General information
The work of the Department concentrates on functions of cholinergic neurons and on cholinergic synaptic transmission. It proceeds in collaboration with laboratories within the country (Charles University First Medical Faculty Department of Physiology and Central Isotope Laboratory) and abroad (University of Minnesota Medical School in Minneapolis, CNRS Laboratoire de Neurobiologie Cellulaire et Moléculaire at Gif-sur-Yvette, University of Tokyo Institute of Brain Research, University of Freiburg Department of Pharmacology, and other laboratories).

Main results
Much effort has been devoted to investigations of the allosteric regulation of muscarinic acetylcholine receptors. We have discovered earlier that the affinity of muscarinic receptors for their antagonist N-methylscopolamine may be allosterically enhanced by the neuromuscular blocker alcuronium, and that the allosteric binding site for alcuronium and related allosteric modulators is probably located close to but more extracellularly than the binding site for classical ligands. Recent findings include the following.

a. The positive allosteric action of alcuronium is subtype-selective  with positive effects occurring at the M₂ (and weakly at the M₄) muscarinic receptor subtype, and negative effects occurring at the M₁, M₃ and M₅ muscarinic receptor subtypes. Two carboxyl groups in the molecule of the receptor are probably important for the binding and the allosteric effect of alcuronium. The positive effect of alcuronium on the binding of N-methylscopolamine can also be revealed on solubilized muscarinic receptors

b. Alcuronium is able to enhance the affinity of the M₂ muscarinic receptors not only to N-methylscopolamine, but also to atropine and N-methylpiperidinylbenzilate, although not (from among the antagonists tested) to quinuclidinyl benzilate or N-methylquinuclidinyl benzilate. Strychnine and eburnamonine (compounds structurally similar to alcuronium) also act as positive allosteric modulators. Alcuronium, strychnine and gallamine (a much investigated negative allosteric modulator of muscarinic receptors) compete for the same binding domain on muscarinic M₂ receptors. Structural features of the classical and the allosteric ligands which determine the direction (positive or negative) of the cooperative interaction between a given couple of ligands remain unknown. Two esters of truxillic acid are extremely potent negative allosteric modulators of the M₂ subtype of muscarinic receptors.

c. Allosteric modulators can enhance the affinity of muscarinic receptors not only toward muscarinic antagonists, but also towards muscarinic agonists. In an investigation of interactions between four allosteric modulators and twelve muscarinic agonists, the affinity for each agonist tested could be enhanced by at least one modulator on at least one muscarinic receptor subtype. The positive allosteric interaction between an allosteric modulator and a classical muscarinic ligand could be demonstrated not only in experiments with radioligand binding, but also in functional experiments measuring the effect of presynaptic receptors on the release of acetylcholine.

d. The conformational change which the allosteric modulators induce in muscarinic receptors concerns not only the binding domain for the classical ligands, but also the domain responsible for the interaction between the receptors and the G proteins. This was revealed by the finding that the allosteric modulators applied to CHO cells (stably transfected with defined subtypes of muscarinic receptors) in the absence of muscarinic agonists induced weak agonist-like effects which could not be blocked by quinuclidinyl benzilate (a strong classical antagonist). Apparently, muscarinic receptors can be activated from domains outside their classical binding site. Such activation could be reproduced on liposomes incorporating purified receptors and purified G protein, but it was not fully identical with the activation induced by a classical agonist.

Working with muscarinic receptors in genetically engineered CHO cells, we discovered (simultaneously with several other laboratories) that the receptors display a strong constitutive activity and that atropine acts as a negative antagonist (inverse agonist), while quinuclidinyl benzilate is a neutral antagonist. Evidence has been obtained suggesting that the classical binding site of muscarinic receptors is composed of two subsites with a tandem arrangement. A comparison of the effects which various subtypes of muscarinic receptors have on the intracellular concentrations of Ca²⁺ ions revealed that, in genetically modified CHO cells, muscarinic receptors of the M₁, M₃ and M₅ subtypes bring about substantial increases of [Ca²⁺]_i , while the effects of the M₂ and M₄ receptors are negligible.

Much effort has been spent on investigations of the control of neurotransmitter release by presynaptic neurotransmitter receptors and of calcium channels involved in the release. Evidence has been obtained (in collaboration with the Department of Pharmacology of the University of Freiburg, Germany) indicating that that presynaptic (2adrenoceptors on the nerve terminals of sympathetic neurons inhibit the release of norepinephrine from the nerve terminals by a selective action on the N-type calcium channels. The activation of presynaptic nicotinic neurons on the nerve terminals of sympathetic neurons brings about an increase in the concentration of Ca²⁺ ions within the nerve terminals. It also leads to an increase in the release of noradrenaline from the terminals, and this effect appears to be caused by the influx of Ca²⁺ ions through the open channels of the activated nicotinic receptors.

Work performed in collaboration with the Department of Pharmacology of the University of Mainz (Germany) has shown that the release of acetylcholine from a motor nerve is also enhanced by presynaptic nicotinic receptors, and that these receptors can be blocked with d-tubocurarine, but not with a-bungarotoxin and related snake toxins. It could be demonstrated that presynaptic muscarinic receptors can strongly inhibit the release of acetylcholine from the cholinergic nerve terminals in human bronchi, and that treatment with inhaled steroids has no substantial influence on the biochemical parameters of the cholinergic innervation of the bronchi in human patients.

It is well known that the release of acetylcholine from striatal cholinergic neurons may be inhibited by presynaptic muscarinic receptors on the terminals of these neurons. We have found that the stimulation-evoked release of acetylcholine from the striatum depends on the function of the N- and P/Q-types of calcium channels (but not on the L-type channels), and that the muscarinic inhibition of the release depends on muscarinic modulation of the N- and (to a lesser degree) the P/Q-types of calcium channels. The inhibitory effects of muscarinic agonists furmethide, oxotremorine-M and bethanechol could be potentiated by brucine, a compound known to act as an allosteric modulator of muscarinic receptors.

We found earlier that the evoked release of acetylcholine from cholinergic nerve terminals can be inhibited by the compound tacrine, known as an inhibitor of cholinesterases. An analysis of the effects of tacrine, performed with the Fura-2 fluorescence method of calcium imaging in the cholinergic SN56 neuronal cell line, revealed that tacrine inhibits the N-type calcium channels and that this effect of tacrine is independent of its inhibitory action on cholinesterases. This finding is important for the evaluation of the therapeutic potential of tacrine. An investigation of the antipsychotic drug clozapine has shown that clozapine has a high affinity for muscarinic receptors and that it acts as a partial agonist at the M₄ and M₂ subtypes of muscarinic receptors.

The effects of muscarinic receptors on the function of the heart are opposite to those of b-adrenergic receptors, and the results of our experiments indicate that these two types of receptors mutually influence also their density in the heart. In experiments in vivo, inhibition of cholinesterases brought about biphasic changes (increases followed by decreases) in the densitiy of both muscarinic receptors and ß-adrenoceptors in the heart. In experiments on cardiomyocytes in culture, muscarinic agonists brought about biphasic changes in the density of ß-adrenoceptors and ß-adrenergic agonists affected the density of muscarinic receptors, although in a less conspicuous manner.

Current work
Current work concerns the following main topics: (a) allosteric regulation of muscarinic receptors and interactions between muscarinic receptors and G proteins; (b) regulation of the development of cholinergic properties of cholinergic neurons and functional mechanisms of presynaptic receptors; (c) functions of cholinergic receptors in the heart and control of their expression; (d) function of cholinergic neurons in Alzheimer´s disease; (e) antibodies against nicotinic receptors in patients with myasthenia gravis.

Publications