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 contribute to the integrity of this barrier and are dynamically regulated by transitions in cAMP. Within PMVECs calcium sensitive adenylyl cyclase activity has been resolved in a phosphodiesterase 4 regulated membrane fraction. However, compartmentation of adenylyl cyclase and other signaling molecules involved in barrier regulation within the PMVEC membrane has not been described. Here we utilize a detergent free discontinuous sucrose density gradient centrifugation technique to separate caveolae/lipid raft fractions from the bulk plasma membrane. AC5/6 immunoreactivity was detected in the caveolin rich light-density fractions while beta adaptin immunoreactivity was detected in the high-density fraction of non-caveolae regions within the plasma membrane. TRPC4 a putative molecular component of the store operated calcium entry channel also resides in the caveolin rich fraction. In addition, cell adhesion molecules N-cadherin and ALCAM and the scaffolding molecule Dlg localize to caveolae. Therefore, it appears that AC6 compartmentalizes to caveolae with other signaling components that dynamically regulate endothelial barrier. Supported by the Welcome Trust, HL66299 and HL60024

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