectivity for mu 1 (Lys7-dermorphin), mu 2 (Trp4-Asn7-dermorphin) and delta 2 (D-Ala2-deltorphin II) opioid receptors would modify gastric secretion (after 2 hr pylorus ligature) and transit (after a phenol red meal) in the rat. Neither i.c.v. nor s.c. injections of the delta 2 opioid agonist affected the gastric functions. In contrast, the mu opioid agonists decreased gastric acid secretion and emptying, i.c.v. injections inducing more potent inhibition than s.c. administration. The mu 1 selective opioid antagonist naloxonazine had no effect on the inhibition of the gastric secretory and motor response to these peptides but naloxone completely blocked their effects. Our findings suggest (1) that in rats, stimulation of central naloxonazine insensitive opioid receptors (mu 2 sites) inhibits gastric acid secretion and emptying; and (2) that delta opioid receptors take no part in mediating these functions.     

Changes of mu opioid receptor binding sites in rat brain following electroacupuncture

With [3H]-ohmefentanyl as a ligand, autoradiographic technique was used to observe the effect of electroacupuncture (EA) on mu opioid receptor binding sites in the brain areas related to pain modulation. The results were as follows: (1) The distribution of the mu receptor in the rat central nervous system was consistent in general with the results reported previously. (2) After EA of Tsu-San-Li, the mu receptor binding sites were increased significantly in the following examined structures: the caudate nucleus, septal nucleus, medial preoptic area, amygdalaoid nucleus, periaqueducal gray, interpeduncular nucleus, nucleus raphe magnus, and cervical and lumbar enlargements. The results indicate that EA is able to increase mu binding sites in the brain areas related to analgesia, suggesting the enhancement of mu receptor function by EA.     

Differential modulation of kappa and mu opioid antinociception by the glycine/NMDA receptor agonist D-serine

D-Serine, a selective agonist for the strychnine-insensitive glycine allosteric site associated with the NMDA receptor-ion channel complex, was found to modulate differentially the antinociception produced by kappa and mu-opioid receptor agonists in the rat formalin test. D-Serine (100 micrograms, i.c.v.) attenuated the antinociception produced by the selective kappa-opioid agonist, enadoline (0.003-0.1 mg kg-1, s.c.) against the tonic, but not acute, phase of the formalin response. Conversely, D-serine potentiated the antinociception produced by morphine (0.3-10 mg kg-1, s.c.) against both the acute and tonic phases. These results demonstrate an important interaction between the opioid and NMDA/glycine systems in the control of nociceptive information possibly at different levels of the neuraxis.     

Replacement of threonine 394 by alanine facilitates internalization and resensitization of the rat mu opioid receptor

Signaling of G protein-coupled receptors is terminated by phosphorylation of intracellular serine and threonine residues. Resensitization of these receptors requires internalization and subsequent dephosphorylation. We have recently shown that the resensitization rate of the rat micro opioid receptor (MOR) isoforms MOR1 and MOR1B is mainly determined by the amino acid composition of their alternatively spliced C-terminal tails. Upon agonist stimulation, MOR1B passes through an accelerated cycle of receptor endocytosis and reactivation, which in turn promotes a greater resistance to agonist-induced desensitization, as compared with MOR1. Given the fact that MOR1B lacks only one putative phosphorylation site (T394 of MOR1), we replaced this threonine by an alanine and stably expressed the wild-type MOR1 and its T394A mutant in mouse neuroblastoma Neuro2a cells. We show that during prolonged [D-Ala2, MePhe4, Gly5-ol]enkephalin exposure (5 h), the T394A receptor mutant desensitized at a slower rate than MOR1. In contrast, T394A is more rapidly removed from the cell surface than MOR1, as determined by flow cytometry using epitope-tagged receptors. This fast internalization was followed by immediate resensitization of T394A during 20 min of agonist removal while the wild-type MOR1 remained inactive. Similar to MOR1B, rapid internalization and reactivation of T394A may explain its delayed desensitization. These findings suggest that T394 represents a negative regulatory signal for MOR1 internalization. Furthermore, phosphorylation of this threonine residue may influence the time course of micro opioid receptor resensitization.     

Spinal opioid mu receptor expression in lumbar spinal cord of rats following nerve injury

The oxidation of inulin with Pt/C as catalyst was studied. Methyl alpha-D-fructofuranoside was used as a model compound for the monomeric unit of inulin. Oxidation occurred selectively at the C-6 position in a high yield (79%). The rate of oxidation and the degree of oxidation obtained for inulin oligosaccharides decreased upon increase of the chain length of the substrate. Inulin could only be oxidized partially: the oxidation degree obtained was 20% of the primary hydroxy groups for inulin with an average dp 30. Possible explanations for these relatively low conversions are discussed. Adsorption and desorption phenomena appear to play and important role during the oxidation process.     

Intake of high-fat food is selectively enhanced by mu opioid receptor stimulation within the nucleus accumbens

The present study was designed to further investigate the nature of feeding induced by opioid stimulation of the nucleus accumbens through an examination of the effects of intra-accumbens (ACB) opioids on macronutrient selection. In 3-hr tests of free-feeding (satiated) rats, intra-ACB administration of the mu receptor agonist D-Ala2,N,Me-Phe4, Gly-ol5-enkephalin (DAMGO; 0, 0.025, 0.25 and 2.5 micrograms bilaterally) markedly enhanced the intake of fat or carbohydrate when the diets were presented individually (although the effect on fat intake was much greater in magnitude). Intra-ACB injections of DAMGO, however, produced potent preferential stimulatory effects on fat ingestion with no effect on carbohydrate ingestion when both fat and carbohydrate diets were present simultaneously. Moreover, this selective stimulation of fat intake was independent of base-line diet preference and could be blocked by systemic injection of naltrexone (5 mg/kg). We also examined the effect of 24-hr food deprivation on the pattern of macronutrient intake in rats with access to both carbohydrate and fat. In contrast to the DAMGO-induced selective enhancement of fat intake, food deprivation significantly increased the intake of both diets to the same extent; however, in this case, only the stimulated fat intake was blocked by systemic naltrexone. Intra-ACB administration of DAMGO in hungry rats produced an effect similar to that observed in free-feeding rats; preference was strongly shifted to fat intake. Similarly, the opioid antagonist naltrexone (20 micrograms) infused directly into ACB preferentially decreased fat intake in hungry rats. These findings suggest that endogenous opioids within the ventral striatum may participate in the mechanisms governing preferences for highly palatable foods, especially those rich in fat.     

Affinity of naloxone and its quaternary analogue for avian central delta and mu opioid receptors

Quaternary narcotic antagonists that are assumed not to penetrate the blood-brain barrier following systemic administration are commonly used to distinguish between peripheral and central actions of opiates. In mammals, these antagonists have a lower affinity for opioid receptors than their tertiary parent compounds. The relative affinity of quaternary vs. tertiary antagonists either for opioid receptors in non-mammalian species or for specific receptor subtypes has, however, not been determined. Using brain tissues from a passerine songbird (Junco hyemalis), we found the affinity of the quaternary antagonist, naloxone methiodide (Nal MI), for brain opioid receptors to be less than 10% that of Nal HCl. Further, Nal MI affinity for mu and delta receptors is 8.7% and 3.7%, respectively, that of Nal HCl. These results confirm that tertiary narcotic antagonist quaternization substantially reduces the affinity of these derivatives for central opioid receptors. They show that this reduction is receptor-type selective, and they extend previous reports demonstrating functional similarities between mammalian and non-mammalian central opioid receptors.     

Protein kinase A maintains cellular tolerance to mu opioid receptor agonists in hypothalamic neurosecretory cells with chronic morphine treatment: convergence on a common pathway with estrogen in modulating mu opioid receptor/effector coupling

The present study examined protein kinase A (PKA) and protein kinase C (PKC) involvement in the maintenance of cellular tolerance to mu opioid receptor agonists resulting from chronic opiate exposure in neurosecretory cells of the hypothalamic arcuate nucleus (ARC). The possibility that the diminution of mu opioid receptor/effector coupling produced by acute 17beta-estradiol or chronic opiate exposures is mediated by a common kinase pathway also was investigated. Intracellular recordings were made in hypothalamic slices prepared from ovariectomized female guinea pigs. The mu opioid receptor agonist D-Ala2, N-Me-Phe4, Gly-ol5-enkephalin (DAMGO) produced dose-dependent hyperpolarizations of ARC neurons. Chronic morphine treatment for 4 days reduced DAMGO potency 2.5-fold with no change in the maximal response. This effect was mimicked by a 20-min bath application of the PKA activator cAMP, Sp-isomer, or the PKC activator phorbol-12,13-dibutyrate. A 30-min bath application of the broad-spectrum protein kinase inhibitor staurosporine completely abolished the reduced DAMGO potency seen in morphine-tolerant neurosecretory cells, including those immunopositive for gonadotropin-releasing hormone. The effect of staurosporine was mimicked by the PKA inhibitor cAMP, Rp-isomer, but not by the PKC inhibitor calphostin C. Finally, a 20-min bath application of 17beta-estradiol did not further reduce DAMGO potency in morphine-tolerant ARC neurons. Therefore, increased PKA activity maintains cellular tolerance to mu opioid receptor agonists in ARC neurosecretory cells caused by chronic morphine treatment. Furthermore, acute 17beta-estradiol and chronic opiate treatments attenuate mu opioid receptor-mediated responses via a common PKA pathway.     

Presence of mu and kappa opioid receptor mRNAs in galanin but not in GnRH neurons in the female rat

Using a combination of radioactive and non-radioactive in situ hybridizations, the expression of mu and kappa opioid receptor mRNA was investigated in neurons in the female rat preoptic nucleus expressing galanin and gonadotropin-releasing hormone (GnRH) mRNA. Numerous cells expressing both mu or kappa and galanin were found in the intermediate and rostral regions of the preoptic area whereas little co-localization was observed at the rostral level. The number of kappa/galanin expressing cells was greater than that of mu/galanin cells. mu/galanin co-localization was observed essentially in the anteroventral preoptic nucleus while neurons expressing kappa/galanin were present in both the anteroventral preoptic nucleus and in the periventricular hypothalamic nucleus. Co-localization of GnRH with mu or kappa could not be detected in the preoptic area. These observations showed that galaninergic neurons but not GnRH neurons of the preoptic area might be directly regulated by endogenous opioid peptides.     

Effects of mu and delta opioid receptor agonists and antagonists on absence epilepsy in WAG/Rij rats

A study was made of the effects of 5-hydroxytryptamine (5-HT) on bradycardia induced in vivo by electrical stimulation of the vagus nerves in pithed rats pretreated with atenolol. 5-HT significantly decreased vagally induced, but not acetylcholine-induced, bradycardia. The first effect was blocked by methiothepin, ketanserin or methiothepin with ketanserin. When 5-HT1 and 5-HT2 receptors were blocked, 5-HT produced an increase in vagally induced bradycardia. Both the inhibition and the potentiation were blocked by simultaneous pretreatment with methiothepin, ketanserin and MDL-72222. The 5-HT2 receptor agonist m-CPP (1-(3-chlorophenyl) piperazine dihydrochloride) caused an inhibition of vagally induced bradycardia whereas the 5-HT3 receptor agonist m-CPBG (1-(m-chlorophenyl)biguanide hydrochloride) produced a significant increase. The data suggest the presence of presynaptic and/or ganglionic 5-HT2 receptors in parasympathetic innervation of the rat heart, stimulation of which inhibits the release of acetylcholine. The presence of 5-HT3 receptors is also suggested, stimulation of which induces the release of acetylcholine.     

Changes in densities of hypothalamic mu opioid receptor during cupric acete-induced preovulatory lh surge in rabbit

Decrease in activity of hypothalamic beta-endorphin (beta-EP) is an important factor for inducing the preovulatory LH surge. To study whether hypothalamic mu opioid receptor is involved in this process, changes in densities of hypothalamic mu opioid receptors were observed in this study by autoradiography and image process during cupric acetate (CuAC)-induced preovulatory LH surge in rabbits. New Zealand female rabbits were injected 1% CuAC 0.9 ml or saline 0.9 ml and sacrificed at different times after the injection. The densities of mu opioid receptor in the medial basal hypothalamus (MBH) and the medial preoptic area (MPO) were measured. A transient increase in densities of MPO mu opioid receptor were observed 1 h after CuAC injection (P < 0.05). The densities of MPO mu opioid receptor decreased significantly before the onset of the LH surge (P < 0.05) and remained at a low level during the surge. The change in densities of mu opioid receptor in the MBH was similar to those in the MPO. No change was observed in the saline control group. There was a negative correlation between the changes in densities of MBH mu opioid receptor and serum LH levels in the process of LH surge. The results suggest that the decrease of hypothalamic mu opioid receptor may be involved in the preovulatory LH surge.     

Single-nucleotide polymorphism in the human mu opioid receptor gene alters beta-endorphin binding and activity: possible implications for opiate addiction

Opioid drugs play important roles in the clinical management of pain, as well as in the development and treatment of drug abuse. The mu opioid receptor is the primary site of action for the most commonly used opioids, including morphine, heroin, fentanyl, and methadone. By sequencing DNA from 113 former heroin addicts in methadone maintenance and 39 individuals with no history of drug or alcohol abuse or dependence, we have identified five different single-nucleotide polymorphisms (SNPs) in the coding region of the mu opioid receptor gene. The most prevalent SNP is a nucleotide substitution at position 118 (A118G), predicting an amino acid change at a putative N-glycosylation site. This SNP displays an allelic frequency of approximately 10% in our study population. Significant differences in allele distribution were observed among ethnic groups studied. The variant receptor resulting from the A118G SNP did not show altered binding affinities for most opioid peptides and alkaloids tested. However, the A118G variant receptor binds beta-endorphin, an endogenous opioid that activates the mu opioid receptor, approximately three times more tightly than the most common allelic form of the receptor. Furthermore, beta-endorphin is approximately three times more potent at the A118G variant receptor than at the most common allelic form in agonist-induced activation of G protein-coupled potassium channels. These results show that SNPs in the mu opioid receptor gene can alter binding and signal transduction in the resulting receptor and may have implications for normal physiology, therapeutics, and vulnerability to develop or protection from diverse diseases including the addictive diseases.     

The orphan opioid receptor and its endogenous ligand--nociceptin/orphanin FQ

Following the elucidation of the amino acid sequences of the mu-, delta- and kappa-opioid receptors, a new 'orphan opioid receptor' was cloned with a high degree of homology to the 'classical' opioid receptors. The endogenous opioid peptides show little or no activity at this new receptor; however, a novel endogenous peptide for the orphan opioid receptor has been isolated and sequenced. Here, Graeme Henderson and Sandy McKnight review recent findings on this new receptor and its endogenous ligand, and address the contentious issue of whether activation of this receptor results in hyperalgesia or analgesia.     

Synthesis of willardiine and 6-azawillardiine analogs: pharmacological characterization on cloned homomeric human AMPA and kainate receptor subtypes

Both willardiine and azawillardiine analogs (18-28) have been reported to be potent and selective agonists for either AMPA or kainate receptors. We report here the novel synthesis and pharmacological characterization of a range of willardiine (18-23) and 6-azawillardiine (24-28) analogs on cells individually expressing human homomeric hGluR1, hGluR2, hGluR4, or hGluR5 receptors. Reaction of the sodium salts of substituted uracils (7-12) or 6-azauracils (13-16) with (S)-3-[(tert-butoxycarbonyl)amino]oxetan-2-one (17) in dry DMF, subsequent deprotection in TFA, and purification by ion-exchange chromatography gave mainly the willardiine analog in which alkylation took place on N1 of the uracil ring. We have investigated the subtype selectivity of these compounds by examining their binding affinity for homomeric hGluR1, -2, -4, or -5 (and hGluR6 in the case of 5-iodowillardiine (22)). From this study we have demonstrated that 22 has high affinity for hGluR5 and, compared to kainate, displays excellent selectivity for this receptor over both the AMPA receptor subtypes and the homomeric kainate receptor, hGluR6. 5-Fluorowillardiine (19) has higher affinity than AMPA for both homomeric hGluR1 and hGluR2 and compared to AMPA displays greater selectivity for AMPA receptor subtypes over the kainate receptor, hGluR5. Some structural features required for optimal activity at homomeric AMPA or kainate receptor subtypes have also been identified. It would appear that quite large lipophilic substituents at the 5-position of the uracil ring not only are accommodated by hGluR5 receptors but also lead to enhanced affinity for these receptors. In contrast to this, for optimal binding affinity to hGluR1, -2, or -4, smaller, electron-withdrawing substituents are required. For optimal activity at hGluR4 receptors a 6-aza-substituted willardiine is favored. The subtype-selective compounds described here are likely to be useful tools to probe the distribution and the physiological roles of the various glutamate receptor subunits in the central nervous system.     

Agonist-induced desensitization of the mu opioid receptor is determined by threonine 394 preceded by acidic amino acids in the COOH-terminal tail

To identify the structural determinants necessary for mu opioid receptor desensitization, we serially ablated potential phosphorylation sites in the carboxyl tail of the receptor and examined their effects on [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO)-induced desensitization. First, we replaced Thr394 with alanine (T394A) and stably expressed this mutant receptor in Chinese hamster ovary cells. The T394A receptor did not desensitize after 1 h of treatment with DAMGO, indicating that Thr394 is required for agonist-induced early desensitization. To test whether Thr394 was the only residue necessary, we investigated the importance of 7 potential phosphorylation sites between residues 363 and 383, which were all replaced by alanines with the Thr394 maintained. This mutant (AT) showed partial loss of desensitization (30%), which was attributable to the Ala mutation at Thr383, since complete desensitization was achieved by restoring Thr383 (ATT). These results suggest that Thr394 is the primary recognition site for G protein-coupled receptor kinases, but Thr383 is also required for complete agonist-induced desensitization. The specificity of Thr394 as the primary initiation site appears to be dependent on the preceding acidic amino acid stretch, because in a mutant in which glutamic acid residues at 388, 391, and 393 were replaced by glutamines (EQ), agonist-induced desensitization was completely abolished, identical to the T394A mutant.