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.
Department of Biochemistry, Faculty of Medicine, University of Nijmegen, The Netherlands.
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Benders
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Li
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Lock
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Bindels
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Bonga
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Veerkamp
D001572:Benzofurans; D002352:Carrier Proteins / Q000378:metabolism*; D003300:Copper / Q000494:pharmacology* / Q000633:toxicity; D004988:Ethers, Cyclic; D005029:Ethylenediamines / Q000494:pharmacology; D016257:Fura-2; D006801:Humans; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology; D019831:Sodium-Calcium Exchanger; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D001572:Benzofurans; D002352:Carrier Proteins; D004988:Ethers, Cyclic; D005029:Ethylenediamines; D019831:Sodium-Calcium Exchanger; C061665:sodium-binding benzofuran isophthalate; D010042:Ouabain; D003300:Copper; D000254:Sodium-Potassium-Exchanging ATPase; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine; D016257:Fura-2