IMMUNOHISTOCHEMICAL AND MOLECULAR CHARACTERIZATION OF THE RAT 11-BETA-HYDROXYSTEROID DEHYDROGENASE TYPE-II ENZYME

Mineralocorticoid action is facilitated by 11 beta-hydroxysteroid dehy drogenase type II (11 beta HSD2), which metabolizes glucocorticoids an d allows aldosterone to bind to the nonselective mineralocorticoid rec eptor. We have recently demonstrated the presence of the 11 beta HSD2 protein in a wide range of human epithelia, suggesting that it is the sole isoform endowing specificity in man. In the present study we have used an immunopurified polyclonal antibody (RAH23) raised against a C -terminal peptide derived from the cloned rat 11 beta HSD2 protein to perform immunohistochemical and molecular analysis in rat tissues. In frozen sections of rat kidney, strong staining was seen with the RAH23 antibody in the distal tubule; weaker staining was observed in the th ick ascending loop of Henle and the medullary and papillary collecting ducts. Punctate cortical staining was observed in the fetus at 20 day s gestation and in 8-day-old rats, with a noticeable increase in the s taining pattern at 16 days of age. The kidney did not attain the adult pattern of staining until 28 days of age. Epithelia of ileum and colo n also stained with RAH23, as did excretory ducts of the submandibular gland. Intrahepatic and excretory bile ducts displayed strong immunor eactivity in the epithelial lining. Rat adrenal glands showed evidence of the 11 beta HSD2 antigen in the zona fasciculata and zona reticula ris, but not in the zona glomerulosa or medulla. Western blot analysis with the RAH23 antibody revealed strong bands in the kidney, colon, a drenal gland, and submandibular gland at 40 kDa, colinear with the mig ration of the cloned 11 beta HSD2 enzyme. A band of medium intensity w as also seen at this size in the pancreas, whereas a band of moderate intensity was seen in the bile duct, and weaker bands were noticed in the stomach, small intestine, and liver, with a diffuse band at 36-42 kDa in the prostate. Strong bands were seen in the pancreas and prosta te at 78 kDa, with weaker signals in the colon, adrenal, stomach, and bile duct. A number of tissues also displayed multiple bands at about 30 kDa. Enzymatic assays on tissue homogenates showed extensive conver sion of corticosterone to its Il-dehydro product in an NAD-dependent m anner in the submandibular gland, adrenal gland, and kidney, but not i n the pancreas or prostate. This study confirms the ubiquitous presenc e of 11 beta HSB2 in sodium-transporting epithelia, demonstrates the h igh level of 11 beta HSD2 protein and enzyme activity in the rat adren al, and suggests a possible role for the enzyme in the biliary system. Further studies are required to determine the relevance of the variou s molecular species to the activity, latency, and processing of the en zyme.