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M Sokolovsky

Publications and source records attributed to M Sokolovsky.

At least 109 records · Page 6Linked to original sources
PubMed1985

Rate constants of agonist binding to muscarinic receptors in rat brain medulla. Evaluation by competition kinetics.

The method of competition kinetics, which measures the binding kinetics of an unlabeled ligand through its effect on the binding kinetics of a labeled ligand, was employed to investigate the kinetics of muscarinic agonist binding to rat brain medulla pons homogenates. The agonists studied were acetylcholine, carbamylcholine, and oxotremorine, with N-methyl-4-[3H]piperidyl benzilate employed as the radiolabeled ligand. Our results suggested that the binding of muscarinic agonists to the high affinity sites is characterized by dissociation rate constants higher by 2 orders of magnitude than those of antagonists, with rather similar association rate constants. In contrast, the major differences between the kinetic binding parameters of agonists and antagonists to the low affinity agonist binding sites are in the association rate constants, which were 2-5 orders of magnitude lower for agonists. This demonstrates that there are basic differences in the interactions of agonists with the low and high affinity sites. Our findings also suggest that isomerization of the muscarinic receptors following ligand binding is significant in the case of antagonists, but not of agonists. Moreover, it is demonstrated that in the medulla pons preparation, agonist-induced interconversion between high and low affinity bindings sites does not occur to an appreciable extent.

Animals↗
PubMed1985

Allosteric interactions between muscarinic agonist binding sites and effector sites demonstrated by the use of bisquaternary pyridinium oximes.

Agonist binding to muscarinic receptors from rat brain stem and cerebral cortex was studied using bisquaternary pyridinium oximes for detecting possible interactions between agonist binding sites and sites of the effector guanosine 5' (beta, gamma-imino) triphosphate (Gpp(NH)p) and Co2+. Pretreatment of either brain stem or cortical homogenates with 200 microM 1-(2-hydroxyiminoethylpyridinium) 1-(3-phenylcarboxypyridinium) dimethylether (HGG-12) reduced the affinity of muscarinic agonists. No change was observed in the relative proportions of high (RH) and low (RL) affinity agonist binding sites. However, the oxime affected the processes of interconversion between these sites. Thus, unlike in control membranes, HGG-12 treated brain stem membranes, Gpp(NH)p could not induce conversion of RH to RL, and in cortical membranes Co2+ could not induce conversion of RL to RH. These results suggest that HGG-12 inactivates a component which is involved in both processes of induced-interconversion. Induced-interconversion between RH and RL was not affected in membranes treated with HGG-12 in the presence of carbamylcholine in concentrations at which mainly RH is occupied by the agonist. The occupation of RH by carbamylcholine protected both RH and RL from the effects of the oxime. The possible role of the molecular events involved is discussed.

Animals↗
PubMed1985

Neurotoxicity of dipiperidinoethane due to in vivo conversion to a selective cholinesterase inhibitor.

Dipiperidinoethane (DPE) administration produces seizures and CNS lesions. Here we elucidate the cholinergic origin of DPE toxicity. DPE is both an acetylcholinesterase (AChE) inhibitor and a muscarinic antagonist. This dual action negates most of the toxic effects of the compound in vivo. The neurotoxicity is believed to arise from oxidative conversion to DPE-N-oxide, which selectively inhibits AChE. Cytotoxicity does not involve muscarinic neurons, since binding parameters were unchanged following in vivo exposure.

Animals↗
PubMed1985

Interactions of quinidine and lidocaine with rat brain and heart muscarinic receptors.

We have studied the effect of quinidine and lidocaine on binding to rat brain and cardiac muscarinic receptors. Both drugs had a higher affinity to brain stem and cardiac receptors, as compared with cerebral cortex, coinciding with the distribution of high-affinity agonist binding sites in the above tissues. The effects of the drugs on muscarinic antagonist and agonist binding did not fit simple competition to one receptor site, suggesting either preferential binding to high affinity agonist binding sites, or allosteric interactions. Batrachotoxin, which opens voltage sensitive sodium channels, had an opposite effect on agonist binding. The possibility of allosteric interactions between the muscarinic receptors and a site analogous to the sodium channel is discussed.

Animals↗
PubMed1985

Reversible and irreversible inhibition of rat brain muscarinic receptors is related to different substitutions on bisquaternary pyridinium oximes.

The role of the functional substituents on the pyridinium ring of bisquaternary pyridinium compounds, mostly oximes, in exerting reversible and irreversible inhibition of binding of [3H]-N-methyl-4-piperidyl benzilate [( 3H]-4NMPB) to rat brain stem muscarinic receptors was studied. The drugs tested, i.e. HGG-42, HGG-12, HGG-52, HI-6, obidoxim, SAD-128 and TMB-4, could reversibly inhibit binding of [3H]-4NMPB, with the highest potency (KI = 1.7 - 6 microM) exhibited by analogs possessing hydrophobic substituents at position 3 or 4 of the pyridinium ring. Bisquaternary drugs possessing an oxime moiety at position 2, but not at position 4 of the pyridinium ring, could also induce about 30% reduction of maximal binding capacity (Bmax) (loss of muscarinic receptors) in addition to their reversible effect. Thus the structural correlates of the reversible and the irreversible effects of these drugs are different.

Animals↗
PubMed1985

Heterogeneity of solubilized muscarinic cholinergic receptors: binding and hydrodynamic properties.

Previous studies have described the conversion, after detergent solubilization, of the multiple populations of membrane-bound muscarinic agonist binding sites to a population of uniform affinity. This paper describes the solubilization of at least two receptor species, distinct in their agonist binding characteristics, which are capable of interconversion by transition metal ions. This finding enabled a more detailed examination of the molecular properties and regional differences of brain muscarinic receptors than was previously possible. Muscarinic receptors (mAChR) obtained from the rat cerebral cortex or medulla pons were solubilized using digitonin or the zwitterion detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps). The equilibrium binding of the antagonist [3H]-4-N-methylpiperidyl benzilate ([3H]4NMPB) to detergent-solubilized receptors resembled binding to neural membranes and exhibited subnanomolar affinity, saturability, and simple mass action kinetics. Agonist binding to soluble preparations was measured by competition of [3H]4NMPB binding sites. Saturation isotherms for agonist binding to digitonin- and Chaps-solubilized mAChR obtained from various brain regions appear flattened and have Hill coefficients in the range 0.52-0.78. Computerized modelling techniques indicate that the best fit to the experimental data is provided by a model specifying two soluble muscarinic agonist binding sites with differing dissociation constants, KH and KL, respectively. Solubilization of cerebral cortex membranes with Chaps or digitonin resulted in a population with a composition of high- and low-affinity sites similar to that found in the membrane-bound state. In contrast, solubilization of the medulla pons resulted in an approximately 40% loss of high-affinity sites. Solubilized receptors retained the sensitivity to transition metals ions, but were insensitive to guanine nucleotides. Density gradient centrifugation indicated that Chaps-solubilized mAChR are composed of two molecular forms with S20,W equal to 9.9 S and 14.9 S. The 14.9 S species comprises approximately 30% of the total binding activity in the cortex and approximately 40% in the medulla. We identify the 14.9 S species as being associated with a guanylnucleotide binding protein because treatment of medulla membranes with guanylylimidodiphosphate prior to solubilization results in disappearance of 14.9 S with 9.9 S unchanged. Sedimentation of cortical mAChR in the presence of Cu+2 leads to an increase in 14.9 S to almost 50% of the total binding activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Batrachotoxin changes the properties of the muscarinic receptor in rat brain and heart: possible interaction(s) between muscarinic receptors and sodium channels.

The effects of Na+-channel activator batrachotoxin (BTX) on the binding properties of muscarinic receptors in homogenates of rat brain and heart were studied. BTX enhanced the affinity for the binding of the agonists carbamoylcholine and acetylcholine to the muscarinic receptors in brainstem and ventricle, but not in the cerebral cortex. Analysis of the data according to a two-site model for agonist binding indicated that the effect of BTX was to increase the affinity of the agonists to the high-affinity site. Guanyl nucleotides, known to induce interconversion of high-affinity agonist binding sites to the low-affinity state, canceled the effect of BTX on carbamoylcholine and acetylcholine binding. BTX had no effect on the binding of the agonist oxotremorine or on the binding of the antagonist [3H]-N-methyl-4-piperidyl benzilate. The local anesthetics dibucaine and tetracaine antagonized the effect of BTX on the binding of muscarinic agonists at concentrations known to inhibit the activation of Na+ channels by BTX. On the basis of these findings, we propose that in specific tissues the muscarinic receptors may interact with the BTX binding site (Na+ channels).

Aconitine↗
PubMed1985

Effect of Ca2+ on the binding characteristics of muscarinic receptors in rat adenohypophysis--variation during the estrous cycle.

The effect of Ca2+ on the biochemical characteristics of muscarinic receptors in the adenohypophysis of male and female rats at the various stages of the estrous cycle was investigated in binding experiments using the specific muscarinic antagonist N-methyl-4-piperidyl benzylate ( [3H]-4NMPB) and the muscarinic agonist oxotremorine. By using Ca2+ chelators such as EGTA, and Ca2+ channel blockers such as D-600, we showed that Ca2+ profoundly alters the binding characteristics of both antagonists and agonists to the muscarinic receptors. In female rats the effect of Ca2+ on antagonist binding is mainly on the maximal binding capacity of the receptors, while changes in the dissociation constants are much more moderate. The effect is expressed in the ability of Ca2+ to expose or to eliminate binding sites as a function of the estrous cycle. In agonist binding, the presence of Ca2+ has a pronounced effect on the proportion of high-affinity binding sites, which parallels the changes induced in antagonist binding throughout the estrous cycle. Interestingly, the natural progression of the cycle from diestrus 2 to the estrous stage undergoes a change identical to that occurring in vitro upon Ca2+ removal. D-600 can completely block the effect of Ca2+ on the binding of both [3H]-4NMPB and oxotremorine. The concentration of D-600 required in order to induce such blocking is also dependent on the estrous cycle. It appears that the progression of the estrous cycle is accompanied by changes in the muscarinic receptors which may in turn be coupled to Ca2+ channels.

Animals↗
PubMed1985

Bisquaternary pyridinium oximes as allosteric inhibitors of rat brain muscarinic receptors.

The mode of interaction of bisquaternary pyridinium oximes with rat brain muscarinic receptors in cerebral cortex and brain stem preparations was studied by the use of the tritium-labeled antagonist N-methyl-4-piperidyl benzilate ( [3H] 4NMPB). Binding of the labeled muscarinic antagonist was inhibited by these drugs, the most potent inhibitors being 1-(2-hydroxyiminoethylpyridinium)-1-(3-cyclohexylcarboxypyridin ium)dimethyl-ether (HGG-42) and its 3-phenylcarboxypyridinium analog (HGG-12) (apparent KI = 1.3-1.7 and 1.8-2.2 microM, respectively). Analysis of the binding properties suggested that binding of the muscarinic antagonist and the bisquaternary pyridinium oximes was nonexclusive. Kinetic binding data provide evidence that the drugs inhibit binding of muscarinic antagonists in an allosteric manner, with a resulting decrease in the rates of both association of [3H]4NMPB to the receptor and its dissociation from it. These effects were observed both in brain stem and in cortical preparations even after pretreatment and washing out of the inhibitors. The selective natures of HGG-12 and HGG-42 were apparent from their irreversible effects on the number of muscarinic binding sites. In brain stem, the presence of these drugs resulted in a loss of about 30% of binding sites, which accounts in part for the apparent decrease in maximal binding capacity observed in the equilibrium binding of [3H]4NMPB. In the cortex, however, only approximately 10% of the muscarinic receptors were lost upon exposure to these drugs. The decrease in the muscarinic receptor population of the brain stem was dependent on both concentration and time and occurred both in vitro and in vivo following injection of HGG-12 into rats. Unlike the in vitro loss of receptor sites, which was irreversible, the in vivo effect was restored 2 hr after the injection. Taken together, the results suggest that the bisquaternary oximes are allosteric inhibitors of the muscarinic acetylcholine receptor and may be capable of distinguishing between receptor states and inducing specific irreversible effects. Because of these properties, the drugs may prove extremely useful as sensitive probes in studies on the nature of the agonist-receptor-effector relationship.

Animals↗
PubMed1985

Muscarinic receptor heterogeneity revealed by interaction with bretylium tosylate. Different ligand-receptor conformations versus different receptor subclasses.

The interaction of the antiarrhythmic drug, bretylium tosylate, with the muscarinic receptor in tissue homogenates from regions of rat brain and heart and from submandibular gland and ileal wall was investigated. Competition binding experiments were carried out using the highly specific tritiated antagonist N-methyl-4-piperidyl benzilate. Bretylium displayed heterogeneous binding characteristics. The binding of the drug to neural and glandular preparations was found to be best fitted by a one-site model in each case. On the other hand, in the case of muscle preparations (heart and ileum), a two-site model yielded a significantly better fit for the binding data than that given by a single site model. High affinity sites for the drug were detected in the muscle tissue only, with equilibrium binding constants of 0.24 +/- 0.12, 0.97 +/- 0.27, and 0.57 +/- 0.41 microM for the atrium, ventricle, and ileum, respectively. The low affinity binding constants in the muscle tissues were similar (approximately 10 microM) to those in the neural and glandular tissues examined, namely, the cortex, the hippocampus, the medulla pons, and the submandibular gland. The drug had no effect on agonist-binding characteristics. The heterogeneous binding of bretylium is compared to that of another nonclassical antagonist, pirenzepine. The results are discussed in relation to two alternative hypotheses put forward to account for antagonist heterogeneity in binding, the one involving ligand-receptor conformations and the other receptor subclasses.

Animals↗
PubMed1985

High affinity binding of [3H]acetylcholine to muscarinic receptors. Regional distribution and modulation by guanine nucleotides.

The interaction of [3H]acetylcholine ([3H]AcCh) with the muscarinic receptor was studied in seven distinct rat brain regions and in heart atrium by employing 10 microM atropine to define specific binding. The specific binding exhibited by the labeled neurotransmitter was found to be sensitive to muscarinic but not to nicotinic drugs. The muscarinic high affinity agonist-binding sites were characterized with respect to their binding properties, regional distribution, pharmacology, and modulation by guanyl nucleotides and by transition metal ions. In all tissues examined, specific binding of [3H]AcCh was saturable over the range of 4-200 nM and occurred in a receptor population that was apparently homogeneous and had a dissociation constant of approximately 19-39 nM in most of the regions. The ratio of muscarinic receptors labeled by [3H]AcCh to those labeled by the potent antagonist [3H]N-methyl-4-piperidylbenzilate varied markedly among tissues, from 0.15 in the hippocampus to 0.71 in the atrium. This ratio was lower in brain regions rich in muscarinic receptors, where smaller sensitivity of [3H]AcCh binding to guanyl nucleotides was also observed. In the presence of the latter [3H]AcCh binding was decreased by 25 to 90% in different tissues, with the greatest decreases occurring in the atrium and brainstem. In the latter preparations, transition metal ions do not affect [3H] AcCh binding, while in the other preparations studied they induce an increase in the binding capacity for the labeled neurotransmitter, which is sensitive to guanine nucleotides.

Acetylcholine↗
PubMed1984

Interactions between Ca2+-antagonist binding sites in rat adenohypophysis: dependence on estrous cycle.

The Ca2+ antagonist [3H]-nitrendipine [( 3H]-NDP) displayed high affinity binding in a saturable manner to a homogeneous population of sites when measured in rat adenohypophysis homogenates prepared from males or from females at the proestrous and estrous stages. Kd values were 0.7 +/- 0.1 nM, 0.75 +/- 0.08 nM and 1.1 +/- 0.1 nM, respectively. Maximal binding capacities (Bmax) were 11 +/- 1 fmole/mg protein for males and 20 +/- 1 fmole/mg protein for females at proestrus and 23 +/- 2 fmole/mg protein at estrus. In none of these preparations was the binding of [3H]-NDP dependent on the presence of Ca2+. The Ca2+ antagonist methoxy verapamil (also known as D-600), which belongs to a class of Ca2+-antagonists different from that of [3H]-NDP, could displace [3H]-NDP in a pattern suggesting possible allosteric interactions between the sites of these two antagonists. The displacement of [3H]-NDP by D-600 was affected by the presence of Ca2+ and varied with the estrous cycle. Our results suggest the existence of interactions between binding sites for NDP and for D-600. These interactions are affected by Ca2+, which might exert its effect through binding to a site of its own. In female adenohypophysis the interactions between these systems vary with the estrous cycle, suggesting that the coupling between them is modulated during this cycle.

Animals↗
PubMed1984

Copper ions and diamide induce a high affinity guanine-nucleotide-insensitive state for muscarinic agonists.

The binding capacity of [3H]-acetylcholine for muscarinic receptors of rat cerebral cortex membranes is increased in the presence of Cu2+ ions from 690 to 1320 fmol/mg protein with no significant change in affinity. Membranes treated with 50 microM Cu2+ and washed retain the increased binding capacity. Agonist binding in copper-treated membranes is insensitive to guanylylimidodiphosphate even at high concentrations (greater than 200 microM). Similar results were obtained when the sulfhydryl oxidizing agent, diamide (2 mM) was substituted for Cu2+ in the treatment of membranes. These data suggest the involvement of inter- or intra-molecular SH/S-S transitions in the interaction between the muscarinic receptor and a guanine nucleotide binding regulatory protein.

Animals↗
PubMed1984

Expression of muscarinic binding sites in primary human brain tumors.

The expression of muscarinic binding sites was examined in a collection of primary brain tumors of different cellular origins and various degrees of dedifferentiation, as compared to control specimens. Eleven gliogenous tumors were examined, all of which contained substantial amounts of muscarinic binding sites. Most of the other tumor types examined did not display detectable binding of [3H]N-methyl-4-piperidyl benzilate ([3H]4NMPB). Scatchard analysis indicated the existence of homogeneous antagonist sites in both normal forebrain and glioblastoma multiforme, with Kd values of 1.2 nM and 0.9 nM, respectively. The density of muscarinic binding sites varied between tumors from different patients, and also between specimens prelevated from different areas of the same tumor. This variability, as well as the average density of binding sites, appeared to be larger in highly malignant tumors than in less malignant ones. In contrast, the density of muscarinic receptors from control specimens was invariably high, but within the same order of magnitude. To test whether the muscarinic binding activity in the brain tumors is correlated to other cholinoceptive properties, cholinesterase activity was also examined. Individual data for density of [3H]4NMPB binding sites were then plotted against corresponding values of cholinesterase activity. The pattern of distribution of these values was clearly different in tumor specimens, when compared to that observed in samples derived from non-malignant brain. Our observations indicate that human brain cells of gliogenous origin are capable of expressing muscarinic binding sites, and that, if a correlation exists between muscarinic receptors and cholinesterase levels in gliogenous tumors, it differs from that of non-malignant brain tissue.

Adult↗
PubMed1984

Recognition of the muscarinic receptor by its endogenous neurotransmitter: binding of [3H]acetylcholine and its modulation by transition metal ions and guanine nucleotides.

Agonist binding to the muscarinic receptor in rat cerebral cortex membranes was studied by using the neurotransmitter itself, [3H]acetylcholine [( 3H]AcCho). By using 10 microM atropine or oxotremorine to define specific binding, it was possible to demonstrate specific binding of [3H]AcCho that was sensitive to muscarinic but not to nicotinic ligands. Equilibrium binding experiments with 5-240 nM [3H]AcCho indicated specific binding of the ligand to a saturable population of muscarinic receptors (361 +/- 29 fmol/mg of protein; Kd = 76 +/- 17 nM). This value represented 25% of the available binding sites for a labeled antagonist in the same preparation and corresponds to the proportion of high-affinity agonist binding sites observed previously in competition experiments with labeled antagonists. Inclusion of transition metal ions (e.g., 2 mM Ni2+) in the assay increased the equilibrium binding of [3H]AcCho (628 +/- 38 fmol/mg of protein, Kd = 86 +/- 21 nM) but did not affect equilibrium binding of 3H-labeled antagonists, indicating conversion of low- into high-affinity muscarinic agonist binding sites. The increase developed slowly over 30 min of incubation at 25 degrees C but could be reversed rapidly (approximately equal to 2 min) by the chelating agent EDTA or by guanine nucleotides. These data directly reveal a slow though quickly reversible interconversion of low- into high-affinity muscarinic agonist binding sites.

Acetylcholine↗
PubMed1984

Interaction of bretylium tosylate with rat cardiac muscarinic receptors. Possible pharmacological relevance to antiarrhythmic action.

The interaction of the antifibrillatory antiarrhythmic drug, bretylium tosylate, with the muscarinic receptor in tissue homogenates from regions of rat brain and heart was investigated. Competition-binding experiments were carried out with the highly specific tritiated antagonist N-methyl-4-piperidyl benzilate. Bretylium tosylate competitively displaced the labeled antagonist from the muscarinic receptor. The binding of the drug to the two brain preparations was found to be best fitted by a one-site model in each case. On the other hand, in the case of both heart preparations, a two-site model yielded a significantly better fit for the binding data than that given by a single-site model. The low affinity-binding constants in the atrium and the ventricle were similar (approximately 10 microM) to those in the brain regions examined, namely, the cortex and the medullapons. Sites with relatively higher affinity for the drug were detected in the heart only, with equilibrium-binding constants of 0.24 +/- 0.12 microM and 0.97 +/- 0.27 microM for the atrium and the ventricle, respectively. The drug also exerted anti-acetylcholine activity (K1 = 14 +/- 2 microM) measured physiologically in the guinea pig atrium, which correlated well with the concentration of the drug observed to be efficacious clinically (approximately 10 microM).

Animals↗
PubMed1984

Localization of phencyclidine binding sites on alpha and beta subunits of the nicotinic acetylcholine receptor from Torpedo ocellata electric organ using azido phencyclidine.

A photolabile derivative of phencycliding (PCP), azido phencyclidine (AZ-PCP), was synthesized and used to localize PCP binding sites on the acetylcholine receptor from Torpedo ocellata electric organ. In the dark, the binding of micromolar concentrations of [3H]AZ-PCP to a receptor-enriched membrane preparation fits a single dissociation constant (Kd = 2.65 microM) and is very similar to the binding of [3H]PCP. The agonist carbamylcholine increases the association rate (and the affinity) of these ligands to the receptor, but it does not alter the total number of available binding sites. Following UV irradiation and gel electrophoresis, [3H]AZ-PCP was found to label specifically the alpha and beta subunits of the receptor. The labeling of the alpha subunit band was heavier, and it was inhibited by tetracaine and PCP but not by alpha-bungarotoxin (alpha-Bgt). The addition of carbamylcholine enhanced the labeling of the beta subunit; this effect was diminished by alpha-Bgt. The labeling of the beta subunit was also inhibited by tetracaine and PCP. The effect of carbamylcholine, which binds to the alpha subunit, could be the result of an induced conformational change, which is propagated to the beta subunit and increases its labeling by [3H]AZ-PCP. A simple model which accommodates the binding and photoaffinity labeling data is described. According to the model, the high affinity PCP binding site is located between the alpha and beta receptor subunits, and the drug thus becomes attached simultaneously to both. Hypothetical overlapping recognition sites for PCP on these receptor subunits would allow binding (and labeling) with increased affinity in the presence of carbamylcholine with no increase in the number of available sites.

Affinity Labels↗