Abstract
Xanomeline is a unique agonist of muscarinic receptors that possesses functional selectivity at the M1 and M4 receptor subtypes. It also exhibits wash-resistant binding to and activation of the receptor. In the present work we investigated the consequences of this type of binding of xanomeline on the binding characteristics and function of the M1 muscarinic receptor. Pretreatment of CHO cells that stably express the M1 receptor for 1 hr with increasing concentrations of xanomeline followed by washing and waiting for an additional 23 hr in control culture media transformed xanomeline-induced inhibition of [3H]NMS binding from monophasic to biphasic. The high-affinity xanomeline binding site exhibited three orders of magnitude higher affinity than in the case of xanomeline added directly to the binding assay medium containing control cells. These effects were associated with a marked decrease in maximal radioligand binding and attenuation of agonist-induced increase in PI hydrolysis and were qualitatively similar to those caused by continuous incubation of cells with xanomeline for 24 hr. Attenuation of agonist-induced PI hydrolysis by persistently-bound xanomeline developed with a time course that parallels the return of receptor activation by prebound xanomeline towards basal levels. Additional data indicated that blockade of the receptor orthosteric site or the use of a non-functional receptor mutant reversed the long-term effects of xanomeline, but not its persistent binding at an allosteric site. Furthermore, the long-term effects of xanomeline on the receptor are mainly due to receptor down-regulation rather than internalization.
Highlights
There are five subtypes of muscarinic acetylcholine receptors that vary in their distribution and function
The specific binding of 0.2 nM [3H]NMS to the human M1 muscarinic acetylcholine receptor (hM1) muscarinic receptor expressed in intact Chinese hamster ovary (CHO) cells was measured in the continuous presence of xanomeline in naıve cells, or following various xanomeline pretreatment and washing conditions
We have shown that pretreatment with xanomeline for 24 h or 1 h followed by washing and waiting for 23 h resulted in an increase in the EC50 of carbachol-mediated PI hydrolysis (Fig. 4A)
Summary
There are five subtypes of muscarinic acetylcholine receptors that vary in their distribution and function. Of the five cloned subtypes of the muscarinic receptor, the M1 subtype is vital for processes involved in learning and memory. Memory deficits such as those seen in Alzheimer’s disease are currently treated with acetylcholinesterase inhibitors. This pharmacological approach is endowed with serious untoward effects due to activation of all subtypes of muscarinic receptors by the elevated levels of acetylcholine. A proposed alternative is to administer M1-selective muscarinic receptor agonists. Development of such agonists, has been hampered by the highly conserved nature of the orthosteric binding domain among the five receptor subtypes [1,3,4]
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