Differential regulation of sigma and PCP receptors after chronic administration of haloperidol and phencyclidine in mice

The existence of multiple receptor sites for the psychotomimetic agents phencyclidine (PCP) and some opiate-benzomorphans such as (+)N-allylnormetazocine ([+]SKF 10,047) in the mammalian central nervous system is well documented. These are: 1) sigma/PCP (sigma p) site, which binds both PCP and psychotomimetic opiates but not antipsychotics such as haloperidol, 2) PCP site, which selectively binds PCP analogs, and 3) sigma/haloperidol (sigma h) site, for which certain antipsychotics and (+)SKF 10,047, but not PCP analogs, display high affinity. In this study we examined the regulation of these receptor sites after chronic treatment of mice with either PCP or haloperidol. The following radiolabeled ligands were used to assess binding to the various receptor subtypes: [3H]-1-[1-[3-hydroxyphenyl)cyclohexyl]piperidine ([3H]PCP-3-OH; sigma p and PCP sites), [3H]thienyl-phencyclidine ([3H]TCP; PCP site), (+)-[3H]SKF 10,047 (sigma p and sigma h sites), and [3H]haloperidol (sigma h and D-2 dopamine receptors). Treatment of mice for 1, 7, 14, and 21 days with PCP (10 mg.kg-1.day-1) failed to induce variations in sigma p, sigma h, and PCP receptor binding. However, similar treatment with haloperidol (4 mg.kg-1.day-1) induced: 1) complete elimination of the binding to sigma h sites, 2) up-regulation of D-2 dopamine receptors, and 3) no change in sigma p and PCP receptor binding after 14 or 21 days of treatment. However, a single day of haloperidol treatment or in vitro incubation of mouse brain membranes with haloperidol failed to alter receptor binding. This study suggests that prolonged treatment of mice with haloperidol induces a loss in sigma h receptors that are presumably associated with certain psychotomimetic effects. This phenomenon is accompanied by an up-regulation of D-2 dopamine receptors.     

Evidence that the potential antipsychotic agent rimcazole (BW 234U) is a specific, competitive antagonist of sigma sites in brain

Rimcazole (BW 234U) is a potential antipsychotic agent which in open-clinical trials appears to be effective in acute schizophrenic patients. In the present study, rimcazole was found to block the specific binding of [3H]-(+)-SKF 10,047 to sigma sites in rat and guinea pig brain (IC50 = 5.0 X 10(-7) M). The compound was 100 times weaker as a blocker of phencyclidine sites (IC50 = 4.3 X 10(-5) M). At 1 X 10(-5) M, rimcazole had only weak effects on mu, delta, kappa and epsilon opioid receptors. Scatchard analysis of the binding data from guinea pig brain revealed an apparent KD for [3H]-(+)-SKF 10,047 of 85 +/- 5 nM and a Bmax of 824 +/- 27 fmole/mg protein. In the presence of 5 X 10(-7) M BW 234U, the apparent KD was 165 +/- 35 nM, but the Bmax (892 +/- 146 fmoles/mg protein) was not affected. This suggests that rimcazole is a competitive inhibitor of sigma sites. The agent was also capable of blocking sigma sites in vivo (ID50 = 6 mg/kg i.p., mice) as judged by an in vivo sigma receptor binding assay. Thus, if the antipsychotic activity of rimcazole is confirmed in double-blind, placebo-controlled trials, it would be the first compound whose mechanism of antipsychotic activity may best be explained by a direct blockade of sigma sites and not by a direct blockade of dopamine (D2) receptors in brain.     

Visualization and solubilization of rat brain opiate receptors with a “κ” ligand selectivity pattern

1. Specific binding of [3H]ethylketocyclazocine (EkappaC), a prototype kappa-opiate agonist, to slide-mounted rat striatal sections is increased in the presence of 100 mM NaCl at 4 degrees C. 2. Under similar incubation conditions, binding of mu and delta prototype opiates is reduced to almost undetectable levels. 3. Correlation (P less than 0.01) of the ligand selectivity pattern of [3H]EKC displacement with the potencies of various opiate drugs in inhibiting the contractions of the rabbit vas deferens, a kappa-opiate receptor bioassay, suggests that the binding site under study represents the pharmacologically relevant kappa-opiate receptor. 4. Visualization of these kappa-opiate receptors with tritium-sensitive film reveals a striking, highly discrete brain distribution pattern (e.g., striatal patches, habenular stripe) which is similar to that of [3H]dihydromorphine and [3H]naloxone. 5. Soluble [3H]EKC binding sites obtained from rat membranes also possess a kappa-like ligand selectivity pattern, with bremazocine being a potent displacer while mu and delta ligands are almost inactive. 6. A possible explanation of these data is that the "kappa"-opiate binding site in rat brain is one transitional state of an opiate receptor capable of assuming distinct conformations with characteristic ligand selectivity patterns. Other possibilities such as pre and post-synaptic locations should also be considered.     

Quantitative autoradiography of [3H]CTOP binding to mu opioid receptors in rat brain

[3H]H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 ([3H]CTOP), a potent and highly selective mu opioid antagonist, was used to localize the mu receptors in rat brain by light microscopic autoradiography. Radioligand binding studies with [3H]CTOP using slide-mounted tissue sections of rat brain produced a Kd value of 1.1 nM with a Bmax value of 79.1 fmol/mg protein. Mu opioid agonists and antagonists inhibited [3H]CTOP binding with high affinity (IC50 values of 0.2-2.4 nM), while the delta agonist DPDPE, delta antagonist ICI 174,864, and kappa agonist U 69, 593 were very weak inhibitors of [3H]CTOP binding (IC50 values of 234-3631 nM). Light microscopic autoradiography of [3H]CTOP binding sites revealed regions of high density (nucleus of the solitary tract, clusters in the caudate-putamen, interpeduncular nucleus, superior and inferior colliculus, subiculum, substantia nigra zona reticulata, medial geniculate, locus coeruleus and dorsal motor nucleus of the vagus) and regions of moderate labeling (areas outside of clusters in the caudate-putamen, cingulate cortex, claustrum and nucleus accumbens). The cerebral cortex (parietal) showed a low density of [3H]CTOP binding.     

Opioid receptors in human neuroblastoma SH-SY5Y cells: evidence for distinct morphine (mu) and enkephalin (delta) binding sites

Human neuroblastoma SH-SY5Y cells exhibited a heterogeneous population of mu and delta types of opioid binding sites. These specific binding sites displayed the characteristic saturability, stereospecificity and reversibility, expected of a receptor. Scatchard analysis of [3H]-D-Ala2-D-Leu5-enkephalin (DADLE) in the presence of 10(-5) M D-Pro4-morphiceptin (to block the mu receptors) and the competitive displacement by various highly selective ligands yielded the binding parameters of delta sites which closely resemble those of the delta receptors in brain and mouse neuroblastoma clones. Similarly, the high affinity binding of [3H]-dihydromorphine, together with the higher potency of morphine analogues to displace [3H]-naloxone binding established the presence of mu sites. Guanine nucleotides and NaCl significantly inhibited the association and increased the dissociation of [3H]-DADLE binding. The observed heterogeneity of opioid receptors in cultured SH-SY5Y cells would serve as an excellent model for the biochemical and pharmacological characterization of brain opiate receptors.     

Differing stereospecificities distinguish opiate receptor subtypes

Paired stereoisomers of compounds active at the proposed mu, kappa and sigma classes of opiate receptors display differing stereoselectivity patterns at the receptor subtypes. The (-) isomers of cyclazocine and SKF-10047 are far more potent than the (+) isomers as displacers of [3H]dihydromorphine from receptors. However, the (-) isomers are only moderately more potent than the (+) isomers at displacing [3H]ethylketocyclazocine from kappa receptors in an assay controlled for radioligand binding to mu receptors, and the (+) and (-) isomers are similar in potency for displacement of [3H]phencyclidine (PCP) from sigma receptors. At the sigma/PCP receptor, (+) ketamine proved four times as potent as (-) ketamine, while the dioxalan derivative dexoxadrol is far more potent than its nearly inactive enantiomer levoxadrol. The results for the sigma/PCP receptor are in agreement with those of behavioral studies. Stereospecificity patterns may provide support for the concept of the opiate receptor subclasses as biochemically distinct entities.     

Endogenous ligands for sigma opioid receptors in the brain ("sigmaphin"): evidence from binding assays

Two endogenous ligands which interact preferentially with the sigma opioid receptors were identified from the guinea-pig brain extract in a Sephadex G-50 fractionation. These two ligands inhibited more potently the binding of [3H]SKF-10047 to sigma opioid receptors than [3H]naloxone to mu opioid receptors, [3H]ethylketocyclazocine to kappa opioid receptors and [3H]DADLE to delta opioid receptors. In the phencyclidine receptor assay, these two ligands were almost inactive. Incubation of these ligands with trypsin destroyed at least 50% of the activities in the sigma opioid receptor assay. Both ligands inhibited the sigma binding in a dose-dependent manner. The inhibition could be eliminated when the two ligands were removed from incubation media by extensive washings. It is therefore concluded that sigma opioid receptors are not phencyclidine receptors and that endogenous ligands for sigma opioid receptors may exist in the brain.     

Opiate receptor binding in the brain of rat during stress

Two types of opioid receptors were studied in the brain of rats: Delta (for endogenous opiate) and mu (for exogenous opiates). 3H derivates: D-Ala2-enkephalin and Naloxone were used as labeled ligands. The results obtained were calculated by computer program for automatic estimation of the data using approximation equations. An increase of binding delta receptors is observed in both types of stress (2-8 times), while to the mu receptors the binding is less effective mainly after irradiation. These data suggest that a close interaction exists between sympathoadrenal system and opioid mechanisms during stress.     

Selective Alterations of Opiate Receptor Subtypes in Mono sodium Glutamate-Treated Rats

Neonatal treatment of rats with monosodium glutamate (MSG) has been demonstrated to destroy cell bodies of neurons in the arcuate nucleus including the brain beta-endorphin (B-END) system. The effects on opiate receptors of the loss of B-END is unknown. Neonatal rats were treated with MSG as previously described. After reaching maturity (7-9 months), MSG-treated rats and litter-matched untreated control rats were decapitated and brains dissected into brain regions. Opiate receptor assays were run with [3H]morphine (mu receptor ligand) and [3H]D-alanine2-D-leucine5 (DADL) enkephalin (delta receptor ligand) for each brain region for both MSG and control rats simultaneously. Scatchard plot analyses showed a selective increase in delta receptors in the thalamus only. No corresponding change in mu receptors in the thalamus was found. The cross-competition IC50 data supported this conclusion, showing a loss in the potency of morphine in displacing [3H]DADL enkephalin in the thalamus of MSG-treated rats. This shift in delta receptors produced an IC50 displacement pattern in thalamus, ordinarily a mu-rich area, similar to that of striatum or cortex, delta-rich areas, again indicating an increase in delta receptors. Similar changes in delta receptors in other brain regions were not found. These results represent one of the few examples of a selective and localized shift in delta with no change in mu sites. Furthermore, the delta increase may reflect an up-regulation of the receptors in thalamus after chronic loss of the endogenous opioid B-END.     

Characterization of [3H]Met-enkephalin-Arg6-Phe7 binding to multiple sites in rat and guinea pig cerebellum

[3H]Met-enkephalin-Arg6-Phe7 (MERF) has been shown to label opioid (kappa2 and delta) and sigma2 sites in rat and frog brain membrane preparations, and no specific binding to kappa1 opioid receptors could be established (refs. 6 and 8). In this study the binding was examined in rat cerebellar membranes which are relatively rich in kappa2-sites, and in guinea pig cerebellar preparations where kappa1 opioid receptors are almost exclusively present. In accordance with our previous results, [3H]MERF binding could not be displaced in guinea pig cerebellar membranes neither with U-69,593 nor with naloxone or levorphanol suggesting no interaction with opioid sites, nevertheless a Kd of 2.8 nM was calculated in cold saturation experiments. In rat cerebellar membrane fractions about the half of the specific [3H]MERF binding sites was inhibited by opiate alkaloids such as naloxone, ethylketocyclazocine, or bremazocine. This portion of the heptapeptide binding sites was stereoselective as demonstrated by the difference in the affinities of the enantiomeric compounds levorphanol and dextrorphan, therefore it would represent an opioid site. In both tissues (-)N-allyl-normetazocine (SKF-10,047), which is also considered as sigma2 ligand, displayed the highest affinities. Among opioid peptides beta-endorphin and dynorphin(1-13) showed the highest potencies, displacing [3H]MERF also from its non-opioid sites. It was concluded therefore that [3H]MERF does not bind to kappa1 sites, and besides kappa2-opioid sites substantial binding to peptide preferring non-opioid sites, and/or sigma2 receptors also occurs.     

Interaction of dextrorotatory opioids with phencyclidine recognition sites in rat brain membranes

The potencies of several dextrorotatory opioids, including four pairs of enantiomers, as inhibitors of specific [³H]PCP binding to rat brain synaptic membranes has been determined. Of the compounds tested unlabeled phencyclidine (PCP) was the most potent followed by (?)? cyclazocine > dextrorphan > (+) ketamine > (+) cyclazocine > (+)? SKF10,047 > levorphanol > dextromethorphan > (?) SKF10,047 > (?)? ketamine > (±) pentazocine and > (±) ethylketocyclazocine. The opiate mu receptor ligands, morphine, naloxone and naltrexone were virtually inactive as competitors of specific [³H]PCP binding. Unlike the stereostructural requirements for opiate mu receptors where activity resides predominantly in the levorotatory enantiomers, the present results support the contention that binding to the [³H]PCP labeled recognition site may reside in either the levorotatory or the dextrorotatory enantiomer. The specific binding of [³H]PCP which was defined as total binding minus that occurring in the presence of 10μM dextrorphan was found to be of a high affinity, saturable, reversible and sensitive to thermal degradation. These results suggest that certain dextrorotatory morphian derivatives may prove to be useful probes in further investigations of the molecular characteristics of the [³H]PCP binding site in brain membrane preparations.     

Biphasic competition between opiates and enkephalins: does it indicate the existence of a common high affinity ("mu-1") binding site?

Displacement from brain membranes of labeled opiates by low concentrations of enkephalins and of labeled enkephalins by low concentrations of opiates has been previously explained by the existence of a common high affinity site termed mu-1. An alternative interpretation of the same results is that the trough seen in the low concentration zone of the displacement curves represents cross binding of mu and delta opioid ligands to delta and mu receptors, respectively. In three sets of experiments with brain membranes, the size of the trough is shown to be dependent on the labeled ligand used: The ratio between the size of troughs seen with [3H]D-Ala, D-Leu enkephalin and with [3H]morphine varies with experimental conditions (storage of membranes at 4 degrees C for 72 h), with ratio of mu:delta receptors (e.g. in thalamus and cortex which are enriched in mu and delta sites, respectively) and with pretreatment of membranes with naloxonazine. These results can not be explained by a common high affinity site, but rather by binding of [3H]D-Ala, D-Leu enkephalin to mu and of [3H]morphine to delta opioid receptors.     

Cystamine-enkephalin dimer. Syntheses and biological activities of enkephalin analogs containing cystamine and cysteamine

A cystamine-enkephalin dimer, containing two molecules of [D-Ala2, Leu5] enkephalin cross-linked at the COOH-terminal leucine residue with cystamine, (NH2-CH2-CH2-S-)2, has been synthesized in order to examine directly the dimerization effect of an enkephalin molecule on the opiate receptor interactions. In a comparison of potencies against [3H]-[D-Ala2,D-Leu5] enkephalin (3H-DADLE) and [3H]-[D-Ala2,MePhe4,Gly-ol5] enkephalin (3H-DAGO) as delta and mu tracers, respectively, enkephalin dimer showed a very high affinity, especially for the delta opiate receptors. Dimer was almost threefold more potent than DADLE, which is one of the most utilized delta ligand to date. When the binding affinity of cystamine-dimer was compared with that of its reduced thiol-monomer, namely [D-Ala2,Leu5,cysteamine6] enkephalin, the increment in affinity was four to fivefold for both delta and mu receptors. The results strongly indicate that the dimeric enkephalin is more potent presumably due to the simultaneous interaction with the two binding sites of the opiate receptors.     

Synthesis and evaluation of optically pure [3H]-(+)-pentazocine, a highly potent and selective radioligand for sigma receptors

Tritium-labeled (+)-pentazocine ([3H]-1b) of specific activity 26.6 Ci/mmol was synthesized in 3 steps starting with (+)-normetazocine (2) of defined optical purity. [3H]-1b has been characterized as a highly selective ligand for labeling of sigma receptors. Competition data revealed that [3H]-1b could be displaced from guinea pig brain membrane preparations with a number of commonly used sigma receptor ligands. [3H]-1b exhibited saturable, enantioselective binding with a Kd of 5.13 +/- 0.97 nM and a Bmax of 1146 +/- 122 fmol/mg protein. Phencyclidine (PCP) displaced [3H]-1b with low affinity while MK-801 was inactive, thus indicating insignificant activity at the PCP-binding site; apomorphine failed to displace [3H]-1b indicating lack of dopamine receptor cross-reactivity. Since the affinity of [3H]-1b is about 6 times that of the two commonly employed sigma ligands ((+)-3-[3H]PPP and [3H]DTG) and since it is more selective for sigma receptors than the benzomorphan [3H]SKF-10,047, it represents the first example of a highly selective benzomorphan based sigma receptor ligand. [3H]-1b should prove useful for further study of the structure and function of sigma receptors.     

Biologically active MK-801 and SKF-10,047 binding sites distinct from those in rat brain are expressed on human lung cancer cells.

We have shown previously that cultured human lung cancer cells of different histologic types express multiple opioid receptors that can regulate their growth. In this report, we show that these cells also express specific, saturable, and high-affinity binding sites (Kd approximately 1 nM) for the non-opioid phencyclidine (PCP), [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohepten-5,10-imine hydrogen maleate] (MK-801) and sigma N-allylnormetazocine (SKF-10,047) receptor ligands. Characterization of these binding sites showed them to be protein in nature and sensitive to the guanine nucleotide GTP. Pharmacological studies showed that (+) MK-801 and (+) SKF-10,047 competed with each other for their binding sites and also for the methadone binding site present in these cells. However, the mu and delta opioid ligands did not compete for (+) MK-801 and (+) SKF-10,047 binding sites. In addition, these binding sites on lung cancer cells appear to be distinct from the N-methyl D-aspartate/PCP receptor ionophore complex reported to be present in rat brain. MK-801 and SKF-10,047, at nM concentrations, were found to inhibit the growth of these cells in culture within a few hours of exposure, and this effect was irreversible after 24 h. The growth effects of these ligands could not be reversed by the opioid antagonist naloxone, suggesting involvement of nonopioid type receptors in the actions of these ligands. The abundant expression of biologically active MK-801 and SKF-10 047 binding sites in these cell lines, distinct from those in rat brain, suggests that these cell lines may prove to be a valuable source for further characterization and purification of these binding sites.     

Autoradiographic Visualization of Kappa Opioid Receptors with Labelled Dynorphins in Guinea Pig Brain

Abstract

The distribution of kappa opioid receptors in guinea pig brain was measured by in vitro receptor autoradiography using [³H]dynorphin A_1–9, [³H]dynorphin A_1–8 and [³H]bremazocine as ligands. The sites labelled by the two dynorphins had identical, heterogeneous distributions in brain sections. High levels of kappa receptors were seen in striatum, claustrum, nucleus accumbens and laminae V and VI of the cerebral cortex. The substantia nigra and superior colliculus also had high dynorphin binding levels. The [³H]dynorphin autoradiographs were closely similar to those obtained using [³H]bremazocine in the presence of mu and delta receptor displacers. It is concluded that tritiated dynorphin A fragments can be used for autoradiographic studies of kappa opioid receptors in brain.     

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.     

Evidence for a mu-delta opioid receptor complex in CHO cells co-expressing mu and delta opioid peptide receptors

Based on non-competitive binding interactions we suggested that mu and delta receptors associate as a mu/delta receptor complex in rat brain. We hypothesized that the same non-competitive binding interactions observed in rat brain will be seen in CHO cells that co-express mu and delta receptors, but not in cells that express just mu or delta receptors. We used CHO cells expressing the cloned human mu receptor, cloned human delta receptor, or cloned mouse delta/human mu ("dimer cell"). Cell membranes were prepared from intact cells pretreated with 100nM SUPERFIT. [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding assays followed published procedures. SUPERFIT, a delta-selective irreversible ligand, decreased [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to delta receptors by approximately 75% and to mu receptors by approximately 50% in dimer cells. SUPERFIT treatment did not decrease [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to mu cells. The IC(50) values observed in SUPERFIT-treated dimer cells were: [d-Pen(2),d-Pen(5)]enkephalin (1820nM) and morphine (171nM). Saturation binding experiments with SUPERFIT-treated dimer cells showed that [d-Pen(2),d-Pen(5)]enkephalin (5000nM) was a competitive inhibitor. In contrast, morphine (1000nM) lowered the B(max) from 1944fmol/mg to 1276fmol/mg protein (35% decrease). Both [d-Pen(2),d-Pen(5)]enkephalin and morphine competitively inhibited [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to SUPERFIT-treated mu cells. The results indicate that the mu-delta opioid receptor complex defined on the basis of non-competitive binding interactions in rat brain over 20 years ago likely occurs as a consequence of the formation of mu-delta heterodimers. SUPERFIT-treated dimer cells may provide a useful model to study the properties of mu-delta heterodimers.     

Solubilization and Characterization of σ-Receptors from Guinea Pig Brain Membranes

The sigma-receptor, a distinct binding site in brain tissue that may mediate some of the psychotomimetic properties of benzomorphan opiates and phencyclidine, has been solubilized using the ionic detergent sodium cholate. Binding assays were performed with the solubilized receptor using vacuum filtration over polyethyleneimine-treated glass fiber filters. The pharmacological specificity of the solubilized binding site for sigma-receptor ligands is nearly identical to the membrane-bound form of the receptor, with the order of potencies for displacement of the selective sigma-ligand [3H]di-o-tolylguanidine ([3H]DTG) closely correlated. The stereoselectivity for (+)-benzomorphan opiate enantiomers was retained by the solubilized receptor. The soluble receptor retained high affinity for binding of [3H]DTG (KD = 28 +/- 0.5 nM) and (+)-[3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [(+)-[3H]3-PPP] (KD = 36 +/- 2 nM). Photoaffinity labeling of the solubilized receptor by [3H]p-azido-DTG, a sigma-selective photoaffinity label, resulted in labeling of a 29-kilodalton polypeptide identical in size to that labeled in intact membranes. Estimation of the Stokes radius of the [3H]DTG binding site was obtained by Sepharose CL-6B chromatography in the presence of 20 mM cholate and calculated to be 8.7 nm. This value was identical to the molecular size found for the binding sites of the sigma-selective ligands (+)-[3H]3-PPP and (+)-[3H]SKF-10,047, supporting the hypothesis that all three ligands bind to the same macromolecular complex.     

The occurrence and receptor specificity of endogenous opioid peptides within the pancreas and liver of the rat. Comparison with brain.

Our observations that opioid peptides have direct effects on islet insulin secretion and liver glucose production prompted a search for endogenous opiates and their receptors in these peripheral tissues. Mu-, delta- and kappa-receptor-active opiates were demonstrated in brain, pancreas and liver extracts by displacement studies using selective ligands for the three opiate receptor subtypes [( 3H][D-Ala2,MePhe4,Gly5-ol]enkephalin, [3H][D-Ala2,D-Leu5]enkephalin and [3H]dynorphin respectively). Receptor-active opiates in brain extracts exhibited a stronger preference for delta-opiate-receptor sites than for mu and kappa sites. Pancreatic extract opiates demonstrated a similar activity at mu and delta sites, but substantially less at kappa sites. Liver extracts displayed similar selectivity for all three sites. The affinities of the receptor-active opiates for mu-, delta- and kappa-receptor subtypes displayed a rank order of potency: brain much greater than pancreas greater than liver. Total immunoreactive beta-endorphin and [Met5]enkephalin levels in liver and hepatocytes were greater than those in brain. Immunoreactive [Met5]enkephalin levels in pancreas were similar to, but beta-endorphin levels were substantially higher than, those in brain. Delta and kappa opiate-binding sites of high affinity were identified in crude membrane preparations of islets of Langerhans, but no specific opiate-binding sites could be demonstrated in liver membrane preparations. Immunoreactive dynorphin and beta-endorphin were demonstrated by immunogold labelling in rat pancreatic islet cells. No positive staining of liver sections for opioids was observed. These results suggest that the tissue content of opiate-receptor-active compounds in the pancreas and the liver is very significant and could contribute to the regulation of normal blood glucose levels.