Pharmacological specificity of some psychotomimetic and antipsychotic agents for the sigma and PCP binding sites

The pharmacological specificity of representative psychotomimetic agents such as phencyclidine (PCP) analogs, opiate benzomorphans and several antipsychotic agents was assessed for the sigma and PCP binding sites. In a series of binding experiments, in rat brain membranes, sigma and PCP binding sites were labeled with [3H]-1-[1-(3-hydroxyphenyl)cyclohexyl]piperidine [( 3H]PCP-3-OH), (+) [3H]-N-allylnormetazocine [(+) [3H]SKF 10047] and (+) [3H]-3-[3-hydroxy-phenyl]-N-(1-propyl)piperidine [(+) [3H]-3-PPP]. PCP analogs inhibit potently high affinity [3H]PCP-3-OH binding and (+) [3H]SKF 10047 binding, moderately the low affinity binding component of [3H]PCP-3-OH and very weakly (+) [3H]-3-PPP binding. (+)SKF 10047 and cyclazocine are potent to moderate inhibitors of (+) [3H]SKF 10047, high affinity [3H]PCP-3-OH and (+) [3H]-3-PPP binding, but extremely weak inhibitors of low affinity [3H]PCP-3-OH binding. The antipsychotic agents display high affinity for (+) [3H]-3-PPP binding sites, moderate affinity for (+) [3H]SKF 10047 sites and have no effect on either the high or low affinity [3H]PCP-3-OH binding. The present data further support the existence of multiple sigma and PCP binding sites.     

A Comparison of [3H]MK-801 and N-[1-(2-Thienyl)cyclohexyl]-3,4-[3H]Piperidine Binding to the N-Methyl-D-Aspartate Receptor Complex in Human Brain

The binding of (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate ([3H]MK-801) and N-[1-(2-thienyl)cyclohexyl]-3,4-[3H]piperidine ([3H]TCP) to the N-methyl-D-aspartate (NMDA) receptor complex of human brain has been investigated. Significant differences were noted between the binding of the two ligands in the same tissue samples. Binding of both ligands was stimulated by addition of glutamic acid or glycine. However, addition of both compounds resulted in an additional effect with [3H]MK-801 but not [3H]TCP binding. Saturation analysis revealed approximately twice as many high-affinity sites for [3H]MK-801 (Bmax, 1,500 +/- 300 fmol/mg of protein) than for [3H]TCP (Bmax, 660 +/- 170 fmol/mg of protein). In addition, a low-affinity site was detected for [3H]MK-801 binding but not [3H]TCP binding. The pharmacology of the high-affinity [3H]MK-801 and [3H]TCP binding sites was similar with rank order of potency of inhibitors being MK801 greater than TCP greater than phencyclidine greater than N-allylnormetazocine (SKF 10047). 2-Amino-5-phosphonopentanoate inhibited binding of both ligands with comparable potency whereas both 7-chlorokynurenic acid and ZnCl2 were more potent inhibitors of [3H]MK-801 than of [3H]TCP binding. All compounds examined exhibited Hill coefficients of significantly less than unity. Saturation analysis performed in the striatum revealed that the number of binding sites was the same for both [3H]MK-801 (Bmax, 1,403 +/- 394 fmol/mg) and [3H]TCP (Bmax, 1,292 +/- 305 fmol/mg). Addition of glutamate or glycine stimulated striatal binding but there was no further increase on addition of both together.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

SKF 525-A and cytochrome P-450 ligands inhibit with high affinity the binding of [3H]dextromethorphan and sigma ligands to guinea pig brain.

The DM1/sigma 1 site binds dextromethorphan (DM) and sigma receptor ligands. The broad binding specificity of this site and its peculiar subcellular distribution prompted us to explore the possibility that this site is a member of the cytochrome P-450 superfamily of enzymes. We tested the effects of the liver microsomal monooxygenase inhibitor SKF 525-A (Proadifen), and other P-450 substrates on the binding of [3H]dextromethorphan, [3H]3-(-3-Hydroxyphenyl)-N-(1-propyl)piperidine and (+)-[3H]1,3-Di-o-tolyl-guanidine ([3H]DTG) to the guinea pig brain. SKF 525-A, l-lobeline and GBR-12909 inhibited the binding of the three labeled ligands with nM affinity. Each drug has identical nM Ki values for the high-affinity site labeled by the three ligands. This indicated that they displaced the labeled ligands from the common DM1/sigma 1 site. Debrisoquine and sparteine, prototypical substrates for liver debrisoquine 4-hydroxylase, displayed Ki values of 9-13 and 3-4 microM respectively against the three labeled ligands. These results, the broad specificity of the DM1/sigma 1 binding site, and its peculiar subcellular distribution, raises the possibility that this binding site is a member of the cytochrome P-450 superfamily of isozymes, rather than a neurotransmitter receptor. These findings may have important implications for the understanding of the therapeutic, side effects and toxicity of several neurotropic drugs.     

Specific binding of N-allylnormetazocine (SKF 10047), a ligand of sigma-opioid receptors, with liver membranes

A sigma-opioid receptor ligand, N-allylnormetazocine (SKF 10047), binds specifically and reversibly to rat liver membranes. The rat liver binding sites for SKF 10047 are similar to sigma-opioid CNS receptors. They fail to interact with classical opiates (morphine, naloxone) and opioid peptides but bind with high affinity benzomorphans (bremazocine, SKF 10047) and various psychotropic drugs (haloperidol, imipramine, phencyclidine etc).     

Identification of a Novel N-Methyl-D-Aspartate Receptor Population in the Rat Medial Thalamus

To evaluate the possibility of pharmacologically distinct N-methyl-D-aspartate (NMDA) receptor subtypes, quantitative autoradiography was used to determine the potency of several compounds as inhibitors of L-[3H]glutamate or [3H]MK-801 binding to rat brain NMDA receptors in 10 brain regions. Competitive NMDA receptor antagonists displayed differing pharmacological profiles in the forebrain, cerebellum, and medial regions of the thalamus (midline nuclei). For example, compared with other competitive antagonists, 3-[(+/-)-2-carboxypiperazin-4-yl]propyl-1-phosphonate (CPP) and LY-233536 were especially weak displacers of L-[3H]glutamate binding in the cerebellum. In the the medial thalamus, CPP and D-2-amino-5-phosphonopentanoate displayed relatively low affinities, whereas LY-233536 was relatively potent. The noncompetitive NMDA receptor antagonists also displayed regional variations in their pharmacological profiles. Relative to other regions, [3H]MK-801 binding in the cerebellum was weakly displaced by MK-801 and potently displaced by dextromethorphan and SKF-10047. In the medial thalamus, 1-[1-(2-thienyl)-cyclohexyl]piperidine was relatively potent and SKF-10047 was relatively weak. These results confirm previous suggestions that the cerebellum contains a distinct NMDA receptor subtype and indicate that nuclei of the medial thalamus contain a novel NMDA receptor subtype that is distinct from both those found in the cerebellum and in the forebrain.     

Characterization of binding sites for [3H]-DTG, a selective sigma receptor ligand, in the sheep pineal gland

Specific binding sites for [3H]-1,3 di-ortho-tolylguanidine ([3H]-DTG), a selective radiolabeled sigma receptor ligand, were detected and characterized in sheep pineal gland membranes. The binding of [3H]-DTG to sheep pineal membranes was rapid and reversible with a rate constant for association (K+1) at 25 degrees C of 0.0052 nM-1.min-1 and rate constant for dissociation (K-1) 0.0515 min-1, giving a Kd (K-1/K+1) of 9.9 nM. Saturation studies demonstrated that [3H]-DTG binds to a single class of sites with an affinity constant (Kd) of 27 +/- 3.4 nM, and a total binding capacity (Bmax) of 1.39 +/- 0.03 pmol/mg protein. Competition experiments showed that the relative order of potency of compounds for inhibition of [3H]-DTG binding to sheep pineal membranes was as follows: trifluoperazine = DTG greater than haloperidol greater than pentazocine greater than (+)-3-PPP greater than (+/-)SKF 10,047. Some steroids (testosterone, progesterone, deoxycorticosterone) previously reported to bind to the sigma site in brain membranes were very weak inhibitors of [3H]-DTG binding in the present study. The results indicate that [3H]-DTG binding sites having the characteristics of sigma receptors are present in sheep pineal gland. The physiological importance of these sites in regulating the synthesis of the pineal hormone melatonin awaits further study.     

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.     

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.     

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.     

Different effects of lectins on the ligand binding of the NMDA receptors and sigma sites in rat brain hippocampus synaptic membranes

The effects of the lectins concanavalin A, WGA, ricin, abrin, and the mistletoe lectins from Viscum album MLI, MLII, and MLIII on the binding of ligands of the NMDA and sigma receptors in rat hippocampus synaptic plasma membranes were investigated. Binding of [³H]MK-801, [³H]glutamate, [³H]5,7-DCKA, and [³H]glycine to the membranes was decreased by 40-60% after addition of galactose-specific lectins (mistletoe lectins MLI, MLII, ricin, abrin) at concentrations of 0.01 mg/ml, but was not affected by the glucose- and mannose-specific lectin Con A, an acetylglucosamine-specific lectin WGA, or an acetylgalactosamine-specific lectin MLIII. The binding of [³H]SKF 10047 was decreased only in the presence of MLIII and did not change after addition of the other lectins. It is suggested that lectin-sensitive ligand binding sites of sigma- and NMDA receptors are located separately, and that the carbohydrate side chains of the sigma receptor do not participate in the modulation of the NMDA-receptor.     

Calcium-Independent γ-Aminobutyric Acid Release from Growth Cones: Role of γ-Aminobutyric Acid Transport

Neuronal growth cones isolated in bulk from neonatal rat forebrain have uptake and K(+)-stimulated release mechanisms for gamma-aminobutyric acid (GABA). Up to and including postnatal day 5, the K(+)-stimulated release of [3H]GABA and endogenous GABA is Ca2+ independent. At these ages, isolated growth cones neither contain synaptic vesicles nor stain for synaptic vesicle antigens. Here we examined the possibility that the release mechanism underlying Ca2(+)-independent GABA release from isolated growth cones is by reversal of the plasma membrane GABA transporter. The effects of two GABA transporter inhibitors, nipecotic acid and an analogue of nipecotic acid, SKF 89976-A, on K(+)-stimulated release of [3H]GABA from superfused growth cones were examined. Nipecotic acid both stimulated basal [3H]GABA release and enhanced K(+)-stimulated release of [3H]GABA, which indicates that this agent can stimulate GABA release and is, therefore, not a useful inhibitor with which to test the role of the GABA transporter in K(+)-stimulated GABA release from growth cones. In contrast, SKF 89976-A profoundly depressed both basal and K(+)-stimulated [3H]GABA release. This occurred at similar concentrations at which uptake was blocked. These observations provide evidence for a major role of the GABA transporter in GABA release from neuronal growth cones.     

MS-377, a selective sigma receptor ligand, indirectly blocks the action of PCP in the N-methyl-D-aspartate receptor ion-channel complex in primary cultured rat neuronal cells

MS-377 ((R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone L-tartrate) is a antipsychotic agent that binds to sigma-1 receptor. MS-377 showed anti-dopaminergic and anti-serotonergic activities and antagonistic action against phencyclidine (PCP)-induced behaviors in an animal model. These anti-psychotic activities of MS-377 are attributable to association with sigma-1 receptor. However, the mechanism by which the sigma-1 receptor ligands exact those numerous effects remains to be elucidated. In the present study, we evaluated the effect of MS-377 on N-methyl-D-aspartate (NMDA) receptor ion-channel complex in primary cultured rat neuronal cells. First, we examined the effect of MS-377 on NMDA-induced Ca2+ influx with fura-2/ AM loaded cells. MS-377 showed no effects on the basal Ca2+ concentration and NMDA-induced Ca2+ influx by itself PCP and SKF-10047 reduced the NMDA-induced increase in intracellular Ca2+ concentration. Pre-incubation of 1 microM MS-377 was found to significantly block the reduction by PCP or SKF-10047 of the NMDA-induced Ca2+ influx. Second, the effect of MS-377 on [3H]MK-801 intact cell binding was examined. PCP, haloperidol and (+)-pentazocine inhibited [3H]MK-801 binding, although MS-377 showed no effect by itself Pre-treatment of MS-377 markedly reversed the inhibition of [3H]MK-801 binding by PCP in a dose-dependent manner. These effects of MS-377 may depend on its affinity for the sigma-1 receptor, because MS-377 is a selective sigma-1 receptor ligand without any affinity for NMDA receptor ion-channel complex. These observations suggest that the MS-377 indirectly modulated the NMDA receptor ion-channel complex, and the anti-psychotic activities of MS-377, in part, are attributable to such on action via sigma-1 receptor.     

[3H]MK-801 Labels a Site on the N-Methyl-D-Aspartate Receptor Channel Complex in Rat Brain Membranes

The potent noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist [3H]MK-801 bound with nanomolar affinity to rat brain membranes in a reversible, saturable, and stereospecific manner. The affinity of [3H]MK-801 was considerably higher in 5 mM Tris-HCl (pH 7.4) than in previous studies using Krebs-Henseleit buffer. [3H]MK-801 labels a homogeneous population of sites in rat cerebral cortical membranes with KD of 6.3 nM and Bmax of 2.37 pmol/mg of protein. This binding was unevenly distributed among brain regions, with hippocampus greater than cortex greater than olfactory bulb = striatum greater than medulla-pons, and the cerebellum failing to show significant binding. Detailed pharmacological characterization indicated [3H]MK-801 binding to a site which was competitively and potently inhibited by known noncompetitive NMDA receptor antagonists, such as phencyclidine, thienylcyclohexylpiperidine (TCP), ketamine, N-allylnormetazocine (SKF 10,047), cyclazocine, and etoxadrol, a specificity similar to sites labelled by [3H]TCP. These sites were distinct from the high-affinity sites labelled by the sigma receptor ligand (+)-[3H]SKF 10,047. [3H]MK-801 binding was allosterically modulated by the endogenous NMDA receptor antagonist Mg2+ and by other active divalent cations. These data suggest that [3H]MK-801 labels a high-affinity site on the NMDA receptor channel complex, distinct from the NMDA recognition site, which is responsible for the blocking action of MK-801 and other noncompetitive NMDA receptor antagonists.     

Specific binding of (+)-3H-SKF 10047 by mouse splenocytes

Stereoselective, saturable, reversible and temperature-dependent binding of ligand of sigma opioid receptors (+)-3H-SKF 10047 to mouse splenocytes has been demonstrated. The estimated Kd value of this interaction (20-100 nM) was similar to that of (+)-3H-SKF 10047-sigma receptor complex. The binding capacity was 100 molecules per cell. A comparatively low affinity, non-stereoselective and temperature-independent binding of (+)-3H-SKF 10047 to mouse splenocytes has also been found.     

Unsaturated fatty acids as endogenous inhibitors of tamoxifen binding to anti-oestrogen-binding sites.

It is known that triphenylethylene anti-oestrogens such as tamoxifen bind to specific high-affinity anti-oestrogen-binding sites, which are distinct from oestrogen receptors. These binding sites are widely distributed in human and animal tissues, but their function and endogenous ligands are unknown. By using [3H]tamoxifen and a rat liver microsomal fraction, a radio-ligand-binding assay was developed in an attempt to identify endogenous ligands for the anti-oestrogen-binding sites in the rat. An ether extract of rat serum inhibited [3H]tamoxifen binding to rat liver binding sites in a dose-dependent manner. Identification of the active serum constituents that inhibited [3H]tamoxifen binding was achieved by g.l.c.-mass spectrometry after preliminary purification of a rat serum extract by silica-gel t.l.c. Three unsaturated fatty acids (oleic, linoleic and arachidonic) accounted for about 50% of the total inhibiting activity of the serum extract. The concentrations of these fatty acids required to inhibit [3H]tamoxifen binding were in the range of 10-100 microM, comparable with those found in the rat circulation under physiological conditions. Saturated fatty acids present in rat serum (palmitic and stearic) did not inhibit [3H]tamoxifen binding. A survey of other fatty acids revealed that, in general, unsaturated fatty acids were far more potent than saturated fatty acids in inhibiting [3H]tamoxifen binding. These studies demonstrate that unsaturated fatty acids are quantitatively the most important circulating inhibitors of [3H]tamoxifen binding to the anti-oestrogen-binding sites. The biological significance of their interaction with these sites, however, remains to be clarified.     

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.     

Identification of leukotriene D4 specific binding sites in the membrane preparation isolated from guinea pig lung

A radioligand binding assay has been established to study leukotriene specific binding sites in the guinea pig and rabbit tissues. Using high specific activity [3H]-leukotriene D4 [( 3H]-LTD4), in the presence or absence of unlabeled LTD4, the diastereoisomer of LTD4 (5R,6S-LTD4), leukotriene E4 (LTE4) and the end-organ antagonist, FPL 55712, we have identified specific binding sites for [3H]-LTD4 in the crude membrane fraction isolated from guinea pig lung. The time required for [3H]-LTD4 binding to reach equilibrium was approximately 20 to 25 min at 37 degrees C in the presence of 10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. The binding of [3H]-LTD4 to the specific sites was saturable, reversible and stereospecific. The maximal number of binding sites (Bmax), derived from Scatchard analysis, was approximately 320 +/- 200 fmol per mg of crude membrane protein. The dissociation constants, derived from kinetic and saturation analyses, were 9.7 nM and 5 +/- 4 nM, respectively. The specific binding sites could not be detected in the crude membrane fraction prepared from guinea pig ileum, brain and liver, or rabbit lung, trachea, ileum and uterus. In radioligand competition experiments, LTD4, FPL 55712 and 5R,6S-LTD4 competed with [3H]-LTD4. The metabolic inhibitors of arachidonic acid and SKF 88046, an antagonist of the indirectly-mediated actions of LTD4, did not significantly compete with [3H]-LTD4 at the specific binding sites. These correlations indicated that these specific binding sites may be the putative leukotriene receptors in the guinea-pig lung.     

Binding of [3H]SCH23390 in Rat Brain: Regional Distribution and Effects of Assay Conditions and GTP Suggest Interactions at a D1-Like Dopamine Receptor

The compound [9-3H]SCH23390 [R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7- ol] was synthesized, and the binding of this purportedly selective antagonist of D1 3,4-dihydroxyphenylethylamine (dopamine) receptors was characterized. The regional distribution of high-affinity, specific [3H]SCH23390 binding sites in the rat brain correlated well with levels of endogenous dopamine. Receptor densities were greatest in corpus striatum, nucleus accumbens, and olfactory tubercle; intermediate levels were found in several limbic and cortical areas, whereas few sites were detectable in cerebellum, brainstem, and ol-factory bulb. Specific binding in caudate-putamen was found to be both temperature- and pH-dependent, with optima at 25-30 degrees C and pH 7.8-8.0. Scatchard or Woolf analyses of binding in caudate-putamen suggest that most of the sites are either of a single class or of classes with similar characteristics (KD = 0.7 +/- 0.1 nM; Bmax = 347 +/- 35 fmol/mg of protein). Both dopamine and cis-flupenthixol altered the slope but not the intercept of lines generated by Scatchard analysis, suggesting a competitive mode of inhibition of [3H]SCH23390 binding. Competition for binding by dopamine or the D1 agonist SKF38393 was inhibited by guanine nucleotides, whereas GTP had little effect on the competition for binding by the antagonist cis-flupenthixol. The competition for [3H]SCH23390 binding sites by dopamine was much more sensitive to GTP than was competition for [3H]spiperone binding. These data support the hypotheses that [3H]SCH23390 binds to recognition sites that differ from those previously described using other radiolabeled dopamine antagonists and that these sites have the characteristics expected of dopamine receptors.     

Binding of [3H]SKF 38393 to Dopamine D-l Receptors in Rat Striatum In Vitro; Estimation of Receptor Molecular Size by Radiation Inactivation

[3H]SKF 38393 (2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine) binds with high affinity to 3,4-dihydroxyphenylethylamine (dopamine) D-1 receptors in rat striatum in vitro (KD = 7 and 14 nM in nonfrozen and frozen striatum, respectively). The number of binding sites (Bmax) was approximately 80.0 pmol/g of original tissue, a value similar to the Bmax for the dopamine D-1 antagonist SCH 23390. Nondisplaceable [3H]SKF 38393 binding was approximately 45% of total binding. Irradiation (0-4 Mrad) of frozen whole striata decreased the number of [3H]SKF 38393 binding sites monoexponentially without changing the binding affinity. The functional molecular mass for the agonist dopamine D-1 binding site was 132,800 daltons, which is higher than the functional molecular mass of the antagonist dopamine D-1 binding site (approximately 80,000 daltons).