Copper accumulation by cultured astrocytes

Abstract

To study copper transport in brain astrocytes, we have used astrocyte-rich primary cultures as model system. Cells in these cultures contained a basal copper content of 1.1+/-0.4 nmol per mg protein. The cellular copper content increased strongly after application of copper chloride in a time and concentration-dependent manner. Analysis of the linear copper accumulation during the first 5 min of copper exposure revealed that cultured astrocytes accumulated copper with saturable kinetics with apparent K(M)- and V(max)-values of 9.4+/-1.8 microM and 0.76+/-0.13 nmol/(min x mg protein), respectively. In contrast, incubation of astrocytes with copper in the presence of ascorbate caused a linear increase of the copper accumulation rates for copper concentrations of up to 30 microM. In addition, copper accumulation was strongly inhibited by the presence of an excess of zinc or of various other divalent metal ions. The presence of mRNA and of immunoreactivity of the copper transport protein Ctr1 in astrocyte cultures suggests that Ctr1 contributes to the observed copper accumulation. However, since some characteristics of the observed copper accumulation are not consistent with Ctr1-mediated copper transport, additional Ctr1-independent mechanism(s) are likely to be involved in astrocytic copper accumulation.