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.
Gay
C V
CV
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802, USA.
Lloyd
Q P
QP
D015720:5'-Nucleotidase / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001842:Bone and Bones / Q000166:cytology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002462:Cell Membrane / Q000201:enzymology; D002645:Chickens; D010006:Osteoblasts / Q000378:metabolism*; D010521:Periosteum / Q000166:cytology*; D011794:Quercetin / Q000494:pharmacology; D013385:Succinate Dehydrogenase / Q000378:metabolism; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014268:Trifluoperazine / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
D013729:Terpenes; D014268:Trifluoperazine; D000001:Calcimycin; D014638:Vanadates; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D011794:Quercetin; D013385:Succinate Dehydrogenase; D015720:5'-Nucleotidase; D000252:Calcium-Transporting ATPases; D002118:Calcium