Medizinische Klinik und Poliklinik D, Experimentelle Nephrologie, Universität Münster, Domagkstrasse 3a, Münster, Germany.
Pflugers Arch. 2011 Aug;462(2):359-69. doi: 10.1007/s00424-011-0969-7. Epub 2011 Apr 27.
The main elimination site of organic cations (OCs) is the renal proximal tubule (PT). OC transporters (OCT) accept endogenous and exogenous substances and xenobiotics. As transgenic mouse models are increasingly used in translational medicine, functional properties with special focus on regulation of OCT of isolated mouse PTs were studied with a new fluorescence reader-based method, which allows studying larger numbers of tubules per kidney. OC transport across the basolateral membrane of PTs from male mice was measured as initial uptake of the fluorescent dye 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP). A microtiter plate fluorescence reader was used to semi-automatically analyze OC transport in freshly isolated tubules. Relative mRNA expression of OCT1/OCT2/OCT3 in PTs was 1/0.3/0.01 and did not vary from S1 to S3 segments. ASP was transported by PTs with a K (m) of 6 μM. It was inhibited by TEA, TPA, or cimetidine (IC(50)=5, 19, or 53 μM, respectively). Angiotensin II stimulated ASP uptake (+63%), while stimulation of PKC reduced (-37%) OC transport. Inhibition of p56(lck) tyrosine kinase (-60%), of PI3K (-36%), of Ca(2+)/calmodulin (-25%), or of PKA (-33%) reduced OC transport. In PTs from OCT1/2(-/-) mice ASP uptake was reduced to ~20%. Using this fluorescence reader-based method, we report substrate specificities and a complex pattern of acute regulation of OC transport in isolated mouse PTs. Compared to isolated human PTs or rat and human OCT isoforms expressed in HEK293-cells, OC transport across the basolateral membrane of freshly isolated mouse PTs shows similarities but also specific differences.
有机阳离子 (OC) 的主要排泄部位是肾脏近端小管 (PT)。OC 转运体 (OCT) 可接受内源性和外源性物质及外源性物质。随着转基因小鼠模型在转化医学中的应用日益增多，我们使用一种新的荧光阅读器为基础的方法研究了分离的小鼠 PT 中 OCT 的功能特性，特别关注其调节作用，该方法允许对每只肾脏的更多肾小管进行研究。OC 跨雄性小鼠 PT 基底外侧膜的转运通过荧光染料 4-(4-(二甲氨基) 苯乙烯基)-N-甲基吡啶鎓 (ASP) 的初始摄取来测量。微量滴定板荧光阅读器用于半自动分析新鲜分离的小管中的 OC 转运。PT 中 OCT1/OCT2/OCT3 的相对 mRNA 表达为 1/0.3/0.01，并且在 S1 到 S3 段之间没有变化。ASP 由 PT 转运，其 K(m)为 6 μM。它被 TEA、TPA 或西咪替丁抑制 (IC(50)=5、19 或 53 μM)。血管紧张素 II 刺激 ASP 摄取 (+63%)，而 PKC 刺激减少 (-37%) OC 转运。p56(lck) 酪氨酸激酶抑制剂 (-60%)、PI3K 抑制剂 (-36%)、Ca(2+)/钙调蛋白抑制剂 (-25%) 或 PKA 抑制剂 (-33%) 均可减少 OC 转运。在 OCT1/2(-/-) 小鼠的 PT 中，ASP 摄取减少到约 20%。使用这种基于荧光阅读器的方法，我们报告了底物特异性和分离的小鼠 PT 中 OC 转运的复杂急性调节模式。与分离的人 PT 或在 HEK293 细胞中表达的大鼠和人 OCT 同工型相比，新鲜分离的小鼠 PT 中基底外侧膜上的 OC 转运具有相似性，但也存在特异性差异。
