Smith R E, Li K X, Andrews R K, Krozowski Z
Laboratory of Molecular Hypertension, Baker Medical Research Institute, Prahran, Australia.
Endocrinology. 1997 Feb;138(2):540-7. doi: 10.1210/endo.138.2.4926.
Mineralocorticoid action is facilitated by 11 beta-hydroxysteroid dehydrogenase type II (11 beta HSD2), which metabolizes glucocorticoids and allows aldosterone to bind to the nonselective mineralocorticoid receptor. 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 thick ascending loop of Henle and the medullary and papillary collecting ducts. Punctate cortical staining was observed in the fetus at 20 days gestation and in 8-day-old rats, with a noticeable increase in the staining 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 colon also stained with RAH23, as did excretory ducts of the submandibular gland. Intrahepatic and excretory bile ducts displayed strong immunoreactivity in the epithelial lining. Rat adrenal glands showed evidence of the 11 beta HSD2 antigen in the zona fasciculata and zona reticularis, but not in the zona glomerulosa or medulla. Western blot analysis with the RAH23 antibody revealed strong bands in the kidney, colon, adrenal gland, and submandibular gland at 40 kDa, colinear with the migration of the cloned 11 beta HSD2 enzyme. A band of medium intensity was 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 prostate 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 conversion of corticosterone to its 11-dehydro product in an NAD-dependent manner in the submandibular gland, adrenal gland, and kidney, but not in the pancreas or prostate. This study confirms the ubiquitous presence of 11 beta HSD2 in sodium-transporting epithelia, demonstrates the high level of 11 beta HSD2 protein and enzyme activity in the rat adrenal, and suggests a possible role for the enzyme in the biliary system. Further studies are required to determine the relevance of the various molecular species to the activity, latency, and processing of the enzyme.
11β-羟类固醇脱氢酶2型（11βHSD2）可促进盐皮质激素的作用，它使糖皮质激素代谢，从而让醛固酮能够与非选择性盐皮质激素受体结合。我们最近证实，11βHSD2蛋白存在于多种人类上皮细胞中，这表明它是赋予人类特异性的唯一同工型。在本研究中，我们使用了一种针对从克隆的大鼠11βHSD2蛋白衍生的C末端肽制备的免疫纯化多克隆抗体（RAH23），对大鼠组织进行免疫组织化学和分子分析。在大鼠肾脏的冰冻切片中，RAH23抗体在远曲小管中染色强烈；在髓袢升支粗段以及髓质和乳头集合管中染色较弱。在妊娠20天的胎儿和8日龄大鼠的皮质中观察到点状染色，16日龄时染色模式有明显增加。直到28日龄，肾脏才达到成年期的染色模式。回肠和结肠的上皮细胞也被RAH23染色，下颌下腺的排泄管也是如此。肝内胆管和排泄胆管的上皮衬里显示出强烈的免疫反应性。大鼠肾上腺在束状带和网状带显示有11βHSD2抗原的证据，但在球状带或髓质中没有。用RAH23抗体进行的蛋白质印迹分析显示，在肾脏、结肠、肾上腺和下颌下腺中出现了40 kDa的强条带，与克隆的11βHSD2酶的迁移一致。在胰腺中也在这个大小处看到一条中等强度的条带，而在胆管中看到一条中等强度的条带，在胃、小肠和肝脏中看到较弱的条带，在前列腺中在36 - 42 kDa处有一条弥散条带。在胰腺和前列腺中在78 kDa处看到强条带，在结肠、肾上腺、胃和胆管中有较弱的信号。许多组织在约30 kDa处也显示出多条条带。对组织匀浆的酶活性测定表明，在下颌下腺、肾上腺和肾脏中，皮质酮以NAD依赖的方式大量转化为其11 - 脱氢产物，但在胰腺或前列腺中则不然。本研究证实了11βHSD2在钠转运上皮细胞中普遍存在，证明了大鼠肾上腺中11βHSD2蛋白和酶活性水平很高，并提示该酶在胆道系统中可能发挥作用。需要进一步研究来确定各种分子形式与该酶的活性、潜伏性和加工过程的相关性。
