Delta-opioid Receptor Density Research Articles

Effects of Imipramine Treatment on δ-Opioid Receptors of the Rat Brain Cortex and Striatum

Imipramine (CAS 113-52-0) is being utilized widely for the treatment of major depression. In recent years, there has been evidence of the involvement of the endogenous opioid system in major depression and its treatment. There is some evidence indicating that opioid receptors could be involved in the antidepressant mechanism of action. Regarding this topic, mood-related behavior of endogenous enkephalins seems to be mediated by delta-opioid receptors. In this work, the effects of subacute (5 day) and chronic (15 day) treatments of imipramine on the density and the affinity of the delta-receptors in the striatum and in the parietal and frontal cortices of the rat brain are described. Studied parameters (Bmax and Kd) were calculated by a saturation binding assay with the delta-opioid agonists [3H]-DPDPE (tyrosyl-2,6-3H(N)-(2-D-penicillamine-5-D-penicillamine)-enkephalin) as specific ligand and DSLET ([D-serine2]-D-leucine-enkephalin-threonine) as non-radioactive competing ligand. It was found that 15 days treatment significantly decreased the delta-opioid receptor density,without changing the affinity, in the frontal cortex of the rat brain. That decrease was confirmed by delta-opioid receptor immunostaining. These results suggest that delta-opioid receptors could play a role in the chronic action mechanism of imipramine.

Prolonged Morphine Treatment Targets δ Opioid Receptors to Neuronal Plasma Membranes and Enhances δ-Mediated Antinociception

Opioid receptors are known to undergo complex regulatory changes in response to ligand exposure. In the present study, we examined the effect of morphine on the in vitro and in vivo density and trafficking of delta opioid receptors (deltaORs). Prolonged exposure (48 hr) of cortical neurons in culture to morphine (10 microm) resulted in a robust increase in the internalization of Fluo-deltorphin, a highly selective fluorescent deltaOR agonist. This effect was mu-mediated because it was entirely blocked by the selective mu opioid receptor antagonist d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH(2) and was reproduced using the selective mu agonist fentanyl citrate. Immunogold electron microscopy revealed a marked increase in the cell surface density of deltaORs in neurons exposed to morphine, indicating that the increase in Fluo-deltorphin internalization was caused by increased receptor availability. Prolonged morphine exposure had no effect on deltaOR protein levels, as assessed by immunocytochemistry and Western blotting, suggesting that the increase in bioavailable deltaORs was caused by recruitment of reserve receptors from intracellular stores and not from receptor neosynthesis. Complementary in vivo studies demonstrated that chronic treatment of adult rats with morphine (5-15 mg/kg, s.c., every 12 hr) similarly augmented targeting of deltaORs to neuronal plasma membranes in the dorsal horn of the spinal cord. Furthermore, this treatment markedly potentiated intrathecal d-[Ala(2)]deltorphin II-induced antinociception. Taken together, these results demonstrate that prolonged stimulation of neurons with morphine markedly increases recruitment of intracellular deltaORs to the cell surface, both in vitro and in vivo. We propose that this type of receptor subtype cross-mobilization may widen the transduction repertoire of G-protein-coupled receptors and offer new therapeutic strategies.

Effects of antisense oligonucleotides on brain delta-opioid receptor density and on SNC80-induced locomotor stimulation and colonic transit inhibition in rats.

1. To reduce the density of delta-opioid receptor protein, five antisense phosphorothioate oligodeoxynucleotides (aODN), targeting the three exons of rat delta-opioid receptor mRNA (DOR), were injected twice daily for 4 days or continuously infused for 7 days into brain lateral ventricles (i.c.v.) of Sprague-Dawley rats. Rats acting as controls were infused or injected with a mismatch sequence (mODN) of each aODN. The density of opioid receptors in rat brain membranes was measured by saturation binding experiments using selective ligands for delta, mu and kappa opioid receptors. 2. aODNs injected twice a day for 4 days left rat brain delta-opioid receptor density unchanged. The ODN targeting the DOR nucleotide sequence 280 - 299 (aODN280 - 299, exon 2), decreased brain delta-opioid receptor density significantly more than aODNs targeting exon 1 (aODN239 - 258), exon 2 (aODN361 - 380), or exon 3 (aODN741 - 760) (to 52% vs 79, 72, and 68%). None of the aODNs to the DOR changed the brain density of mu- or k-opioid receptors. 3. When in a novel environment (but not when kept in their home cages), the locomotor activity of aODN280 - 299 treated rats was significantly lower than that of saline or mODN treated rats. The delta-opioid agonist SNC80 (5 mg kg-1, s.c.) significantly and potently stimulated locomotion and delayed colonic propulsion in saline- and mODN-infused rats, but left motor behaviour and colonic transit of delta-knockdown rats unchanged. 4. The baseline nociceptive threshold and the antinociceptive response to morphine were unchanged in delta-knockdown rats.

Affinity Labeling of δ Opioid Receptors by an Enkephalin-Derivative Alkylating Agent, DSLET-Mal

Opioid binding properties of Tyr-D-Ser-Gly-Phe-Leu-Thr-NH-NH-Gly-Mal (DSLET-Mal), a novel enkephalin-framed affinity label, was determined in rat brain membranes. In competition studies the ligand showed high affinity for the delta opioid sites, labelled by [3H][Ile5,6]deltorphin II (Ki = 8 nM), whereas its binding to the mu ([3H]DAMGO) and kappa ([3H]EKC) sites was weaker. Preincubation of the rat brain membranes with DSLET-Mal at micromolar concentrations resulted in a wash-resistant and dose-dependent inhibition of the [3H][Ile5,6]deltorphin II binding sites (96% blocking at 10 μM concentration). Intracerebroventricular (ICV) administration of DSLET-Mal reduced the density of delta opioid receptors and had no effect on mu and kappa receptors, as determined by saturation binding studies. [Ile5,6]deltorphin II-stimulated [35S]GTPγS binding was determined in membrane preparations of different brain areas of the ICV-treated animals. In both frontal cortex and hippocampus DSLET-Mal significantly decreased G protein activation by the delta agonist, having no effect on DAMGO stimulated [35S]GTPγS binding. DSLET-Mal had qualitatively similar effects on both receptor binding and G protein activation. These characteristics of the compound studied suggest that DSLET-Mal can serve as an affinity label for further studies of the delta-opioid receptors.

Quantification of delta-opioid receptors in human brain with N1'-([11C]methyl) naltrindole and positron emission tomography.

The regional binding of N1'-([11C]methyl)naltrindole (MeNTI), a selective delta-opioid antagonist, was studied in healthy human subjects with positron emission tomography (PET). After the bolus intravenous administration of high specific activity [11C]MeNTI, PET was performed over 90 minutes. Arterial plasma samples were obtained during the scanning period and assayed for the presence of radiolabeled metabolites. The data were analyzed with various kinetic (two- and three-compartment models, Patlak graphical analysis) and nonkinetic (apparent volume of distribution and activity at a late scanning time) approaches. This tracer showed irreversible binding characteristics during the scanning period used. The results of the analyses also were compared with the density and distribution of delta-opioid receptors in the human brain in vitro. Additionally, computer simulations were performed to assess the effects of changes in receptor binding and tracer transport changes on the perceived binding parameters obtained with the models. A constrained three-compartment kinetic model was demonstrated to be superior to other quantification models for the description of MeNTI kinetics and quantification of delta receptor binding in the human brain with 11C-labeled MeNTI.

The effect of repeated administration of morphine, cocaine and ethanol on mu and delta opioid receptor density in the nucleus accumbens and striatum of the rat

The present study was carried out to evaluate the effect of morphine, cocaine and ethanol on the density of opioid receptors in the nucleus accumbens and striatum of rat brain. The animals were injected i.p. with morphine in a single dose 20 mg/kg, or twice daily for 10 days in increasing doses of 20–100 mg/kg. Cocaine was administered in a dose of 60 mg/kg/day following the “binge” paradigm, every hour for 3 h, one day (single treatment) or five days (chronic treatment). Ethanol was administered in drinking water at increasing concentrations of 1–6% v/v, for one month. As shown by receptor autoradiography, single morphine and cocaine administration did not influence the binding density of the selective ligand of δ2 receptors [ 3H]Ile 5,6deltorphin b, but single administration of cocaine decreased binding density of a highly selective antagonist of δ receptors, [ 3H]H-Tyr-TicΨ[CH 2-NH]Phe-Phe-OH. Repeated morphine administration decreased the receptor density after both ligands of the δ receptor in the nucleus accumbens after 3, 24 and 48 h, and in the striatum after 24 and 48 h. The density of [ 3H]Ile 5,6deltorphin b binding remained unchanged in both structures following repeated cocaine administration. After repeated cocaine administration either no changes (3 h) or a decrease in the binding of [ 3H]H-Tyr-TicΨ[CH 2-NH]Phe-Phe-OH in the nucleus accumbens and striatum were observed after 24 and 48 h. Ethanol did not influence the binding density of [ 3H]H-Tyr-TicΨ[CH 2-NH]Phe-Phe-OH and [ 3H]Ile 5,6deltorphin b in the nucleus accumbens and striatum at any time-point studied. In the nucleus accumbens and striatum, no changes were found in the binding density of [ 3H]Tyr- d-Ala-Gly-MePhe-Gly-ol following single or repeated morphine administration. At 3 h after single or repeated “binge” cocaine administration, the binding of [ 3H]Tyr- d-Ala-Gly-MePhe-Gly-ol was not changed in either structure, but after 24 h the density of μ opioid receptors was decreased in both structures. Ethanol given to rats in drinking water decreased the binding of [ 3H]Tyr- d-Ala-Gly-MePhe-Gly-ol at the time of exposure to ethanol, yet in the nucleus accumbens only. Ethanol withdrawal decreased the density of the μ receptor in both structures after 24, 48 and 96 h. The above data indicate that repeated administration of morphine evokes a long-lasting down-regulation of the density of δ1 and δ2 opioid receptors, whereas cocaine affects in a similar way only the δ1 subtype in the nucleus accumbens, and to a lesser extent in the striatum. A long-term intake of ethanol solution down-regulates μ opioid receptors in both structures, but has no effect on any type of δ receptors. Thus changes in the particular opioid receptor depend on the type of drug used. Furthermore, the most profound changes are observed after late withdrawal, which may play some role in maintaining the state of dependence.

Electrophysiological studies on the postnatal development of the spinal antinociceptive effects of the delta opioid receptor agonist DPDPE in the rat.

1. The antinociceptive effects of the delta opioid receptor selective agonist, DPDPE [(D-Pen2,D-Pen5)-enkephalin] was studied in rats aged postnatal day (P) 14, P21, P28 and P56. 2. Antinociceptive effects of DPDPE were measured as percentage inhibition of the C-fibre evoked response and post-discharge of dorsal horn neurones evoked by peripheral electrical stimulation. DPDPE was administered by topical application, akin to intrathecal injection. 3. DPDPE (0.1-100 microg) produced dose-related inhibitions at all ages; these inhibitions were reversed by 5 microg of the opioid antagonist naloxone. 4. The dose-response curves for C-fibre evoked response and post-discharge of the neurones were not different in rats aged P14 and P21. DPDPE was significantly more potent at P14 and P21 compared with its inhibitory effects on these responses at P28 and P56. 5. DPDPE produced minor inhibitions of the A-fibre evoked response of the neurones at P14, P21, P28 and P56, suggesting that the inhibitory effects of DPDPE are mediated via presynaptic receptors on the terminals of C-fibre afferents. 6. Since spinal delta opioid receptor density changes little over this period, the increased antinociceptive potency of DPDPE in the rat pups compared with the adult is likely to be due to post-receptor events, or in developmental changes in the actions of other transmitter/receptor systems within the spinal cord.

Age- and sex-related differences in opioid receptor densities in the songbird vocal control system

Avian vocal control regions of adult male songbirds contain opioid peptides and receptors, suggesting that opioids play a role in avian vocal behavior control. In a previous study, we found no difference in opioid receptor densities in singing versus nonsinging adult male dark-eyed juncos (Junco hyemalis), leading us to hypothesize that opioids are not involved in controlling song production. To assess whether opioids may be involved in other aspects of vocal behavior, we used quantitative in vitro autoradiography to compare mu and delta opioid receptor densities in vocal control regions of singing adult males with those of adult females and adolescent (about 3 months old) males and females. We found mu and delta receptors in all vocal control regions measured. Adolescents had significantly higher opioid receptor densities than did adults in area X (delta), robust n. of the archistriatum (delta and mu), and n. intercollicularis (mu), suggesting a developmental role for opioids in the vocal control system. Based on opioid roles in other animal models, we propose that opioids may be involved in song learning, auditory processing, and/or vocal control system development.

Characterization of the μ and δ Opioid Receptors in the Brain of the C57BL/6 and DBA/2 Mice, Selected for Their Differences in Voluntary Ethanol Consumption

Genetically determined differences in the activity of the hypothalamic beta-endorphin system have been demonstrated between the C57BL/6 (alcohol-preferring) and DBA/2 (alcohol-aversive) inbred strains of mice. The present studies examined the distribution and density of the mu and delta receptors in specific brain regions that may mediate the rewarding and reinforcing effects of ethanol, using quantitative autoradiography and the specific mu agonist FK 33-824 and delta agonist DPDPE, in their iodinated form. 125I-FK 33-824 recognizes a high affinity binding site in brain membrane preparations from both the C57BL/6 (Kd = 1.37 +/- 0.22 nM; Bmax = 80 +/- 12.3 fmol/mg protein) and DBA/2 mice (Kd = 1.02 +/- 0.16 nM; Bmax = 39.5 +/- 9.6 fmol/mg protein), whereas 125I-DPDPE binds to a high affinity binding site in brain membranes from both the C57BL/6 (Kd = 1.08 +/- 0.34 nM; Bmax = 24.4 +/- 4.5 fmol/mg protein) and DBA/2 mice (Kd = 0.68 +/- 0.24 nM; Bmax = 15.3 +/- 3.7 fmol/mg protein). The autoradiographic studies demonstrated differences in the density of the mu opioid receptors between the two strains of mice in brain nuclei that are not directly related to the brain reward system. However, strain-related differences in the density of delta opioid receptors were observed in regions of the limbic system known to mediate the positive reinforcing effects of many drugs of abuse. The density of delta receptors was significantly higher in the ventral tegmental area and nucleus accumbens of the C57BL/6 mice. The results of the present study support the hypothesis that genetically determined differences exist in the density of opioid receptors in distinct regions of the brain between the C57BL/6 and DBA/2 inbred strains of mice, which may play a role in controlling their voluntary ethanol consumption.

IMPLICATIONS OF ENDOGENOUS OPIOIDS AND DOPAMINE IN ALCOHOLISM: HUMAN AND BASIC SCIENCE STUDIES

We investigated the endogenous opioid system and its role in mediating the reinforcing effects of ethanol that lead to high ethanol consumption as a biochemical marker of an individual's vulnerability to excessive ethanol consumption. We performed studies using human subjects with [high risk (HR)] and without [low risk (LR)] a family history of alcoholism to supplement our studies with experimental animals bred selectively for high- or low-ethanol consumption. HR subjects had lower basal plasma beta-endorphin levels as compared with LR subjects, but they had a more pronounced release of beta-endorphin after exposure to ethanol. Findings from animal studies indicated that ethanol-preferring (C57BL/6) mice (analogous to the HR human subjects) had higher levels of hypothalamic beta-endorphin activity than did ethanol-avoiding (DBA/2) mice (analogous to the LR human subjects) under basal conditions. However, the C57BL/6 mice had a more pronounced release of hypothalamic beta-endorphin than did DBA/2 mice after exposure to ethanol. Thus, although hypothalamic beta-endorphin system activity in human and animal models of alcoholism differs under basal conditions, there is enhanced hypothalamic beta-endorphin system activity after exposure to ethanol in both models. We have also performed studies comparing the density and distribution of opioid receptors in brains of ethanol-preferring animals, such as C57BL/6 mice and ALKO-alcohol (AA) rats, and ethanol-avoiding animals, such as DBA/2 mice and ALKO-non-alcohol (ANA) rats. Interestingly, it was observed that in distinct brain regions known to be important for mediating the process of reinforcement, the C57BL/6 mice had a higher density of delta-opioid receptors than the DBA/2 mice, while the AA rats had a higher density of mu-opioid receptors than the ANA rats. Thus, in the ethanol-preferring animals, the increased release of beta-endorphin following exposure to ethanol was associated with a higher density of delta- or mu-opioid receptors in brain regions important for reinforcement, such as the nucleus accumbens and the ventral tegmental area, and may interact with the dopaminergic system and promote ethanol's reinforcing properties, leading to excessive drinking and alcoholism.

Continuous infusion of clozapine increases Mu and delta opioid receptors and proenkephalin mRNA in mouse brain

The biochemical mechanisms involved in the actions of the atypical antipsychotic clozapine are still unclear. Because elevated levels of enkephalin in certain areas of the central nervous system may be necessary for antipsychotic activity, we have examined the effect of clozapine on certain receptors and mRNA transcripts involved in the opioid peptidergic system. Clozapine was infused continuously into mice for 21 days and the density of mu and delta opioid receptors was measured in the brains by quantitative receptor autoradiography, and the level of proenkephalin mRNA and dopamine D1 and D2 receptor mRNA were measured by in situ hybridization histochemistry. The results showed that continuous infusion of clozapine increased the density of D1 but not D2 receptors. However, it failed to alter the levels of either D1 or D2 dopamine receptor mRNA. By contrast, clozapine increased the density of mu and delta opioid receptors and increased the levels of proenkephalin mRNA. These results indicate that continuous treatment with clozapine increases opioid peptidergic activity in mouse brain and suggest that alteration of peptidergic activity as well as alteration of dopaminergic activity may be involved in its antipsychotic action.

Human δ opioid receptor

A human delta opioid receptor cDNA clone (pREP10/hDOR) was transfected into Chinese hamster ovary (CHO) cells. The stable cell line expressed a high density of delta opioid receptors (137,000 +/- 21,600 receptors/cell). DPDPE inhibited 90% of the forskolin-stimulated cAMP accumulation in these cells with high potency (EC50 = 1.3 nM). This effect of DPDPE was antagonized by naltrindole. The pseudo-pA2 value (Ke) of 155 pM for naltrindole is consistent with that measured for delta receptor antagonism in the mouse vas deferens. This is the first detailed characterization of DPDPE activity on forskolin-stimulated cAMP accumulation mediated through a human delta opioid receptor. The data support the use of the recombinant cell line for functional studies of opioid drugs.

Kappa and delta opioid receptor densities following chronic repeated cocaine administration

Although cocaine has a vinety of pharmacological actions, it is thought that the reinforcing properties of cocaine are mediated by its ability to inhibit the reuptake of dopamine. Cocaine binds to the dopamine transporter and prevents the reuptake of dopamine into presynaptic terminals, thereby increasing the concentration of dopamine in the synapse. In animals models of drug reinforcement, dopamine receptor antagonists block cocaine-induced reward (1) supporting the role of dopamine in cocaine reinforcement. In a similar manner, opiate receptor antagonists can also attenuate the rewarding effects of cocaine (2,3,4) suggesting an interaction between central opioid and dopaminergic systems in cocaine reinforcement

Characterization and localization of delta opioid binding sites in the bovine pineal gland

Opioid binding sites in the bovine pineal were characterized using the highly selective delta opioid agonist 3H-[D-Pen2,pCl-Phe4,D-Pen5] enkephalin (DPDP(C1)E). Pineal membranes possess a single class of high affinity binding sites for this delta ligand (Kd = 0.26 nM; Bmax = 250 fmol/mg protein). The specific opioid antagonist naloxone dose dependently inhibited 3H-DPDP(C1)E binding, confirming that this ligand is indeed binding to opioid receptors. The delta selective ligands deltorphin and [D-Pen2,5]enkephalin (DPDPE) were much more potent than the mu selective compounds dermorphin and [D-Ala2,MePhe4,Gly5- ol]enkephalin (DAMGO) in inhibiting 3H-DPDP(C1)E binding. These results demonstrate that in bovine pineal membranes, DPDP(C1)E binds to delta opioid sites. Autoradiographic studies showed a uniform distribution of 3H-DPDP(C1)E binding over the bovine pineal in the sections we analyzed. This distribution suggests that delta opioid binding sites are associated with pinealocytes which account for the majority of cell types in the pineal. However, it is not possible to rule out that these receptors may also be associated with other cell types which are present in the bovine pineal. The density and widespread distribution of delta opioid receptors supports the hypothesis that endogenous opioid peptides directly modulate pineal function.

Opioid receptor changes in weaver mouse striatum

The dopaminergic projection from the substantia nigra to the patch and matrix compartments of the caudate-putamen undergoes a spontaneous, early postnatal degeneration in mice which carry the weaver gene. The projection to nucleus accumbens is relatively spared. Dopaminergic afferents have been shown to be important modulators of striatal opioid receptor expression. In the present study, opioid receptor localization in the caudate-putamen and nucleus accumbens of control and weaver mice was examined by quantitative autoradiography. Mu, delta and kappa opioid receptors were differentially distributed in nucleus accumbens and in patch and matrix compartments of the caudate-putamen. In animals which were homozygous for the weaver gene, the density of mu opioid receptors in both patch and matrix compartments was unchanged with respect to control mice. In contrast, the density of delta and kappa opioid receptors was significantly decreased in weaver caudate-putamen and nucleus accumbens. The significance of these results with respect to opioid receptor expression and Parkinson's disease is discussed.

Comparison of the butyrate effects on neurotransmitter receptors in neurohybrids NG 108-15 and NCB-20 cells

Our previous study demonstrated that long term treatment of NCB-20 cells with sodium butyrate resulted in a marked increase in the density of delta-opioid receptors with a much lesser effect on muscarinic cholinergic and no effect on alpha 2-adrenergic receptors. In the present study we investigated the effect of sodium butyrate on these three types of receptors in NG108-15 cells whose neuroblastoma parent is the same as that of NCB-20 cells. Long term treatment of NG108-15 cells with sodium butyrate (0.5 nM) induced a 2-fold increase in the density of the specific binding of 3H-clonidine. A comparable increase in the number of binding sites was detected when 3H-yohimbine was used as the receptor ligand. The butyrate-induced increase in the alpha 2-adrenergic receptor binding could be totally abolished by treatment with a protein synthesis inhibitor, cycloheximide, suggesting that synthesis of receptor protein is involved. The same butyrate treatment had no significant effect on opioid and muscarinic cholinergic receptor bindings. Thus, butyrate effects on the expression of these types of receptors in NG108-15 and NCB-20 cells are dramatically different. These data suggest that induction by butyrate of neurotransmitter receptors requires concerted action of genetic factors of both parents of the neurohybrids.