| Research Abstract |
We showed that P2Y purinoceptors regulate the size of endothelial cells via [Ca^<2+>]i derived from Ca^<2+> stores. Also, we have shown that P2Y receptor stimulation accelerate the macromolecular permeation through endothelial cell layer. In order to approach the mechanism of this acceleration, we examine the effects of ML-9, a myosin light chain kinase inhibitor, and Y-27632, a Rho-kinase inhibitor, on fluorescein isothiocyanate (FITC) dextran (FD-4 ; MW 4400) permeation human across umbilical vein endothelial cell (HUVEC) monolayer. FD-4 permeation was analyzed by high performance liquid chromatography (HPLC)-fluorescence detection. 2meS-ATP, a P2Y receptor agonist, enhanced the permeability of FD-4, which was inhibited PPADS, a P2Y-receptor antagonist. 2meS-ATP-induced increase in the permeability of FD-4 was prevented by ML-9 significantly. Also, Y-27632 prevented 2meS-ATP-induced increase in the permeability of FD-4. Both ML-9 and Y-27632 did not influence the spontaneous permeati
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on of FD-4. These results suggest that phosphorylation of myosin light chain may play important role in the purinergic regulation of macromolecular permeation through vascular endothelium
Then, in order to determine whether the P2Y receptor participates in the regulation of permeability in intact microvessels, we examined the effects of exogenous and endogenous ATP on the permeation of the surface tissue of perfused rat caudal artery using FD-4 (1.0 mg/mL). The permeation of FD-4 was assessed by a confocal fluorescence imaging system. We found that 2meS-ATP, a P2Y receptor agonist, enhanced the fluorescence intensity of FD-4 in the surface of the rat caudal artery tissue and that it was inhibited by PPADS. Also, noradrenaline, a sympathetic neurotransmitter, and bradykinin, an inflammatory autacoid, enhanced the fluorescence intensity of FD-4 in the surface tissue of the rat caudal artery. The enhancement by noradrenaline was significantly inhibited by the P2 receptor antagonist. In addition, noradrenaline and bradykinin caused the release of ATP, ADP, AMP and adenosine from the endothelium of the rat caudal artery. These results indicate that the exogenous and endogenous ATP increase the macromolecular permeability of blood capillaries via the P2Y receptor. Such purinergic regulation of endothelial permeability may function in physiological and pathophysiological conditions. Less
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