Figure 1. β2AR regulation of M₃ mAChR signaling.

Upon activation of M₃ mAChR by ligand binding, the G_αq subunit is released to bind and activate PLC, which hydrolyzes PIP₂ into IP₃ and DAG. IP₃ induces Ca²⁺ release from intracellular stores via the IP₃R whereas DAG activates PKC. The initial IP₃-induced release of Ca²⁺, as well as cADP-ribose (not shown), both acting via RyR (not shown) contribute to a more sustained increase of intracellular Ca²⁺, also promoted by additional influx of Ca²⁺ from the extracellular space through Ca²⁺ channels on the cell membrane. Ca²⁺ binds calmodulin to form the Ca²⁺/calmodulin complex which activates MLCK to subsequently phosphorylate MLC allowing activation of myosin ATPase (not shown) which effectively leads to force generation by cross-bridge cycling (contraction). PKC promotes the influx of Ca²⁺ from the extracellular compartment by increasing the open probability of Ca²⁺ channels. PKC phosphorylation of the MLCP inhibitor CPI-17 mediates Ca²⁺ -sensitization as MLCP limits contraction by dephosphorylating MLC. Similarly, M₃ mAChR activates RhoA and its downstream effector Rho kinase which also phosphorylates CPI-17 and thereby mediates Ca²⁺ -sensitization. PKC-mediated phosphorylation of MLCK may constitute an additional mechanism for Ca²⁺ -sensitization. Activation of the β2AR counteracts M₃ mAChR-induced signaling and contraction at several levels. Ligand-induced conformation change of the β2AR results in the release of the G_αs subunit which subsequently activates AC to hydrolyze ATP to cAMP. cAMP binds to the PKA regulatory units (not shown) which then release the catalytically active PKA. PKA phosphorylates the M₃ mAChR (1) (although relevance of this for desensitization in ASM is not certain), the G_αq subunit (2) and PLC (3) diminishing M₃ mAChR activation and IP₃ production (and possibly Rho kinase activation). The release of intracellular Ca²⁺ is inhibited through PKA-mediated phosphorylation of IP₃R (4) and RyR (not shown), whereas phosphorylation of the K_Ca2+ (5) channels results in K⁺ efflux and hyperpolarization of the cell. PKA abrogates Ca²⁺ -sensitization by phosphorylating CPI-17 (6). PKA phosphorylation of HSP20 (7) renders it capable of inhibiting contraction through ill-defined mechanisms possibly involving regulation of actin dynamics and actin-myosin binding. Another potential effector downstream of cAMP, Epac (8), has been suggested as an inhibitor of contraction, however its role in ASM as an effector downstream of β2AR remains to be determined.