THE BISPHOSPHONATE TILUDRONATE IS A POTENT INHIBITOR OF THE OSTEOCLAST VACUOLAR H-ATPASE()

Although bisphosphonates have been shown to be potent inhibitors of os teoclast-mediated bone resorption in vivo and in vitro and are used as therapeutic agents in hyper-resorptive bone diseases such as Paget di sease or hypercalcemia of malignancy, their exact biochemical target(s ) and mode(s) of action are for the most part still unknown. Tbe resor ption of bone requires solubilization of the mineral component of the matrix, achieved by acidification of the resorbing compartment by a va cuolar-type proton ATPase (V-ATPase) present in the ruffled border mem brane of osteoclasts. Since we have shown that the V-ATPase is inhibit ed by both ADP and phosphate, which share structural characteristics w ith bisphosphonates, we hypothesized that inhibition of the osteoclast V-ATPase could be one of the mechanism(s) by which bisphosphonates in hibit bone resorption. Pyrophosphate and the bisphosphonates etidronat e, alendronate, and YM-175 inhibited proton transport in membrane vesi cles derived from chicken kidney and osteoclasts but with very low pot ency (IC50 greater than or equal to 5 mM). In contrast, the ability of tiludronate to inhibit proton transport was 5-fold higher in kidney-d erived vesicles (IC50 = 1.1 mM) and 10,000-fold higher in vesicles der ived from osteoclasts (IC50 = 466 nM). Tiludronate also potently inhib ited proton transport in yeast microsomal preparations (IC50 = 3.5 mu M) and inhibited the activity of purified yeast V-ATPase. The inhibiti on of the osteoclast V-ATPas-mediated proton transport by tiludronate was rapid, pa-dependent, and reversible. No change in membrane vesicle permeability to protons was detected. The inhibition was noncompetiti ve with respect to ATP, and tiludronate did not protect the pump from inactivation by N-ethylmaleimide, strongly suggesting that tiludronate does nob: bind to the catalytic site of the enzyme. It is concluded t hat tiludronate is a significantly more potent inhibitor of V-ATPases than other bisphosphonates and that it has a significant degree of sel ectivity for the avian osteoclast V-ATPase relative to the avian kidne y V-ATPase.