Department of Biology and Biochemistry, University of Houston, Houston, Texas, USA.
Mol Pharmacol. 2012 Oct;82(4):728-37. doi: 10.1124/mol.112.079376. Epub 2012 Jul 24.
Atypical N-methyl-D-aspartate (NMDA) receptors are expressed in podocytes. Sustained (≥24 h) application of 50 to100 μM NMDA to immortalized mouse podocytes evoked a marked increase in the production of reactive oxygen species(ROS) such as H₂O₂. This effect of NMDA was associated with increased cell-surface expression of p47(phox), a cytosolic regulatory subunit of the NADPH oxidase NOX2. NMDA-evoked generation of ROS drove an increase in steady-state surface expression of transient receptor potential canonical (TRPC) 6 channels, which was blocked by the NMDA antagonist dizocilpine(MK-801) and by a membrane-permeable scavenger of ROS. The effect of NMDA on TRPC6 was observed using cell surface biotinylation assays and also with whole-cell recordings made under conditions designed to facilitate detection of current through TRPC6. NMDA mobilization of TRPC6 channels was blocked by concurrent treatment with the NMDA antagonist MK-801 and by a membrane-permeable scavenger ofROS. Mobilization of TRPC6 was also evoked by L-homocysteic acid. NMDA treatment also increased nuclear localization of endogenous nuclear factor of activated T cells, which could be blocked by MK-801, by scavenging ROS, by the calcineurin inhibitor cyclosporine, and by the TRPC channel inhibitor 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl]imidazole (SKF-96365). NMDA treatment also evoked robust activation of Rho but not Rac,consistent with previous studies of downstream effectors of TRPC6 activation. Exposing cells to NMDA for 24 h reduced total and cell surface expression of the podocyte markers nephrin and podocin, but there was no loss of cells. With longer NMDA exposure (72 h), we observed loss of cells associated with nuclear fragmentation and increased expression of caspase-3, caspase-6, and Bax, suggesting an apoptotic process.
非典型 N-甲基-D-天冬氨酸 (NMDA) 受体存在于足细胞中。持续（≥24 小时）应用 50 至 100 μM NMDA 于永生化的小鼠足细胞可引起活性氧（ROS）如 H₂O₂的产生显著增加。NMDA 的这种作用与 NADPH 氧化酶 NOX2 的胞质调节亚基 p47(phox)的细胞表面表达增加有关。NMDA 诱导的 ROS 生成驱动瞬时受体电位经典（TRPC）6 通道的稳态表面表达增加，该增加被 NMDA 拮抗剂地卓西平（MK-801）和 ROS 膜通透性清除剂阻断。使用细胞表面生物素化测定和在设计用于促进 TRPC6 电流检测的全细胞记录中观察到 NMDA 对 TRPC6 的作用。TRPC6 通道的 NMDA 动员被同时用 NMDA 拮抗剂 MK-801 和 ROS 膜通透性清除剂处理阻断。L-高半胱氨酸酸也可引发 TRPC6 动员。NMDA 处理还增加了核因子激活 T 细胞的核定位，该定位可被 MK-801、ROS 清除、钙调神经磷酸酶抑制剂环孢菌素和 TRPC 通道抑制剂 1-[2-(4-甲氧基苯基）-2-[3-(4-甲氧基苯基）丙氧基]乙基]咪唑（SKF-96365）阻断。NMDA 处理还引起 Rho 的强烈激活，但不引起 Rac 的激活，这与 TRPC6 激活的下游效应物的先前研究一致。将细胞暴露于 NMDA 24 小时可降低足细胞标志物足突蛋白和 podocin 的总表达和细胞表面表达，但没有细胞丢失。用更长时间的 NMDA 暴露（72 小时），我们观察到与核片段化和 caspase-3、caspase-6 和 Bax 表达增加相关的细胞丢失，表明发生了凋亡过程。
