Study on regulatory system of vascular permeability by ATP (Aim at pharmacological DDS)

我们发现P2 Y嘌呤受体通过来自Ca^2+库的[Ca^2+]i调节内皮细胞的大小。此外，我们已经表明，P2 Y受体刺激加速大分子渗透通过内皮细胞层。为了探讨这种加速的机制，我们研究了ML-9，一种肌球蛋白轻链激酶抑制剂，和Y-27632，一种Rho激酶抑制剂，对异硫氰酸荧光素（FITC）葡聚糖（FD-4 ; MW 4400）透过人脐静脉内皮细胞（HUVEC）单层的影响。通过高效液相色谱（HPLC）-荧光检测来分析FD-4的渗透。P2 Y受体激动剂2 meS-ATP可增加FD-4的通透性，而P2 Y受体拮抗剂PPADS则可抑制这种作用。ML-9可显著抑制2 meS-ATP诱导的FD-4细胞通透性增加。此外，Y-27632阻止2 meS-ATP诱导的FD-4渗透性增加。ML-9和Y-27632均不影响细胞的自发渗透， ...更多信息 在FD-4上。这些结果表明，肌球蛋白轻链的磷酸化可能在大分子通过血管内皮的嘌呤调节中起重要作用。为了确定P2 Y受体是否参与完整微血管通透性的调节，我们用FD-4（1.0mg/mL）灌流大鼠尾动脉，检测了外源性和内源性ATP对表面组织通透性的影响。通过共聚焦荧光成像系统评估FD-4的渗透。我们发现P2 Y受体激动剂2 meS-ATP可增强大鼠尾动脉组织表面FD-4的荧光强度，而PPADS可抑制这种荧光强度。此外，去甲肾上腺素，交感神经递质，缓激肽，炎症autacoid，增强FD-4的荧光强度在大鼠尾动脉的表面组织。去甲肾上腺素的增强作用可被P2受体拮抗剂显著抑制。此外，去甲肾上腺素和缓激肽引起大鼠尾动脉内皮细胞释放ATP、ADP、AMP和腺苷。这些结果表明，外源性和内源性ATP通过P2 Y受体增加毛细血管大分子的通透性。这种内皮通透性的嘌呤能调节可能在生理和病理生理条件下起作用。少

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 … More 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 endotheliumThen, 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