Pavel Michal

Publications (2)2.59 Total impact

• Article: Membrane cholesterol content influences binding properties of muscarinic M2 receptors and differentially impacts activation of second messenger pathways.

  Pavel Michal, Vladimír Rudajev, Esam E El-Fakahany, Vladimír Dolezal
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  ABSTRACT: We investigated the influence of membrane cholesterol content on preferential and non-preferential signaling through the M(2) muscarinic acetylcholine receptor expressed in CHO cells. Cholesterol depletion by 39% significantly decreased the affinity of M(2) receptors for [(3)H]-N-methylscopolamine ([(3)H]-NMS) binding and increased B(max) in intact cells and membranes. Membranes displayed two-affinity agonist binding sites for carbachol and cholesterol depletion doubled the fraction of high-affinity binding sites. In intact cells it also reduced the rate of agonist-induced receptor internalization and changed the profile of agonist binding from a single site to two affinity states. Cholesterol enrichment by 137% had no effects on carbachol E(max) of cAMP synthesis inhibition and on cAMP synthesis stimulation and inositolphosphates (IP) accumulation at higher agonist concentrations (non-preferred pathways). On the other hand, cholesterol depletion significantly increased E(max) of cAMP synthesis inhibition or stimulation without change in potency, and decreased E(max) of IP accumulation. Noteworthy, modifications of membrane cholesterol had no effect on membrane permeability, oxidative activity, protein content, or relative expression of G(s), G(i/o), and G(q/11) alpha subunits. These results demonstrate distinct changes of M(2) receptor signaling through both preferential and non-preferential G-proteins consequent to membrane cholesterol depletion that occur at the level of receptor/G-protein/effector protein interactions in the cell membrane. The significant decrease of IP accumulation by cholesterol depletion was also observed in cells expressing M(3) receptors and by both cholesterol depletion and enrichment in cells expressing M(1) receptors indicating relevance of reduced G(q/11) signaling for the pathogenesis of Alzheimer's disease.
  European journal of pharmacology 02/2009; 606(1-3):50-60. · 2.59 Impact Factor
• Article: Clozapine interaction with the M2 and M4 subtypes of muscarinic receptors

  Pavel Michal, Michaela Lysı́ková, Esam E El-Fakahany, Stanislav Tuček
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  ABSTRACT: Available evidence indicates that the antipsychotic drug clozapine acts as a partial agonist at the muscarinic M4 and as an antagonist at the M2 receptors. We wondered whether there is indeed a fundamental difference between its action on these two receptor subtypes, and whether it interacts with their classical or allosteric binding sites. In experiments on Chinese hamster ovary cells stably expressing the M2 or M4 receptors, clozapine inhibited the binding of the specific muscarinic ligand []N-methylscopolamine to either receptor subtype. The affinity of the high-affinity sites for clozapine was diminished by GTP in the way expected for agonists on both the M2 and the M4 receptor subtypes. Arunlakshana–Schild plots of data obtained in saturation binding experiments with []N-methylscopolamine at different concentrations of clozapine were linear with a slope of unity. Clozapine did not alter the time course of []N-methylscopolamine dissociation from muscarinic M2 or M4 receptors. It inhibited the synthesis of cyclic AMP in cells expressing the M4 receptor subtype, but did not measurably inhibit the synthesis of cyclic AMP in cells expressing the M2 receptor subtype. We conclude that clozapine has a high affinity for muscarinic M2 and M4 receptor subtypes, that it associates with the classical and not with the allosteric binding site, and that it acts as a partial agonist on both the M2 and the M4 receptor subtype.
  European Journal of Pharmacology.