Investigation of 11beta-Hydroxysteroid Dehydrogenase in Salivary Gland

The enzyme responsible for catalysing the oxidation-reduction of the 11-oxygen functions of steroids is 11beta-hydroxysteroid dehydrogenase (11beta-HSD). Previous physiological and histochemical studies have shown that the enzyme occurs in salivary gland, as demonstrated by the conversion of cortisol to cortisone in significant quantities. The aims of this thesis were to report more fully on the investigation of 11beta-HSD in rat submandibular gland. A reverse isotope dilution technique using cortisol-4-14C as substrate was used to assay the enzyme in homogenate, spectrophotometric and fluoro-metric assays being unsuitable. Cortisol was converted to cortisone, as identified by formation of cortisone derivatives and recrystallisation to constant specific activity. A variety of assay parameters were investigated to find the optimal conditions of enzyme activity. Nicotinamide adenine dinucleotide was the preferred cofactor to nicotinamide adenine dinucleotide phosphate with the optimal pH conditions for the cofactor-linked enzyme being pH8.1-8.9 and pH7.6 respectively. Both cofactors were in saturating amounts at 1.25mM in the assay volume of 11ml. A phosphate buffer showed no side effects whereas borate and tris buffer showed chemical reactions and therefore phosphate buffer was used to control pH. The optimal temperature for the NAD- and NADP-linked 11beta-hydroxysteroid dehydrogenase was 37C for incubations of 2 mins. and 7 mins. respectively. Km and Vmax values for the NAD-linked 11beta-hydroxysteroid dehydrogenase were 0.48muM and 1.27 n moles/min., the NADP-linked 11beta-hydroxysteroid dehydrogenase values being 0.95muM and 0.37n moles/min. The substrate concentration chosen for enzyme assay was approximately 2xKm of the NAD-linked enzyme. Verification of enzyme assay for 100mg gland homogenates was shown by increasingly proportional enzyme activity with enzyme (gland) concentration around this value. Reversibility of the reaction using cortisone-4-14C as substrate could not be shown although reduced cofactors and a variety of pH values and incubation periods were used. One of the effects of 113-HSD in salivary gland is, therefore, to convert physiologically active steroids into inactive steroids by 11beta-hydroxyl group transfer to 11-oxo group steroids. Corticosterone, the naturally secreted adrenocorticoid in the rat, was metabolised by the submandibular gland 11beta-HSD. Rat kidney was also used to investigate 11beta-hydroxysteroid dehydrogenase activity. The conversion of cortisol to cortisone was confirmed but using cortisone as substrate it was converted to a product other than cortisol thus indicating the difference between kidney and salivary gland metabolism of cortisone. The intracellular localisation of 11beta-hydroxysteroid dehydrogenase was studied by incubating subcellular particles of the rat submandibular salivary gland for the NAD-linked 11beta-hydroxysteroid dehydrogenase. The subcellular particles were isolated from homogenate by differential centrifugation in sucrose. The main enzyme activity was found in the crude nuclear fraction and this led to the investigation of what part of the nuclear fraction was responsible for the enzyme activity. Nuclei were isolated using 1.8M Sucrose and these were found to retain the major enzyme activity. Attempts to solubilise the nuclear enzyme using detergent and hypotonic media were unsuccessful. It is of interest to see that the enzyme is nuclear bound as the liver enzyme has been located in the microsomal (105,000g/hr.) fraction. The enzyme in the salivary gland may influence nucleic acid metabolism. However, further work will have to be undertaken to investigate this possibility. It was recognised that the rat submandibular gland was a tissue of differentiated cell types. In the study of 11beta-hydroxysteroid dehydrogenase it was considered important to investigate the activity of the steroid enzyme in separated tubules using the enzyme, collagenase, to break up the gland. However, little activity was recovered in the tubules probably because the collagenase used included a number of protease and peptidase impurities. The histochemical localisation of the enzyme was in the duct cells. It was not located in any single component of the salivary duct system but was evident in striated, lobular and interlobular excretory ducts. Acinar tissue did not exhibit any activity. Enzymes involved in central metabolic pathways were present principally in the ducts whereas alkaline phosphatase was present in the acinar tissue and not in the duct system. Acid phosphatase appeared in equal amounts in both the acinar and ductal tissue. The presence of these enzymes gives further proof of the concept of a highly active metabolic duct epithelium. The culture of salivary gland ducts was investigated but no 11beta-hydroxysteroid dehydrogenase activity was present in cultures which were 7 days and 3 weeks old. The enzyme may require the addition of various cofactors and hormones which are lacking in the tissue culture