Characterization of calcium efflux by osteoblasts derived from long bone periosteum

Inside-out vesicles were prepared from the plasma membrane of osteoblasts which had been isolated from the periosteum of 2-3-week-old chicken tibia and cultured for 6-8 days. Calcium uptake was initiated by adding 0.4 mM ATP and detected as a reduction in fluorescence from the reaction medium using the Ca(2+)-specific fluoroprobe, fluo-3. The reaction medium contained ouabain (1 mM) to block Na+, K(+)-ATPase activity and oligomycin (20 micrograms/ml) to block mitochondrial activity. Thapsigargin (5 microM) had little effect, indicating that contributions to Ca2+ uptake by endoplasmic reticulum derived microsomes were minimal. The Ca2+ uptake rate was 9.9 +/- 2.3 nmol/mg protein/min. Trifluoperazine (0.1 mM), which impairs the capacity of calmodulin to activate Ca(2+)-ATPase, substantially inhibited transport, as did quercetin (10 mM) and vanadate (10 microM), inhibitors of Ca(2+)-ATPases. This study has shown the presence of an outwardly directed, calmodulin-sensitive calcium transport system in the primary osteoblast plasma membrane. The pumping rate is substantially less than rates found in the intestine, a tissue which is involved in massive transport of Ca2+, but is similar to rates found in many other tissues. It is concluded that the enzyme does not support calcium translocation to sites of mineralization.