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The carbonic anhydrase inhibitor, acetazolamide, has been found to elicit a hypocalcemic response in acutely nephrectomized rats. This effect cannot be attributed to changes in pCCh, pH citrate, phosphate or protein. Administration of acetazolamide to parathyroidectomized-nephrectomized rats has been shown to inhibit the hypercalcemic response to parathyroid extract and dibutyryl cyclic 3',5'-AMP. The hypercalcemic response to epinephrine was not inhibited in the same preparation. These data are consistent with the hypothesis that cyclic 3',5'-AMP is the second messenger of parathyroid hormone in its action on bone, and that carbonic acid production may be a later, or possibly the final, messenger of the hormone.

Zinc deficiency is associated with a reduced rate of bone formation that can be corrected by supplementation of the deficient diet with adequate amounts of zinc. This study was conducted to examine the effects of zinc on bone cell parameters associated with bone formation. Tibiae were removed from 19-d-old chicken embryos and incubated for 48 h in Dulbecco's modified Eagle's medium supplemented with antibiotics, bovine serum albumin, and HEPES. The addition of zinc (25-200 g/dL) to tibial cultures resulted in a concentration-dependent increase in alkaline phosphatase activity, an increase in the incorporation of proline into bone protein and an increase in the post-translational oxidation of proline to peptidyl hydroxyproline. These effects of zinc were all diminished by the addition of 2,6-pyridine dicarboxylic acid, a chelator of zinc. The addition of either cycloheximide (10(-5)M), dactinomycin (10(-8)M), or hydroxyurea (10(-3)M) to tibial cultures also attenuated the effects of zinc. The effect of zinc on bone cell DNA synthesis was measured by following the incorporation of 3H-thymidine into DNA and by fluorometric measurement of cellular DNA content. These methods revealed that the addition of zinc to cultured tibiae resulted in a concentration-dependent increase in tibial DNA content and synthesis rate. The magnitude of the zinc-induced DNA increase was similar to the magnitude of the zinc-induced increases in alkaline phosphatase activity, proline incorporation, and hydroxyproline synthesis. Normalization of these latter responses to tibial DNA content yield data indicating that the effect of zinc on bone formation results from a zinc-induced increase in bone cell proliferation.

Many investigations have indicated a functional role for carbonic anhydrase in the mediation of hormone-stimulated bone resorption. These studies depend heavily on the use of heterocyclic sulfonamide inhibitors of carbonic anhydrase. These drugs have effects on many tissues other than bone, and some of these effects confound the interpretation of studies of the role of carbonic acid in bone metabolism. A novel, "bone-targeted" sulfonamide has been produced to obviate these extraosseous effects. This compound (designated WP-1) is the combination of tetracycline and acetazolamide, such that the acetazolamide is not an active inhibitor. Hydrolysis of WP-1 yields an active carbonic anhydrase inhibitor. WP-1 has a marked affinity for bone mineral, allowing deposition of the drug in bone. At a concentration of 1 0-5 M , WP-1 attenuates parathyroid hormone stimulated net release of calcium from neonatal rat calvaria in culture. WP-1 is the first member of a class of drugs which may prove useful as pharmacological probes in the study of bone metabolism. 0 1987 Society for Experimental Biology and Medicine. 96

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