Asymmetric Distribution of Functional Sodium-Calcium Exchanger in Primary Osteoblasts

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Abstract
To understand calcium translocation in osteoblasts, we have determined the location of sodium-calcium (Na-Ca) exchanger (NCX) in relation to actin and alpha-tubulin in primary cultures of avian osteoblasts. Osteoblasts derived from the periosteal surface of tibias from growing chickens were cultured for 8 days in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Lysates immunoblotted with antibodies raised against the canine cardiac Na-Ca antibodies revealed a 70 kDa exchanger protein. Cross-reactivity of the anti-NCX antibody was confirmed by enriching for NCX in protein samples derived from plasma membrane vesicles by affinity chromatography using the exchanger inhibitory peptide. Fractions enriched for the exchanger were eluted from the column and subjected to immunoblotting with the anti-NCX antibody, revealing an intense single band at 70 kDa. Examination of live cells loaded with Calcium Green-1 AM ester by confocal microscopy demonstrated sodium-dependent calcium uptake, confirming the presence of functional NCX in intact cells. Immunolocalization studies of osteoblasts stained with anti-NCX antibodies revealed asymmetric localization of the exchanger in cultured osteoblasts, residing almost entirely within two 0.5-microm optical sections along the substrate adherent side of the cells. Since NCX is known to be a low-affinity, high-capacity calcium translocating molecule and also appears to be asymmetrically positioned, it is likely to play a key role in bone formation.