Jp. Stains et Cv. Gay, ASYMMETRIC DISTRIBUTION OF FUNCTIONAL SODIUM-CALCIUM EXCHANGER IN PRIMARY OSTEOBLASTS, Journal of bone and mineral research, 13(12), 1998, pp. 1862-1869
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.