ASYMMETRIC DISTRIBUTION OF FUNCTIONAL SODIUM-CALCIUM EXCHANGER IN PRIMARY OSTEOBLASTS

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, Ost eoblasts derived from the periosteal surface of tibias from growing ch ickens were cultured for 8 days in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Lysates immunoblotted with antibodi es raised against the canine cardiac Na-Ca antibodies revealed a 70 kD a exchanger protein. Cross-reactivity of the anti-NCX antibody was con firmed by enriching for NCX in protein samples derived from plasma mem brane vesicles by affinity chromatography using the exchanger inhibito ry peptide. Fractions enriched for the exchanger were eluted from the column and subjected to immunoblotting with the anti-NCX antibody, rev ealing an intense single band at 70 kDa. Examination of live cells loa ded 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 shined with anti-NCX antibodies revealed asymmetric localization of the exch anger in cultured osteoblasts, residing almost entirely within hive 0. 5-mu m optical sections along the substrate adherent side of the cells . Since NCX is known to be a low-affinity, high-capacity calcium trans locating molecule and also appears to be asymmetrically positioned, it is likely to play a key role in bone formation.