Histamine H1-Receptor in Heart: Unique Electrophoretic Mobility and Autoradiographic Localization

Histamine H1-receptors, visualized in the guinea pig heart by autoradiography using [125I]iodobolpyramine as a specific probe, are abundant in the nodal tissue and cardiac vessels but also occur heterogeneously in the myocardium. Following photoaffinity labeling with [125I]iodoazidophenpyramine and electrophoresis, the ligand binding domain of the heart H1-receptor was shown to be present on a major 68-kDa and a less abundant 54- to 58-kDa protein. The 68-kDa protein displayed a molecular size higher in heart than in all other tissues (56 kDa). This indicates the existence of at least two isoforms of the H1-receptor; the cardiac isoform, however, was pharmacologically indistinguishable from the common isoform studied in cerebellar membranes using available ligands. Its distinct electrophoretic properties suggest that the cardiac isoform may have a unique function.     

Identification of the D2--Dopamine Receptor Binding Subunit in Several Mammalian Tissues and Species by Photoaffinity Labeling

Photoaffinity labeling of the D2-dopamine receptor in plasma membrane preparations of various tissues from several mammalian species was performed using the recently developed D2-dopaminergic antagonist probe [125I]N-(p-azidophenethyl)spiperone ([125I]N3-NAPS). In tissues containing D2-receptors such as the corpus striatum from rat, dog, calf, hamster, guinea pig, and rabbit as well as the anterior pituitary of rat, bovine, and hamster, the probe covalently labels a peptide of Mr = 94,000. Specificity of the labeling is typically D2-dopaminergic in character. The covalent labeling is blocked by (+)-butaclamol but not by the inactive (-)isomer. Agonists block incorporation with the order of potency: N-n-propylnorapomorphine greater than apomorphine greater than dopamine. The D2-selective antagonist spiperone blocks labeling of the Mr = 94,000 peptide whereas the D1-selective antagonist SCH-23390 is ineffective. Thus, these results indicate that the ligand binding subunit of the D2-dopamine receptor resides on a Mr = 94,000 peptide in these various tissues from several species. Under conditions where proteolysis is not stringently controlled, peptides of lower Mr (32-38,000) are labeled at the expense of the Mr = 94,000 peptide. The most efficient protease inhibitor tested in these systems was EDTA, suggesting that the generation of these lower Mr receptor fragments might be the result of a metal-dependent proteolysis in the membrane preparations. In the rat neurointermediate lobe, a tissue containing D2-receptors, [125I]N3-NAPS specifically labels a major peptide of Mr approximately equal to 120,000 in addition to the Mr = 94,000 peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological Characterization and Autoradiographic Localization of Histamine H2 Receptors in Human Brain Identified with [125I]Iodoaminopotentidine

125I-Aminopotentidine (125I-APT), a reversible probe of high specific radioactivity and high affinity and selectivity for the H2 receptor, was used to characterize and localize this histamine receptor subtype in human brain samples obtained at autopsy. On membranes of human caudate nucleus, specific 125I-APT binding at equilibrium revealed a single component, with a dissociation constant of 0.3 nM and maximal capacity of about 100 fmol/mg of protein. At 0.2 nM, 125I-APT specific binding, as defined with tiotidine, an H2-receptor antagonist chemically unrelated to iodoaminopotentidine, represented 40-50% of the total. Specific 125I-APT binding was inhibited by a series of typical H2-receptor antagonists that displayed apparent dissociation constants closely similar to corresponding values at the reference biological system, i.e., guinea pig atrium. This indicates that the pharmacology of the H2 receptor is the same in the human brain as on this reference system. However, histamine was about 10-fold more potent in inhibiting 125I-APT binding to membranes of human brain than of guinea pig brain. 125I-APT binding was also inhibited by amitriptyline and mianserin, two antidepressant drugs, in micromolar concentrations corresponding to effective plasma concentrations of treated patients. The distribution of H2 receptors was established autoradiographically with 125I-APT on a series of coronal sections of human brain after assessing the pharmacological specificity of the labeling. The highest density of 125I-APT sites was found in the basal ganglia, various parts of the limbic system, e.g., hippocampus or amygdaloid complex, and the cerebral cortex. H2 receptors displayed a laminar distribution in cerebral cortex and hippocampal formation. A low density of sites was found in cerebellum as well as in hypothalamus, the brain area where all the perikarya and the largest number of axons of histaminergic neurons are found. The widespread distribution of H2 receptors in the human brain is consistent with the alleged modulatory role of histamine mediated by this subtype of receptor.     

Photoaffinity Labeling of Benzodiazepine Receptor Proteins with the Partial Inverse Agonist [3H]Ro 15–4513: A Biochemical and Autoradiographic Study

Photolabeling of the benzodiazepine receptor, which to date has been done with benzodiazepine agonists such as flunitrazepam, can also be achieved with Ro 15-4513, a partial inverse agonist of the benzodiazepine receptor. [3H]Ro 15-4513 specifically and irreversibly labeled a protein with an apparent molecular weight of 51,000 (P51) in cerebellum and at least two proteins with apparent molecular weights of 51,000 (P51) and 55,000 (P55) in hippocampus. Photolabeling was inhibited by 10 microM diazepam but not by 10 microM Ro 5-4864. The BZ1 receptor-selective ligands CL 218872 and beta-carboline-3-carboxylate ethyl ester preferentially inhibited irreversible binding of [3H]Ro 15-4513 to protein P51. Not only these biochemical results but also the distribution and density of [3H]Ro 15-4513 binding sites in rat brain sections were similar to the findings with [3H]flunitrazepam. Thus, the binding sites for agonists and inverse agonists appear to be located on the same proteins. In contrast, whereas [3H]flunitrazepam is known to label only 25% of the benzodiazepine binding sites in brain membranes, all binding sites are photolabeled by [3H]Ro 15-4513. Thus, all benzodiazepine receptor sites are associated with photolabeled proteins with apparent molecular weights of 51,000 and/or 55,000. In cerebellum, an additional protein (MW 57,000) unrelated to the benzodiazepine receptor was labeled by [3H]Ro 15-4513 but not by [3H]flunitrazepam. In brain sections, this component contributed to higher labeling by [3H]Ro 15-4513 in the granular than the molecular layer.     

Irreversible Binding of [3H]Flunitrazepam to Different Proteins in Various Brain Regions

Irreversible photolabeling by [3H]flunitrazepam of four proteins with apparent molecular weights 51,000 (P51), 53,000 (P53), 55,000 (P55), and 59,000 (P59) was investigated in various rat brain regions by SDS-polyacrylamide gel electrophoresis, fluorography, and quantitative determination of radioactivity bound to proteins. On maximal labeling of these proteins, only 15-25% of [3H]flunitrazepam reversibly bound to membranes becomes irreversibly attached to proteins. Results presented indicate that for every [3H]flunitrazepam molecule irreversibly bound to membranes, three molecules dissociate from reversible benzodiazepine binding sites. This seems to indicate that these proteins are either closely associated or identical with reversible benzodiazepine binding sites, and supports the hypothesis that four benzodiazepine binding sites are associated with one benzodiazepine receptor. When irreversible labeling profiles of proteins P51, P53, P55, and P59 were compared in different brain regions, it was found that labeling of individual proteins varied independently, supporting previous evidence that these proteins are associated with distinct benzodiazepine receptors.     

Identification of the 5-HT1A Receptor Binding Subunit in Rat Brain Membranes Using the Photoaffinity Probe [3H]8-Methoxy-2-[N-n-Propyl, N-3-(2-Nitro-4-Azidophenyl)Aminopropyl]Aminotetralin

The synthesis of a tritiated derivative of the 5-HT1A photoaffinity probe 8-methoxy-2-[N-n-propyl, N-3-(2-nitro-4-azidophenyl)aminopropyl]aminotetralin ([3H]8-methoxy-3'-NAP-amino-PAT) allowed the use of this probe for attempting the irreversible labeling of specific binding sites in rat brain membranes. Sodium dodecyl-sulfate-polyacrylamide gel electrophoresis of proteins solubilized from hippocampal microsomal membranes that had been incubated with 20 nM [3H]8-methoxy-3'-NAP-amino-PAT under UV light revealed a marked incorporation of 3H label into a 63-kilodalton protein termed PI. As expected of a possible correspondence between PI and 5-HT1A receptor binding sites, 3H labeling by the photoaffinity probe could be prevented by selective 5-HT1A ligands such as 8-hydroxy-2-(di-n-propylamino)tetralin, ipsapirone, buspirone, and gepirone and by N-ethylmaleimide, but not by the 5-HT2 antagonist ketanserin, noradrenaline- and dopamine-related drugs, monoamine oxidase inhibitors, and chlorimipramine. Furthermore, the regional and subcellular distributions of PI were identical to those of specific 5-HT1A binding sites. These results indicated that the binding subunit of the 5-HT1A receptor is a 63-kilodalton protein with a functionally important sulfhydryl group(s).     

Photoaffinity Labeling with Progesterone-11α-Hemisuccinate-(2-[125I]Iodohistamine) Identifies Four Protein Bands in Mouse Brain Membranes

Abstract: The radiolabeled progesterone (PG) analogue progesterone-11α-hemisuccinate-(2-[¹²⁵I]iodohistamine) was used to label PG binding proteins in brain membranes from mouse cerebellum. Photoaffinity labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified specific PG binding protein bands 1–4 of 64−29 kDa. Bands 1 and 4 were well resolved on the gel and easily quantified. Preincubation with PG inhibited photolabeling in a dose-dependent manner. The labeling was specific with respect to steroid structure. For band 1, the extent of inhibition of labeling by PG and 3α,5α-pregnanolone (3α) was pronounced. Other steroids such as testosterone (Tes), estradiol (Est), and corticosterone (Cor) were less effective, whereas pregnenolone sulfate (PS) and cholesterol (Cho) were ineffective. With respect to band 4, Est was the most effective; PG, 3α, and Tes were intermediate; and PS, Cho, and Cor were ineffective. The results describe specific membrane proteins that bind PG (band 1) and Est (band 4).     

[3H]Dopamine Labeling of D3 Dopaminergic Sites in Human, Rat, and Calf Brain

Abstract: The binding of [³H]dopamine to brain regions of calf, rat, and human was investigated. The calf caudate contained the highest density of [³H]dopamine binding sites, with a B_max value of 185 fmol/mg protein, whereas rat and human striatum contained one-third this number of sites. The K_D values for [³H]dopamine in all tissues were 2–3 nM. Dopaminergic catecholamines (dopamine, apomorphine, 6,7-dihydroxy-2-aminotetralin, and N-propylnorapomorphine) inhibited the binding of [³H]dopamine in all three species, at low concentrations, with IC₅₀ values of 1.5 to 6 nM. Neuroleptics, in contrast, inhibited the binding at high concentrations (with IC₅₀ values of 200 to 40,000 nM). The [³H]dopamine binding sites were saturable, heat-labile, and detectable only in dopamine-rich brain regions; these sites differed from D₂ dopamine sites (labeled by [³H]butyrophenone neuroleptics), and from D_l dopamine sites (labeled by [³H]thioxanthene neuroleptics) associated with the dopamine-stimulated adenylate cyclase. We have, therefore, called these high-affinity [³H]dopamine binding sites D₃ sites. [³H]Apomorphine and [³H]ADTN also appeared to label D₃ sites. These ligands however, were less selective than [³H]dopamine, and labeled sites other than D₃ as well. Assay conditions were important in determining the parameters of [³H]dopamine binding. The optimum conditions for selective labeling of the D₃ dopaminergic sites, using [³H]dopamine, required the presence of EDTA and ascorbate.     

Mapping of Histamine H1 Receptors in the Human Brain Using [11C]Pyrilamine and Positron Emission Tomography

We have studied the characteristics of carbon-11 labeled pyrilamine as a radioligand for investigating histamine H1 receptors in human brain with positron emission tomography (PET). [11C]Pyrilamine is distributed evenly in proportion to cerebral blood flow at initial PET images. Later (after 45-60 min), 11C radioactivity was observed at high concentrations in the frontal and temporal cortex, hippocampus, and thalamus, and at low concentrations in the cerebellum and pons. The regional distribution of the carbon-11 labeled compound in the brain corresponded well with that of the histamine H1 receptors determined in vitro in autopsied materials. In six controls, the frontal and temporal cortices/cerebellum ratio increased during the first 60 min to reach a value of 1.22 +/- 0.071. Intravenous administration of d-chlorpheniramine (5 mg) completely abolished the specific binding in vivo in the frontal cortex and temporal cortex (cortex/cerebellum ratio, 0.955 +/- 0.015). The availability of this method for measuring histamine H1 receptors in vivo in humans will facilitate studies on neurological and psychiatric disorders in which histamine H1 receptors are thought to be abnormal.     

Photoaffinity Labeling of Adenylate Cyclase-Linked Serotonin Receptors in Aplysia Neurons

Serotonin stimulated adenylate cyclase in Aplysia neurons with a Kact of 0.7 microM. Under the same conditions, 1-[2-(4-aminophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine stimulated adenylate cyclase with a Kact of 20 microM. The azido derivative of this compound, 1-[2-(4-azidophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine, or of serotonin, (4-amino, 3-nitrophenylazido-serotonin), also stimulated the cyclase in the dark, but with lower efficiency (Kact greater than 10(-4) M). Irradiation of the membranes in the presence of 100 microM 1-[2-(4-azidophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine abolished 75% of the cyclase activity stimulated by 5 microM serotonin. Under the same conditions, 100 microM 4-amino, 3-nitrophenylazido-serotonin did not inhibit serotonin-stimulated adenylate cyclase activity. When [3H]1-[2-(4-azidophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine (20 microM) was irradiated with membranes for 5 min at 4 degrees C, a dozen peptides were labeled, as revealed by a fluorogram of sodium dodecyl sulfate-polyacrylamide gels. Among them, the labeling of five polypeptides (molecular weights of 45,000, 55,000, 63,000, 80,000, and 94,000) was protected by the presence of 0.2 mM serotonin during photolysis. These peptides may be related to serotonin receptors.     

Histamine H1-Receptor Binding Sites in Guinea Pig Brain Membranes: Regulation of Agonist Interactions by Guanine Nucleotides and Cations

Abstract: Agonist, but not antagonist, interactions with histamine H₂-receptors labeled by [³H]mepyramine are regulated selectively by sodium, divalent cations, and guanine nucleotides. Sodium decreases the affinity of histamine and the agonist 2-amino-ethylpyridine for [³H]mepyramine sites in guinea pig brain membranes up to 10-fold. The effect of sodium is exerted to a lesser extent by lithium, while potassium and rubidium are much weaker. Guanine nucleotides also decrease the affinity of histamine for H₁ binding sites about twofold. GTP and its nonmetabolized analogue GMP-PNP as well as GDP exert similar effects, while GMP, ATP, ADP, and AMP are inactive. The effects of GTP and sodium on histamine interactions with H₁-receptors are additive. By contrast, certain divalent cations enhance the potency of histamine at H₁-receptors. Manganese is most potent, while magnesium is almost as active as manganese and calcium is essentially inactive. Sodium, divalent cations, and guanine nucleotides have negligible effects on the interactions of antihistamines with H₁-receptors.     

Ontogenetic Development of Histamine H1- Receptor Binding in Rat Brain

Abstract: Histamine H_1- receptors labeled with [³H]mepyramine in rat brain show an age-dependent development. [³H]Mepyramine receptor density and histidine decarboxylase activity in whole rat brain reach adult levels at 25–30 days after birth and they attain 50% of adult level at day 10 and 17, respectively. The apparently later development of histidine decarboxylase activity in whole rat brain is partly accounted for by a biphasic developmental increase of this enzymatic activity in cerebral cortex. For all other brain regions examined, the development of histamine H_1- receptors parallels that of histidine decarboxylase. The increase in [³H]mepyramine binding can be accounted for by an absolute increase in the numbers of the receptor sites, with no change in affinity. Subcellular fractionation studies indicate that histamine H_1- receptors are predominantly associated with synaptosomal fractions derived from both newborn and adult rat.     

In Vivo Labeling of Serotonin Uptake Sites with [3H]Paroxetine

Previous work has shown that [3H]paroxetine is a potent and selective in vitro label for serotonin uptake sites in the mammalian brain. In the present study, [3H]paroxetine was tested in mice as an in vivo label for serotonin uptake sites. Maximum tritium concentration in the whole brain (1.4% of the intravenous dose) was reached 1 h after injection into a tail vein. Distribution of the tracer at 3 h after injection followed the distribution of serotonin uptake sites known from previous in vitro binding studies (r = 0.85). The areas of highest [3H]paroxetine concentration, in decreasing order, were: hypothalamus greater than frontal cortex greater than olfactory tubercles greater than thalamus greater than upper colliculi greater than brainstem greater than hippocampus greater than striatum greater than cerebellum. Preinjection of carrier paroxetine (1 mg/kg) significantly decreased [3H]paroxetine concentration in all areas except in the cerebellum, which is known to contain a relatively low number of specific binding sites. Kinetic studies showed highest specific [3H]paroxetine binding (tissue minus cerebellum) at 2 h after injection and slow clearance of activity thereafter (half-time of dissociation from the hypothalamus, 215 min). The specificity of in vivo [3H]paroxetine binding was studied by preinjecting monoamine uptake blockers or receptor antagonists 5 min before administration of [3H]paroxetine. Serotonergic or muscarinic cholinergic receptor antagonists and dopamine or norepinephrine uptake blockers did not reduce the in vivo binding of [3H]paroxetine. In contrast, there was an excellent correlation (r = 0.99) between the in vivo inhibitory potencies of serotonin uptake blockers in this study and previously published in vitro data on inhibition of [3H] serotonin uptake in brain synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

H-Pro-[3H]Leu-Gly-NH2: Uptake and Metabolism in Rat Brain

The uptake and metabolism of H-Pro-[3H]Leu-Gly-NH2 ([3H]PLG) in rat brain was investigated by reverse-phase paired-ion high pressure liquid chromatography. Following in vitro incubation of [3H]PLG with rat brain subcellular preparations, the microsomal-cytosol fraction was about twice as active in degrading PLG as the crude mitochondrial-synaptosomal fraction. For both enzyme preparations the pH optimum was found at pH 7-7.5. The major labeled metabolite was [3H]leucine, whereas 3H]labeled Leu-Gly-NH2 as the only labeled peptide intermediate was found in trace amounts. After intravenous injection of [3H]PLG the uptake of unmetabolized peptide in the brain appeared to be very low: 0.008% and 0.001% of the administered dose/g tissue at 2 and 5 min after injection respectively, while at longer survival times intact peptide was below the detection limit. Compared with the intravenous route of administration, intracerebroventricular injection of [3H]PLG yielded much higher brain concentrations of unmetabolized PLG. Following both routes of administration, the metabolite profile was in agreement with that obtained after in vitro incubation. However, the in vivo experiments also showed considerable incorporation of [3H]leucine liberated from [3H]PLG into proteins. Both the in vitro and in vivo results indicate that the initial cleavage of PLG in rat brain occurs at the NH2-terminus and that the dipeptide intermediate H-Leu-Gly-NH2 is subsequently hydrolyzed to its constituent amino acids very rapidly.     

Density of NMDA-Coupled and Uncoupled 1-[1-(2-[3H]Thienyl)cyclohexyl]piperidine Recognition Sites in the Brain and Spinal Cord: Differential Effects of NMDA Agonists and Antagonists

Abstract: Binding of 1-[1-(2-[³H]thienyl)cyclohexyl]piperidine ([³H]TCP) to mouse brain and spinal cord membranes was studied using compounds selective for the NMDA-coupled 1-(1-phenylcyclohexyl)piperidine (PCP) and/or σ recognition sites. In both tissues, [³H]TCP labeled two populations of binding sites. Density of the low-affinity sites was approximately the same in both tissues, but the population of the high-affinity [³H]TCP sites was three times bigger in the brain than in the spinal cord. Self- and cross-displacement studies showed that the high-affinity [³H]TCP binding sites could be identical with NMDA receptor-coupled PCP sites, whereas the low-affinity [³H]TCP sites may be associated with σ binding sites in both tissues. The NMDA-coupled PCP sites labeled in the presence of 6.25 n M [³H]TCP constituted a much higher percentage of the total binding in the brain (75%) than in the spinal cord (44%). Consistent with this, reintroduction of glycine and glutamate significantly increased, but DA antagonists significantly inhibited [³H]TCP binding in the brain but not in the spinal cord. Together, these data suggest that a large component of [³H]TCP-labeled binding sites in the spinal cord may be associated with σ but not the NMDA receptor-coupled PCP sites.     

Characterization of the A1 Adenosine Receptor-Adenylate Cyclase System of Cerebral Cortex Using an Agonist Photoaffinity Ligand

Endogenous adenosine acting via A1 adenosine receptors is capable of inhibiting adenylate cyclase activity and neurotransmitter release in the brain. In this report, we describe the synthesis and attributes of a new series of A1 adenosine receptor agonists. One of these, [125I]N6-2-(4-amino-3-iodophenyl)ethyladenosine, can be used as a radioligand and another, [125I]N6-2-(4-azido-3-iodophenyl)ethyladenosine, as a photoaffinity probe. The unlabeled ligand, N6-2-(4-aminophenyl)ethyladenosine, and its iodinated product are full agonists, inhibiting cyclic AMP production in rat cerebral cortex membranes to the same extent as the prototypic A1 agonist N6-R-1-phenyl-2-propyladenosine. These new ligands are not substrates for adenosine deaminase. The new photoaffinity azide described here labels an Mr 38,000 protein that displays all the pharmacological characteristics expected of the A1 adenosine receptor. This is the same molecular-weight protein previously described using a cross-linking radioligand. This new azide compound demonstrates a 15-fold higher efficiency of incorporation, making it the photoaffinity probe of choice for tissues containing low concentrations of A1 adenosine receptors.     

H-Pro-[3H]Leu-Gly-NH2: Plasma Profile and Brain Uptake Following Subcutaneous Injection in the Rat

Following subcutaneous injection of the tripeptide H-Pro-[3H]Leu-Gly-NH2 ([3H]PLG) in rats, the profile of intact peptide and its radioactively labeled metabolites was examined both in plasma and in brain tissue. [3H]PLG and metabolites were determined in trichloroacetic acid extracts by reverse-phase paired-ion HPLC. Maximal plasma levels of unmetabolized PLG were reached 6-8 min after administration, after which they decreased with an elimination half-life of 20 min. The uptake of [3H]PLG in the brain ranged from 0.0013% to 0.0017% of the administered dose per g tissue at 6-30 min following subcutaneous injection. After comparing these results with our previous findings with intravenous injection of [3H]PLG, it seemed likely that the subcutaneous route of administration might be more effective in eliciting CNS effects of PLG than the intravenous route of administration. The metabolite profiles in plasma and brain point to an initial cleavage of PLG at the NH2-terminal side and a very rapid degradation of the peptide intermediate H-Leu-Gly-NH2.     

[3H]Citalopram Binding to Brain and Platelet Membranes of Human and Rat

Citalopram, a selective serotonin (5-HT) uptake inhibitor with antidepressant properties, was found to bind with high affinity to the 5-HT transporter from human neuronal and platelet membranes. At 20 degrees C, KD was about 1.5 nM in both tissues. [3H]Citalopram bound to rat neuronal membranes with higher affinity than to human neuronal and platelet membranes; at 20 degrees C KD was about 0.7 nM. The Bmax value for the binding of [3H]citalopram to platelet membranes was close to that found using the 5-HT uptake inhibitors [3H]imipramine and [3H]paroxetine, suggesting that all three 5-HT uptake inhibitors bind to the 5-HT transporter. The dissociation rate of [3H]citalopram increased twofold with each 4-5 degree C increase in temperature in both human and rat membranes, although at any given temperature, the dissociation rate was about four times faster in the human neuronal and platelet membranes than in rat neuronal membranes.     

Opposite Age-Dependent Changes of α2A-Adrenoceptors and Nonadrenoceptor [3H]Idazoxan Binding Sites (I2-Imidazoline Sites) in the Human Brain: Strong Correlation of I2 with Monoamine Oxidase-B Sites

Abstract: In the postmortem human brain (27 specimens of frontal cortex, Brodmann area 9), the specific binding of the antagonists [³H]RX 821002 (2-methoxyidazoxan) to α_2A-adrenoceptors and that of [³H]idazoxan to l₂-imidazo-line sites (a nonadrenoceptor mitochondrial site) were determined in parallel to study the effect of aging (range, 4–89 years) on both brain proteins. The density of α_2A-adrenoceptors and age were negatively correlated (r=-0.71; p < 0.001). In contrast, the density of l₂-imidazo-line sites was positively correlated with aging (r= 0.59; p < 0.005). The ratio of receptor densities (α_2A/l₂) also showed a marked negative correlation with age (r=-0.76; p < 0.001). In an age-selected group (range, 10–89 years), the density of monoamine oxidase (MAO)-B sites labeled by [³H]Ro 19–6327 (lazabemide) also showed a positive correlation with age (r= 0.80; p < 0.005). In these subjects, the density of l₂-imidazoline sites correlated well with the density of MAO-B sites (r= 0.70; p < 0.005). The ratio of the density of these sites (MAO-B/l₂) did not correlate with the age of the subject at death (r=-0.15). In the human frontal cortex, idazoxan displayed very low affinity (K_i= 89 μM) against the binding of [³H]Ro 19–6327 to MAO-B, which discounted a direct interaction of [³H]idazoxan with the active center of the enzyme and indicated that the l₂-imidazoline site cannot be identified with MAO-B. However, l₂-imidazoline sites and MAO-B show a clear coexpression not only in the human frontal cortex during the process of aging, but also in various brain regions of the human and rat brains. It is suggested that the l₂-imidazoline site has a specific location on glial (astrocyte) cells.