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S Tucek

Publications and source records attributed to S Tucek.

At least 37 records · Page 2Linked to original sources
PubMed1995

Competition between positive and negative allosteric effectors on muscarinic receptors.

Alcuronium allosterically increases the affinity of cardiac muscarinic receptors for methyl-N-scopolamine (NMS), whereas gallamine has the opposite effect. We discovered that strychnine also increases the affinity of muscarinic receptors in rat heart atria for NMS. It is not known whether the positive and the negative allosteric effectors bind to the same binding site. To investigate this question, we elaborated on a theoretical model predicting changes in the binding of a classic radiolabeled ligand occurring in the presence of a positive and a negative allosteric effector that compete for the allosteric binding site. The model is based on data obtained at equilibrium and avoids uncertainties associated with the use of nonequilibrium methods for the evaluation of interactions between allosteric ligands. We examined changes in the binding of [3H]NMS to membranes of rat heart atria exposed to various concentrations of a positive allosteric effector (alcuronium or strychnine) and of a negative allosteric effector (gallamine) simultaneously. The binding data obtained were in perfect agreement with the model assuming competition between gallamine and alcuronium and gallamine and strychnine, strongly suggesting that these positive and negative allosteric effectors bind to identical or overlapping sites.

Alcuronium↗
PubMed1995

Subtype selectivity of the positive allosteric action of alcuronium at cloned M1-M5 muscarinic acetylcholine receptors.

The neuromuscular blocking drug alcuronium was found earlier to increase the affinity of muscarinic receptors for methyl-N-scopolamine (NMS). This effect could be observed in some but not in other tissues. Subtype selectivity of the positive allosteric action of alcuronium was now investigated in radioligand binding experiments in Chinese hamster ovary (CHO) cells stably transfected with the genes encoding the M1-M5 subtypes of muscarinic receptors. Alcuronium had a particularly high affinity for the M2 receptor subtype (Kd = 0.6 microM) and its affinity for muscarinic receptor subtypes diminished in the order M2 > M4 = M3 > M1 > M5. Alcuronium allosterically increased the binding of (3H)NMS to membranes containing receptors of the M2 (cooperativity factor alpha = 0.38) and M4 subtypes (alpha = 0.72) and inhibited the binding of (3H)NMS to membranes containing receptors of the M1, M3 and M5 subtypes (alpha = 3.35-4.35). The positive effects of alcuronium could also be observed in experiments with (3H)NMS binding to intact whole cells, indicating that the positive allosteric action of alcuronium occurs by alcuronium binding to receptor domains that are accessible from the extracellular space. Alcuronium diminished the affinity for (3H)quinuclidinyl benzilate [(3H)QNB] at all five subtypes of muscarinic receptors and slowed down the dissociation of both (3H)NMS and (3H)QNB; its decelerating effect on radioligand dissociation was most pronounced at the M2 receptor subtype. Differences between the effects of alcuronium on individual muscarinic receptor subtypes are apparently responsible for differences between the allosteric effects of alcuronium on muscarinic receptors in various tissues that had been described previously.

Alcuronium↗
PubMed1994

Protection by alcuronium of muscarinic receptors against chemical inactivation and location of the allosteric binding site for alcuronium.

We have found earlier that the neuromuscular blocker alcuronium binds to cardiac muscarinic receptors simultaneously with their specific antagonist [3H]methyl-N-scopolamine ([3H]NMS) and allosterically increases their affinity to this ligand. Nothing is known about the allosteric site with which alcuronium interacts. To gain an insight, we have now investigated how the binding of [3H]NMS is affected by agents known to modify specific residues in proteins and how their effects are altered by alcuronium. Reagents that covalently modify the tyrosyl residues (p-nitrobenzenesulfonyl fluoride and 4-chloro-7-nitrobenzofurazan) and the carboxyl groups of aspartate and glutamate [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N'-dicyclohexylcarbodiimide, and N-ethyl-5-phenylisoxazolium-3'-sulfonate] blocked the binding of [3H]NMS to receptors in rat heart atria. Their action was probably due to the modification of tyrosyl and aspartyl residues directly in the muscarinic binding sites because it was antagonized by atropine and carbamoylcholine. Alcuronium and gallamine, another allosteric ligand, also protected the [3H]NMS binding sites against the inactivation by tyrosine- and carboxyl-directed chemical modifiers just as well as by benzilylcholine mustard, known to attach covalently to the muscarinic binding sites. Protection by alcuronium has also been observed on cerebrocortical muscarinic receptors. The effect of alcuronium indicates that the drug interferes with the access of chemical modifiers to the muscarinic sites. In view of the unspecific nature of most of the modifiers used (with regard to muscarinic mechanisms), the protection by alcuronium appears to be best explained on the assumption that the drug binds in close vicinity of the "classical" muscarinic site and sterically blocks the access to this site.

Acetylcholine↗
PubMed1994

Two populations of muscarinic binding sites in the chick heart distinguished by affinities for ligands and selective inactivation.

1. By measuring the binding of N-[3H-methyl]-scopolamine ([3H]-NMS) and of unlabelled subtype-specific muscarinic antagonists, two populations of muscarinic binding sites can be distinguished in the membranes of cardiac ventricles taken from 1-day-old chicks. One of them, corresponding to approximately 80% of [3H]-NMS binding sites, has higher affinities for AF-DX116 (pKi = 6.42) and methoctramine (pKi = 7.33); the rate of [3H]NMS dissociation from these sites is fast. The other population, corresponding to approximately 20% of [3H]-NMS binding sites, has lower affinities for AF-DX116 (pKi = 5.00) and methoctramine (pKi = 6.19); the rate of [3H]-NMS dissociation from these sites is slow. Both populations have high affinities for pirenzepine, but the affinity of the former (major) population is lower (pKi = 7.99) than that of the latter (minor) population (pKi = 10.14). 2. Since it has been shown earlier that two mRNAs for muscarinic receptors are expressed in the chick heart, one of them close to the genetically defined m2 and the other to the m4 subtype, we propose that the major population of binding sites with high affinities for AF-DX116 and methoctramine and the lower affinity for pirenzepine represents the M2-like receptors, while the minor population represents the M4-like receptors. 3. It proved possible to obtain isolated samples of either population by selectively protecting the M2-like sites with AF-DX116 and the M4-like sites with pirenzepine, and by inactivating the unprotected sites with benzilylcholine mustard. The properties of the isolated populations corresponded to those derived from the analysis of [3H]-NMS binding to the original mixed population.4 Alcuronium exerted positive allosteric action on the binding of [3H]-NMS both to the M2-like and the M4-like population and severely slowed down [3H]-NMS dissociation from them; its affinity for the M2-like sites was 3-10 times higher.

Alcuronium↗
PubMed1994

Mechanisms of steric and cooperative actions of alcuronium on cardiac muscarinic acetylcholine receptors.

Kinetics of the interactions between the neuromuscular blocker alcuronium, the specific muscarinic antagonist N-[methyl-3H] methyl scopolamine ([3H]NMS), and muscarinic receptors were investigated in homogenates of rat heart atria. Two effects of alcuronium on the binding of [3H]NMS could be distinguished. (a) Alcuronium concentration-dependently slowed the association of [3H]NMS with receptors and the dissociation of [3H]NMS from receptors so that, at high alcuronium concentrations, equilibrium binding could not be reached, even after 20 hr, without special precautions. (b) Alcuronium increased the affinity of receptors for [3H]NMS, which was manifested by a decrease of the apparent Kd (> 3-fold) with no change in the Bmax for [3H]NMS binding. The effects of alcuronium on the rates of [3H]NMS association and dissociation can be explained only by a reaction mechanism in which [3H]NMS binds only to free receptors (not occupied by alcuronium), whereas alcuronium binds both to free receptors and to receptors occupied by [3H]NMS. Similarly, [3H]NMS cannot dissociate from receptors as long as alcuronium is attached to them. Experimental data agree with corresponding mathematical models. It is proposed that alcuronium blocks entry to the pocket containing the [3H]NMS binding site. In addition to this blocking effect, alcuronium has a positive allosteric effect on [3H]NMS binding, presumably by inducing a conformational change of the orthosteric muscarinic binding site. Earlier observations suggesting that, at high concentrations, alcuronium also competes for [3H]NMS binding sites can be explained by insufficient equilibration of the system.

Alcuronium↗
PubMed1993

Presynaptic muscarinic receptors and the release of acetylcholine from cerebrocortical prisms: roles of Ca2+ and K+ concentrations.

The mechanism by which presynaptic muscarinic autoreceptors inhibit the release of acetylcholine (ACh) from cerebrocortical cholinergic fibres has not been clarified. To test the view that muscarinic autoreceptors act by decreasing Ca2+ influx, we performed experiments in which rat cerebrocortical prisms were preloaded with (14C)choline, washed, depolarized with 14-65 mM K+ in the absence of Ca2+ and then exposed (still under depolarization) to various concentrations of Ca2+ to evoke the release of (14C)ACh. The muscarinic agonist, oxotremorine, used at a 100 microM concentration, inhibited the release of (14C)ACh by 59-86% in experiments with 14 and 26.5 mM K+ but had no significant effect at 65.5 mM K+. No systematic changes in the inhibitory effects of oxotremorine could be found at any of the K+ concentrations used when the concentration of Ca2+ was varied in the range of 0.25-4.0 mM. At 2 mM Ca2+ and K+ concentrations above 14 mM, the inhibitory effect of oxotremorine was inversely related to the concentration of K+. The inhibitory effect of oxotremorine on (14C)ACh release was not blocked by 100 microM 4-amino-pyridine. The fact that the inhibitory effect of oxotremorine could not be overcome by an increase in the concentration of Ca2+ suggests that, under the conditions used, a restriction of the influx of Ca2+ did not play a major role in the muscarinic inhibition of ACh release; rather, oxotremorine appeared to act by decreasing membrane depolarization.2+ of the Ca(2+)-voltage hypothesis of neurotransmitter release, supposing

Acetylcholine↗
PubMed1992

Acetylcarnitine, carnitine and glucose diminish the effect of muscarinic antagonist quinuclidinyl benzilate on striatal acetylcholine content.

The content of acetylcholine (ACh) in the striatum, brain cortex and hippocampus of rats was lowered 20-180 min after intraperitoneal injection of the muscarinic antagonist quinuclidinyl benzilate (QNB). The depletion of ACh content in the striatum was diminished in animals treated with a single dose of acetyl-L-carnitine, L- or D,L-carnitine, or D-glucose. It is likely that QNB stimulates ACh release by blocking presynaptic muscarinic autoreceptors and that acetylcarnitine, carnitine and glucose support the resynthesis of ACh by increasing the availability of acetylcoenzyme A. They do not have the same consistent effect in the brain cortex and hippocampus; this difference may be related to the lower turnover rate of ACh and to the difference in the anatomical arrangement of cholinergic structures in these parts of the brain.

Acetylcarnitine↗
PubMed1992

Quest for agonist and antagonist selectivity at muscarinic receptors in guinea-pig smooth muscles and cardiac atria.

Potencies of 11 muscarinic agonists in eliciting contraction of smooth muscle in guinea-pig ileum, trachea, urinary bladder and uterus and in inhibiting the rate of contractions of cardiac atria were compared. While acetylcholine (ACh) was the most potent agonist on the ileum, uterus and cardiac atria, cis-L(+)-dioxolane was equally as potent as ACh on the ileum and more potent on the urinary bladder and trachea. Compared to ACh, methylfurmethide, oxotremorine, acetoxybut-2-inyl-trimethylammonium and cis-L(+)-dioxolane acted weakly on the atria. Aceclidine, arecoline and acetyl-beta-methylcholine displayed selectivity for the urinary bladder and pilocarpine for the tracheal and urinary bladder smooth muscles. Oxotremorine had very low activity on the uterus. The stereoselectivity of muscarinic ACh receptors (mAChRs) for cis-L(+)-and cis-D(-)-dioxolane was low in the urinary bladder and uterus and high in the ileum and trachea. Most antagonists showed little selectivity between different organs, but S(-)-phenylcyclohexylglycoloyl choline was 6 times more active on the urinary bladder than on the ileum and AF-DX 116 was 12-30 times more active on the atria than on the smooth muscles. Among the N-alkyl derivatives of benzilylcholine, the octyl derivative as 400 times more active on the ileum than on the atria, while among the N-alkyl derivatives of QNB, the N-decyl derivative was 41 times more active on the ileum. The observed differences in the potency of various agonists and their stereoisomers on different smooth muscles cannot be explained by differences in the accessibility of receptors or in receptor reserve.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Investigation of the mechanism of the effect of tacrine (tetrahydroaminoacridine) on the metabolism of acetylcholine and choline in brain cortical prisms.

The mechanism by which tacrine increases the content and synthesis of acetylcholine (ACh) in cerebrocortical prisms exposed to an irreversible inhibitor of cholinesterases and incubated under resting conditions (Dolezal and Tucek, 1991) is not known. As found in the present experiments, this effect of tacrine is only apparent if its application had been preceded by a period of preincubation, but the preincubation is ineffective if it occurs in the presence of hemicholinium-3. Apparently, choline or a choline-containing compound accumulates in the slices during the preincubation and is then utilized for the enhanced synthesis of ACh in the presence of tacrine. Tacrine did not induce a decrease in the amount of radiolabel that had been incorporated from choline into acid-insoluble compounds, which suggests that the choline which is used for the synthesis of additional ACh does not originate from choline lipids. However, tacrine was found to diminish the efflux of choline from prisms which had been preincubated with an increased concentration of choline in the medium, and from prisms incubated in the presence of hemicholinium-3. It also diminished the efflux of radioactive choline that had accumulated in the prisms during preincubation with a very low concentration of tacrine, when the prisms were subsequently incubated with 4-aminopyridine. It is proposed that the potency of tacrine to increase the content and synthesis of ACh in cerebrocortical prisms whose cholinesterases had been inhibited is due to its ability to diminish the efflux of endogenous choline from the nerve terminals.

4-Aminopyridine↗
PubMed1992

Sensitive method for HPLC determination of acetylcholine, choline and their analogues using fluorometric detection.

A method is described for reversed-phase HPLC separation of acetylcholine and choline and of their homologues in tissue extracts or perfusion fluids, combined with postcolumn enzymatic derivatization and fluorometric quantification. The separation occurs on a polymeric resin derivatized with hydrophobic moiety and the mobile phase consists of Na2HPO4, 3-(p-hydroxyphenyl)propionic acid and sodium dodecylsulphate; postcolumn enzyme reactor contains immobilized acetylcholinesterase, choline oxidase, and peroxidase. The limits of detection are 1 pmol choline and 3 pmol acetylcholine per sample. The method is free of interferences encountered with electrochemical detection and well suited for non-attended automatic operation.

Acetylcholine↗
PubMed1992

Effects of tetrodotoxin, Ca2+ absence, d-tubocurarine and vesamicol on spontaneous acetylcholine release from rat muscle.

1. Rat hemidiaphragms were incubated in a physiological low-K+ medium without stimulation and the amount of acetylcholine (ACh) released was measured radioenzymatically. Cholinesterases were inhibited by paraoxon. 2. In the presence of 1 microM tetrodotoxin (TTX), the amount of ACh released during a 2 h incubation was lowered by 40%. A similar decrease was observed in the absence of Ca2+ and in the presence of 10 microM-d-tubocurarine (dTC). The effects of TTX combined with Ca2+ removal, and of TTX combined with dTC were no greater than those of TTX, dTC or Ca2+ removal alone. TTX and dTC had no effect on the release of ACh from diaphragms 4 days after denervation. 3. The reduction of spontaneous ACh release observed in the presence of TTX or dTC or in the absence of Ca2+ is best interpreted on the assumption that about 40% of the ACh release was due to the impulse activity known to be generated in intramuscular motor nerve branches by the ACh which accumulates after the inhibition of cholinesterases. 4. In the presence of 1 and 10 microM vesamicol (AH5183, 2-(4-phenylpiperidino)-cyclohexanol), the release of ACh was also diminished by approximately 40%. Vesamicol did not augment the inhibition of release produced by TTX or by the omission of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1992

[The effect increased choline levels on the synthesis and release of acetylcholine in heart atria in white rats].

The paper addresses the problem of (1) the relationship of choline in extracellular fluid and acetylcholine (ACh) synthesis in chambers of the heart of white rats, and (2) the possibility of physiological involvement of this ACh in the control of cardiac activity. Within 60 min after s.c. administration of choline in the dose of 300 mg/kg body weight the ACh content rose to 136% of the control value in isolated heart chambers and after administration of 400 mg/kg body weight to 159% of the control value. Increased ACh synthesis failed to affect the heart rate, nor did it increase the tonic effect of vagus innervation of the heart. Excessive ACh could however be released by electric transmural stimulation of isolated heart chambers as well as by potassium depolarization. The release depended on the presence of calcium ions. The obtained results demonstrate that an increase in the level of choline in extracellular fluid results in increased ACh synthesis in nerve terminals. Enhanced release of this ACh is a precondition for high choline levels to induce an increase in parasympathetic control of cardiac function.

Acetylcholine↗
PubMed1991

The binding of cholinesterase inhibitors tacrine (tetrahydroaminoacridine) and 7-methoxytacrine to muscarinic acetylcholine receptors in rat brain in the presence of eserine.

Cholinesterase inhibitor tacrine (1,2,3,4-tetrahydro-9-aminoacridine) is known to interfere with the binding of specific ligands to muscarinic receptors with unusually steep binding inhibition curves. We investigated whether the concentration dependence of the inhibition of binding is associated with the inhibitory effect of tacrine on the activity of cholinesterases, and compared the effect of tacrine with that of 7-methoxytacrine. Tacrine was found to inhibit the specific binding of [3H]quinuclidinyl benzilate (QNB) in rat brain cortex with IC50 values of 11 microM both in the absence and in the presence of 100 microM eserine, which had been added to ensure complete inhibition of cholinesterases at all concentrations of tacrine; in the cerebellum, the IC50 value was 10 microM in the absence and 14 microM in the presence of eserine; Hill slope factors (nH) were in the range of 1.55-1.79 and were not significantly affected by the presence of eserine. 7-Methoxytacrine inhibited the binding of [3H]QNB with an IC50 value of 2.3 microM in the cortex and of 2.6 microM in the cerebellum. The results indicate that the degree and the steep course of the inhibition of [3H]QNB binding to M1 and M2 muscarinic receptors by tacrine do not depend on its inhibitory effect on cholinesterases, and that 7-methoxytacrine is likely to interfere with the function of muscarinic receptors 4-5 times more strongly than tacrine.

Animals↗
PubMed1991

Positive and negative effects of tacrine (tetrahydroaminoacridine) and methoxytacrine on the metabolism of acetylcholine in brain cortical prisms incubated under "resting" conditions.

The effects of tacrine (1,2,3,4-tetrahydro-9-aminoacridine) and 7-methoxytacrine on the metabolism of acetylcholine were investigated in experiments on prisms of rat cerebral cortex incubated in vitro in low-potassium (3 mmol/L K+) media; cholinesterases were inactivated by paraoxon to avoid any action of tacrine and methoxytacrine via their inhibition. Under "resting" conditions, tacrine and methoxytacrine increased the synthesis of unlabeled acetylcholine in the prisms; at the same time, they inhibited the uptake of [14C]choline from the medium and the synthesis of [14C]acetylcholine. The concentration of free choline was not increased by tacrine or methoxytacrine in either the tissue or the medium. The contradiction between the increased synthesis of unlabeled and the diminished synthesis of labeled acetylcholine indicates that the utilization of intracellular choline (which is presumably mobilized from intracellular choline esters) for the synthesis of acetylcholine is increased by tacrine and methoxytacrine. This conclusion is supported by the observation that the inhibition of acetylcholine synthesis during incubation with hemicholinium-3 (an inhibitor of choline transport into cholinergic nerve terminals) was overcome when tacrine was present simultaneously with hemicholinium-3. When the prisms were preincubated with [14C]choline and incubated with tacrine or methoxytacrine only after this, the amount of [14C]acetylcholine recovered in the tissue plus the medium was higher at the end of incubation with tacrine or methoxytacrine than without them, again suggesting that the drugs were able to increase the utilization of intracellular [14C]choline or its esters for acetylcholine synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Aminopyridine↗
PubMed1991

Negative effects of tacrine (tetrahydroaminoacridine) and methoxytacrine on the metabolism of acetylcholine in brain slices incubated under conditions stimulating neurotransmitter release.

The effects of tacrine (1,2,3,4-tetrahydro-9-aminoacridine) and 7-methoxytacrine on the metabolism of brain acetylcholine were investigated in experiments in which acetylcholine turnover was stimulated by tissue depolarization or by 4-aminopyridine. Rat cerebrocortical prisms were preincubated under "resting" conditions (Krebs-Ringer buffer with 3 mmol/L K+ and with paraoxon to inhibit cholinesterases) and then incubated in the presence of tacrine or methoxytacrine and of 50 mmol/L K+. Both drugs diminished the amount of acetylcholine released by depolarization and the amount of acetylcholine synthesized during incubation; in experiments in which [14C]choline was present in the incubation medium simultaneously with tacrine or methoxytacrine, the drugs diminished the uptake of [14C]choline by the tissue and the amount of [14C]-acetylcholine synthesized and released into the medium. In these experiments, it was not possible to distinguish whether the inhibitory effects of tacrine and methoxytacrine were primarily on the process of acetylcholine synthesis (particularly on the uptake of choline), or whether the drugs also acted directly on the process of neurotransmitter release. In subsequent experiments the prisms were preincubated with [14C]choline and only then subjected to a short depolarization in the presence of hemicholinium-3 and tacrine or methoxytacrine. Both drugs severely inhibited the release of preformed [14C]acetylcholine and prevented the diminution of tissue [14C]acetylcholine stores. Methoxytacrine was also found to diminish the release of acetylcholine induced by 4-aminopyridine while increasing the content of acetylcholine in the tissue. Tacrine and methoxytacrine had no effect on the activity of choline acetyltransferase (EC 2.3.1.6).(ABSTRACT TRUNCATED AT 250 WORDS)

4-Aminopyridine↗