期刊
PHYSIOLOGICAL REPORTS
卷 1, 期 5, 页码 -出版社
WILEY
DOI: 10.1002/phy2.121
关键词
Biotinylation; dynasore; ECIS; Ventilatorinduced lung injury; vesicles
类别
资金
- [P01 HL066299]
- [NS10RR027535]
- [R01 HL092992]
In previous studies, blockade or gene deletion of either myosin light chain kinase (MLCK) or the mechanogated transient receptor potential vanilloid 4 (TRPV4) channel attenuated mechanical lung injury. To determine their effects on calcium entry, rat pulmonary microvascular endothelial cells (RPMVEC) were labeled with fluo-4 and calcium entry initiated with the TRPV4 agonist, 4 alpha-phorbol 12, 13-didecanoate (4 alpha PDD). Mean calcium transients peaked at similar to 25 sec and persisted similar to 500 sec. The 4 alpha PDD response was essentially abolished in calcium-free media, or after pretreatment with the MLCK inhibitor, ML-7. ML-7 also attenuated the 4 alpha PDD-induced inward calcium current measured directly using whole-cell patch clamp. Pretreatment with dynasore, an inhibitor of dynamin produced an initial calcium transient followed by a 4 alpha PDD transient of unchanged peak intensity. Automated averaging of areas under the curve (AUC) of calcium transients in individual cells indicated total calcium activity with a relationship between treatment groups of ML-7 + 4 alpha PDD < 4 alpha PDD only < dynasore + 4 alpha PDD. Measurement of biotinylated surface TRPV4 protein indicated a significant reduction after ML-7 pretreatment, but no significant change with dynasore treatment. RPMVEC monolayer electrical resistances were decreased by only 3% with 10 mu mol/L 4 alpha PDD and the response was dose-related. Dynasore alone produced a 29% decrease in resistance, but neither ML-7 nor dynasore affected the subsequent 4 alpha PDD resistance response. These studies suggest that MLCK may inhibit mechanogated calcium responses through reduced surface expression of stretch activated TRPV4 channels in the plasma membrane.
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