Role of carbonic anhydrase in bone resorption induced by 1,25 dihydroxyvitamin D3 in vitro

Summary The present investigation was undertaken to study the role of carbonic anhydrase in 1,25 dihydroxyvitamin D₃-induced bone resorption. Calvaria were removed from 5- to 6-day-old mice and cultured for periods up to 96 h in Dulbecco's Modified Eagle Medium (high glucose, 4,500 mg/dl) supplemented with antibiotics and either heat-inactivated horse and fetal calf sera or bovine serum albumin. The experimental cultures contained 1×10⁻⁸ M 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃). All cultures were incubated at 37°C in 5% CO(in2)/95% air. Bone resorption was assessed by release of stable calcium into the medium. Bone enzymes (acid and alkaline phosphatases and carbonic anhydrase) were determined following homogenization in 0.25 M sucrose. The effects of 1,25(OH)₂D₃ were studied in the presence and absence of the carbonic anhydrase inhibitor acetazolamide and its analogue (CL 13,850), which lacks inhibitory activity. Acetazolamide inhibited 1,25(OH)₂D₃-induced calcium release in a dose-dependent fashion from 10⁻⁵–10⁻⁴ M. When added to the cultures at a concentration of 1×10⁻⁴ M, acetazolamide completely blocked the 1,25(OH)₂D₃-induced calcium release, a phenomenon not seen with an equimolar concentration of CL 13,850. The most significant finding was that 1,25(OH)₂D₃-induced calcium release was accompanied by a significant increase in the carbonic anhydrase activity of bone at both 48 (treated/control ratio=2.05) and 96 (treated/control ratio=2.59) hours. Bone alkaline phosphatase activity decreased and acid phosphatase activity increased in response to 1,25(OH)₂D₃. These findings support the concept that carbonic anhydrase is involved in bone resorption inducedin vitro by certain calcemic hormones and related compounds.