Characterization of sodium +/calcium 2+ exchanger in osteoblasts and investigation into its role in mineralization

Osteoblasts are the cells that control bone formation. Osteoblasts synthesize and secrete a complex, primarily collagenous, extracellular matrix that has the capacity to nucleate hydroxyapatite crystal formation when sufficient amounts of calcium and phosphate are supplied. The precise mechanism of mineral formation in bone has yet to be defined. One aspect of mineralization that is poorly understood is how Ca2+is translocated into the bone matrix for calcification to occur. In these studies, the role of Na +/Ca2+ exchanger as a mechanism used by osteoblasts to deliver Ca2+ into the bone matrix for mineralization was examined.;Na+/Ca2+ exchanger was shown by immunoblotting to be present in primary osteoblast cultures. The demonstration of the ability to transport Ca2+ into the cell in a Na+-dependent manner revealed that the Na+/Ca2+ exchanger was also functional. RT-PCR revealed that the NCX3 was the primary exchanger isoform present in these cells. Confocal microscopy of osteoblasts stained with antibodies against the exchanger demonstrated it to be deployed primarily along the bone matrix facing surface of the osteoblast The localization of Na+/Ca2+ exchanger on the surface of the osteoblast adjacent to the site of calcification made it a candidate as a regulator of Ca2+ transport required for bone mineralization. Furthermore, the expression of Na+/Ca2+ exchanger RNA and protein was consistent with a possible role in mineralization.;A role for Na+/Ca2+ exchanger in directing Ca2+ efflux for mineralization by osteoblasts was tested using two unrelated inhibitors of Na+/Ca2+ exchanger, bepridil and KB-R7943. Treatment of confluent cultures of osteoblasts for up to 14 days with KB-R7943 or bepridil diminished in a dose-dependent manner the ability of the cells to form a calcified matrix, without apparent effect on cell morphology, viability or ability of the cells to differentiate. These data indicate that inhibition of Na+/Ca2+ exchanger impacts mineral accumulation in the bone matrix by blocking the efflux of Ca2+ from the osteoblast into the bone fluid. This suggests that Na+/Ca2+ exchanger is an important regulator of the bone fluid microenvironment, and that Na+/Ca 2+ exchanger function appears critical to the mineralization process.