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

Abstract

Cytosolic Ca2+ concentration ([Ca2+](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 Ca2+ 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 μM) produced higher [Ca2+](i) levels in RPAECs than in RPMVECs and increased permeability in RPAEC but not in RPMVEC monolayers. Due to the attenuated [Ca2+](i) response in RPMVECs, we investigated whether reduced activation of store-operated Ca2+ entry was responsible for the insensitivity to thapsigargin. Addition of the drug in media containing 100 nM extracellular Ca2+ followed by readdition media with 2 mM extracellular Ca2+ increased RPMVEC [Ca2+](i) to a level higher than that in RPAECs. Under these conditions, RPMVEC permeability was not increased, suggesting that [Ca2+](i) in RPMVECs does not initiate barrier disruption. Also, ionomycin (1.4 μM) did not alter RPMVEC permeability, but the protein phosphatase inhibitor calyculin A (100 nM) induced permeability in RPMVECs. These data indicate that, whereas increased [Ca2+](i) promotes permeability in RPAECs, it is not sufficient in RPMVECs, which show an apparent uncoupling of [Ca2+](i) signaling pathways or dominant Ca2+-independent mechanisms from controlling cellular gap formation and permeability.