Relationship Between [3H]Mazindol Binding to Dopamine Uptake Sites and [3H]Dopamine Uptake in Rat Striatum During Aging

Certain drugs exhibit a remarkable correlation between their ability to inhibit synaptosomal uptake of dopamine and the binding of [3H]mazindol to striatal membranes. To investigate the role of mazindol binding sites in the dopamine uptake process and the fate of these sites (labeling dopaminergic neurons) during aging, we have examined the properties of mazindol binding and dopamine uptake in individual young and old rats. There was a 48% decrease (p = 0.0001) in the Bmax of mazindol binding and a 23% decrease (p = 0.0166) in the Vmax of dopamine uptake with no apparent change in their affinities with age. Regression analysis of the relationship between Bmax and Vmax exhibited a significant correlation in old (p = 0.0156) but not young rats (p = 0.1398). These data suggest that the number of mazindol binding sites decreases with age and that the number of sites on the dopamine transporter complex far exceeds the number required to elicit maximal dopamine uptake.     

5-Hydroxytryptamine Stimulates [3H]Dopamine Release from the Fish Retina

Abstract: Serotonin (5-hydroxytryptamine, 5-HT; 0.5 μM and above) stimulated the release of [³H]dopamine ([³H]DA) from particulate fractions of the carp ( Cyprinus carpio ) retina. The 5-HT effect was dose- and Ca²⁺-dependent, and was structurally specific. A similar response was not elicited by the other indoles (5,6-dihydroxytryptamine, 5,7-dihydroxytryptamine, 5-hydroxytrypto-phan, or 5-hydroxyindoleacetic acid) examined. An increase in [³H]DA release was elicited by addition of 5-HT agonists (5-methoxytryptamine, 5-methoxy- N,N- dimethyltryptamine, and tryptamine), but not antagonized by three 5-HT antagonists (metergolin, methysergide, and spiperone). Either DA alone or noradrenaline (0.5 m M ) produced a large increase in [³H]DA release from the particulate fractions, but this action was Ca²⁺-independent. Further, no significant release of [³H]γ-aminobutyric acid could be evoked by 5-HT (0.5 mM) under similar experimental conditions. Taken together, the present data suggest that 5-HT stimulates [³H]DA release from the fish retina through a specific receptor-mediated mechanism on dopaminergic terminals, but not through an exchange or nonspecific phenomenon.     

[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.     

Comparison of the Binding of [3H]Spiperone and [3H]Domperidone in Homogenates of Mammalian Retina and Caudate Nucleus

Abstract— The specific binding of [³H]spiperone and [³H]domperidone, as defined by 1 μ m -(+)butaclamol, was compared in homogenates of bovine retina and caudate nucleus. Scatchard analyses of saturation data for [³H]spiperone binding yielded dissociation constants ( K _d) of 0.35 n m in the retina and 0.64 n m in the caudate nucleus. Comparison of the maximum number of binding sites (B_max) present in each tissue indicated that the density of sites in bovine caudate nucleus (270 fmol/mg protein) was approximately three times higher than in bovine retina (92 fmol/mg protein). This difference was even more marked in guinea pig tissues, with a ratio of 7:1 between corpus striatum and retina. The pharmacological analysis of [³H]spiperone binding in both the bovine retina and caudate nucleus indicated an interaction with dopaminergic rather than serotonergic sites. However, inhibition curves obtained to dopaminergic agonists in the bovine retina were significantly steeper than those observed in the bovine caudate nucleus, as reflected in the greater Hill coefficients obtained for these agents in the retina. Furthermore, only a small amount of specific [³H]domperidone binding was observed in either the bovine caudate nucleus or the guinea pig striatum, whilst no specific [³H]domperidone binding was detectable in homogenates of either bovine or guinea pig retina. These data suggest that the retina possesses only a small population of dopaminergic D2 sites and that these binding sites may differ from those present in the caudate nucleus.     

Influence of Sodium and Sulphydryl Groups on [3H]Sulpiride Binding Sites in Rat Striatal Membranes

Abstract: The effect of thiol reagents on the specific binding of the atypical neuroleptic, sulpiride, to rat striatal membranes was examined. Pretreatment of membranes with N -ethylmaleimide (NEM), but not with iodoacetamide or dithiothreitol (DTT), diminishes [³H]sulpiride binding. The effect is dependent on time, temperature, and the concentration of NEM. The reaction proceeds with pseudo-first-order rate kinetics, indicating the involvement of a single essential SH group in the binding of [³H]sulpiride to striatal membranes sites. Both sodium and sulpiride, but not nucleotides, protect the [³H]sulpiride sites from NEM inactivation in a dose-dependent fashion. As sodium is essential for [³H]sulpiride binding it is possible that in the presence of sodium and sulpiride a conformational change takes place that makes the essential SH group less accessible t o alkylation.     

[3H]GBR 12935 Binding to Dopamine Uptake Sites in the Human Brain

Binding of 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine ([3H]GBR 12935) was studied in membrane preparations of several human brain regions. In putamen, the substituted piperazine derivates cis- and trans-flupenthixol displaced 90% of the total [3H]GBR 12935 binding. Computer-assisted analysis of the competition curves revealed a high-affinity site (30%; KiH = 54 nM) and a low-affinity site (60%; KiL = 4.5 microM). The dopamine uptake blockers mazindol and nomifensine only displaced 30% of the total [3H]GBR 12935 binding in a monophasic way. Binding of [3H]GBR 12935 to the dopamine uptake sites, i.e., that displaced by dopamine uptake blockers, corresponded to part of the binding having low affinity for flupenthixol and was only detected in putamen, nucleus caudatus, nucleus accumbens, and substantia nigra. Even after masking the high-affinity binding site for flupenthixol by including 1 microM cis-flupenthixol in the binding assays, no dopamine uptake sites could be detected in globus pallidus, amygdala, thalamus, hippocampus, and cerebral cortex. Binding of [3H]GBR 12935 to dopamine uptake sites was lost in the nucleus caudatus ipsilateral to ventral midbrain infarctions, confirming their location on nigrostriatal nerve endings. Gross unilateral lesions of the striato- and pallidonigral pathways did not affect the number of dopamine uptake sites in the ipsilateral substantia nigra, suggesting that they may reside on the soma or dendrites of nigral neurons.     

Ontogeny of Receptor Binding Sites for [3H]Glutamic Acid and [3H]Kainic Acid in the Rat Cerebellum

The development of the specific binding sites for L-[3H]glutamic acid (KD = 370 nM) and for [3H]kainic acid (KD = 39 nM) was studied in the rat cerebellum. Specific binding at both sites remains low during the first week after birth but increases markedly during the second and third weeks after birth, when glutamatergic parallel fiber synaptogenesis occurs. The development of the kainate site lags behind that of the glutamate site, indicating their autonomy.     

Comparison of [3H]WIN 35,428 Binding, a Marker for Dopamine Transporter, in Embryonic Mesencephalic Neuronal Cultures with Striatal Membranes of Adult Rats

Abstract: In contrast to striatal membranes of adult rats, where high- ( K _D1= 34 n M ) and low- ( K _D2= 48,400 n M ) affinity binding sites for [³H]WIN 35,428 are present, in primary cultures of ventral mesencephalon neurons (CVMNs) only low-affinity binding sites were found ( K _D= 336,000 n M ). The binding of [³H]WIN 35,428 in CVMNs prepared from rat embryos was reversible, saturable, and located in cytosol. Although dopamine (DA) uptake blockers inhibited [³H]DA uptake at nanomolar concentrations in CVMNs, the displacement of [³H]WIN 35,428 binding in CVMNs by DA uptake inhibitors required 100-8,000 times higher concentrations than were needed to displace [³H]WIN 35,428 binding in striatal membranes. Piperazine derivatives, e.g., GBR-12909, GBR-12935, and rimcazole, inhibited [³H]WIN 35,428 binding in CVMNs more effectively than did cocaine, WIN 35,428, mazindol, nomifensine, or benztropin. A positive correlation ( r = 0.779; p < 0.001) was found between drug affinities for the striatal membrane sites labeled by [³H]WIN 35,428 and their abilities to inhibit DA uptake in CVMNs, whereas no correlation existed between the IC₅₀ values of drugs that inhibited [³H]WIN 35,428 binding and [³H]DA uptake in CVMNs. The cytosolic [³H]WIN 35,428 binding sites may be a piperazine acceptor and may not be involved in the regulation of the DA transporter.     

High- and Low-Affinity Binding Components for [3H]Imipramine in Rat Cerebral Cortex

The present study demonstrates that [3H]imipramine binds to both high- and low-affinity imipramine binding components on membranes prepared from rat cerebral cortex. Scatchard and computer analyses of saturation experiments using a wide range of [3H]imipramine concentrations (0.5 nM-50 nM) revealed the presence of two binding components. Inhibition experiments in which membranes were incubated with [3H]imipramine and various concentrations of unlabelled imipramine gave shallow inhibition curves with a Hill coefficient of 0.60 +/- 0.04. When dissociation rates of imipramine were studied, biphasic dissociation curves were obtained with apparent half-times of dissociation of 2.5 +/- 0.4 min and 18.5 +/- 2.5 min. Thus analysis of saturation, competition, and dissociation experiments indicate that [3H]imipramine binds to low as well as high-affinity binding sites in rat cortex.     

Specific [3H]Piflutixol Binding to CHAPS-Solubilised Rat Striatal Preparations Involves Dopamine D-2 but Not D-1 Binding Sites

[3H]Piflutixol binding to rat striatal membrane preparations identifies both D-1 and D-2 sites. We used [3H]piflutixol to characterise those binding sites present in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)-solubilised rat striatal preparations. The specific binding of [3H]piflutixol, as defined using cis-flupenthixol, to CHAPS-solubilised rat striatal tissue was saturable and of high affinity. Specific [3H]piflutixol binding to the solubilised preparations was displaced stereoselectively by the isomers of butaclamol and to an equal extent by both cis-flupenthixol and (+/-)-sulpiride. A positive correlation was found between the capacity of a range of drugs to displace [3H]piflutixol binding and the displacement of [3H]spiperone to the same preparations. The Bmax of [3H]piflutixol binding was not different from that of [3H]spiperone binding to the same preparation. These studies suggest that, in contrast to specific binding of membrane preparations, the specific binding of [3H]piflutixol to CHAPS-solubilised preparations involves mainly D-2 sites. Specific [3H]piflutixol binding, in contrast to [3H]spiperone binding, showed only slow dissociation from soluble preparations. The binding of [3H]piflutixol to CHAPS-solubilised preparations was retained during passage through a gel filtration column. This prelabelling of solubilised striatal preparations using [3H]piflutixol may aid in the purification of CHAPS-solubilised rat striatal D-2 sites.     

Comparison of the In Vitro Receptor Binding Properties of N-[3H]Methylspiperone and [3H]Raclopride to Rat and Human Brain Membranes

The aim of the present investigation was to study and compare the in vitro binding properties of the two radioligands N-[3H]methylspiperone ([3H]NMSP) and [3H]raclopride. These compounds, labeled with 11C, have been extensively used in positron emission tomography studies on central dopamine D2 receptors in schizophrenic patients, although with diverging results. One study (using [11C]NMSP) showed an increased dopamine receptor density in drug-naive schizophrenic patients, whereas in another study (using [11C]raclopride) the density in schizophrenic patients was no different from that in healthy controls. In the present study, using in vitro binding techniques, the density of the binding sites was found to be similar irrespective of which of the two radioligands was used (20 fmol/mg wet weight in rat striatum and 10 fmol/mg in human putamen; the 5-hydroxytryptamine 2 receptors were blocked with 40 nM ketanserin). [3H]NMSP had a 10-fold higher affinity (KD, 0.3 nM in rat striatum and 0.2 nM in human putamen) than [3H]raclopride (KD, 2.1 nM in rat striatum and 3.9 nM in human putamen), which was consistent with the longer dissociation half-life of [3H]NMSP compared with [3H]raclopride (14.8 and 1.19 min, respectively). There was an approximate overall similarity between the inhibition constants for five dopamine antagonists, chlorpromazine, haloperidol, raclopride, remoxipride, and NMSP, when using either radioligand. The Ki values were, however, two- to four-fold higher when using [3H]NMSP as the radioligand, irrespective of inhibiting compound, except for chlorpromazine (and haloperidol in human putamen). NMSP was found to inhibit the binding of [3H]raclopride competitively, whereas raclopride inhibited the binding of [3H]NMSP both competitively and noncompetitively. This difference suggests that part of the binding site is exclusively used by NMSP and can only be allosterically interfered with by raclopride. It is proposed that [3H]NMSP binds to an additional set of accessory binding sites, presumably located more distantly from the agonist binding active site than the sites to which [3H]raclopride binds.     

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.     

[3H] GBR-12935 Binding to Human Cerebral Cortex Is Not to Dopamine Uptake Sites

Abstract: The binding of the dopamine uptake inhibitor [³H] GBR-12935 to 16 regions of the human brain was investigated in competition experiments with increasing concentrations of GBR-12909, mazindol, and dopamine. The methodology used included a relatively high tissue concentration (8 mg/ml) and addition of 5 m M KCI in the assay buffer. GBR-12909 inhibited 80–90% of the binding in most regions, whereas dopamine only inhibited the binding in the striatum. Mazindol inhibited only part of the cortical binding at concentrations of >1 μ M , whereas the inhibition in the caudate and the putamen also contained a high-affinity component representing the dopamine uptake site. It is concluded that the [³H] GBR-12935 binding sensitive to GBR-12909 cannot be regarded as specific binding to the dopamine uptake site because the displaceable binding most likely is not related to the dopamine uptake site.     

Formation of [3H]Inositol Metabolites in Rat Hippocampal Formation Slices Prelabelled with [3H]Inositol and Stimulated with Carbachol

Rat hippocampal formation slices were prelabelled with [3H]inositol and stimulated with carbachol for times between 7 s and 3 min. The [3H]inositol metabolites in an acid extract of the slices were resolved with anion-exchange HPLC. Carbachol dramatically increased the concentration of [3H]inositol monophosphate, [3H]inositol bisphosphate (two isomers), [3H]inositol 1,3,4-trisphosphate, [3H]inositol 1,4,5-trisphosphate, and [3H]inositol 1,3,4,5-tetrakisphosphate. The levels of [3H]inositol 1,4,5-trisphosphate rose most rapidly; they were maximally elevated after only 7 s and declined toward control levels in 1 min followed by a more sustained elevation in levels for up to 3 min. When [3H]inositol 1,4,5-trisphosphate was incubated with hippocampal formation homogenates in an ATP-containing buffer it was very rapidly metabolised. After 5 min [3H]inositol 1,4-bisphosphate, [3H]inositol 1,3,4-trisphosphate, and [3H]inositol 1,3,4,5-tetrakisphosphate could be detected in the homogenates. Under similar experimental conditions [3H]inositol 1,3,4,5-tetrakisphosphate is metabolised to [3H]inositol 1,3,4-trisphosphate and an inositol bisphosphate isomer that is not [3H]inositol 1,4-bisphosphate. We conclude that like other tissues the primary event in the hippocampus following carbachol stimulation is the activation of phosphatidylinositol 4,5-bisphosphate selective phospholipase C.     

Binding of [3H] γ-Aminobutyric Acid and [3H]Muscimol in Purified Rat Brain Synaptic Plasma Membranes and the Effects of Bicuculline

Abstract: The binding of [³H] γ-aminobutyric acid ([³H]GABA) and [³H]muscimol has been studied in purified synaptic plasma membrane (SPM) preparations from rat brain. Scatchard analysis of specific binding (defined as that displaced by 100 μMγ-aminobutyrate) indicated that the binding of both radiolabelled ligands was best described by a two component Langmuir adsorption isotherm. The apparent K_D and B_max values for [³H]GABA at 4°C were K_D1, 20 nM; K_D2,165 nM; B_max1, 0.48 pmol;B_max2, 6.0 pmol. mg^?1; for [³H]muscimol at 4°C they were: K_D1, 1.75 nM; K_D2, 17.5 nM; B_maxl, 0.84 pmol. mg^?1; B_max2, 4.8 pmol.mg^?1; and for [³H]muscimol at 37°C they were: K_D1, 7.0 nM; K_m, 60 nM; B_max], 0.5 pmol-mg^?1; B_max2, 7.2 pmol-mg¹. Under the experimental conditions used, the similar B_milx values for [³H]GABA and [³H]muscimol binding to the SPM preparations suggests that the high- and low-affinity components for the two radiolabeled ligands are identical. The effects of the GAB A antagonist bicuculline on the binding of [³H]muscimol at 4^CC and 37°C were studied. At 4°C, antagonism of muscimol binding appeared to be competitive at the high-affinity site but noncompetitive at the low-affinity site. At 37°C, antagonism was again competitive at the high-affinity site but was of a mixed competitive/noncompetitive nature at the low-affinity site. Assuming that binding to the high-affinity site is associated with the pharmacological actions of bicuculline, the apparent K_D values obtained suggest a pA₂ value of 5.3 against [³H]muscimol at 4°C and 37°C. This figure is in good agreement with several estimates of the potency of bicuculline based on pharmacological measurements. Results from displacement studies using [³H]GABA and [³H]muscimol suggest that [³H]GABA might be a more satisfactory ligand than [³H]muscimol in GABA radioreceptor assays.     

Characterization of [3H]Dopamine Uptake Sites and [3H]Cocaine Recognition Sites in Primary Cultures of Mesencephalic Neurons During In Vitro Development

[3H]Dopamine uptake and [3H]cocaine binding sites were studied in primary cultures of ventral mesencephalon from 14-day-old rat embryos. Specific binding sites for [3H]cocaine and [3H]mazindol were detected only in intact cell cultures of ventral mesencephalon, and were absent in sonicated, washed membranes prepared from these cell cultures. [3H]Cocaine was not taken up by the cells through an active transport process because [3H]cocaine binding occurred also at 4 degrees C. Moreover, the possibility of [3H]cocaine entering the cells by passive diffusion and ion trapping was also excluded because extensive washing failed to remove [3H]cocaine from the cells. [3H]Cocaine binding was reduced to 6% of control when cells were permeabilized with streptolysin O (0.2 U/ml, 5 min). Taken together, these results suggest that in cultured mesencephalic neurons, [3H]cocaine may enter the cell by passive diffusion and then be sequestered by a cytosolic compartment that is lost in the process of permeabilization or sonication and washing of membrane preparations. Permeabilization of cultured neurons failed to alter the storage of [3H]dopamine. When cells were permeabilized with streptolysin O (0.2 U/ml; 5 min) after [3H]dopamine was taken up, [3H]dopamine was retained by the cells and did not leak into the incubation medium, indicating that [3H]dopamine was stored in sites that could not pass through the perforated membranes. In contrast, [3H]dopamine uptake into already permeabilized cells was reduced by 33%, suggesting that a cytosolic protein that had leaked out may play a functional role in the uptake process. In contrast to striatal membrane preparations of adult rats, [3H]cocaine binding in intact mesencephalic cell cultures was Na+ independent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of Tryptic Peptides of Benzodiazepine Binding Proteins Photolabeled with [3H]Flunitrazepam or [3H]Ro 15–4513

When rat brain membranes were incubated with the benzodiazepine agonist [3H]flunitrazepam or the partial inverse benzodiazepine agonist [3H]Ro 15-4513 in the presence of ultraviolet light one protein (P51) was specifically and irreversibly labeled in cerebellum and at least two proteins (P51 and P55) were labeled in hippocampus. After digestion of the membranes with trypsin, protein P51 was degraded into several peptides. When P51 was photolabeled with [3H]Ro 15-4513, four peptides with apparent molecular weights of 39,000, 29,000, 21,000, and 17,000 were observed. When P51 was labeled with [3H]flunitrazepam, only two peptides with apparent molecular weights of 39,000 and 25,000 were obtained. Protein P55 was only partially degraded by trypsin, and whether it was labeled with [3H]flunitrazepam or [3H]Ro 15-4513 it yielded the same two proteolytic peptides with apparent molecular weights of 42,000 and 45,000. These results support the existence of at least two different benzodiazepine receptor subtypes associated with proteins P51 and P55. The different receptors seem to be differentially protected against treatment with trypsin. In addition, these results indicate that in the benzodiazepine receptor subtype associated with P51 benzodiazepine agonists and partial inverse benzodiazepine agonists irreversibly bind to different parts of the molecule.     

Comparison of [3H]Choline and d-[3H]Aspartate Efflux from Guinea Pig and Human Neocortex

The outflow of [3H]choline ([3H]Ch) evoked by electrical field stimulation and the efflux of D-[3H]Asp induced by 35 mM KCl and 1-10 microM ouabain were studied in human and guinea pig cortical slices, kept under identical experimental conditions. [3H]Ch outflow was significantly lower whereas D-[3H]Asp efflux was significantly higher in humans than in guinea pigs. This suggests a different proportion of the two neuronal systems in these two species. Blockade of muscarinic autoreceptors with atropine increased, whereas stimulation of alpha 2 receptors with norepinephrine (NE) reduced, the evoked [3H]Ch outflow to the same extent in human and guinea pig cortical slices. Conversely, NE did not affect ouabain-induced D-[3H]Asp efflux, suggesting that an alpha 2-mediated control is not operative in the glutamatergic cortical structures. Desmethylimipramine, 2-5 microM, was able to increase [3H]Ch outflow through atropine-like mechanisms only in the human. This drug at 20-50 microM inhibited [3H]Ch and D-[3H]Asp efflux in both species, through mechanisms unrelated to its monoamine reuptake blocking properties. Thus, similarities and differences can be detected between humans and guinea pigs with regard to (a) the relative potency of the cholinergic and acidic amino acidergic signals and (b) the modulation of neurotransmitter outflow by drugs acting on auto- and the heteroreceptors.     

[3H]GBR-12935 Binding to the Dopamine Transporter Is Decreased in the Caudate Nucleus in Parkinson''s Disease

The specific binding of [3H]GBR-12935 to membranes prepared from human caudate nucleus is saturable (Bmax 1.36 +/- 0.18 pmol/mg protein), sodium dependent and of high affinity (KD 2.34 +/- 0.18 nM). Freezing of tissue from rat brain, or refrigeration followed by freezing, results in a small but significant (less than or equal to 20%) decrease in specific [3H]GBR-12935 binding when compared to the binding observed in fresh (nonfrozen) tissue, and this decrease may account, in part, for the differences in specific binding between rat and human brain membranes. Despite small differences in binding site density between fresh and frozen tissue there is a good correlation (r = 0.98; p less than 0.01) between the potencies of a series of drugs in displacing specific [3H]GBR-12935 binding to human caudate membranes and rat striatum as well as in inhibiting dopamine uptake in rat striatal synaptosomes (r = 0.96; p less than 0.01). The specific binding of [3H]GBR-12935 to membranes prepared from the caudate nuclei of patients with Parkinson's disease is decreased compared to membranes prepared from age- and sex-matched controls. These data suggest that [3H]GBR-12935 binds in a sodium-dependent fashion to the dopamine transport complex in human brain and that specific binding is decreased by a pathological degeneration of dopaminergic neurons to the caudate nucleus.