Pulmonary microvascular and macrovascular endothelial cells:: differential regulation of Ca2+ and permeability

Cytosolic Ca(2+) concentration ([Ca(2+)](i)) plays an important role in control of pulmonary vascular endothelial cell (ECs) barrier function. In this study, we investigated whether thapsigargin-and ionomycin-induced changes in cytosolic Ca(2+) induce permeability in rat pulmonary microvascular (RPMV) versus macrovascular (RPA) ECs. In Transwell cultures, RPMVECs formed a tighter, more restrictive barrier than RPAECs to 12,000-, 72,000-, and 150,000-molecular-weight FITC-labeled dextrans. Thapsigargin (1 mu M) produced higher [Ca(2+)](i) levels in RPAECs than in RPMVECs and increased permeability in RPAEC but not in RPMVEC monolayers. Due to the attenuated [Ca(2+)](i) response in RPMVECs, we investigated whether reduced activation of store-operated Ca(2+) entry was responsible for the insensitiv ity to thapsigargin. Addition of the drug in media containing 100 nM extracellular Ca(2+) followed by readdition media with 2 mM extracellular Ca(2+) increased RPMVEC [Ca(2+)](i) to a level higher than that in RPAECs. Under these conditions, RPMVEC permeability was not increased, suggesting that [Ca(2+)](i) in RPMVECs does not initiate barrier disruption. Also, ionomycin (1.4 mu M) did not alter RPMVEC permeability, but the protein phosphatase inhibitor calyculin A (100 nM) induced permeability in RPMVECs. These data indicate that, whereas increased [Ca(2+)](i) promotes permeability in RPAECs, it is not sufficient in RPMVECs, which show an apparent uncoupling of [Ca(2+)](i) signaling pathways or dominant Ca(2+)-independent mechanisms from controlling cellular gap formation and permeability.