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.