Effects of variable copper availability on the ion transport kinetics in B104 neuroblastoma cell line

Mechanisms of copper transport in mammalian nervous cells have not been completely defined. A pivotal role in Cu uptake is ascribed to the high-affinity membrane transporter CTR1 although we can’t exclude a contribute of DCT1 carrier and prion protein. In this study a neuroblastoma cell model was used in order to investigate how cell culture media, low or high in copper content, can affect the rate of ion influx. Methods: B104 rat neuroblastoma cells were pre-treated for 48-96 h with cuprizone, a selective copper chelator, and with an higher than physiological amount of copper. Then the effect of media Cu concentrations on the rate of copper uptake in suspended cells was determined by a fluorimetric method employing the Cu(I) sensitive dye Phen Green SK (PG). An additional approach consisting of a confocal microscopy method applied to PG loaded cells was devised to pursue the same purposes. Results: Fluorimetric measurements of copper uptake in 48- 96 h Cu-depleted cells didn’t allow us to detect any change in kinetic parameters. Analogously no significant differences were observed in the rate of Cu influx in cells treated with an excess of copper chloride. In order to uncover possible inadequacy of the fluorimetric technique respect to our intents, Cu transport phenomena were evaluated by confocal microscopy in cells grown on pre-treated cover glasses. A diffuse decrease in the cell internalized PG dye signal was observed as a consequence of its binding to copper entering the cells. Conclusion: Exposure of B104 neuroblastoma cells to variable copper availability in culture media didn’t affect the kinetics of Cu cellular intake, thus enlightening strictly regulated mechanisms of copper homeostasis. Meeting FIRB - Lecce http://