Molecular studies of cross-talk between M2 and M3 muscarinic acetylcholine receptor subtypes
thesisposted on 15.12.2014, 10:34 by David Charles. Hornigold
Many cell-types in the body express mixed populations of muscarinic acetylcholine (mACh) receptors. For example, various types of smooth muscle express both M2 and M3 mACh receptors. While M3 mACh receptor sub-population is implicated in pharmacomechanical coupling in smooth muscle, the role of the M2 mACh receptor remains unclear. The aim of the studies described here was to investigate if there is 'cross-talk' between the signalling pathways regulated by M2 and M3 mACh receptors, co-expressed in either model (CHO) cells, or a smooth muscle tissue preparation.;Based on [3H]-NMS radioligand binding, cyclic AMP and inositol 1,4,5-trisphosphate (IP3) mass measurements, and single cell Ca2+-imaging experiments, stable and functional co-expression of M2 and M3 mACh receptors was demonstrated in two CHO-m2m3 cell lines. Initial experiments provided little evidence for M2/M3 mACh receptor cross-talk in CHO-m2m3 cells, as co-stimulation of the M2 receptor had no discernible effect on M3 receptor-mediated phospholipase. C activation, assessed at the level of IP3 and [Ca2+]i. Although both M2 and M3 mACh receptor stimulation modulated adenylyl cyclase activity, the biphasic modulation of cyclic AMP concentration in the CHO-m2m3 cells could be explained as additivity between the responses observed in CHO-m2 and -m3 cells. M2 and M3 mACh receptor stimulation caused essentially opposite effects on cell proliferation, assessed by measuring [3H]-thymidine incorporation into DNA, in CHO-m2 and CHO-m3 cells. In CHO-m2m3 cells, the inhibitory M3 mACh receptor-mediated effect appeared to dominate with respect to cell growth, however, a proliferative M2 mACh receptor-mediated effect could be 'unmasked' using the subtype-selective mACh receptor antagonist, darifenacin.