Relationship between force and regulatory myosin light chain phosphorylation in airway smooth muscle

We tested the hypothesis that increases in force at a given cytosolic Ca²⁺ concentration (i.e., Ca²⁺ sensitization) produced by muscarinic stimulation of canine tracheal smooth muscle (CTSM) are produced in part by mechanisms independent of changes in regulatory myosin light chain (rMLC) phosphorylation. This was accomplished by comparing the relationship between rMLC phosphorylation and force in α-toxin-permeabilized CTSM in the absence and presence of acetylcholine (ACh). Forces were normalized to the contraction induced by 10 μM Ca²⁺ in each strip, and rMLC phosphorylation is expressed as a percentage of total rMLC. ACh (100 μM) plus GTP (1 μM) significantly shifted the Ca²⁺-force relationship curve to the left (EC₅₀: 0.39 ± 0.06 to 0.078 ± 0.006 μM Ca²⁺) and significantly increased the maximum force (104.4 ± 4.8 to 120.2 ± 2.8%; n = 6 observations). The Ca²⁺-rMLC phosphorylation relationship curve was also shifted to the left (EC₅₀: 1.26 ± 0.57 to 0.13 ± 0.04 μM Ca²⁺) and upward (maximum rMLC phosphorylation: 70.9 ± 7.9 to 88.5 ± 5.1%; n = 6 observations). The relationships between rMLC phosphorylation and force constructed from mean values at corresponding Ca²⁺ concentrations were not different in the presence and absence of ACh. We find no evidence that muscarinic stimulation increases Ca²⁺ sensitivity in CTSM by mechanisms other than increases in rMLC phosphorylation.