Inhibition of alkaline phosphatase activity by competition between bisphosphonates and divalent metal ions

Recent investigations have demonstrated that bisphosphonate (BP) inhibits ALP activities, but there are still many points that remain uncovered. We tested the inhibitions of human bone, placental, and liver type ALP activity in the presence of Mg, Zn and Ca (as Mg, Zn, and Ca-ALP respectively), with paranitrophenyl phosphate (p NPP) or inorganic pyrophosphate (PPi) as substrates, using various BPs: non- nitrogen-containing BP clodronate and nitrogen-containing BP risedronate and alendronate for inhibitors. Using p NPP as a substrate, the relationship between the effects of clodronate, risedronate or alendronate on bone or liver Mg, Zn and Ca-ALP activities, and the concentrations of coexisting Mg, Zn and Ca were examined. Mg, Zn, and Ca-ALP activities of both isozymes decreased to the concentration dependency of each BP ; this was required as higher concentrations of BPs as the coexisting Mg concentration was higher for the suppression of Mg-ALP activity by BPs. In addition, risedronate and alendronate showed stronger Mg-ALP inhibitory action compared with clodronate. These results suggest that BP inhibits human bone and liver Mg-ALP activity competitively with Mg. Also, the strength of the inhibition by nitrogen-containing BPs are stronger than those of non-nitrogen-containing BPs. For Zn and Ca-ALP activities, BPs also showed a tendency of competition with Zn or Ca, but not as significant as Mg. Clodronate suppressed the Ca-ALP activities of MC3T3-E1 and human placental ALP using PPi as substrates, to the concentration dependency competitively with Ca. Considering the possibility that BP with a P-C-P structure might compete with the substrates containing a P-O-P structure, we measured the effects of clodronate, risedronate, and alendronate on p NPP concentration dependency of human bone and liver Mg, Zn and Ca-ALP activities. BPs inhibited the maximum activity depending on the concentration, but did not significantly affect the 50 % activtity concentration by p NPP. This suggests that BPs do not compete with the substrate of ALP, p NPP. These results suggest that BP inhibits ALP activity by competition with bivalent metal, regardless of the type of ALP, substrate, and the type of bivalent metal for the activation. In addition, its action of nitrogen-containing BP is stronger than non-nitrogen-containing BP.