P2Y Receptor-Mediated Ca²⁺ Signaling Increases Human Vascular Endothelial Cell Permeability

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We investigated the effects of P2-receptor agonists on cell size, intracellular calcium levels ([Ca²⁺]_i), and permeation of FITC-labeled dextran (FD-4) as well as the relationship between these effects in human umbilical vein endothelial cells (HUVEC). FD-4 concentration, cell size, and [Ca²⁺]_i were analyzed by HPLC with fluorescence, phase contrast microscopic imaging, and fluorescent confocal microscopic imaging, respectively. The P2Y₁-receptor agonists 2-methylthio ATP (2meS-ATP) and ADP decreased cell size and increased [Ca²⁺]_i in HUVEC. The P2Y₂-receptor agonist UTP increased [Ca²⁺]_i, but did not influence cell size. The P2X-receptor agonist α,β-methylene ATP did not induce either response. The decrease in size and increase in [Ca²⁺]_i by 2meS-ATP were blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, P2Y₁-antagonist), thapsigargin (Ca²⁺-pump inhibitor), and U73122 (phospholipase C inhibitor). Furthermore, 2meS-ATP (P2Y₁-receptor agonist) enhanced permeation of FD-4 through the endothelial cell monolayer. The 2meS-ATP-induced enhancement of the permeation was also prevented by PPADS, thapsigargin, and U73122. These results indicate that activation of P2Y receptors induces a decrease in cell size, an increase in [Ca²⁺]_i, and may participate in facilitating macromolecular permeability in HUVEC.