Opioid Binding Sites Research Articles

Biased, Bitopic, Opioid-Adrenergic Tethered Compounds May Improve Specificity, Lower Dosage and Enhance Agonist or Antagonist Function with Reduced Risk of Tolerance and Addiction.

This paper proposes the design of combination opioid–adrenergic tethered compounds to enhance efficacy and specificity, lower dosage, increase duration of activity, decrease side effects, and reduce risk of developing tolerance and/or addiction. Combinations of adrenergic and opioid drugs are sometimes used to improve analgesia, decrease opioid doses required to achieve analgesia, and to prolong the duration of analgesia. Recent mechanistic research suggests that these enhanced functions result from an allosteric adrenergic binding site on opioid receptors and, conversely, an allosteric opioid binding site on adrenergic receptors. Dual occupancy of the receptors maintains the receptors in their high affinity, most active states; drops the concentration of ligand required for full activity; and prevents downregulation and internalization of the receptors, thus inhibiting tolerance to the drugs. Activation of both opioid and adrenergic receptors also enhances heterodimerization of the receptors, additionally improving each drug’s efficacy. Tethering adrenergic drugs to opioids could produce new drug candidates with highly desirable features. Constraints—such as the locations of the opioid binding sites on adrenergic receptors and adrenergic binding sites on opioid receptors, length of tethers that must govern the design of such novel compounds, and types of tethers—are described and examples of possible structures provided.

Adrenergic Agonists Bind to Adrenergic-Receptor-Like Regions of the Mu Opioid Receptor, Enhancing Morphine and Methionine-Enkephalin Binding: A New Approach to "Biased Opioids"?

Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR) were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists.

Mu Opioid Pharmacology: 40 Years to the Promised Land.

Opioids continue to play a major role in medicine, but not without problems. Side effects limit their utility medically, while the potential of addiction has had a major societal impact. Pharmacologists have been trying to develop opioids lacking side effects since the first derivative, heroin, was synthesized in the 1870s. The identification of opioid receptors about 40 years ago opened up new insights into our understanding of opioid action, fueled by the molecular biology revolution of the 1980s and 1990s. A major result of these studies was the discovery that the mu opioid receptor gene, Oprm1, undergoes extensive alternative splicing in mice, rats, and humans. This single gene generates three sets of proteins, each containing many variants. The object of this review is to describe these variants and how they can be targeted to generate safer, effective analgesic drugs. Mu opioid receptor multiplicity was first suggested over 35 years ago based upon a series of selective antagonists and detailed binding assays. The identification of the different classes of mu opioid receptor splice variants enabled us to target one of the classes of splice variants to obtain potent analgesics lacking respiratory depression, physical dependence, and reward behavior. They also displayed no cross tolerance to morphine analgesia and had diminished effects on gastrointestinal transit. Forty years after the identification of opioid-binding sites in brain the promised land of safer, nonaddictive analgesics is in sight.

Effects of prolonged-release oxycodone/naloxone on pain control, bowel function and quality of life: A prospective observational study

Background and aimStrong opioids including oxycodone are amongst the most effective analgesics to combat moderate to severe pain of various aetiologies, but opioid-induced bowel dysfunction (OIBD) represents a relevant problem. The rationale for development of a prolonged-release (PR) fixed combination of oxycodone and naloxone was to counteract OIBD. Due to its negligible oral bioavailability, the μ-opioid receptor antagonist naloxone is able to selectively displace opioids from local μ-receptors in the gastrointestinal tract without affecting central opioid binding sites. Pivotal trials of PR oxycodone/naloxone not only demonstrated improved bowel function but also equivalent analgesic efficacy compared to PR oxycodone alone. Controlled clinical trials comparing PR oxycodone/naloxone with strong opioids other than oxycodone are not available. The present study is the first data set aimed at comparing pain control, bowel function, and quality of life (QoL) in patients newly treated with or switched to PR oxycodone/naloxone or other strong opioids during routine clinical practice. MethodsIn this three-arm, prospective observational study, 588 patients with moderate to severe pain of varying aetiologies received either PR oxycodone/naloxone (OXN group and OXN 40/20 group with indicated use of the 40mg/20mg dose strength at baseline) or other strong opioids (control group), dosed according to pain severity, for 4–6 weeks. Data documented include pain intensity (NRS), bowel function (Bowel Function Index, BFI), pain-related functional impairment (BPI-SF), QoL (EuroQol EQ-5D-3L), and a global assessment of treatment. ResultsPatients receiving PR oxycodone/naloxone experienced a clinically important reduction in pain intensity and pain-related functional impairment of approximately 40%. The reductions of pain intensity (−2.9±2.3) and pain-related functional impairment (−2.4±2.3) in the OXN group were significantly more pronounced than in the control group (−2.1±2.1 and −1.8±1.7). In the control group, mean reductions in pain intensity did not reach the threshold of ≥30% for at least moderate clinically important differences, although patients were prescribed higher doses of morphine equivalents than OXN group patients. Improvements in bowel function (OXN: −16.0±27.6; control: 3.1±24.4) and QoL (OXN: 20.8±24.2; control: 13.2±23.1) were also significantly more pronounced in the OXN group, with BFI scores reduced to a level that reflects normal bowel function. Results for the OXN 40/20 group receiving higher doses of PR oxycodone/naloxone were in line with those for the OXN group. In the control group, more frequent gastrointestinal adverse events and less favourable ratings of tolerability resulted in a higher rate of treatment discontinuations due to adverse events. ConclusionsIn patients receiving PR oxycodone/naloxone, more favourable outcomes compared with other strong opioids regarding pain control, bowel function, and QoL were observed. ImplicationsThe present findings underline the value of PR oxycodone/naloxone in the management of patients with moderate to severe chronic pain. The data set further adds to our understanding of the benefits and risks of opioid treatment in routine clinical practice.

Sandmeyer reaction repurposed for the site-selective, non-oxidizing radioiodination of fully-deprotected peptides: Studies on the endogenous opioid peptide α-neoendorphin

Standard radioiodination methods lack site-selectivity and either mask charges (Bolton-Hunter) or involve oxidative reaction conditions (chloramine-T). Opioid peptides are very sensitive to certain structural modifications, making these labeling methods untenable. In our model opioid peptide, α-neoendorphin, we replaced a tyrosyl hydroxyl with an iodine, and in cell lines stably expressing mu, delta, or kappa opioid receptors, we saw no negative effects on binding. We then optimized a repurposed Sandmeyer reaction using copper(I) catalysts with non-redoxing/non-nucleophilic ligands, bringing the radiochemical yield up to around 30%, and site-selectively incorporated radioactive iodine into this position under non-oxidizing reaction conditions, which should be broadly compatible with most peptides. The 125I- and 131I-labeled versions of the compound bound with high affinity to opioid receptors in mouse brain homogenates, thus demonstrating the general utility of the labeling strategy and of the peptide for exploring opioid binding sites.

The Dilemma of Opioid-Induced Hyperalgesia and Tolerance in Chronic Opioid Therapy = معضلة فرط التألم و حالة التحمل الناتج عن العلاج طويل الأمد بالأدوية ( المسكنات ) الأفيونية

The Dilemma of Opioid-Induced Hyperalgesia and Tolerance in Chronic Opioid Therapy = معضلة فرط التألم و حالة التحمل الناتج عن العلاج طويل الأمد بالأدوية ( المسكنات ) الأفيونية

CNR1 gene deletion affects the density of endomorphin-2 binding sites in the mouse brain in a hemisphere-specific manner

Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) are two endogenous tetrapeptides with very high affinities for the μ-opioid receptor. Until recently, the precise neuroanatomical localization of the binding sites for these peptides was unknown. However, the recent synthesis of tritiated forms of these molecules has permitted these binding sites to be analysed with a very high degree of neuroanatomical specificity. Preliminary studies demonstrated a superior binding profile for EM-2, with less non-specific binding than EM-1. As the endogenous cannabinoid and opioid systems interact at several levels, we investigated how deletion of the CNR1 gene, which encodes the cannabinoid receptor 1 (CB1R) protein, affects the brain distribution of EM-2 binding sites. Our results revealed no differences in the average density of EM-2 binding sites in CB1 receptor knockout (CB1R KO) and WT mice. However, when both hemispheres were analysed separately, we detected specific alterations in the distribution of EM-2 binding sites in the right hemisphere of CB1R KO mice. While, the density of EM-2 binding sites in CB1R KO mice was higher in the CA3 hippocampal field and in the pontine tegmental nuclei, it was lower in the superior colliculus and ventral tegmental area than in WT controls. No differences were observed in the left hemisphere for any of the regions analysed. For the first time these findings demonstrate a lateralization effect on cerebral opioid binding sites that may be mediated by the central cannabinoid system.

Generation of novel radiolabeled opiates through site-selective iodination

Tritiated opioid radioligands have proven valuable in exploring opioid binding sites. However, tritium has many limitations. Its low specific activity and limited counting efficiency makes it difficult to examine low abundant, high affinity sites and its disposal is problematic due to the need to use organic scintillants and its relatively long half-life. To overcome these issues, we have synthesized both unlabeled and carrier-free radioiodinated iodobenzoyl derivatives of 6β-naltrexamine ((125)I-BNtxA, 18), 6β-naloxamine ((125)I-BNalA, 19) and 6β-oxymorphamine ((125)I-BOxyA, 20) with specific activities of 2100Ci/mmol. To optimize the utility of the radioligand, we designed a synthesis in which the radiolabel is incorporated in the last synthetic step, which required the selective iodination of the benzoyl moiety without incorporation into the phenolic A ring. Competition studies demonstrated high affinity of the unlabelled compounds for opioid receptors in transfected cell lines, as did the direct binding of the (125)I-ligands to the opioid receptors. The radioligand displayed very high sensitivity, enabling a marked reduction in tissue, as well as excellent signal/noise characteristics. These new (125)I-radioligands should prove valuable in future studies of opioid binding sites.

Facilitation of μ-Opioid Receptor Activity by Preventing δ-Opioid Receptor-Mediated Codegradation

δ-opioid receptors (DORs) form heteromers with μ-opioid receptors (MORs) and negatively regulate MOR-mediated spinal analgesia. However, the underlying mechanism remains largely unclear. The present study shows that the activity of MORs can be enhanced by preventing MORs from DOR-mediated codegradation. Treatment with DOR-specific agonists led to endocytosis of both DORs and MORs. These receptors were further processed for ubiquitination and lysosomal degradation, resulting in a reduction of surface MORs. Such effects were attenuated by treatment with an interfering peptide containing the first transmembrane domain of MOR(MOR(TM1)), which interacted with DORs and disrupted the MOR/DOR interaction. Furthermore, the systemically applied fusion protein consisting of MOR(TM1) and TAT at the C terminus could disrupt the MOR/DOR interaction in the mouse spinal cord, enhance the morphine analgesia, and reduce the antinociceptive tolerance to morphine. Thus, dissociation of MORs from DORs in the cell membrane isa potential strategy to improve opioid analgesic therapies.

Chronic pain and prescription opioid misuse

Tolerance is the phenomenon where identical doses of an opioid result in a decreasing effect; higher doses are required to produce the same effect. Tolerance can occur either to the drug’s therapeutic effect or to its unwanted sideeffects. Pharmacological tolerance develops because of a decrease in the number of opioidbinding sites or an acute depletion of the neurotransmitter released by the drug or by a decreased activity in intracellular secondmessenger systems. Behavioural tolerance occurs when the patient learns to compensate over time for the expected pharmacological effects of the drug.

Kappa2 opioid receptor subtype binding requires the presence of the DOR-1 gene

Over the past several years substantial evidence has documented that opioid receptor homo- and heterodimers form in cell lines expressing one or more of the opioid receptors. We used opioid receptor knockout mice to determine whether in vivo pharmacological characteristics of kappa1 and kappa2 opioid receptors changed following knockout of specific opioid receptors. Using displacement of the general opioid ligand diprenorphine, we observed that occupancy or knockout of the DOR-1 gene increases the binding density of kappa1 receptors and eliminates kappa2 receptors in crude membrane preparations while the total density of kappa opioid binding sites is unchanged. Further, the analgesic potency of U69,593 in cumulative dose response curves is enhanced in mice lacking the DOR-1 gene. These results demonstrate that the DOR-1 gene is required for the expression of the kappa2 opioid receptor subtype and are consistent with the possibility that a KOR-1/DOR-1 heterodimer mediates kappa2 pharmacology.

Cholinergic, opioid and glycine receptor binding sites localized in human spinal cord by in vitro autoradiography. Changes in amyotrophic lateral sclerosis.

Binding sites for the receptor ligands 3H-quinuclidinylbenzilate, 3H-alpha-bungarotoxin (3H-alpha-Btx), 3H-etorphine and 3H-strychnine were localized autoradiographically at cervical, thoracic and lumbar levels of spinal cords from post-mortem human control subjects and subjects with amyotrophic lateral sclerosis (ALS). The highest densities of muscarinic binding sites were found in the motor neuron areas and in the substantia gelatinosa, while the grey matter binding was very low within Clarke's column. Both 3H-alpha-Btx and opioid receptor binding sites were numerous within the substantia gelatinosa, while glycine receptor binding sites were more uniformly distributed within the spinal grey matter. In ALS cases, muscarinic receptor binding sites were markedly reduced in motor neuron areas and slightly reduced in the dorsal horn, while the other binding sites studied were relatively unchanged.

The Effects of Fentanyl-Like Opioids and Hydromorphone on Human 5-HT3A Receptors

5-HT(3) receptors are involved in various physiologic functions, including the modulation of emesis. 5-HT(3) antagonists are clinically widely used as potent antiemetics. Emesis is also a side effect of opioid analgesics. Intriguingly, the natural opioid morphine shows specific interactions with human 5-HT(3) receptors at clinically relevant concentrations. In the present study, we investigated whether this is a general effect of opioids, even when they are structurally diverse. Therefore, another morphine (phenanthrene-type) derivative, hydromorphone, and fentanyl including its (4-anilinopiperidine-type) derivatives were tested. Whole-cell patches from human embryonic kidney-293 cells, stably transfected with the human 5-HT(3A) receptor cDNA, were used to determine the opioid effects on the 5-HT (3 microM)-induced currents using the patch clamp technique (voltage-clamp). None of the fentanyl derivatives affected currents through the 5-HT(3A) receptor (3 microM 5-HT) significantly in the clinically relevant nanomolar concentration range (IC(50) values >30 microM). In contrast, hydromorphone was considerably more potent (IC(50) = 5.3 microM), slowing the current activation- and desensitization-kinetics significantly (at 3 microM by a factor of 1.9 and 2.4, respectively), similar to morphine. At concentrations much higher than clinically relevant, but within the range predicted from Meyer-Overton correlations for nonspecific interactions, the fentanyl derivatives all showed at least a tendency to suppress current amplitudes, but they had diverse effects on the activation- and desensitization-kinetics of 5-HT(3A) receptors. Only morphine and hydromorphone, but not the fentanyl derivatives, reduced 5-HT-induced current amplitudes and slowed current kinetics near clinically relevant concentrations. The high potencies of morphine and hydromorphone, when compared to their lipophilicities, suggest a specific interaction with 5-HT(3A) receptors. In contrast, the effects of fentanyl-type opioids appear to be of unspecific nature. Because the rank order of opioid potencies for human 5-HT(3A) receptors is opposite of that for opioid receptors, the site involved is structurally different from opioid receptor binding sites. In agreement with recent data on different phenols, a phenolic OH-group (which morphine and hydromorphone possess) may contribute to specific interactions of morphine and hydromorphone with the 5-HT(3A) receptor. Future clinical studies could test whether corresponding differences in emetogenicity between different classes of opioids will be found.

Comparison of opioid receptor binding in horse, guinea pig, and rat cerebral cortex and cerebellum

ObjectiveTo compare the density and binding characteristics of opioid receptor subtypes in horse, rat, and guinea pig cerebral cortex and cerebellum. Study designProspective receptor binding study. AnimalsWhole brains were obtained from four neurologically normal adult horses during necropsy. Rat and guinea pig brains were obtained commercially. MethodsThe cerebellum and cerebral cortex were dissected from each brain, and tissue homogenates prepared. A radioligand binding technique with the highly selective ligands [3H]-DAMGO, [3H]-U69593, and [3H]-DPDPE was used to identify the mu- (μ), kappa- (κ) and delta- (δ) opioid receptors, respectively. Competitive binding assays were performed with these ligands and varying concentrations of one of multiple unlabeled ligands. ResultsWhile there were marked species differences in relative densities of opioid receptors, all radioligands interacted with their binding sites with high, nanomolar affinity in both the cerebral cortex and cerebellum. In the horse cerebral cortex, the percentages of total opioid binding sites for the μ-, κ- and δ-receptors were 71%, 14% and 15%, respectively. In the rat and guinea pig cerebral cortex, the corresponding values were 56%μ-, 4%κ- and 40%δ-receptors, and 25%μ-, 37%κ- and 38%δ-receptors, respectively. In horse and guinea pig cerebellum, the binding was 37%μ-, 59%κ- and 4%δ-receptors, and 15%μ-, 76%κ- and 10%δ-receptors, respectively. For competitive analysis, all competitors of the μ-, κ- and δ-receptors completely displaced [3H]-DAMGO, [3H]-U69593, and [3H]-DPDPE and had inhibitory constants in the nanomolar range. Conclusion and clinical relevanceHorses used in this study had a greater density of μ-receptors in the cerebral cortex compared with rats and guinea pigs but without further characterization of the functional role of these receptors it is impossible to determine the clinical significance of these data.

An Emerging Role for the Delta Opioid Receptor in the Regulation of Mu Opioid Receptor Function

Morphine and related opiates are commonly used in the clinical management of various types of pain. However, the antinociceptive properties of morphine are often overshadowed by the development of tolerance and dependence following its chronic use. The mechanisms underlying opiate tolerance are not fully understood, but appear to involve numerous and complex physiological adaptations. Recently, a role for the heterodimerization of mu and delta opioid receptors in the development of morphine tolerance has been proposed. This novel mechanism could help us to understand several observations, such as the critical role of delta opioid receptor regulation, the impact of delta opioid receptor binding site occupancy, and the participation of beta-arrestin2, in the development of morphine tolerance.

Striatal delta opioid receptor binding in experimental models of Parkinson's disease and dyskinesia

Enhanced delta opioid receptor transmission may represent an endogenous compensatory mechanism in parkinsonism to reduce the activity of the indirect striatopallidal pathway following dopamine depletion. Furthermore, increased delta opioid receptor transmission may be causative in the production of dyskinesia following repeated dopaminergic treatment in Parkinson's disease. The present study employed radioligand receptor autoradiography, using [3H]naltrindole, a ligand selective for the delta opioid receptor, to assess delta opioid receptor binding sites in forebrain regions of reserpine-treated rats, and in parkinsonian nondyskinetic, and dyskinetic MPTP-lesioned macaques. In reserpine-treated animals, specific delta opioid binding was increased in premotor cortex (+30%), sensorimotor striatum (+20%), and associative striatum (+17%) rostrally, but was not changed in caudal forebrain. In contrast, delta opioid receptor binding was not significantly altered at any region analyzed, in either nondyskinetic or dyskinetic, MPTP-lesioned macaques, compared to normal. These results suggest that transient changes in delta opioid receptor binding may occur in motor circuits following acute dopamine depletion. However, in the more chronic MPTP-lesioned macaque model, simple changes in delta opioid receptor number or affinity are unlikely to contribute to mechanisms for abnormal opioid transmission in Parkinson's disease and dyskinesia.

Microenvironment-Induced Endothelial Heterogeneity Stimulates Retinal Neovascularization in Sickle Mice.

Retinopathy is a serious complication of sickle cell disease (SCD) which is accompanied by endothelial activation and a pro-inflammatory microenvironment. We hypothesized that endogenous and exogenous factors modulate the endothelial microenvironment and endothelial specific alterations leading to retinal neovascularization. While VEGF receptor 2/Flk1 is a key player in the process of angiogenesis, opioids used to treat pain in SCD also promote angiogenesis. Therefore, we examined the role of opioids in the regulation of endothelial microenvironment, Flk1 expression and endothelial activity, using wild type (WT) and NY1DD sickle mice with transgenes for human α and βS globin. We injected 9–11 month old mice with morphine subcutaneously (50 mg/day/70 Kg with 10 mg increments every 2 weeks and/or equimolar amount of naloxone) for 10 to 16 months. NY1DD mouse retina exhibited appreciably higher expression of phospho-Flk1 vs WT which was further upregulated by morphine treatment for 10 and 16 months. Naloxone treatment antagonized morphine induced upregulation of phospho-Flk1, suggesting an opioid receptor mediated effect. Upregulation of phospho-Flk1 was accompanied by an increase in cell cycle regulatory protein cyclin D1 and stimulation of phospho-MAPK/ERK and phospho-Akt in morphine vs PBS treated mouse retina. Furthermore, morphine treatment led to a 5 and 20 fold increase in plasma IL6 and TNFα, respectively as compared to PBS treatment and promoted retinal neovascularization. Thus morphine treatment exaggerates the pro-inflammatory micro-environment and orchestrates a pro-angiogenic tone by upregulating Flk1 and activating the growth and survival promoting signaling in NY1DD mouse retina. To specifically delineate the role of opioids in endothelial activity leading to retinopathy, we isolated retinal endothelial cells (REC) from WT and NY1DD mice. Both VEGF and morphine stimulated REC proliferation in a dose-dependent fashion. Maximum proliferation was achieved with 1 μm morphine or 10 ng/ml VEGF in WT REC, but it was unsaturable upto 10 μm morphine or 1 μg/ml VEGF in NY1DD REC. Optimum morphine and VEGF-induced REC proliferation was 1.5 and 3 fold, respectively in NY1DD Vs WT REC, suggestive of a heterogeneity between WT and NY1DD REC. Saturation kinetics and opioid receptor binding studies showed that the Bmax for 3H-diprenorphine and the number of opioid receptor binding sites were 3-fold higher in NY1DD vs WT REC. RT-PCR analysis showed a higher expression of both Flk1 and μ opioid receptor (MOR) in NY1DD vs WT REC. Incubation of WT REC with IL6 as well as morphine for 48h upregulated Flk1 protein expression. Morphine-induced upregulation of Flk1 was abrogated by naloxone, anti-IL6 antibodies and STAT3 inhibitor Ac-PpYLKTK-OH. On the other hand, incubation of WT-REC with VEGF or IL6 for 48h upregulated MOR expression. Together, these data suggest that morphine stimulates Flk1 expression directly via opioid receptor(s) and also by upregulating IL6 via a STAT3 signaling pathway, while VEGF and IL6 also stimulate MOR expression. Therefore, a pro-inflammatory microenvironment in SCD upregulates endothelial Flk1 and opioid receptors, which is further exaggerated by addition of morphine leading to increased retinal neovascularization. It is likely that opioid use in SCD promotes retinopathy and that opioid receptor antagonists may play a preventive role.

Synthesis and Preliminary In vitro Investigation of Bivalent Ligands Containing Homo- and Heterodimeric Pharmacophores at μ, δ, and κ Opioid Receptors

A series of homo- and heterodimeric ligands containing kappa agonist and mu agonist/antagonist pharmacophores joined by a linker chain of varying lengths was synthesized and evaluated in vitro by their binding affinity at mu, delta, and kappa opioid receptors. The functional activities of these compounds were measured in the [(35)S]GTPgammaS binding assay. The data suggest that the stereochemistry of the pharmacophores, the N-substituents of the pharmacophore, ester linkages, and the spacer length were crucial factors for optimum interactions of such ligands at opioid receptor binding sites. These novel ligands as well as their pharmacological properties will serve as the basis for our continuing investigation of such bivalent ligands as probes of the opioid receptor oligomerization phenomena and for in vivo studies as analgesics.

Characterization of Opioid-Binding Sites in Zebrafish Brain

The pharmacological profile of opioid-binding sites in zebrafish brain homogenates has been studied using radiolabeled binding techniques. The nonselective antagonist [(3)H]diprenorphine binds with high affinity (K(D) = 0.27 +/- 0.08 nM and a B(max) = 212 +/- 14.3 fmol/mg protein), displaying two different binding sites with affinities of K(D1) = 0.08 +/- 0.02 nM and K(D2) = 17.8 +/- 9.18 nM. The nonselective agonist [(3)H]bremazocine also binds with high affinity to zebrafish brain membranes but only displays one single binding site with a K(D) = 1.1 +/- 0.09 nM and a B(max) = 705 +/- 19.3 fmol/mg protein. Competition binding assays using [(3)H]diprenorphine and several unlabeled ligands were performed. The synthetic selective agonists for mammalian opioid receptors DPDPE ([DPen(2),D-Pen(5)]-enkephalin), DAMGO ([D-Ala(2),NMe-Phe(4),Gly(5)-ol]-enkephalin), and U69,593 [(5alpha,7alpha,8beta)-(+)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide] failed to effectively displace [(3)H]diprenorphine binding, whereas nonselective ligands and the endogenous opioid peptides such as dynorphin A showed good affinities in the nanomolar range, although several of the endogenous peptides only displaced approximately 50% of the specifically bound [(3)H]diprenorphine. Our results provide evidence that, although the selective synthetic compounds for mammalian receptors do not fully recognize the opioid-binding sites in zebrafish brain, the activity of the endogenous zebrafish opioid system might not significantly differ from that displayed by the mammalian opioid system. Hence, the study of zebrafish opioid activity may contribute to an understanding of endogenous opioid systems in higher vertebrates.

Stimulatory effect of morphine on rat pineal melatonin synthesis via a cyclic AMP-dependent transcription pathway

The expression of mRNA of opioid receptors and the existence of opioid binding site in the rat pineal gland have been demonstrated previously. A major finding was that morphine enhanced the activity of the rate-limiting enzyme, N-acetyltransferase (NAT) and increased the level of melatonin in rat pineal gland. An attempt has been made in order to clarify the mechanism of this induction. In the present study, the stimulatory effect of morphine on the expression of NAT mRNA in the rat pineal gland has been demonstrated using semi-quantitative RT-PCR technique. The results showed that both acute and chronic morphine treatments significantly increased NAT mRNA expression in rat pineal gland. In addition, the effect of morphine on the phosphorylation of the transcription factors, cyclic AMP responsive element-binding protein (CREB) was investigated. Western blot analysis showed that morphine significantly increased phosphorylation of CREB. These results indicate that at least one downstream messenger pathway for the activation of opioidergic system on the induction of melatonin synthesis in the rat pineal gland acts via cyclic AMP-dependent cascade and transcription mechanism.