Mwanjewe James, Grover Ashok K
Department of Medicine, HSC 4N41, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5.
Biochem J. 2004 Mar 15;378(Pt 3):975-82. doi: 10.1042/BJ20031187.
Cells take up transferrin-bound iron or NTBI (non-transferrin-bound iron). After treatment with NGF (nerve growth factor), PC12 cells exhibited a neuronal phenotype and an increase in the NTBI uptake (55Fe2+ or 55Fe3+). We loaded the cells with the dye calcein, whose fluorescence increases in the presence of Ca2+ but is quenched with Fe2+ or Fe3+. When examined using calcein fluorescence or radioactive iron, DAG (diacylglycerol)-stimulated NTBI entry was more in NGF-treated PC12 cells compared with untreated cells. All experiments were performed at 1.5 mM extracellular Ca2+. Nramp2 (natural-resistance-associated macrophage protein 2) mRNA expression did not change after the NGF treatment. Expression of the bivalent cation entry protein TRPC6 (transient receptor potential canonical 6) was detected only in the NGF-treated cells. To verify that increased NTBI uptake depended on TRPC6, we examined whether transfecting HEK-293 (human embryonic kidney 293) cells with TRPC6 also increased the NTBI (55Fe) uptake. We also cotransfected HEK-293 cells with two plasmids, one expressing TRPC6 and the other expressing the fluorescent protein DsRED2 to identify the transfected cells. Challenging the calcein-loaded HEK-293 cells (which intrinsically express the a1-adrenergic receptors) with phenylephrine or a cell-permeant DAG increased the fluorescence signal more rapidly in transfected cells compared with untransfected cells. However, when iron (Fe2+ and Fe3+) was added before adding phenylephrine or DAG, the fluorescence intensity decreased more rapidly in transfected cells compared with untransfected cells, thereby indicating a greater stimulation of the NTBI uptake in cells expressing TRPC6. We postulate that the increase in the NTBI entry into neuronal PC12 cells is through TRPC6, a pathway that is unique since it is receptor-stimulated. Since neuronal cells express TRPC6, this pathway may have a role in neurotoxicity.
细胞摄取转铁蛋白结合铁或非转铁蛋白结合铁（NTBI）。用神经生长因子（NGF）处理后，PC12细胞表现出神经元表型，且NTBI摄取增加（55Fe2+或55Fe3+）。我们用钙黄绿素标记细胞，其荧光在Ca2+存在时增强，但会被Fe2+或Fe3+淬灭。当用钙黄绿素荧光或放射性铁检测时，与未处理的细胞相比，二酰基甘油（DAG）刺激的NTBI进入在经NGF处理的PC12细胞中更多。所有实验均在1.5 mM细胞外Ca2+条件下进行。NGF处理后，天然抗性相关巨噬细胞蛋白2（Nramp2）的mRNA表达没有变化。仅在经NGF处理的细胞中检测到二价阳离子进入蛋白瞬时受体电位香草酸亚型6（TRPC6）的表达。为了验证NTBI摄取增加是否依赖于TRPC6，我们检测了用TRPC6转染人胚肾293（HEK-293）细胞是否也会增加NTBI（55Fe）的摄取。我们还用两个质粒共转染HEK-293细胞，一个表达TRPC6，另一个表达荧光蛋白DsRED2以识别转染细胞。用去氧肾上腺素或细胞渗透性DAG刺激加载了钙黄绿素的HEK-293细胞（其本身表达α1肾上腺素能受体），与未转染的细胞相比，转染细胞中的荧光信号增加得更快。然而，在添加去氧肾上腺素或DAG之前添加铁（Fe2+和Fe3+）时，与未转染的细胞相比，转染细胞中的荧光强度下降得更快，从而表明表达TRPC6的细胞中NTBI摄取受到更大刺激。我们推测，NTBI进入神经元PC12细胞的增加是通过TRPC6实现的，这是一条独特的途径，因为它是受体刺激的。由于神经元细胞表达TRPC6，这条途径可能在神经毒性中起作用。
