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A novel organic cation transporter involved in paeonol transport across the inner blood-retinal barrier and changes in uptake in high glucose conditions
Experimental Eye Research ( IF 3.0 ) Pub Date : 2020-12-08 , DOI: 10.1016/j.exer.2020.108387
Asmita Gyawali , Myung-Hee Kim , Young-Sook Kang

Paeonol exerts various pharmacological effects owing to its antiangiogenic, antioxidant, and antidiabetic activities. We aimed to investigate the transport mechanism of paeonol across the inner blood-retinal barrier both in vitro and in vivo. The carotid artery single injection method was used to investigate the retina uptake index of paeonol. The retina uptake index (RUI) value of [³H]paeonol was dependent on both concentration and pH. This value decreased significantly in the presence of imperatorin, tramadol, and pyrilamine when compared to the control. However, para-aminohippuric acid, choline, and taurine had no effect on the RUI value. Conditionally immortalized rat retina capillary endothelial cells (TR-iBRB cell lines) were used as an in vitro model of the inner blood-retinal barrier (iBRB). The uptake of [³H]paeonol by the TR-iBRB cell lines was found to be time-, concentration-, and pH-dependent. However, the uptake was unaffected by the absence of sodium or by membrane potential disruption. Moreover, in vitro structural analog studies revealed that [³H]paeonol uptake was inhibited in the presence of organic cationic compounds including imperatorin, clonidine and tramadol. This is consistent with the results obtained in vivo. In addition, transfections with OCTN1, 2 or plasma membrane monoamine transporter (PMAT) small interfering RNA did not affect paeonol uptake in TR-iBRB cell lines. Upon pre-incubation of these cell lines with high glucose (HG) media, [3H]paeonol uptake decreased and mRNA expression levels of angiogenetic factors, such as hypoxia inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) increased. However, after the pretreatment of unlabeled paeonol in HG conditions, the mRNA levels of VEGF and HIF-1 were comparatively reduced, and the [3H]paeonol uptake rate was restored. After being exposed to inflammatory conditions induced by glutamate, TNF-α, and LPS, paeonol and propranolol pretreatment significantly increased the uptake of both [3H]paeonol and [3H]propranolol in TR-iBRB cell lines compared to their respective controls. Our results demonstrate that the transport of paeonol to the retina across the iBRB may involve the proton-coupled organic cation antiporter system, and the uptake of paeonol is changed by HG conditions.



中文翻译:

一种新型的有机阳离子转运蛋白,参与丹皮酚跨内血-视网膜屏障的转运并在高葡萄糖条件下吸收发生变化

丹皮酚具有抗血管生成,抗氧化和抗糖尿病的作用,因此具有多种药理作用。我们的目的是在体外体内研究丹皮酚跨内血视网膜屏障的转运机制。颈动脉单次注射法用于研究丹皮酚的视网膜摄取指数。[3 H]丹皮酚的视网膜摄取指数(RUI)值取决于浓度和pH。与对照组相比,在存在欧前胡素,曲马多和吡拉明的情况下,该值显着降低。但是,对氨基马尿酸,胆碱和牛磺酸对RUI值没有影响。有条件永生的大鼠视网膜毛细血管内皮细胞(TR-iBRB细胞系)用作体外内血视网膜屏障(iBRB)模型。发现TR-iBRB细胞系对[3 H]丹皮酚的吸收是时间,浓度和pH依赖性的。然而,摄取不受钠的缺乏或膜电位破坏的影响。此外,体外结构类似物研究表明,在含有欧前胡素,可乐定和曲马多的有机阳离子化合物的存在下,[3 H]丹皮酚的摄取受到抑制。这与体内获得的结果一致。此外,用OCTN1、2或质膜单胺转运蛋白(PMAT)小干扰RNA进行的转染不会影响丹皮酚在TR-iBRB细胞系中的摄取。将这些细胞系与高葡萄糖(HG)培养基预孵育后,[ 3H]酚的摄取减少,血管生成因子如缺氧诱导因子-1(HIF-1)和血管内皮生长因子(VEGF)的mRNA表达水平增加。但是,在HG条件下对未标记的pa药进行预处理后,VEGF和HIF-1的mRNA水平相对降低,[ 3 H] pa药的吸收率得以恢复。暴露于谷氨酸,TNF-α和LPS诱导的炎症后,pa药和普萘洛尔的预处理显着增加了[ 3 H] pa药和[ 3与它们各自的对照相比,TR-iBRB细胞系中的H]普萘洛尔。我们的结果表明,跨iBRB的丹皮酚向视网膜的转运可能涉及质子偶联的有机阳离子逆转运体系统,并且由于HG条件而改变了丹皮酚的吸收。

更新日期:2020-12-08
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