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Myosin light chain kinase regulates the surface expression and calcium entry through transient receptor potential vanilloid 4 channels in rat pulmonary microvascular endothelial cells
Author(s) -
Parker James C.,
Kelley Sarah,
Meyer Angela,
Mouner Marc,
Hashizume Masahiro,
Townsley Mary I.
Publication year - 2011
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.25.1_supplement.1101.1
Subject(s) - myosin light chain kinase , trpv4 , transient receptor potential channel , chemistry , microbiology and biotechnology , voltage dependent calcium channel , calcium in biology , calcium , myosin , biophysics , receptor , biochemistry , biology , organic chemistry
Ventilator induced lung injury (VILI) is a significantly significant contributor to mortality during treatment for the acute respiratory distress syndrome (ARDS). Experimental VILI was initiated by calcium influx through stretch activated cation channels identified as transient receptor potential vanilloid 4 (TRPV4) channels and could be attenuated by inhibition of myosin light chain kinase (MLCK). We recently observed that ML‐7, a specific MLCK inhibitor, blocked Ca2+ entry induced by the TRPV4 agonist, 4α‐phorbol 12, 13‐didecanoate (4αPDD). Ca2+ transients were measured in Flou‐4 labeled rat pulmonary microvascular endothelial cells (RPMVEC) using a confocal fluorescence microscope either with or without ML‐7 pre‐treatment. After 10 uM 4αPDD, cytosolic Ca2+ in RPMVEC increased to 2.2±0.3 times baseline, reached a peak at 36±10 sec and decayed with a T1/2 of 65 ±15 sec, but was completely blocked by ML‐7 pre‐treatment. We hypothesized that ML‐7 might regulate Ca2+ entry by surface expression of TRPV4, so monolayers were biotinylated, lysed and incubated with Streptavadin beads. Western blots indicated a 9‐fold greater TRPV4 protein in untreated compared to ML‐7 pretreated cells. These studies suggest a continuous traffic of TRPV4 channel containing vesicles to and from the cell membrane mediated by MLCK dependent phosphorylation.