Bisphosphonates directly inhibit the bone resorption activity of isolated avian osteoclasts in vitro.

Bisphosphonates are useful in treatment of disorders with increased osteoclastic activity, but the mechanism by which bisphosphonates act is unknown. We used cultures of chicken osteoclasts to address this issue, and found that 1-hydroxyethylidenediphosphonic acid (EHDP), dichloromethylidenediphosphonic acid (Cl2MDP), or 3-amino-1-hydroxypropylidene-1,1-diphosphonic acid (APD) all cause direct dose-dependent suppression of osteoclastic activity. Effects are mediated by bone-bound drugs, with 50% reduction of bone degradation occurring at 500 nM to 5 microM of the different agents. Osteoclastic bone-binding capacity decreased by 30-40% after 72 h of bisphosphonate treatment, despite maintenance of cell viability. Significant inhibition of bone resorption in each case is seen only after 24-72 h of treatment. Osteoclast activity depends on ATP-dependent proton transport. Using acridine orange as an indicator, we found that EHDP reduces proton accumulation by osteoclasts. However, inside-out plasma membrane vesicles from osteoclasts transport H+ normally in response to ATP in high concentrations of EHDP, Cl2MDP, or APD. This suggests that the bisphosphonates act as metabolic inhibitors. Bisphosphonates reduce osteoclastic protein synthesis, supporting this hypothesis. Furthermore, [3H]leucine incorporation by the fibroblast, which does not resorb bone, is also diminished by EHDP, Cl2MDP and APD except when co-cultured with bisphosphonate-binding bone particles. Thus, the resorption-antagonizing capacities of EHDP, Cl2MDP and APD reflect metabolic inhibition, with selectivity for the osteoclast resulting from high affinity binding to bone mineral.

Receptor-mediated uptake of a mannose-6-phosphate bearing glycoprotein by isolated chicken osteoclasts.

We have recently shown that degradation of bone collagen by osteoclasts occurs via proteolytic enzyme activity that depends on an acidic milieu. Since bone resorption occurs in an extracellular, acidic compartment located at the cell-matrix attachment site, the osteoclast must deliver the acid collagenolytic enzymes to the cell surface. These observations raise the possibility that the mannose-6-phosphate (M-6-P) receptor, known to sort acidic proteases in other cells, is involved in trafficking lysosomal enzymes to the plasmalemma of bone resorbing cells. To this end we studied receptor-mediated uptake, distribution and release, by isolated chicken osteoclasts, of 125I-hexosaminidase, a M-6-P bearing enzyme. We found that at 4 degrees C, the bone-resorbing polykaryons bind approximately 10,000 molecules of radioligand/cell with a Kd of 0.7 nM, which is endocytosed by osteoclasts at 37 degrees C by a calcium-independent process. Furthermore, 125I-hexosaminidase uptake is unaffected by mannosylated albumin, documenting specificity of the receptor-mediated event. Release of endocytosed enzyme from the cell is also much more rapid than its degradation, attesting to a pathway of uptake and secretion. By autoradiography, the M-6-P bearing ligand is concentrated at the site of osteoclast-bone attachment. Thus, osteoclasts also have the capacity to deliver M-6-P bearing degradative enzymes to their surface at the site of matrix degradation.