Muscarinic acetylcholine receptor signalling in model cells and smooth muscle
thesisposted on 15.12.2014, 10:34 by Donna Kirsty. Boxall
In this Thesis, experiments are described in which the signalling of M2- and M3-mACh receptors as homogeneous receptor populations and as a co-expressed population in mammalian cell-lines have been studied. These experiments set out to investigate the hypothesis that M2-mACh receptors can influence signalling by M3-mACh receptors via 'cross-talk' at the level of second messenger signalling. This may have particular significance with respect to mACh receptors in smooth muscle. In many smooth muscle tissues mACh receptor subtypes are co-expressed, with the major population often being M2-mACh receptors and the minor population being M3-mACh receptors. It is the M3-mACh receptors which have been implicated in the direct mediation of contraction via activation of phosphoinositide metabolism. Whilst it is known that M2-mACh receptors signal via inhibition of adenylyl cyclase, it is not known whether M2-mACh receptors can modulate signalling via M3-mACh receptors to influence smooth muscle tone. By studying 'cross-talk' of these two receptors in a model cell system, this possibility can be evaluated.;Chinese hamster ovary (CHO) cells expressing M2-, M3- or co-expressing M2- and M3- mACh receptors were characterised with respect to agonist-stimulation of inhibition of forskolin-stimulated cyclic AMP levels, and agonist-stimulation of inositol 1,4,5-trisphosphate (InsP3; time-course and concentration-dependence). These two responses have been shown to be representative of M2- and M3-mAaCh receptor-mediated responses, respectively. Whilst inhibition of cAMP production in CHO-SSLLM2 cells was pertussis toxin-sensitive and was blocked by tripitramine (30 nM), neither stimulation of cAAMP, nor InsP3 production in CHO-M3 cells was sensitive to either pretreatment.;The studies indicate that cross-talk can occur between the signalling pathways of M2- and M3-mACh receptors in model cells. Further investigations to establish the mechanism of this cross-talk should include studies of signalling in a smooth muscle tissue which co-expresses these mACh receptors, to establish whether similar cross-talk occurs in a more physiological context.