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

我们发现，P2Y嘌呤受体通过[Ca^&lt；2+&gt；]i来调节内皮细胞的大小。此外，我们还发现，P2Y受体的刺激加速了大分子通过内皮细胞层的渗透。为了探讨这种加速的机制，我们检测了肌球蛋白轻链激酶抑制剂ML-9和Rho激酶抑制剂Y-27632对异硫氰酸荧光素葡聚糖(FD-4；MW4400)渗透人脐静脉内皮细胞单层的影响。采用高效液相色谱-荧光检测法分析FD-4的渗透性。2P2Y受体激动剂MES-ATP增强FD-4的通透性，而P2Y受体拮抗剂PPADS则抑制FD-4的通透性。2 ML-9能明显抑制甲硫氨酸-三磷酸腺苷诱导的FD-4细胞通透性增加。此外，Y-27632还可拮抗2meS-三磷酸腺苷引起的FD-4细胞通透性增加。ML-9和Y-27632均不影响…的自发渗透性更多关于FD-4的信息。这些结果提示肌球蛋白轻链的磷酸化可能在嘌呤能调节大分子血管内皮细胞通透性中起重要作用。为了确定P2Y受体是否参与完整微血管通透性的调节，我们用FD-4(1.0 mg/mL)观察了外源性和内源性ATP对大鼠尾动脉表面组织通透性的影响。用共聚焦荧光成像系统评价FD-4的渗透性。我们发现，P2Y受体激动剂2meS-ATP能增强大鼠尾动脉组织表面FD-4的荧光强度，并被PPADS抑制。此外，交感神经递质去甲肾上腺素和炎性蜗牛蛋白缓激肽也增强了大鼠尾动脉表面组织中FD-4的荧光强度。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可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，外源性和内源性三磷酸腺苷通过P2Y受体增加毛细血管大分子通透性。这种对内皮通透性的嘌呤能调节可能在生理和病理生理条件下发挥作用。较少

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