Store-operated calcium entry and increased endothelial cell permeability

We hypothesized that myosin light chain kinase (MLCK) links calcium release to activation of store-operated calcium entry, which is important for cont rol of the endothelial cell barrier. Acute inhibition of MLCK caused calciu m release from inositol trisphosphate-sensitive calcium stores and prevente d subsequent activation of store-operated calcium entry by thapsigargin, su ggesting that MLCK serves as an important mechanism linking store depletion to activation of membrane calcium channels. Moreover, in voltage-clamped s ingle rat pulmonary artery endothelial cells, thapsigargin activated an inw ard calcium current that was abolished by MLCK inhibition. F-actin disrupti on activated a calcium current, and F-actin stabilization eliminated the th apsigargin-induced current. Thapsigargin increased endothelial cell permeab ility in the presence, but not in the absence, of extracellular calcium, in dicating the importance of calcium entry in decreasing barrier function. Al though MLCK inhibition prevented thapsigargin from stimulating calcium entr y, it did not prevent thapsigargin from increasing permeability. Rather, in hibition of MLCK activity increased permeability that was especially promin ent in low extracellular calcium. In conclusion, MLCK links store depletion to activation of a store-operated calcium entry channel. However, inhibiti on of calcium entry by MLCK is not sufficient to prevent thapsigargin from increasing endothelial cell permeability.