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PubMed1991

Glycyrrhetinic acid, an inhibitor of 11 beta-hydroxysteroid dehydrogenase, alters local cerebral glucose utilization in vivo.

11 beta-Hydroxysteroid dehydrogenase (11 beta-OHSD) metabolizes corticosterone (B) to inactive 11-dehydrocorticosterone and thus protects the non-specific renal mineralocorticoid receptor from exposure to B in vivo. There is regional 11 beta-OHSD mRNA expression and bioactivity in brain in vitro, but any in vivo function is unknown. We used the [14C]2-deoxyglucose technique in conscious rats to investigate whether 11 beta-OHSD inhibition with glycyrrhetinic acid alters local cerebral metabolic activity. We found increased glucose use in subregions of the hypothalamus, hippocampus, neocortex and subthalamus. Thus, 11 beta-OHSD may play a role in regulating the effects of B in the brain, in vivo.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1992

Tissue localization of 11 beta-hydroxysteroid dehydrogenase and its relationship to the glucocorticoid receptor.

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) dictates specificity for the mineralocorticoid receptor (MR) by converting the active steroid cortisol to cortisone in man (corticosterone to 11-dehydrocorticosterone in rodents), leaving aldosterone to occupy the MR. However cortisol is the principal circulating glucocorticoid in man and 11 beta-HSD, distributed in a tissue specific fashion, may represent a powerful mechanism in regulating exposure of active steroid to the glucocorticoid receptor (GR). A detailed localization study of 11 beta-HSD gene expression and activity in numerous rat tissues has been performed and compared with the presence of GR mRNA. 11 beta-HSD mRNA (1.4 kB) measured by hybridization to a cDNA derived from hepatic 11 beta-HSD, and enzyme activity, measured by percentage conversion of [3H]corticosterone to [3H]11-dehydrocorticosterone by tissue homogenate, was widespread, present in all tissues studied except spleen, brain cortex and heart. There was a close correlation between tissue 11 beta-HSD mRNA levels and activity (r = 0.91, P less than 0.001) suggesting pretranslational regulation of the enzyme at a tissue level. There was also close co-localization of GR mRNA (7 kB), measured by hybridization to a rat GR cRNA probe, and enzyme mRNA/activity in every tissue studied except heart and brain cortex in which GR mRNA was found. In the mineralocorticoid target tissues kidney and colon, additional 11 beta-HSD mRNA bands were seen (kidney 1.8 kB, colon 3.4 kB), suggesting the presence of multiple dehydrogenase species. 11 beta-HSD is widely distributed and suitably placed to modulate ligand occupancy of the GR. The possibility of multiple dehydrogenase species in mineralocorticoid target tissues is consistent with the hypothesis that the ubiquitous 'native' 1.4 kB hepatic enzyme regulates the GR, and these separate dehydrogenases regulate the MR.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1993

The forms and functions of 11 beta-hydroxysteroid dehydrogenase.

In this review, we consider the relationship between the structure and function of 11 beta-hydroxysteroid dehydrogenase (11-HSD) purified from rat liver. The rat liver enzyme is a single domain glycoprotein with a unique active site and belongs to the short chain alcohol dehydrogenase family. Evidence supporting the presence in other tissues of 11-HSD isoforms is discussed.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1993

Tissue specific effects of thyroid hormone on 11 beta-hydroxysteroid dehydrogenase gene expression.

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) by converting active glucocorticoid to an inactive metabolite confers specificity upon the mineralocorticoid receptor (MR) and regulates ligand access to the glucocorticoid receptor (GR). Factors which influence 11 beta-HSD activity seem likely to be of considerable importance in the modulation of both mineralocorticoid and glucocorticoid hormone action. The administration of tri-iodothyronine (T3) to rats has previously been shown to reduce 11 beta-HSD activity in liver but not in kidney. We have studied the effect of T3 on 11 beta-HSD gene expression in vivo in rat liver, kidney, distal colon and pituitary. In addition the effects of T3 on 11 beta-HSD gene expression in vitro in the rat pituitary GH3 cell line have been studied. T3 administration to normal adult rats (40 micrograms/day, s.c. for 1, 3 and 7 days) resulted in a marked decline in liver and pituitary 11 beta-HSD mRNA levels and activity following 3 and 7 days of treatment. These reduced levels were maintained for 3 days following withdrawal of T3 treatment, but returned to control levels after 7 days. In contrast 11 beta-HSD mRNA and activity in kidney and distal colon were unaffected by T3 treatment at each time point studied. In vitro, levels of 11 beta-HSD mRNA and activity in GH3 cells were unchanged following 8, 24 and 72 h treatment with T3 (10(-8) to 10(-6) M). T3 bio-activity was confirmed by a marked dose-dependent decline in the expression of the T3 and glucocorticoid responsive gene, prolactin. T3 inhibits 11 beta-HSD gene expression in both liver and pituitary at a pre-translational level. This effect is absent in the predominantly mineralocorticoid target tissues, kidney and distal colon, i.e. it is tissue specific and as such is consistent with the existence of multiple differentially regulated isoforms of 11 beta-HSD. The time course of the T3 effect in liver and pituitary in vivo and the lack of any effect in vitro suggests that this action is indirect, and not as a result of interaction between the T3 receptor and the putative thyroid hormone response element on the rat 11 beta-HSD gene.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1993

Differential inhibition of 11 beta-hydroxysteroid dehydrogenase by carbenoxolone in rat brain regions and peripheral tissues.

Carbenoxolone (CX), the succinyl ester of glycyrrhetinic acid, causes hypokalemia and hypernatremia. Its pharmacological effects are believed to be due to its inhibition of 11 beta-hydroxysteroid dehydrogenase (11-HSD). There was a marked inhibition of this enzyme in the liver, kidney, pituitary, hippocampus, hypothalamus and amygdala 1 h after intraperitoneal administration of CX (100 mg kg-1) to intact male rats. Intracerebral injection of CX (1.5 mg kg-1) into the 3rd ventricle inhibited the oxidation of corticosterone to 11-dehydrocorticosterone by 11-HSD in the pituitary and hippocampus and produced marked behavioral hyperactivity but had no effect in the liver or kidney. Lower amounts of CX (10-50 micrograms/rat) given intracerebroventricularly (i.c.v) were without significant effect on 11-HSD in the pituitary or amygdala 1 h after infusion but inhibited this enzyme differentially in the hippocampus and hypothalamus. Inhibition of 11-HSD activity in the hippocampus and hypothalamus was observed up to 6 h after i.c.v. administration of CX (50 micrograms/rat) together with some decrease in activity of this enzyme in the pituitary at 3 h. The findings that low doses of CX given i.c.v. can alter the activity of 11-HSD in specific brain regions without affecting its activity in peripheral tissues, and only marginally in the pituitary, provides a method to study the central role of this enzyme independently of systemic effects.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1993

Barbiturates modulate the activity of the pituitary 3 alpha-hydroxysteroid oxidoreductases and inhibit their production of 3 alpha,5 alpha-tetrahydroprogesterone.

The effects of sodium phenobarbital and sodium barbital on the activity of the particulate and cytosolic 5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductases (3 alpha-HSORs) of female rat anterior pituitary were investigated. By altering the 3 alpha-HSOR catalyzed conversion of 5 alpha-dihydroprogesterone (5 alpha-DHP) to 3 alpha,5 alpha-tetrahydroprogesterone (3 alpha,5 alpha-THP), these barbiturates could influence the in situ production of 3 alpha,5 alpha-THP. 3 alpha,5 alpha-THP has potent barbiturate-like effects on brain GABAA receptors. Both phenobarbital and 3 alpha,5 alpha-THP can affect gonadotropin release in female rats. In vitro incubations of each 3 alpha-HSOR activity were assayed in the presence of sodium phenobarbital (0.1 to 10.0 mM) or sodium barbital (1.0 to 10.0 mM). Since both 3 alpha-HSOR activities catalyze the reversible oxidoreduction of 5 alpha-DHP and 3 alpha,5 alpha-THP, we examined the effect of these barbiturates not only on the conversion of 5 alpha-DHP to 3 alpha,5 alpha-THP (reductive reaction) but also on the "back conversion" of 3 alpha,5 alpha-THP to 5 alpha-DHP (oxidative reaction). The results indicate that both phenobarbital and, to a lesser extent barbital, significantly affected the activities of the two 3 alpha-HSORs in both reductive and oxidative directions. In the reductive direction, phenobarbital inhibited the activity (33%) of both cytosolic and particulate enzymes which would presumably decrease the levels of 3 alpha,5 alpha-THP. In the oxidative direction, a pattern of stimulation was observed (20 to 100%). Thus, this stimulatory effect on the oxidative conversion of 3 alpha,5 alpha-THP to 5 alpha-DHP, which would presumably also decrease 3 alpha,5 alpha-THP levels, appears correlated with the inhibitory effect of these barbiturates on the reductive conversion of 5 alpha-DHP to 3 alpha,5 alpha-THP. Sodium barbital exhibited somewhat similar effects. These changes suggest that barbiturates can lower 3 alpha,5 alpha-THP levels in the anterior pituitary. The results also suggest the possibility that lowered 3 alpha,5 alpha-THP levels may be involved, at least in part, in the reduction of gonadotropin release by barbiturates.

3-Hydroxysteroid Dehydrogenases↗
PubMed1995

Changes in the levels of 11 beta-hydroxysteroid dehydrogenase mRNA over the oestrous cycle in the rat.

The NADP dependent enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) metabolizes glucocorticoids to their inactive 11-keto-metabolites in a wide range of tissues. To date very little is known about the regulation of this enzyme at the level of gene transcription. In this study we show significant changes in the uterine, renal, ovarian and hepatic levels of 11 beta HSD1 mRNA over the oestrous cycle. Uterine and renal message levels followed the same pattern, with the highest levels observed at dioestrus and the lowest levels at oestrus, a pattern that correlates with plasma oestrogen levels during the cycle. In both the uterus and kidney 11 beta HSD1 message levels more than halved from dioestrus to oestrus, while renal levels than doubled at metoestrus. In contrast, hepatic 11 beta HSD1 message levels at prooestrus were twice those observed at metoestrus. Ovarian levels remain constant until metoestrus when a marked decrease in message levels was seen. 11 beta HSD1 mRNA levels are thus differentially regulated in a tissue specific manner throughout the oestrous cycle.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1995

Cloning, sequencing and tissue-distribution of mouse 11 beta-hydroxysteroid dehydrogenase-1 cDNA.

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) reversibly converts physiological glucocorticoids (cortisol, corticosterone) to inactive 11-dehydro forms, and thus controls glucocorticoid access to receptors in a variety of tissues. We have cloned a cDNA encoding 'liver-type' 11 beta-HSD (11 beta-HSD1) from the mouse using PCR, and have determined its nucleotide sequence. Mouse 11 beta-HSD1 cDNA showed 91% identity to rat 11 beta-HSD1 cDNA. There was 87% amino acid identity with rat 11 beta-HSD1 with conservation of the putative cofactor and substrate binding domains. Northern blot analysis of mouse tissues demonstrated abundant 11 beta-HSD1 message in the liver, kidney and lung, with lower expression in brain subregions and gonads. 11 beta-HSD1 mRNA was below the level of detection in the murine colon. 11 beta-HSD1 mRNA levels in kidney was higher in males than in females, but in contrast to the rat, there was no sexual dimorphism in the mouse liver. Although males and females showed different mRNA levels in the kidney, there was no sex difference in 11 beta-HSD enzyme activity. Thus, despite the high inter-species conservation of 11 beta-HSD1, there are clear species and tissue-specific differences in its expression.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1995

Co-expression of two distinct isoforms of 11 beta-hydroxysteroid dehydrogenase in the ovine placenta.

We have previously described two distinct isoforms of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) with respect to enzymatic activity in the ovine liver and kidney. To determine which isoform(s) is expressed in the ovine placenta, we studied the characteristics of 11 beta-HSD activity in placental tissues collected at days 140-143 of pregnancy. 11 beta-HSD activity was determined by a radiometric conversion assay using cortisol and cortisone as physiological substrates. At 100 nM cortisol, the placental 11 beta-HSD utilized NAD as cofactor, but displayed preference for NADP at 10 microM cortisol. Kinetic characteristics were examined in the presence of alternate cofactors, in order to determine whether this difference in the cofactor requirement represents distinct enzymes. With NAD as cofactor, the placental 11 beta-dehydrogenase had a Km (110 +/- 18 nM) compatible with the kidney enzyme, but displayed a Km (12 +/- 2 microM) similar/identical to the liver 11 beta-HSD when NADP was used. By contrast, the placental 11-oxoreductase showed preference for NADPH regardless of cortisone concentration. Kinetic analysis, using NADPH as cofactor, revealed a single species of 11-oxoreductase activity with a Km of 4 +/- 0.9 microM and a Vmax of 3.1 +/- 0.5 pmol/mg/min. Finally, since the NAD-dependent 11 beta-HSD in the ovine placenta displayed similar/identical kinetic characteristics to the enzyme described previously in the ovine kidney where a truncated 11 beta-HSD transcript was identified, we have also determined whether this transcript is expressed in the placenta by Northern blotting. It was found that the truncated 11 beta-HSD transcript was undetectable in the total RNA samples. These results demonstrate that both liver- and kidney-types of 11 beta-HSD activities are expressed in the ovine placenta, thus providing further evidence for the existence of a NAD-dependent 11 beta-HSD distinct from the well-characterized hepatic NADP-dependent enzyme. Furthermore, the lack of the truncated 11 beta-HSD transcript in the placenta suggests that the NAD-dependent enzyme identified in placenta and kidney is the product of a gene distinct from 11 beta-HSD.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1994

11 beta-Hydroxysteroid dehydrogenase and tissue specificity of androgen action in human prostate cancer cell LNCaP.

Incubation of whole LNCaP cells in suspension with tritium labeled cortisol revealed two major and one minor radioactive product. Of the major products, one migrated with an Rf value identical to cortisol (Kendall's compound "F"), and the second migrated with an Rf value similar to nonradioactive cortisone (Kendall's compound "E"); the third minor product comigrated with 21-acetylated cortisol. The conversion of cortisol to cortisone was linear with respect to cell number, and conversion reached a plateau after 120 min of incubation at 37 degrees C. One half of the cortisol was converted to cortisone within 2 h of incubation at 37 degrees C. This conversion was nicotine amide dinucleotide (NAD) dependent. Low levels of transcription activation by cortisol were documented in LNCaP cells transfected with glucocorticoid and androgen responsive mouse mammary tumor virus-bacterial chloramphenicol acetyltransferase chimeric gene (MMTV-CAT). Hormone binding assay and transactivation analysis revealed the presence of a functional mineralocorticoid receptor in LNCaP cells. Treatment of transfectants with F in the presence of carbenoxolone, a potent inhibitor of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), resulted in a two orders of magnitude increase in measurable CAT activity. The addition of the reduced form of nicotine amide dinucleotide (NADH) in the presence of 10(-7) M E stimulated measurable CAT activity in LNCaP cells. In conferring aldosterone specificity in mineralocorticoid target tissues, 11 beta-HSD may have an important role as "gate keeper" in allowing a specific androgen response in hormone responsive LNCaP prostate cancer cells.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1994

Evidence for distinct isoforms of 11 beta-hydroxysteroid dehydrogenase in the ovine liver and kidney.

We have previously identified a unique 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) transcript in the ovine kidney. To examine whether this is indicative of a distinct isoform with respect to enzymatic activity, we studied and compared the characteristics of 11 beta-HSD activity in the ovine liver and kidney. 11 beta-HSD activity was determined by a radiometric conversion assay using cortisol and cortisone as physiological substrates. Although in both liver and kidney, the enzyme was localized by subcellular fractionation in the microsomes, the renal 11 beta-HSD displayed distinct characteristics in that it expressed only dehydrogenase activity and utilized almost exclusively NAD as cofactor (the respective activity in the presence of NAD and NADP was 190 +/- 26 and 12 +/- 2 pmol/min/mg protein). By contrast, the liver enzyme contained both dehydrogenase and reductase activities, and displayed preference for NADP and NADPH, respectively. Furthermore, with cortisol as substrate, the kidney 11 beta-HSD had a Km of 68 +/- 7 nM which was over 100 times lower than the hepatic enzyme (8 +/- 1 microM). In addition, the renal 11 beta-HSD activity was inhibited in a dose-dependent fashion by both carbenoxolone, a potent inhibitor of 11 beta-HSD, and the end product cortisone, whereas the liver enzyme showed little inhibition by either substance. In summary, these results provide strong evidence for the existence of distinct isoforms of 11 beta-HSD with respect to enzymatic activity in the ovine liver and kidney. In addition, the characteristics of the kidney enzyme closely resemble those of that described previously in the rabbit renal aldosterone target cells, and thus further demonstrating the presence of an isoform of 11 beta-HSD distinct from the NADP-dependent enzyme purified and cloned from the rat liver.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1994

The purification of 11 beta-hydroxysteroid dehydrogenase from mouse liver microsomes.

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) is a microsomal enzyme complex responsible for the interconversion of active 11-hydroxy glucocorticoids to inactive 11-oxo metabolites. It has long been controversially discussed whether 11-dehydrogenation and 11-oxoreduction are catalysed by a single bidirectional enzyme or if the 11 beta-HSD system comprises 2 kinetically distinct microsomal enzyme activities, 11-dehydrogenase and 11-oxoreductase. However, 11-oxoreduction of homogeneously purified 11 beta-HSD could not be demonstrated under in vitro conditions until today. We have purified 11 beta-HSD from mouse liver microsomes to homogeneity by a purification method which affords a gentle membrane protein solubilization as well as providing a favourable detergent surrounding during the various chromatographic steps. Following 11-dehydrogenation of corticosterone and 11-oxoreduction of dehydrocorticosterone simultaneously throughout the entire purification procedure we could demonstrate that 11 beta-HSD retains both oxidative and reductive activities in almost the same ratio, which is also true for the homogeneously purified enzyme. Deducing from the coincidentally increasing specific activities of 11-dehydrogenation and 11-oxoreduction the conclusion can be drawn that both activities reside within the same protein. Furthermore, in addition to NADP(H) also NAD(H) can serve as cosubstrate, which is mainly true for the oxidative direction. In conclusion, our results provide evidence that the oxidative and reductive behaviour of 11 beta-HSD can be explained by the concept of a unique, reversible oxidoreductase thus disproving the two enzyme theory.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1994

Inhibition of 11 beta-hydroxysteroid dehydrogenase obtained from guinea pig kidney by furosemide, naringenin and some other compounds.

Inhibition of 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) can cause excess mineralocorticoid effects and hypokalemia. Several substances causing hypokalemia (glycyrrhizic acid in licorice and gossypol) inhibit this enzyme. We tested other compounds for activity to inhibit 11 beta-OHSD in guinea pig kidney cortex microsomes with NADP as cofactor and cortisol as substrate. Furosemide was an inhibitor while bumetanide was not, indicating a mechanism for the increase K+ excretion caused by furosemide compared with bumetanide. Naringenin (found in grapefruit juice), ethacrynic acid, and chenodeoxycholic acid had inhibitor IC50 values similar to glycyrrhizic acid. We conclude that various compounds can inhibit this enzyme and may play a role in K+ metabolism and adrenocorticosteroid action.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1995

Identification and tissue distribution of a novel variant of 11 beta-hydroxysteroid dehydrogenase 1 transcript.

A novel variant of 11 beta-hydroxysteroid dehydrogenase 1 (11 beta-HSD1) mRNA was identified from the ovine liver by reverse transcription-polymerase chain reaction (RT/PCR), and was named 11 beta-HSD1C mRNA. Sequence analysis of the RT-PCR product revealed that 11 beta-HSD1C mRNA was the product of an alternative exon-splicing within the 11 beta-HSD1 gene in which exon 5 was spliced out. Although it caused a deletion of 48 amino acids in the deduced 11 beta-HSD1 protein, this alternative splicing did not result in a shift within the predicted open reading frame of 11 beta-HSD1 cDNA. Thus, 11 beta-HSD1C mRNA was predicted to code for a protein of 244 amino acids. Using RT-PCR, we also examined the expression of 11 beta-HSD1C mRNA in ovine fetal organs and in maternal myometrium, endometrium, chorion, amnion and placenta. The 11 beta-HSD1C mRNA was expressed ubiquitously, similar to 11 beta-HSD1A mRNA, but at a lower abundance. Furthermore, since levels of 11 beta-HSD1C mRNA were directly related to those of 11 beta-HSD1A mRNA, there is no tissue-specificity for this shorter transcript and the only factor regulating its production appears to be 11 beta-HSD1A mRNA itself. To determine whether 11 beta-HSD1C mRNA encoded a functional enzyme, we inserted the cDNA into the expression vector pRc/CMV, and transfected the construct into Chinese hamster ovary cells. The transfected cells expressed a mRNA of expected size but contained no detectable 11 beta-HSD activity. When combined with cellular extracts of 11 beta-HSD1A cDNA transfected cells, they also did not alter either the dehydrogenase or reductase activity. The functional significance of the 11 beta-HSD1 transcript lacking exon 5 (11 beta-HSD1C mRNA) remains to be determined.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1995

Placental 11 beta-hydroxysteroid dehydrogenase and the programming of hypertension.

Excessive foetal exposure to glucocorticoids retards growth and "programmes" adult hypertension in rats. Placental 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), which catalyses the conversion of corticosterone and cortisol to inert 11 keto-products, normally protects the foetus from excess maternal glucocorticoids. In both rats and humans there is considerable natural variation in placental 11 beta-HSD, and enzyme activity correlates with birth weight. Moreover, inhibition of placental 11 beta-HSD in the rat reduces birth weight and produces hypertensive adult offspring, many months after prenatal treatment with enzyme inhibitors; these effects are dependent upon maternal adrenal products. These data suggest that placental 11 beta-HSD, by regulating foetal exposure to maternal glucocorticoids, crucially determines foeto-placental growth and the programming of hypertension. Maternal protein restriction during pregnancy also produces hypertensive offspring and selectively attenuates placental 11 beta-HSD activity. Thus, deficiency of the placental barrier to maternal glucocorticoids may represent a common pathway between the maternal environment and foeto-placental programming of later disease. These data may, at least in part, explain the human epidemiological observations linking early life events to the risk of subsequent hypertension. The recent characterization, purification and cDNA cloning of a distinct human placental 11 beta-HSD (type 2) will aid the further study of these intriguing findings.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1995

The human 11 beta-hydroxysteroid dehydrogenase type II enzyme: comparisons with other species and localization to the distal nephron.

Effective glucocorticoid inactivation is currently thought to be an indispensable feature of mineralocorticoid target cells. The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) inactivates glucocorticoids and prevents them from binding to the non-selective mineralocorticoid receptor. In the kidney it is the NAD dependent high affinity isoform (11 beta-HSD2) which is thought to endow specificity on the receptor. The recent cloning of the human, sheep and rabbit 11 beta-HSD2 enzymes permits a comparison of the enzyme from the three species. Human and rabbit enzymes are 87% identical and of similar length, while the human and sheep enzymes have only 75% identity. The last 12 residues in all three species were found to be highly divergent, but most of the ovine dishomology can be accounted for by the deletion of a single nucleotide toward the C-terminus of the protein resulting in a shift in reading frame generating a protein 27 residues longer than the human isoform. Numerous other deletions were also observed in this region of the sheep cDNA sequence. Furthermore, the rabbit cDNA also displayed a large degree of dishomology with the human sequence a short distance downstream from the termination codon. Conserved overlapping cytoplasmic translocation signals were observed in all three species, suggesting a topology whereby the enzyme is anchored into the endoplasmic reticulum by multiple hydrophobic regions in the N-terminus and the bulk of the 11 beta-HSD2 peptide is sited in the cytoplasm. A polyclonal antibody generated against the C-terminus of human 11 beta-HSD2 was used to localize the enzyme within the kidney. A high level of immunoreactive was observed in distal tubules and collecting ducts, localizing the enzyme to the same part of the nephron as the mineralocorticoid receptor. Moderate levels of staining were also seen in vascular smooth muscle cells. These results support the notion that 11 beta-HSD2 is an autocrine protector of the mineralocorticoid receptor and that it plays an important role in cardiovascular homeostatic mechanisms.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1995

Type 2 11 beta-hydroxysteroid dehydrogenase in foetal and adult life.

Two isoforms of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) catalyse the interconversion of active cortisol to inactive cortisone; 11 beta-HSD1 is a low affinity, NADP(H)-dependent dehydrogenase/oxo-reductase, and 11 beta-HSD2 a high affinity, NAD-dependent dehydrogenase. Because of the importance of 11 beta-HSD in regulating corticosteroid hormone action, we have analysed the distribution of the 11 beta-HSD isoforms in human adult and foetal tissues (including placenta), and, in addition have performed a series of substrate specificity studies on the novel, kidney 11 beta-HSD2 isoform. Using an RT-PCR approach, we failed to detect 11 beta-HSD1 mRNA in any human mid-gestational foetal tissues. In contrast 11 beta-HSD2 mRNA was present in foetal lung, adrenal, colon and kidney. In adult tissues 11 beta-HSD2 gene expression was confined to the mineralocorticoid target tissues, kidney and colon, whilst 11 beta-HSD1 was expressed predominantly in glucocorticoid target tissues, liver, lung, pituitary and cerebellum. In human kidney homogenates, 11-hydroxylated progesterone derivatives, glycyrrhetinic acid, corticosterone and the "end products" cortisone and 11-dehydrocorticosterone were potent inhibitors of the NAD-dependent conversion of cortisol to cortisone. Finally high levels of 11 beta-HSD2 mRNA and activity were observed in term placentae, which correlated positively with foetal weight. The tissue-specific distribution of the 11 beta-HSD isoforms is in keeping with their differential roles, 11 beta-HSD1 regulating glucocorticoid hormone action and 11 beta-HSD2 mineralocorticoid hormone action. The correlation of 11 beta-HSD2 activity in the placenta with foetal weight suggests, in addition, a crucial role for this enzyme in foetal development, possibly in mediating ontogeny of the foetal hypothalamo-pituitary-adrenal axis.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1996

3 Alpha-hydroxysteroid oxidoreductase activities in dihydrotestosterone degradation and back-formation in rat prostate and epididymis.

The metabolism of dihydrotestosterone (DHT) and 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-Adiol) was assessed in full homogenates of rat prostate and epididymis. The major degradational route of DHT was catalysed by the enzyme(s) 3 alpha-hydroxysteroid oxidoreductase (HSOR). Enzyme kinetic characteristics Vmax, Km and Vmax/Km ratio, were obtained for the NADP(H)- and NAD(H)-dependent interconversion of DHT and 3 alpha-Adiol at pH 7.0 and at saturated co-factor concentration. For both the reduction of DHT and the oxidation of 3 alpha-Adiol, NAD(H) was the preferred co-factor when activities were rated by their Vmax and Vmax/Km ratio. Combining the data with the earlier established Vmax/Km ratios for the 5 alpha-reductase isozyme type I and II activities in rat prostate and epididymis indicated that DHT, at saturated co-factor concentrations, would not be sustained in either tissue considering the reported enzyme characteristics. The reported exclusive bioavailability of the co-factors NADPH and NAD+ in vivo, however, will direct the metabolic pathways in these tissues to sustain the formation of DHT.

3-Hydroxysteroid Dehydrogenases↗