Wash - resistantly bound xanomeline inhibits acetylcholine release by persistent activation of

We studied the effects of xanomeline (3-[3-hexyloxy-1,2,5-thiadiazo-4-yl]-1,2,5,6-tetrahydro1-methylpyridine) wash-resistant binding on presynaptic muscarinic regulation of electricallyevoked [H]acetylcholine (ACh) release from rat brain slices. In both cortical and striatal tissues that possess M2 and M4 autoreceptors, respectively, immediate application of 10 μM xanomeline had no effect on evoked [H]ACh release or its inhibition by 10 μM carbachol. In contrast, preincubation with 1, 10 or 100 μM xanomeline for 15 min decreased evoked release of ACh measured after 53 minutes of washing in xanomeline-free medium in a concentrationdependent manner. The maximal inhibitory effect equaled the immediate effect of the muscarinic full agonist carbachol, and was completely (at 1 and 10 μM xanomeline) or partially (at 100 μM xanomeline) blocked by 1 μM N-methylscopolamine. Neither presence of N-methylscopolamine during 100 μM xanomeline treatment nor previous irreversible inactivation of the classical receptor binding site using propylbenzylcholine mustard in cortical slices prevented the inhibitory effect of wash-resistantly bound xanomeline. Treatment of cortical slices with xanomeline slightly decreased the number of muscarinic binding sites and markedly decreased affinity for N-methylscopolamine. When applied as in acetylcholine release experiments xanomeline did not impair presynaptic α2 adrenoceptormediated regulation of noradrenaline release. The functional studies in brain tisse reported in this work demonstrate that xanomeline can function as a wash-resistant agonist of native presynaptic muscarinic M2 and M4 receptors with both competitive and allosteric components of action. This article has not been copyedited and formatted. The final version may differ from this version. JPET Fast Forward. Published on April 19, 2007 as DOI: 10.1124/jpet.107.122093 at A PE T Jornals on A ril 2, 2017 jpet.asjournals.org D ow nladed from