[3H]Harman Binding Experiments. II: Regional and Subcellular Distribution of Specific [3H]Harman Binding and Monoamine Oxidase Subtypes A and B Activity in Marmoset and Rat

[3H]Harman (1-[3H]methyl-beta-carboline) was used in a novel radioligand binding assay to label selectively and with high affinity monoamine oxidase (MAO) type A. The concentration of the enzyme was determined in six CNS regions of the primate species marmoset (Callithrix jacchus) and of the rat: hypothalamus, hippocampus, cerebellum, cerebral cortex, striatum, and spinal cord. The specific [3H]harman binding in the CNS of the marmoset reveals the same pharmacological profile and other characteristics (affinity, saturability, and reversibility) as in the CNS of the rat. The regional distribution of the [3H]harman binding density (Bmax) in the CNS exhibits a distinct pattern in the marmoset and the rat and a 35 (hypothalamus) to 75% (hippocampus) lower Bmax in the marmoset than in the rat. The Bmax values of [3H]harman binding in the CNS of the marmoset and the rat combined as well as those from visceral organs of the rat (liver, heart, lung, thymus, spleen, and kidney) correlated positively and highly significantly with the respective Vmax values of specific MAO activity of the A type but not of the B type, determined with kynuramine as the substrate. In subcellular fractionation experiments with rat cerebral cortex, the highest [3H]harman binding density (Bmax) and MAO-A activity (Vmax) were detected in mitochondrial fractions and severalfold lower values in the synaptosomal membrane fraction. In conclusion, we suggest that [3H]harman binding is a biochemical tool as a selective marker to quantify MAO-A in the CNS of different mammalian species as well as in extraneuronal tissues.     

Neuropeptide Y receptor in the rat brain

The specific binding of the chloramine-T iodinated neuropeptide Y (125I-NPY) to membranes from rat cerebral cortex was investigated using equilibrium binding and kinetic methods. The equilibrium binding of 125I-NPY at 37 degrees C was characterized by a Kd value of 0.38 nM. The receptor densities in the cerebral cortex, hypothalamus and cerebellum were 0.45 pmol/mg, 0.47 pmol/mg and 0.04 pmol/mg protein respectively. The binding site for 125I-NPY was sensitive to treatment with proteolytic enzymes and thiol reagents. The binding showed a sharp optimum at pH 7-7.7 and was inhibited by increasing concentrations of Mg2+.     

Evidence for Distinct 5-Hydroxytryptamine2 Binding Site Subtypes in Cortical Membrane Preparations

5-Hydroxytryptamine (5-HT) displays a sixfold higher affinity for 5-HT2 binding sites labeled by [3H]ketanserin in rat (IC50 = 200 +/- 40 nM) and human (IC50 = 190 +/- 50 nM) cortex than for 5-HT2 sites in bovine cortex (IC50 = 1,200 +/- 130 nM). The Hill slopes of the 5-HT competition curves are 0.67 +/- 0.04 in rat, 0.69 +/- 0.08 in human, and 0.96 +/- 0.02 in bovine cortex. Scatchard analysis of (+/-)-[3H]4-bromo-2,5-dimethoxyamphetamine ([3H]DOB) binding in the rat indicates a population of binding sites with a KD of 0.38 +/- 0.04 nM and a Bmax of 1.5 +/- 0.05 pmol/g tissue. In contrast, specific [3H]DOB binding cannot be detected in bovine cortical membranes. These data indicate that species variations exist in 5-HT2 binding site subtypes and that [3H]ketanserin appears to label a homogeneous population of 5-HT2 binding site subtypes in bovine cortex.     

Buprenorphine: High-Affinity Binding to Dorsal Spinal Cord

The binding of the mixed opiate agonist-antagonist [3H]buprenorphine was compared with [3H]naloxone and [3H]dihydromorphine binding in membranes prepared from rat whole brain and dorsal spinal cord. Scatchard analysis of binding to whole brain yielded KD values close to 1.0 nM for all three 3H-ligands studied, although [3H]buprenorphine labelled five times as many binding sites. [3H]Naloxone and [3H]dihydromorphine bound to dorsal spinal cord with approximately the same affinity as to whole brain, although both 3H-ligands labelled fewer sites in the spinal cord. In contrast, Scatchard analysis of [3H]buprenorphine binding to spinal cord yielded curvilinear Scatchard plots, suggesting the presence of a very high-affinity (KD = 0.12 nM) binding site in addition to the high-affinity site (KD = 1.0 nM) present in the brain. Studies on the displacement of [3H]buprenorphine by opiates and D-Ala2,Met5-enkephalinamide supported the presence of two binding sites for this ligand in the spinal cord.     

Characterization of L-Glutamate Binding Sites in Rat Spinal Cord Synaptic Membranes: Evidence for Multiple Chloride Ion-Dependent Sites

The effects of various ions on L-glutamate (L-Glu) binding sites (Na+-dependent, Cl(-)-dependent, and Cl(-)-independent) in synaptic plasma membranes (SPM) isolated from rat spinal cord and forebrain were examined. Cl(-)-dependent binding sites were over twofold higher in spinal cord (Bmax = 152 +/- 34 pmol/mg protein) as compared to forebrain SPM (Bmax = 64 +/- 12 pmol/mg protein). Na+-dependent binding, on the other hand, was nearly sixfold less in spinal cord (Bmax = 74 +/- 10 pmol/mg protein) compared to forebrain SPM (408 +/- 26 pmol/mg protein). Uptake of L-Glu (Na+-dependent) was also eightfold less in the P2 fraction from spinal cord relative to forebrain (Vmax of 2.89 and 22.3 pmol/mg protein/min, respectively). The effects of Na+, K+, NH4+, and Ca2+ on L-Glu binding sites were similar in both regions of the CNS. In addition, in spinal cord membranes, Br-, I-, and NO3- were equivalent to Cl- in their capacity to stimulate L-Glu binding, whereas F- and CO3- were less effective. Cl(-)-dependent L-Glu binding in spinal cord membranes consisted of two distinct sites. The predominant site (74% of the total) had characteristics similar to the Cl(-)-dependent binding site in forebrain membranes [i.e., Ki values of 5.7 +/- 1.4 microM and 119 +/- 38 nM for 2-amino-4-phosphonobutyric acid (AP4) and quisqualic acid, (QUIS), respectively]. The other Cl(-)-dependent site was unaffected by AP4 but was blocked by QUIS (Ki = 14.2 +/- 4.8 microM).     

Binding sites of a novel neuropeptide pituitary-adenylate-cyclase-activating polypeptide in the rat brain and lung

Pituitary-adenylate-cyclase-activating polypeptide (PACAP) is a novel 38-amino-acid neuropeptide isolated from ovine hypothalamic tissues based on its activity of stimulating adenylate cyclase of rat pituitary cells. Binding sites for PACAP were studied in rat tissue membranes using a 27-amino-acid N-terminal derivative of PACAP [PACAP(1-27)] labelled with 125I. Particularly high specific binding sites of 125I-PACAP(1-27) were noted in the hypothalamus, brain stem, cerebellum and lung. Specific binding sites are also present in the pituitary gland, but at a lower concentration, and mainly in the anterior lobe. Very low concentration of 125I-PACAP(1-27)-binding sites were found in the colon, aorta and kidney membranes and no binding sites were detected in the pancreas and testis. Maximal binding of 125I-PACAP(1-27) was observed at pH 7.4. Interaction of 125I-PACAP(1-27) with its binding site was rapid, specific and saturable as well as time, pH and temperature dependent. PACAP(1-27) is more potent than PACAP in displacing the binding of 125I-PACAP(1-27) with brain membranes [concentration that inhibits 50% of the binding (IC50) = 7.45 +/- 1.52 nM and 11.45 +/- 3.65 nM, respectively; mean +/- SEM, n = 4] and lung membranes (IC50 = 4.41 +/- 0.87 nM and 10.68 +/- 3.09 nM, respectively). Vasoactive intestinal peptide displaced the binding of 125I-PACAP(1-27) in lung membrane (IC50 = 16.88 +/- 5.14 nM) but not in brain membranes. The equilibrium binding of 125I-PACAP(1-27) at 4 degrees C was characterized by a single class of binding site for the brain membrane with a dissociation constant (Kd) of 2.46 +/- 0.53 nM and a maximal binding capacity (Bmax) of 8.44 +/- 3.13 pmol/mg protein, but there were two classes of binding site for lung membranes with Kd of 1.02 +/- 0.51 nM and 5.19 +/- 0.99 nM, and Bmax of 2.84 +/- 0.72 pmol/mg protein and 9.13 +/- 1.89 pmol/mg protein, respectively. These findings suggest that subtypes of PACAP-binding sites exist and PACAP may have a physiological role in the hypothalamus/pituitary axis as well as in other regions of the brain and lung.     

Characterization of the Binding of [3H]SR 95531, a GABAA Antagonist, to Rat Brain Membranes

A synthetic derivative of gamma-aminobutyric acid (GABA), SR 95531 [2-(3'-carboxy-2'-propyl)-3-amino-6-p-methoxyphenylpyridazinium bromide], has recently been reported, on the basis of biochemical and in vivo microiontophoretic studies, to be a potent, selective, competitive, and reversible GABAA antagonist. In the present study, the binding of [3H]SR 95531 to washed, frozen, and thawed rat brain membranes was characterized. Specific binding was linear with tissue concentrations, had a pH optimum at neutrality, and was maximal at 4 degrees C after 30 min of incubation. Pretreatment of the membranes with Triton X-100 resulted in a 50% decrease of specific binding. Addition of iodide, thiocyanate, or nitrate to the incubation mixture decreased the affinity of [3H]SR 95531 for its binding site; Na+ had no effect. Subcellular fractionation showed that 74% of the P2 binding was in synaptosomes; 31% of the total homogenate binding was in P2 and 50% in P3. The binding of [3H]SR 95531 was saturable; Scatchard analysis of the saturation isotherm revealed two apparent populations of binding sites (KD of 6.34 nM and Bmax of 0.19 pmol/mg of protein; KD of 32 nM and Bmax of 0.81 pmol/mg of protein). The binding of [3H]SR 95531 was reversible, and association and dissociation kinetics confirmed the existence of two binding sites. Only GABAA ligands were effective displacers of [3H]SR 95531. GABAA antagonists were relatively more potent in displacing [3H]SR 95531 than [3H]GABA; the inverse was true for GABAA agonists. There were marked regional differences in the distribution of binding sites: hippocampus = cerebral cortex greater than thalamus = olfactory bulb = hypothalamus = amygdala = striatum greater than pons-medulla and cerebellum. The surprisingly low density of binding sites in the cerebellum was owing to a marked reduction of Bmax values at both the high- and the low-affinity binding sites. In conclusion, the present results demonstrate specific, high-affinity, saturable, and reversible binding of [3H]SR 95531 to rat brain membranes and strongly suggest that this radioligand labels the GABAA receptor site in its antagonist conformation.     

CL 218,872 Binding to Benzodiazepine Receptors in Rat Spinal Cord: Modulation by γ-Aminobutyric Acid and Evidence for Receptor Heterogeneity

The binding of the triazolopyridazine CL 218,872 to central benzodiazepine receptors identified with [3H]Ro 15-1788 was studied in extensively washed homogenates of rat spinal cord and cerebral cortex. CL 218,872 displacement curves were shallow in both spinal cord (nH = 0.67) and cortex (nH = 0.54), suggesting the presence of type 1 and type 2 benzodiazepine receptors in both tissues. CL 218,872 had lower affinity in spinal cord (IC50 = 825 nM) than cortex (IC50 = 152 nM), possibly reflecting the presence of fewer type 1 sites in the cord. Activating gamma-aminobutyric acid (GABA) receptors with 10 microM muscimol resulted in a two- to threefold increase in CL 218,872 affinity in both tissues without changes in the displacement curve slope. This indicates that GABA enhances CL 218,872 affinity for both type 1 and type 2 sites in both spinal cord and cerebral cortex.     

Opiate antagonist binding sites in discrete brain regions of spontaneously hypertensive and normotensive Wistar-Kyoto rats

The binding of 3H-naltrexone, an opiate receptor antagonist, to membranes of discrete brain regions and spinal cord of 10 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. The brain regions examined were hypothalamus, amygdala, hippocampus, corpus striatum, pons and medulla, midbrain and cortex. 3H-Naltrexone bound to membranes of brain regions and spinal cord at a single high affinity site with an apparent dissociation constant value of 3 nM. The highest density of 3H-naltrexone binding sites were in hippocampus and lowest in the cerebral cortex. The receptor density (Bmax value) and apparent dissociation constant (Kd value) values of 3H-naltrexone to bind to opiate receptors on the membranes of amygdala, hippocampus, corpus striatum, pons and medulla, midbrain, cortex and spinal cord of WKY and SHR rats did not differ. The Bmax value of 3H-naltrexone binding to membranes of hypothalamus of SHR rats was 518% higher than WKY rats but the Kd values in the two strains did not differ. It is concluded that SHR rats have higher density of opiate receptors labeled with 3H-naltrexone in the hypothalamus only, in comparison with WKY rats, and that such a difference in the density of opiate receptors may be related to the elevated blood pressure in SHR rats.     

Specific High-Affinity Binding of L-[3H]Aspartate to Rat Brain Membranes

Abstract: The binding of L-[³H]aspartate was investigated in washed membranes prepared from whole rat brain. We were able to differentiate two separate binding sites differing in their Na dependence. The Na-independent binding was saturable, reversible, and optimal at 20°C and at pHs in the neutral range. The dissociation constant (K_d) at 20°C was about 200 n M . This binding site seemed to be modulated by magnesium and calcium at physiological concentrations. None of the amino acids tested was a potent competitor for Na-independent L-[³H]aspartate binding. This binding site was unevenly distributed in the rat central nervous system: cerebellum = cerebral cortex > ponsmedulla > spinal cord. Destruction of the intrinsic neurons of the cerebellum by injecting kainic acid 30 days before sacrifice resulted in a 53% reduction in Na-independent binding in this region. The Na-dependent binding of L-[³H]-aspartate (K_d= 484 n M ) was strongly inhibited by D-aspartate, L-glutamate, D,L-aspartate β-hydroxamate; was unaffected by calcium and magnesium; and showed a different pattern of distribution: cerebral cortex > cerebellum = pons-medulla = spinal cord. This binding in cerebellum was unaffected by injections of kainic acid.     

n-[3H]Butyl-β-Carboline-3-Carboxylate, a Putative Endogenous Ligand, Binds Preferentially to Subtype 1 of Central Benzodiazepine Receptors

Synthetic n-butyl beta-carboline-3-carboxylate, an endogenous central benzodiazepine receptor inhibitor found in brain, was tritium-labeled from the butenyl ester. Binding of this [3H]beta-carboline was concentrated particularly in the synaptosomal membrane fraction of the cerebral cortex; this fraction showed a single type of high-affinity site (KD = 2.7 +/- 0.1 nM) with a Bmax of 1.16 +/- 0.08 pmol/mg of protein. The number of sites labeled was about half of that obtained with [3H]flunitrazepam binding (Bmax = 2.36 +/- 0.06 pmol/mg of protein). On the other hand, in the cerebellum, both ligands bound to practically the same number of sites. When [3H]flunitrazepam binding was done in the presence of 10(-11)-10(-5) M butyl beta-carboline, the differences between the two brain regions were more apparent. In cerebellar membranes the data fitted a straight line in the Eadie-Hofstee plot; this finding and a Hill number near unity suggest a single type of binding site. In the cortical membranes the data of binding fitted a concave curve, and the Hill number was 0.6. These are characteristics of two types of binding sites with different affinities (KD1 = 0.6-1.5 nM and KD2 = 12-18 nM). The differentiation of a high- and low-affinity site in the cerebral cortex was corroborated by experiments in which [3H]butyl beta-carboline binding was displaced by the triazolopyridazine CL 218,872. These results demonstrate that in the cerebral cortex there are two subtypes of sites (1 and 2) of central benzodiazepine receptors and that CL 218,872 binds preferentially to subtype 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycine Binding to Rat Cortex and Spinal Cord: Binding Characteristics and Pharmacology Reveal Distinct Populations of Sites

Glycine is the principal inhibitory neurotransmitter in posterior regions of the brain. In addition, glycine serves as an allosteric regulator of excitatory neurotransmission mediated by the N-methyl-D-aspartate (NMDA) acidic amino acid receptor subtype. The studies presented here characterize [3H]glycine binding to washed membranes prepared from rat spinal cord and cortex, areas enriched in glycine inhibitory and NMDA receptors, respectively, in an attempt to define the glycine recognition sites on the two classes of receptors. Specific binding for [3H]glycine was seen in both cortex and spinal cord. Saturation analyses in cortex were best fitted by a two-site model with respective equilibrium dissociation constants (KD values) of 0.24 and 5.6 microM and respective maximal binding constants (Bmax values) of 3.4 and 26.7 pmol/mg of protein. Similar analyses in spinal cord were best fitted by a one-site model with a KD of 5.8 microM and Bmax of 20.2 pmol/mg of protein. Na+ had no effect on [3H]glycine binding to cortical membranes but increased the binding to spinal cord membranes by greater than 15-fold. This Na+-dependent binding may reflect glycine binding to the recognition site of the high-affinity, Na+-dependent glycine uptake system. Several short-chain, neutral amino acids displaced [3H]glycine binding from both cortical and spinal cord membranes. The most potent displacers of [3H]glycine binding to cortical membranes were D-serine and D-alanine, followed by the L-isomers of serine and alanine and beta-alanine. In contrast, D-serine and D-alanine were similar in potency to L-serine in spinal cord membranes. Compounds active at receptors for the acidic amino acids had disparate effects on the binding of [3H]glycine. At 10 microM, NMDA resulted in a 25% increase, whereas D- and L-2-amino-5-phosphonovaleric acid at 100 microM resulted in a 30% decrease, in [3H]glycine binding to cortical membranes. Kynurenic acid was the most potent of the acidic amino acid-related compounds at displacing [3H]glycine binding. In cortical membranes, kynurenic acid displacement was resolved into a high- and a low-affinity component; the high-affinity component displaced the high-affinity component of [3H]glycine binding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increased γ-Aminobutyric Acid Receptor Function in the Cerebral Cortex of Myoclonic Calves with an Hereditary Deficit in Glycine/Strychnine Receptors

Inherited congenital myoclonus (ICM) of Poll Hereford cattle is a neurological disease in which there are severe alterations in spinal cord glycine-mediated neurotransmission. There is a specific and marked decrease, or defect, in glycine receptors and a significant increase in neuronal (synaptosomal) glycine uptake. Here we have examined the characteristics of the cerebral gamma-aminobutyric acid (GABA) receptor complex, and demonstrate that the malfunction of the spinal cord inhibitory system is accompanied by a change in the major inhibitory system in the cerebral cortex. In synaptic membrane preparations from ICM calves, both high-and low-affinity binding sites for the GABA agonist [3H]muscimol were found (KD = 9.3 +/- 1.5 and 227 +/- 41 nM, respectively), whereas only the high-affinity site was detectable in controls (KD = 14.0 +/- 3.1 nM). The density and affinity of benzodiazepine agonist binding sites labelled by [3H]diazepam were unchanged, but there was an increase in GABA-stimulated benzodiazepine binding. The affinity for t-[3H]butylbicyclo-o-benzoate, a ligand that binds to the GABA-activated chloride channel, was significantly increased in ICM brain membranes (KD = 148 +/- 14 nM) compared with controls (KD = 245 +/- 33 nM). Muscimol-stimulated 36Cl- uptake was 12% greater in microsacs prepared from ICM calf cerebral cortex, and the uptake was more sensitive to block by the GABA antagonist picrotoxin. The results show that the characteristics of the GABA receptor complex in ICM calf cortex differ from those in cortex from unaffected calves, a difference that is particularly apparent for the low-affinity, physiologically relevant GABA receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the binding sites for nimodipine and (-)-desmethoxyverapamil in bovine cardiac sarcolemma

The bovine cardiac sarcolemmal binding sites for the dihydropyridine nimodipine and the phenylalkylamine (-)-desmethoxyverapamil were studied. The density of the nimodipine and (-)-desmethoxyverapamil binding sites increased 8.3-fold and 3.4-fold with the sarcolemma. The binding sites for both compounds were destroyed by trypsin. Nimodipine bound in the presence of 1 mM free calcium to a high-affinity and a low-affinity site with apparent Kd values of 0.35 +/- 0.09 nM (n = 9) and 33 +/- 6.0 nM (n = 9) and with apparent densities of 0.3 +/- 0.05 pmol/mg (n = 9) and 8.2 +/- 1.0 pmol/mg (n = 9). The binding to the high-affinity site was abolished by 1 mM EGTA. The binding sites were specific for dihydropyridines. The (-)-isomers of several phenylalkylamines inhibited nimodipine binding by an apparent allosteric mechanism. (-)-Desmethoxyverapamil bound in the presence of 5 mM EGTA to a high-affinity and a low-affinity site with apparent Kd values of 1.4 +/- 0.3 nM (n = 6) and 171 +/- 26 nM (n = 6) and with apparent densities of 0.16 +/- 0.02 pmol/mg (n = 6) and 13.6 +/- 2.7 pmol/mg (n = 6). The binding to both sites was inhibited by calcium with a half-maximal concentration of 4.3 mM. The binding sites were specific for the other phenylalkylamines and had a higher affinity for the (-)-isomers than for the (+)-isomers. Nimodipine inhibited the binding of (-)-desmethoxyverapamil by an apparent allosteric mechanism. d-cis-Diltiazem inhibited non-competitively the binding of (-)-[3H]desmethoxyverapamil with a Ki of 3.7 microM. Diltiazem up to concentrations of 10 microM did not affect the amount of nimodipine bound at equilibrium at 20 degrees C. However, but in agreement with this result, diltiazem decreased threefold at 20 degrees C the dissociation and association rates for the high-affinity nimodipine receptor. These rates were only marginally affected at 4 degrees C and 37 degrees C. d-cis-Diltiazem reversed in a competitive manner the inhibition of nimodipine binding elicited by the addition of (-)-desmethoxyverapamil with a Ka value of 1.6 microM. The amount of nimodipine bound was inhibited by 50% by the adenosine uptake inhibitors nitrobenzylthioinosine and hexobendine with apparent median inhibitory concentrations of 1 nM and 3 nM, respectively. Nitrobenzylthioinosine completely abolished binding of nimodipine to the low-affinity site, but did not affect binding to the high-affinity site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pharmacological Differentiation and Characterization of 5-HT1A, 5-HT1B, and 5-HT1C Binding Sites in Rat Frontal Cortex

Drug interactions with 5-HT1 (5-hydroxytryptamine type 1) binding site subtypes were analyzed in rat frontal cortex. 8-Hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) displays high affinity (Ki 3.3 +/- 1 nM) for 29 +/- 3% of total [3H]5-HT binding in rat frontal cortex and low affinity (Ki 9,300 +/- 1,000) for 71 +/- 4% of the remaining 5-HT1 sites. Therefore, non-5-HT1A binding in rat frontal cortex was defined as specific [3H]5-HT binding observed in the presence of 100 nM 8-OH-DPAT. 5-Methoxy 3-(1,2,3,6-tetrahydro-4-pyridinyl) 1 H indole (RU 24969), 1-(m-trifluoromethylphenyl)piperazine (TFMPP), mianserin, and methysergide produce shallow competition curves of [3H]5-HT binding from non-5-HT1A sites. Addition of 10(-3) M GTP does not increase the apparent Hill slopes of these competition curves. Computer-assisted iterative curve fitting suggests that these drugs can discriminate two distinct subpopulations of non-5-HT1A binding sites, each representing approximately 35% of the total [3H]5-HT binding in the rat frontal cortex. All three 5-HT1 binding site subtypes display nanomolar affinity for 5-HT and 5-methoxytryptamine. A homogeneous population of 5-HT1A sites can be directly labeled using [3H]8-OH-DPAT. These sites display nanomolar affinity for 8-OH-DPAT, WB 4101, RU 24969, 2-(4-[4-(2-pyrimidinyl)-1-piperazinyl] butyl)-1,2-benzisothiazol-3-(2H)one-1, 1-dioxidehydrochloride (TVX Q 7821), 5-methoxydimethyltryptamine, and d-lysergic acid diethylamide. The potencies of RU 24969, TFMPP, and quipazine for [3H]5-HT binding are increased by addition of 100 nM 8-OH-DPAT and 3,000 nM mianserin to the [3H]5-HT binding assay. Moreover, the drugs have apparent Hill slopes near 1 under these conditions. This subpopulation of total [3H]5-HT binding is designated 5-HT1B. By contrast, methysergide and mianserin become more potent inhibitors of residual [3H]5-HT binding to non-5-HT1A sites in the presence of 100 nM 8-OH-DPAT and 10 nM RU 24969. The drug competition curves under these conditions have apparent Hill slopes of near unity and these sites are designated 5-HT1C. Drug competition studies using a series of 24 agents reveals that each 5-HT1 subtype site has a unique pharmacological profile. These results suggest that radioligand studies can be used to differentiate three distinct subpopulations of 5-HT1 binding sites labeled by [3H]5-HT in rat frontal cortex.     

Characterization of a neurokinin B receptor site in rat brain using a highly selective radioligand

We have recently characterized a tachykinin receptor subtype (SP-N) whose preferred ligand is the mammalian neuropeptide, neurokinin B (Laufer, R., Wormser, U., Friedman, Z. Y., Gilon, C., Chorev, M., and Selinger, Z. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7444-7448). To investigate this novel tachykinin receptor, we have now prepared a radiolabeled peptide, N alpha-[( 125I]desamino-3-iodotyrosyl)-[Asp5,6, N-methyl-Phe8]substance P (5-11) heptapeptide (125I-BH-NH-Senktide), which selectively interacts with the SP-N receptor subtype. The binding of 125I-BH-NH-Senktide to rat cerebral cortex membranes was studied under conditions that minimized nonspecific binding. Unlike other tachykinin receptor probes, this radioligand is not degraded during the binding experiment. Binding of 125I-BH-NH-Senktide is reversible, saturable, and of high affinity (KD = 0.9 nM). The radioligand labels a single class of binding site (122 fmol binding sites/mg of protein), as indicated by a linear Scatchard plot and a Hill coefficient close to unity (nH = 1.05). The pharmacological specificity of this binding site corresponds to that of the neuronal SP-N receptor in guinea pig ileum myenteric plexus, which was determined by a functional bioassay. Among various rat brain regions, the highest binding was observed in the cerebral cortex, olfactory bulb, hypothalamus, and hippocampus. These results suggest the existence and specific distribution of a neurokinin B receptor site of the SP-N type in rat brain. 125I-BH-NH-Senktide is the first selective and potent probe for this receptor and is thus an important tool for further studies of its distribution, regulation, and functional role.     

Characterization of rat hypothalamic progestin binding by spheroidal hydroxylapatite chromatography.

The progestin-high-affinity-binding components in rat target tissues have been assayed by a simple and precise procedure by using spheroidal hydroxylapatite. The progestin 'receptors' in the uterus and hypothalamus of female rats are highly specific for progestins, which they bind with high affinity (Kd for [3H]progesterone in hypothalamus is 1.9 nM and in uterus is 3.7 nM). The dissociation of [3H]progesterone from receptor in vitro is rapid: t1/2 6 degrees C = 45 min in uterine cytosol; t1/2 6 degrees C = 160 min in hypothalamic cytosol. The binding is destroyed by proteinase. In the cytosol of hypothalamus and cortex of developing rats, progestin 'receptors' were present in both male and female rats by 2-3 days after birth; subsequent changes in concentration of these 'receptors' appeared to be independent of sex. Concentrations of progestin 'receptor' were close to adult values by 8-9 days, and thereafter changed relatively little.     

Properties and distribution of receptors for pituitary adenylate cyclase activating peptide (PACAP) in rat brain and spinal cord.

A high density (in the pmol/mg protein range) of specific functional receptors for PACAP (pituitary adenylate cyclase activating polypeptide) was observed in membranes from rat brain cortex, olfactory bulb, hypothalamus, hippocampus, striatum, cerebellum, pons and cervico-dorsal spinal cord, using [125I]PACAP-27 (PACAP 1-27). The tracer bound rapidly, specifically and reversibly. Competition binding curves were compatible with the coexistence, in the eight central nervous areas explored, of high and low affinity binding sites for PACAP-27 (Kd of 0.2 nM and 3.0 nM, respectively), and of only one class of binding sites for PACAP-38 (PACAP (1-38), Kd 0.2-0.9 nM). VIP inhibited only partially the binding of [125I]PACAP-27, and PHI, GRF(1-29)NH2 and secretin were ineffective at 1 microM. Chemical [125I]PACAP-27 cross-linking revealed a single specific 64 kDa protein species. In rat brain cortical membranes, saturation and competition experiments, using [125I]PACAP-38 as radioligand, indicated the presence of both high (Kd 0.13 nM) and low (Kd 8-10 nM) affinity binding sites for PACAP-38 and of low affinity (Kd 30 nM) binding sites for PACAP-27. These data taken collectively suggest the coexistence of PACAP-A receptors with a slight preference for PACAP-27 over PACAP-38 and of PACAP-B receptors that recognize PACAP-38 with a high affinity and PACAP-27 with low affinity. Both PACAP-27 and PACAP-38 stimulated adenylate cyclase with similar potency and efficacy. VIP was markedly less potent in this respect and also less efficient, except on cerebellar membranes.     

Characterization of Two Classes of Opioid Binding Sites in Drosophila melanogaster Head Membranes

Opioid receptors have been characterized in Drosophila neural tissue. [3H]Etorphine (universal opioid ligand) bound stereospecifically, saturably, and with high affinity (KD = 8.8 +/- 1.7 nM; Bmax = 2.3 +/- 0.2 pmol/mg of protein) to Drosophila head membranes. Binding analyses with more specific ligands showed the presence of two distinct opioid sites in this tissue. One site was labeled by [3H]dihydromorphine ([3H]DHM), a mu-selective ligand: KD = 150 +/- 34 nM; Bmax = 3.0 +/- 0.6 pmol/mg of protein. Trypsin or heat treatment (100 degrees C for 15 min) of the Drosophila extract reduced specific [3H]DHM binding by greater than 80%. The rank order of potency of drugs at this site was levorphanol greater than DHM greater than normorphine greater than naloxone much greater than dextrorphan; the mu-specific peptide [D-Ala2,Gly-ol5]-enkephalin and delta-, kappa-, and sigma-ligands were inactive at this site. The other site was labeled by (-)-[3H]ethylketocyclazocine ((-)-[3H]EKC), a kappa-opioid, which bound stereospecifically, saturably, and with relatively high affinity to an apparent single class of receptors (KD = 212 +/- 25 nM; Bmax = 1.9 +/- 0.2 pmol/mg of protein). (-)-[3H]EKC binding could be displaced by kappa-opioids but not by mu-, delta-, or sigma-opioids or by the kappa-peptide dynorphin. Specific binding constituted approximately 70% of total binding at 1 nM and approximately 50% at 800 nM for all three radioligands ([3H]etorphine, [3H]EKC, and [3H]DHM). Specific binding of the delta-ligands [3H][D-Ala2,D-Leu5]-enkephalin and [3H][D-Pen2,D-Pen5]-enkephalin was undetectable in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycine Receptors in the Human Substantia Nigra as Defined by [3H]Strychnine Binding

Abstract: Specific [³H]strychnine binding was used to identify the glycine receptor macromolecular complex in human spinal cord, substantia nigra, inferior olivary nucleus, and cerebral cortex. In material from control patients a high-affinity K d (3–8 n m ) was observed in the spinal cord and the substantia nigra, both the pars compacta and the pars reticulata. This is very similar to the values observed in the rat and bovine spinal cord (8 and 3 n m , respectively) and rat substantia nigra (12 n m ). In the human brain the distribution of [³H]strychnine binding (at 10 n m ) was: spinal cord – substantia nigra, pars compacta > substantia nigra, pars reticulata = inferior olivary nucleus > cerebral cortex. The binding capacity ( B _max) of the rat brain (substantia nigra or spinal cord) was approximately 10-fold that of the human brain. [ ³ H]Strychnine binding was significantly decreased in the substantia nigra from Parkinson's disease patients, both in the pars compacta (67% of control) and the pars reticulata (50% of control), but not in the inferior olivary nucleus. The results were reproduced in a preliminary experiment in rats with unilateral 6-hydroxydopamine lesions of the medial forebrain bundle. In the substantia nigra from patients who died with Huntington's disease, [³H]strychnine binding tended to be high (150% of control, NS) in both the pars compacta and the reticulata. [³H]Strychnine binding was unaltered in the substantia nigra of patients with senile dementia. Together with previous neurophysiological and neuropharmacological findings, those results support the hypothesis of glycine receptors occurring on dopamine cell bodies and/or dendrites in the substantia nigra.