Abstract
Extracellular β‐NAD is known to increase intracellular calcium concentrations [Ca2+]I, inositol 1,4,5‐trisphate (IP3) and cAMP in different cell types and by different mechanisms. Since β‐NAD is released extracellularly in EC, we hypothesized that it could participate in the regulation of EC permeability. Our data demonstrate that β‐NAD increases the transmonolayer electrical resistance (TER) of human pulmonary artery EC (HPAEC) monolayers in a concentration‐dependant manner indicating barrier enhancement. Importantly, simultaneous addition of β‐NAD and the potent edemagenic agent, thrombin, significantly attenuated thrombin‐induced EC permeability representing the barrier‐protective effect of β‐NAD. Immunofluorescence studies reveal more pronounced staining of cell‐cell junctional protein VE‐Cadherin at the cellular periphery signifying increased tightness of cell‐cell contacts after β‐NAD stimulation. Recent reports suggest that extracellular β‐NAD is an agonist for P2Y1 and P2Y11 purinoceptors and our data demonstrate their moderate (P2Y1) and high expression (P2Y11) in HPAEC. Interestingly, specific siRNA studies indicate the participation of both of these purinoceptors in β‐NAD‐induced TER increase. In addition, β‐NAD restored microtubule disruption induced by Nocodazole as evidenced by ECIS (Electrical cell‐substrate impedance sensing) and Immunofluorescence.
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