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S Tucek

Publications and source records attributed to S Tucek.

At least 37 records · Page 2Linked to original sources
PubMed1997

Positive cooperativity of acetylcholine and other agonists with allosteric ligands on muscarinic acetylcholine receptors.

It is well known that allosteric modulators of muscarinic acetylcholine receptors can both diminish and increase the affinity of receptors for their antagonists. We investigated whether the allosteric modulators can also increase the affinity of receptors for their agonists. Twelve agonists and five allosteric modulators were tested in experiments on membranes of CHO cells that had been stably transfected with genes for the M1-M4 receptor subtypes. Allosterically induced changes in the affinities for agonists were computed from changes in the ability of a fixed concentration of each agonist to compete with [3H]N-methylscopolamine for the binding to the receptors in the absence and the presence of varying concentrations of allosteric modulators. The effects of allosteric modulators varied greatly depending on the agonists and the subtypes of receptors. The affinity for acetylcholine was augmented by (-)-eburnamonine on the M2 and M4 receptors and by brucine on the M1 and M3 receptors. Brucine also enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pilocarpine, 3-(3-pentylthio-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1- methylpyridine (pentylthio-TZTP), oxotremorine-M, and McN-A-343 on the M1, M3, and M4 receptors, for pentylthio-TZTP on the M2 receptors, and for arecoline on the M3 receptors. (-)-Eburnamonine enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pentylthio-TZTP, pilocarpine, oxotremorine and oxotremorine-M on the M2 receptors and for pilocarpine on the M4 receptors. Vincamine, strychnine, and alcuronium displayed fewer positive allosteric interactions with the agonists, but each allosteric modulator displayed positive cooperativity with at least one agonist on at least one muscarinic receptor subtype. The highest degrees of positive cooperativity were observed between (-)-eburnamonine and pilocarpine and (-)-eburnamonine and oxotremorine-M on the M2 receptors (25- and 7-fold increases in affinity, respectively) and between brucine and pentylthio-TZTP on the M2 and brucine and carbachol on the M1 receptors (8-fold increases in affinity). The discovery that it is possible to increase the affinity of muscarinic receptors for their agonists by allosteric modulators offers a new way to subtype-specific pharmacological enhancement of transmission at cholinergic (muscarinic) synapses.

Acetylcholine↗
PubMed1996

Activation of muscarinic acetylcholine receptors via their allosteric binding sites.

Ligands that bind to the allosteric-binding sites on muscarinic acetylcholine receptors alter the conformation of the classical-binding sites of these receptors and either diminish or increase their affinity for muscarinic agonists and classical antagonists. It is not known whether the resulting conformational change also affects the interaction between the receptors and the G proteins. We have now found that the muscarinic receptor allosteric modulators alcuronium, gallamine, and strychnine (acting in the absence of an agonist) alter the synthesis of cAMP in Chinese hamster ovary (CHO) cells expressing the M2 or the M4 subtype of muscarinic receptors in the same direction as the agonist carbachol. In addition, most of their effects on the production of inositol phosphates in CHO cells expressing the M1 or the M3 muscarinic receptor subtypes are also similar to (although much weaker than) those of carbachol. The agonist-like effects of the allosteric modulators are not observed in CHO cells that have not been transfected with the gene for any of the subtypes of muscarinic receptors. The effects of alcuronium on the formation of cAMP and inositol phosphates are not prevented by the classical muscarinic antagonist quinuclidinyl benzilate. These observations demonstrate for the first time that the G protein-mediated functional responses of muscarinic receptors can be evoked not only from their classical, but also from their allosteric, binding sites. This represents a new mechanism of receptor activation.

Alcuronium↗
PubMed1996

Positive allosteric action of eburnamonine on cardiac muscarinic acetylcholine receptors.

It was discovered recently that alcuronium and strychnine (which is a precursor of alcuronium) allosterically increase the affinity of cardiac muscarinic receptors for the antagonist, N-methylscopolamine. We have now investigated the effects of l-eburnamonine and vincamine, which are both closely related to strychnine. In experiments on rat heart atria, l-eburnamonine was found to increase the binding of [3H]N-methylscopolamine with Ehlert's cooperativity coefficient alpha = 0.35, which indicates that the strength of its allosteric action is close to that of alcuronium and strychnine (alpha = 0.31 and 0.44, respectively). However, the affinity of l-eburnamonine for the cardiac muscarinic receptors is lower than the affinities of alcuronium and strychnine (KAR = 22.6 microM, 0.15 microM, and 3.4 microM, respectively). In spite of its extremely close similarity to l-eburnamonine, vincamine has a negative allosteric effect on the binding of [3H]N-methylscopolamine (alpha = 4.1; KAR = 22.8 microM). It is likely that a systematic investigation of the allosteric effects of the analogues of strychnine will not only yield new allosteric effectors on muscarinic receptors, but also clarify the structural features responsible for the direction (positive or negative) of their allosteric effect.

Alcuronium↗
PubMed1996

Biphasic changes in the density of muscarinic and beta-adrenergic receptors in cardiac atria of rats treated with diisopropylfluorophosphate.

Chronic treatment with organophosphate inhibitors of cholinesterases is known to bring about down-regulation of muscarinic acetylcholine receptors in the heart while its effect on the functionally antagonistic beta-adrenergic receptors is not known. We describe experiments in which rats were exposed to daily injections of diisopropylflurophosphate (DFP) and the density of muscarinic and beta-adrenergic binding sites in their cardiac atria was measured according to the binding of (3H)quinuclidinyl benzilate ((3H)QNB) and (-)-4-(3-tert-butylamino-2-hydroxy)-propoxy-(5,7-3H) benzimidazol-2-one ((3H)CGP 12177) as subtype non-specific muscarinic and beta-adrenergic ligands, respectively. Biphasic course of changes was discovered with both ligands. With the dosage scheme applied, the density of beta-adrenoceptors was augmented 24 h after the first dose of DFP and decreased to about one half of control values after 5 days of treatment with DFP. The density of muscarinic receptors was augmented after two days of treatment with DFP and decreased to about one half of control values after 5 days of treatment. Comparatively small changes in the heart rate were observed during the treatment, they reflected changes in the muscarinic and beta-adrenergic receptor density. The finding of DFP-induced changes in the binding of (3H)CGP 12177 suggests that the mechanisms responsible for the control of the density of muscarinic and beta-adrenergic receptors in the heart cells are interconnected but potential roles of other factors involved in in vivo experiments deserve further analysis. The transient increase in the density of muscarinic receptors after two days of DFP treatment appears related to published data on transient stimulation by cholinergic agonists of the transcription of mRNAs for muscarinic receptors.

Animals↗
PubMed1995

Constitutive activity of the M1-M4 subtypes of muscarinic receptors in transfected CHO cells and of muscarinic receptors in the heart cells revealed by negative antagonists.

We investigated whether muscarinic receptors of the M1-M4 receptor subtypes are constitutively active. We have found that the synthesis of cyclic AMP was enhanced by the muscarinic antagonists atropine and N-methylscopolamine (NMS) in Chinese hamster ovary (CHO) cells stably transfected with human m2 and m4 muscarinic receptor genes and in rat cardiomyocytes expressing the M2 receptor subtype, and that the production of inositol phosphates was inhibited by atropine and NMS in CHO cells stably transfected with human m1 and m3 and with rat m1 muscarinic receptor genes. The muscarinic antagonists quinuclidinyl benzilate and AF-DX 116 had no effect in some cases and acted like atropine and NMS in others. We conclude that the M1-M4 subtypes of muscarinic receptors are constitutively active in the CHO cell lines expressing them and in cardiomyocytes and that atropine and NMS act as negative antagonists on these receptor subtypes by stabilizing them in the inactive conformation.

Animals↗
PubMed1995

Positive and negative allosteric interactions on muscarinic receptors.

It was previously found that alcuronium increases the binding of [3H]methyl-N-scopolamine to cardiac muscarinic receptors by a positive allosteric action while its effect on the binding of [3H]quinuclidinyl benzilate is negative. The, features of the antagonist's molecule which decide whether its allosteric interaction with alcuronium is positive or negative are not known. In the present work, it was found that alcuronium has a positive allosteric effect also on the binding of [3H]atropine and [3H]methyl-N-piperidinyl benzilate to muscarinic receptors in rat heart atria and that its effect on the binding of [3H]methyl-N-quinuclidinyl benzilate is negative. A comparison of the five radiolabelled antagonists that have been investigated so far indicates that the type of allosteric interaction (positive or negative) is not determined by the presence or absence of the quaternary nitrogen or of the benzilyl moiety in the molecule of the antagonist. Apparently, features of the N-bearing moiety of muscarinic antagonists other than the presence of a charge on nitrogen play a key role in the determination of the type of interaction.

Alcuronium↗
PubMed1995

Positive allosteric interactions on cardiac muscarinic receptors: effects of chemical modifications of disulphide and carboxyl groups.

Changes in the allosteric effects of alcuronium on rat cardiac muscarinic receptors were investigated after chemical modifications of S-S bonds or free carboxyl groups. In membranes pretreated with dithiothreitol, alcuronium lost its positive action on the binding of [3H]methyl-N-scopolamine while its inhibitory effect on radioligand dissociation was preserved. In membranes pretreated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), known to modify free carboxyl groups in proteins, the ability to bind [3H]methyl-N-scopolamine was preserved if the pretreatment had been performed in the presence of alcuronium, methyl-N-scopolamine or carbachol, while the positive cooperative effect of alcuronium on [3H]methyl-N-scopolamine binding was only preserved in membranes that had been exposed to EDC in the presence of alcuronium. Methyl-N-scopolamine, carbachol and alcuronium differed in their ability to protect (against EDC) the action of alcuronium on the rate of [3H]methyl-N-scopolamine dissociation. The results suggest that the disulphide bridge connecting the first two extracellular loops of muscarinic receptors is important for the positive allosteric action of alcuronium and that three carboxyl groups (presumably aspartate residues) are involved in receptor interactions with alcuronium and methyl-N-scopolamine. The first group is important for the effect of alcuronium on the affinity for methyl-N-scopolamine, the second is critical for the effect of alcuronium on the rate of methyl-N-scopolamine dissociation, and the third is critical for methyl-N-scopolamine binding. Presumably, the two charged nitrogens of alcuronium associate with the first and the second of the three groups involved.

Alcuronium↗
PubMed1995

Allosteric modulation of muscarinic acetylcholine receptors.

Five subtypes of muscarinic acetylcholine receptors have been identified in mammalian tissues, but the selectivity of ligands that are active at these receptors is low. It is possible, however, that selective compounds may be developed by targeting their allosteric site(s). Important new insights into the mechanism of allosteric control of muscarinic receptors have been obtained recently in investigations of the allosteric effects of neuromuscular blockers, and competition between ligands for the allosteric binding site has now been demonstrated. It is now apparent that the binding site for most allosteric ligands is close to the binding site for acetylcholine but that it is located at a more extracellular position. Stanislav Tucek and Jan Proska discuss the pharmacological implications of ligand interaction at these two sites and the therapeutic possibilities.

Allosteric Regulation↗
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↗