Comparison of the filtration and centrifugation methods for assaying [3H](d-ala2,d-leu5)enkephalin binding to mouse brain membranes

The binding of [³H](d-ala²,d-leu⁵)enkephalin (DADLE) to mouse brain membranes was studied via rapid filtration with GF/C filters and via the centrifugation method. The amount of specific binding determined by filtration was found to be dependent on the length of time between the initiation of vacuum and the actual time of filtration. Vacuum strength alone also significantly affected the amount of specifically bound [³H]DADLE measured by filtration. The centrifugation method increased the number of experimentally determined binding sites when compared to the filtration method. The difference in the number of binding sites was only partially attributable to passage of binding material through the GF/C filter. The ability of morphine to displace [³H]DADLE was also found to differ slightly between the two methods. These data indicate that centrifugation is a better method of determining the binding parameters of [³H]DADLE in mouse brain membrane preparations.     

Consequences of 125I-labeled insulin degradation by hepatocytes on the interpretation of receptor binding studies

The association of ¹²⁵I-labeled insulin with hepatocytes was assayed by filtration or microcentrifugation. Assay by centrifugation resulted in a greater amount of retained radioactive label throughout the course of association of ¹²⁵I-labeled insulin with hepatocytes. Similarly, saturation experiments assayed by microcentrifugation suggested greater binding than filtration. During dissociation, cells isolated by centrifugation released a greater amount of rapid-dissociating radioactive label. Control experiments of [³H]-inulin exclusion with cell pellets, which were isolated during microcentrifugation, demonstrated that the difference between the methods was not due to extracellular trapping of radioactivity. Therefore, the data suggested that there was more low-affinity retention when binding was assayed by centrifugation than filtration. The integrity of the ¹²⁵I-labeled insulin extracted from hepatocytes was determined by column chromatography. A substantially greater proportion of the extracted radioactivity was fragments of ¹²⁵I-labeled insulin in cells isolated by centrifugation. It is suggested that the extensive washing of the cells during filtration removes more fragments than does centrifugation. During dissociation, the low-affinity component of radioactivity, which was observed in the centrifugal assay, resulted from the transient retention of insulin fragments. The extensive degradation of insulin, which was assayed by either method, and the differences observed between these methods, should be considered in the interpretation of binding experiments with cells.     

[3H]cocaine labels a binding site associated with the serotonin transporter in guinea pig brain: Allosteric modulation by paroxetine

We studied the characteristics of [³H]cocaine binding to membranes prepared from whole guinea pig brain. Cocaine binding was specific and saturable. A one-site binding model fit the data adequately: the Kd value of [³H]cocaine was 44 nM with a Bmax value of 280 fmol/mg protein. The rank order of potency for the [³H]cocaine binding site was paroxetine > clomipramine > (–)-cocaine > fluoxetine > mazindol > desipramine > GBR12909 > phencyclidine > benztropine > GBR12935 > (+)-cocaine. The IC50 values of these drugs for inhibition of [³H]cocaine binding were highly correlated with their IC50 values for inhibition of [³H]5-HT uptake into synaptosomes prepared from whole guinea pig brain. High affinity 5-HT uptake inhibitors produced dose-dependent wash-resistant (pseudoirreversible) inhibition of [³H]cocaine binding. The wash-resistant inhibition produced by paroxetine was due to an increase in the Kd of [³H]cocaine binding sites, and was accompanied by an increase in the dissociation rate, consistent with an allosteric mechanism. These studies suggest that, using membranes prepared from whole guinea pig brain, [³H]cocaine labels a binding site associated with serotonin transporter and that paroxetine and cocaine bind to different sites on the serotonin transporter.Abbreviations GBR12909 1-(2-{bis(4-fluorophenyl)methoxy}ethyl)-4-{3-phenylpropyl}piperazine - TCP 1-{1-(2-thienyl)cyclohexyl}piperidine - BTCP N-{1-(2-benzo(b)thiophenyl)cyclohexyl}piperidine - PCP 1-(1-phenylcyclohexyl)piperidine - GBR12935 (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine) - CMI clomipramine     

Binding of imipramine and cocaine to a model lipid membrane

[³H]Imipramine and [³H]cocaine were concentrated at membranes of liposomes prepared from phosphatidylcholine, cholesterol, and dicetylphosphate. This binding has an apparent dissociation constant in the micromolar range and a density close to 2 pmol/g of phosphatidylcholine. The potencies of various drugs in inhibiting the binding ot liposomes correlated only weakly with those in inhibiting the high-affinity binding of [³H]imipramine and [³H]cocaine to brain membranes. However, there was a highly significant correlation between the potencies of drugs in inhibiting binding to liposomes and their lipophilic character, indicating the involvement of hydrophobic bonding. Although the amounts of phosphatidylcholine and cholesterol in brain preparations in assays for high-affinity binding to brain membranes were in the same range as those used in our assays with liposomes, the inhibition of the high-affinity binding to brain membranes was only weakly dependent upon the lipophilicity of the inhibiting drug. These results indicate that lipophilicity is but one of the factors in the complex binding interactions between lipophilic substances and integral brain membranes. In addition, the results are in agreement with the suggestion that phosphatidylcholine and cholesterol are not the primary sites of high-affinity binding [³H]imipramine and [³H]cocaine to brain membranes, although it cannot be ruled out that these lipids have different properties in natural biological membranes and in artificial liposome membranes.     

Rat mesencephalic neuronal cells cultured for different periods as a model of dopamine transporter ontogenesis

Ventral mesencephalic neurons contained only low-affinity and sodium-independent binding sites of [³H]WIN 35,428 (marker of dopamine transporter) during the first 10d in primary cultures. These sites were present in cytosol, and they are not very probably related to dopamine transporter. After 12 d in culture, membrane-bound, high-affinity, and sodium-dependent [³H]WIN 35,428 binding sites were detected. In membranes prepared from cells 14 d in culture, cocaine displaced [³H]WIN 35,428 binding with similar potency to that in striatal membranes of adult rat brain. The high-affinity [³H]WIN 35,428 binding sites in mesencephalic neuronal cell cultures are very probably related to dopamine transporter. The development of high-affinity [³H]WIN 35,428 binding sites in neurons cultured for different time periods could be a useful model of dopamine transporter ontogenesis.     

Binding Sites for [3H]Cocaine in Mouse Striatum and Cerebral Cortex Have Different Dissociation Kinetics

[3H]Cocaine dissociates from its binding sites in the mouse cerebral cortex with a half-time of 25 s. The dissociation kinetics in the striatum is consonant with the presence of two populations of sites with dissociation half times of 2 s and 27 s, comprising 88% and 12%, respectively, of the total binding sites. On the basis of previous pharmacological characterization of [3H]cocaine binding, we propose that the slowly dissociating component represents the sites associated with 5-hydroxytryptamine (serotonin) uptake, and the rapidly dissociating component the 3,4-dihydroxyphenylethylamine (dopamine)-related sites. Evidence is presented that the extremely high dissociation rates do not preclude the measurement of [3H]cocaine binding by rapid filtration. The dissociation of [3H]cocaine from cerebrocortical membranes is slowed to a small but statistically significant extent by serotonin.     

Demonstration and Characterization of two Classes of Cardiac Glycoside Binding Sites to Rat Heart Membrane Preparations Using Quantitative Computer Modeling

Abstract

Cardiac glycoside binding to rat heart membrane preparations was measured by rapid filtration technique. The binding data were analyzed using quantitative computer analysis. The experimental results using [³H]-ouabain as the labeled ligand were consistent with a model in which cardiac glycoside specific binding occurs at two independent classes of sites. The high affinity sites were characterized by a dissociation constants of 40 nM, 50 nM, and 61 nM for ouabain, digoxin and digitoxin, respectively, with a binding capacity of 1.3 pmoles/mg protein. The lower affinity sites for ouabain were characterized by dissociation constants of 2.3 µM, 67 nM and 71 nM for ouabain, digoxin and digitoxin, respectively, with a binding capacity of 3 pmoles/mg protein. Potassium ions inhibit [³H]-ouabain binding in a dose dependent manner with an IC₅₀ of 500 µM. Quantitative computer modelling indicated that potassium inhibits ouabain binding at both binding sites.     

[3H]-BIBP3226 and [3H]-NPY Binding to Intact SK-N-MC Cells and CHO Cells Expressing the Human Y1 Receptor

Abstract

We have studied the binding of [³H]-NPY and the newly developed non-peptide Y₁ receptor antagonist [³H]-BIBP3226 to intact SK-N-MC cells and CHO-K1 cells transfected with the human NPY Y₁ receptor gene i.e. CHO-Y1 cells. Whereas the association and dissociation of the specific [³H]-NPY binding was slow, the binding kinetics of [³H]-BIBP3226 binding was very rapid. Saturation binding of both radioligands reveal the presence of an apparently homogeneous population of high affinity binding sites in both cell lines. The corresponding equilibrium dissociation constants are similar for the two cell lines and are close to those obtained from previous competition binding experiments. The specific binding of both radioligands was completely and with high affinity displaced by BIBP3226 and its inactive (S)-enantiomer BIBP3435 was much less potent. Whilst the NPY Y₁ agonists NPY, PYY and [Leu³¹-Pro³⁴]-NPY completely and potently displaced [³H]-NPY binding, they could only displace 70 to 80 % of the [³H]-BIBP3226 binding sites in CHO-Y1 and SK-N-MC cells. A possible explanation can be that only part of the receptors are G-protein coupled. In agreement pertussis toxin was found to reduce high affinity [³H]-NPY binding sites in CHO-Y1 cells whereas [³H]-BIBP3226 binding parameters remained unchanged.     

The Dissociation Rate of Unlabelled Dopamine Antagonists and Agonists from the Dopamine-D2 Receptor,Application of an Original Filter Method

Abstract

A filtration technique was developed to measure the dissociation rate of unlabelled drugs from membrane receptors. Receptor preparations saturated with unlabelled drug were adsorbed to glass fibre filters positioned on a filtration apparatus. Dissociation of the drug from the receptor sites was achieved by repeatedly applying small buffer samples on the filter, this was followed by short incubation on the filter with a [³H]ligand. Rat striatal membrane preparations adsorbed on filters retained the same dopamine-D² receptor binding properties as the tissue in aqueous suspension. Stereospecific [³H]haloperidol binding was maximal with 20 mg tissue per filter and 5 min incubation, K_D = 2.8 nM and B_max = 24 fmoles/mg tissue was found. The dissociation from the dopamine-D₂ receptor for 18 dopamine antagonists and 3 agonists followed first order reaction kinetics. Half-lifes of dissociation ranged from 3.5 min for azaperone to 233 min for metitepine. There was no strict relationship between dissociation half-life and apparent equilibrium binding affinity or lipophilicity of the drugs. Half-life of receptor dissociation appeared neither to be a primary determining factor in the duration of pharmacological action of the drugs. The importance of the drug receptor dissociation rate for binding experiments in vitro as well as for chronic drug treatment is discussed.     

Polyamines Modulate the Binding of [3H]MK-801 to the Solubilized N-Methyl-D-Aspartate Receptor

Spermine and spermidine enhance the binding of [³H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a, d]cyclohepten-5,10-imine ([³H]MK-801) to N-methyl-D-aspartate (NMDA) receptors in membranes prepared from rat brain. These polyamines also enhance binding of [³H]MK-801 to NMDA receptors that have been solubilized with deoxycholate. Other polyamines selectively antagonize this effect, a finding indicating that the polyamine recognition site retains pharmacological and structural specificity after solubilization. In the presence of spermidine, an increase in the affinity of the solubilized NMDA receptor for [³H]MK-801 is observed. However, the rates of both association and dissociation of [³H]MK-801 binding to solubilized NMDA receptors are accelerated when assays are carried out in the presence of spermidine. When kinetic data are transformed, pseudo-first-order association and first-order dissociation plots are nonlinear in the presence of spermidine, an observation indicating a complex binding mechanism. Effects of spermidine on solubilized NMDA receptors are similar to effects previously described in studies of membrane-bound receptors. The data indicate that polyamines interact with a specific recognition site that remains associated with other components of the NMDA receptor complex after detergent solubilization.     

Characterisation of [3H]MK-801 Binding and its Cooperative Modulation by PIG Brain Membranes

Abstract

Cooperative modulation of [³H]MK-801 binding to extensively washed pig cortical brain membranes in the presence of various concentrations of L-glutamate, glycine, spermine, CPP and DCKA was evaluated in association experiments. In saturation experiments [³H]MK-801 labelled a homogeneous population of binding sites with a K_d-value of 1.26 ± 0.18 nmol 1^?1 and a B_max-value of 2130 ± 200 fmol/mg protein. The pharmacological profile of this site was further evaluated in competition experiments with known NMDA receptor channel blockers. In nonequilibrium binding experiments EC₅₀-values of reference compounds acting at the L-glutamate, at the glycine, and at the polyamine site, were determined by increasing or decreasing [³H]MK-801 binding. Ifenprodil reduced [³H]MK-801 binding in a biphasic manner. All the data obtained are in agreement with results from [³H]MK-801 binding to rodent as well as human brain membranes. This study therefore strongly suggests, that pig cortical membranes are a suitable alternative to rodent brain membranes, and an acceptable substitute for human brain membranes in [³H]MK-801 binding experiments.     

[3H]WIN 35,065–2: A Ligand for Cocaine Receptors in Striatum

[3H]WIN 35,065-2 binding to striatal membranes was characterized, primarily by centrifugation assay. Like [3H]cocaine, [3H]WIN 35,065-2 binds to both high- and low-affinity sites. [3H]WIN 35,065-2, however, exhibits consistently higher affinities than [3H]cocaine. Saturation experiments indicate a low-affinity binding site with an apparent KD of approximately 160 nM and a Bmax of 135 fmol/mg of tissue. A high-affinity site has also been identified with an apparent KD of 5.6 nM and a Bmax of 5.2 fmol/mg of tissue. The specific-to-nonspecific binding ratios with [3H]WIN 35,065-2 were higher than with [3H]cocaine in both centrifugation and filtration assays. Pharmacological characterization suggests that [3H]WIN 35,065-2 binds to the dopamine transporter. Mazindol, GBR 12909, nomifensine, and (-)-cocaine are potent inhibitors of [3H]WIN 35,065-2 binding. In contrast, the norepinephrine transporter ligand desipramine is a weak inhibitor, and the serotonin transporter ligand citalopram does not inhibit binding. The effect of sodium on binding was examined under conditions in which (a) the low-affinity site was primarily (87%) occupied and (b) approximately 50% of both sites were occupied. The results indicate that both sites are sodium dependent. Injection of 6-hydroxydopamine into the striatum results in a significant loss of both high- and low-affinity sites, a finding suggesting that both sites are on dopaminergic nerve terminals. Taken together, these data are consistent with the presence of multiple cocaine binding sites associated with the dopamine transporter.     

High Affinity Stereospecific Binding of [3H] Cocaine in Striatum and its Relationship to the Dopamine Transporter

A high affinity (K_D 35 nM) binding site for [³H]cocaine is detected in rat brain Striatum present at 2–3 pmol/mg protein of synaptic membranes. This binding is displaced by cocaine analogues with the same rank order as their inhibition of [³H]dopamine ([³H]DA) uptake into striatal synaptosomes (r = 0.99), paralleling the order of their central stimulant activity. The potent DA uptake inhibitors nomifensine, mazindol, and benztropine are more potent inhibitors of this high affinity [³H]cocaine binding than desipramine and imipramine. Cathinone and amphetamine, which are more potent central stimulants than cocaine, displace the high affinity [³H] cocaine binding stereos-pecifically, but with lower potency (IC₅₀ ～ 1μM) than does cocaine. It is suggested that the DA transporter in Striatum is the putative “cocaine receptor.

Binding of [³H] cocaine, measured in 10 mM Na₂HPO₄-0.32 M sucrose, pH 7.4 buffer, is inhibited by physiologic concentrations of Na⁺ and K⁺ and by biogenic amines. DA and Na⁺ reduce the affinity of the putative “cocaine receptor” for [³H]cocaine without changing the B_max, suggesting that inhibition may be competitive. However, TRIS reduces [³H]cocaine binding non-competitively while Na⁺ potentiates it in TRIS buffer. Binding of [³H]mazindol is inhibited competitively by cocaine. In phosphate-sucrose buffer, cocaine and mazindol are equally potent in inhibiting [³H]mazindol binding, but in TRIS-NaCl buffer cocaine has 10 times lower potency. It is suggested that the cocaine receptor in the striatum may be an allosteric protein with mazindol and cocaine binding to overlapping sites, while Na⁺ and DA are allosteric modulators, which stabilize a lower affinity state for cocaine.     

Characterization of quisqualate recognition sites in rat brain tissue usingDl-[3H]α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and a filtration assay

The binding of [³H]AMPA (Dl--amino-3-hydroxy-5-methylisoxazole-4-propionic acid), a ligand for the putative quisqualate excitatory amino acid receptor subtype, was evaluated using centrifugation and filtration receptor binding techniques in rat brain crude synaptosomal membrane preparations. Maximal specific binding of [³H]AMPA occurred in Triton X-100 treated membranes in the presence of the chaotropic agent potassium thiocyanate (KSCN). The effects of KSCN on binding were reversible and optimal at 100 mM. Supernatant obtained from detergent-treated membranes inhibited specific [³H]AMPA and [³H]kainic acid binding, suggesting the presence of an inhibitory agent which was tentatively identified as glutamate. Using centrifugation, saturation analysis revealed two distinct binding sites in both the absence and presence of KSCN. The chaotrope was most effective in increasing binding at the low affinity binding site, enhancing the affinity (K _d) without a concommitant change in the total number of binding sites. Using filtration, a single binding site was detected in Triton-treated membranes. Like the data obtained by centrifugation, KSCN enhanced the affinity of the receptor (K _d value=10 nM) without altering the number of binding sites (B _max=1.2 pmol/mg protein). The rank order of potency of various glutamate analogs in the [³H]AMPA binding assay was quisqualate > AMPA > l-glutamate > kainate > d-glutamate, consistent with the labeling of a quisqualate-type excitatory amino acid receptor subtype.l-glutamic acid diethylester, and 2-amino-7-phosphonoheptanoic acid (AP₇) were inactive. The present technique provides a rapid, reliable assay for the evaluation of quisqualate-type excitatory amino acid agonists and/or antagonists that may be used to discover more potent and selective agents.     

A novel effect of molybdate on the binding of [3H]aldosterone to gel-filtered type I receptors in brain cytosol

Recently we reported that adding molybdate to crude steroid-free cytosol at 0°C results in a dose-dependent reduction in the binding of [³H]aldosterone ([³H]ALDO), to Type I adrenocorticosteroid receptors. In the experiments outlined here, we found that addition of molybdate to steroid-free brain cytosol produces a 30–50% increase in the subsequently measured maximal specific binding capacity (B _MAX) of [³H]ALDO-Type I receptors if the cytosol is subjected to Sephadex G-25 gel filtration prior to steroid addition. These manipulations were found to have no effect on the equilibrium dissociation constant (K _d) of the receptors. In contrast, when gel filtration of steroid-free cytosol was performed in the absence of molybdate, there was a 2-fold increase in the K_d and over a 50% reduction in the subsequently measuredB _MAX of [³H]ALDO-Type I receptors. When molybdate was added to this steroid-free cytosol immediately following gel filtration, there was no reduction (or increase) in Type I receptor [³H]ALDO binding capacity compared with nongel-filtered controls. The addition of as little as 2 mM molybdate to crude steroid-free cytosol was found to stabilize the binding capacity of Type I receptors during exposure to 22°C incubations; however, when gel-filtered steroid-free cytosol was exposed to these conditions at least 10 mM molybdate was required to stabilize Type I receptor binding capacity. Adding the sulfhydryl reducing reagent, dithiothreitol, to the various steroid-free cytosols had little effect on [³H]ALDO-Type I receptor binding. The effects of molybdate, revealed in this study, on Type I receptors in brain cytosol subjected to gel filtration are clearly different from those seen with receptors in crude cytosol preparations, as well as from those reported in the literature for other steroid receptors. Possible mechanisms of action of molybdate on unoccupied Type I receptors in crude and gel-filtered cytosol are discussed.     

Errors Introduced in Radioligand Binding Studies Due to Displaceable,Cation Dependent, [3H]prazosin Binding to Glass-Fibre Filters and Glass Surfaces

Abstract

[³H]prazosin not only specifically and homogeneously labels α₁-adrenoceptors, but also binds to glass surfaces and non-linearly to the glass-fibre filters, commonly used in radioligand binding experiments. Binding to filters can be modulated by unlabeled α-adrenergic compounds and cations. If no correction is applied for displaceable filter binding, analysis of [³H]prazosin binding experiments leads to erroneous results. Analysis of [³H]prazosin saturation experiments on guinea-pig cerebral cortex membranes with correction for filter binding before the non-linear fit procedure indicated that [³H]prazosin labels a homogeneous population of α₁-adrenoceptors (R_tot: 8.33 fmol˙mg^?1 wet tissue) with a dissociation constant of 1.28×10^?10 M. However, analysis of the same data after correction for non-specific binding, (determined in parallel experiments by adding 10 μM phentolamine to the incubation medium) resulted in a best fit to a model in which [³H]prazosin labels two α₁-adrenoceptor subpopulations (R₁: 15.0 fmol˙mg^?1 and R₂: 14.6 fmol˙mg^?1 wet tissue) with dissociation constants of respectively 1.78×10^?10 and 5.63×10^?9 M. The discrepancy between the two methods of analysis is due to displacement of the radioligand from the filters by phentolamine.

Prazosin and oxymetazoline are also able to displace filter-bound [³H]prazosin. The extent to which displaceable filter binding distorts the proper results depends on the actual magnitude of the error and also on the method of analysis.     

Reconstitution of Solubilized Serotonin-S2 Receptors into Phospholipid Vesicles

Abstract

Serotonin-S₂ receptors from rat frontal cortex were solubilized using CHAPS/sodium chloride. Reconstitution of the solubilized receptors was achieved by dilution of the soluble preparation, followed by centrifugation to remove the detergent. The receptors were truly reconstituted as judged by sedimentation, increased thermostability and electron microscopy. The reconstituted preparation showed high-affinity binding of [³H]7-aminoketanserin. The binding characteristics resembled those obtained for membrane-bound receptors.     

Endogenous peptide(s) inhibiting [3H]cocaine binding in mouse brain

The supernatant fraction of centrifuged homogenate of brain tissue contains material that inhibits the saturable binding of [³H]cocaine to crude mouse brain membranes. This material was subjected to heat treatment to remove protein; further purification was achieved by filtering through an Amicon UM-10 membrane ultrafilter and gel filtration of the ultrafiltrate on Sephadex G-25. Sensitivity to acid hydrolysis and peptidase action indicates that the inhibitory activity resides in peptide material with a low molecular weight. The partially purified inhibitor has similar effects to that of cocaine on the specific binding of various ligands to opiate and nonopiate receptors in mouse brain membranes.     

Binding of [3H]batrachotoxinin A-20-α-benzoate and [3h]saxitoxin to receptor sites associated with sodium channels in trout brain synaptoneurosomes

1. [³H]Batrachotoxinin A-20-α-benzoate ([³H]BTX-b) and [³H]saxitoxin ([³H]STX), radioligands that bind to distinct sites on the voltage-sensitive sodium channel, were bound specifically to saturable sites in rainbow trout (Oncorhynchus mykiss) brain synaptoneurosomes.2. Specific [³H]BTX-B binding was temperature dependent with highest levels of specific [³H]BTX-B binding observed at 7°C. Specific binding was inversely correlated with assay temperature at temperatures above 7°C.3. Saturating concentrations of scorpion (Leiurus quinquestriatus) venom (ScV) stimulated specific [³H]BTX-B binding at 27°C, but not at 7°C. The dihydropyrazole insecticide RH 3421 inhibited specific [³H]BTX-B binding at 7°C but had no effect on specific binding at 27°C. The sodium channel activators veratridine and aconitine and the local anesthetic dibucaine inhibited specific [³H]BTX-B binding at both 7°C and 27°C.4. Displacement experiments in the presence of ScV at 27°C gave an equilibrium dissociation constant (K_d) for [³H]BTX-B of 710 nM and a maximal binding capacity (B_max) of 11.3 pmol/mg protein. Kinetic experiments established the rates of association (1.17 × 10⁵min⁻¹ nM⁻¹) and dissociation (0.0514min⁻¹) of the ligand-receptor complex.5. The binding of [³H]STX reached apparent saturation at 7.5 nM. Scatchard analysis of the saturation data indicated a K_d of 3.8nM and a B_max of 1.9 pmol/mg protein.6. These studies provide evidence for high affinity, saturable binding sites for [³H]BTX-B and [³H]STX in trout brain preparations. Whereas certain neurotoxins modified the specific binding of [³H]BTX-B in trout brain synaptoneurosomes in a predictable fashion, other compounds known to affect specific [³H]BTX-B binding in mammalian brain preparations had no effect on specific [³H]BTX-B binding in the trout.     

Muscarinic binding to mouse brain receptor sites

In mouse brain the binding of [³H]-Atropine to the muscarinic receptor seems to be a simple mass-action determined process as gauged both by approach to equilibrium kinetics and binding at equilibrium. In contrast, using isotopic dilution technique, dissociation measurements indicate the existence of two receptor-ligand complexes. It would appear that association and dissociation rates of binding of the muscarinic antagonists atropine, scopolamine, N-methyl-4-piperidyl benzilate (4NMPB) and 3-quinuclidinyl benzilate (QNB) decrease with increasing affinity based on comparisons of kinetic binding data. The differences between the association rate constants are small whereas those between the dissociation rate constants differ markedly. This kinetic behavior is similar to the well-known time profile of antimuscarinic activity in isolated tissues. These phenomena are discussed in terms of possible isomerization of the receptor-ligand complex, as has been proposed recently for [³H]-scopolamine and [³H]-4NMPB binding.