ATP reduces macromolecule permeability of endothelial monolayers despite increasing [Ca2+]i.

We investigated the relationship between the ATP-evoked rise of cytosolic Ca2+concentration ([Ca2+]i) and barrier function in porcine aortic endothelial monolayers. ATP (0.01-100 μM) induced a transient rise of [Ca2+]iand reduced permeability in a concentration-dependent manner. In contrast, the Ca2+ ionophore ionomycin (1 μM) elicited a rise in [Ca2+]icomparable to that induced by ATP (10 μM), but it increased permeability. For the reduction of permeability, nucleotides were found to be in the following order of potency: ATP = ATPγS > ADP = UTP. Blockade of adenosine receptors by 8-phenyltheophylline (10 μM) did not affect ATP (10 μM)-induced reduction of permeability. ATP reduced permeability even in endothelial monolayers that had been loaded with the Ca2+ chelator BAPTA to prevent the rise in [Ca2+]i. U-73122 (1 μM), an inhibitor of phospholipase C (PLC), completely abolished the effect of ATP (10 μM) on permeability. It also abolished the translocation of protein kinase C (PKC) in response to ATP, which could also be achieved by the PKC inhibitors Gö-6976 (100 nM) or bisindolylmaleimide I (1 μM). In the presence of PKC inhibitors, however, the permeability effect of ATP was not affected. The presence of inhibitors of adenylate or guanylate cyclase (50 μM SQ-22536 or 20 μM ODQ) prevented changes in cyclic nucleotides but did not affect the permeability effects of ATP. The study shows that ATP reduces macromolecule permeability via a PLC-mediated mechanism that is independent of the concomitant effects of ATP on cytosolic Ca2+, cyclic nucleotides, or PKC.