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Relationship between force and regulatory myosin light chain phosphorylation in airway smooth muscle
T Kai1, H Yoshimura, K A Jones
1Department of Anesthesiology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA.
Abstract:
We tested the hypothesis that increases in force at a given cytosolic Ca(2+) concentration (i.e., Ca(2+) 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 alpha-toxin-permeabilized CTSM in the absence and presence of acetylcholine (ACh). Forces were normalized to the contraction induced by 10 microM Ca(2+) in each strip, and rMLC phosphorylation is expressed as a percentage of total rMLC. ACh (100 microM) plus GTP (1 microM) significantly shifted the Ca(2+)-force relationship curve to the left (EC(50): 0.39 +/- 0.06 to 0.078 +/- 0.006 microM Ca(2+)) and significantly increased the maximum force (104.4 +/- 4.8 to 120.2 +/- 2.8%; n = 6 observations). The Ca(2+)-rMLC phosphorylation relationship curve was also shifted to the left (EC(50): 1.26 +/- 0.57 to 0.13 +/- 0.04 microM Ca(2+)) 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 Ca(2+) concentrations were not different in the presence and absence of ACh. We find no evidence that muscarinic stimulation increases Ca(2+) sensitivity in CTSM by mechanisms other than increases in rMLC phosphorylation.
Insights
Muscarinic stimulation increases canine tracheal smooth muscle force by enhancing regulatory myosin light chain (rMLC) phosphorylation. This study found no evidence for force increases independent of rMLC phosphorylation changes.
Area of Science:
- Physiology
- Muscle Biology
- Pharmacology
Background:
- Muscarinic stimulation enhances airway smooth muscle contraction.
- The role of regulatory myosin light chain (rMLC) phosphorylation in this process is debated.
- Investigating Ca(2+) sensitization mechanisms is crucial for understanding airway hyperresponsiveness.
Purpose of the Study:
- To test if muscarinic stimulation increases force in canine tracheal smooth muscle (CTSM) via mechanisms independent of rMLC phosphorylation.
- To compare the Ca(2+)-force and Ca(2+)-rMLC phosphorylation relationships in the presence and absence of acetylcholine (ACh).
Main Methods:
- Utilized alpha-toxin-permeabilized CTSM strips.
- Measured force and rMLC phosphorylation at varying cytosolic Ca(2+) concentrations.
- Administered acetylcholine (ACh) and guanosine triphosphate (GTP) to stimulate muscarinic receptors.
Main Results:
- ACh significantly shifted the Ca(2+)-force relationship leftward and increased maximum force.
- ACh also shifted the Ca(2+)-rMLC phosphorylation relationship leftward and upward.
- The relationship between rMLC phosphorylation and force was not altered by ACh.
Conclusions:
- Muscarinic stimulation increases Ca(2+) sensitivity in CTSM solely through increased rMLC phosphorylation.
- No evidence supports Ca(2+) sensitization mechanisms independent of rMLC phosphorylation in this model.
- Findings clarify the molecular basis of muscarinic-induced airway smooth muscle contraction.