Evidence that [3H]-alpha,beta-methylene ATP may label an endothelial-derived cell line 5'-nucleotidase with high affinity.

1. In membranes prepared from a permanent cell line of endothelial origin (WEC cells), [3H]-alpha, beta-methylene ATP ([3H]-alpha, beta-meATP) labelled high (pKd = 9.5; Bmax = 3.75 pmol mg-1 protein) and low (pKd = 7.2; Bmax = 23.3 pmol mg-1 protein) affinity binding sites. The high affinity [3H]-alpha, beta-meATP binding sites in the WEC cell membranes could be selectively labelled with a low concentration of the radioligand (1 nM). In competition studies performed at a radioligand concentration of 1 nM, 88.6% of the sites possessed high affinity (pIC50 = 8.26) for alpha, beta-meATP. 2. The high affinity [3H]-alpha, beta-meATP binding sites appeared heterogeneous since in competition studies a number of nucleotide analogues (alpha, beta-meADP, ATP, ADP, AMP, GTP, GppNHp, GMP) and adenosine identified two populations of the sites labelled by 1 nM [3H]-alpha, beta-meATP. The proportion of sites with high affinity for these compounds was found to vary between 42 and 69%. 3. Approximately 60-69% of the binding sites labelled with 1 nM [3H]-alpha, beta-meATP possessed high affinity for alpha, beta-meADP (pIC50 = 8.87), AMP (pIC50 = 7.12), GMP (pIC50 = 7.34), UTP (pIC50 = 6.12), GTP (pIC50 = 7.59), GppNHp (pIC50 = 7.35) and adenosine (pIC50 = 5.45).(ABSTRACT TRUNCATED AT 250 WORDS)     

[3H] sertraline binding to rat brain membranes

Tritiated sertraline, a radiolabeled form of a potent and selective inhibitor of serotonin uptake, was found to bind with high affinity to rat whole brain membranes. Characterization studies showed that [³H] sertraline binding occurred at a single site with the following parameters:K _d 0.57 nM,B _max 821 fmol/mg protein,n _h 1.06. This binding was reversible; the dissociation constant calculated from kinetic measurements (K _d 0.81 nM) agreed with that determined by saturation binding experiments. [³H] Sertraline binding in the presence of serotonin, paroxetine, fluoxetine or imipramine suggested competitive inhibition of binding (large increase inK _d with little change inB _max). The rank order of potency of inhibition of [³H] sertraline binding was similar to that of inhibition of serotonin uptake for known uptake inhibitors and the 1-amino-4-phenyltetralin uptake blockers. A marked decrease in ex vivo [³H] sertraline binding in the brain of rats 7 days after treatment withp-chloroamphetamine was consistent with the loss of serotonin uptake sites induced by this agent. The results of our study indicated that [³H] sertraline labels serotonin uptake sites in rat brain.     

Estradiol-induced increase of specific [3H]ketanserin binding sites on rat uterine membranes

[3H]Ketanserin, a specific serotonin (5-HT) antagonist, was used to investigate whether 5-HT receptors increased in the uterine membranes of ovariectomized rats on administration of 17 beta-estradiol-3-benzoate (estradiol) and also to investigate the characteristics of specific [3H]ketanserin binding to the uterine membranes from estradiol-treated ovariectomized rat. Administration of estradiol significantly increased the amount of [3H]ketanserin specifically bound at equilibrium but did not change the apparent affinity of specific [3H]ketanserin binding. The specific [3H]ketanserin binding to estradiol-treated ovariectomized preparations was rapid and reversible. The Scatchard plots of the saturation curves of specific [3H]ketanserin binding to untreated and estradiol-treated ovariectomized preparations were convex. The apparent Ki values of various serotonergic agents deduced from displacements by these compounds of specific [3H]ketanserin binding to estradiol-treated ovariectomized preparations were two to four orders of magnitude smaller than those of adrenergic, dopaminergic and histaminergic agents. These results suggest that [3H]ketanserin binds mainly to 5-HT receptors in the uterine membranes of estradiol-treated ovariectomized rats.     

Alpha 2-adrenoceptor and catecholamine-insensitive binding sites for [3H]rilmenidine in membranes from rat cerebral cortex.

The kinetic and pharmacological characteristics of the binding of the oxazoline antihypertensive drug, [3H]rilmenidine, to membranes of rat cerebral cortex have been determined. Computerised resolution of curvi-linear, equilibrium binding isotherms was consistent with the existence of two distinct binding sites for [3H]rilmenidine: Kd 17.3 +/- 7.41 nM, Bmax 0.197 +/- 0.06 pmol/mg protein and Kd 254 +/- 48 nM, Bmax 1.59 +/- 0.08 pmol/mg protein. Moreover, the resolution of two association and dissociation rates also suggested the existence of two binding site populations. Drug inhibition studies revealed that specific binding of [3H]rilmenidine (2 nM) was only inhibited by a maximum of 50% by the catecholamines, adrenaline and noradrenaline, but was completely inhibited by some oxazolines, by guanabenz (a guanidino drug) and by several imidazoline compounds including naphazoline, oxymetazoline and clonidine. Binding isotherms for these drugs were also best fit by a two-site model. The relative Ki values at the high affinity site for [3H]rilmenidine and the number of these high affinity sites are consistent with this site being an alpha 2-adrenoceptor. The high affinity of oxymetazoline and low affinity of prazosin for high affinity [3H]rilmenidine binding sites together with the rank order of potency of oxymetazoline greater than phentolamine greater than SKF 104078 greater than ARC-239 greater than prazosin suggest that [3H]rilmenidine binds to the alpha 2A sub-type of adrenoceptor. Computer-resolved Ki values for drugs at the larger number of lower affinity binding sites were very similar to Ki values determined in the presence of 10 microM adrenaline (used to block alpha 2-adrenoceptor binding).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of avermectins with [3H]beta-carboline-3-carboxylate ethyl ester and [3H]diazepam binding sites in rat brain cortical membranes

The binding of [3H] beta-carboline-3-carboxylate ethyl ester ([3H] beta-CCE), a ligand for the benzodiazepine receptor in the mammalian CNS, to rat cortical membranes, is enhanced by avermectin B1a and its therapeutic formulation, Ivermectin. In contrast to the effects of the avermectins on [3H]diazepam binding, which involve changes in both receptor affinity and number, increases in beta-CCE binding, which are much less than those observed for the benzodiazepine ligand, involve only alterations in receptor number. This Bmax increase is bicuculline insensitive whereas Ivermectin effects on benzodiazepine binding are partially antagonized by GABA antagonist. The data suggest a differential interaction by the avermectins on benzodiazepine and beta-CCE binding sites in rat cortical membranes and indicate that these macrolide anthelmintics may be a useful tool for characterizing benzodiazepine/anxiolytic receptor subtypes.     

Acetylcholinesterase potentiates [3H]fluorowillardiine and [3H]AMPA binding to rat cortical membranes

In addition to its action at cholinergic synapses acetylcholinesterase (AChE) has been proposed to modulate neuronal activity by mechanisms unrelated to the hydrolysis of acetylcholine. We have investigated the effects of AChE on the binding of the specific AMPA receptor agonists (S)-[3H]5-fluorowillardiine ([3H]FW) and [3H]AMPA to rat cortical membranes. Pretreatment of membranes with AChE causes a dose-dependent increase in the binding of both radiolabelled agonists with a maximal increase to approximately 60% above control. This increase is completely blocked by the specific AChE inhibitors propidium, physostigmine, DFP and BW 284C51. AChE pretreatment had no effect on [3H]kainate binding. [3H]FW binding to membranes from young (15-day-old) rats is four orders of magnitude more sensitive to AChE modulation than membranes from adult rats (EC50 values of 4x10(-5) and 0.1 unit/ml, respectively) although the total percentage increase in binding is similar. Furthermore, the AChE-induced potentiation of [3H]FW binding is Ca2+ - and temperature-dependent suggesting an enzymatic action for AChE in this system. Saturation binding experiments with [3H]FW to adult membranes reveal high and low affinity binding sites and demonstrate that the main action of AChE is to increase the Bmax of both sites. These findings suggest that modulation of AMPA receptors could provide a molecular mechanism of action for the previously reported effects of AChE in synapse formation, synaptic plasticity and neurodegeneration.     

Modulation by nucleotides of binding sites for [3H]glibenclamide in rat aorta and cardiac ventricular membranes

Radioligand binding techniques were employed to determine the modulation by nucleotides of the specific [3H]glibenclamide (Gli) binding to rat aortic and cardiac ventricular preparations. Saturation analysis revealed a single binding site with K(D) value of 31.3 nM and Bmax of 180 fmol/mg wet weight in aortic preparations. We also observed that [3H]Gli bound reversibly and specifically to cardiac membranes. Unlabeled glibenclamide displaced [3H]Gli-specific binding of cardiac membranes completely with K(I) of 54.4 nM. In cardiac membranes, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and uridine diphosphate (UDP) (from 0.01-5 mM) concentration dependently inhibited [3H]Gli binding independent of Mg2+. The values of K(I) were 0.47, 0.22, and 0.58 mM, respectively. However, in aortic preparations, [3H]Gli-specific binding was increased by ATP of 5 and 10 mM and showed a biphasic response to ADP. At concentrations to 1 mM, ADP inhibited binding; above 5 mM, the specific [3H]Gli binding was increased. UDP did not alter the binding up to 5 mM. In the presence of Mg2+ (20 mM), the inhibitory effects of ATP (0.01-1 mM) or ADP (0.01-5 mM) on the binding in cardiac membranes were abolished, whereas the facilitatory effects of ATP or ADP in aortic preparations were strengthened. Analysis of kinetics showed that the time of [3H]Gli association and dissociation in cardiac and aortic preparations was monophasic. The association was delayed with dissociation unchanged by ATP, ADP, and UDP of 1 mM, respectively, in cardiac membranes. In aorta, however, at the same concentration ATP accelerated association and retarded dissociation and vice versa for ADP. Association and dissociation were not changed by UDP of 5 mM. We conclude that ATP, ADP, and UDP are all major allosteric modulators of K(ATP) channels and they affect the antagonist binding to heart (sulfonylurea receptor 2A) and aorta (sulfonylurea receptor 2B) differently.     

Characterization of [3H]-imidazenil binding to rat brain membranes.

1. The binding of [3H]-imidazenil, an imidazobenzodiazepine carboxamide, to rat cerebellar membranes was characterized at different temperatures. 2. Specific binding was linear with tissue concentrations and reached maximum after 90, 30 and 5 min incubation at 0, 21 and 37 degrees C, respectively. The binding was of high affinity, specific and saturable; non linear regression and Scatchard analysis of the data was compatible with the presence of a single population of receptor sites with Bmax of 0.74 +/- 0.020, 0.90 +/- 0.011 and 1.0 +/- 0.036 pmol mg-1 protein at 0, 21 and 27 degrees C, respectively. Binding affinity decreased with increasing temperature: Kd were 0.29 +/- 0.051 nM (0 degrees C), 1.0 +/- 0.080 nM (21 degrees C) and 2.4 +/- 0.38 nM (37 degrees C). 3. At all tested temperatures, [3H]-imidazenil binding was reversible and the Kd calculated from the dissociation and association rate constants approximated the equilibrium Kd. 4. In the presence of gamma-aminobutyric acid (GABA), Kd increased 4 fold at 0 degrees C, whereas Bmax increased, albeit slightly, at all temperatures. 5. Benzodiazepines (BZDs), imidazopyridines and methyl-beta-carboline-3-carboxylate (beta CCM) were effective inhibitors of [3H]-imidazenil binding. Conversely, GABAA antagonists, barbiturates, picrotoxin and peripheral BZD receptor ligands were devoid of any activity. 6. Comparing [3H]-imidazenil to [3H]-flumazenil binding in various brain areas, similar densities of recognition sites as well as like regional differences in the distribution of binding sites for both radioligands were observed (cortex = striatum > cerebellum > spinal cord).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the slow calcium channel binding sites for [3H]SR 33557 in rat heart sarcolemmal membranes.

SR 33557 represents a new class of compounds (indolizine sulfone) that inhibit L-type Ca2+ channels. [3H]SR 33557 has been shown to bind with high affinity (Kd congruent to 0.36 nM, calculated from saturation isotherms and association/dissociation kinetics) to a single class of sites in a purified preparation of rat cardiac sarcolemmal membranes. The binding was found to be saturable and reversible. The maximal binding capacity was in approximately 1:1 stoichiometry with that of other Ca2+ channel antagonists. Various divalent cations (Mg2+, Mn2+, Ca2+, Ba2+, and Cd2+) were shown to inhibit specific [3H]SR 33557 binding, with Cd2+ being the most potent. Among several receptor or channel ligands (including omega-conotoxin and Na+ and K+ channel modulators), only the L-type Ca2+ channel antagonists were found to displace [3H]SR 33557. However, dihydropyridines, phenylalkylamines, benzothiazepines, and diphenylbutylpiperidines were found to inhibit [3H]SR 33557 in a noncompetitive manner as demonstrated by displacement and saturation experiments in addition to dissociation kinetics. From these results, we suggest that SR 33557 binds with high affinity to a unique site on the L-type Ca2+ channel found in rat cardiac sarcolemmal membranes.     

Specific [(3)H]-guanosine binding sites in rat brain membranes

1. Extracellular guanosine has diverse effects on many cellular components of the central nervous system, some of which may be related to its uptake into cells and others to its ability to release adenine-based purines from cells. Yet other effects of extracellular guanosine are compatible with an action on G-protein linked cell membrane receptors. 2. Specific binding sites for [(3)H]-guanosine were detected on membrane preparations from rat brain. The kinetics of [(3)H]-guanosine binding to membranes was described by rate constants of association and dissociation of 2.6122 x 10(7) M(-1) min(-1) and 1.69 min(-1), respectively. A single high affinity binding site for [(3)H]-guanosine with a K(D) of 95.4 +/- 11.9 nM and B(max) of 0.57 +/- 0.03 pmol mg(-1) protein was shown. This site was specific for guanosine, and the order of potency in displacing 50 nM [(3)H]-guanosine was: guanosine=6-thio-guanosine > inosine > 6-thio-guanine > guanine. Other naturally occurring purines, such as adenosine, hypoxanthine, xanthine caffeine, theophylline, GDP, GMP and ATP were unable to significantly displace the radiolabelled guanosine. Thus, this binding site is distinct from the well-characterized receptors for adenosine and purines. 5. The addition of GTP produced a small concentration-dependent decrease in guanosine binding, suggesting this guanosine binding site was linked to a G-protein. 6. Our results therefore are consistent with the existence of a novel cell membrane receptor site, specific for guanosine.     

Alteration of binding sites for [3H]P1075 and [3H]glibenclamide in renovascular hypertensive rat aorta

Aim: The alterations of the binding sites for ATP-sensitive K channel (KATP) openers and blockers in aortic strips were investigated in hypertensive rats. Methods: Radioligand binding techniques were used to compare the specific binding properties of [3H]P1075 and [3H]glibenclamide (Gli) in normotensive (NWR) and reno-vascular hypertensive rat (RVHR) aortic strips. Results: The KD values of [^3H]P1075 binding were increased by 1.5-fold, while the Bmax values were unchanged in RVHR. The IC50 values of P1075 and pinacidil (Pin) for displacing the [^3H]P1075 binding in RVHR were increased by 1.8-and 1.7-fold, respectively. The kinetic processes of association and dissociation of [^3H]P1075 binding were slower in RVHR. Glibenclamide pretreatment slowed down the kinetic processes of the association and dissociation of [3H]P1075 binding in NWR, but failed to alter the kinetic processes of [^3H]P1075 binding in RVHR. The IC50 values of Gli for displacing the [^3H]Gli binding at high-affinity sites were increased by 3-fold, while those at low-affinity sites remained to be unchanged in RVHR. The kinetic processes of association of [^3H]Gli binding were decreased and those of the dissociation were accelerated in RVHR. The treatment with Pin slowed down the association kinetic processes but accelerated the process of the dissociation of [^3H]Gli binding in NWR, but did not alter the kinetics of [3H]Gli binding in RVHR. Conclusion: The affinity of binding sites for [3H]p 1075 and of high-affinity binding sites for [^3H]Gli are decreased, and the negative allosteric interactions between the two binding sites are impaired in RVHR aorta.     

Multiple binding sites of [3H]clonidine on hepatic plasma membranes

The binding of [3H]clonidine on mouse liver plasma membrane was a rapid, saturable and reversible process. It was characterized by two types of population: high affinity receptors with KD of 6.76 +/- 1.02 nM and Bmax of 106.15 +/- 24.05 fmol/mg protein, and low affinity receptors with KD of 63.66 +/- 12.85 nM and Bmax of 818.06 +/- 128.49 fmol/mg protein. Displacement of [3H]clonidine from its binding sites by various ligands indicated that alpha 1--as well as alpha 2--adrenoceptors were involved in the high affinity system. The respective participation of these two types of receptors was discussed.     

Phenylethanolaminotetralines compete with [3H]dihydroalprenolol binding to rat colon membranes without evidencing atypical beta-adrenergic sites.

[3H]Dihydroalprenolol ([3H]DHA) specific binding (determined by the difference in the presence and absence of 20 microM (-)isoprenaline) to rat colon membranes was saturable (Bmax = 39.6 fmol/mg protein), of high affinity (Kd = 0.87 nM) and stereospecific (IC50 330 and 3510 nM for (-)- and (+)isoprenaline, respectively); the Hill coefficient was close to one, indicating binding homogeneity. [3H]DHA (0.6 nM) specific binding was potently inhibited (Ki range 1.9-3.3 nM) by the non-selective beta-adrenoceptor antagonists pindolol, alprenolol, but not by the non-adrenergic compounds 5-hydroxytryptamine, 8-hydroxydipropylaminotetraline, methysergide, dopamine and verapamil (Ki greater than 10,000 nM). The selective beta 1- and beta 2-adrenoceptor antagonists CGP 20,712A and ICI 118,551 resulted in biphasic competition binding curves, whose low and high affinity components were compatible with two populations of binding sites accounting for about 75 (beta 2) and 25% (beta 1) of total sites. The relative competing potencies of reference adrenergic agonists also suggested a prevalence of beta 2-adrenergic sites. The new agonists phenylethanolaminotetralines (PEATs), highly selective for the atypical beta-adrenoceptors whose abundance in rat colon has been confirmed by comprehensive functional studies, had variable affinity for the [3H]DHA-labelled sites depending on chirality, but with no substantial correlation with their pharmacological potency. Only 40% of [3H]DHA binding, at a concentration about 10 times its Kd for high affinity sites (beta 1 and beta 2), was prevented by saturating concentrations of isoprenaline. Under this condition, the representative PEAT, SR 58611A, highly potent and selective for atypical beta-adrenoceptors in functional tests, and its pharmacologically inactive enantiomer, both inhibited the residual binding equipotently. In conclusion, [3H]DHA binding did not detect atypical beta-adrenoceptor sites in rat colon membranes, most probably because of its weaker affinity for them than for the coexisting beta 1 and beta 2 sites. PEAT stereoisomers proved essential for assessing both the stereospecificity and the functional significance of this atypical binding and to compare their affinity for [3H]DHA-labelled sites and pharmacological potency.     

Three distinct binding sites for [3H]-prazosin in the rat cerebral cortex.

1. The putative alpha 1-adrenoceptor subtypes of rat cerebral cortex membranes were characterized in binding. 2. Specific binding of [3H]-prazosin was saturable between 20-5000 pm. Scatchard plots of the binding data were non-linear, indicating the presence of two distinct affinity sites for prazosin (pKD, high = 10.18, Rhigh = 308 fmol mg-1 protein; pKD, low = 8.96, Rlow = 221 fmol mg-1 protein). 3. In the membranes pretreated with chlorethylclonidine (CEC) two affinity sites for prazosin were also observed: the affinities were similar to those without CEC pretreatment, but the maximum numbers of binding sites were reduced by CEC pretreatment to 23 and 62% for prazosin-high (Rhigh) and low affinity sites (Rlow), respectively. 4. The prazosin-high affinity sites were further subdivided into two subclasses by WB4101(2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane) and phentolamine; the low affinity sites for WB4101 and phentolamine were more potently inactivated by CEC as compared with the high affinity sites. On the other hand, prazosin, HV723 (alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)- amino )-propyl)benzeneacetonitrile fumarate) and yohimbine inhibited [3H]-prazosin binding to prazosin-high affinity sites monophasically. 5. In addition to the high affinity sites, the prazosin-low affinity sites were labelled at high concentrations of [3H]-prazosin. Thus, prazosin and WB4101 showed shallow displacement curves. On the other hand, HV723 and yohimbine did not discriminate between prazosin-high and low affinity sites. 6. Two distinct alpha 1-adrenoceptor subclassifications have been recently proposed (alpha 1A, alpha 1B subtypes and alpha 1H, alpha 1L, alpha 1N subtypes).(ABSTRACT TRUNCATED AT 250 WORDS)     

The characterization of [3H]sulpiride binding sites in pig striatal membranes

Pig striatal membranes have [3H]sulpiride-binding sites similar to those identified in rat striatal membranes. The pharmacological profile indicates that this binding is to dopamine receptors. Agonist displacement of [3H]sulpiride binding in pig striatal membranes is subject to guanine nucleotide regulation. This effect is mimicked by heat treatment. N-ethyl maleamide (20 microM) and dithioerythritol (3 mM) decrease agonist affinity for the [3H]sulpiride-binding site in pig striatal membranes without significantly affecting maximal displacement.     

Desipramine and the phencyclidine derivative BTCP differently inhibit [3H]TCP binding to high- and low-affinity sites.

The effects of N-[1-(2-benzo(b)thiophenyl)cyclohexyl]piperidine (BTCP) and desipramine on [3H]N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding were investigated in vivo and in vitro. In the cerebrum, both drugs were competitive inhibitors of high-affinity [3H]TCP binding. Conversely, in the cerebellum, they were non-competitive inhibitors of low-affinity [3H]TCP binding. These results imply that the different [3H]TCP binding sites have distinct pharmacological properties, and show that, although chemically related to TCP, BTCP has an effect similar to that of desipramine.     

Identification and characterisation of L-[3H]aspartate binding sites on rat spinal cord synaptic membranes

The binding of L-[3H] aspartate to extensively-washed rat spinal cord synaptic membranes was investigated. Specific binding was enriched in synaptic membranes and was optimal under physiological conditions of temperature and pH. Equilibrium binding was established relatively slowly over a period of 30 min, and was totally reversible within 40 min. Saturation analysis revealed complex binding patterns. Two sites were clearly demonstrable, only one of which was shown to be saturable over the ligand concentration range employed in the study (0.1-10 microM). There was also some indication of the presence of a higher affinity site, although this was not investigated in any detail. Saturable binding demonstrated a KD = 1.4 microM and Bmax = 105 pmole/mg protein. Structure-activity studies with a range of amino acid analogues indicated that binding was stereospecific and was inhibited by a very restricted range of compounds. The most potent inhibitors of binding were L-glutamate and L-aspartate. There was no evidence for the involvement of NMDA receptors. Effects of possible endogenous modulators, including ions and guanosine nucleotides were investigated, and the chemical nature of the binding site probed with a number of protein-modifying agents.     

Specific [3H]neurotensin binding to rat spinal cord membranes

The binding of [3H]neurotensin to membranes prepared from rat spinal cord has been studied in vitro. Scatchard analysis of saturation binding data indicated that [3H]neurotensin binds with high affinity (Kd = 6.3 nM) to a single, saturable population of binding sites (Bmax = 12.4 pmol/g tissue). Neurotensin1-13 (IC50 = 5.9 nM) and neurotensin8-13 (IC50 = 3.7 nM) were potent inhibitors of [3H]neurotensin binding whereas neurotensin1-8 was virtually inactive at concentrations up to 10(-5) M. Sodium chloride (150 mM) significantly inhibited binding, while potassium chloride (5 mM), magnesium chloride (10 mM), manganese chloride (1 mM) and GMP-PNP (0.1 mM) were without effect. The characteristics of the binding of [3H]neurotensin obtained in this study are consistent with this ligand binding to a physiologic neurotensin receptor in rat spinal cord membranes.