DPDPE Binding Research Articles

3H]DPDPE binding to δ opioid receptors in the rat mesocorticolimbic and nigrostriatal pathways is transiently increased by acute ethanol administration

Dopaminergic transmission in the mesolimbic and nigrostriatal pathways plays a key role in the reinforcement mechanisms and brain sensitivity to ethanol, respectively. Ethanol reinforcement and high alcohol drinking behaviour have been postulated to be partially mediated by a neurobiological mechanism involving the ethanol-induced activation of the endogenous opioid system. Activation of opioid neural pathways by ethanol may include alterations in the processing, release and/or the receptor binding of opioid peptides. The aim of this work was to investigate the effects of acute ethanol administration on δ opioid receptors in the rat mesocortical, meso-accumbens and nigrostriatal pathways by quantitative receptor autoradiography, using [ 3H] (2- d-penicillamine, 5- d-penicillamine)-enkephalin as radioligand. A significant increase in [ 3H] (2- d-penicillamine, 5- d-penicillamine)-enkephalin binding was observed in the substantia nigra pars reticulata 1 h after ethanol treatment. Two hours after drug exposure, ligand binding was significantly increased in the frontal and prefrontal cortices, the core and shell regions of the nucleus accumbens, and in the anterior-medial and medial-posterior regions of the caudate–putamen. In contrast, ligand binding was significantly decreased in the posterior region of the caudate–putamen 30 min after ethanol administration. The observed effects may reflect ethanol-induced changes in ligand binding affinity and/or in receptor density. Our results suggest that transitory changes in δ opioid receptors with different kinetic patterns may be involved in ethanol reinforcement and brain sensitivity to the drug. Ethanol-induced δ receptor up- and down-regulation mechanisms may participate in modulation of dopaminergic transmission in the mesocorticolimbic and nigrostriatal pathways.

Anti-mu opioid antiserum against the third external loop of the cloned mu-opioid receptor acts as a mu receptor neutral antagonist

The region from the third external loop to the C terminus of MOR-1 appeared to be critical to the selective binding of MOR-1 ligands as DAMGO and morphine to MOR-1. To study the pharmacological properties of the third extracellular loop an antibody was raised in rabbits against the sequence 304–316 which is unique to MOR-1 and includes the third external loop; the anti-MOR-1 antibody was affinity purified against the immunogen sequence and characterized by [ 3H]DAMGO and Western blotting; [ 3H]DPDPE binding assay remained unchanged in the presence of the antibody. Anti-MOR-1 IgG was characterized as a neutral antagonist in Chinese hamster ovary (CHO) cells hyperexpressing constitutively active MOR-1s; in fact, anti-MOR-1 IgG completely reversed the inhibition induced by the MOR-1 agonist endomorphin1, endomorphin2, DAMGO and morphine on forskolin stimulated cyclic AMP (cAMP) accumulation and attenuated both the action of the selective MOR-1 agonist DAMGO to increase [ 35S]GTPγS binding and the action of the MOR-1 inverse agonist beta-chlornaltrexamine (CNA) to decrease [ 35S]GTPγS binding. Radioligand binding assay using membrane suspensions from CHO cells hyperexpressing MOR-1 revealed a significant decreased binding affinity and capacity of all the tested MOR-1 selective ligands after preincubation with anti-MOR-1 IgG. Therefore, the third extracellular loop of MOR-1 appeared to be a key element for the binding of MOR-1 ligands.

Effects of NMDA receptor antagonists on δ1- and δ2-opioid receptor agonists-induced changes in the mouse brain [ [formula omitted]]DPDPE binding

Male Swiss-Webster mice were injected intracerebroventricularly (i.c.v.) with [ d-Pen 2, d-Pen 5]enkephalin (20 μg/mouse) twice a day for 2 days. This procedure resulted in down-regulation of binding sites for [ 3 H ][ d-Pen 2, d-Pen 5]enkephalin as evidenced by a 52% decrease in the B max value. Twice daily injections of (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]-cyclohepten-5,10-imine (MK-801) (0.1 mg/kg, i.p.) or [(−)3- SR,4a- RS,8a- SR-6-(phosphonomethyl)-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid] (LY 235959) (2 mg/kg, i.p.), the noncompetitive and competitive antagonist of the N-methyl- d-aspartate (NMDA) receptor, respectively, for 2 days did not alter the B max or K d value of [ 3 H ][ d-Pen 2, d-Pen 5]enkephalin binding to the mouse brain. Concurrent treatment of MK-801, but not of LY 235959 with [ d-Pen 2, d-Pen 5]enkephalin, reversed the decreases in B max value of [ 3 H ][ d-Pen 2, d-Pen 5]enkephalin. Twice daily injections of [ d-Ala 2,Glu 4] deltorphin II (20 μg/mouse) for 2 days caused an increase in the K d value, but not the B max value of [ 3 H ][ d-Pen 2, d-Pen 5]enkephalin to bind to brain membranes. Concurrent treatment of [ d-Ala 2,Glu 4]deltorphin II with LY 235959 reversed the increase in K d value of [ 3 H ][ d-Pen 2, d-Pen 5]enkephalin binding induced by multiple injections of [ d-Ala 2,Glu 4]deltorphin II, but MK-801 had no effect. The results suggest that multiple injections of δ 1- and δ 2-opioid receptor agonists down-regulate δ 1-opioid receptors of the brain by modifying B max and K d values of [ 3 H ][ d-Pen 2, d-Pen 5]enkephalin binding, respectively. MK-801 and LY 235959 reverse δ 1- and δ 2-opioid receptor agonists-induced down-regulation of brain δ 1-opioid receptor, respectively, apparently by different mechanisms. It is concluded that short term treatment of mice with δ 1-opioid receptor agonist down-regulates brain δ 1-opioid receptors by decreasing B max of the ligand which is partially reversed by concurrent treatment with MK-801 but not by LY 235959. On the other hand, short term treatment of mice with δ 2-opioid receptor agonist down-regulates brain δ 1-opioid receptors by increasing K d of the ligand which is partially reversed by concurrent treatment with LY 235959 but not by MK-801.

Localization of Mu and Delta Opioid Receptors to Anterior Cingulate Afferents and Projection Neurons and Input/Output Model of Mu Regulation

Anterior cingulate cortex (ACC) has one of the highest densities of opioid receptors in the CNS and it has been implicated in acute and chronic pain responses. Little is known, however, about which neurons express opioid receptors in their dendrites and axon terminals. The present studies employed experimental techniques to remove afferent axons or classes of projection neurons from ratACC area 24 followed by coverslip autoradiography to localize changes in binding of [3H]Tyr-d-Ala-Gly-MePhe-Gly-ol (DAMGO) to mu receptors and 2-[3H]d-penicillamine-5-d-penicillamine-en-kephalin (DPDPE) to delta receptors. Removal of all afferents to area 24 with undercut lesions did not alter DPDPE binding, but significantly reduced binding of DAMGO in layers I, III, and V. In contrast, removal of all cortical neurons with the excitotoxin ibotenic acid almost abolished DPDPE binding in all layers. The same lesions reduced DAMGO binding in most layers; however, there was a postlesion bimodal distribution in binding with high levels of binding in layer I and moderate levels in layer VI. These data suggest that delta receptors are expressed by cortical neurons, while mu receptors are expressed by both cortical neurons and afferent axons. To explore the distribution of postsynaptic receptors, immunotoxin lesions were made in area 24 by injection of OX7-saporin into the caudate and/or thalamic nuclei. Almost complete removal of projection neurons to these targets in layers Vb and VIa did not alter DPDPE binding, while the lesions reduced DAMGO binding in all but layer II. Removal of layer Vb corticostriatal projection neurons with caudate OX7-saporin injections reduced binding only in this layer. It is proposed that opioidergic circuits in area 24 are organized according to an input/output model for mu opioid regulation. In this model mu receptors regulate axon terminal activity from the thalamus in layer Ia and the locus coeruleus in layers Ic and II, whereas cortical outputs to the thalamus are modulated via postsynaptic receptors expressed in all layers by thalamocortical projection neurons with somata in layer VI. These opioidergic circuits in ACC are of particular importance because they may regulate responses to chronic nociceptive activity and associated pain perceptions.

TIPP[ψ], a highly selective δ ligand

TIPP[ψ] is a new δ-selective opioid peptide antagonist. In the current study, we have explored its selectivity against the μ and κ receptor subtypes. Against [ 3H]DPDPE binding, TIPP[ψ] is quite potent, with a K i value of < 1 nM, confirming its potent activity at δ receptors. In contrast, its K i values against μ 1, μ 2, κ 1, κ 2 and κ 3 binding sites are all >5 μM. DPDPE also is δ-selective. It labels δ sites >25-fold more potently than μ 1 receptors and is even more selective against the other subtypes. However, this selectivity does not compare to the δ μ 1 selectivity of TIPP[ψ] which exceeds 15,000. This far higher selectivity, coupled with its antagonist properties, gives TIPP[ψ] a number of advantages over previously reported δ-selective compounds. We have utilized these advantages to develop an improved μ 1 binding assay using TIPP[ψ].

Synthesis of enkephalin-based affinity labels for delta opioid receptors

Affinity labels (irreversible ligands) are useful pharmacological tools to study receptor structure and function. As part of a program to prepare affinity labels based on opioid peptides, we developed a general synthetic strategy to introduce different electrophilic labeling moieties (i.e. isothiocyanate and bromoacetamide) via a p-amino functionality on a phenylalanine residue. The affinity labelling groups were incorporated into the Phe 4 position of the delta opioid receptor antagonists ICI-174,864 (1) and N,N-dibenzyl leucine enkephalin (2). Using an orthogonal Boc/Fmoc protection strategy, the common peptide precursors were assembled in solution, the affinity labels introduced and the peptides deprotected. In cloned δ receptors expressed in CHO cells (3), only the isothiocyanate derivative of N,N-dibenzyl leucine enkephalin exhibited wash-resistant inhibition of [ 3H]DPDPE binding.

Sylvian fissure length and angle asymmetries in normal MZ twins and in MZ twins discordant for schizophrenia

Anterior cingulate cortex (ACC) has one of the highest densities of opioid receptors in the CNS and it has been implicated in acute and chronic pain responses. Little is known, however, about which neurons express opioid receptors in their dendrites and axon terminals. The present studies employed experimental techniques to remove afferent axons or classes of projection neurons from ratACC area 24 followed by coverslip autoradiography to localize changes in binding of [3H]Tyr-d-Ala-Gly-MePhe-Gly-ol (DAMGO) to mu receptors and 2-[3H]d-penicillamine-5-d-penicillamine-en-kephalin (DPDPE) to delta receptors. Removal of all afferents to area 24 with undercut lesions did not alter DPDPE binding, but significantly reduced binding of DAMGO in layers I, III, and V. In contrast, removal of all cortical neurons with the excitotoxin ibotenic acid almost abolished DPDPE binding in all layers. The same lesions reduced DAMGO binding in most layers; however, there was a postlesion bimodal distribution in binding with high levels of binding in layer I and moderate levels in layer VI. These data suggest that delta receptors are expressed by cortical neurons, while mu receptors are expressed by both cortical neurons and afferent axons. To explore the distribution of postsynaptic receptors, immunotoxin lesions were made in area 24 by injection of OX7-saporin into the caudate and/or thalamic nuclei. Almost complete removal of projection neurons to these targets in layers Vb and VIa did not alter DPDPE binding, while the lesions reduced DAMGO binding in all but layer II. Removal of layer Vb corticostriatal projection neurons with caudate OX7-saporin injections reduced binding only in this layer. It is proposed that opioidergic circuits in area 24 are organized according to an input/output model for mu opioid regulation. In this model mu receptors regulate axon terminal activity from the thalamus in layer Ia and the locus coeruleus in layers Ic and II, whereas cortical outputs to the thalamus are modulated via postsynaptic receptors expressed in all layers by thalamocortical projection neurons with somata in layer VI. These opioidergic circuits in ACC are of particular importance because they may regulate responses to chronic nociceptive activity and associated pain perceptions.

Autoradiographic evidence for decrease in binding of μ- and δ-opioid receptors after subchronic [D-Ala 2,D-Leu 5]enkephalin treatment in rats

Tolerance to the antinociceptive effect of [D-Ala 2,D-Leu 5]enkephalin (DADLE) develops in Sprague-Dawley rats given the peptide chronically. δ-Opioid receptor binding is significantly reduced in P 2 membranes from various brain areas after 1–3 days' treatment. μ-Opioid receptor binding, however, is reduced only in striatum, and only after 5 days. To study this finding further, receptor autoradiography was used to quantity μ- and δ-opioid binding sites in rat brain sections after subchronic DADLE treatment. Autoradiograms were made following equilibrium binding of the highly selective opioid radioligands, [ 3H]Tyr-D-Ala-Gly-MePhe-Gly-ol ([ 3H]DAMGO) and [ 3H][D-Pen 2,5]enkephalin ([ 3H]DPDPE) to brain sections. Computerized grain counting was applied to discrete regions of the autoradiograms corresponding to caudate and interpeduncular nuclei. We found that [ 3H]DAMGO binding decreased in caudate after 3 days of DADLE treatment and [ 3H]DPDPE binding decreased in the interpeduncular nucleus, rostal portion, after 1-day DADLE treatment. These autoradiographic changes are consistent with our earlier results for biochemical binding, although we now detected an earlier change in μ-opioid receptor binding, by using autoradiography.

Differential antagonism by naltrindole-5′-isothiocyanate on [ 3H]DSLET and [ 3H]DPDPE binding to striatal slices of mice

Naltrindole-5′-isothiocyanate (5′-NTII), a nonequilibrium δ opioid receptor antagoniist, has been shown to antagonize differentially the antinociception induced by DSLET without affecting that induced by DPDPE. In the present study, we investigated whether or not 5′-NTII can differentially affect DSLET and DPDPE binding to receptor sites in striatal slices of mouse brain. We found that 5′-NTII only changed the binding characteristics of [ 3H]DSLET andnot that of [ 3H]DPDPE. Saturation binding studies revealed that 5′-NTII treatment in vitro inhibited [ 3H]DSLET binding by decreasing the affinity but not the number of binding sites. This finding was supported by saturation studies with [ 3H]DSLET in striatal slices from mice that were pretreated with 5′-NTII (10 nmol, i.c.v. or 10 mg/kg, s.c.). Thus, the results suggest that 5′-NTII can antagonize differentially the binding to striatal slices of mice of [ 3H]DSLET but not that of [ 3H]DPDPE. The binding parameters also suggest that 5′-NTII may not antagonize [ 3H]DSLET binding by alkylating at the receptor recognition site because the number of binding sites did not decrease. 5′-NTII may bind to some other part of the membrane to indirectly desensitize the receptor to a low affinity form. Lastly, the differential alteration of binding sites between [ 3H]DSLET and [ 3H]DPDPE by 5′-NTII strongly support the postulated existence of δ opioid receptor subtypes.

Dopamine depletion produces augmented behavioral responses to a mu-, but not a delta-opioid receptor agonist in the nucleus accumbens: lack of a role for receptor upregulation.

Microinjection of either mu- or delta-opioid agonists into the nucleus accumbens produces an increased locomotor activity, and when the dopaminergic innervation of the nucleus accumbens is bilaterally lesioned, the locomotor response to the microinjection of mixed mu- and delta-opioid agonists is augmented. To determine whether the lesion-induced augmentation to opioids is specific to mu- or delta-opioid receptor activation, dopamine innervation of the nucleus accumbens was lesioned with 6-hydroxydopamine (6-OHDA), and the motor stimulant response to intra-accumbens microinjection of the selective mu-opioid agonist, Tyr-D-Ala-Gly-mePhe-Gly-OH (DAMGO), was compared to that of the delta-opioid agonist, [D-penicillamine2,5]-enkephalin (DPDPE). The lesions caused a 95% depletion of tissue dopamine levels in the nucleus accumbens of the DAMGO-injected rats compared to sham-lesioned rats. Horizontal and vertical photocell counts were significantly increased in response to DAMGO in 6-OHDA-lesioned compared to the sham-lesioned rats. This behavioral augmentation was dose dependent and blocked by naloxone. In rats with similar accumbal dopamine depletions (94%), the locomotor response to DPDPE was not enhanced. The augmentation in the behavioral response to DAMGO was not associated with a change in the Bmax or Kd of [125I]DAMGO binding in nucleus accumbens homogenates from lesioned rats. Likewise, using quantitative receptor autoradiography, no difference between 6-OHDA- and sham-lesioned rats was observed in [125I]DAMGO or [125I]DPDPE binding. Therefore, the augmented behavioral response to opioids in the nucleus accumbens following dopamine depletion relies predominately on mu-opioid receptor stimulation. However, this augmentation is not mediated by an alteration in the number or affinity of these receptors.

Effects of intracerebroventricular β-funaltrexamine on μ and δ opioid receptors in the rat: dichotomy between binding and antinociception

The effects of intracerebroventricutar (i.c.v.) β-funaltrexamine (β-FNA) pretreatment at −24 or −6 h were studied on μ and β opioid receptor binding and on antinociception produced by i.c.v. morphine in rats. μ and δ opioid receptor binding in brain membrane preparations was performed with [ 3H][D-Ala 2,MePhe 4Gly-ol 5]enkephalin (DAGO) and [ 3H][D-Pen 2,D-Pen 5]enkephalin (DPDPE) as radiolabeled ligands, respectively. Effects of i.c.v. β-FNA (24 h) on μ, and δ binding depended on dosage. For [ 3h]dago binding, 3 μg, β-FNA did not affect either the K d or B max, whereas 10 μg increased the K dout changing the B max· pretreatment for 24 h did not alter [ 3h]dpdpe binding at 3 μat 10 μ, the K dincreased with no change in the B maxeatment with 10 μg β-FNA for 6 h gave similar results to the 24-h treatment in μ binding, but did not change δ binding. When μ binding was performed on various brain regions, pretreatment with 10 μg β-FNA for 24 h increased the K d in all regions studied (the penaqueductal gray, thalamus, striatum and cortex). However, this pretreatment decreased the B max only in the periaqueductal gray (by 22%) and cortex (by 14%). Pretreatment of rats with β-FNA (3 or 10 μg at −24 h), which by itself caused some hyperalgesia, greatly antagonized the antinociceptive effect of morphine (10 μg i.c.v.) in the hot-plate test. Our work with β-FNA has revealed an apparent discrepancy between binding and behavioral results. This dichotomy may, in part, be the result of the limited distribution of β-FNA to the periventricular area. It may also be due to the presence of uncoupled receptors and/or may be related to the finding that high affinity [ 3h]dago binding sites in vitro may not represent functional receptors in vivo.

Receptor changes in the spinal cord of sheep associated with exposure to chronic pain

There is evidence that post-injury hypersensitivity is partly due to changes in the central nervous system. Sheep with footrot were used to investigate the effect of chronic pain on some receptors thought to be involved in spinal nociceptive processing systems (alpha 2 adrenoceptor and mu and delta opioid receptors). Saturation binding studies showed a variable distribution of [ 3H] clonidine (alpha 2 adrenoceptor agonist) in the spinal cord of normal sheep. The number of receptors (B max) present in areas thought to be involved in nociceptive processing, laminae I and II and lamina X, increased to 131% and 169% of control sheep values respectively in animals exposed to chronic pain. The affinity of the receptors ( K D ), however, remained unchanged at approximately 2 nM. There was less [ 3H]DAGO (mu opioid agonist) and [ 3H]DPDPE (delta opioid agonist) binding in the sheep spinal cord. Both opioid receptor types being mainly located in the superficial dorsal horn. The [ 3H]DPDPE binding was unchanged in the sheep with footrot, whilst the number (B max), but not the affinity, of the [ 3H]DAGO binding sites increased in laminae I and II in lame animals to 130% of the control sheep values. Hence, in animals in chronic pain, the number of alpha 2 adrenoceptors and mu opioid receptors increased mainly in areas of the sheep spinal cord associated with nociception.

Evidence for δ‐Opioid Binding and GTP‐Regulatory Proteins in 5‐Day‐Old Rat Brain Membranes

The availability of the bispenicillamine enkephalin [3H] [D-Pen2,D-Pen5]enkephalin ([3H]DPDPE) a highly selective ligand for delta-opioid receptors, has made possible a more definitive examination of the ontogeny of this receptor subtype. In this report, the binding characteristics of [3H]DPDPE in 5-day-old neonatal (P-5) and adult rat brain are compared. Analysis of saturation curves as well as homologous displacement data revealed no significant difference in the binding affinity of [3H]DPDPE between P-5 animals and adults. Conversely, the binding capacity increased fivefold during this period. The delta-specificity of the sites was further proven by competition experiments with mu- and delta-selective ligands. Mn2+ (0.5 mM) elevated [3H]DPDPE specific binding by lowering the Kd, whereas 50 microM 5'-guanylylimidodiphosphate inhibited it by decreasing the total number of high-affinity binding sites in both P-5 animals and adults. Pertussis toxin-catalyzed ADP ribosylation experiments revealed the presence of 40-kDa proteins, with a molecular mass corresponding to G protein subunits alpha i/alpha o, as early as 1 h after birth. There was a low, but detectable, basal low-Km GTPase activity in P-5 animals, which increased fivefold during postnatal development. The present report establishes the existence of high-affinity [3H]DPDPE binding as well as GTP-regulatory proteins 5 days after birth. Yet, heterologous competition studies and ionic effects suggest that neonatal binding sites differ from adult receptors. Whether the neonatal sites are newly synthesized, incompletely processed sites or a developmentally programmed isoform remains to be determined.

Dog cerebral cortex contains μ-, δ- and κ-opioid receptors at different densities: apparent lack of evidence for subtypes of the κ-receptor using selective radioligands

The biochemical and pharmacological properties of mu (μ), kappa (κ) and delta (δ) opioid receptors were ascertained in dog cerebral cortex homogenates. The selective peptides, [ 3H] d-Pen 2- d-Pen 5enkephalin ([ 3H]DPDPE) and [ 3H] d-Ala 2-MePhe 4-Glyol 5-enkephalin ([ 3H]Glyol; [ 3H]DAMGO), bound to δ- and μ-opioid receptors with high affinity (dissociation constants, K dvalues= 4.7and1.6nM) but to different densities of binding sites (B max values of 49.2 and 6.6 fmol/mg protein, respectively) in washed homogenates of dog cerebral cortex. In contrast, the non-peptides, [ 3H]U69593 ([ 3H]U69) and [ 3H]etorphine ([ 3H]ET), labeled a high concentration of κ-opioid receptors (respectiveB max values of 67.2 and 76.6 fmol/mg protein) of high affinity (respectiveK ds of 1.4 and 0.47 nM) in the same tissue homogenates. Thus, the relative rank order of opioid receptor densities was:κ > δ ≫ μ. The selective labeling of the κ-receptors with two different drugs ([ 3H]U69 and [ 3H]ET) failed to reveal the possible existence of multiple κ-sites based on the relativeB max values of the two radioglands. This conclusion was further supported by the similarity of pharmacological specificity of both [ 3H]U69 and [ 3H]ET binding, where all the opioids tested produced 100% inhibition of these labels and where the rank order of potency of opioids at inhibiting the binding of these probes was:U50488 to >U69593 >dynophin-(1–8) >naloxone≫morphine≫Glyol (DAMGO)>DPDPE. The fact that [ 3H]Glyol selectively bound μ-receptors was borne-out by the relative rank order of inhibitory potency of different compounds against [ 3H]Glyol binding to dog cortex membranes, thus:Glyol (DAMGO)>morphine>naloxone>dynorphin-(1–8) ≫U50488 >U69593. The pharmacological selectivity of [ 3H]DPDPE binding to δ-receptors was different to the other receptor subtypes, being:DPDPE>dynorphin-(1–8) >naloxone>morphine⩾glyol≫U69 >U50488. These data have therefore helped identify the presence of μ-, δ and κ-opioid receptors in the dog cerebral cortex, and have shown the lack of existence of subtypes of the κ-receptor in this tissue.

Opioid receptors in rat cardiac sarcolemma: effect of phenylephrine and isoproterenol

The present study demonstrates the presence of opioid receptors in the rat cardiac sarcolemma isolated by the hypotonic LiBr-shock procedure. Opioid binding was measured by using [3H]U69 593, [3H](2-D-penicillamine,5-D-penicillamine)enkephalin ([3H]DPDPE) or [3H][D-Ala2,MePhe4,Gly-(ol)5]enkephalin ([3H]DAGO) as selective radioligands for K, delta and mu opioid receptors, respectively. Both the K- and delta-selective ligands exhibited highly specific (75-86%) binding, saturable at a concentration of about 20 nM. No specific binding for the selective agonist DAGO was observed. A marked increase in both [3H]U69 593 and [3H]DPDPE binding was observed after incubation of the sarcolemma with the alpha-adrenoceptor agonist phenylephrine or with the beta-adrenoceptor agonist isoproterenol. These stimulatory effects were associated with an increase in the Bmax values, a decrease in the Kd values, and were completely antagonized by the respective antagonists phentolamine and propranolol.

Discrete mapping of brain mu and delta opioid receptors using selective peptides: Quantitative autoradiography, species differences and comparison with kappa receptors

The opioid peptides, [ 3H]DAGO and [ 3H]DPDPE, bound to rat and guinea pig brain homogenates with a high, nanomolar affinity and to a high density of mu and delta receptors, respectively. [ 3H]DAGO binding to mu receptors was competitively inhibited by unlabelled opioids with the following rank order of potency: DAGO > morphine > DADLE > naloxone > etorphine >> U50488 >> DPDPE. In contrast, [ 3H]DPDPE binding to delta receptors was inhibited by compounds with the following rank order of potency: DPDPE > DADLE > etorphine > dynorphin(1–8) > naloxone >> U50488 >> DAGO. These profiles were consistent with specific labelling of the mu and delta opioid receptors, respectively. In vitro autoradiographic techniques coupled with computer-assisted image analyses revealed a discrete but differential anatomical localization of mu and delta receptors in the rat and guinea pig brain. In general, mu and delta receptor density in the rat exceeded that in the guinea pig brain and differed markedly from that of kappa receptors in these species. However, while mu receptors were distributed throughout the brain with “hotspots” in the fore-, mid- and hindbrain of the two rodents, the delta sites were relatively diffusely distributed, and were mainly concentrated in the forebrain with particularly high levels within the olfactory bulb (OB), n.accumbens and striatum. Notable regions of high density of mu receptors in the rat and guinea pig brain were the accessory olfactory bulb, striatal “patches” and “streaks”, amygdaloid nuclei, ventral hippocampal subiculum and dentate gyrus, numerous thalamic nuclei, geniculate bodies, central grey, superior and inferior colliculi, solitary and pontine nuclei and s.nigra. Tissues of high delta receptor concentration included, OB (external plexiform layer), striatum, n.accumbens, amygdala and cortex (layers I–II and V–VI). Delta receptors in the guinea pig were, in general, similarly distributed to the rat, but in contrast to the latter, the hindbrain regions such as the thalamus, geniculate bodies, central grey and superior and inferior colliculi of the guinea pig were apparently more enriched than the rat. These patterns of mu and delta site distribution differed dramatically from that of the kappa opioid sites in these species studied with the peptide [ 125I]dynorphin(1–8).

Autoradiographic localization of δ opioid receptors in the rat brain using a highly selective bis-penicillamine cyclic enkephalin analog

A pioneering approach to the design of a ligand selective for the 8 opioid receptors was implemented by Mosberg et al. (1983) who demonstrated that the cyclic conformationally restricted enkephalin analog (2-D-penicillamine,5-D-penicillamine)enkephalin (DPDPE) displayed extremely high specificity toward the 8 opioid receptors. Recently Akiyama et al. (1985) provided the first in vitro characterization of tritium labeled DPDPE binding to 8 opioid receptors in the rat brain and neuroblastoma-glioma hybrid (NG 108-15) cells. In the present study we examined the light microscopic autoradiographic distribution of opioid receptors in the rat central nervous system employing the highly selective 8 agonist [3H]DPDPE (S.A.= 40 Ci /mmol , Amersham Corp, Arlington Heights, IL.). Male Sprague-Dawley rats (150-200 g) were killed by decapitation, and the brain was removed and coated with plastic embedding medium (OCT Compound, Lab-Tek Products) onto microtome chucks and frozen by immersion into liquid nitrogen. Saggital and coronal sections (20 #m) were cut, thawed and moun ted onto chromealum/gelatin coated glass slides and air dried at room temperature. Slide-mounted sections were preincubated for 15 min at 25°C in 50 mM TrisHCI buffer (pH 7.4 at 25°C) containing 5 mM MgC12, 2 m g /ml BSA, 20 g g / m l bacitracin and 100 mM NaC1, to reduce any endogenous opioids