Single Population Of Sites Research Articles

Characterisation and partial purification of the GABAB receptor from the rat cerebellum using the novel antagonist [[formula omitted]]CGP 62349

The novel GABAB receptor antagonist [3H]CGP 62349 binds rat cerebellar synaptosomal membranes with high affinity at a single population of sites (Kd=0.9 nM, Bmax=760 fmol/mg protein). Solubilisation with 1% Triton X-100/0.5 M NaCl/10% glycerol resulted in a marked increase in [3H]CGP 62349 binding (Kd=0.5 nM, Bmax=1285 fmol/mg protein). Competition of [3H]CGP 62349 binding with a range of GABAergic ligands yielded the same rank order at membrane-bound and soluble receptors: CGP 54626=CGP 55845≫GABA>(−)-baclofen>CGP 35348=CGP 36742. The GABAA ligand isoguvacine did not displace [3H]CGP 62349 binding. Partial purification of [3H]CGP 62349 binding sites was obtained by sucrose density centrifugation and a predominant protein in the peak binding fraction was recognised by an anti-GABAB receptor antibody and had a molecular weight similar to the recombinant expressed GABABR1a. These results demonstrate that [3H]CGP 62349 provides a useful additional tool for further characterisation of the pharmacology and biochemistry of the native GABAB receptor.

High affinity binding of [3H]epibatidine to rat brain membranes.

Several compounds, such as epibatidine, A-85380, and their analogs, have been identified recently as nAChR ligands whose affinities lie in the low picomolar range. Accurate measurement of such high affinities is fraught with certain technical difficulties, which may account for the inconsistency of previously reported affinities of epibatidine, ranging from 4 to 60 pM. Here, we demonstrate that (+/-)-[3H]epibatidine (1-500 pM) binds to a single population of sites in rat brain with KD of 8 +/- 2 pM. This affinity was confirmed in both kinetic experiments and competition assays with (+/-)-[3H]epibatidine and (-)-[3H]cytisine, which were performed under experimental conditions developed specifically for ligands with subnanomolar affinities. Variations from these conditions decreased the observed affinities.

Preclinical profile of befloxatone, a new reversible MAO-A inhibitor

Befloxatone, a novel oxazolidinone derivative, is a potent, selective and reversible monoamine oxidase A (MAO-A) inhibitor in vitro ( K iA=1.9–3.6 nM) and ex vivo (ED 50 MAO-A=0.02 mg/kg, p.o.). It does not interact with a large number of receptors, monoamine transporters or other amine oxidases. Binding studies with [ 3H]-befloxatone in rat brain sections show that it labels with high affinity ( K d=1.3 nM) a single population of sites with the pharmacological characteristics and regional distribution of MAO-A. In the rat brain, befloxatone (0.75 mg/kg, i.p.) increases tissue levels of monoamines and decreases levels of their deaminated metabolites. Acute administration of befloxatone (0.75 mg/kg, i.p.) induces an increase in extracellular striatal dopamine and cortical norepinephrine but not cortical serotonin levels in the rat. Befloxatone (1 mg/kg, i.p.) potently inhibits the firing rate of serotonergic neurons, partially decreases the firing of noradrenergic neurons and has no effect on the firing of dopaminergic neurons (a mirror image of its effects on monoamine release in terminal regions), suggesting that the relative effects of befloxatone on monoamine release may be governed by autoreceptor-mediated control of monoaminergic neurons at the cell body level. Befloxatone (0.03–0.3 mg/kg, p.o.) exhibits potent activity in behavioural models predictive of antidepressant activity. Befloxatone (up to 1.5 mg/kg, p.o.) does not potentiate the pressor effects of orally administered tyramine at centrally active doses and duodenal [ 3H]-befloxatone binding is displaced by increasing doses of orally administered tyramine (0.1–40 mg/kg, i.p.). These results suggest that befloxatone is a potent reversible MAO-A inhibitor with antidepressant potential and a wide safety margin with regard to the potentiation of the pressor effect of tyramine.

Selective sites for polyamine binding to rabbit intestinal brush-border membranes

The intestinal polyamine transporters have not yet been identified. Our aim was to characterize specific polyamine binding sites in rabbit intestinal brush-border membranes (IBBM) as a starting step for identification of polyamine transporters. This was investigated at 4° and at low membrane concentration. Saturation isotherms for [ 3H]putrescine (PUT) binding indicated a single population of sites (puT) with a dissociation equilibrium constant K d of 3.8 μ M and a density of sites B max of 58 pmol/mg of protein. [ 3H]spermidine (SPD) binding also involved only one class of sites (spD), albeit with a lower affinity ( K d = 106 μ M) and higher abundance ( B max = 1240 pmol/mg of protein) than puT. On the contrary, [ 14C]spermine (SPM) bound two classes of sites (spM 1 and spM 2) differing in their affinity ( K d = 2.5 and 31.4 μ M) and abundance ( B max = 467 and 1617 pmol/mg of protein, respectively). Membrane association of SPM at 4° was much faster than that of SPD and PUT, both of which proceeded at a similar rate. In contrast to PUT and SPD dissociation, SPM dissociation at 23° did not follow a first-order reaction. Specifically bound [ 3H]PUT, unlike [ 3H]SPD and [ 14C]SPM, dissociated at 23° independently of the addition of nonradioactive polyamine. Methylglyoxal- bis-(guanylhydrazone) was an extremely potent inhibitor of PUT binding ( K i = 3.2 ± 1.5 n M), but as with PUT and cadaverine (CAD), it did not alter [ 3H]SPD and [ 14C]SPM binding substantially. The intestinal brush-border membrane may contain at least three sites specific for polyamine binding and exhibiting different ligand selectivity. Site puT might be associated with the transport system already described for intestinal uptake of PUT.

Binding of ADP to sarcoplasmic reticulum Ca 2+-ATPase in the absence of Mg 2+ is specifically inhibited by thapsigargin: observations on the ligand stoichiometry

The conditions of nucleotide binding to native, though partly purified, Ca 2+-ATPase from SR as well as the stoichiometry of nucleotide and strontium binding and the phosphorylation capacity was reevaluated. Binding of MgADP appeared to be aberrant whereas even high-affinity binding of [ 14C]-ADP took place in the absence of Mg 2+. Also low-affinity ATP binding was possible in the absence of divalent cations. A heterogeneity in ADP binding compatible with a two-component model in the absence of thapsigargin was changed to an apparent homogeneity of low-affinity receptors following a mole:mole interaction of enzyme and thapsigargin. Since the affinity of both components was reduced by thapsigargin, high- as well as low-affinity ADP binding seem to be specific and probably to the substrate receptor proper. Analysis of ADP binding isotherms in the absence of Mg 2+ according to a model of two independent populations of sites was compatible with a binding capacity of 8.49 ± 0.43 nmoles/mg protein corresponding to a molecular mass of 118 ± 6 kD per ADP site. The same total binding capacity was found for ATP. The phosphorylation capacity corresponded to more than one and less than two ≈P per two 110-kD peptides (formally one ≈P per 154 kD protein). Specific binding of Ca 2+ and the congener Sr 2+ to SR Ca 2+-ATPase was compatible with their interaction with a single population of sites. The binding capacity was equal to one divalent cation per nucleotide binding peptide. The binding of one nucleotide and one divalent cation per ≈110 kD peptide and the absence of cooperativity in divalent cation binding might imply that Ca 2+-ATPase works as a monomer.

Binding and internalization of neurotensin in hybrid cells derived from septal cholinergic neurons.

Autoradiographic studies from our laboratory have previously demonstrated a selective association of high affinity neurotensin (NT) binding sites with basal forebrain cholinergic neurons. In search of an in vitro model for further characterization of the role and regulation of these sites, we have examined the binding and internalization of 125I-Tyr3-NT (125I-NT) and fluorescein isothiocyanate (FITC)-conjugated NT (fluo-NT) on SN17 hybrid cells, produced by fusion of embryonic murine septal cells with neuroblastoma. 125I-NT binding to SN17 membrane preparations was specific and saturable. Scatchard analysis of the data was suggestive of an interaction with a single population of sites, the affinity (Kd = 1.7 nM) and pharmacological profile of which were comparable to those of neural NT receptors. No specific binding was observed on the parent neuroblastoma cell line, confirming that the expression of those sites is a neuronal trait. Incubation of whole SN17 cells with 125I-NT resulted in a time- and temperature-dependent internalization of the specifically bound peptide. The t1/2 of this internalization was estimated at 13 min, a value nearly identical to that reported for neurons in culture. Confocal microscopic analyses using fluo-NT indicated that the internalization process was endocytic in nature in that: 1) it was entirely blocked by the endocytosis inhibitor phenylarsine oxide; and 2) it was mediated through small intracytoplasmic particles the size and maturation of which corresponded to that of endosomes. It is proposed that the expression and internalization of NT receptors by SN17 hybrid cells represent a new facet of these cells' cholinergic phenotype that makes them amenable to the study of NT interactions with cholinergic cells.

125I]5-iodo-6-nitroquipazine: a potent and selective ligand for the 5-hyfroxytryptamine uptake complex. I. In vitro studies

In search of a potent and selective radioiodinated ligand for the 5-hydroxytryptamine (serotonin or 5-HT) uptake complex, we synthesized and evaluated the in vitro properties of [ 125I]5-iodo-6-nitroquipazine. The binding properties and pharmacological profile of this radioligand were studied in rat brain homogenates, and it was found to display high affinity and selectivity for the serotonin uptake complex. Scatchard analysis of the binding data indicated a single population of sites with a K d of23 ± 6pM and aB max of 430 ± 50 fmol/mg protein (mean ±S.E.M., n - 7). Inhibitors of serotonin uptake were the most efficient competitors for [ 125I]5-iodo-6-nitroquipazine binding with K i values similar in rank order and magnitude to those obtained in studies of other established serotonin uptake blockers. Inhibitors of dopamine and norepinephrine uptake as well as a wide variety of postsynaptic receptor agents were relatively ineefective in inhibiting [ 125I]5-iodo-6-nitroquipazine binding to rat brain membranes. Serotonin was the only monoaminergic neurotransmitter capable of effectively competing for [ 125I]5-iodo-6-nitroquipazine binding sites and gave a K i value of 2.8 ± 0.6 μM. Lesions of the serotonergic system with p-chloroamphetamine resulted in a dramatic loss 90%of [ 125I]5-iodo-6-nitroquipazine binding to rat cortical membranes. Non-radiolabeled 5-iodo-6-nitroquipazine potently inhibited the binding of [ 3H]paroxetine to serotonin reuptake sites in rat cortical membranes with a K i of 0.17 ± 0.06n M. These results demonstrate that 5-iodo-6-nitroquipazine is a potent and selective ligand for the serotonin uptake complex, and the [ 125I]-labeled radioligand has an advantage of detecting low levels of binding sites.

An epithelial high-affinity amiloride-binding site, different from the Na+ channel.

Specific binding of the radioactive amiloride analogues [3H]phenamil and [3H]benzamil was studied in plasma membrane from chicken lower intestine. A single population of sites whose affinities and specificities towards pyrazinecarboxamides roughly resemble those of the epithelial Na+ channel, was identified. However, a matched comparison of pyrazinecarboxamide binding and Na+ transport inhibition revealed substantial differences between the high-affinity [3H]phenamil-binding site detected, and the site whose occupancy by phenamil blocks Na+ transport. First, 5-(N-ethyl-N-isopropyl)-amiloride was found to displace bound [3H]phenamil at concentrations that are at least 10-fold lower than those needed to block the channel. Second, the rates at which [3H]phenamil associates and dissociates from this site are lower than the rates at which Na+ channels are inhibited and reactivated, under similar conditions. A site with high affinity to both amiloride and 5-(N-ethyl-N-isopropyl)-amiloride was detected also in membranes from other epithelia. We conclude that tight epithelia contain a major high-affinity amiloride receptor other than the Na(+)-conducting channel, the Na+/H+ antiport or the Na+/Ca2+ exchanger. This site could be associated with a pool of nonconducting channels, another (but structurally related) channel, or a totally unrelated protein.

Autoradiographic localization of dopamine uptake sites in the rat brain with 3H-GBR 12935.

The regional distribution of dopamine (DA) uptake sites in the rat brain has been studied by quantitative autoradiography using [3H]GBR 12935 as a ligand. The binding of [3H]GBR 12935 to striatal sections was saturable and of high affinity (Kd = 1.6 nM); it occurred at a single population of sites and possessed the pharmacological features of the DA uptake sites. The highest densities of [3H]GBR 12935 binding sites were found in the caudate-putamen, nucleus accumbens, olfactory tubercle, ventral tegmental area and substantia nigra (especially in the pars compacta). Moderate levels of [3H]GBR 12935 binding were observed in globus pallidus, thalamus, hypothalamus, hippocampus, amygdala (basolateral nucleus) and prefrontal and singular cortices. This regional distribution of [3H]GBR 12935 binding closely correlated with the reported distribution of dopaminergic nerve terminals. The topographical distribution of [3H]GBR 12935 has also been studied in detail in striatal subregions and this distribution was compared, using quantitative TH immunoreactivity, to the density of striatal dopaminergic nerve terminals. There is good overlapping between these two regional distributions, the highest density of both markers was found in the lateral part of the striatum and a similar rostro-caudal gradient has been observed. A dopaminergic denervation caused a complete loss of [3H]GBR 12935 in basal ganglia ipsilateral to the lesion.

Evidence for heterogenous glycine domains but conserved multiple states of the excitatory amino acid recognition site of the NMDA receptor: regional binding studies with [3H]glycine and [3H]L-glutamate

The possible heterogeneity of the agonist and glycine sites of the N-methyl-D-aspartate (NMDA) receptor-complex was examined using receptor binding techniques. Binding of [3H]L-glutamate [( 3H]GLU) and [3H]glycine to synaptic membranes of cerebral and cerebellar cortices, and membranes of a granule cell preparation of rat cerebellum, was characterized. [3H]Glycine always labelled a single population of sites; densities of binding sites (Bmax) in cortical, cerebellar and "granule" membranes were 3.1, 0.87 and 3.6 pmol/mg protein, respectively. Dissociation constants (Kd) in the same three preparations were 0.13, 0.31 and 1.9 microM, respectively. In competition studies, D-cycloserine, but not D-serine and 7-chlorokynurenate, showed varying potency between the membrane preparations, and analysis of variance (ANOVA) revealed a significant interaction between ligands and membrane fractions. Binding of [3H]GLU was saturable and to a single population of sites: Kd 0.5-0.9 microM and Bmax 3.2-3.6 pmol/mg protein. In all three membrane preparations the rank order of potency of NMDA agonists as inhibitors of the binding of [3H]GLU was always L-aspartate greater than L-cysteate greater than L-cysteinesulphinate greater than L-serine-O-sulphate greater than ibotenate greater than L-homocysteate. NMDA, quinolinate and competitive NMDA antagonists were only weak inhibitors of the binding of [3H]GLU and never fully inhibited specific binding. Other subtype-selective excitatory amino acids were very weak or ineffective inhibitors of binding. Binding of NMDA agonists was better described by a two site model whereby the proportion of high affinity sites did not vary significantly across the three membrane preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiographic localization of α1‐adrenoceptors in the dog prostate and urethra with 3H‐prazosin

AbstractThe regional distribution of α1‐adrenoceptors in the dog urethra has been studied by quantitative autoradiography using the specific α1‐antagonist 3H‐prazosin as a ligand. The binding of 3H‐prazosin to urethral tissue sections was saturable, reversible, and of high affinity (Kd = 0.7 nM); it occurred at a single population of sites and possessed the pharmacological features of the α1‐adrenoceptor. The binding parameters of 3H‐prazosin on urethral tissue sections were found similar to those obtained on urethral membranes. Autoradiographic results revealed that the α1‐adrenoceptors are heterogeneously distributed along the dog urethral axis. The highest densities of 3H‐prazosin binding sites were found in the preprostatic urethra. Comparison between autoradiographic and histological slides revealed that the 3H‐prazosin binding sites were only localized on the smooth muscle fibres of the dog urethra.

Calcium ions inhibit the allosteric interaction between the dihydropyridine and phenylalkylamine binding site on the voltage-gated calcium channel in heart sarcolemma but not in skeletal muscle transverse tubules

Calcium channel blockers bind with high affinity to sites on the voltage-sensitive Ca2+ channel. Radioligand binding studies with various Ca2+ channel blockers have facilitated identification and characterization of binding sites on the channel structure. In the present study we evaluated the relationship between the binding sites for the Ca2+ channel blockers on the voltage-sensitive Ca2+ channel from rabbit heart sarcolemma and rabbit skeletal muscle transverse tubules. [3H]PN200-110 binds with high affinity to a single population of sites on the voltage-sensitive Ca2+ channel in both rabbit heart sarcolemma and skeletal muscle transverse tubules. [3H]PN200-110 binding was not affected by added Ca2+ whereas EGTA and EDTA noncompetitively inhibited binding in both types of membrane preparations. EDTA was a more potent inhibitor of [3H]PN200-110 binding than EGTA. Diltiazem stimulates the binding of [3H]PN200-110 in a temperature-sensitive manner. Verapamil inhibited binding of [3H]PN200-110 to both types of membrane preparations in a negative manner, although this effect was of a complex nature in skeletal muscle transverse tubules. The negative effect of verapamil on [3H]PN200-110 binding in cardiac muscle was completely reversed by Ca2+. On the other hand, Ca2+ was without effect on the negative cooperativity seen between verapamil and [3H]PN200-110 binding in skeletal muscle transverse tubules. Since Ca2+ did not affect [3H]PN200-110 binding to membranes, we would like to suggest that Ca2+ is modulating the negative effect of verapamil on [3H]PN200-110 binding through a distinct Ca2+ binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Putative M 2 muscarinic receptors of rat heart have high affinity for organophosphorus anticholinesterases

The M 2 subtype of muscarinic receptor is predominant in heart, and such receptors were reported to be located in muscles as well as in presynaptic cholinergic and adrenergic nerve terminals. Muscarinic receptors of rat heart were identified by the high affinity binding of the agonist (+)-[ 3H] cis-methyldioxolane ([ 3H]CD), which has been used to label a high affinity population of M 2 receptors. A single population of sites ( K D 2.74 n m; B max of 82 fmol/mg protein) was detected and [ 3H]CD binding was sensitive to the M 2 antagonist himbacine but much less so to pirenzepine, the M 1 antagonist. These cardiac receptors had different sensitivities to NiCl 2 and N-ethylmaleimide from brain muscarinic receptors, that were also labeled with [ 3H]CD and considered to be of the M 2 subtype. Up to 70% of the [ 3H]CD-labeled cardiac receptors had high affinities for several organophosphate (OP) anticholinesterases. [ 3H]CD binding was inhibited by the nerve agents soman, VX, sarin, and tabun, with K 0.5 values of 0.8, 2, 20, and 50 n m, respectively. The apparent competitive nature of inhibition of [ 3H]CD binding by both sarin and paraoxon suggests that the OPs bind to the acetylcholine binding site of the muscarinic receptor. Other OP insecticides had lower potencies, inhibiting <50% of 5 n m [ 3H]CD binding by 1 μM of EPN, coumaphos, dioxathion, dichlorvos, or chlorpyriphos. There was poor correlation between the potencies of the OPs in reversibly inhibiting [ 3H]CD binding, and their anticholinesterase activities and toxicities. Acetylcholinesterases are the primary targets for these OP compounds because of the irreversible nature of their inhibition, which results in building of acetylcholine concentrations that activate muscarinic and nicotinic receptors and desensitize them, thereby inhibiting respiration. Nevertheless, the high affinities that cardiac muscarinic receptors have for these toxicants point to their extra vulnerability. It is suggested that the success of iv administration of the muscarinic receptor inhibitor atropine in initial therapy of poisoning by OP anticholinesterases may be related in part to the extra sensitivity of M 2 receptors to certain OPs.

Reoxygenation, But Neither Hypoxia nor Intermittent Ischemia, Increases [125I]Endothelin-1 Binding to Rat Cardiac Membranes

Standard binding techniques were used to establish whether either hypoxia, reoxygenation, perfusion under acidotic conditions, or "stunning" of the myocardium resembles ischemia and postischemic reperfusion in increasing cardiac membrane [125I]endothelin-1 (ET-1) binding site density (Bmax). Membranes from aerobically perfused rat hearts bound [125I]ET-1 to a single population of sites, with an affinity (KD) of 0.093 +/- 0.004 nM and a Bmax of 98.8 +/- 5.2 fmol/mg of protein. Bmax was increased (p less than 0.01) after 30 min of global ischemia, and further increased upon reperfusion, without changes in KD or selectivity. Neither three 10 min episodes of ischemia separated by 15 min of perfusion, nor perfusion at pH 6.8 instead of 7.4, nor 60 min of hypoxia altered Bmax, KD, or selectivity. Reoxygenation after 60 min of hypoxia increased Bmax (p less than 0.01) and KD (p less than 0.01) without changing selectivity. These results are interpreted to mean that the ischemia-induced increase in Bmax for [125I]ET-1 cannot be explained simply in terms of the ischemia-induced acidosis, or the accompanying reduction in tissue adenosine triphosphate and creatine phosphate.

Characterization and regional distribution of strychnine-insensitive [ 3H]glycine binding sites in rat brain by quantitative receptor autoradiography

Recent evidence suggests that a strychnine-insensitive glycine modulatory site is associated with the N-methyl- d-aspartate receptor-channel complex. A quantitative autoradiographic method was used to characterize the pharmacological specificity and anatomical distribution of strychnine-insensitive [ 3H]glycine binding sites in rat brain. [ 3H]Glycine binding was specific, saturable, reversible, pH and temperature-sensitive and of high affinity. [ 3H]Glycine interacted with a single population of sites having a K D of approximately 200 nM and a maximum density of 6.2 pmol/mg protein (stratum radiatum, CA1). Binding exhibited a pharmacological profile similar to the physiologically defined strychnine-insensitive glycine modulatory site. Binding was stereoselective; the rank order of potency of simple amino acids as displacers of binding was: glycine > d-serine > d-alanine > l-serine > l-alanine > l-valine > d-valine. Binding was not altered by the inhibitory glycine receptor ligand, strychnine, by the glutamate agonists, quisqualate and kainate, or by GABA receptor selective ligands. Most competitive agonists or antagonists of the N-methyl- d-aspartate recognition site were ineffective displacers of glycine binding. The exceptions were the aminophosphono series of antagonists, d-alpha-aminoadipate, gamma- d-glutamylglycine and beta- d-aspartylaminomethylphosphonic acid. However, the inhibition of [ 3H]glycine binding produced by the aminophosphono compounds could be accounted for by the level of glycine contamination present in these compounds. The non-competitive NMDA receptor-channel blockers, phencyclidine, its thienyl derivative, and MK-801 did not alter glycine binding. Kynurenate, glycine methylester, l-serine-O-sulfate , l-homocysteic acid, and several glycine-containing dipeptides were effective displacers of glycine binding. Structure-activity relations of agonists and antagonists of the strychnine-insensitive glycine binding site are discussed. The distribution of strychnine-insensitive [ 3H]glycine binding was heterogeneous with the following rank order of binding densities: hippocampus > cerebral cortex > caudate-putamen ⩾ thalamus > cerebellum > brain stem. This distribution of binding was correlated with N-methyl- d-aspartate-sensitive [ 3H]glutamate binding ( r 2 = 0.77; P< 0.001; Pearson product-moment) and [ 3H]thienylcyclohexylpiperidine binding ( r 2 = 0.72; P < 0.001 ). These observations are consistent with the hypothesis that the strychnine-insensitive glycine binding site is closely associated with the N-methyl- d-aspartate receptor-channel complex. These data also suggest that the stoichiometry between these binding sites is approximately 1:3:4 (thienylcyclohexylpiperidine: glycine:glutamate) in stratum radiatum of area CA1. However, in other brain regions, there were more glycine than glutamate binding sites suggesting that there may be heterogeneity in the N-methyl- d-aspartate receptor-channel complex.

Quantitative autoradiographic characterization of l-[3H] glutamate binding sites in rat vestibular nuclei

Quantitative autoradiography has been used to characterize L-[3H] glutamate binding sites and to describe their distribution in frozen sections of rat vestibular nuclei. Scatchard plots and Hill coefficients of glutamate binding suggest that glutamate interacts with a single population of sites having a KD of about 126 nM and a capacity of 2.5 pmol/mg of protein. Although the level of glutamate binding was not very high compared to the highest levels described for some other brain regions, it was nonetheless substantial. The sites were distributed unevenly in the four vestibular nuclei and their distribution correlated well with the projection areas of the vestibular nerve, which has been described as a glutamate-mediated pathway. The highest numbers of glutamate binding sites were observed in the medial vestibular nuclei. This technique provides a very sensitive assay for characterizing the pharmacological subtypes of glutamate binding in the vestibular nuclei and for analyzing changes in these sites during development or after deafferentation of the vestibular nuclei.

Properties of benzodiazepine binding sites in peripheral blood lymphocytes

Benzodiazepine (BDZ) binding sites were studied by using 3H-diazepam and 3H-Ro 5-4864 in intact lymphocytes from peripheral blood (PBL), in comparison to kidney and cerebellum. Experiments with 3H-diazepam performed at equilibrium and measuring kinetics revealed that BDZ binding sites are indeed present in rat PBL. The binding is saturable (Bmax 557 fmoles/10(6) cells), with high affinity (KD = 9.3 nM) and reversible. Specific binding sites are also observed by saturation experiments with 3H-Ro 5-4864 (Bmax 175 fmoles/10(6) cells, KD 2.2 nM). In addition, analysis of saturation isotherms obtained with 3H-diazepam indicates that BDZ binding sites are also present in human PBL. Scatchard plot of binding isotherms revealed an apparent single population of sites in all cases. The pharmacological characterization of BDZ binding sites in PBL, as compared with those of kidney and cerebellum, showed that these sites belong to the so-called "peripheral type."

Characterization of neurotensin binding sites in intact and solubilized bovine brain membranes.

Analysis of the equilibrium binding of [3H]-neurotensin(1-13) at 25 degrees C to its receptor sites in bovine cortex membranes indicated a single population of sites with an apparent equilibrium dissociation constant (KD) of 3.3 nM and a density (Bmax) of 350 fmol/mg protein (Hill coefficient nH = 0.97). Kinetic dissociation studies revealed the presence of a second class of sites comprising less than 10% of the total. KD values of 0.3 and 2.0 nM were obtained for the higher and lower affinity classes of sites, respectively, from association-dissociation kinetic studies. The binding of [3H]neurotensin was decreased by cations (monovalent and divalent) and by a nonhydrolysable guanine nucleotide analogue. Competition studies gave a potency ranking of [Gln4]neurotensin greater than neurotensin(8-13) greater than neurotensin(1-13). Smaller neurotensin analogues and neurotensin-like peptides were unable to compete with [3H]neurotensin. Stable binding activity for [3H]neurotensin in detergent solution (Kd = 5.5 nM, Bmax = 250 fmol/mg protein, nH = 1.0) was obtained in 2% digitonin/1 mM Mg2+ extracts of membranes which had been preincubated (25 degrees C, 1 h) with 1 mM Mg2+ prior to solubilization. Association-dissociation kinetic studies then revealed the presence of two classes of sites (KD1 = 0.5 nM, KD2 = 3.6 nM) in a similar proportion to that found in the membranes. The solubilized [3H]-neurotensin activity retained its sensitivity to cations and guanine nucleotide.

Binding of muscarine receptor antagonists to pig coronary smooth muscle.

In order to characterize the muscarinic binding site on coronary smooth muscle, we investigated the binding properties of (3H)quinuclidinyl benzilate (QNB) in membrane preparations of pig coronary arteries and atria. Scatchard analysis and Hill plot showed that (3H)QNB binds to a single population of sites in both tissues. The binding profiles of the muscarine receptor antagonists atropine, 11-[2-[dimethylamino)methyl)-1-piperidinyl)acetyl)-5,11-dihydro-6H-pyrid o(2,3- b) (1,4)benzodiazepine-6-one (AF-DX 116), pirenzepine, and 4-diphenylacetoxy-N-methylpiperidine methiobromide (4-DAMP) in both tissues were compared with binding data from other tissues, representative for different muscarinic binding site subtypes. It is concluded that the pig coronary smooth muscle muscarinic binding site is different from M1 and M2 binding sites investigated so far.

Autoradiographic distribution of the D 1 agonist [ 3H]SKF 38393, in the rat brain and spinal cord. Comparison with the distribution of D 2 dopamine receptors

The regional distribution of the specific D 1 agonist [ 3H]SKF 38393 (SKF 38393, 2,3,4,5-tetra-hydro-7, 8-dihydroxy-1-phenyl-1H-3-benzazepine) has been studied autoradiographically in the rat CNS. The binding of [ 3H]SKF 38393 to striatal sections was saturable, stereospecific, reversible, of high affinity (K d = 9.9 nM) and partly sodium sensitive; it occurred at a single population of sites and possessed the pharmacological characteristics of the dopamine D 1 receptor. The highest levels of [ 3H]SKF 38393 binding sites were found in the caudate-putamen, nucleus accumbens, olfactory tubercle and substantia nigra. Moderately high concentrations of the [ 3H]ligand were observed in the amygdala, endopyriform nucleus, nucleus olfactorius anterior, lateral septum, primary olfactory cortex, cerebellum (molecular layer) and spinal cord. An intermediate labelling was found in the thalamus, habenula, subthalamic nucleus, hypothalamus, ventral tegmental area, superior colliculus, hippocampus and cerebral cortex. Moderate levels of [ 3H]SKF 38393 binding were observed in the globus pallidus and arcuate nucleus. The autoradiographic distribution of [ 3H]SKF 38393 overlapped with that of [ 3H]N,n-propylnorapomorphine, a radioligand which labels the D 2 dopamine receptors, in a number of dopamine-rich brain areas but there were several areas which exhibited a high density of [ 3H]SKF 38393 binding sites but undetectable concentrations of [ 3H]N,n-propylnorapomorphine. Moreover, in the spinal cord, the subregional localization of these [ 3H]ligands clearly differed. Intrastriatal injection of ibotenic acid caused a large decrease in [ 3H]SKF 38393 and [ 3H]N,n-propylnorapomorphine binding in the striatum and provoked a reduction of [ 3H]SKF 38393 but not [ 3H]N,n-propylnorapomorphine binding in the substantia nigra confirming the view that nigral D 1 but not D 2 receptors are located on striatonigral fibres.