Muscarinic acetylcholine receptors (mAChRs) (nomenclature as agreed by the NC-IUPHAR Subcommittee on Muscarinic Acetylcholine Receptors [50]) are activated by the endogenous agonist acetylcholine. All five (M1-M5) mAChRs are ubiquitously expressed in the human body and are therefore attractive targets for many disorders. Functionally, M1, M3, and M5 mAChRs preferentially couple to Gq/11 proteins, whilst M2 and M4 mAChRs predominantly couple to Gi/o proteins. Both agonists and antagonists of mAChRs are clinically approved drugs, including pilocarpine for the treatment of elevated intra-ocular pressure and glaucoma, and atropine for the treatment of bradycardia and poisoning by muscarinic agents such as organophosphates. I
Abstract Die Umsetzung des Xanthins (I) mit H 2 O bei Raumtemperatur liefert bei pH 5 Harnsäure (II) (12% Ausbeute), deren Ausbeute mit der Reaktionsdauer zunimmt; die Reaktion verläuft zugleich unter hydrolytischer Umkehrung zu 3‐Hydroxy‐xanthin (III) sowie unter Reduktion zu Xanthin (IVa).
1. Membranes from rat cerebral cortex, myocardium and extraorbital lacrimal gland were used as sources of M1, M2 and M3 muscarinic acetylcholine receptors respectively and the affinities of seven antagonists for the three subtypes were examined under different experimental conditions. 2. The affinities for the membrane-bound receptors were measured at different ionic strengths and temperatures and compared with those determined on the receptor solubilised in the neutral detergent digitonin or the zwitterionic detergent, CHAPSO. 3. The range of measured affinity constants of a given antagonist for a specific subtype varied from 2 (atropine at M1 receptors) to 1000 (AF-DX 116 at M2 receptors). 4. As a consequence of these changes in affinity, which were dependent on the drug, the subtype and the experimental conditions, both the structure-binding relationships of a given subtype can be markedly changed as well as the selectivity of a drug for the different subtypes. For example it is possible to change the relative affinities of AF-DX 116 and gallamine at membrane-bound M1 receptors from 50:1 to 1:60. 5. Experimental conditions for the observation of high selectivity of pirenzepine, AF-DX 116, gallamine and hexahydrosiladiphenidol for the three subtypes are given. 6. When the receptors are removed from their membrane environment by solubilisation in detergent, antagonist affinities are changed but the subtypes still retain different structure-binding relationships. 7. In general, AF-DX 116 and the allosteric antagonist, gallamine, behave differently from the other antagonists, suggesting that they bind in different ways to muscarinic receptors. Careful attention should therefore be paid to the experimental conditions in binding assays used to assess the affinities and selectivities of new muscarinic antagonists in order to avoid misleading results. 9. The ability to produce enhanced or attenuated affinities and selectivities of antagonists, resulting from the induction of different conformations of the receptor by a variety of physical, chemical or molecular biological perturbations may lead to a better understanding of the structural basis of drug receptor interactions.
[3H]Telenzepine has been shown to bind with high affinity (3 x 10(9) M-1) to a subpopulation of muscarinic binding sites in rat cerebral cortex, which have a high affinity for pirenzepine. The binding kinetics were very slow at 30 degrees. Only 50% of the [3H] telenzepine was found to be capable of binding to the receptors with high affinity. This suggested the presence of optical isomers of telenzepine. These were partially resolved on the picomole scale by using cortical muscarinic receptors to selectively bind the active isomer. It was then possible to measure the temperature and time dependence of the racemization of the inactive to the active enantiomer. The energy barrier for the inversion was 35 kcal/mol, and racemization was very slow even at 90 degrees. The affinity and selectivity of the unlabeled enantiomers for the different muscarinic receptor subtypes present on membranes from rat cerebral cortex, heart, and lacrimal gland was measured. The selectivity of active (+)-isomer was considerably greater than that of the (-)-isomer. As a consequence, the stereoselectivity of the enantiomers varied from 500 (M1 receptors in cerebral cortex) to 75 (cardiac receptors).
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