Na+/Ca2+ exchange in rat osteoblast-like UMR 106 cells.
Ca2+ efflux from osteoblasts is thought to be mediated by Na+/Ca2+ exchange and by a plasma membrane Ca(2+)-ATPase. The presence of plasma membrane Na+/Ca2+ exchange was determined in rat UMR 106 osteosarcoma cells by functional and molecular studies. Na+/Ca2+ exchange activity was tested by measuring changes of [Ca2+]i in single cells. After Na+ loading the cells and removing extracellular Na+, the direction of exchange was reversed and [Ca2+]i increased by 100%. Multiple isoforms of the NCX1 gene product, encoding plasma membrane Na+/Ca2+ exchangers, were cloned from UMR 106 cells and a sample of primary human osteoblasts using homology-based RT-PCR. Isoforms NACA3, NACA7, and NACA10 were found in UMR 106 cells, whereas human osteoblasts expressed NACA3 and NACA7. Transcripts for NCX2 and the Na+/Ca2+, K+ exchanger were not detected. Northern analysis of UMR 106 cells with a probe to the NCX1 gene product revealed the presence of a transcript of 7 kb, the size of the exchanger message. Western analysis of UMR 106 cell membrane preparations with a polyclonal antibody specific for the NCX1 exchanger showed the presence of reacting proteins consistent with the reported masses of the exchanger at 125 and 85 kD. These results demonstrate Na(+)-dependent Ca2+ efflux from UMR 106 cells and the presence of several NACA isoforms in UMR 106 and primary human osteoblasts.
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire, USA.
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White
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Gesek
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Friedman
D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D019831:Sodium-Calcium Exchanger; C074530:potassium-dependent sodium-calcium exchanger; D012964:Sodium; D002118:Calcium