Inhibition of bone resorption in vitro by serine-esterase inhibitors

Abstract

The effect of two synthetic serine esterase inhibitors, N- α-dansyl( p-guanidino)phenylalaninepiperidine hydrochloride (I 2581) and d-phenylalanyl- l-prolyl- l-arginine chloromethyl ketone ( d-Phe-Pro-Arg-CH 2Cl), on bone resorption in organ cultured mouse calvaria from neonatal mice has been examined. Mineral mobilization was assessed by analyzing the release of 45Ca, stable calcium (Ca 2+) and inorganic phosphate (P i). Organic matrix degradation was studied by analyzing the release of 3H from [ 3H]proline-labelled bones, and by quantifying the amounts of hydroxyproline in bone after culture. It was found that I 2581, at and above 30 μmol/l, dose-dependently inhibited 45Ca release induced by thrombin, parathyroid hormone (PTH), prostaglandin E 2 and 1-α-hydroxyvitamin D-3. I 2581 (50 μmol/l) inhibited PTH-stimulated release of 3H from [ 3H]proline-labelled bones, and this effect was reversible after withdrawal of I 2581. I 2581 (50 μmol/l) inhibited the release of Ca 2+, P i, β-glucuronidase and β- N-acetylglucosaminidase in bones stimulated by PTH and 1-α-hydroxyvitamin D-3, without affecting the release of lactate dehydrogenase. In parallel, I 2581 decreased PTH and 1-α-hydroxyvitamin D-3 induced reduction of hydroxyproline levels in bones after culture. I 2581 (50 μmol/l) did not affect the basal release of 45Ca, Ca 2+, β-glucuronidase and β- N-acetylglucosaminidase, nor the basal amounts of hydroxyproline in bones after culture. d-Phe-Pro-Arg-CH 2Cl (100 μmol/l) significantly inhibited PTH- and PGE 2-induced release of 45Ca without affecting basal release of radioactive calcium. These data indicate that activation of serine proteinase(s) may be a necessary step in the mechanism of action of several stimulators of bone resorption.