SPECIFIC ACTIVITY OF SKELETAL ALKALINE-PHOSPHATASE IN HUMAN OSTEOBLAST-LINE CELLS REGULATED BY PHOSPHATE, PHOSPHATE-ESTERS, AND PHOSPHATE ANALOGS AND RELEASE OF ALKALINE-PHOSPHATASE ACTIVITY INVERSELY REGULATED BY CALCIUM

We assessed the significance of Ca and phosphate (P(i)) as determinant s of (1) the amount of skeletal alkaline phosphatase (ALP) activity in SaOS-2 (human osteosarcoma) cells and normal human bone cells, and (2 ) the release of ALP activity from the cells into the culture medium. After 24 h in serum-free BGJ(b) medium containing 0.25-2 mM P(i), the specific activity of ALP in SaOS-2 cells was proportional to P(i) conc entration (r = 0.99, p < 0.001). The P(i)-dependent increase in ALP ac tivity was time dependent (evident within 6 h) and could not be attrib uted to decreased ALP release, since P(i) also increased the amount of ALP activity released (r = 0.99, p < 0.001). Parallel studies with Ca (0.25-2.0 mM) showed that the amount of ALP activity released from Sa OS-2 cells was inversely proportional to the concentration of Ca (r = -0.85, p < 0.01). This effect was rapid (i.e., observed within 1 h) an d could not be attributed to a decrease in the amount of ALP activity in the cells. Phase distribution studies showed that the effect of low Ca to increase ALP release reflected increases in the release of both hydrophilic ALP (i.e., anchorless ALP, released by phosphatidylinosit ol-glycanase activity) and hydrophobic ALP (i.e., phosphatidylinositol -glycan-anchored ALP, released by membrane vesicle formation). The ran ge of Ca-dependent changes in ALP-specific activity was much smaller t han the range of P(i)-dependent changes. The observed correlation betw een skeletal ALP-specific activity and P(i) was not unique to osteosar coma cells or to P(i). Similar effects were seen in normal human bone cells in response to P(i) (r = 0.99, p < 0.001) and in SaOS-2 cells in response to a variety of P(i) esters and analogs (e.g., beta-glycero- P(i) and molybdate). Further studies indicated that the effects of pho sphoryl compounds on ALP-specific activity could not be correlated wit h effects on ALP reaction kinetics, cell proliferation, or acid phosph atase activity and that the beta-glycero-P(i)-dependent increase in AL P activity was blocked by cycloheximide but not actinomycin D. Togethe r these data suggest that the function of skeletal ALP may be regulate d by P(i) and that Ca may be involved in ALP release.