Physiological versus pharmacological signalling to myosin phosphorylation in airway smooth muscle

Abstract

Ca²⁺/calmodulin activation of myosin light chain kinase (MLCK) initiates myosin regulatory light chain (RLC) phosphorylation for smooth muscle contraction with subsequent dephosphorylation for relaxation by myosin light chain phosphatase (MLCP) containing regulatory (MYPT1) and catalytic (PP1cδ) subunits. RLC phosphorylation-dependent force development is regulated by distinct signalling modules involving protein phosphorylations. We investigated responses to cholinergic agonist treatment versus neurostimulation by electric field stimulation (EFS) in bovine tracheal smooth muscle. Concentration/temporal responses to carbachol demonstrated tight coupling between force development and RLC phosphorylation, but sensitivity differences in MLCK, MYPT1 T853, MYPT1 T696, MBS85, paxillin, and CPI-17 phosphorylations. EFS increased force and phosphorylation of RLC, CPI-17, and MLCK. In the presence of the cholinesterase inhibitor neostigmine, EFS led to an additional increase in phosphorylation of MYPT1 T853 and MYPT1 T696, MBS85, and paxillin. Thus, there were distinct pharmacological versus physiological responses in signalling modules acting on RLC phosphorylation and force responses, probably related to degenerate G protein signalling networks.

Studies with genetically modified mice were performed. Expression of another MYPT1 family member, MBS85, was enriched in mouse, as well as bovine tracheal smooth muscle. Carbachol concentration/temporal-force responses were similar in trachea from MYPT1^SM+/+, MYPT1^SM-/− and the knockin mutant mice containing nonphosphorylatable MYPT1 T853A with no differences in RLC phosphorylation. Thus, MYPT1 T853 phosphorylation was not necessary for regulation of RLC phosphorylation in tonic airway smooth muscle. Furthermore, MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction.

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