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Use of Phase Loop Plots to Assess Ca 2+ Sensitivity of Force Generation in Airway Smooth Muscle
Author(s) -
Han Young Soo,
Sieck Gary
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.690.1
Subject(s) - biophysics , skeletal muscle , chemistry , isometric exercise , actin , muscle contraction , myosin , myosin light chain kinase , biochemistry , anatomy , biology , medicine
In excitation‐contraction coupling, a transient elevation of cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ) triggers a force response. The Ca 2+ sensitivity of muscle reflects the relationship between [Ca 2+ ] cyt and force, and in skeletal and cardiac muscle is often defined as the [Ca 2+ ] cyt at which 50% force is generated. In smooth muscle, Ca 2+ sensitivity has been assessed as the ratio of peak force to peak cytosolic [Ca 2+ ] cyt . However, this measurement overlooks the dynamic nature of Ca 2+ regulation of force generation. In the present study, in order to provide a dynamic index of Ca 2+ sensitivity in airway smooth muscle (ASM), we simultaneously measured [Ca 2+ ] cyt and force and determined their temporal dependence using a phase loop plot. We hypothesized that [Ca 2+ ] cyt /force phase loop plot in ASM would be altered by conditions in which the extent of regulatory myosin light chain (rMLC) phosphorylation was affected. Intact ASM strips were isolated from porcine tracheas obtained from local abattoir. ASM strips, were loaded with Fura‐2 AM (Ca 2+ fluorescent indicator) and stimulated using 1 μM ACh. Isometric force and [Ca 2+ ] cyt responses were simultaneously measured using a Guth Muscle Research System. In the first set of experiments we examined the effect of Y27623, a Rho‐kinase inhibitor, and calyculin A, a protein phosphatase inhibitor that affected Ca 2+ ‐induced rMLC phosphorylation. In a second set of experiments, we examined the effect of cytochalasin D (Cyto‐D), an inhibitor of actin polymerization, which reduces force independent of Ca 2+ ‐induced rMLC phosphorylation. As expected, Y27623 decreased ACh‐induced isometric force but had no effect on evoked [Ca 2+ ] cyt transients. Cyto‐D also decreased force with no effects on evoked [Ca 2+ ] cyt . By comparison, calyculin A increased force with no effect on [Ca 2+ ] cyt . Although these results suggest changes in Ca 2+ sensitivity, the phase loop plots showed no significant shift indicative of changes in dynamic Ca 2+ sensitivity. These results do not support our hypothesis that dynamic Ca 2+ sensitivity in ASM is modulated by the extent of rMLC phosphorylation. Under these experimental conditions, it appears that isometric force generation in ASM is not entirely dependent on [Ca 2+ ] cyt . Support or Funding Information Supported by NIH grant HL126451 (GCS)