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M Sokolovsky

Publications and source records attributed to M Sokolovsky.

At least 91 records · Page 5Linked to original sources
PubMed1988

Kinetics of choline uptake into isolated rat forebrain microvessels: evidence of endocrine modulation.

The active uptake of [methyl-3H]choline into isolated rat brain microvessel suspension was studied as a likely guide to the transport of choline across the blood-brain barrier. The method consisted primarily of incubation of the suspension with a fixed concentration of labeled choline in the presence of increasing concentrations of unlabeled choline or any other inhibitor (I) of active uptake, defined as the difference in uptake at 37 degrees and 0 degrees C. From the linear regression of (1/V) against [I], the following values of Vmax (nmol g-1 min-1) and Km (microM) were obtained for choline: 2-month-old males, 10.6 +/- 3.8 and 6.1 +/- 0.9; 3-month old random females, 28.4 +/- 5.9 and 12.6 +/- 4.0; females at metaestrus, 17.8 +/- 10.3 and 8.3 +/- 5.0; at diestrus, 31.1 +/- 9.3 and 13.0 +/- 2.6; at proestrus, 54.9 +/- 2.2 and 14.0 +/- 1.5; at estrus, 19.2 +/- 2.2 and 2.6 +/- 1.7. The differences between males and random females (p less than 0.018) and between females at proestrus and estrus (p less than 0.005) are significant. It is suggested that these inter- and intrasex variations in choline uptake reflect a dynamic adjustment of supply in accordance with brain demand for choline at the time of assay. Hemicholinium-3 was an effective inhibitor of choline uptake, Ki = 14.0 +/- 8.5 microM; dimethylaminoethanol was much less effective; and imipramine had no measurable effect.

Animals↗
PubMed1988

Solubilization of rat brain phencyclidine receptors in an active binding form that is sensitive to N-methyl-D-aspartate receptor ligands.

Phencyclidine (PCP) receptors were successfully solubilized from rat forebrain membranes with 1% sodium cholate. Approximately 58% of the initial protein and 20-30% of the high-affinity PCP binding sites were solubilized. The high affinity toward PCP-like drugs, the stereo-selectivity of the sites, and the sensitivity to N-methyl-D-aspartate (NMDA) receptor ligands were preserved. Binding of the potent PCP receptor ligand N-[3H][1-(2-thienyl)cyclohexyl] piperidine ([3H]TCP) to the soluble receptors was saturable (KD = 35 nM), and PCP-like drugs inhibited [3H]TCP binding in a rank order of potency close to that observed for the membrane-bound receptors; the most potent inhibitors were TCP (Ki = 31 nM) and the anticonvulsant MK-801 (Ki = 50 nM). The NMDA receptor antagonist 2-amino-5-phosphonovaleric acid inhibited binding of [3H]TCP to the soluble receptors; glutamate or NMDA diminished this inhibition in a dose-dependent manner. Taken together, the results indicate that the soluble PCP receptor preparation contains the glutamate recognition sites and may represent a single receptor complex for PCP and NMDA, as suggested by electrophysiological data. The successful solubilization of the PCP receptors in an active binding form should now facilitate their purification.

2-Amino-5-phosphonovalerate↗
PubMed1987

Binding studies and photoaffinity labeling identify two classes of phencyclidine receptors in rat brain.

Binding and photoaffinity labeling experiments were employed in order to differentiate 1-(1-phenylcyclohexyl)piperidine (PCP) receptor sites in rat brain. Two classes of PCP receptors were characterized and localized: one class binds [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) with high affinity (Kd = 10-15 nM) and the other binds the ligand with a relatively low affinity (Kd = 80-100 nM). The two classes of sites have different patterns of distribution. Forebrain regions are characterized by high-affinity sites (hippocampus greater than frontal cortex greater than thalamus greater than olfactory bulb greater than hypothalamus), but some parts (e.g., hippocampus, hypothalamus) contain low-affinity sites as well. In the cerebellum only low-affinity sites were detected. Binding sites for [3H]PCP and for its photolabile analogue [3H]azido-PCP showed a regional distribution similar to that of the [3H]TCP sites. The neuroleptic drug haloperidol did not block binding to either the high- or the low-affinity [3H]TCP sites, whereas Ca2+ inhibited binding to both. Photoaffinity labeling of the PCP receptors with [3H]AZ-PCP indicated that five specifically labeled polypeptides of these receptors (Mr 90,000, 62,000, 49,000, 40,000, and 33,000) are unevenly distributed in the rat brain. Two of the stereoselectively labeled polypeptides (Mr 90,000 and 33,000) appear to be associated with the high- and low-affinity [3H]TCP-binding sites; the density of the Mr 90,000 polypeptide in various brain regions correlates well with the localization of the high-affinity sites, whereas the density of the Mr 33,000 polypeptide correlates best with the distribution of the low-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1987

Localization of azidophencyclidine-binding site on the nicotinic acetylcholine receptor alpha-subunit.

Nicotinic acetylcholine receptors in receptor-rich membranes from Torpedo californica and from T. marmorata electric tissue were photolabeled with the non-competitive inhibitor [3H]azidophencyclidine. The receptor subunits were separated on SDS-polyacrylamide gels and the alpha-subunits recovered from the gel, were subjected to Staphylococcus aureus V8 protease cleavage. The proteolytic fragments were resolved by SDS-polyacrylamide gel electrophoresis and were identified on protein blots by 125I-labeled alpha-bungarotoxin binding and by staining with concanavalin A. The site of specific azidophencyclidine labeling has been localized to the V8-18 kDa fragment which binds toxin. Labeling of the V8-18 kDa fragment was observed in the absence and in the presence of carbamylcholine. This was found for both the species of Torpedo used here.

Affinity Labels↗
PubMed1987

Multiple mode of binding of phencyclidines: high affinity association between phencyclidine receptors in rat brain and a monovalent ion-sensitive polypeptide.

Two populations of phencyclidine (PCP) binding sites are shown to exist in the rat brain: a high-affinity monovalent ion-sensitive site (Kd of 10-14 nM for [3H]TCP, [3H]N-[1-(2-thienyl)cyclohexyl]piperidine), which exists in both the frontal cortex and the hippocampus, and a lower affinity site (Kd of 80-130 nM for [3H]TCP) which is found in the hippocampus but not in the frontal cortex. The nature of the interactions between the ion-binding sites and the high affinity PCP receptors depend on both ligand structure (PCP or TCP) and the ion involved (K' or Na'). The high-affinity sites are associated with an Mr 90,000 polypeptide whose labeling by [3H]azido phencyclidine is selectively inhibited by monovalent ions.

Animals↗
PubMed1987

Dual pathways in muscarinic receptor stimulation of phosphoinositide hydrolysis.

The relationships between phosphoinositide hydrolysis induced by various muscarinic agonists and by membrane depolarization agents were investigated in rat cerebral cortex and heart atrium slices. In both preparations, phosphoinositide hydrolysis was stimulated by a combination of carbamylcholine and membrane depolarization with 40 mM K+ in a synergistic fashion. The synergism was more pronounced at lower external calcium ion concentrations and was sensitive to verapamil. Lower external calcium ion concentrations were required for demonstration of the synergism in heart atrium slices than in cerebral cortex slices. The carbamylcholine-induced stimulation was only partially additive with membrane depolarization via Na+ channel gating by batrachotoxin. In addition, K+ depolarization eliminated the sensitivity of carbamylcholine-stimulated phosphoinositide hydrolysis to the sodium channel blocker tetrodotoxin. Our results suggest that muscarinically stimulated phosphoinositide hydrolysis in rat cerebral cortex and heart atrium slices may occur by dual pathways which interact synergistically and that only one of the pathways is depolarization-dependent. Different muscarinic agonists could preferentially utilize these pathways, thus perhaps explaining their different potencies in stimulating phosphoinositide hydrolysis.

Animals↗
PubMed1986

Role of lipids in age-related changes in the properties of muscarinic receptors in cultured rat heart myocytes.

The effects of the culture's age and of liposome treatments on the properties of muscarinic receptors in cultured rat heart myocytes prepared from the hearts of newborn (1-3 days old) rats were investigated. In these studies we investigated the binding characteristics of antagonists and agonists to the myocyte muscarinic receptors in young (5 days after plating) vs. older (14 days after plating) cultures. Our findings demonstrate that the aging of the cells in culture is accompanied by a reduction in the muscarinic binding capacity and by alterations in the proportion of high- and low-affinity states toward muscarinic agonists, as well as by striking changes in the mode of coupling of the receptors with guanine nucleotide binding protein(s) [G protein(s)]. The above effects of the culture's age occur concomitantly with alterations in the lipid composition of the cultured myocytes (in 14-day old cultures, the phosphatidylcholine/sphingomyelin ratio is reduced, and the cholesterol level is elevated). In order to explore whether the lipid composition is involved in the mechanism that alters the properties and coupling of the muscarinic receptors, we treated aging cultures with liposomes containing egg phosphatidylcholine. This treatment resulted in 14 day old cultures with a lipid composition similar to that of young cultures, and the treated myocytes demonstrated muscarinic receptor properties similar to those of young myocyte cultures. The implications for the role of membrane lipid composition and organization in determining the properties of the muscarinic receptors and their coupling with G proteins are discussed.

Acetylcholine↗
PubMed1986

Interactions between the muscarinic receptors, sodium channels, and guanine nucleotide-binding protein(s) in rat atria.

The effects of the voltage-sensitive sodium channel activator batrachotoxin (BTX) on the binding properties of muscarinic receptors were studied in homogenates of rat atria. Also studied were the effects of muscarinic ligands on the binding of tritium-labeled batrachotoxin ([3H]BTX) to the same preparation. BTX (1 microM), which induces an open state in sodium channels, enhanced the affinity of binding of several agonists to the muscarinic receptors. Analysis of the data indicated that the effect of BTX was to increase the affinity of the agonists toward the high-affinity sites. Binding of antagonists was not affected by BTX. At higher concentrations of toxin, the density of the high affinity muscarinic sites was also affected. The binding of agonists (but not of antagonists) to muscarinic receptors in turn enhanced the specific binding of [3H]BTX to sodium channels. These effects on the muscarinic receptors and on the sodium channels were inhibited in the presence of Gpp(NH)p at concentrations lower than those bringing about conversion of binding sites from the high affinity to the low affinity conformation. On the basis of these findings we suggest that the opening of sodium channels and the binding of agonists to muscarinic receptors in rat atrial membranes are coupled events which are mediated by guanine nucleotide-binding protein(s). Such a hypothesis is consistent with previously proposed models for signal transduction in the membrane.

Acetylcholine↗
PubMed1986

Acetylcholine rhythm in the preoptic area of the rat hypothalamus is synchronized with the estrous cycle.

Acetylcholine levels in the preoptic area of rat hypothalamus were found to change rhythmically, in synchronization with the estrous cycle. During the proestrous critical period, these levels demonstrated a drop followed by a sharp rise. The absence of this phenomena in males indicates sexual dimorphism. The close association between this newly detected hypothalamic acetylcholine rhythm and the estrous cycle suggests the possible involvement of acetylcholine in regulation of the estrous cycle.

Acetylcholine↗
PubMed1986

Identification of polypeptides of the phencyclidine receptor of rat hippocampus by photoaffinity labeling with [3H]azidophencyclidine.

Polypeptide components of the phencyclidine (PCP) receptor present in rat hippocampus were identified with the photolabile derivative of phencyclidine [3H]azidophencyclidine ( [3H]AZ-PCP). The labeled affinity probe was shown to reversibly bind to specific sites in the dark. The number of receptor sites bound is equal to those labeled by [3H]PCP, and their pharmacology and stereospecificity are identical with those of the PCP/sigma-opiate receptors. The dissociation constant of [3H]AZ-PCP from these receptors is 0.25 +/- 0.08 microM. Photolysis of hippocampus membranes preequilibrated with [3H]AZ-PCP, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed the existence of five major labeled bands of which a Mr 90 000 band and a Mr 33 000 band were heavily labeled. Inhibition experiments, in which membranes were incubated with [3H]AZ-PCP in the presence of various PCP analogues and opiates, indicate that labeling of both the Mr 90 000 band and the Mr 33 000 band is sensitive to relatively low concentrations (10 microM) of potent PCP/sigma receptor ligands, while similar concentrations of levoxadrol, naloxone, morphine, D-Ala-D-Leu-enkephalin, atropine, propranolol, and serotonin were all ineffective. Stereoselective inhibition of labeling of the Mr 90 000 band and of the Mr 33 000 band was also observed by the use of dexoxadrol and levoxadrol. The Mr 33 000 band was not as sensitive as the Mr 90 000 band to inhibition by the selective PCP receptor ligands N-[1-(2-thienyl)cyclohexyl]piperidine and PCP.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1986

Divergent regulation of muscarinic binding sites and acetylcholinesterase in discrete regions of the developing human fetal brain.

The expression of muscarinic acetylcholine binding sites and of cholinesterases was studied in extracts prepared from discrete regions of the human fetal brain, between the gestational ages of 14 and 24 weeks. The specific binding of [3H]N-methyl-4-piperidyl benzilate [( 4H]-4NMPB) to muscarinic binding sites ranged between 0.05 and 1.30 pmol/mg protein in the different brain regions, with Kd values of 1.2 +/- 0.2 nM. Binding of the cholinergic agonist oxotremorine fitted, in most of the brain regions examined, with a two-site model for the muscarinic binding sites. The density of muscarinic binding sites increased with development in most regions, with different rates and onset times. It was higher by about sixfold in some areas destined to become cholinergic, such as the cortex and midbrain, than in noncholinergic areas such as the cerebellum. In other areas destined to become cholinergic, such as the hippocampus and the caudate putamen, the receptor density remained low. Average density values increased from 0.1 +/- 0.1 at 14 weeks up to 0.7 +/- 0.4 pmol/mg protein at 24 weeks. The variability in the specific activities of cholinesterase was relatively low, and extracts from different brain regions hydrolyzed from 5 to 30 nmol of [3H]acetylcholine/min/mg protein. These were mostly "true" acetylcholinesterase (EC 3.1.1.7) activities, inhibited by 10(-5) M BW284C51, with minor pseudocholinesterase (EC 3.1.1.8) activities, inhibited by 10(-5) M iso-OMPA. The enzyme from different brain regions and developmental stages displayed similar Km values toward [3H]acetylcholine (ca. 4 X 10(-4) M-1). The ontogenetic changes in cholinesterase specific activities had no unifying pattern and/or relationship to the cholinergic nature of the various brain areas. In most of the brain regions, the arbitrary ratio between the specific activity of cholinesterase and the density of muscarinic binding sites decreased with development, with average values and variability ranges of 83 +/- 50 and 19 +/- 19 at 14 and 24 weeks, respectively. Our findings suggest divergent regulation for cholinergic binding sites and cholinesterase in the fetal human brain and imply that the expression of muscarinic receptors is related to the development of cholinergic transmission, while acetylcholinesterase is also involved in other functions in the fetal human brain.

Acetylcholine↗
PubMed1986

Bisquaternary pyridinium oximes as presynaptic agonists and postsynaptic antagonists of muscarinic receptors.

A study of the effects of bisquaternary pyridinium oximes on calcium-dependent potassium-evoked [3H]acetylcholine release from rat brain slices revealed that at presynaptic autoreceptors these drugs function like muscarinic agonists, as they mimic the effects of acetylcholine in their inhibition of the evoked [3H]-acetylcholine release in an atropine-sensitive and dose-dependent manner. Since the bisquaternary pyridinium oximes are mild muscarinic antagonists at postsynaptic muscarinic receptors, they constitute a category of muscarinic ligands that are characterized by inverse dual activity at pre- and postsynaptic muscarinic receptors. These drugs may have dual function on cholinergic transmission by acting as presynaptic agonists and as postsynaptic antagonists. The most potent inhibitor of the evoked [3H]acetylcholine release was 1,1'-(4-hydroxyiminopyridinium)trimethylene (TMB-4) (I50 = 8 microM) and the weakest were 1-(2-hydroxyiminoethylpyridinium) 1-(3-cyclohexylcarboxypyridinium) dimethylether (HGG-42) and 1-(2-hydroxyiminoethylpyridinium) 1-(3-phenylcarboxypyridinium) dimethylether (HGG-12) (I50 = 150 microM). As postsynaptic antagonists, the latter drugs are more potent (K1 = 1.3-3.3 microM) than TMB-4 (K1 = 50 microM). Combined therapy with two drugs such as TMB-4 and HGG-12 might be effective in blocking severe hyperactivity of the cholinergic system.

Acetylcholine↗
PubMed1985

Increased agonist affinity is induced in tetranitromethane-modified muscarinic receptors.

Tetranitromethane (TNM) modifies the muscarinic receptors from rat cerebral cortex. The modified receptor possesses an increased binding affinity (6-9-fold) toward several agonists such as acetylcholine, carbamoylcholine, arecoline, etc. The binding of antagonists (Bmax and Kd) is only slightly altered. The effects of TNM treatment can be prevented by atropine, thus indicating that TNM modifies residue(s) at the binding site. We carried out a series of successive chemical modifications which indicated that the modified residue(s) is (are) most probably a tyrosyl and not a cysteinyl residue. This conclusion gains support from the pH profile of agonist binding, which suggests the involvement of a residue with an apparent pK comparable to that of the phenolic hydroxyl of a nitrotyrosyl residue. The binding properties of the modified receptor, when compared to those of the native one, clearly indicate that the response to TNM modification with respect to the binding of agonists such as acetylcholine and carbamoylcholine is different from that when oxotremorine and its analogue are employed. This is interpreted as being the result of different binding modes exhibited by the various agonists. Nitration of the receptors can be prevented by the presence of an antagonist but not by an agonist. We propose that this differential response is due to the formation of ligand-receptor complexes that differ with respect to the microenvironment of the modified tyrosyl residue.

Acetylcholine↗
PubMed1985

Regional heterogeneity of rat brain phencyclidine (PCP) receptors revealed by photoaffinity labeling with [3H] azido phencyclidine.

Photoaffinity labeling of rat brain phencyclidine (PCP) receptors with [3H] azido phencyclidine ([3H]AZ-PCP) reveals the existence of five polypeptides which are specifically labeled by the affinity probe (Mr's 90,000, 62,000, 49,000, 40,000 and 33,000). These labeled components are unevenly distributed in rat brain. In the frontal cortex, thalamus and olfactory bulb, the major bands labeled are the Mr's 90 K and 62 K polypeptides; in the cerebellum most of the labeling is in the 90 K and 33 K bands; and in the hippocampus all but the Mr 40 K band are heavily labeled. Together with dexoxadrol/[3H]PCP competition binding data, which indicated the existence of high and low affinity dexoxadrol/PCP binding sites, these results suggest regional heterogeneity of PCP receptors. The regional distribution of the high affinity dexoxadrol binding sites correlates best with that of the Mr 90 K polypeptide.

Affinity Labels↗
PubMed1985

Rat brain and heart muscarinic receptors: modification with tetranitromethane.

Tetranitromethane at a concentration of 50 microM modifies the muscarinic receptors in membrane preparations from rat striatum, hippocampus and heart atrium, but not from the rat brain stem. While the binding of antagonists is only slightly altered, the modified receptor possesses an increased affinity of up to 8-fold for [3H]-acetylcholine binding to the high affinity state. This effect is absent if the nitration is carried out in the presence of an antagonist, but not in the presence of an agonist. The affinity for carbamylcholine is increased for both the high and the low affinity state of the receptor, as is evident from its ability to compete with a labeled antagonist. In addition, the proportion of binding sites (alpha) exhibiting the high affinity state for [3H]-acetylcholine or for carbamylcholine is increased upon nitration. This increase cannot be protected against by an antagonist, and is enhanced when nitration takes place in the presence of an agonist. With the agonists oxotremorine and [3H]-oxotremorine-M only the latter effect (i.e., increase in alpha) is observed following nitration, while their dissociation constants for the receptor are unchanged. Data are discussed with respect to the proposed existence of subtypes of muscarinic receptors, as well as the importance of the agonist chosen for studies of ligand-receptor interactions.

Acetylcholine↗
PubMed1985

Analysis of ligand binding to receptors by competition kinetics. Application to muscarinic antagonists in rat brain cortex.

We propose a method for obtaining kinetic parameters of unlabeled ligands which is based on analyzing the effect of their competition on the binding kinetics of a labeled ligand. In this method (competition kinetics), the binding kinetics of a labeled ligand are measured in the absence and in the presence of given concentrations of a competing unlabeled ligand. The rate equations appropriate to the particular kinetic model may be solved employing linear homogeneous differential equations. The rate constants appearing in the final integrated expressions can be evaluated by nonlinear regression. We have demonstrated the validity and applicability of the method to study receptor systems, using the muscarinic receptors in rat brain cortex as an experimental system. We have determined the various kinetic parameters of two muscarinic antagonists (N-methyl-4-[3H]piperidyl benzilate and (-)-N-[3H]methylscopolamine) by following directly their binding kinetics. These parameters were compared with those obtained for the same unlabeled ligands in homogeneous and heterogeneous competition studies. In all cases, the parameters obtained by the competition kinetics method were close to those extracted from direct binding kinetics, and in cases where both types of studies were performed on the same preparations, the parameters obtained by the two methods were essentially identical.

Animals↗