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Maturation Decreases Fractional Activation of Myosin Light Chain Kinase in Ovine Common Carotid Arteries
Author(s) -
Williams James,
Adeoye Olayemi,
Pearce William
Publication year - 2015
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.29.1_supplement.1052.7
Subject(s) - myosin light chain kinase , contractility , myosin , anatomy , chemistry , biology , medicine , endocrinology , microbiology and biotechnology
The present study tests the hypothesis that postnatal changes in contractile protein organization drive changes in MLC20 phosphorylation that contribute to age‐related changes in contractility. In homogenates, MLCK exhibited similar enzyme velocities in fetal and adult ovine carotid arteries. To compare MLCK activity in situ, common carotids were prepared and mounted in a custom‐made organ bath that enabled solenoid control of tissue freezing in methacarn after 0, 1.0, 2.0 or 3.0 s stimulation at optimal voltage. Fixed segments were divided to measure MLC20, ser19 pMLC and MLCK by Westerns and confocal microscopy. Postnatal maturation significantly decreased in situ MLCK activity (F: 1.05±0.28: A: 0.42±0.08 ng pMLC/ng MLCK/sec), while significantly increasing MLCK (∼100%) and MLC20 (∼60%) abundances. The ratio of Mander's colocalization coefficients (m1 to m2), an index of the ratio of MLCK to MLC20 masses, exhibited identical values in adult and fetal arteries. However, when the mass of MLCK used to normalize MLCK activity was corrected to calculate only the mass of MLCK colocalized with MLC20, the corrected estimates of MLCK velocity, in situ , were identical in fetal and adult arteries (F: 1.70±0.12; A: 1.75±0.09 ng pMLC/ng MLCK/sec) at peak activation. These data strongly suggest that postnatal differences in contractility are due to changes in contractile protein organization, that proportionately more of total MLCK is colocalized with MLC20 in fetal than adult arteries, and that maturation significantly decreases fractional activation of MLCK (68.5±5.0 F, 27.3±1.8% A).