Copper toxicity in cultured human skeletal muscle cells: the involvement of Na+/K(+)-ATPase and the Na+/Ca(2+)-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 isophatalate (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 microM. The cytoplasmic free Ca2+ 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+/Ca(2+)-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+/Ca(2+)-exchanger reverses and [Ca2+]i rises.