Effects of four bisphosphonates on bone resorption, lysosomal enzyme release, protein synthesis and mitotic activities in mouse calvarial bones in vitro

Abstract

The effects of 3-amino-1-hydroxy-propylidene-1,1-bisphosphonate (AHPrBP), 1-hydroxyethylidene-1,1-bisphosphonate (HEBP), dichloromethylenebisphosphonate (Cl 2MBP) and azacycloheptylidene-2,2-bisphosphonate (AHBP) on bone were examined in organ culture using newborn mice calvaria. AHPrBP, HEBP and Cl 2MBP caused a dose-dependent inhibition of PTH-stimulated ( 10 nmol l ) release of 45Ca from the calvaria, at and above a concentration of 3 μmol 1 , whereas AHBP only caused a slight inhibition, at and above 100 μmol l . AHPrBP inhibited PTH-stimulated release of 3H from bones prelabelled with [ 3H]-proline. AHPrBP ( 30 μmol l ) diminished the stimulatory effect of 1α(OH)vitamin D 3 ( 10 nmol l ), prostaglandin E 2 ( 0.1 μmol l ) and renal tumor conditioned media on 45Ca release. AHPrBP and Cl 2MBP, at and above 3 μmol l , decreased PTH-stimulated mobilization of Ca 2+ and P i and in parallel the release of β-glucuronidase without affecting the release of lactate dehydrogenase. The inhibitory effect of AHPrBP ( 30 μmol l ) on PTH-induced 45Ca release was irreversible. The inhibition by AHPrBP ( 30 μmol l ) on spontaneous and PTH-stimulated release of 45Ca can be seen first after 24 h of culture. Similarly the inhibitory effect by HEBP ( 30 μmol l ) and Cl 2MBP ( 30 μmol l ) was delayed and could be observed after 36 and 24 h of culture, respectively. PTH-stimulated release of Ca 2+, P i, β-glucuronidase and β-N-acetylglucosaminidase was reduced by AHPrBP first after 24 h of culture. AHPrBP, HEBP and Cl 2MBP, at concentrations which are inhibitory on bone resorption, do not affect protein synthesis and mitotic activities in mouse calvaria. These data show that AHPrBP, HEBP and Cl 2MBP inhibit bone resorption in vitro and in parallel decrease lysosomal enzyme release by a mechanism, which is not related to cytotoxicity. In addition, the delayed inhibitory effect on bone resorption and lysosomal enzyme release by all the compounds suggest that bisphosphonates inhibit bone resorption indirectly and not by a direct effect on existing osteoclasts. The delayed inhibition by bisphosphonates on bone resorption may be due to decreased recruitment of new osteoclasts as a consequence of an inhibitory action on mononuclear osteoclast precursor cells.