Biochemical and Pharmacological Characterization of Serotonin-O-Carboxymethylglycyl[125I]Iodotyrosinamide5 a New Radioiodinated Probe for 5-HT1B and 5-HT1D Binding Sites

There is a lack of radioactive probes, particularly radioiodinated probes, for the direct labeling of serotonin-1B (5-HT1B) and serotonin-1D (5-HT1D) binding sites. Serotonin-O-carboxymethylglycyltyrosinamide (S-CM-GTNH2) was shown previously to be specific for these two subtypes; we, therefore, linked a 125I to its tyrosine residue. Biochemical and pharmacological properties of S-CM-G[125I]TNH2-binding sites were studied by quantitative autoradiography on rat and guinea pig brain sections. S-CM-G[125I]TNH2 binding is saturable and reversible with a KD value of 1.3 nM in the rat and 6.4 nM in the guinea pig. Binding is heterogeneous, paralleling the anatomical distribution of 5-HT1B sites in the rat and of 5-HT1D sites in the guinea pig. The binding of 0.02 nM S-CM-G[125I]TNH2 was inhibited by low concentrations of 5-HT, S-CM-GTNH2, CGS 12066 B, 5-methoxytryptamine, and tryptamine in both species. Propranolol inhibited the radioligand binding with a greater affinity in the rat than in the guinea pig. Conversely, 8-hydroxy-2-(di-n-propylamino)tetralin inhibited S-CM-G[125I]TNH2 binding with a greater affinity in the guinea pig than in the rat. Other competitors, specific for 5-HT1C, 5-HT2, 5-HT3, and adrenergic receptors, inhibited S-CM-G[125I]TNH2 binding in rat and guinea pig substantia nigra and in other labeled structures known to contain these receptors, but only at high concentrations. S-CM-G[125I]TNH2 is then a useful new probe for the direct study of 5-HT1B and 5-HT1D binding sites.     

Existence of Three Subtypes of Bradykinin B2 Receptors in Guinea Pig

We describe the binding of [3H]bradykinin to homogenates of guinea pig brain, lung, and ileum. Analysis of [3H]bradykinin binding kinetics in guinea pig brain, lung, and ileum suggests the existence of two binding sites in each tissue. The finding of two binding sites for [3H]bradykinin in ileum, lung, and brain was further supported by Scatchard analysis of equilibrium binding in each tissue. [3H]Bradykinin binds to a high-affinity site in brain, lung, and ileum (KD = 70-200 pM), which constitutes approximately 20% of the bradykinin binding, and to a second, lower-affinity site (0.63-0.95 nM), which constitutes the remaining 80% of binding. Displacement studies with various bradykinin analogues led us to subdivide the high- and lower-affinity sites in each tissue and to suggest the existence of three subtypes of B2 receptors in the guinea pig, which we classify as B2a, B2b, and B2c. Binding of [3H]bradykinin is largely to a B2b receptor subtype, which constitutes the majority of binding in brain, lung, and ileum and represents the lower-affinity site in our binding studies. Receptor subtype B2c constitutes approximately 20% of binding sites in the brain and lung and is equivalent to the high-affinity site in brain and lung. We suggest that a third subtype of B2 receptor (high-affinity site in ileum), B2a, is found only in the ileum. All three subtypes of B2 receptors display a high affinity for bradykinin, whereas they show different affinities for various bradykinin analogues displaying agonist or antagonist activities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific Binding of [3H]Pyrilamine to Histamine H1 Receptors in Guinea Pig Brain In Vivo: Determination of Binding Parameters by a Kinetic Four-Compartment Model

The binding of [3H]pyrilamine, a selective ligand of histamine H1 receptors, to guinea pig brain in vivo was compared with its binding to a brain homogenate. The pharmacological properties (regional distribution, saturability, and stereoselectivity) of the [3H]pyrilamine binding in vivo were similar to those of the in vitro binding to brain homogenate. A dynamic four-compartment model was proposed for the analysis of the kinetics of [3H]pyrilamine binding in vivo. The receptor constants in vivo were determined by a computer-fitting method after correcting the radioactivity of arterial plasma and brain for the presence of radioactive metabolites. The in vivo association and dissociation were 213 and 42 times, respectively, slower than those of in vitro binding at 37 degrees C. A possible mechanism for slow association and dissociation in vivo is discussed.     

[3H]Nitrobenzylthioinosine Binding to the Guinea Pig CNS Nucleoside Transport System: A Pharmacological Characterization

The binding of [3H]nitrobenzylthioinosine (NBMPR) to specific membrane sites in guinea pig brain was rapid, reversible, and saturable, and was dependent upon protein concentration, pH, and temperature. Mass law analysis of the binding data for cortical membranes indicated that NBMPR bound with high affinity to a single class of sites at which the equilibrium dissociation constant (KD) for NBMPR was 0.10-0.25 nM and which possessed a maximum binding capacity (Bmax) per mg of protein of 300 fmol of NBMPR. Kinetic analysis of the site-specific binding of NBMPR yielded an independent estimate of the KD of 0.16 nM. A relatively homogeneous subcellular distribution of the sites for NBMPR was found in cortical tissue. Recognized inhibitors of nucleoside transport were potent, competitive inhibitors of the binding of NBMPR in guinea pig CNS membranes whereas benzodiazepines and phenothiazines have low affinity for the sites. NBMPR sites in guinea pig cortical membranes have characteristics similar to those for NBMPR in human erythrocytes, the occupation of which is associated with inhibition of nucleoside transport. The comparable affinities for a range of agents for sites in human erythrocytes and guinea pig CNS membranes suggest that NBMPR also binds to transport inhibitory elements of the guinea pig CNS nucleoside transport system. It is proposed that the study of the binding of NBMPR provides an effective method by which to examine drug interactions with the membrane-located nucleoside transport system in CNS membranes.     

Characterization of histamine H1 binding sites on human T lymphocytes by means of 125I-iodobolpyramine. Preferential expression of H1 receptors on CD8 T lymphocytes

The presence of histamine H1 receptors on lymphocytes has been indirectly suggested by the various effects of agonists or antagonists on the functionally distinct T lymphocyte subsets. Recently, a new H1 antagonist, 125I-iodobolpyramine, whose structure is similar to mepyramine, has become available for the detection of H1 receptors in guinea pig brain. When using 125I-iodobolpyramine on human T lymphocytes, the presence of a single highly specific H1 binding site was evidenced. The binding of 125I-iodobolpyramine to human T cells was reversible when using 1000-fold excess of the cold H1 antagonist, d-chlorpheniramine. Binding saturation was achieved at 0.60-0.65 nM of 125I-iodobolpyramine, the binding equilibrium was reached in 20-30 min at 27 degrees C. The dissociation constant was KD = 0.41 +/- 0.07 (mean +/- SE) and the number of receptors per T cell was 3407 +/- 592 (mean +/- SE) as deduced from saturation and kinetic curves. In competition experiments using a panel of H1 ligands, the T cell binding sites detected by 125I-iodobolpyramine showed a pharmacological behavior characteristic of histamine H1 receptors. It was of particular interest that 125I-iodobolpyramine binding displayed clearcut stereoselectivity as assessed by the higher affinity of the d-configuration of chlorpheniramine than the l form. Study of purified CD4 and CD8 T cells showed that twice as much H1 histamine receptors were expressed by CD8 T lymphocytes (6615 +/- 1125) as compared to CD4 T cells (3545 +/- 459). These results underline the need for studying the functional properties of such pharmacologically defined T lymphocyte H1 binding sites.     

Design of an Affinity Matrix for Purification of the Histamine H1 Receptor from Guinea Pig Cerebellum

H1 receptors from guinea pig cerebellum were solubilized using digitonin, and [125I]iodobolpyramine was used as a probe. [125I]Iodobolpyramine binding to this solubilized preparation occurred with a KD of 0.1 nM and a Bmax of 220 fmol/mg of protein and was inhibited by various H1 ligands with the expected potencies. Using a gel filtration procedure, a very sensitive radioassay was set up for detecting H1 activity in the solubilized preparation: 0.1 nM [125I]iodobolpyramine specific binding represented greater than 90% of total binding. Moreover, the synthesis is described of potent H1 antagonists that are mepyramine derivatives with an amino alkyl acylamido alkyl spacer arm. One of them, UCL 1057 (Ki = 0.5 nM), has been coupled to a Sepharose epoxy-activated resin. The resulting affinity matrix adsorbed selectively [125I]iodobolpyramine binding sites from the guinea pig cerebellum soluble preparation. In contrast, a Sepharose-glycine matrix was not able to adsorb these sites.     

Biexponential Kinetics of (R)-α-[3H]Methylhistamine Binding to the Rat Brain H3 Histamine Receptor

The H3 histamine receptor is a high-affinity receptor reported to mediate inhibition of CNS histidine decarboxylase activity and depolarization-induced histamine release. We have used (R)-alpha-[3H]methylhistamine, a specific, high-affinity agonist, to characterize ligand binding to this receptor. Saturation binding studies with rat brain membranes disclosed a single class of sites (KD = 0.68 nM; Bmax = 78 fmol/mg of protein). Competition binding assays also yielded an apparently single class of sites with a rank order of potency for ligands characteristic of an H3 histamine receptor: N alpha-methylhistamine, (R)-alpha-methylhistamine greater than histamine, thioperamide greater than impromidine greater than burimamide greater than dimaprit. In contrast, kinetic studies disclosed two classes of sites, one with fast, the other with slow on-and-off rates. Density of (R)-alpha-[3H]methylhistamine binding followed the order: caudate, midbrain (thalamus and hippocampus), cortex greater than hypothalamus greater than brainstem greater than cerebellum. These data are consistent with an H3 histamine receptor, distinct from H1 and H2 receptors, that occurs in two conformations with respect to agonist association and dissociation or with multiple H3 receptor subtypes that are at present pharmacologically undifferentiated.     

Characterization of a Digitonin-Solubilized Bovine Brain H3 Histamine Receptor Coupled to a Guanine Nucleotide-Binding Protein

The H3 receptor is a high-affinity histamine receptor that inhibits release of several neurotransmitters, including histamine. We have characterized H3 receptor binding in bovine brain and developed conditions for its solubilization. Particulate [3H]histamine binding showed an apparently single class of sites (KD = 4.6 nM; Bmax = 78 fmol/mg of protein). Of the detergents tested, digitonin at a detergent/protein ratio of 1:1 (wt/wt) yielded the greatest amount of solubilized receptors, typically 15-30% of particulate binding. Neither equilibrium binding of [3H]histamine to receptors (KD = 6.1 nM; Bmax = 92 fmol/mg of protein) nor the inhibitor profile was substantially altered by digitonin solubilization. However, solubilization did increase the rate of [3H]histamine association with and dissociation from the receptor. Size-exclusion chromatography indicated an apparent molecular weight of 220,000 for the solubilized receptor, and peak binding from this column retained its guanine nucleotide sensitivity. These last two observations are consistent with the solubilized receptor occurring in complex with a guanine nucleotide-binding protein.     

N1-Methyl-2-125I-Lysergic Acid Diethylamide, a Preferred Ligand for In Vitro and In Vivo Characterization of Serotonin Receptors

Methylation of 2-125I-lysergic acid diethylamide (125I-LSD) at the N1 position produces a new derivative, N1-methyl-2-125I-lysergic acid diethylamide (125I-MIL), with improved selectivity and higher affinity for serotonin 5-HT2 receptors. In rat frontal cortex homogenates, specific binding of 125I-MIL represents 80-90% of total binding, and the apparent dissociation constant (KD) for serotonin 5-HT2 receptors is 0.14 nM (using 2 mg of tissue/ml). 125I-MIL also displays a high affinity for serotonin 5-HT1C receptors, with an apparent dissociation constant of 0.41 nM at this site. 125I-MIL exhibits at least 60-fold higher affinity for serotonin 5-HT2 receptors than for other classes of neurotransmitter receptors, with the dopamine D2 receptor as its most potent secondary binding site. Studies of the association and dissociation kinetics of 125I-MIL reveal a strong temperature dependence, with very slow association and dissociation rates at 0 degree C. Autoradiographic experiments confirm the improved specificity of 125I-MIL. Selective labeling of serotonin receptors was observed in all brain areas examined. In vivo binding studies in mice indicate that 125I-MIL is the best serotonin receptor label yet described, with the highest frontal cortex to cerebellum ratio of any serotonergic radioligand. 125I-MIL is a promising ligand for both in vitro and in vivo labeling of serotonin receptors in the mammalian brain.     

Guinea pig hippocampal 5-HT1E receptors: a tool for selective drug development

Recent studies have indicated that the serotonin [5-hydroxytryptamine (5-HT)] 1E receptor, originally discovered in human brain tissue, is not expressed in rat or mouse brain. Thus, there have been few reports on 5-HT_1E receptor drug development. However, expression of 5-HT_1E receptor mRNA has been shown in guinea pig brain. To establish this species as an animal model for 5-HT_1E drug development, we identified brain regions that exhibit 5-carboxyamidotryptamine, ritanserin, and LY344864 – insensitive [³H]5-HT binding (characteristic of the 5-HT_1E receptor). In hippocampal homogenates, where 5-HT_1E receptor density was sufficiently high for radioligand binding analysis, 100 nM 5-carboxyamidotryptamine, 30 nM ritanserin, and 100 nM LY344864 were used to mask [³H]5-HT binding at non-5-HT_1E receptors. The K _d of [³H]5-HT was 5.7 ± 0.7 nM and is indistinguishable from the cloned receptor K _d of 6.5 ± 0.6 nM. The affinities of 16 drugs for the cloned and hippocampal-expressed guinea pig 5-HT_1E receptors are essentially identical ( R ² = 0.97). These findings indicate that using these conditions autoradiographical distribution and signal transduction studies of the 5-HT_1E receptor in guinea pig brain are feasible. Using the guinea pig as an animal model should provide important insights into possible functions of this receptor and the therapeutic potential of selective human 5-HT_1E drugs.     

Cloning and characterization of the hamster and guinea pig nicotinic acid receptors

In this study, we present the identification and characterization of hamster and guinea pig nicotinic acid receptors. The hamster receptor shares approximately 80-90% identity with the nucleotide and amino acid sequences of human, mouse, and rat receptors. The guinea pig receptor shares 76-80% identity with the nucleotide and amino acid sequences of these other species. [(3)H]nicotinic acid binding affinity at guinea pig and hamster receptors is similar to that in human (dissociation constant = 121 nM for guinea pig, 72 nM for hamster, and 74 nM for human), as are potencies of nicotinic acid analogs in competition binding studies. Inhibition of forskolin-stimulated cAMP production by nicotinic acid and related analogs is also similar to the activity in the human receptor. Analysis of mRNA tissue distribution for the hamster and guinea pig nicotinic acid receptors shows expression across a number of tissues, with higher expression in adipose, lung, skeletal muscle, spleen, testis, and ovary.     

Autoradiographic Localization of [3H]Zolpidem Binding Sites in the Rat CNS: Comparison with the Distribution of [3H]Flunitrazepam Binding Sites

The regional distribution of [3H]zolpidem, a novel imidazopyridine hypnotic possessing preferential affinity for the BZD1 (benzodiazepine subtype 1) receptor, has been studied autoradiographically in the rat CNS and compared with that of [3H]flunitrazepam. The binding of [3H]zolpidem to rat brain sections was saturable, specific, reversible, and of high affinity (KD = 6.4 nM). It occurred at a single population of sites whose pharmacological characteristics were similar to those of the benzodiazepine receptors labeled with [3H]flunitrazepam. However, ethyl-beta-carboline-3-carboxylate and CL 218,872 were more potent displacers of [3H]zolpidem than of [3H]flunitrazepam. The autoradiographic brain distribution of [3H]zolpidem binding sites was qualitatively similar to that previously reported for benzodiazepine receptors. The highest levels of [3H]-zolpidem binding sites occurred in the olfactory bulb (glomerular layer), inferior colliculus, ventral pallidum, nucleus of the diagonal band of Broca, cerebral cortex (layer IV), medial septum, islands of Calleja, subthalamic nucleus, and substantia nigra pars reticulata, whereas the lowest densities were found in parts of the thalamus, pons, and medulla. Comparative quantitative autoradiographic analysis of the binding of [3H]zolpidem and [3H]flunitrazepam [a mixed BZD1/BZD2 (benzodiazepine subtype 2) receptor agonist] in the CNS revealed that the relative density of both 3H-labeled ligands differed in several brain areas. Similar levels of binding for both ligands were found in brain regions enriched in BZD1 receptors, e.g., substantia nigra pars reticulata, inferior colliculus, cerebellum, and cerebral cortex lamina IV. The levels of [3H]zolpidem binding were five times lower than those of [3H]flunitrazepam binding in those brain regions enriched in BZD2 receptors, e.g., nucleus accumbens, dentate gyrus, and striatum. Moreover, [3H]zolpidem binding was undetectable in the spinal cord (which contains predominantly BZD2 receptors). Finally, like CL 218,872 and ethyl-beta-carboline-3-carboxylate, zolpidem was a more potent displacer of [3H]flunitrazepam binding in brain regions enriched in BZD1 receptors than in brain areas enriched in BZD2 receptors. The present data add further support to the view that zolpidem, although structurally unrelated to the benzodiazepines, binds to the benzodiazepine receptor and possesses selectivity for the BZD1 receptor subtype.     

Autoradiographic comparison of [I]LSD-labeled 5-HT2A receptor distribution in rat and guinea pig brain

Although the density and distribution of 5-HT_2A(5-hydroxytryptamine-2A) receptors is well established for rat brain, the 5-HT_2A receptor distribution and density in guinea pig brain has not been extensively studied. In the present in vitro study, we have utilized ¹²⁵I-lysergic acid diethylamide ([¹²⁵I]LSD) to quantify and compare 5-HT_2A receptor density in coronal sections of rat and guinea pig brain. Spiperone (1 μM) and sulpiride (1 μM) were used to displace [¹²⁵I]LSD binding from 5-HT_2A and D₂ binding sites, respectively. Ligand binding was quantified by computer-aided image analysis densitometry (MCID). Similar to the rat, areas of highest specific 5-HT_2A receptor binding (fmol/mg protein) in guinea pig brain included the claustrum and Layer 4 of the cerebral cortex. Significant binding was also found in remaining neocortical layers, islands of Calleja, caudate putamen, olfactory bulb, nucleus accumbens, and choroid plexus. While the rat brain exhibited a high level of specific binding in the tenia tecta and mammillary nuclei, little binding was observed in these regions in the guinea pig. In both rat and guinea pig, low specific binding was found in amygdaloid, thalamic, or cerebellar areas. These studies indicate a general similarity between 5-HT_2A binding site distribution and relative density in guinea pig and rat brain but point to a few brain regions where significant differences exist.     

The binding of [3H]mepyramine to histamine H1 receptors in monkey brain

Several laboratories have reported ligand binding studies using radioactive histamine H1 antagonists to label the H1 receptors in mammalian brain. We have extended these studies to a detailed examination of the binding of [3H]mepyramine to monkey brain and have shown that the distribution is similar to that in man, with specific binding sites being concentrated in the frontal cortex with relatively low binding to the pons and basal ganglia. The binding shows a single saturable component with a KD of about 1 nM and a Hill plot slope close to unity. These observations are the same for all structures tested. Comparison with data from other laboratories suggests that in this species, the histamine receptor is the same as that in peripheral tissues. From Ki values for various ligands and comparison of KD estimates in other species, the receptor seems to be essentially identical to the H1 receptor in central and peripheral tissues of the guinea pig and also to that in human brain. The rat and possibly the dog have minor differences from the monkey in terms of KD values for [3H]mepyramine binding.     

Affinities of Histamine H1-Antagonists in Guinea Pig Brain: Similarity of Values Determined from [3H]Mepyramine Binding and from Inhibition of a Functional Response

Affinity constants for five antagonists at histamine H1-receptors in guinea pig brain have been determined from inhibition of the potentiation by histamine of the adenosine-induced accumulation of cyclic AMP in cerebral cortical slices. This action of histamine appeared to be mediated solely through H1-receptors. The affinity constants obtained were similar to those determined on peripheral H1-receptors and from inhibition of high-affinity [3H]mepyramine binding. This provides strong evidence that at least some of the [3H]mepyramine binding sites in guinea pig brain can be identified with functional H1-receptors.     

Comparison of [125I]Iodolysergic Acid Diethylamide Binding in Human Frontal Cortex and Platelet Tissue

The human platelet contains a functional 5-hydroxytryptamine (5-HT) receptor that appears to resemble the 5-HT2 subtype. In this study, we have used the iodinated derivative [125I]iodolysergic acid diethylamide ([125I]iodoLSD) in an attempt to label 5-HT receptors in human platelet and frontal cortex membranes under identical assay conditions to compare the sites labelled in these two tissues. In human frontal cortex, [125I]iodoLSD labelled a single high-affinity site (KD = 0.35 +/- 0.02 nM). Displacement of specific [125I]iodoLSD binding indicated a typical 5-HT2 receptor inhibition profile, which demonstrated a significant linear correlation (r = 0.97, p less than 0.001, n = 17) with that observed using [3H]ketanserin. However, [125I]iodoLSD (Bmax = 136 +/- 7 fmol/mg of protein) labelled significantly fewer sites than [3H]ketanserin (Bmax = 258 +/- 19 fmol/mg of protein) (p less than 0.001, n = 6). In human platelet membranes, [125I]iodoLSD labelled a single site with affinity (KD = 0.37 +/- 0.03 nM) similar to that in frontal cortex. The inhibition profile in the platelet showed significant correlation with that in frontal cortex (r = 0.96, p less than 0.001, n = 16). We conclude that the site labelled by [125I]iodoLSD in human platelet membranes is biochemically similar to that in frontal cortex and most closely resembles the 5-HT2 receptor subtype, although the discrepancy in binding capacities of [125I]iodoLSD and [3H]ketanserin raises a question about the absolute nature of this receptor.     

Studies on Muscarinic Binding Sites in Human Brain Identified with [3H]Pirenzepine

Pirenzepine, a potent antimuscarinic agent with apparent selectivity for a subtype (M1) of muscarinic receptors, was used in tritiated form to characterize its binding to human brain tissue. Specific [3H]pirenzepine binding showed rapid association and dissociation. From kinetic and competitive binding experiments, its KD was 5.5 nM and 9 nM, respectively. Regional distribution of [3H]pirenzepine binding determined in parallel with [3H]quinuclidinyl benzilate binding, a nonselective muscarinic antagonist, indicated a significant correlation for the maximum number of binding sites for the two radioligands in 13 brain regions, with the highest amount of binding for each in the putamen and the least in the cerebellum. Binding for [3H]pirenzepine averaged 57% of that for [3H]quinuclidinyl benzilate, with a range of 20% (cerebellum) to 77% (frontal cortex). Most antidepressants and neuroleptics tested had affinities for [3H]pirenzepine binding sites that were not significantly different from their previously reported values obtained with the use of [3H]quinuclidinyl benzilate.     

[3H]1-[2-(4-Aminophenyl)Ethyl]-4-(3-Trifluoromethylphenyl)Piperazine: A Selective Radioligand for 5-HT1A Receptors in Rat Brain

1-[2-(4-Aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (PAPP) inhibits [3H]5-hydroxytryptamine (5-HT, serotonin) binding to 5-HT1A and 5-HT1B sites in rat brain with apparent equilibrium dissociation constants (KD) of 2.9 and 328 nM, respectively. [3H]PAPP was synthesized, its binding to central serotonin receptors was examined, and its potential usefulness as a 5-HT1A receptor radioligand was evaluated. With either 10 microM 5-HT or 1 microM 8-hydroxy-2-(di-n-propylamino)tetralin to define nonspecific binding, [3H]PAPP bound to a single class of sites in rat cortical membranes with a KD of 1.6 nM and a maximal binding density (Bmax) of 162 fmol/mg of protein. d-Lysergic acid diethylamide and 5-HT, two nonselective inhibitors of [3H]5-HT binding, displaced 1 nM [3H]PAPP with a potency that matched their affinity for 5-HT1 receptors. Spiperone and 8-hydroxy-2-(di-n-propylamino)tetralin, two compounds that discriminate [3H]5-HT binding to 5-HT1A and 5-HT1B sites, inhibited [3H]PAPP binding in accordance with their much higher affinities for the 5-HT1A receptor subtype. Furthermore, the ability of N-(m-trifluoromethylphenyl)piperazine and ketanserin to inhibit [3H]PAPP binding reflected their low affinities for the 5-HT1A receptor. Several nonserotonergic compounds were also found to be relatively poor displacers of [3H]PAPP binding. The regional distribution of serotonin-sensitive [3H]PAPP sites correlated with the densities of 5-HT1A receptors in the cortex, hippocampus, corpus striatum, and cerebellum of the rat. These results indicate that [3H]PAPP binds selectively and with high affinity to 5-HT1A receptor sites in rat brain.     

H3-Receptors Control Histamine Release in Human Brain

The regulation of histamine release was studied on slices prepared from pieces of human cerebral cortex removed during neurosurgery and labeled with L-[3H]histidine. Depolarization by increased extracellular K+ concentration induced [3H]histamine release, although to a lesser extent than from rat brain slices. Exogenous histamine reduced by up to 60% the K+-evoked release, with an EC50 of 3.5 +/- 0.5 X 10(-8) M. The H3-receptor antagonists impromidine and thioperamide reversed the histamine effect in an apparently competitive manner and enhanced the K+-evoked release, indicating a participation of endogenous histamine in the release control process. The potencies of histamine and the H3-receptor antagonists were similar to those of these agents at presynaptic H3-autoreceptors controlling [3H]histamine release from rat brain slices. It is concluded that H3-receptors control histamine release in the human brain.     

Binding of the Adenosine A2 Receptor Ligand [3H]CGS 21680 to Human and Rat Brain: Evidence for Multiple Affinity Sites

A new radiolabeled adenosine receptor agonist, 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadeno sin e (CGS 21680), apparently specific for high-affinity binding sites of the A2 subtype in rat brain, was used to identify and pharmacologically characterize adenosine receptors in human brain. The binding of [3H]CGS 21680, as determined by standard radioligand binding technique in the presence of exogenously added adenosine deaminase, reached equilibrium after 40 min at 25 degrees C. In saturation studies, a single class of high-affinity binding sites with values for KD of 22 +/- 0.5 nM and Bmax of 444 +/- 63 fmol/mg of protein were observed. Similar binding characteristics were observed regardless of whether rapid filtration or centrifugation was used to separate bound versus free ligand. Of the 14 brain regions examined, [3H]CGS 21680 binding was highest in putamen, followed by globus pallidus and caudate nucleus. The level of [3H]CGS 21680 binding in these areas of basal ganglia was identical to 5'-N-[3H]ethylcarboxamidoadenosine ([3H]NECA) binding in the presence of 50 nM N6-cyclopentyladenosine (CPA). The rank order of agonist potencies as determined by a series of competition experiments was NECA greater than or equal to CGS 21680 greater than 2-chloroadenosine greater than N6-(R)-phenylisopropyladenosine greater than N6-cyclohexyladenosine greater than N6-(S)-phenylisopropyladenosine. This potency order was the same for the binding of [3H]CGS 21680 to rat, and of [3H]NECA in the presence of 50 nM CPA to rat and human, brain membranes.(ABSTRACT TRUNCATED AT 250 WORDS)