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

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Abstract
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.