Dopamine D2 receptors selectively labeled by a benzamide neuroleptic: [3H]-YM-09151-2

Summary In order to label dopamine D₂ receptors selectively we tritiated the potent benzamide neuroleptic, YM-09151-2 (26.7 Ci/mmol). The binding of [³H]-YM-09151-2 to canine striatal membranes was saturable and specific with a K _D of 57 pmol/l and B _max of 36 pmol/g tissue as determined by Scatchard analysis. The K _D, but not the B _max, of [³H]-YM-09151-2 increased 6-fold in the absence of sodium chloride. [³H]-YM-09151-2 labeled 40% more sites than [³H]-spiperone in the same tissue homogenate. [³H]-YM-09151-2 binding was inhibited by dopaminergic drugs in a concentration and stereoselective manner with the appropriate dopamine D₂ receptor profile. Thus, dopamine agonists inhibited [³H]-YM-09151-2 binding to canine striatal membranes with the following rank order of potency: (–)-N-n-propylnorapomorphine > apomorphine > (±)-6,7-dihydroxy-2-aminotetralin > (+)-N-n-propylnorapomorphine > dopamine > (–)-noradrenaline > serotonin > (–)-isoprenaline. Dopaminergic antagonists competed for [³H]-YM-09151-2 binding with the following order of potency: spiperone > (+)-butaclamol > haloperidol > clebopride > (–)-sulpiride > SCH-23390 > (–)-butaclamol. Furthermore, dopamine agonists recognized 2 states of the receptor labeled by [³H]-YM-09151-2, D ₂ ^high and D ₂ ^low . The D ₂ ^high state of the receptor could be converted to D ₂ ^low by guanine nucleotides and sodium ions as is the case for [³H]-spiperone binding to D₂ receptors. [³H]-YM-09151-2 appears to be a more selective ligand for dopamine D₂ receptors than [³H]-spiperone, since YM-09151-2 displays approximately 9-fold lower affinity than spiperone for cortical serotonergic (S₂) receptors. [³H]-YM-09151-2 may become a useful tool for the selective characterization of dopamine D₂ receptors.Abbreviations used (±)ADTN (±)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene - NPA N-n-propylnorapomorphine - Gpp(NH)p 5-guanylylimidodiphosphate     

A pharmacological comparison of [3H]-granisetron binding sites in brain and peripheral tissues of the mouse

The affinities of a range of structurally diverse 5-HT₃ receptor agonists and antagonists for [³H]-granisetron binding sites have been measured in membrane homogenates prepared from central and peripheral tissues of the mouse. By comparing the affinities of compounds across these tissues, the question of whether intea-species 5-HT₃ receptor subtypes exist in the mouse has been addressed.In entorhinal cortex and brainstem, [³H]-granisetron bound to a single high affinity saturable binding site (K_d 0.47 ± 0.14 and 0.60 ± 0.05 nM; B _max 20 ± 6 and 7 ± 2 fmol (mg protein)^–1 respectively; mean ±SEM; n = 3). In distal and proximal colon, the specific binding of [³H]-granisetron was best fitted to a 2-site model. K_d values obtained for the high affinity site were similar to those obtained in brain tissue (distal colon: 0.47 ± 0.09 nM, n = 4; proximal colon: 0.39 ± 0.09 nM, n = 4). In salivary gland, 2-sites were evident in 2 out of 4 experiments. The K_d value (calculated from the high affinity site in the 2-site model) was approximately 10-fold less than in brain or colon (3.3 ± 1.1 nM, n = 4). B _max values were 7 ± 2, 4 ± 1 and 71 ± 16 fmol (mg protein)^–1 for distal colon, proximal colon and salivary gland respectively. For all tissues the estimated affinity of the low affinity site was variable, and B _max values could not be reliably calculated.Extensive comparative studies performed with 17 different 5-HT₃ receptor agonists and antagonists in the five tissues did not reveal differences in affinity for any compound between the entorhinal cortex and the brainstem nor between the two regions of the colon. However, MDL72222, R-zacopride, d-tubocurarine, and GR80284 apparently had significantly lower affinity for colon than brain binding sites. Also, MDL72222, 2-methyl-5-HT, GR80284, 1-(m-chlorophenyl)-biguanide, metoclopramide, and granisetron had significantly lower affinity for the salivary gland binding sites than the brain binding sites. In an attempt to replicate these observations, we conducted a second study using the compounds which had shown the largest inter-tissue differences in affinity keeping as many variables as possible constant. Simultaneous comparative assays on entorhinal cortex, colon and salivary gland homogenates taken from the same mice showed that the differences that were apparent in the initial comparative study were not maintained. In conclusion, we can find no clear evidence for the existence of tissue-specific subtypes of the 5-HT₃ high affinity binding site for [³H]-granisetron in the mouse in the tissues tested. However, a low affinity binding site for [³H]-granisetron was detected in peripheral tissues.     

Platelet 5-HT uptake sites in depression: three concurrent measures using [3H] imipramine and [3H] paroxetine

Platelet 5-HT uptake sites were measured in 40 depressed patients and 40 controls using [³H] imipramine binding, defined with desmethylimipramine (DMI) and Na⁺ dependence, and [³H] paroxetine binding. In control subjects the B_max of DMI defined [³H] imipramine binding was significantly higher than both Na⁺ dependent [³H] imipramine (by 30%) and [³H] paroxetine binding (by 22%). The B_max of Na⁺ dependent [³H] imipramine and [³H] paroxetine binding did not differ significantly. The K_d of Na⁺ dependent [³H] imipramine binding was significantly lower than the K_d of DMI defined [³H] imipramine binding. The binding of DMI defined and Na⁺ dependent [³H] imipramine and [³H] paroxetine did not differ significantly between depressed patients and controls in the total group, in those depressed patients who had never taken antidepressants or in those depressed patients who had been recently with-drawn from antidepressants. This study provides no support for the view that the number of platelet 5-HT uptake sites are reduced in depression.     

[3H]-Domperidone labels only a single population of receptors which convert from high to low affinity for dopamine in rat brain

Summary Dopamine recognized and competed for a single population of [³H]-domperidone-binding sites in rat striatum and olfactory tubercle when tested in the presence of sodium ions and guanine nucleotide [Gpp(NH)p]. In the absence of Na⁺ and Gpp(NH)p, however, dopamine recognized two components of [³H]-domperidone binding. Thus, [³H]-domperidone labelled only a single population of dopamine receptors (type D₂) which fully converted from high to low affinity for dopamine. These results agree with those found previously using [³H]-spiperone and [³H]-YM-09151-2.     

Binding studies with [3H]cis-methyldioxolane in different tissues

Summary Special conditions - tricine buffer containing Ca²⁺ and Mg²⁺, 22°C (TCM) — allow to label a much higher proportion of muscarinic receptors by [³H]cis-methyldioxolane (CD) than hitherto described (Vickroy et al. 1984 a). Taking the maximum number of binding sites, B _max, of [³H]QNB as 100%, B _max of [³H]CD amounts to 83% in the rat heart instead of the reported 17%, 33% in the cerebral cortex instead of 6%, 20% in hippocampus and 55% in pons/medulla. In the salivary glands specific binding was negligible. The affinities of a number of muscarinic agonists and antagonists to [³H]CD and [³H]QNB binding sites in different tissues of the rat are compared. Apparent affinities of agonists are much higher in the [³H]CD system, affinities of antagonists are slightly higher in the [³H]QNB system. In both assay systems receptors of heart and pons/ medulla membranes seem to have similar drug specificity. They differ somewhat from those in the cortex. Receptors in the salivary glands, however, seem to be completely different from those in the other three tissues. In the heart [³H]CD binding can be abolished almost completely by GppNHp. In the cortex about half of the [³H]CD binding is susceptible to GppNHp. The reduction of binding in the cortex is due to a change in B _max and not in the dissociation constant K _D. Competition of unlabelled pirenzepine with [³H]CD: In heart and pons/medulla only low affinity sites for pirenzepine (M₂-receptors) are labelled by [³H]CD. In regions rich in M₁ receptors like hippocampus (80% M₁ receptors) or cortex (65–70% M₁ receptors) the proportion of M₁ receptors labelled by [³H]CD is smaller than expected considering the concentration of M₁ receptors present in these tissues. Thus [³H]CD, under the conditions described in this paper, seems to label preferentially but not exclusively M₂ receptors in their agonist high affinity form. Send offprint requests to A. Closse at the above address     

Studies on the binding of [3H]flumazenil and [3H]sarmazenil in post-mortem human brain

Two benzodiazepine analogues, [³H]flumazenil and [³H]sarmazenil, were used to study the GABA_A/benzodiazepine receptor complex in human post-mortem brain using in vitro receptor assays on homogenates and whole hemisphere autoradiography. Both radioligands bound in a saturable manner to single binding sites in the tissue preparations from any brain region. The highest levels of binding were found in the cortical regions and in cortex cerebelli. Both [³H]flumazenil and [³H]sarmazenil were excellent radioligands for autoradiography with high binding in cerebral and cerebellar cortex with no or very low binding in areas with white matter. The addition of a high concentration of flumazenil or clonazepam did not inhibit the binding of [³H]sarmazenil to granule cells in the cerebellum while the binding of [³H]flumazenil was abolished completely in all regions. The results show that with the two different radioligands, one an antagonist and one a partial inverse agonist, the binding pattern to GABA_A/benzodiazepine receptor complex is approximately similar in most brain regions. The additional binding seen in the cerebellum with [³H]sarmazenil is suggested to be due to binding to an α₆-containing complex.     

[3H]MDL 100,907: a novel selective 5-HT2A receptor ligand

In studies using standard radioligands, unlabeled MDL 100,907 (R-(+)--(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidinemethanol) has been shown to have a high degree of selectivity for the 5-HT_2A receptor. The present study was undertaken to investigate the receptor binding characteristics of [³H]MDL 100,907 in rat cortical homogenates. [³H]MDL 100,907 was found to reach equilibrium at 37°C after 15 min. Saturation experiments indicated binding to a single site with a K_D of 0.56 nM, Hill slope of 1.15, and a B_max of 512 fmol/mg protein. In parallel experiments with the standard 5-HT_2A receptor radioligand, [³H]ketanserin, with prazosin added to block ₁ receptors, a similar Hill slope and B_max was noted but a two-fold higher K_D was found. In competition binding studies using 0.5 nM [³H]MDL 100,907, some 19 standard ligands to various receptors including the 5HT_1A, D₂, ₁, and receptors resulted in estimated K_I values that were consistent with [³H]MDL 100,907 selectively binding to the 5-HT_2A receptor. A comparison of the K_I values for 17 standard 5-HT_2A receptor agonists and antagonists displacing [³H]MDL 100,907 versus [³H]ketanserin resulted in a highly significant linear correlation (R² = 0.96, P<0.001). Taken together these results suggest that [³H]MDL 100,907 is binding to the 5-HT_2A receptor with a sub-nanomolar affinity without the use of secondary blocking agents.     

Nitroimipramines — Synthesis and pharmacological effects of potent long-acting inhibitors of [3H] serotonin uptake and [3H] imipramine binding

Summary A series of nitro derivatives of imipramine have been prepared by nitration of imipramine. Several of the products, especially 2-nitroimipramine (2) and 2,8-dinitroimipramine (4) were found to be very potent inhibitors of [³H] serotonin uptake and high affinity [³H] imipramine binding in human platelets. In contrast to the parent antidepressant, imipramine, the inhibition of platelet [³H] serotonin uptake and [³H] imipramine binding by the nitro derivatives of imipramine was long-acting and essentially irreversible at low temperatures. These compounds should prove to be valuable tools for subsequent studies on the purification and characterization of the transport protein(s) involved in serotonin uptake and may have novel behavioral and clinical effects.     

Cholinergic modulation of [3H]dopamine release from dendrosomes of rat substantia nigra

Summary Dendrosomes prepared from substantia nigra are able to take up and release [³H]dopamine in a Ca²⁺-dependent manner. The V_max values of [³H]dopamine uptake in substantia nigra dendrosomes was about 5 times lower than that in caudate putamen synaptosomes. The pattern of the K⁺-dependency of the [³H]dopamine release in substantia nigra dendrosomes was significantly different from that found in caudate putamen synaptosomes. The release of [³H]dopamine evoked by 15 mmol/l KCl from superfused dendrosomes was increased in a concentration-dependent manner by acetylcholine. The maximal potentiation produced by acetylcholine was about 40%. The potentiation of [³H]dopamine release by 10 µmol/l acetylcholine was insensitive to mecamylamine but antagonized by atropine and by pirenzepine. The effects of acetylcholine on the release of [³H]acetylcholine from substantia nigra nerve endings was also studied. Exogenous acetylcholine added to the superfusion medium decreased in a concentration-dependent manner the release of acetylcholine. This effect was not antagonized by mecamylamine or pirenzepine but fully antagonized by atropine. The data suggest the existence, in the substantia nigra of the rat, of two distinct muscarinic receptor subtypes regulating respectively dopamine release from dopamine dendrites and acetylcholine release from cholinergic nerve terminals.Part of this work was presented at a satellite meeting of the 11th International Congress of Pharmacology: Dopamine '90 held in Como, Italy (July 1990) Send offprint requests to M. Raiteri at the above address     

[3H] GR67330, a very high affinity ligand for 5-HT3 receptors

Summary GR67330 potently inhibited 5-hydroxytryptamine (5-HT)-induced depolarizations of the rat isolated vagus nerve. At the higher concentrations used (0.3 nmol/l–1 nmol/l) this was accompanied by a marked reduction in the maximum response to 5-HT. The calculated pK_B value was 10.2.The binding of the tritiated derivative of GR67330 to homogenates of rat entorhinal cortex was examined. Kinetic analysis revealed that specific [³H] GR67330 (0.1 nmol/l) binding was rapid and reversible. Association and dissociation rate constants were 1.48 ± 0.36 × 10⁸ mol/l^–1 s^–1 and 7.85 ± 0.41 × 10^–3 s^–1 respectively. Equilibrium saturation analysis revealed specific binding was to a single site (Bmax 22.6±0.21 fmol/mg protein) of high affinity (Kd 0.038±0.003 nmol/l). At low ligand concentrations, specific binding was up to 90% of total binding. If unlabelled GR67330 was used to define non-specific binding two sites were evident (Kd₁ 0.066 ± 0.007 nmol/l, Kd₂ 20.1 ± 9.7 nmol/l; Bmax₂ 31.5 ± 3.2 fmol/mg protein, Bmax₂ 1110 ± 420 fmol/mg protein). [³H] GR67330 binding was inhibited potently by 5-HT₃ antagonists and agonists. Ligands for other 5-HT receptors and other neurotransmitter receptors were either only weakly active or inactive at inhibiting binding. Hill numbers for antagonist inhibition of binding were close to unity, except for quipazine which was significantly greater than one. In common with other 5-HT₃ binding studies, all 5-HT₃ agonist tested had Hill numbers greater than one (1.51–1.71). GR38032 and GR65630 inhibited a greater proportion of binding than other 5-HT₃ antagonists, this additional binding was interpreted as inhibition from a second saturable site unrelated to the 5-HT₃ receptor.Homogenates of five areas of rat brain were examined for specific [³H]-GR67330 binding (entorhinal cortex, cingulate cortex, parietal cortex, hippocampus and nucleus accumbens/olfactory tubercle). In each brain area a site of very high affinity was labelled. Drug inhibition profiles were also very similar in each brain area. It is concluded that, because of its high affinity, [³H] GR67330 will be a useful ligand to label 5-HT₃ receptors especially in tissues with low receptor densities and to map 5-HT₃ receptors autoradiographically.Abbreviations 5-HT 5-Hydroxytryptamine - 8-OH-DPAT 8-hydroxy-2-di-N-propylaminotetralin - 5-CT 5-carboxyamidotryptamine - GR38032 (±) 1,2,3,9-tetrahydro-9-methyl-3[(2-methyl-1H-imidazol-1-yl)methyl]-4H-carbazol-4-one - GR65630 3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl) -1-propanone - GR67330 (±)1,2,3,9 - tetrahydro-9-methyl-3-[(5-methyl-1H-imidazol-4-yl)methyl]-4H-carbazol-4-one - MDL72222 1H,3,5H-tropan-3-yl-3,5-dichlorobenzoate - ICS 205–930 (3-tropanyl)-1H-indole-3-carboxylic acid ester - BRL24924 endo-4-amino-5-chloro-2-methoxy-N-(1-azabicyclo[3,3,1]non-4-yl)benzamide - BRL43694 endo-N-(9-methyl-9-azabicyclo[3,3,1]non-3-yl)-1-methyl-indazole-3-carboxamide - SDZ 206-830 (3-homotropanyl)-1-methyl-5-fluoro-indole-3-carboxylic acid ester - mCPP meta-chlorophenylpiperazine Send offprint requests to G. J. Kilpatrick at the above address     

Autoradiographic localisation of [3H]2-BFI imidazoline I2 binding sites in mouse brain

Imidazoline I2 binding sites are heterogeneous in nature and have been observed in the brain of a number of species. Development of specific imidazoline I2 radioligands, such as [3H]2-BFI and [3H]BU224, that have a high affinity for the imidazoline I2 binding site, has enabled the central distribution of these sites to be mapped. Extensive studies have been conducted on the rat brain with a number of radioligands. However, to date a comprehensive analysis of imidazoline I2 ligand binding in mouse brain has not been completed. In the present work we describe levels of [3H]2-BFI specific binding found throughout the mouse brain. [3H]2-BFI (2 nM) showed discrete regional distribution which was readily displaced by saturating concentrations of the specific imidazoline I2 ligand BU224. The highest levels of [3H]2-BFI specific binding were found in the dorsal raphe, paraventricular thalamus and nucleus accumbens. Moderate levels were found throughout the lining of the aqueduct, lateral ventricle, lateral 4th ventricle, 4th ventricle, 3rd ventricle, but not the dorsal 3rd ventricle. Based on the loss of [3H]idazoxan binding in brain homogenates from monoamine oxidase-A and B (MAO-A and MAO-B) deficient mice it has been suggested that imidazoline I2 binding sites are predominantly on MAO. Consistent with this hypothesis the regional distribution of [3H]2-BFI shows some overlap with that previously reported for MAO. However, in the rat imidazoline I2 binding sites have been shown to be heterogeneous in nature and it is likely [3H]2-BFI is binding to multiple imidazoline I2 binding sites within mouse brain.     

Autoradiographic localisation of D3-dopamine receptors in the human brain using the selective D3-dopamine receptor agonist (+)-[3H]PD 128907

The selective D₃-dopamine receptor agonist 4aR,10bR-(+)-trans-3,4,4a,10b-tetrahydro-4-[N-propyl-2,3-³H]-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol ([³H]PD 128907) was used to visualise D₃-dopamine receptors in whole hemisphere cryosections from post-mortem human brain. [³H]PD 128907 has an 18- to 40-fold selectivity for D₃- over D₂-dopamine receptors as compared to a 7- to 24-fold selectivity of the more commonly used ligand [³H]7-OH-DPAT. [³H]PD 128907 accumulated markedly in the nucleus accumbens and in the ventral parts of caudate nucleus and putamen, with a slightly heterogeneous (patch-matrix like) distribution. The binding in the lateral parts of caudate nucleus and putamen was much less dense. No binding was obtained in any other regions. A very high proportion of [³H]PD 128907 was specifically bound, as judged from the low binding remaining in the presence of the D₂/D₃-dopamine receptor antagonist raclopride. This gives the ligand a potential for the detection of low density D₃-dopamine receptors in the human brain. The binding obtained with [³H]PD 128907 was qualitatively similar to that using [³H]7-OH-DPAT in the presence of GTP. However, [³H]7-OH-DPAT labelled, in contrast to [³H]PD 128907, also D₃-dopamine receptors in neocortex. The new compound [³H]PD 128907 appears to be a suitable radioligand for autoradiographic examination of the D₃-dopamine receptor localisation in the human brain, and should also be useful for pharmacological studies of this receptor subtype. Received: 20 November 1995/Final version: 2 May 1996     

Synaptic concentration of dopamine in rat striatal slices in relationship to [3H]raclopride binding to the dopamine D2 receptor

The in vivo binding of dopamine (DA) radioligands to D2 receptors can be affected by competition with endogenous dopamine. In the present study, we used a brain slice preparation that provides more controlled conditions than in vivo preparations in order to examine the relationship between synaptic DA and the binding of [3H]raclopride to D2 receptors. We also estimated the synaptic DA concentration in rat striatal slices by determining the changes in [3H]raclopride binding. To correlate the changes in [3H]raclopride binding with the concentration of synaptic DA, the kinetic parameters were determined. [3H]Raclopride reached equilibrium binding conditions within two hours. The Ki value for DA in inhibiting [3H]raclopride binding was about 2.2 nM. The increase in synaptic DA evoked by electrical stimulation decreased the striatal binding of [3H]raclopride in a frequency-dependent manner. Increases in the DA concentration evoked by amphetamine (AMPH) or cocaine decreased [3H]raclopride binding by 74% or 20%, respectively, corresponding to increases in the synaptic DA concentrations of 1.6 nM or 0.6 nM, respectively. Pargyline also decreased [3H]raclopride binding by 36% corresponding at a concentration of 1.2 nM. In contrast, the depletion of synaptic DA by alpha-methyl-p-tyrosine (alpha-MpT) increased the specific binding of [3H]raclopride by 43% when the DA concentration was decreased to 0.7 nM. The changes in the DA concentration at the synapse were responsible for the changes in the striatal binding of [3H]raclopride. The values calculated in this study may therefore approximate the changes in the synaptic DA concentration in rat striatal slices following manipulation.     

Effect of repetitive electroconvulsive treatment on sensitivity to pain and on [3H]nitrendipine binding sites in cortical and hippocampal membranes

The effect of electroconvulsive shock (ECS) on the responsiveness to pain (measured by the hot-plate test) and on the characteristics ofl-type calcium channels (measured as [³H]nitrendipine binding sites) in the cortex and hippocampus was tested on the Wistar rat. In animals receiving a single ECS, the calcium channel density and affinity 24 h after treatment did not differ from the controls; the response to pain was also at the control level. Repeated ECS (eight once-daily shocks) resulted in an increased responsiveness to pain (shortening of response latency) and in an increase in the density of cortical, but not hippocampal, calcium channels. The K_D value for [³H]nitrendipine binding sites in either brain region remained unaltered by ECS. The calcium channel antagonist nifedipine, which by itself did not significantly alter the response to pain, prevented the enhancement of pain sensitivity brought about by ECS. The results suggest activation of calcium-dependent mechanisms by repeated ECS and confirm the involvement of calcium channels in pain mechanisms.     

Unaltered 5-HT- and desipramine-sensitive [3H]imipramine binding and [3H]5-HT uptake in rat brain after chronic imipramine and norzimeldine treatment

Several reports have shown heterogeneity of [³H]imipramine binding to brain membranes. Recently, a high affinity and 5-HT sensitive [³H]imipramine binding site of protein nature, that was suggested to be identical to the substrate recognition site for 5-HT uptake, was demonstrated. Since most studies on the regulation of the [³H]imipramine binding sites by antidepressants have used desipramine displaceable binding, which is heterogenous in nature and contains binding not related to 5-HT uptake sites, the present report studies the possible effects of chronic (3 weeks) administration of imipramine or norzimeldine (10 mg/kg intraperitoneally twice daily) on 5-HT sensitive [³H]imipramine binding sites. For comparison, desipramine sensitive binding was also studied, as well as the physiological correlate 5-HT uptake. There were no changes in either [³H]imipramine binding or 5-HT uptake after the antidepressant treatment.Supported by the Swedish Medical Research Council Offprint requests to: J. Marcusson at Dept. of Geriatric Medicine     

Characterization of the [3H]-desipramine binding site of the bovine adrenomedullary plasma membrane

Summary The specific (i.e. nisoxetine-sensitive) binding of [³H]desipramine was studied in membranes prepared from bovine adrenal medullae. (1) [³H]desipramine bound reversibly and with high affinity (K _D = 2.8 nmol/l) to a single class of non-interacting binding sites (Hill coefficient = 0.96); the maximal number of binding sites (B_max) was 2.1 pmol/mg protein. (2) Binding of [³H]desipramine was dependent on [Na⁺] and [Cl^–]. Increasing the concentrations of these ions increased binding. (3) Substrates and inhibitors of the neuronal noradrenaline transport system (uptake,) inhibited binding of [³H]desipramine with a rank order of potency typical for an interaction with the uptake, carrier.The characteristics of [³H]desipramine binding remained essentially unchanged after solubilization of adrenomedullary membranes with the non-ionic detergent digitonin.The results indicate that the plasma membrane of bovine adreno-medulary cells is endowed with the neuronal uptake₁ transporter. Correspondence to: H. Bönisch     

新抗胆碱药[3H]三环哌酯对人脑M受体的作用

用放射配体受体结合试验法,研究了新化合物三环哌酯与人大脑皮质M受体的结合特性,并与QNB作了比较。饱和实验结果显示,[³H]三环哌酯的结合参数与[³H]QNB相近,两种配体的作用均符合单位点模型。竞争性抑制实验结果表明二者作用强度相当。[³H]三环哌酯的结合和解离速率常数均较[³H]QNB大,且其与皮质M受体的解离受季铵酚的变构调节,结果提示,两种配体与M受体有一些不同的结合特性,在M受体研究中,[³H]三环哌酯可以作为[³H]QNB的补充工具。     

Saturable uptake of [3H]-tryptamine in rabbit platelets is inhibited by 5-hydroxytryptamine uptake blockers

Summary [³H]-tryptamine is taken up by rabbit platelets through an active and staurable process which is temperture sensitive, sodium-dependent and inhibited by imipramine and non-tricyclic 5-hydroxytryptamine (5-HT) uptake blockers.There is an excellent correlation between the Ki for the inhibition of [³H]-tryptamine and [³H]-5-HT uptake in rabbit platelets for a series of 5-HT uptake blockers. These results indicate that [³H]-tryptamine is actively transported through the membrane of blood platelets by the same carrier that transports 5-HT.     

[3H]ICS 205-930 labels 5-HT3 recognition sites in membranes of cat and rabbit vagus nerve and superior cervical ganglion

Summary The binding characteristics of [³H]ICS 205-930, a 5-hydroxytryptamine 5-HT₃ receptor antagonist, were investigated in membranes prepared from cat and rabbit vagus nerve (VN) and superior cervical ganglion (SCG). The autoradiographic localisation of 5-HT₃ recognition sites was also assessed using [³H]ICS 205-930 in slices from cat medulla oblongata, nodose ganglion and vagus nerve.[³H]ICS 205-930 bound to a homogeneous population of high affinity recognition sites in cat VN: B_max = 201 ± 43 fmol/mg protein, pK_D = 9.26 ± 0.17 and SCG: B_max = 291 ± 40 fmol/mg, pK_D = 9.35 ± 0.80 (n = 3). Competition experiments performed in membranes from cat VN and SCG with agonists and antagonists suggested the presence of a homogeneous population of [³H]ICS 205-930 recognition sites. Competition curves were steep and monophasic and were best fitted by a 1 receptor site model. The following rank order of affinity for [³H]ICS 205-930 binding sites was observed with antagonists: SDZ 206-830 = ICS 205-930 > BRL 43694 > SDZ 206–792 > quipazine > MDL 72222 > metoclopramide > mCPP and agonists: 2-methyl-5-HT = 5-HT > phenylbiguanide. A similar profile was observed for a limited series of compounds in rabbit membranes. Drugs acting at 5-HT₁, 5-HT₂ and dopamine receptors (domperidone, spiperone and metergoline) showed very low affinities for [³H]ICS 205-930 recognition sites. The sites labelled with [³H]ICS 205-930 in vagus nerve and superior cervical ganglion of both species displayed the pharmacological profile of a 5-HT₃ receptor. There was a significant correlation between the rank order of affinity of the tested compounds for [³H]ICS 205-930 recognition sites in cat and rabbit membranes and their rank order of affinity for 5-HT₃ receptors from neuroblastoma-glioma NG 108-15 cells. Autoradiographic studies suggest that [³H]ICS 205-930 binding sites are present over and around the nodose ganglion cell somata, along certain fibers of the vagus nerve and in the terminal areas of this nerve in the medullar nucleus of the vagus.The present data demonstrate that [³H]ICS 205-930 identifies 5-HT₃ receptors in preparations of cat and rabbit vagus nerve and superior cervical ganglion.Send offprint requests to D. Hoyer at the above addressThe present results have been presented in part at the Winter Meeting of the British Pharmacological Society, London, December 20–22, 1988 (Hoyer et al. 1989)     

Bay K 8644 enhances the outflow of [3H]-noradrenaline and [3H]-DOPEG from isolated rat atria

Summary The effect of Bay K 8644 (a dihydropyridine Ca²⁺-channel activator), was examined on spontaneous and stimulus-evoked release of tritium from isolated rat atria prelabelled with [³H]-noradrenaline. Bay K8644 (3mol/l) significantly increased atrial rate from 206±7 to 259±9 beats·min^–1 (P<0.05) and also tritium outflow (expressed as fractional rate of loss in min^– × 103) from 6.49±0.35 to 8.61±0.74 (P<0.05). Neither the maximal rate nor the overflow of tritium induced by stimulation of sympathetic nerve terminals was changed by the compound. The increase in basal tritium outflow produced by Bay K 8644 was calcium-dependent. However, it could not be antagonized by nitrendipine. The overflow of tritium induced by Bay K 8644 consisted mainly of 3,4-dihydroxyphenylglycol ([³H]-DOPEG), indicating that the compound produces a leakage from the storage vesicles of sympathetic nerve terminals of the isolated rat atria.Members of Consejo Nacional de Investigaciones Científicas - Técnicas (CONICET), Argentina Send offprint requests to M. C. Camilión de Hurtado at the above address