Abstract
Strongly adherent PMVECs interact to form the pulmonary capillary barrier, necessary to maintain efficient gas exchange in the lung. Tight cell‐cell junctions between adjacent pulmonary capillary endothelial cells and cytoskeletal elements control barrier integrity and are dynamically regulated by cAMP. Within PMVECs, calcium sensitive adenylyl cyclase (AC) activity has been resolved in a phosphodiesterase 4 regulated membrane fraction. However, compartmentation of AC and other signaling molecules involved in barrier regulation within PMVEC membranes has not been described. Utilizing detergent free discontinuous sucrose density gradient centrifugation to separate caveolae/lipid raft fractions from the bulk plasma membrane we detect AC5/6 in the caveolin rich light density fractions. TRPC4 a putative molecular component of the store operated calcium entry channel also resides in caveolin fractions. In addition, filamin, an actin binding protein that when PKA phosphorylated stabilizes the actin cytoskeleton, also localizes to caveolae with cell adhesion molecules N‐ and E‐cadherin, and ALCAM. In contrast, VE‐cadherin is found outside of lipid rafts in the bulk plasma membrane compartment. Therefore, it appears that AC6 compartmentalizes to caveolae with other signaling components that dynamically regulate endothelial barrier. Supported by the Welcome Trust, HL66299 and F32HL091651
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