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.     

Comparison of the endogenous heptapeptide Met-enkephalin-Arg6-Phe7 binding in amphibian and mammalian brain

In previous communications [4, 38] we published that [3H]Met-enkephalin-Arg6-Phe7 (MERF) binds to opioid (kappa2 and delta) and sigma2 sites in frog and rat brain membrane preparations, however no binding to kappa1 sites could be established. In the present paper we compare the frog, rat and guinea pig brain membrane fractions with respect to their MERF binding data. No qualitative differences were found between the three species but specific binding of labelled MERF was maximal in frog brain and lowest in guinea pig brain, which corresponds to their kappa2 opioid receptor distribution. The naloxone resistant binding was also present in all investigated species and varied from 25% in frog and guinea pig cerebrum, to 50% in rat cerebrum and cerebellum, but no naloxone inhibition was found in guinea pig cerebellum where no kappa2 opioid receptors have been found. The presence of sigma2-like receptor was demonstrated in each investigated membrane fraction with displacement experiments using (-)N-allyl-normetazocine as competitor of tritiated MERF. It was shown that this site was responsible for 60-80% of [3H]MERF binding. The remaining part of the naloxone resistant labelled MERF binding could be displaced only with endogenous opioid peptides as met-enkephalin, dynorphin and beta-endorphin. The eventual physiological role of multiple MERF receptors is discussed.     

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.     

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.     

A Glycine Site Associated with N-Methyl-d-Aspartic Acid Receptors: Characterization and Identification of a New Class of Antagonists

Membranes from rat telencephalon contain a single class of strychnine-insensitive glycine sites. That these sites are associated with N-methyl-D-aspartic acid (NMDA) receptors is indicated by the observations that [3H]glycine binding is selectively modulated by NMDA receptor ligands and, conversely, that several amino acids interacting with the glycine sites increase [3H]N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding to the phencyclidine site of the NMDA receptor. The endogenous compound kynurenate and several related quinoline and quinoxaline derivatives inhibit glycine binding with affinities that are much higher than their affinities for glutamate binding sites. In contrast to glycine, kynurenate-type compounds inhibit [3H]TCP binding and thus are suggested to form a novel class of antagonists of the NMDA receptor acting through the glycine site. These results suggest the existence of a dual and opposite modulation of NMDA receptors by endogenous ligands.     

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 characterization of binding sites for [3H]NE-100 in guinea pig brain

The receptor binding specificity and neuroanatomical distribution of [³H]NE-100 (N, N- dipropyl-2- [4- methoxy-3- (2- phenylethoxy) phenyl] ethylamine monohydrochloride)-labeled sigma receptor in guinea pig brain were examined using quantitative autoradiography. NE-100 potently inhibited [³H]NE-100 binding to slide-mounted sections of guinea pig brain with the IC₅₀ value of 1.09 nM, therefore, NE-100 apparently has high affinity binding sites. Competition studies, under conditions similar to those used to visualize the receptor, yielded the following rank order of potency: NE-100 > haloperidol > DuP734 > (+)pentazocine ⪢ (−)pentazocine. Non-sigma ligands such as phencyclidine (PCP), MK-801 and (−)sulpiride had negligible affinities for [³H]NE-100 binding sites. High densities of [³H]NE-100 binding sites displaceable by haloperidol were present in the granule layer of the cerebellum, the cingulate cortex, the CA3 region of the hippocampus, the hypothalamus and the pons. The distribution of [³H]NE-100 binding sites was consistent with that of [³H](+)pentazocine, a sigma₁ ligand. These sigma sites may possibly be related to various aspects of schizophrenia.     

Interaction of [3H](–)-SKF-10,047 with Brain σ Receptors: Characterization and Autoradiographic Visualization

The sigma opiates differ from other opiates in their stimulatory and psychotomimetic actions. The sigma opiate [3H](-)-SKF-10,047 has been used to characterize sigma receptors in rat nervous tissue. Binding of [3H](-)-SKF-10,047 to rat brain membranes was of high affinity, saturable, and reversible. Scatchard analysis revealed the apparent interaction of this drug with two distinct binding sites characterized by affinities of 0.03 and 75 nM (5 mM Tris-HCl buffer, pH 7.4, at 4 degrees C). Competition analyses involving rank order determinations for a series of opiates and other drugs indicate that the high-affinity binding site is the mu opiate receptor. The lower-affinity site (revealed after suppression of mu and delta receptor binding) has been identified as the sigma opiate/phencyclidine receptor. In vitro autoradiography has been used to visualize neuroanatomical patterns of receptors labeled using [3H](-)-SKF-10,047 in the presence of normorphine and [D-Ala2,D-Leu5]enkephalin to block mu and delta interactions, respectively. Labeling patterns differ markedly from those for mu, delta, or kappa receptors. The highest densities (determined by quantitative autoradiography) are found in the medial portion of the nucleus accumbens, amygdaloid nucleus, hippocampal formation, central gray, locus coeruleus, and the parabrachial nuclei. Receptors in these structures could account for the stimulatory, mood-altering, and analgesic properties of the sigma opiates. Although not the most selective sigma opiate ligand, [3H](-)-SKF-10,047 binds to sigma opiate receptors in brain, and this interaction can be readily distinguished from its interactions with other classes of brain opiate receptors.     

14 beta-(Bromoacetamido)morphine irreversibly labels mu opioid receptors in rat brain membranes

The binding properties of 14 beta-(bromoacetamido)morphine (BAM) and the ability of BAM to irreversibly inhibit opioid binding to rat brain membranes were examined to characterize the affinity and selectivity of BAM as an irreversible affinity ligand for opioid receptors. BAM had the same receptor selectivity as morphine, with a 3-5-fold decrease in affinity for the different types of opioid receptors. When brain membranes were incubated with BAM, followed by extensive washing, opioid binding was restored to control levels. However, when membranes were incubated with dithiothreitol (DTT), followed by BAM, and subsequently washed, 90% of the 0.25 nM [3H] [D-Ala2,(Me)Phe4,Gly(ol)5]enkephalin (DAGO) binding was irreversibly inhibited as a result of the specific alkylation of a sulfhydryl group at the mu binding site. This inhibition was dependent on the concentrations of both DTT and BAM. The mu receptor specificity of BAM alkylation was demonstrated by the ability of BAM alkylated membranes to still bind the delta-selective peptide [3H] [D-penicillamine2,D-penicillamine5]enkephalin (DPDPE) and (-)-[3H]bremazocine in the presence of mu and delta blockers, selective for kappa binding sites. Under conditions where 90% of the 0.25 nM [3H]DAGO binding sites were blocked, 80% of the 0.8 nM [3H]naloxone binding and 50% of the 0.25 nM 125I-labeled beta h-endorphin binding were inhibited by BAM alkylation. Morphine and naloxone partially protected the binding site from alkylation with BAM, while ligands that did not bind to the mu site did not afford protection.2+hese studies have demonstrated that when a disulfide bond     

Focal Stimulation of the Mossy Fibers Releases Endogenous Dynorphins That Bind K1-Opioid Receptors in Guinea Pig Hippocampus

Physiological release of endogenous opioids in guinea pig hippocampal slices was detected in an in vitro competition binding assay using [3H]U69,593, a kappa 1-selective radioligand. Veratridine-induced opioid release caused a decrease in [3H]U69,593 binding that was blocked by either tetrodotoxin addition or the removal of calcium from the incubation buffer. Focal electrical stimulation of opioid peptide-containing afferent pathways resulted in a decrease in [3H]U69,593 binding, whereas stimulation of a major afferent lacking endogenous opioid immunoreactivity had no effect. The addition of 6-cyano-7-nitroquinoxaline-2,3-dione blocked the reduction in [3H]U69,593 binding caused by perforant path stimulation, but not the reduction caused by mossy fiber stimulation, suggesting that the primary source of endogenous kappa ligands was likely to be the dentate granule cells. Antisera against dynorphin A(1-8) or dynorphin B peptides inhibited the effects of mossy fiber stimulation in the [3H]U69,593 displacement assay. Antisera against other prodynorphin- and proenkephalin-derived opioid peptides had no effect. As shown by receptor autoradiography, the distribution of kappa 1 binding sites was limited to the molecular layer of the dentate gyrus and the presubiculum region of temporal hippocampal slices. These results indicate that prodynorphin-derived opioids released under physiological conditions from the mossy fibers act at kappa 1 receptors in the guinea pig dentate gyrus.     

Characterization of [3H]desmethylimipramine binding in bovine adrenal medulla: interactions with sigma- and (or) phencyclidine-receptor ligands.

High-affinity binding sites (apparent KD 2.87 nM) for [3H]desmethylimipramine ([3H]DMI), have been demonstrated and characterized in membrane preparations of bovine adrenal medulla. The binding of [3H]DMI improved upon pretreatment of the membrane with KCl and was saturable, sodium dependent, and potently inhibited by nisoxetine and imipramine. [3H]DMI binding was also inhibited by various phencyclidine (PCP)- and (or) sigma-receptor ligands, with the following order of potency: haloperidol > rimcazole > (-)-butaclamol > dextromethorphan > MK-801 > (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP) > PCP > N-(2-thienyl)cyclohexyl-3,4-piperidine (TCP) > (+)-SKF-10047 > (-)-SKF-10047. The inhibition produced by sigma ligands was not attributed to stimulation of either sigma 1- or sigma 2-receptors, owing to inactivity of the selective sigma-receptor ligands (+)-pentazocine and 1,3-di(2-tolyl)guanidine (DTG). The inhibition of [3H]DMI binding by sigma- and PCP-receptor ligands was not attributed to PCP1- or PCP2-receptor stimulation, owing to the decreased potency (100-fold) of these ligands in [3H]DMI assays compared with the affinity for brain PCP1 sites, and the ineffectiveness of the PCP2-ligand N-(1-(2-benzo(b)thiophenyl)cyclohexyl)piperidine (BTCP). Scatchard analysis of the inhibition by the sigma-ligands haloperidol and (+)-3-PPP, as well as the PCP1 receptor ligand MK-801, demonstrated noncompetitive interaction with the site bound by [3H]DMI. These studies indicate that bovine adrenomedullary membranes possess a specific receptor for the noradrenaline uptake inhibitor [3H]DMI, which is sensitive to allosteric modulation produced by PCP and sigma-ligands.     

A Monoclonal Antibody Recognizing K- but Not γ- and δ-Opioid Receptors

A monoclonal antibody (mAb), KA8 that interacts with the kappa-opioid receptor binding site was generated. BALB/c female mice were immunized with a partially purified kappa-opioid receptor preparation from frog brain. Spleen cells were hybridized with SP2/0AG8 myeloma cells. The antibody-producing hybridomas were screened for competition with opioid ligands in a modified enzyme-linked immunosorbent assay. The cell line KA8 secretes an IgG1 (kappa-light chain) immunoglobulin. The mAb KA8 purified by affinity chromatography on protein A-Sepharose CL4B was able to precipitate the antigen from a solubilized and affinity-purified frog brain kappa-opioid receptor preparation. In competition studies, the mAb KA8 decreased specific [3H]ethylketocyclazocine ([3H]EKC) binding to the frog brain membrane fraction in a concentration-dependent manner to a maximum to 72%. The degree of the inhibition was increased to 86% when mu- and delta-opioid binding was suppressed by 100 nM [D-Ala2,NMe-Phe4,Gly-ol]-enkephalin (DAGO) and 100 nM [D-Ala2,L-Leu5]-enkephalin (DADLE), respectively, and to 100% when mu-, delta-, and kappa 2-sites were blocked by 5 microM DADLE. However, the mu-specific [3H]DAGO and the delta-preferring [3H]DADLE binding to frog brain membranes cannot be inhibited by mAb KA8. These data suggest that this mAb is recognizing the kappa- but not the mu- and delta-subtype of opioid receptors. The mAb KA8 also inhibits specific [3H]naloxone and [3H]EKC binding to chick brain cultured neurons and rat brain membranes, whereas it has only a slight effect on [3H]EKC binding to guinea pig cerebellar membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Monoclonal anti-idiotypic antibodies to opioid receptors

Two monoclonal anti-idiotypic antibodies (anti-Id-135 and anti-Id-14, both of the IgM class) which interact with the binding site of opioid receptors were generated. A monoclonal anti-beta-endorphin antibody (3-E7) which displays binding characteristics for opioid ligands similar to opioid receptors served as the antigen (Gramsch, C., Meo, T., Riethmüller, G., and Herz, A., (1983) J. Neurochem. 40, 1220-1226; Meo, T., Gramsch, C., Inan, R., H?llt, V., Weber, E., Herz, A., and Riethmüller, G. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4048-4088) and the hybridomas obtained were screened for anti-idiotypic antibodies with Fab fragments of 3-E7. The anti-idiotypes were then screened for opioid binding to rat brain membrane receptors, yielding several positive clones two of which were more intensively studied. Both anti-idiotypic antibodies were about equally potent in displacing the mu- and delta-opioid receptor ligands [3H]dihydromorphine, 125I-labeled beta-endorphin, [D-Ala2, D-Leu5-3H]enkephalin and [3H]naloxone from rat brain membrane opioid receptors; no interaction was observed with the kappa-ligands [3H]ethylketazocine or [3H]bremazocine. The anti-idiotypic antibodies were able to precipitate [3H] diprenorphine binding sites from solubilized opioid receptor preparations. In addition, both antibodies showed opioid antagonistic properties as demonstrated by their abilities to block the inhibitory effect of [D-Ala2, D-Leu5-3H]enkephalin on prostaglandin E1-stimulated cAMP accumulation in NG 108-15 hybrid cells. Our findings demonstrate the successful generation of monoclonal antibodies interacting with membrane-bound and solubilized opioid receptors of the mu- and delta-type.     

GBR-12909 and fluspirilene potently inhibited binding of [3H] (+)3-PPP to sigma receptors in rat brain

Fluspirilene and GBR-12909, two compounds structurally similar to BMY-14802 and haloperidol, were assessed for their ability to interact with sigma receptors. Fluspirilene, an antipsychotic agent that interacts potently with dopamine receptors, inhibited the binding of [3H]-(+) 3-PPP (IC50 = 380 nM) more potently than rimcazole, a putative sigma antagonist that was tested clinically for antipsychotic activity. GBR-12909, a potent dopamine uptake blocker, also inhibited the binding of [3H]-(+) 3-PPP with an IC50 of 48 nM. However, other compounds that block the re-uptake of catecholamines, such as nomifensine, desipramine, imipramine, xylamine, benztropine and cocaine, were much weaker than GBR-12909 as sigma ligands. Thus, GBR-12909 and fluspirilene, compounds structurally similar to BMY-14802, are potent sigma ligands.     

Effects of stress and beta-funal trexamine pretreatment on morphine analgesia and opioid binding in rats

This study was essentially an in vivo protection experiment designed to test further the hypothesis that stress induces release of endogenous opioids which then act at opioid receptors. Rats that were either subjected to restraint stress for 1 hr or unstressed were injected ICV with either saline or 2.5 micrograms of beta-funaltrexamine (beta-FNA), an irreversible opioid antagonist that alkylates the mu-opioid receptor. Twenty-four hours later, subjects were tested unstressed for morphine analgesia (tail-flick assay) or were sacrificed and opioid binding in brain was determined. [3H]D-Ala2NMePhe4-Gly5(ol)enkephalin (DAGO) served as a specific ligand for mu- opioid receptors, and [3H]-bremazocine as a general ligand for all opioid receptors. Rats injected with saline while stressed were significantly less sensitive to the analgesic action of morphine 24 hr later than were their unstressed counterparts. Beta-FNA pretreatment attenuated morphine analgesia in an insurmountable manner. Animals pretreated with beta-FNA while stressed were significantly more sensitive to the analgesic effect of morphine than were animals that received beta-FNA while unstressed, consistent with the hypothesis that stress induces release of endogenous opioids that would protect opioid receptors from alkylation by beta-FNA. beta-FNA caused small and similar decreases in [3H]-DAGO binding in brain of both stressed and unstressed animals. Stressed rats injected with saline tended to have increased levels of [3H]DAGO and [3H]-bremazocine binding compared to the other groups. This outcome may be relevant to the tolerance to morphine analgesia caused by stress.     

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.     

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.     

Pharmacological Characteristics and Distributions of σ and Phencyclidine Receptors in the Animal Kingdom

The phylogenetic distributions ofσ- and phencyclidine receptors in neural tissues of 13 species and the pharmacological characteristics of these receptors in whole sea anemone and neural tissues of the guinea pig, chicken, and frog were studied. Specific binding of [³H]haloperidol and [³H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine, ligands that bind with high affinity to σ- and phencyclidine receptors, respectively, was detected in all organisms examined. The order of potencies of various ligands to inhibit 1 nM [³H]haloperidol binding in brains of frogs and guinea pigs or 1 nM [³H]N-[1-(2-thienyl)cyclohexyl]-3,4–piperidine in chicken or guinea pig brain homogenates was very similar. However, the characteristics and stereospecificity of binding of the two radioligands in sea anemone were different than in higher organisms. The results suggest that σ– and phencyclidine binding sites are evolutionarily old, as the characteristics of the two sites are well preserved over a range of vertebrate phyla.     

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.