Increased Potency of Muscarinic Agonists at Muscarinic M3 Receptor Following Co‐expression with b2 Adrenoceptor in CHO‐K1 Cells

Abstract

Both muscarinic (M2 and M3 subtypes) and b2 adrenoceptors (b2 AR) are known to co-localize and interact in many contractile tissues. We investigated the signaling properties of CHO cells co-expressing the human M3 receptor and b2AR. These cells (M3/b2) exhibited significantly increased (20–30 fold) potency in [Ca2+]i release for various muscarinic agonists; EC50 (nM)- 0.8, 0.6, 4.8, 134, 0.2 and 4.4 nM for carbachol, oxotremorine, pilocarpine, McN-A-343, 5-mehtylfurmethiodide, and aceclidine) compared with control cells expressing a comparable level of M3 receptor alone. However, M3 receptor binding affinity ([3H]NMS) for both agonists and antagonists was similar between M3/b2 and M3 clones. In addition, in cells co-expressing the human M2 subtype with b2 adrenoceptors, muscarinic agonist activity was unaltered when tested in cAMP inhibition assays, suggesting that the change of agonist potency in the presence of b2 adrenoceptors is more likely M3 specific. Furthermore, this enhanced agonist potency at M3 receptors was not affected by pretreatment with either the b2AR antagonist alprenolol (10 mM) or agonist (1 mM isoproterenol). Our in vitro findings of enhanced muscarinic agonist potency in M3/b2 receptor co-expressed cells compared with cells expressing the M3 receptor alone recapitulate published findings in transgenic mice over-expressing b2 adrenoceptors in airway smooth muscle [McGraw et al. 2003], where the constrictor response to muscarinic receptors was enhanced due to an increase in PLCb activity and expression by increased b2 adrenoceptor expression. These results suggest that the expression level of the b2 adrenoceptor in a cell, rather than its stimulation by agonist, is a requisite for enhancement of M3 receptor agonist potency.