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Dynamic Assessment of Ca 2+ Sensitivity of Isometric Force in Intact Airway Smooth Muscle Using Phase Loop Plots
Author(s) -
Osorio Sara,
Han Young,
Sieck Gary
Publication year - 2018
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.2018.32.1_supplement.770.6
Subject(s) - isometric exercise , biophysics , chemistry , muscle contraction , skeletal muscle , anatomy , medicine , biology
A transient elevation of cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ) triggers a force response in excitation‐contraction coupling. 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 (ED50). In smooth muscle, Ca 2+ sensitivity has been assessed as the ratio of peak force to peak cytosolic [Ca 2+ ] cyt . However, this conventional 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 examined the phase loop plot assessment of Ca 2+ sensitivity in ASM under the condition in which the conditional assessment indicated an increase ( Calyculin A treatment) in Ca 2+ sensitivity . 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 mM ACh. Isometric force and [Ca 2+ ] cyt responses were simultaneously measured using a Guth Muscle Research System. ASM strips were exposed to Calyculin A (phosphatase inhibitor; 100 nM for 10 min), which was previously reported to alter static Ca 2+ sensitivity. Phase loop plots were calculated from simultaneous [Ca 2+ ] cyt and force responses. The extent of rMLC phosphorylation was determined at different time periods after ACh stimulation using Phos‐tag SDS‐PAGE (SuperSep Phos‐tag; Wako Pure Chemical Industries, Ltd.) and Western blotting. Calyculin A increased ACh‐induced isometric force in ASM strips, but it had no effect on the amplitude of evoked [Ca 2+ ] cyt transients. These results suggested that Calyculin A increased static Ca 2+ sensitivity. However, the phase loop plots showed no significant shift in dynamic Ca 2+ sensitivity with Calyculin A. These results indicate that the phase loop plot provides a valuable tool to assess dynamic Ca 2+ sensitivity of isometric force generation in ASM, which better reflects the complex process of excitation‐contraction coupling in smooth muscle. These results further indicate that dynamic Ca 2+ sensitivity of force generation in ASM is independent of rMLC phosphorylation. Support or Funding Information Supported by NIH grant HL126451 (GCS) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .