Interactions of D600 (methoxyverapamil) and local anesthetics with rat brain α-adrenergic and muscarinic receptors

D600 (methoxyverapamil) was found to inhibit the specific binding assayed in rat brain homogenates of the antagonist agents [³H]WB 4101 and [³H]QNB to the α-adrenergic and muscarinic receptors respectively. The IC₅₀ concentrations of D600 in standard binding experiments were 1.7 × 10_?6 M and 1.4 × 10 _?5M, with calculated k₁ values of 0.98 × 10_?6 M and 8.83 × 10_?6M.Scatchard analyses showed these inhibitions to be competitive. Lidocaine and tetracaine also inhibited radioligand binding to these receptors, with K₁ values of 5.25 × 10^?4M and 4.85 × 10^?5 M for the α-receptor and 8.2 × 10^t-5 M and 6.94 × 10^?6 M for the muscarinic receptor; these inhibitions also appeared to be competitive. Increasing the Ca²⁺ concentration in the assays to 10 mM did not influence the effects of D600 or the anesthetics. Analyses of inhibitions of muscarinic receptor binding produced by D600 and lidocaine over a range of pH indicated that the inhibitory species of D600 is the uncharged form, whereas the charged form of lidocaine is inhibitory. Interactions of D600 and lidocaine with the agonist site on the muscarinic receptor were studied by measuring the effects of these agents on the displacement of [³H]QNB by the muscarinic agonist carbachol. Comparison of these results with a theoretical model indicates that carbachol, [³H]QNB, and D600 or lidocaine competitively displace one another at the same agonist site. The binding of labeled naloxone to the opiate receptor was also inhibited by D600, the IC₅₀g being 4 × 10^t?6 M. These inhibitory effects of D600 and the local anesthetics on different receptors suggest that these agents may act by a common mechanism, namely by perturbing membrane structures. These results suggest caution in interpreting experiments in which D600 and verapamil are used analytically as Ca antagonists to assess the involvement of Ca in a biological system.     

BIOCHEMICAL CHARACTERISTICS OF MUSCARINIC CHOLINORECEPTORS IN SWINE TRACHEAL SMOOTH MUSCLE

• 1 The tritiated muscarinic cholinoreceptor antagonist quinuclidinyl benzilate, [³H]QNB, was used to characterize the muscarinic receptors associated with homogenized membrane of the smooth muscle from swine trachea. Based on receptor binding assays, the homogenate had specific, saturable, high-affinity receptors for [³H]QNB.
• 2 Specific binding was time- and temperature-dependent. The association of [³H]QNB with the muscarinic receptor reached equilibrium much sooner at 37°C than 25°C at a [³H]QNB concentration of 180 pM (30 min and 2 h, respectively). Equilibrium at both temperatures was attained within 5 min at a [³H]QNB concentration of 1800 pM. All remaining experiments were performed at 37°C.
• 3 Binding was saturable with respect to [³H]QNB and tissue concentrations. Analysis of binding isotherms yielded an apparent equilibrium dissociation constant (K_D) of 51±20 pM and a maximum receptor density (B_max) of 2.17±0.27 pmole/mg protein. The Hill coefficient for [³H]QNB binding was 1.07±0.16. The association (K₁) and dissociation (K_-1) rate constants were determined to be (5.51±0.16) × 10⁸ M^?1 min^?1 and (1.41±0.18) × 10^?2 min^?1, respectively. K_D calculated from the ratio of K₁ and K_-1 was 26.3±3.8 pM; this value is close to the value of K_D calculated from Scatchard plots of binding isotherms.
• 4 The density of muscarinic receptor binding sites was 10-fold greater in tracheal smooth muscle than in tracheal epithelium (0.20±0.03 pmole/mg protein). There is no difference between weanling and young adult swine in the density of muscarinic receptors in tracheal smooth muscle.
• 5 The nonselective muscarinic antagonists atropine, scopolamine and quinuclidinyl benzilate (QNB) competitively inhibited [³H]QNB binding to the homogenate with Hill coefficients of 0.9-1.0 and inhibition constants (K_i) of nanomolar range.
• 6 Competition with selective muscarinic antagonists pirenzepine and 3-quinuclidinyl xanthene-9-carboxylate (QNX) gave K_i values, 0.26 M and 0.78 nM, respectively, and Hill coefficients of approximately 1. There was a single population of [³H]QNB binding sites of the M₂ subtype for all tested muscarinic antagonists.
• 7 Competition with selective muscarinic agonists pilocarpine and carbachol yielded K_i values of micromolar range, Hill coefficients of less than 1, and revealed the existence of two binding sites (P < 0.01).

     

Cholinergic elements in a human choriocarcinoma cell line

Components of the cholinergic system have been identified in the JEG choriocarcinoma cell line. [³H]Quinuclidinyl benzilate (QNB) was used to identify high affinity muscarinic binding sites in a whole cell preparation. Specific binding was saturable with respect to QNB concentration and revealed a binding site density of 27 fmoles/mg protein. The bimolecular rates of association, 2.24 × 10⁷ M^?1 min^?1, and dissociation, 4.2 × 10^?3min^?1, revealed a dissociation constant (K_d) of 180 pM which agreed closely with that derived from saturation isotherms, 245 pM. Muscarinic antagonists and agonists were able to compete effectively for these binding sites, whereas non-muscarinic compounds were not. Cholinesterase activity was also demonstrated with substrate preference consistent with that of acetyl-cholinesterase (acetylcholine > acetyl-β-methylcholine > butyrylcholine) hydrolyzing 2.42 ± 0.19 × 10^?3μmoles acetylcholine·min^?1 ·(mg protein)^?1. No choline acetyltransferase activity was detected in these cells, however.     

The influence of guanyl-5′-yl imidodiphosphate and sodium chloride on the binding of the muscarinic agonist, [3H] cis methyldioxolane

[³H]Quinuclydinyl benzylate([³H]QNB) binding was carried out on crude synaptosomal membranes isolated from cat cerebral cortex. The specific binding showed a single type of site with K_D 0.25 nM, Hill number 0.89 and B_max 114 pmol/g protein. The local anesthetics procaine, tetracaine, and the adrenergic antagonists phentolamine and propranolol, in concentrations between 1 nM and 500 μM, inhibited [³H]QNB binding with K_i varying between 9 μM for procaine and 80 μM for propranolol. The Hill coefficients obtained from logit/log plots suggested that there was no cooperativity between the binding sites for local anesthetics. At various concentrations the inhibition by procaine and propranolol may appear as competitive or non-competitive. The Hill numbers obtained from the saturation curves suggest that there was no cooperativity between anesthetics and [³H]QNB binding sites. Neither 1 mM Ca²⁺ nor Mg²⁺ affected [³H]QNB binding or the action of the drugs. The effect of local anesthetics and adrenergic antagonists was reversible and these drugs did not protect the muscarinic receptor from the deleterious effect of Triton X-100 as was the case with muscarinic agents. Our findings suggest that local anesthetics inhibit [³H]QNB binding to the muscarinic receptor by acting at some accessory site but not on the true receptor site. The possible mechanism of action of local anesthetics on synaptic transmission is discussed.     

Pharmacology of high-affinity binding of [3H](±)2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN) to bovine caudate nucleus tissue

High-affinity binding of [³H](±)2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene ([³H]-ADTN) was improved by use of a subcellular fraction (P₄) of tissue obtained from calf brain. The highest concentration of binding sites was found in caudate nucleus which was evaluated extensively. Binding of 0.5 nM [³H]-ADTN was optimal at 25° and pH 7.5 to 8.0 when a cation-free medium containing antioxidants was used and was 82–87% displaceable (“specific”). The T_{$\text{1}\text{2}$} for association was 20 min, and for dissociation 38 min, under these conditions. Analysis of association-dissociation kinetics and of ligand saturation isotherms revealed an apparent affinity (K_d) of 1–2 nM and a binding maximum (B_max) of 422 fmoles/mg protein. The process proved to be reversible by, and monophasically competitive with, several potent dopamine agonists, with Hill constants dose to unity. Stereoselectivity was found with eight isomer-pairs. Binding of [³H]-ADTN was selective for 3,4-dihydroxyphenethylamines and 10,11-dihydroxyaporphines with potent dopamine-agonist actions, but not for adrenergic catecholamines or other catechols, or blockers of dopamine uptake. Dopamine antagonists competed more weakly and in poor correspondence with their in vivo activities. There was a close correspondence between IC₅₀ values obtained for fifty of the agents tested with both [³H]-ADTN and [³H]-(?)apomorphine (r and slope > 0.9), supporting impressions of the structure-activity characteristics of dopamine agonist binding sites based on prior studies with [³H]-apomorphine.     

Differential toxicity of carrier-bound methotrexate toward human lymphocytes,marrow and tumor cells

Methotrexate that was covalently linked to poly-l-lysine (mol. wt 3,000 and 60,000) (MTX-PLL 3K and 60K) was more inhibitory to the growth of five cell lines from human solid tumors (IC₅₀ 5?10 × 10^?8 M and 1?2.6 × 10^?8 M respectively) than to the growth of five lines of human lymphocytes (IC₅₀ 5?8 × 10^?7 M and 2?5 × 10^?7 M). In contrast, both methotrexate that was covalently linked to human serum albumin (MTX-HSA), and the free drug, were equally toxic to the two classes of cells, with IC₅₀ of 3?15 × 10^?7 M and 2?7 × 10^?8 M, respectively, for the cell types. Uptake studies showed that, whereas MTX and MTX-HSA were transported equally well into WI-L2 lymphocytes, human bone marrow cells, and an astrocytoma tumor line, uptake of MTX-PLL by the astrocytoma cells at 37° was three to four times greater than uptake by WI-L2 lymphocytes or marrow cells. [³H]Deoxyuridine ([³H]-Urd) incorporation studies indicated that low concentrations of MTX-PLL 60K (5 × 10^?7 M) resulted in inhibition of the target enzyme dihydrofolate reductase (DHFR) in the astrocytoma cells, but no iinhibition of DHFR occurred in WI-L2 lymphocytes or marrow cells until concentrations were reached where the carrier itself became toxic (5 × 10^?6 M). Two inhibitors of the lysosomal enzymes, chloroquine and lupuptin, were able to reverse the toxicity of MTX-PLL 60K against the astrocytoma cell line, increasing its IC₅₀ from 2 × 10^?8 to 2 × 10^?7 M. Both lysosomal inhibitors had no effect on the toxicity of MTX-PLL 60K against the WI-L2 lymphocytes or of MTX or MTX-HSA against either cell type, indicating that the increased toxicity of MTX-PLL 60K against the tumor cells was due, in part, to the ability of the lysosomes of these cells to convert MTX-PLL 60K either to the free drug or to a derivative that was effective in inactivating DHFR. These results suggest that comparable differential toxicity between marrow and tumor cells might also be achieved in vivo if MTX-PLL is infused over long periods at a rate that would maintain a constant serum concentration sufficient to kill tumor cells without affecting bone marrow cells.     

3H]Quinuclidinyl benzilate binding to muscarinic receptors and [3H]WB-4101 binding to alpha-adrenergic receptors in rabbit iris. Comparison of results in slices and microsomal fractions

The binding characteristics of [³H]-(l)-quinuclidinyl benzilate (QNB) and [³H]WB-4101 to microsomal fractions and slices from rabbit iris muscle were compared. [³H] QNB binding to both microsomal fractions and muscle slices was of high affinity and low capacity and was displaced by muscarinic ligands. The equilibrium dissociation constants (K_D) for [³H]QNB binding to microsomes and slices were 0.069 nM and 1.97nM, respectively. This shift to a higher value for the K_p of the microsomal fraction compared with that of the slices was also observed lor the association rate constants (K_I) and inhibition constants (K_J), but not for the dissociation rate constants (K_?1). Kinetic studies on the binding characteristics of [³H]WB-4101 revealed high affinity sites with K_D values of 2.33 and 10.19 nM for microsomal fractions and slices, respectively. The findings of comparable binding patterns for [³H]QNB and [³H]WB-4101 binding to microsomal fractions and intact muscle slices argue against the possibility of alterations in receptor properties following tissue disruption. It is proposed that the differences in receptor-mediated biochemical responses that are seen between intact tissue and cell-free homogenates, such as the ‘phosphoinositide effect’, are more likely to be due to alterations in receptor function, e.g. changes in ionic permeabilities, rather than to actual changes in receptor properties.     

Binding of a labeled antidepressant to rat brain tissue

[³H]desipramine. a labeled tricyclic antidepressant, binds specifically to rat brain crude synaptosomal and membranal preparations. The binding is rapid, displaceable and saturable with a IC₅₀ value of 4 × 10^?6M. The maximal number of sites is 18–26 nmoles/g original tissue in the synaptosomal preparation and only 6–8 nmoles/g in the membranal preparation. Scatchard plots of the binding data are not linear, suggesting a heterogenous set of binding sites. Pargyline and a number of neurotransmitters and agonists have only a small effect on desipramine binding; whereas various neurotransmitter antagonists inhibit the binding at the μM concentration range.     

Muscarinic receptors on rat ileal villus and crypt cells

Ileal villus and crypt cells exhibit morphological and biochemical differences which may be responsible for functional differences in relation to ion transport. Cholinergic agonists act directly on epithelial cell muscarinic receptors, but it is not known if these receptors exist on both villus and crypt cells. Using the potent muscarinic antagonist [³H](?)-quinuclidinyl benzilate (QNB) we have determined the distribution of muscarinic receptors in rat ileal villus and crypt cells. Plasma membrane preparations from ileal villus and crypt cells possessed a specific, saturable, and high affinity QNB binding site with apparent dissociation constants of 0.23 ± 0.05 and 0.21 ± 0.04 nM (mean ± s.e., n = 6) and densities of 92.2 ± 2.8 and 90.1 ± 16.2 fmol (mg protein)^?1, respectively. Both types of cells showed similar potencies for agonist and antagonist competition of QNB binding. The muscarinic receptors in membrane fractions from villus cells were found primarily on the basolateral membrane rather than on the brush border membrane. Secretion induced by cholinergic stimulation of the small intestine might, therefore, be due to an effect on both villus and crypt cells as both types contain muscarinic receptors. Furthermore, such stimulation also may result in mucin secretion, as goblet cells were present in the preparation we studied, and receptors on these cells may have contributed to the amount of binding of [³H]QNB.     

[3H]5′-N-Ethylcarboxamidoadenosine selectively labels the low affinity adenosine binding protein,adenotin, on intact chinese hamster ovary cells

The ability of [³H]5′-N-ethylcarboxamidoadenosine (NECA) to specifically bind recognition sites on intact Chinese hamster ovary (CHO) cells was examined in the present study. Saturation experiments indicated that [³H]NECA bound with moderate affinity (Kd = 400 nM) and large capacity (apparent Bmax = 3.2 pmol/10⁵ cells) to intact CHO cells. No specific binding to these cells was observed with the A₁-selective agonist 20 nM [³H]cyclohexyladenosine or with the A₂-selective agonist 20 nM [³H]CGS 21680. Competition studies revealed that close structural analogs of NECA and the xanthine phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) inhibited 20 nM [³H]NECA binding with moderate affinity (IC₅₀s 0.5–15 μM). Adenosine also showed weak activity (IC₅₀ = 100 μM) for inhibiting [³H]NECA binding. However, a wide variety of prototypic adenosine receptor agonists and antagonists did not significantly interact with these [³H]NECA recognition sites on CHO cells. [³H]NECA binding to CHO cell membranes was not sensitive to guanine nucleotides and NECA did not stimulate cAMP formation. These results are consistent with the previously demonstrated ability of [³H]NECA to bind low affinity adenosine binding proteins (adenotin proteins), as well as, adenosine receptors in a variety of mammalian tissues. The present results further indicate that [³H]NECA selectively labels in adenotin-like recognition site on intact CHO cells in the absence of detectable binding to high affinity adenosine receptors. © 1993 Wiley-Liss, Inc.     

Identification of non-serotonergic [3H]ketanserin binding sites on human platelets and their role in serotonin release

[³H]Ketanserin was found to label (besides a low amount of 5-HT₂ receptors) non-serptonergic binding sites on human platelet membranes. The latter binding was detected in the presence of excess of the 5-HT₂ antagonist BW501, and was potently inhibited by tetrabenazine. [³H]Ketanserin revealed a K_D value = 19 ± 4 nM and a B_max = 425 ± 82 fmol/10⁹ platelets for these binding sites. [³H]Ketanserin binding in the presence of BW501 was inhibited by the tetrabenazine derivative RO-4-1284 (IC₅₀ = 1.4 nM), tetrabenazine (IC₅₀ = 8.6 nM) and the ketanserin derivatives R 71 278 (IC₅₀ = 6.3 nM). R 47 288 (IC₅₀ = 17 nM) and R 71 428 (IC₅₀ = 100 nM). Ketanserin revealed an IC₅₀ = 32 nM. The drugs were found to trigger the release of ³H from [³H]5-HT-loaded human platelets in superfusion experiments in vitro. The amount of ³H released by the drugs correlated with their binding affinities for the non-serotonergic sites. The non-serotonergic [³H]ketanserin binding sites on human platelets and their possible role in triggering monoamine release corresponded to the properties of non-serotonergic ketanserin binding sites previously characterized in rat striatum. The possible role of the action of ketanserin on the non-serotonergic sites in the reported partial reduction by ketanserin of the monoamine content in cardiovascular tissues is discussed.     

Characterization of [3H]muscimol binding to mouse brain membranes.

The binding of [³H]muscimol {[methylene-³H(N)]-3-hydroxy-5-aminoethyl isoxazole} to a membrane fraction from mouse brain was studied as a possible model for the GABA recognition site of the γ-aminobutyric acid (GABA)-anionophore receptor complex. Kinetic studies showed two distinct association and dissociation rate constants, 2.3 × 10^?4 and 1.4 × 10^?5 sec^?1 nM^?1 for association and 1.2 × 10^?2 and 1.0 × 10^?3 sec^?1 for dissociation. Equilibrium analysis (Scatchard plot) of binding data also indicated two types of sites with K_D equal to 9 × 10^?9 and 70 × 10^?9 M. Detergents (0.1% Triton X-100, 0.1% Lubrol PX or 5% Tween 20) had no effect on the binding of [³H]muscimol or other conformationally restricted agonists such as imidazoleacetic acid, isoguvacine, THIP (4,5,6,7-tetrahydroisoxazole [5,4-c] pyridine-2-ol), and (±)-isonipecotic acid. In contrast to this, the detergents potentiated the ability of the less conformationally restricted agonists such as GABA and β-alanine in displacing [³H]muscimol. Binding of bicuculline was reduced by all of the detergent treatments.     

Protection by atropine of the inhibition caused by triton X-100 on central muscarinic receptors

Synaptosomal membranes from cat cerebral cortex were labelled with [³H]-quinuclidinyl benzylate ([³H]-QNB). There was shown to be a single type of binding site with Kd = 0.34 nM, Bmax = 2.2 nmol/g protein and Hill No. = 1.01. Triton X-100 at 5 × 10^?4% inhibited the specific binding of [³H]QNB and the inhibition was almost complete at 10^?2%. Treatment with 2.5 × 10^?6M atropine, followed by centrifugation washings protected the receptor site from the inhibitory action of the detergent. The protection afforded by other cholinergic drugs was less effective. The use of this technique has confirmed the results of our previous work on the possible pre- and postsynaptic location of central muscarinic receptors. These findings open the possibility for protection of other detergent-sensitive receptors and for their isolation and purification as well-defined macromolecules.     

Simultaneous evaluation by a double labelling method of drug-induced uptake inhibition and release of dopamine in synaptosomal preparation of rat striatum

Crude synaptosomal preparations from corpora striata of rat were preloaded with [¹⁴C]DA, rinsed, and then incubated with [³H]DA and the drug to be tested. During the 5 min of the second incubation, DA uptake and release rates were time- and temperature-dependent. On this double labelling test, nomifensine, cocaine, benztropine and amphetamines displayed IC₅₀ and release curves similar to those obtained from separate uptake and release studies. In the presence of high concentrations of iprindole, amitriptyline and butriptyline (>3 × 10^?6 M), a [¹⁴C]DA release was observed which closely coincided with an apparent inhibition of [³H]DA uptake. This double labelling test allows the determination of the participation of the releasing effect of drugs in their apparent inhibition of DA uptake.     

Cardiac glycoside receptor, (Na+ + K+)ATPase activity and force of contraction in rat heart

In order to elucidate the role of (Na⁺ + K⁺)ATPase and of the ouabain binding sites in the pharmacological effects of ouabain in the digitalis insensitive species rat, specific [³H]ouabain binding and (Na⁺ + K⁺)ATPase activity were measured simultaneously in a rat heart cell membrane preparation. Specific [³H]ouabain binding, ⁸⁶Rb⁺-uptake and force of contraction were also measured simultaneously in electrically stimulated contracting ventricular strips of rat heart. The following results were obtained: (1) Rat heart cell membranes exhibit two classes of [³H]ouabain binding sites with apparent dissociation constants (K_D) of the [³H]ouabain-receptor complex 1–2.3 × 10^?7 M and 2.8 × 10^?5 M. (2) (Na⁺ + K⁺)ATPase activity of rat heart cell membranes is half-maximally inhibited by ouabain at a concentration of 4 × 10^?5 M when assayed at the same conditions as [³H]ouabain binding. (3) Specific [³H]ouabain binding to electrically stimulated (1 Hz) contracting ventricular strips of rat heart exhibited only one class of receptors (K_D = 3 × 10^?7M). Force of contraction increased half-maximally at 3 × 10^?7 M ouabain when measured simultaneously and ⁸⁶Rb⁺uptake was inhibited half-maximally at 3 × 10^?5 M ouabain. Thus, there is a serious discrepancy between the effect of ouabain on (Na⁺ + K⁺)ATPase activity and ⁸⁶Rb⁺ uptake on one hand and on force of contraction on the other hand, whereas there is a good correlation between [³H]ouabain-receptor binding and increase in force of contraction. These results indicate that inhibition by ouabain of active cation transport is not a mandatory prerequisite of its positive inotropic effect, at least in the rat heart.     

Solubilized receptors for [3H]dopamine (D3 binding sites) from canine brain

The objective of the present study was to solubilize the D₃ site which binds [³H]dopamine, using the same digitonin method that had been successful in solubilizing the D₂ dopamine receptor. Canine brain striatal membranes were solubilized by a final concentration of 1% digitonin. The specific binding of [³H]dopamine to the soluble D₃ binding sites was measured using Sephadex G-50 gel filtration. The density of D₃ sites was identical in the membrane and soluble preparations (82–90 fmoles/mg protein), although the dissociation constant (K_D value) went from 1.2 nM in the membranes to the value of 3.4 nM in the soluble material. The concentrations of various drugs which inhibited the binding of [³H]dopamine were similar in the two preparations. The agonists [dopamine, apomorphine and (±)-6,7-dihydroxy-2-aminotetralin (ADTN)] all inhibited the binding of [³H]dopamine by 50% at concentrations between 2 and 20 nM in both the intact and soluble preparations. The neuroleptics were all equally weak in inhibiting the binding of [³H]dopamine, with IC₅₀ values in the micromolar concentration range, values typical for the D₃ site. Approximately 36% of the D₃ sites were recovered from the original tissue. Since the densities and recoveries of the D₂ and D₃ sites differed upon digitonin solubilization, this provided further indirect evidence that these two sites are distinct and separate entities which might ultimately be separated.     

Allosteric Effects of the Alkane-bis-ammonium Compound W84 and of Tacrine on [3H]Pirenzepine Binding at M1-Receptors in Rat Cerebral Cortex

Abstract: The bis-quaternary W84, hexamethylene-bis-[dimethyl-(3-phthalimidopropyl)-ammonium bromide], is a potent allosteric modulator of M₂-cholinoceptors. In this study we aimed at quantifying its allosteric effect on the dissociation of [³H]pirenzepine from M₁-cholinoceptors in rat cerebral cortex and to measure the effects on association and equilibrium binding of [³H]pirenzepine. For sake of comparison tacrine was included which is known to be a potent allosteric modulator of [³H]pirenzepine binding to M₁-receptors. Under control conditions (3 mM MgHPO₄, 50 mM Tris-HCl, pH 7.4, 23°) [³H]pirenzepine binding was characterized by K_D=5 nM and B_max=965 fmol/mg membrane protein, the rate constants amounting to k₊₁ = 5.0 μM^?1xmin^?1 and k_-1=0.031 min^?1. W84 and tacrine reduced [³H]pirenzepine binding concentration-dependently with IC₅₀-values of 1.9 μM and 2.6 μM, respectively. [³H]pirenzepine association was inhibited by the compounds with EC_50,ass⁼1.8 μM for W84 and EC_50,ass=2.4 μM for tacrine. The concentrations reducing the dissociation rate by 50% amounted to EC₅₀,_diss=21 μM for W84 and to EC_50,diss⁼54 μM for tacrine. Compared with W84, the dose-response curves of tacrine for the investigated effects were significantly steeper. In conclusion, W84 affected [³H]pirenzepine binding to M₁-receptors allosterically with a higher potency than tacrine but probably by a different mechanism.     

3H]Ouabain binding and dissociation in rabbit colon: effect of ions and drugs

²²Na fluxes (J) and [³H]ouabain binding were studied in vitro in the distal portion of the rabbit colon, mounted as a membrane, separating two perspex chambers. J_MS^Na (flux of ²²Na from mucosal to serosal chamber) was 2.1 and J_SM^Na was 1.0 μmoles cm² × hr· J_SM^Na was completely abolished by 10^?3 M ouabain, placed in the serosal side. J_SM^Na was unaffected by ouabain. [³H] Ouabain binding to the serosal surface of the colon was inhibited to the same maximal effect by ‘cold’ ouabain (I₅₀ = 5 × 10^?7 M), by K⁺ placed in the serosal chamber (I₅₀ = 3.5 mM) and by replacement of Na⁺ with choline on the serosal side. Increased tissue concentration of Na⁺ did not affect [³H]ouabain binding, suggesting that extracellular rather than intracellular sodium plays a major role in cardiac glycoside binding. Various cardiac glycosides (digoxin, digitoxin. ouabain) inhibited [³H]ouabain binding to the serosal side of the colon but other steroids (estriol, testosterone, desoxycorticosterone) had not effect.Efflux of [³H]ouabain, bound to the serosal side of the colon, was differentiated into dissociation of nonspecifically bound [³H]ouabain (95 per cent in 60 min) and dissociation of specifically bound [³H]ouabain (20 per cent in 60 min). Dissociation of specifically bound [³H]ouabain was accelerated when Na was replaced by choline (50 per cent in 60 min). The dissociation rate of nonspecifically bound ouabain was unaffected by replacement of Na⁺· [³H]Ouabain binding to mucosal surface was unaffected by ‘cold’ ouabain, by increased K⁺ in the medium or by replacement of Na⁺. It is concluded that there is no specific binding of [³H]ouabain to the mucosal surface. Omission of Ca, Mg or phosphate from the medium did not affect [³H]Ouabain binding to either mucosal or serosal surfaces of rabbit colon.     

3H]dihydroergocryptine binding to bovine striatal membranes defined by a low d-butaclamol concentration: antagonism by substituted benzamides

An apparent dopamine-D2 receptor binding system was characterized using [³H]dihydroergocryptine (DHE) as the ligand. Specific binding to bovine striatal membranes was defined as the difference between binding in the absence and in the presence of 4 nM d-butaclamol. These high affinity binding sites (K_d = 0.24 nM) were saturable (B_max = 75 fmoles/mg protein), apparently homogeneous, non-interacting, and stereospecific. Specific binding was greatest in areas rich in dopaminergic innervation. In competition experiments, apomorphine (ic₅₀ = 100 nM) was fifty times more potent than dopamine which, in turn, was nine times more potent than l-norepinephrine and serotonin. Antipsychotic drugs including the substituted benzamides—metoclopramide, sulpiride and sultopride—were active in competing with [³H]DHE for these binding sites ($\text{ic}{}_{50}\text{= 100}\text{nM}$), and their affinities agreed well with their anti-dopaminergic potencies in vivo. Potencies of antipsychotic drugs in competing for DHE binding sites were increased compared to their potencies in competing for [³H]spiroperidol binding sites using 1 μM d-butaclamol as the masking ligand. This relative increase was about 10-fold greater for the substituted benzamides than for other antipsychotic drugs. The relative inactivity of substituted benzamides in competing for binding sites labeled by spiroperidol was accounted for by their greater selectivity for dopamine receptors. In binding studies using a non-selective radio-ligand, selectivity could be enhanced by defining specific binding sites with a low concentration of masking ligand.     

Interaction of the Hexamethonium Derivative W84, an Allosteric Effector at Muscarinic Cholinoceptors,with Rat Brain Nicotinic Binding Sites

The hexamethonium derivative W84 (hexamethylene-bis-[dimethyl-(3-phthalimidopropyl)-ammonium bromide]) combined with atropine has an overadditive protective action against organophosphorus intoxications. It affects allosterically the binding of (—)[³H]N-methylscopolamine ([³H]NMS) to muscarinic cholinoceptors. Because nicotinic receptors are involved in organophosphorus intoxications, the interaction of W84 with nicotinic cholinoceptors was investigated. (—)[³H]nicotine (2.5 nM) was used to label nicotinic binding sites in rat brain membranes in 50 mM Tris, pH 7.3, at 23°. Under control conditions, (—)[³H]nicotine-binding revealed a K_D of 4×10^-9M and a B_max of 53 fmol/mg membrane protein. W84 inhibited (—)[³H]nicotine-binding with an IC50 of 3 × 10^-5M by reducing the binding affinity. The IC50 of hexamethonium was 20 × 10^-5M. At 10^-4M, W84 did not affect the dissociation rate of (—)[³H]nicotine, suggesting a lack of allosteric activity. For sake of comparison, the action of W84 was checked on [³H]NMS-binding (control: K_D ～ 1 × 10^-9M, B_max ～ 500 fmol/mg prot.). W84 inhibited the binding of [³H]NMS (0.5 nM) with an IC50 of 1.5 × 10^-5M. At 10^-4M, W84 prevented [³H]NMS-dissociation almost completely, thus displaying the allosteric action at muscarinic cholinoceptors. In conclusion, the results of the (—)[³H]nicotine-binding experiments point to a pure competitive action of W84 at nicotine cholinoceptors, lacking any allosteric effect. This competitive action may contribute to the protective effect of W84 in organophosphorus poisoning.