Role of transient receptor potential canonical 6 (TRPC6) in non-transferrin-bound iron uptake in neuronal phenotype PC12 cells

Abstract

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.

New Molecular Mechanisms on the Activation of TRPM2 Channels by Oxidative Stress and ADP-Ribose

“…To confirm the effects of intracellular hydroxyl radical, cells in the study of Ishii et al [53] were pretreated with FeSO 4 for 1 h, and then H 2 O 2 was added after washing out extracellular Fe 2+ in TRPM2-expressing HEK293 cells and beta-cell line RIN-5F cells. Many investigators reported that various types of cells, including HEK293, take up Fe 2+ within a few minutes [55,56]. By pretreatment with FeSO 4 for 1 h, addition of H 2 O 2 significantly activated TRPM2, suggesting that the intracellularproduced hydroxyl radical is responsible for activation of TRPM2 [53].…”

Iron and calcium in the central nervous system: a close relationship in health and sickness

“…In particular, Mwanjewe and Grover [31] investigated the role of the members of the canonical subfamily of TRP (TRPC [32]) that are considered possible candidates for store-and receptor-operated calcium channels. These nonselective cationic channels might therefore account for the calcium influx that is observed after store depletion or phospholipase C activation by G-protein-coupled receptors [30].…”

Proliferating brain cells are a target of neurotoxic CSF in systemic autoimmune disease

“…leukocyte chemotaxis or lymphocyte-target cell conjugation), and has minimal effects on cell function (De Clerck et al, 1994). Therefore, the calcein-release assay and fluorescence microfluorometry (Mwanjewe and Grover, 2004) were used to verify cytotoxicity and screen for qualitative changes in ionic metabolism. In brief, C17.2 cells were cultured in 35-mm petri dishes for 24 h, the center bottom having been removed and replaced with glass coverslips.…”