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

The effects of 3-amino-1-hydroxy-propylidene-1,1-bisphosphonate (AHPrBP), 1-hydroxyethylidene-1,1-bisphosphonate (HEBP), dichloromethylenebisphosphonate (Cl2MBP) and azacycloheptylidene-2,2-bisphosphonate (AHBP) on bone were examined in organ culture using newborn mice calvaria. AHPrBP, HEBP and Cl2MBP caused a dose-dependent inhibition of PTH-stimulated (10 nmol/l) release of 45Ca from the calvaria, at and above a concentration of 3 mumol/l, whereas AHBP only caused a slight inhibition, at and above 100 mumol/l. AHPrBP inhibited PTH-stimulated release of 3H from bones prelabelled with [3H]-proline. AHPrBP (30 mumol/l) diminished the stimulatory effect of 1 alpha(OH)vitamin D3 (10 nmol/l), prostaglandin E2 (0.1 mumol/l) and renal tumor conditioned media on 45Ca release. AHPrBP and Cl2MBP, at and above 3 mumol/l, decreased PTH-stimulated mobilization of Ca2+ and Pi and in parallel the release of beta-glucuronidase without affecting the release of lactate dehydrogenase. The inhibitory effect of AHPrBP (30 mumol/l) on PTH-induced 45Ca release was irreversible. The inhibition by AHPrBP (30 mumol/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 mumol/l) and Cl2MBP (30 mumol/l) was delayed and could be observed after 36 and 24 h of culture, respectively. PTH-stimulated release of Ca2+, Pi, beta-glucuronidase and beta-N-acetylglucosaminidase was reduced by AHPrBP first after 24 h of culture. AHPrBP, HEBP and Cl2MBP, 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 Cl2MBP 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.