Gallium(III) is a new therapeutic agent for hypercalcemia. Ga3+ reduces osteoclast action, but how it inhibits the cell's physiology is unknown. In vivo, 7-12-mu-M Ga(III) reduces calcium release from bone, but surprisingly, 10-100-mu-M Ga3+ added to isolated avian osteoclasts did not reduce their degradation of L-(5-H-3)-proline bone. H-3-proline labels bone collagen specifically, and collagenolysis is an excellent indicator of bone dissolution because collagen is the least soluble component of bone. Ga(III) > 100-mu-M inhibited osteoclasts in vitro, but also killed the cells. To resolve this apparent conflict, we measured Ga-67 distribution between bone, cells, and media. Gallium binds avidly but slowly to bone fragments. One hundred micrograms of bone clears 60% of 1-mu-M gallium from 500-mu-l of tissue culture medium, with steady state at > 24 h. Osteoclasts on bone inhibited gallium binding capacity approximately 40%, indicating a difference in available binding area and suggesting that osteoclasts protect their substrate from Ga binding. Less gallium binds to bone in serum-containing medium than in phosphate-buffered saline; 30% reduction of the affinity constant suggests that the serum containing medium competes with bone binding. Consequently, the effect of [Ga] on bone degradation was studied using accurately controlled amounts of Ga(III) pre-bound to the bone. Under these conditions, gallium sensitivity of osteoclasts is striking. At 2 days, 100-mu-g of bone pre-incubated with 1 ml of 1-mu-M Ga3+, with 10 pmoles Ga3+/mu-g bone, was degraded at 50% the rate of control bone; over 50 pM Ga3+/mu-g bone, resorption was essentially zero. In contrast, pre-treatment of bone with [Ga3+] as high as 15-mu-M had no significant effect on bone resorption rate beyond 3 days, indicating that gallium below approximately 150 pg/mu-g bone acts for a limited time and does not permanently damage the cells. We conclude that bone-bound Ga(III) from medium concentrations < 15-mu-M inhibits osteoclasts reversibly, while irreversible toxicity occurs at solution [Ga3+] > 50-mu-M.
