Role of β-GP-derived pi in mineralization via ecto-alkaline phosphatase in cultured fetal calvaria cells

The permissive effect of beta-GP on mineralization in cultured rat fetal calvaria cells was investigated in relationship with phosphohydrolase activity of ecto-ALP at physiological pH range. Beta-GP present in the culture medium for 8 days exerted a stimulatory effect on 45Ca incorporation into matrix cell layers while the ecto-ALP activity level measured on intact cells with a saturating concentration of pNPP was similar for cells grown either in the presence or absence of beta-GP. In both types of cultures, beta-GP addition inhibited pNPP hydrolysis in a competitive and reversible manner and increased Pi concentration in the medium. The dose dependency of the effect of beta-GP on 45Ca incorporation and generation of Pi was similar (k phi = 3 mM). Levamisole, but not dexamisole, inhibited both pNPP and beta-GP hydrolyses, which were likely catalyzed by the same ecto-enzyme. The rate of 45Ca incorporation into matrix cell layers, which was high (0.90 mumol/4h/mg cell protein) in cells grown in the absence of beta-GP, was inhibited by 50% by levamisole. In cells grown in the absence of beta-GP, the 45Ca incorporation rate increased progressively after beta-GP addition, reaching after 12 h the value of cultures grown in the presence of beta-GP, the increase being totally inhibited by levamisole. In both types of cells, addition of exogenous Pi at concentrations corresponding to medium levels of beta-GP-derived Pi rapidly led to high 45Ca incorporation rate which was unaffected by levamisole. beta-GP removal from cultures grown in its presence reduced by 50% the 45Ca incorporation rate which recovered the initial value after exogenous Pi addition independently of levamisole presence. Thus, mineral deposition did not affect the level and catalytic efficiency of ecto-ALP to hydrolyze beta-GP in cultured fetal calvaria cells, yet it influenced the beta-GP-stimulatory effect on mineralization so as to render this process not sensitive to high medium Pi levels.