Labeling of A1 adenosine receptors in porcine atria with the antagonist radioligand 8-cyclopentyl-1,3-[3H]dipropylxanthine

We have used the antagonist radioligand 8-cyclopentyl-1,3-[3H]dipropylxanthine to label adenosine recognition sites in porcine atrial membranes. 8-Cyclopentyl-1,3-[3H]dipropylxanthine bound saturably, reversibly and with high affinity to an apparently homogeneous population of recognition sites with a Bmax of 32.0 +/- 0.9 fmol/mg protein and a KD of 0.394 +/- 0.049 nM. Prototypic adenosine receptor agonists inhibited the specific binding of 8-cyclopentyl-1,3-[3H]dipropylxanthine in a manner consistent with the labeling of an A1 adenosine receptor. 8-Cyclopentyl-1,3-[3H]dipropylxanthine appears to be a valuable antagonist radioligand for the characterization of cardiac adenosine receptors.     

A1 Adenosine receptors can occur manifesting two kinetic components of 8-cyclopentyl-1,3-[3H]dipropylxanthine ([3H]DPCPX) binding

The results described in this paper show, for the first time, that At adenosine receptors can have two kinetic components for the binding of the antagonist [³H]DPCPX. At low ionic strength ( 42mmo1/l), dissociation of [³H]DPCPX bound to A₁ receptors fitted better to a two kinetic components model than to a one kinetic component model. The kinetic constants were consistent with comparable Kd values for the two components of the antagonist binding, and therefore these two components cannot be distinguished by saturation isotherm analysis. Correspondence to: E.I. Canela at the above address     

Characterization of peripheral-type benzodiazepine binding sites in brain using [3H]Ro 5-4864

The binding of [3H]Ro 5-4864 to the peripheral-type benzodiazepine binding site in brain is characterized. The binding is saturable, high-affinity (KD = 1.6 nM), and reversible. The comparison of [3H]Ro 5-4864 and [3H]diazepam binding sites reveals major differences which include the following. There are about one-fourth as many peripheral-type binding sites than central sites in brain. Peripheral sites are present in many extranervous tissues and have a brain regional distribution distinct from that of the central-type receptor. The [3H]Ro 5-4864 binding site also is apparently highly localized in the nuclear membrane in contrast to the central-type receptor, which is synaptosomal. gamma-Aminobutyric acid has no effect on [3H]Ro 5-8464 binding, again in contrast to its marked effect on [3H]diazepam binding. Various putative benzodiazepine receptor ligands, such as purines, beta-carbolines, and kynurenamines, are also inactive as inhibitors of [3H]Ro 5-4864 binding. Blocking the benzodiazepine receptor by photoaffinity labeling decreases [3H]diazepam binding by more than 80% and has no effect on [3H]Ro 5-4864 binding. These results indicate that the peripheral-type benzodiazepine binding site in brain is a separate entity whose physiological function is probably distinct from that of the central-type benzodiazepine receptor.     

Characterization of binding sites for [3H]spiroperidol

Experiments were designed to investigate the biochemical properties of binding sites for [3H]spiroperidol ([3H]SPD) solubilized from canine caudate and to define the effect of detergent on the binding of the radioligand. Extraction of canine caudate with 0.75-1.0% digitonin was found to generate the maximum yield of binding sites for [3H]SPD while minimizing extraction of membrane proteins. Although binding sites were solubilized with 1.0% digitonin, a 10-fold reduction in detergent concentration was necessary to achieve maximal binding of [3H]SPD. The rank order of affinity for agonists and antagonists was consistent with the pharmacologic properties of the D2 subtype of the dopamine receptor. However, the binding of antagonists was found to be complex. Studies with some preparations of pooled canine caudate resulted in competition curves for the D2-selective antagonists domperidone and sulpiride that best fit a single-site model. Other preparations exhibited biphasic inhibition curves with these antagonists. The class of binding sites for [3H]SPD with low affinity for D2-selective antagonists constituted as much as 30-40% of the binding sites. Enrichment of solubilized binding sites for [3H]SPD was achieved by size exclusion HPLC followed by adsorption to DEAE-Sephadex and elution with buffer of increasing ionic strength. Enrichment of binding sites was accompanied by a decrease in the affinity of solubilized sites for [3H]SPD.     

Characterization of [3H]tryptamine binding sites in brain

[3H]Tryptamine binds with high affinity to sites on rat brain membranes. The sites have the characteristics of tryptamine receptor recognition sites. These sites are widely distributed among rat brain regions with the highest density occurring in the cerebral cortex, striatum and hippocampus. The site is also found in human cerebral cortex. The binding site is localized mainly to the synaptosomal fraction. Drug competition studies indicate that the [3H]tryptamine binding site is distinct from serotonin receptors. Drugs that are potent inhibitors of [3H]tryptamine binding include tetrahydro-beta-carboline, quipazine, phenylethylamine, amphetamine, p-chloroamphetamine and methamphetamine.     

Characterization of L-[3H]glutamate binding sites in bovine brain coated vesicles.

Clathrin-coated vesicles isolated from bovine brain exhibit an L-[3H]glutamate-specific binding. Coated vesicles were purified from bovine brain by differential centrifugation and gel filtration. High purity of coated vesicles was established previously by several enzyme markers and electron microscopy. The binding activity was performed in the absence of Na+, Ca2+, and Cl- ions to avoid binding and/or uptake to uptake sites. Coated vesicles were frozen, thawed, treated with 0.04% Triton X-100 and washed before incubation with L-[3H]glutamate. Saturation binding experiments revealed a single binding site with a Kd = 439 +/- 87 nM and a Bmax = 11.74 +/- 3.4 pmol/mg protein, consistent with kinetics characteristic for glutamate receptors. The glutamate-specific binding was stereospecific for glutamate and aspartate, showing higher affinity for L-forms than D-forms. Pharmacological characterization indicated that specific binding was sensitive to quisqualate and almost insensitive to kainate and N-methyl-D-aspartate. 200 microM guanosine triphosphate (GTP) produced a decrease of 50% in L-[3H]glutamate binding activity and competition experiments produced an affinity shift to the right of the glutamate dose-response curve. These results support the evidence that glutamate receptors are present in bovine brain coated vesicles and, at least in part, are associated to a G-protein.     

The binding of 1,3-[3H]-dipropyl-8-cyclopentylxanthine to adenosine A1 receptors in rat smooth muscle preparations.

1. The binding of 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), an antagonist radioligand selective for adenosine A1 receptors, was studied in rat duodenum, colon muscularis mucosae and longitudinal muscle, urinary bladder and vasa deferentia. 2. [3H]-DPCPX bound with high affinity to a single site in all membrane preparations studied with the exception of the rat urinary bladder in which no specific binding was detected. The affinity (Kd) of the binding site for [3H]-DPCPX was similar in all membrane preparations, the colon longitudinal muscle (1.18 +/- 0.47 nM), colon muscularis mucosae (0.84 +/- 0.15 nM), duodenum (1.59 +/- 0.18 nM) and vasa deferentia (0.93 +/- 0.17 nM). The density of [3H]-DPCPX binding sites was similar in the duodenum (38.8 +/- 4 fmol mg-1 protein), muscularis mucosae (43 +/- 3.5 fmol mg-1 protein) and vasa deferentia (43.3 +/- 12.2 fmol mg-1 protein), but in the longitudinal muscle 6-7 fold more binding sites (295 +/- 70 fmol mg-1 protein) were identified. 3. Inhibition studies using DPCPX (0.1-100 nM), N6-cyclopentyladenosine (CPA) (0.1-100 nM), 5'-N-ethylcarboxamidoadenosine (NECA) (2 nM-10 microM) and (R)-N6-phenylisopropyladenosine (R-PIA) (1 nM-1 microM) to displace the binding of [3H]-DPCPX at a concentration around the Kd value (1 nM), demonstrated an order of potency of displacement in all tissues of DPCPX > or = CPA > R-PIA > NECA. This potency order is characteristic of an A1 receptor, indicating that [3H]-DPCPX binds to adenosine A1 receptors in the rat duodenum, colon and vasa deferentia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of some organophosphorus compounds on the binding of a radioligand (8-cyclopentyl 1, 3-[3H]dipropylxanthine) to adenosine receptors in ovine cardiac membranes.

8-Cyclopentyl-1,3-[3H] dipropylxanthine [( 3H]CPX) is a potent radioligand that specifically binds to the A1 adenosine receptors. Its high specificity makes it a suitable ligand for the characterization of A1 adenosine receptors in tissues with low receptor densities. We have demonstrated that the organophosphorus compounds soman, tabun and sarin, at relatively high concentrations, all bind to the A1 adenosine receptors in ovine cardiac membranes with Ki values of 36.7, 328 and 175 microM, respectively. The binding of soman to the receptor site was found to be totally reversible. We suggested that these organophosphorus compounds affect the mechanical responses of the heart through interaction with a potassium channel that does not seem to be closely linked to the A1 adenosine receptors.     

Characterization of forskolin binding sites in rat brain membranes using [14,15-3H]14,15-dihydroforskolin as a ligand

Summary [14, 15-³H]14, 15-Dihydroforskolin ([³H]DHF) has been used as a radioactive ligand to identify forskolin binding sites in rat brain membranes. The binding was saturable and reversible. The binding sites showed positive cooperative properties as evident from an upward convex Scatchard plot and a Hill coefficient of 1.6. The equilibrium dissociation constants (K _D) were in the range between 10 M and 10 nM as estimated from the limiting slopes of the curved Scatchard plot. Half-maximal saturation of the binding sites was observed at a ligand concentration of 225 nM. The binding kinetics were very rapid: Binding equilibrium was reached in less than 2 min and a large excess of cold forskolin displaced 80% of the radioligand within 2 min. The dissociation reaction was not first order, characterized by a decreasing dissociation rate constant. Bound [³H]DHF could be displaced with forskolin (IC₅₀ 0.3 M), 14,15-dihydroforskolin (IC₅₀ 0.8 M) and 7-desacetylforskolin (IC₅₀ 3 M). However, nucleotides (ATP, GTP) and other receptor ligands (adenosine, isoproterenol) had no effect on the binding. Although the density of the forskolin binding sites (3.2 pmole/mg protein) is similar to those of other adenylate cyclase linked receptors, discrepancies between the K _D and the ED₅₀ obtained in adenylate cyclase studies and the finding that activation of the enzyme by forskolin is negative cooperative makes it difficult to clearly relate the binding sites to adenylate cyclase.     

(+)-[3H]SK&F 10,047 binding sites in rat liver

Stereospecific binding sites for (+)-[3H]SKF 10,047 have been demonstrated in rat liver. These binding sites were shown to be opioid receptors of the sigma type, and this was confirmed by their binding properties: reversibility, saturability, stereospecificity, effects of ions and various pharmacological drugs on (+)-[3H]SK sigma F 10,047 binding. The authors suggest that (+)-[3H]SKF 10,047 binding sites in liver and brain are a part of novel 'sigmergic' regulatory system.     

[3H]-5-carboxamidotryptamine labels 5-HT1D binding sites in bovine substantia nigra.

1. [3H]-5-hydroxytryptamine (5-HT) has been shown to radiolabel at least five types of 5-HT binding sites in mammalian brain tissue, 5-HT1A, 5-HT1C, 5-HT1D and 5-HT1D and 5-HT1E (Frazer et al., 1990). Selective masking of 5-HT1A and 5-HT1C receptors, has uncovered binding sites which display both high (5-HT1D) and low (5-HT1E) affinity for 5-carboxamidotryptamine (5-CT). By utilizing [3H]-5-CT we have eliminated a portion of the complex binding (5-HT1E) seen when [3H]-5-HT is used as a radioligand. 2. [3H]-5-CT binding to 5-HT1D sites in bovine substantia nigra was rapid, reversible and saturable, displaying high affinity (Kd = 0.38 +/- 0.04 nM) and low non-specific binding (> 90% specific binding). 3. In bovine substantia nigra, [3H]-5-CT labelled an equivalent number of binding sites to [3H]-5-CT (403 +/- 18 and 362 +/- 20 fmol mg-1 protein, respectively) and binding was sensitive to guanine nucleotides. 4. A linear correlation (r2 = 0.99) existed between the potency of compounds to displace [3H]-5-HT and [3H]-5-CT in bovine substantia nigra. 5. Therefore, [3H]-5-CT is a novel radioligand for the examination of 5-HT1-like binding sites, which under proper experimental conditions can be used to radiolabel selectively 5-HT-1D-like binding sites.     

A new class of adenosine receptors in brain. Characterization by 2-chloro[3H]adenosine binding

Micromolar concentrations of adenosine and its analogs have profound depressant effects on neuronal firing and synaptic transmission in many brain areas. Using the adenosine agonist 2-chloro[3H]adenosine (Cl[3H]Ado), we have identified a distinct class of micromolar-affinity adenosine binding sites in rat forebrain membranes. Specific Cl[3H]Ado binding was reversible and saturable with an apparent KD of 9.1 microM and a Bmax of 61 pmoles/mg protein. The present studies were conducted using washed brain membrane fractions not treated with adenosine deaminase. Specific Cl[3H]Ado binding under these conditions was insensitive to (-)-N6-(R-phenylisopropyl)adenosine ((-)PIA) and treatment with 3 mM N-ethylmaleimide, unlike high-affinity A1 adenosine receptor binding. Treatment of membranes with adenosine deaminase revealed an additional population of binding sites sensitive to (-)PIA. Inhibition of Cl[3H]Ado binding by adenosine analogs exhibited an order of potency ClAdo greater than 5'-N-ethylcarboxamide adenosine (NECA) greater than (-)PIA which differs from that of both A1 and A2 adenosine receptors. The potent A1 and A2 receptor antagonist 8-phenyltheophylline had no significant effect on binding up to 10 microM. Specific binding, however, was inhibited by the adenosine antagonists 8(p-sulfophenyl)theophylline, isobutylmethylxanthine, theophylline, and caffeine. Micromolar Cl[3H]Ado binding was highly selective for adenosine agonists and antagonists. These results suggest that the micromolar-affinity Cl[3H]Ado binding sites may represent a novel central purinergic receptor, distinct from the A1 and A2 adenosine receptors involved in the regulation of adenylate cyclase.     

Multiple [3H]-nemonapride binding sites in calf brain

[³H]-Nemonapride has been the ligand of choice to label D₄ dopamine receptors. Its specificity was questioned when it was discovered that sigma (σ) sites were also labeled by [³H]-nemonapride. To further characterize the binding of [³H]-nemonapride, three areas of calf brain (striatum, frontal cortex and cerebellum) were examined. In all three areas, [³H]-nemonapride labeled multiple sites. Dopaminergic and σ sites were the most prominent. The σ binding profile was σ-1 like with a K_i binding profile as follows (in order of decreasing potency): haloperidol, PPAP, pentazocine, DTG, U-50488, R(+)-3-PPP. Experiments using sulpiride and pentazocine to block striatal dopaminergic and σ sites, respectively, revealed additional, not previously characterized binding sites for [³H]-nemonapride. One component which was present in striatum but not in frontal cortex or cerebellum, had affinity for some neuroleptics and WB-4101, but not for typical serotonergic agents. Thus, [³H]-nemonapride has no selectivity for dopamine receptors unless stringent experimental conditions are met. Received: 19 September 1996 / Accepted: 12 March 1997     

Type-A cholecystokinin binding sites in cow brain: characterization using (-)-[3H]L364718 membrane binding assays

(-)-[3H]L364718 membrane binding assays were employed to localize and characterize cholecystokinin (CCK)-A binding sites in rat and cow brain. Specific binding was detected in all brain areas tested, but in all areas of rat brain and most areas of cow brain the level was too low to allow characterization of the ligand binding specificity of these sites. Membranes prepared from cow nucleus accumbens and striatum contained higher levels of (-)-[3H]L364718 specific binding which represented 55-70% of total binding. Characterization of the ligand binding properties of (-)-[3H]L364718 binding sites in cow nucleus accumbens revealed that these sites are similar to CCK-A sites found in pancreatic membranes. Binding of (-)-[3H]L364718 was saturable and had high affinity (Kd = 45 pm). Sites labeled by (-)-[3H]L364718 displayed stereospecificity for the stereoisomers of CR1409. The competition curve for CCK8 was shallow and was steepened and shifted to the right by the presence of the stable GTP analog guanosine 5'-(beta,delta-imido)triphosphate. The potency of CCK8, but not (-)-L36478, was also affected by the buffer in which the assay was conducted. Future use of (-)-[3H]L364718 membrane binding assays using cow nucleus accumbens and/or striatum will help explore the possibility of differences in ligand recognition among CCK-A sites found in brain and peripheral tissues.     

Neuronal location of N6-cyclohexyl[3H]adenosine binding sites in rat and guinea-pig brain

N6-cyclohexyl[3H]adenosine, ([3H]CHA), was used to label adenosine A1 receptors in crude synaptic membranes prepared from rat and guinea pig brain. The density of [3H]CHA binding sites was highest in the rat hippocampus and cerebellum and in the guinea-pig hippocampus. A micro-dissection of coronal sections of guinea-pig hippocampus revealed that the specific binding capacity for [3H]CHA in area CA-1 was 20-30% higher than in area CA-3, dentate gyrus and subiculum. Selective neuronal lesions of serotonergic, noradrenergic and cholinergic afferents to the hippocampus failed to alter [3H]CHA binding to hippocampal membranes. These results suggest that [3H]CHA binding sites are not associated with axons or terminals of these neurones in the hippocampus. Intrahippocampal injection of kainic acid reduced the number of [3H]CHA recognition sites by 30% with no alteration in the affinity of [3H]CHA for these receptors. Thus, a significant portion of A1 receptors may be associated with intrinsic neurones of the hippocampus which do not appear to be innervated by noradrenergic, cholinergic or serotonergic axons.     

Interactions of spironolactone with (+)-[3H]-isradipine and (-)-[3H]-desmethoxyverapamil binding sites in vascular smooth muscle.

Spironolactone partially inhibited the specific binding of (+)-[3H]-isradipine and (-)-[3H]-desmethoxyverapamil to vascular smooth muscle membranes. It is suggested that spironolactone interacts at a binding site of the calcium channel complex and allosterically modulates ligand binding at receptor sites in the channel.