Effect of oxygen tension and indomethacin on production of collagenase and neutral proteinase enzymes and their latent forms by porcine gingival explants in culture

Abstract

Porcine gingival explants cultured in a serum-free minimally essential medium produced at least two distinct neutral proteinases which, in combination, digested tissue collagen. One proteolytic activity was typical of collagenases with a specific, limited activity on native gingival collagens. The second degraded denatured collagen. Both enzymes also existed in latent forms which were activated during culture. The enzymes were inhibited by EDTA and O-phenanthroline, and to some extent by dithiothreitol and cysteine. By increasing the O 2 tension of the culture medium from pO 2 of 150mm Hg to pO 2 of 280mm Hg, the production of both enzymes and their precursors was increased two-fold. Conversely. 5 μg/ml indornethacin in the medium significantly depressed the synthesis of these enzymes. When gingival epithelial tissue and connective tissue were cultured separately, neutral proteinases were only produced by the connective tissue. The production of the collagenolytic enzymes was accompanied by a marked general loss of tissue cellularity and cell necrosis. Radioautography with 3H-proline indicated that protein synthesis was most active at the epithelium-connective tissue junction. The collagenase and neutral proteinase appear to have a similar catalytic mechanism and may be secreted as a collagenolytic enzyme package by the connective tissue cells. The inhibition of the production of these enzymes by indomethacin indicates that prostaglandins may have an important role in the degradation of gingival connective tissue.