ANALYSIS OF COPPER TRANSPORT IN B104 NEUROBLASTOMACELLS

Copper is an essential trace metal required as a cofactor in a broad range of enzymatic functions including cellular antioxidant activity, oxidative phosphorylation and neurological function. If in excess, it can initiate a cascade of events leading to cell disruption. Consequently, cells have developed sophisticated homeostatic mechanisms to maintain adequate intracellular copper concentrations (Pena et al., J. Nutr. 129(7): 1251-60, 1999). In the attempt to characterise the components of brain copper homeostasis, we studied the expression and function of copper transport systems in a rat neuroblastoma cell line (B104). In our cell model copper fluxes could be related to the functional expression of a member of the Ctr family named rat Ctr1 and to the primary active copper transport MNK protein. In both cases we demonstrated their mRNA expression in B104 cells using specific primers and the RT-PCR amplification method. Transmembrane copper fluxes were measured by loading cells with the Cu2+-sensitive fluorophore Phen Green SK. Copper addition in the extra cellular medium induced a rapid fluorescence quenching of the entrapped probe, probably due to copper entry via Ctr1 protein, followed by a fast recovery of fluorescence related to a putative MNK-mediated copper efflux. The initial rate of copper uptake was dependent on external ion concentrations and saturated at approximately 1 5M Cu2+. Furthermore, we found that zinc can affect copper transport by partially inhibiting the uptake. In conclusion, our results support the hypothesis that copper transport in neuroblastoma cells is a complex phenomenon presumably mediated by more than a single carrier process.