The effect of diphenylhydantoin upon the biosynthesis and degradation of collagen in cat palatal mucosa in organ culture

Abstract

The effect of diphenylhydantoin (DPH) upon collagen metabolism in cat palatal mucosa was studied in organ culture using a grid technique. The incorporation of [ 3H]proline into [ 3H]hydroxyproline was taken as a measure for collagen synthesis. Using a 24 hr [ 3H]proline pulse the collagen synthesis in the control explants was found to be constant from the third day until the end of the culture period at day 15. DPH, in the concentrations 5 and 20 μg/ml, did not influence collagen or non-collagen protein synthesis. When collagen synthesis was studied using continuous labelling with [ 3H]proline for 6 days, DPH increased the incorporation of radioactive collagen into the tissues. To study the collagen degradation cats were injected with [ 3H]proline and six days later the palatal mucosa was excised and cultured in the absence and presence of DPH for 6 days. The release of radioactive hydroxyproline into the culture medium was taken as a measure for collagen degradation. DPH (20 μg/ml) resulted in about a 50 per cent decreased release of radioactive and non-radioactive hydroxyproline to the medium. To study the effect of DPH on the degradation of in vitro labelled collagen, mucosal explants were cultured in the presence of [ 3H]proline for 24 hr, and post-cultured for another 24 hr period. The release of labelled and unlabelled hydroxyproline from the explants was then followed for 4 days. DPH was found to inhibit the release of radioactive hydroxyproline (from collagen 1–2 days old) only slightly, but caused a strong inhibition of the degradation of non-radioactive collagen. It is concluded that DPH is an inhibitor of collagen degradation and that the inhibition applies mainly to the degradation of older collagen.