Opioid Receptors In Rat Brain Research Articles

Opiorphin highly improves the specific binding and affinity of MERF and MEGY to rat brain opioid receptors

Endogenously occurring opioid peptides are rapidly metabolized by different ectopeptidases. Human opiorphin is a recently discovered natural inhibitor of the enkephalin-inactivating neutral endopeptidase (NEP) and aminopeptidase-N (AP-N) (Wisner et al., 2006). To date, in vitro receptor binding experiments must be performed either in the presence of a mixture of peptidase inhibitors and/or at low temperatures, to block peptidase activity. Here we demonstrate that, compared to classic inhibitor cocktails, opiorphin dramatically increases the binding of [3H]MERF and [3H]MEGY ligands to rat brain membrane preparations. We found that at 0°C the increase in specific binding is as high as 40–60% and at 24°C this rise was even higher. In contrast, the binding of the control [3H]endomorphin-1, which is relatively slowly degraded in rat brain membrane preparations, was not enhanced by opiorphin compared to other inhibitors. In addition, in homologous binding displacement experiments, the IC50 affinity values measured at 24°C were also significantly improved using opiorphin compared to the inhibitor cocktail. In heterologous binding experiments the differences were less obvious, but still pronounced using [3H]MERF and MEGY compared to dynorphin1–11, or naloxone and DAGO competitor ligands.

μ Opioid Receptor Coupling to Gi/oProteins Increases during Postnatal Development in Rat Brain

Mu opioid receptors are densely expressed within rat striatum and are concentrated in anatomically discrete patches called striosomes. The density of striosomal mu receptors remains relatively constant during postnatal development, but little is known about their functional maturation. We examined the extent of G protein coupling by mu opioid receptors in rat brain during development, focusing on striosomes within the striatum because of receptor density. The mu receptors were quantified using [(3)H][d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) autoradiography. Adjacent sections were analyzed for DAMGO-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding to assess mu receptor activation of G(i/o) proteins. Striosomal mu receptor expression increased only slightly between postnatal day 5 and adult. In contrast, mu receptor-stimulated [(35)S]GTPgammaS binding increased from 0.13 to 2.6 fmol/mg tissue over the same period, a 20-fold difference. The ratio of specific DAMGO-stimulated [(35)S]GTPgammaS binding to [(3)H]DAMGO binding, representing the relative number of G proteins activated per receptor, increased 19-fold between postnatal day 5 and adult. Similar patterns were observed throughout the striatum and other brain regions such as the nucleus accumbens, although the extent of change varied from region to region. These data indicate that mu opioid receptors exhibit enhanced function in the adult rat brain compared with the neonate. These data also suggest that this increase in G protein coupling is developmentally regulated and that in the developing rat brain the density of mu opioid receptor expression may not necessarily correlate with receptor activation of G proteins.

CHRONIC BLOCKADE OF HIPPOCAMPAL KAPPA RECEPTORS INCREASES ARTERIAL PRESSURE IN CONSCIOUS SPONTANEOUSLY HYPERTENSIVE RATS BUT NOT IN NORMOTENSIVE WISTAR KYOTO RATS

Previous studies in this laboratory have shown that dynorphins acting on hippocampal kappa opioid receptors in rat brain exert a restraining influence on arterial blood pressure and that a relative deficiency of their production in the spontaneously hypertensive rat (SHR) may be involved in SHR hypertension. Kappa receptor blockade should therefore exacerbate hypertension in SHR. We explored the latter possibility by chronic unilateral infusion of nor-binaltorphimine dihydrochloride (nor-BNI), a selective kappa receptor antagonist, into the right dorsal hippocampus of conscious SHR and normotensive Wistar-Kyoto (WKY) controls over a 14 day period. Additional controls consisted of similar hippocampal infusions of equivalent volumes of drug vehicle in both rat strains. Mean arterial pressure (MAP) and heart rates (HR) were determined in each animal once daily by the tail cuff method during this period. Nor-BNI induced a sustained increase in the basal high level of MAP in SHR from 132 ± 8 to 150 ± 10 mmHg throughout the 14 days and an increased HR from 427 ± 17 to 477 ± 30 on day 3 to 5 following the drug. By contrast, nor-BNI had no significant effects on either MAP or HR in WKY rats and control infusions of drug vehicle were similarly without effect in both strains. The results support our previous suggestion that the kappa opioid system of the hippocampus ordinarily restrains arterial blood pressure in SHR since prolonged hippocampal kappa receptor blockade results in augmented hypertension in this strain.

Gene expression and activity of specific opioid-degrading enzymes in different brain regions of the AA and ANA lines of rats

There is increasing evidence that alcoholism runs in families suggesting that genetic factors may play a role. In support of this hypothesis, the alcohol-preferring (AA) and the alcohol-avoiding (ANA) rat lines have been developed through selective outbreeding. Numerous studies indicate that the endogenous opioid system may be involved in controlling ethanol consumption. Changes in opioid peptides and opioid receptors have been described after ethanol intake. But, the influence of ethanol on peptidolytic degradation of opioid peptides has been largely ignored, although the peptidase-mediated metabolism of neuropeptides is known as an important regulatory site of peptidergic transmission. Neutral endopeptidase 24.11 (NEP) and angiotensin-converting enzyme (ACE) degrade neuropeptides, including enkephalin and are expressed in the brain. Furthermore, a good correspondence between the regional distribution of NEP and opioid receptors in rat brain has already been reported pointing to a possible role of NEP in regulating opioid peptides. For both enzymes studied, the gene expression pattern was found to be in good agreement with the corresponding enzyme activities in the brain regions investigated, showing the highest levels for both specific mRNAs and enzyme activities in the striatum. Differences in both measured parameters were detected in distinct brain regions of AA and ANA rats. Furthermore, in some brain regions discrepancies between ACE and NEP mRNA levels and the corresponding enzyme activities were observed. For example, in olfactory bulb and striatum such discrepancies were found for both enzymes studied. In tegmentum/colliculi a higher NEP gene expression in AA rats was associated with a higher NEP enzyme activity compared to the amounts found in ANA rats.

Discrimination of a novel type of rat brain delta opioid receptors by enkephalin analog containing structurally constrained cyclopropylphenylalanine (inverted delta Phe).

Four different stereoisomers of cyclopropylphenylalanine (inverted delta Phe) were incorporated into [D-Ala2,Leu5]enkephalin at the position 4. These conformationally restricted enkephalin analogs were evaluated for their binding characteristics to mu and delta opioid receptors in rat brain. A striking finding is that the E-(2R,3S)-isomer binds to a novel class of delta receptors and discriminates this receptor from the ordinary delta receptor. This new type of delta receptor suspected to be a receptor which suppresses the thermal analgesia mediated through mu receptor. The Z-(2R,3R)-isomer was very potent with several times more enhanced affinity to delta receptors than to mu receptors, but could not differentiate the delta receptors. The Z-(2S,3S)-isomer was weak, and E-(2S,3R)-isomer was almost inactive.

Pharmacokinetics of pentazocine and its occupancy of opioid receptors in rat brain.

In order to assess quantitatively the pharmacodynamic process of pentazocine (PTZ), time courses of its plasma concentration and of the occupation of specific opioid receptors in the brain were investigated after intravenous (i.v.) administration of PTZ to rats. The plasma concentration of PTZ was determined by HPLC and the pharmacokinetic parameters were analyzed using nonlinear least-squares analysis. Measurement of ex vivo receptor occupation was made by comparing the specific [3H]naloxone (opioid receptor antagonist) binding in vitro to the crude P2-synaptosomal fractions between vehicle-treated rats (control) and PTZ-treated rats. Following the i.v. administration of PTZ, the occupancy of specific opioid receptors decreased rapidly until 10 min, depending on the two pharmacological doses (2.5 and 10 mg/kg). The results strongly suggest the fast binding kinetics of PTZ in terms of its association with and dissociation from specific opioid receptor sites in the brain in addition to its fast rate of disappearance from the brain compartment. Furthermore, we demonstrated that the time profile of receptor occupancy correlated well (r = 0.8650) with that of the unbound concentration in plasma until 120 min after the i.v. administration of PTZ to rats.

Specific affinity labeling of μ opioid receptors in rat brain by S-activated sulfhydryldihydromorphine analogs

S-Activated sulfhydryldihydromorphine analogs 1 and 2 were synthesized. In the rat brain receptor binding assays, both 1 and 2 exhibited considerably high affinities for μ opioid receptors (IC 50; 1= 31.1 nM, 2= 10.7 nM). However, when each analog was incubated with membranes for the purpose of getting disulfide bridgings, 1 ( EC 50 = 58 nM) was found to affinity-label the μ receptors about 30 times more effectively than 2 ( EC 50 = 1700 nM).

Long-term sensitization to the behavioral effects of naltrexone is associated with regionally specific changes in the number of μ and δ opioid receptors in rat brain

Enhanced sensitivity to some of the behavioral effects of the opioid antagonist naltrexone (NTX) develops following once-weekly injections of cumulative doses of the drug. Rats treated with this regimen of NTX injections show enhanced sensitivity to the operant response rate decreasing effects of NTX and NTX-induced salivation. The enhanced sensitivity is long-lasting and appears to be produced through conditioning processes. We have conducted saturation binding assays to assess possible changes in the number and affinity of mu and delta opioid receptors in cortical, midbrain and hindbrain membrane preparations from Long-Evans rats treated once weekly for 8 weeks with cumulative doses of the drug (1, 3, 10, 30 and 100 mg/kg). 3H-DAMGO (0.5-21 nM) and 3H-pCl-DPDPE (0.04-4 nM) were used to characterize mu and delta receptors, respectively. NTX treatment had no effect on 3H-DAMGO binding in cortex, but decreased binding in midbrain and increased binding in hindbrain relative to saline-treated controls. Saturation analyses revealed that these differences reflected changes in the number, but not the affinity of mu receptors. NTX treatment also increased the amount of 3H-pCl-DPDPE bound to delta receptors in midbrain and hindbrain, but not in cortex. Again, these changes were due to changes in the number of receptors. Thus, chronic NTX differentially affects the number of mu and delta opioid receptors in various brain regions.

Chronic ICV infusion of morphine and NPFF down-regulate mu opioid receptors in rat brain: A quantitative autoradiographic study

Regulation of the opioid μ receptor by pharmacological manipulation has been extensively investigated using various radioreceptor binding techniques. However, unlike most receptor systems, where chronic agonist administration induces a characteristic down-regulation in receptor density, prolonged morphine treatment has shown conflicting results. Both up- and down-regulation of μ receptors by morphine have been reported (for review see (1)). Our lab recently demonstrated that morphine modulating peptide, NPFF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH 2), down-regulates opioid μ receptors (2). The purpose of this study was to compare the effects of chronic ICV infusion of morphine and NPFF on μ opioid binding sites using the in vitro techniques of whole brain homogenate receptor binding and autoradiography.

Transient down-regulation of neonatal rat brain μ-opioid receptors upon in utero exposure to buprenorphine

Gestational actions of the mixed agonist-antagonist buprenorphine on μ-and κ 1-opioid binding in neonatal and maternal rat brain were investigated. Upon exposure of pregnant rats to 0.5 mg/kg buprenorphine for 7 days prior to birth, postnatal day-one (P1) and P7 offspring brain μ-binding parameters ( K d and B max) were assessed with 3H-labeled [ d-Ala 2,Mephe 2,Gly-ol 5] enkephalin (DAMGE). DAMGE binding was attenuated by 64% in P1 membranes, whereas P7 preparations showed no changes. The same buprenorphine regimen resulted in diminished DAMGE B max values in mothers' brains, 2 but not 7 days after cessation of drug administration. Receptor density changes were not accompanied by alteration of μ-binding affinities. Although the postnatal developmental profile of κ 1 opioid receptors in rat brain measured with [ 3H]U69593 revealed the presence of an ample number of sites for detection, their binding parameters in P1, P7 pups and mothers were unaffected by 0.5 mg/kg buprenorphine. In summary, buprenorphine administration to pregnant rats transiently down-regulates μ opioid receptors in neonatal and maternal brain.

Selective labeling of κ2 opioid receptors in rat brain by [ 125I]IOXY: Interaction of opioid peptides and other drugs with multiple κ2a binding sites

Recent studies from our laboratory resolved two subtypes of the κ 2 binding site, termed κ 2a and κ 2b, using guinea pig, rat, and human brain membranes depleted of μ and δ receptors by pretreatment with the site-directed acylating agents BIT (μ-selective) and FIT (δ-selective). 6β-Iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5α-epoxymorphinan (IOXY), an opioid antagonist that has high affinity for κ 2 sites, was radioiodinated to maximum specific activity (2200 Ci/mmol) and purified by high pressure liquid chromotography and used to characterize multiple κ 2 binding sites. The results indicated that [ 125I]IOXY, like [ 3H]bremazocine, selectively labels κ 2 binding sites in rat brain membranes pretreated with BIT and FIT. Using 100 n M [ d-Ala 2-MePhe 4,Gly-ol 5]enkephalin to block [ 125I]IOXY binding to the κ 2b site, two subtypes of the κ 2a binding site were resolved, both in the absence and presence of 50 μ M 5′-guanylyimidodiphosphate. Viewed collectively, these results provide further evidence for heterogeneity of the κ opioid receptor, which may provide new targets for drug design, synthesis, and therapeutics.

Characterization of [3H]naltrindole binding to delta opioid receptors in rat brain.

[3H]Naltrindole binding characteristics were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C measured an equilibrium dissociation constant (Kd) value of 37.0 +/- 3.0 pM and a receptor density (Bmax) value of 63.4 +/- 2.0 fmol/mg protein. Association binding studies showed that equilibrium was reached within 90 min at a radioligand concentration of 30 pM. Naltrindole, as well as the ligands selective for delta (delta) opioid receptors, such as pCI-DPDPE and Deltorphin II inhibited [3H]naltrindole binding with nanomolar IC50 values. Ligands selective for mu (mu) and kappa (kappa) opioid receptors were only effective in inhibiting [3H]naltrindole binding at micromolar concentrations. From these data, we conclude that [3H]naltrindole is a high affinity, selective radioligand for delta opioid receptors.

Chronic effect of [D-Pen 2,D-Pen 5]enkephalin on rat brain opioid receptors

In previous studies, we have demonstrated that chronic etorphine or [D-Ala 2,D-Leu 5]enkephalin (DADLE) treatment of rats results in the reduction of μ- and δ-opioid receptor binding activities as tolerance develops. As both etorphine and DADLE are relatively non-specific opioid ligands, interacting with both μ- and δ-rcceptors these studies could not determine whether down-regulation of a specific receptor type occurs. Therefore, in the present studies, animals were rendered tolerant to the δ-opioid receptor-selective agonist [D-Pen 2,D-Pen 5]enkephalin (DPDPE), and receptor binding activities were measured. Treating Sprague-Dawley rats with increasing doses of DPDPE (80-160-240-320 μg/kg) i.c.v. for 1 to 4 days resulted in a time-dependent increase in the ad 50 of DPDPE to elicit an antinociceptivc response. When δ-receptor binding was determined by using [ 3H]DPDPE, a 40–50% decrease in binding in the midbrain and cortex, and 25–35% decrease in binding in the striatum were observed after 3 or 4 days of DPDPE treatment. Scatchard analysis of the [ 3H]DPDPE saturation binding data revealed a decrease in B max values and no significant change in k d values. To our surprise, when μ-receptor hinding was determined by using ( 3H]Tyr-D-Ala-Gly-MePhe-Gly-ol (DAMGO), a 10–15% decrease in binding was also observed in the midbrain and cortex after 4 days of DPDPE treatment. Our conclusion is that chronic DPDPE treatment preferentially reduces δ-opioid receptor binding activity. Its minor effect on the μ-opioid receptor maybe due to an interaction between δ ex and μ ex binding sites.

Evidence for two subtypes of δ opioid receptors in rat brain

Evidence for two subtypes of δ opioid receptors in rat brain

A monoclonal anti-idiotypic antibody to μ and δ opioid receptors

A mouse monoclonal, anti-idiotypic, anti-opioid receptor antibody (Ab2-AOR) has been generated from monoclonal anti-morphine antibodies (Ab1). Hybridoma culture supernatants were screened by a solid phase radioimmunoassay (RIA), based on their competition with radiolabelled morphine for Ab1. One of the Ab2s that gave a positive RIA also competed at rat brain opioid receptors with tritiated opioid ligands dihydromorphine (DHM), naloxone, etorphine, Tyr- d-Ala-Gly-Phe- d-Leu (DADLE), Tyr- d-Ala-Gly-NMe-Phe-Gly-ol (DAMGE) and Tyr- d-Pen-Gly-Phe- d-Pen (DPDPE). SDS-PAGE revealed Ab2-AOR to be highly purified after successive affinity and protein A-Sepharose chromatography. Ab2-AOR at concentrations of 10–100 nM competed with both μ- and δ-selective specific ligands for brain opioid receptors. Less than 13 μg/ml Ab2-AOR completely inhibited specific opioid radioligand binding to both soluble and membrane-bound opioid receptors. To demonstrate its anti-δ receptor activity further, a double-antibody ELISA procedure was developed that is based on the binding of Ab2-AOR to immobilized NG 108-15 cells (which contain only δ opioid receptors). Dose-dependent, opioid peptide- and opiate alkaloid-competitive binding of Ab2-AOR-containing ascites fluid to NG 108-15 cells was observed. A μ opioid agonist effect was demonstrated for Ab2-AOR, in that it decreased by 70% [ 3H]thymidine incorporation into DNA of fetal brain cell aggregates. This agonist-like action of Ab2-AOR was blocked by naltrexone. The antibody bound specifically to brain tissue sections and the presence of diprenorphine blocked this interaction. Hence, an Ab2 with μ and δ specificity has been characterized.

Opioid Activities of Morphiceptin Analogs Derived from Human β-Casein

Abstract Four tetrapeptide amides with the N-terminal Tyr–Pro sequence were synthesized as possible opioid agonists that can be produced by degradation of human β-casein. When these peptides were tested for their ability to bind to the μ and δ opioid receptors in rat brain, only H–Tyr–Pro–Phe–Val-NH2 was active, showing the 60% increased affinity for the μ receptors as compared with morphiceptin (H–Tyr–Pro–Phe–Pro–NH2) derived from bovine β-casein. It was highly μ-selective, as well as morphiceptin, with the μ/δ-selectivity ratio of 285. Other three analogs, H–Tyr–Pro–Ser–Phe–NH2, H–Tyr–Pro–Val–Arg–NH2 and H–Tyr–Pro–Val–Pro–NH2, were almost completely inactive. These results suggested that, for the morphiceptin-like tetrapeptide amides, the presence of Phe at position 3 is essential to elicit an activity to bind to the μ opioid receptors. Conformational considerations by measuring the CD spectra indicated that the sequence of tetrapeptide amide Tyr–Pro–Xxx–Pro(or Val)–NH2 is an important structural requirement to interact with the μ opioid receptors.

Modulation of mu, delta and kappa opioid receptors in rat brain by metal ions and histidine

The effect of zinc (Zn 2+) and several other trace elements was studied on the binding of the opioid receptor agonists [ 3H] DAGO ([Tyr- d-Ala-Gly-Methyl-Phe-Olyol]-enkephalin) a, [ 3H] DSTLE ([Tyr- d-Ser-Gly-Phe-Leu-Thr]-enkephalin) and [ 3H] EKC (ethylketocyclazocine), which are specific for the mu, delta and kappa opioid receptors, respectively, in the cerebral cortex of the rat. Physiological concentrations of zinc were inhibitory to mu receptor binding, whereas the delta and kappa receptors were relatively insensitive to this inhibition. Scatchard analysis, using these opioid agonists, revealed curvilinear plots; concentrations of zinc equal to or less than the IC 50 (the concentration of cation which caused 50% inhibition of the binding of opioid ligand to its receptor), increased the K D (the dissociation constant) of all three subtypes of receptor, with no effect on the B max (the maximum number of binding sites) and abolished the high affinity sites of the delta and kappa receptors. Copper, cadmium and mercury also inhibited the binding of these ligands to their receptors. Histidine was most effective in preventing the inhibitory effects of zinc and copper, whereas it was less effective on cadmium and without any effect on the inhibition caused by mercury. Magnesium and manganese were stimulatory to opioid receptor binding, whereas cobalt and nickel had dual (stimulatory and inhibitory) effects. Non-inhibitory concentrations of zinc significantly decreased the stimulatory effects of magnesium and manganese on the mu and delta receptors, suggesting that part of the effect of zinc was through prevention of the actions of stimulatory cations. The reducing reagent dithiothreitol partially protected against inhibition by zinc and the oxidizing reagent dithiobisnitrobenzoic acid potentiated the inhibitory effects of zinc on the binding of [ 3H] DSTLE and [ 3H] DAGO. These results suggest that physiological concentrations of free zinc can inhibit mu but not delta and kappa receptors. The lower susceptibility of delta and kappa receptors to inhibition by zinc may be due to the presence of fewer sulfhydryl-groups in these receptors, or due to their inaccessibility to binding by zinc. Relative physiological concentrations of free zinc and histidine may govern the extent of binding of the mu ligands to their receptors.

Affinity States of Rat Brain Opioid Receptors in Different Tissue Preparations

The binding of [3H]Tyr-D-Ala-Gly-(N-Me)Phe-Gly-ol ([3H]DAGO) and [3H]Tyr-D-Thr-Gly-Phe-Leu-Thr ([3H]DTLET), selective agonists for mu- and delta-opioid binding sites, respectively, has been investigated using different rat brain tissue preparations and buffer systems. The results were compared with the binding of the ligands to crude membrane fractions in Tris-HCl, the most commonly used preparation for binding studies. In both rat brain membranes and intact cells, Krebs-HEPES induced a decrease in the affinities of [3H]DAGO and [3H]DTLET, but little modification was observed when 20-microns tissue slices were used, whatever the brain area studied. The dissociation rate of [3H]DTLET was clearly dependent on the tissue preparation used, because the koff value of this ligand in Krebs-HEPES was 2.5-fold higher in membrane fractions than that measured in intact cells. The kinetic dissociation constant of [3H]DTLET in membrane fractions in Krebs-HEPES was 6.5-fold greater than that measured in Tris-HCl. In intact cells, the koff value for [3H]DTLET was lower than that found in membrane fractions in Krebs-HEPES and similar to that observed in membrane preparations in Tris-HCl supplemented with 30 mM NaCl. These data suggest (a) that the koff constant of [3H]DTLET was regulated by the ionic environment of the delta-opioid receptor, which is clearly dependent on the preservation of cellular structure, and (b) that opioid receptors could exist under different states that are regulated, in part, by the intracellular Na+ concentration.

New 4-(heteroanilido)piperidines, structurally related to the pure opioidagonist fentanyl, with agonist and/or antagonist properties

A research program based on certain heterocyclic modifications (12-50) of the fentanyl (1) molecule has generated a novel class of opioids. In the mouse hot-plate test, these compounds were weaker analgesics than 1. Two types of antagonists were observed in morphine-treated rabbits: those (e.g., 28) that reversed both respiratory depression and analgesia and those (e.g. 32) that selectively reversed respiratory depression. Evaluation of in vitro binding affinities to rat brain opioid receptors was inconclusive for a common locus of action for the agonist as well as the antagonist component. Further pharmacological evaluation of 32, N-(2-pyrazinyl)-N-(1-phenethyl-4-piperidinyl)-2-furamide, in the rat showed it to be a potent analgesic (ED50 = 0.07 mg/kg, tail-flick test) with little cardiovascular and respiratory depression when compared to fentanyl.

Differential down-regulation of delta opioid binding sites during physical dependence on methionine enkephalin in the rat

Post-synaptic receptor modulation is thought to be one important mechanism involved in the adaptation of a neuronal system during chronic exposure to a drug. However, initial studies of opioid receptor regulation following chronic in vivo administration of narcotic agonists, such as morphine, reported no down-regulation in the number of opioid receptors in the brain. Subsequent studies, employing in vitro preparations, have reported evidence of opioid receptor down-regulation under specific conditions. It remains to be determined whether the in vitro phenomena of opioid receptor plasticity is relevant to the intact mammalian central nervous system. The data in this report shows that chronic in vivo administration the opioid peptide methionine enkephalin, results in a significant, regionally specific down-regulation of delta opioid receptors in rat brain: 30% decrease in receptor density in the striatum; no change in hypothalamus.