COPPER TOXICITY IN CULTURED HUMAN SKELETAL-MUSCLE CELLS - THE INVOLVEMENT OF NA+/K+-ATPASE AND THE NA+/CA2+-EXCHANGER

Copper (Cu2+) intoxication has been shown to induce pathological changes in various tissues. The mechanism underlying Cu2+ toxicity is still unclear. It has been suggested that the Na+/K+-ATPase and/or a change of the membrane permeability may be involved. In this study we examined the effects of Cu2+ on the Na+ and Ca2+ homeostasis of cultured human skeletal muscle cells using the ion-selective fluorescent probes Na-+-binding benzofuran isophtalate (SBFI) and Fura-2 respectively. In addition, we measured the effect of Cu2+ on the Na+/K+-ATPase activity. Cu2+ and ouabain increase the cytoplasmic free Na+ concentration ([Na+](i)). Subsequent addition of Cu2+ after ouabain does not affect the rate of [Na+](i) increase. Cu2+ inhibits the Na+/K+-ATPase activity with an IC50 of 51 mu M. The cytoplasmic free Ca2+ concentration concentration ([Ca2+](i)) remains unaffected for more than 10 min after the administration of Cu2+. Thereafter, [Ca2+](i) increases as a result of the Na+/Ca2+-exchanger operating in the reversed mode. The effects of Cu2+ on the Na+ homeostasis are reversed by the reducing and chelating agent dithiothreitol and the heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN). In conclusion, SBFI is a good tool to examine Na+ homeostasis in cultured human skeletal muscle cells. Under the experimental conditions used, Cu2+ does not modify the general membrane permeability, but inhibits the Na+/K+-pump leading to an increase of [Na+](i). As a consequence the operation mode of the Na+/Ca2+-exchanger reverses and [Ca2+](i) rises.