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11beta-hydroxysteroid dehydrogenase type 2 in mouse aorta: localization and influence on response to glucocorticoids.

Both isozymes of 11beta-hydroxysteroid dehydrogenase, which interconvert active and inactive glucocorticoids, are expressed in the mouse aortic wall. Mice deficient in 11HSD type 2 (which converts active corticosterone into inert 11-dehydrocorticosterone) have hypertension and impaired endothelial nitric oxide activity. It has been suggested that 11HSD2 influences vascular function directly by limiting glucocorticoid-mediated inhibition of endothelium-derived nitric oxide. This study sought to determine (1) the cellular distribution of the 11HSD isozymes within the mouse aortic wall and (2) the influence of 11HSD2 on direct glucocorticoid-mediated changes in aortic function. Mouse aortas were separated into their component layers and RNA extracted for RT-PCR. Both types of corticosteroid (mineralocorticoid and glucocorticoid) receptors and both 11HSD isozymes were expressed in the aortic wall. 11HSD1 expression colocalized with alpha-smooth muscle actin (a marker for smooth muscle cells), whereas 11HSD2 colocalized with TIE-2 (a marker for endothelial cells). Functional relaxation responses of mouse aortic rings were unaltered after exposure to glucocorticoids for 24 hours. In the presence of l-arginine, glucocorticoids produced an endothelium-independent reduction of contraction; similar results were obtained with aortas from mice with genetic inactivation of 11HSD2. Incubation in medium containing l-arginine reversed the endothelial cell dysfunction associated with 11HSD2 inactivation. Thus, 11HSD2 is appropriately sited to modulate endothelial cell function, but endothelial dysfunction in 11HSD2 knockout mice cannot be explained simply by increased access of corticosterone to endothelial cell corticosteroid receptors. Therefore, additional mechanisms, possibly involving indirect effects of enhanced corticosterone action in the kidney and the resultant hypertension, must be involved.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗

Gene expression of 11 beta-hydroxysteroid dehydrogenase in the mesenteric arteries of genetically hypertensive rats.

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) modulates the access of corticosteroids to their receptors and plays an important role in controlling blood pressure. We determined 11 beta-HSD activity and mRNA levels in the mesenteric arteries of genetically hypertensive rats, the Dahl salt-sensitive hypertensive rat, and compared them with Dahl salt-resistant and Sprague-Dawley rats. 11 beta-HSD activity was expressed as the percent conversion of [3H]corticosterone to [3H]11-dehydrocorticosterone. 11 beta-HSD activity was significantly decreased in the mesenteric arteries of 8-week-old Dahl salt-sensitive hypertensive rats (11.4 +/- 1.4%) compared with Dahl salt-resistant rats (17.4 +/- 1.4%) or Sprague-Dawley rats (18.0 +/- 1.5%) of the same age (P < .05). There were no significant differences in 11 beta-HSD activity between 4-week-old Dahl salt-sensitive hypertensive and Dahl salt-resistant rats of the same age (15.3 +/- 1.3% and 15.1 +/- 1.9%, respectively). The concentration of 11 beta-HSD mRNA in the mesenteric arteries of 8-week-old Dahl salt-sensitive hypertensive rats was significantly lower than in Dahl salt-resistant or Sprague-Dawley rats of the same age (P < .05). There were no significant differences in the concentration of 11 beta-HSD mRNA in the mesenteric arteries of 4-week-old Dahl salt-sensitive hypertensive rats, Dahl salt-resistant rats, and Sprague-Dawley rats. These results indicate that 11 beta-HSD in the vascular wall may play a role in the pathogenesis of hypertension in this rat model.

11-beta-Hydroxysteroid Dehydrogenases↗

Evidence of coexisting changes in 11 beta-hydroxysteroid dehydrogenase and 5 beta-reductase activity in subjects with untreated essential hypertension.

We compared corticosteroid metabolite excretion rates and patterns in a group of 68 subjects with untreated essential hypertension and a matched group of 48 normotensive control subjects. The ratio of tetrahydrocortisol plus allotetrahydrocortisol to tetrahydrocortisone and the ratio of allotetrahydrocortisol to tetrahydrocortisol were significantly higher in the hypertensive group. This is qualitatively similar to the situation found in patients with the syndrome of apparent mineralocorticoid excess or subjects treated with licorice or carbenoxolone where hypertension is known to arise from deficiencies of 11 beta-hydroxysteroid dehydrogenase and 5 beta-reductase activities. The equivalent ratios for corticosterone metabolites were not different between groups, but total corticosterone metabolite excretion was higher in the hypertensive group. Plasma cortisol levels were lower in hypertensive than in control subjects, but corticosterone levels were higher. This evidence supports a previous suggestion that the activities of these two enzymes may be reduced in essential hypertension, but the contribution of these changes to hypertension is not known.

11-beta-Hydroxysteroid Dehydrogenases↗

Endogenous renal 11 beta-hydroxysteroid dehydrogenase inhibitory factors in patients with low-renin essential hypertension.

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) modulates the access of corticosteroids to their receptors and is important in blood pressure control. The excretion of renal 11 beta-HSD (ie, NAD(+)-dependent isoform) is thought to protect renal mineralocorticoid receptors from cortisol. To examine whether endogenous renal 11 beta-HSD inhibitory factor(s) may be involved in the pathophysiology of hypertension, we studied the urinary excretion of such inhibitors in 30 patients with low-renin essential hypertension and 20 normotensive control subjects. The effect of sodium restriction on the urinary excretion of the inhibitors wa also evaluated in six normotensive control subjects. Urine was extracted with Sep-Pak cartridges and high-performance liquid chromatography. Endogenous renal 11 beta-HSD inhibitors were measured by the inhibition of 11 beta-HSD bioactivity in microsomes from the human kidney. The urinary excretion of the inhibitors was significantly increased in patients with low-renin essential hypertension (1280 +/- 88 nmol/d, mean +/- SEM) compared with normotensive control subjects (704 +/- 56 nmol/d) (P < .05). Ratios of urinary tetrahydrocortisol+allo-tetrahydrocortisol to tetrahydrocortisone did not differ significantly. Sodium restriction reduced the urinary excretion of the endogenous renal 11 beta-HSD inhibitors but did not affect the ratio of urinary tetrahydrocortisol+allo-tetrahydrocortisol to tetrahydrocortisone. Endogenous renal 11 beta-HSD inhibitory factors may contribute to the pathogenesis of low-renin essential hypertension by modulating the activity of 11 beta-HSD. Sodium intake may directly or indirectly regulate the inhibitory factors.

11-beta-Hydroxysteroid Dehydrogenases↗

Inhibition of 11-beta-hydroxysteroid dehydrogenase in pregnant rats and the programming of blood pressure in the offspring.

Recent epidemiological studies have linked low birth weight with the later occurrence of cardiovascular and metabolic disorders, particularly hypertension. We have proposed that fetal exposure to excess maternal glucocorticoids may underpin this association. Normally, the fetus is protected from maternal glucocorticoids by placental 11beta-hydroxysteroid dehydrogenase (11beta-HSD). We have previously shown that treatment of pregnant rats with dexamethasone, a synthetic glucocorticoid that is poorly metabolized by the enzyme, reduces birth weight and produces elevated blood pressure in the adult offspring. Moreover, low activity of placental 11beta-HSD correlates with low birth weight in rats. Here, we show that maternal administration of carbenoxolone, a potent inhibitor of 11 beta-HSD, throughout pregnancy leads to reduced birth weight (mean 20 percent decrease) and elevated blood pressures (increase in mean arterial pressure, 9 mm Hg in males, 7 mm Hg in females) in the adult offspring of carbenoxolone-treated rats. This effect requires the presence of maternal adrenal products, as carbenoxolone given to adrenalectomized pregnant rats had no effect on birth weight or blood pressure. These data support the hypothesis that excess exposure of the fetoplacental unit to maternal glucocorticoids reduces birth weight and programs subsequent hypertension and indicate a key role for placental 11beta-HSD in controlling such exposure.

11-beta-Hydroxysteroid Dehydrogenases↗

Genetic association of 11 beta-hydroxysteroid dehydrogenase type 2 (HSD11B2) flanking microsatellites with essential hypertension in blacks.

11 beta-Hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) specifically modulates access of the mineralocorticoid aldosterone to the kidney mineralocorticoid type 1 receptors in a physiological environment in which there is a molar excess of cortisol. Cortisol and aldosterone have similar affinities for mineralocorticoid receptors. Mechanistically, 11 beta-HSD2 converts cortisol to cortisone. The other known isoform, 11 beta-HSD1, not only catalyzes the cortisol to cortisone reaction but also the reverse reaction, making it unlikely to play an important role in modulating the access of aldosterone to mineralocorticoid receptors. Mutations in the HSD11B2 gene (both exonic and intronic) have been demonstrated to cause reduced activity of this enzyme in the syndrome of apparent mineralocorticoid excess, a rare autosomal recessive disorder. We hypothesized that this locus is also involved in the etiology of essential hypertension. To test this locus and flanking chromosomal regions for allelic association and genetic linkage to essential hypertension, it is necessary to have informative genetic markers. To this end, we have refined the localization of 11 beta-HSD2 to 16q22.1. We genotyped subjects using the nearest flanking microsatellites (D16S301 and D16S496). We conducted an association study using black subjects with hypertensive end-stage renal disease, black normotensive control subjects, and black and white individuals from the general population. We used chi 2 analysis and Fisher's exact test to test for association with these candidate gene markers. No significant association was found between D16S301 and hypertension. However, a positive association with hypertension was found at the D16S496 microsatellite locus (chi 2 = 6.98, df = 1, P < or = .008). Our data suggest that HSD11B2 is associated with hypertension in our black subjects with hypertensive end-stage renal disease. The 16q22.1 chromosome region potentially harbors a candidate gene for essential hypertension. Confirmation of our findings in another independently ascertained group of hypertensive subjects will provide a basis for proceeding with sib-pair linkage analyses.

11-beta-Hydroxysteroid Dehydrogenases↗

Gene expression of 11beta-hydroxysteroid dehydrogenase type 1 and type 2 in the kidneys of insulin-dependent diabetic rats.

The presence of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) activity in the kidney has been suggested to be important in the regulation of glucocorticoid-induced disorders of electrolyte balance and the control of blood pressure. To assess the possible effect of 11beta-HSD isoforms in diabetes-related hypertension, we measured the mean systolic blood pressure and the 11beta-HSD activity and mRNA levels for both 11beta-HSD1 and 11beta-HSD2 in the kidney of streptozotocin (STZ)-diabetic female rats. Three weeks after injection of STZ (65 mg/kg), the mean systolic blood pressure of diabetic rats was elevated 13.6% above that of normal rats (P<.01). The renal 11beta-HSD2 activity and level of mRNA expression were significantly decreased in diabetic rats (P<.01). However, the treatment of rats with STZ did not decrease the levels of renal 11beta-HSD1 activity and mRNA expression in diabetic rats. Insulin administered subcutaneously to diabetic rats for 2 weeks completely reversed the decrease in renal 11beta-HSD2 activity and gene expression and prevented the elevation in blood pressure in the diabetic rat. These results indicate that alteration of renal 11beta-HSD2 activity and gene expression may be primarily responsible for the changes in blood pressure of STZ-diabetic rats after early treatment with insulin.

11-beta-Hydroxysteroid Dehydrogenases↗

Renal 11beta-hydroxysteroid dehydrogenase in genetically salt-sensitive hypertensive rats.

-Renal 11beta-hydroxysteroid dehydrogenase II (11beta-HSDII) converts glucocorticoids into inactive metabolites and plays an important role in controlling blood pressure and sodium retention. To examine whether this enzyme may be involved in the pathophysiology of salt-sensitive hypertension, we determined 11beta-HSDII activity and mRNA levels in the blood vessel and kidney of Dahl Iwai salt-sensitive (DS) rats and Dahl Iwai salt-resistant (DR) rats. Urinary free corticosterone:free 11-dehydrocorticosterone ratio was measured to estimate renal 11beta-HSD activity. Vascular 11beta-HSDII activity was expressed as percent conversion of [3H]corticosterone to [3H]11-dehydrocorticosterone in homogenized mesenteric arteries. 11beta-HSDII mRNA was estimated with the use of competitive polymerase chain reaction (PCR). Renal 11beta-HSDII activity and mRNA levels were significantly decreased in 8- and 12-week-old high salt DS rats compared with DR, Sprague-Dawley (SD), or low salt DS rats of the same age. Decreased 11beta-HSDII activity and mRNA levels in mesenteric arteries were observed in 8- and 12-week-old high salt DS rats. Urinary excretion of 11beta-HSDII inhibitory factors was measured by inhibition of enzyme activity in microsomes from human kidney. The urinary inhibitors were significantly increased in 8- and 12-week-old high salt DS rats compared with DR, SD, or low salt DS rats of the same age. There were no significant differences in 11beta-HSDII activity and mRNA levels in mesenteric arteries and kidney or in urinary inhibitors between 4-week-old DS, DR, and SD rats. These results indicate that 11beta-HSDII may play a role in salt sensitivity and development of hypertension in the DS rat.

11-beta-Hydroxysteroid Dehydrogenases↗

Mutants of 11beta-hydroxysteroid dehydrogenase (11-HSD2) with partial activity: improved correlations between genotype and biochemical phenotype in apparent mineralocorticoid excess.

Mutations in the kidney isozyme of human 11-hydroxysteroid dehydrogenase (11-HSD2) cause apparent mineralocorticoid excess, an autosomal recessive form of familial hypertension. We studied 4 patients with AME, identifying 4 novel and 3 previously reported mutations in the HSD11B2 (HSD11K) gene. Point mutations causing amino acid substitutions were introduced into a pCMV5/11HSD2 expression construct and expressed in mammalian CHOP cells. Mutations L179R and R208H abolished activity in whole cells. Mutants S180F, A237V, and A328V had 19%, 72%, and 25%, respectively, of the activity of the wild-type enzyme in whole cells when cortisol was used as the substrate and 80%, 140%, and 55%, respectively, of wild-type activity when corticosterone was used as the substrate. However, these mutant proteins were only 0.6% to 5.7% as active as the wild-type enzyme in cell lysates, suggesting that these mutations alter stability of the enzyme. In regression analyses of all AME patients with published genotypes, several biochemical and clinical parameters were highly correlated with mutant enzymatic activity, demonstrated in whole cells, when cortisol was used as the substrate. These included the ratio of urinary cortisone to cortisol metabolites (R(2)=0.648, P<0.0001), age at presentation (R(2)=0.614, P<0.0001), and birth weight (R(2)=0.576, P=0.0004). Approximately 5% conversion of cortisol to cortisone is predicted in subjects with mutations that completely inactivate HSD11B2, suggesting that a low level of enzymatic activity is mediated by another enzyme, possibly 11-HSD1.

11-beta-Hydroxysteroid Dehydrogenases↗

11beta-hydroxysteroid dehydrogenase and corticosteroid action in lyon hypertensive rats.

Adrenocorticosteroid activity in Lyon hypertensive (LH) and low blood pressure (LL) rat strains differ in several respects. Abnormal activity of 11beta-hydroxysteroid dehydrogenase enzymes (11beta-HSD1 and 11beta-HSD2), which interconvert corticosterone and inactive 11-dehydrocorticosterone, might contribute to the LH phenotype by regulating corticosteroid hormone access to receptors. 11beta-HSD2 (expressed in kidney but not liver) prevents endogenous glucocorticoids from binding to mineralocorticoid receptors. 11beta-HSD1 (expressed in liver and kidney) favors active glucocorticoid formation from 11-dehydrocorticosterone. 11beta-HSD properties in LH and LL have been compared by several approaches: (1) 11betaHSD activities have been measured in vitro as corticosterone dehydrogenation and in vivo as interconversion of injected cortisol and cortisone; (2) the effects of cortisol and cortisone on urine electrolytes and volume have been measured; and (3) 11beta-HSD mRNA expression has been measured by in situ hybridization. 11beta-HSD2 enzyme activities in LH and LL rats were similar and urinary cortisone:cortisol ratios were not different after cortisol injection. Cortisol caused a natriuresis and kaliuresis in both strains, with a slightly reduced response in LH rats. Renal 11beta-HSD2 mRNA expression was slightly lower in LH rats. 11beta-HSD1 was less active in LH than LL rats: enzyme activities were lower in tissue extracts; urinary cortisone:cortisol was lower in LL rats after cortisone injections; cortisone increased urine volume in LL but not LH rats; and mRNA levels tended to be lower in LH tissues. We conclude that 11beta-HSD1 is impaired in LH rats. The LH phenotype of heavier adrenals, raised corticosterone, and reduced thymus weight is similar to that described for 11beta-HSD1 knockout mice.

11-beta-Hydroxysteroid Dehydrogenases↗

Role of 11beta-hydroxysteroid dehydrogenase in nongenomic aldosterone effects in human arteries.

The aim of the present study was to demonstrate rapid effects of aldosterone on the Na(+)-H(+) exchanger in strips of human vascular vessels and to determine whether 11beta-hydroxysteroid dehydrogenase enzyme (11beta-HSD) could play a protective role in this response, such as that described for the classic type I mineralocorticoid receptor (MR). The activity of 11beta-HSD isoforms 1 and 2 were measured in fetal and adult arteries. Both isoforms are present in adult and fetal vessels. However, a significant difference in the proportion of each isoform was found. Isoform 1 activity (in pmol x min(-1) x 100 mg(-1) protein) was 42+/-5 in fetal vessels and 29+/-2 in adult arteries, and isoform 2 activity was 78+/-7 in fetal and 12+/-2 in adult tissue. The nongenomic effect of aldosterone on Na(+)-H(+) exchanger activity was measured in strips of chorionic and radial uterine arteries loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein. Recordings of intracellular pH (pH(i)) were made by videofluorescence microscopy. Aldosterone (0.5 nmol/L) rapidly increased pH(i), with a half-maximal effect between 2 and 3 nmol/L in both fetal and adult vessels. Ethylisopropylamiloride, a specific inhibitor of the Na(+)-H(+) exchanger, inhibited this effect. The hormone-mediated increase in pH(i) was unaffected by spironolactone, a classic antagonist of MR, but was completely blocked by RU28318. Cortisol (up to 1 micromol/L) had no effect on pH(i), but when applied in the presence of carbenoxolone, a dramatic increase in Na(+)-H(+) exchanger activity was evident. The increments on pH(i) for each cortisol concentration were similar to those observed for aldosterone. These findings suggest that vascular 11beta-HSD plays an active role in maintaining the specificity of the rapid effects of aldosterone.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗

Endothelial cell dysfunction in mice after transgenic knockout of type 2, but not type 1, 11beta-hydroxysteroid dehydrogenase.

BACKGROUND: 11beta-Hydroxysteroid dehydrogenase (11betaHSD) isozymes catalyze the interconversion of active and inactive glucocorticoids, allowing local regulation of corticosteroid receptor activation. Both are present in the vessel wall; here, using mice with selective inactivation of 11betaHSD isozymes, we test the hypothesis that 11betaHSDs influence vascular function. METHODS AND RESULTS: Thoracic aortas were obtained from weight-matched male wild-type (MF1x129 cross(+/+)), 11betaHSD1(-/-), and 11betaHSD2(-/-) mice. mRNA for both isozymes was detected in wild-type aortas by RT-PCR. 11betaHSD activity in aortic homogenates (48.81+/-4.65% conversion) was reduced in both 11betaHSD1(-/-) (6.36+/-2.47% conversion; P<0.0002) and 11betaHSD2(-/-) (24.71+/-3.69; P=0.002) mice. Functional responses were unaffected in aortic rings isolated from 11betaHSD1(-/-) mice. In contrast, aortas from 11betaHSD2(-/-) mice demonstrated selectively enhanced constriction to norepinephrine (E(max) 4.28+/-0.56 versus 1.72+/-0.47 mN/mm; P=0.004) attributable to impaired endothelium-derived nitric oxide activity. Relaxation responses to endothelium-dependent and -independent vasodilators were also impaired. To control for chronic renal mineralocorticoid excess, MF1 mice were treated with fludrocortisone (16 weeks) but did not reproduce the functional changes observed in 11betaHSD2(-/-) mice. CONCLUSIONS: Although both 11betaHSD isozymes are present in the vascular wall, reactivation of glucocorticoids by 11betaHSD1 does not influence aortic function. Mice with 11betaHSD2 knockout, however, have endothelial dysfunction causing enhanced norepinephrine-mediated contraction. This appears to be independent of renal sodium retention and may contribute to hypertension in 11betaHSD2 deficiency.

11-beta-Hydroxysteroid Dehydrogenases↗

Clinical relevance of airway 11beta-hydroxysteroid dehydrogenase type II enzyme in asthma.

11beta-hydroxysteroid dehydrogenases (11beta-HSD) are responsible for the conversion of bioactive glucocorticoids to and from inactive metabolites. 11beta-HSD2 is generally considered a high-affinity inactivator of natural glucocorticoids, although its activity with synthetic compounds in vivo is unknown. Inhaled corticosteroids (ICS) remain the primary antiinflammatory agents for treating asthma, but little is known about their metabolism in the lung. The aims of this study were to determine whether the 11beta-HSD2 enzyme can be localized to human airway tissue and whether differential expression of this enzyme relates to asthma severity and ICS needs. We studied airway biopsy specimens from 22 asthmatic subjects, in two groups: (1) a group not treated with ICS (n = 7); and (2) a group treated with ICS (range: 200 to 1,500 microg/d; n = 15). A control population consisted of nine nonasthmatic subjects. Immunostaining was done with an immunopurified antibody to human 11beta-HSD2. Immunoreactivity was generally localized to the endothelium of vessels in the lamina propria and to airway epithelium both in asthmatic patients and nonasthmatic controls. There was a statistically significant inverse relationship between the ICS dose required for effective treatment and the extent of epithelial 11beta-HSD2 staining (r = -0.44; p = 0.04). This is consistent with 11beta-HSD2 acting as an oxidoreductase that regenerates rather than inactivates ICS. This study suggests that glucocorticoid sensitivity in the lung is not determined by ICS breakdown, but may be related to 11beta-HSD2 sustaining the activation of synthetic glucocorticoids.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗

Dexamethasone upregulates 11beta-hydroxysteroid dehydrogenase type 2 in BEAS-2B cells.

The actions of natural and synthetic glucocorticoids are in part determined by 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2). We examined whether carbenoxolone, a potent inhibitor of 11beta-HSD, would potentiate the inhibitory action of dexamethasone on interleukin-8 release from BEAS-2B cells, and whether prolonged treatment with dexamethasone at therapeutic doses would upregulate 11beta-HSD2 in the cells. We found that carbenoxolone increased the potency of dexamethasone almost 10-fold. Reverse transcription-polymerase chain reaction and Western blot revealed that BEAS-2B cells expressed 11beta-HSD2, but not 11beta-HSD1. An enzyme activity assay of the cell homogenate demonstrated only NAD+-dependent dehydrogenase activity. The Km value for cortisol in intact BEAS-2B cells was estimated to be 42 nM. When the cells were incubated with dexamethasone for up to 72 hours at increasing concentrations (10(-9) to 10(-5) M), there were considerable increases in mRNA and protein levels of 11beta-HSD2. Prolonged treatment with dexamethasone also increased the enzyme activity of 11beta-HSD in the cells in a dose- and time-dependent manner, with complete inhibition by RU38486. These results suggest that bronchial epithelial cells possess an autoregulatory system for glucocorticoids in the control of their own bioactive levels by inducing the expression of 11beta-HSD2, and that 11beta-HSD2 in the bronchial epithelium may play a role in the local regulation of inhaled glucocorticoid actions.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗

Regulation of the action of hydrocortisone in airway epithelial cells by 11beta-hydroxysteroid dehydrogenase.

11beta-hydroxysteroid dehydrogenase (11betaHSD) reversibly converts hydrocortisone, the predominant active endogenous glucocorticoid in humans, to its inactive metabolite cortisone by oxidizing the 11-hydroxy group to an 11-keto group. Because this enzyme is highly expressed in human bronchial epithelial cells, we hypothesized that it regulates epithelial responses to glucocorticoids by reducing levels of hydrocortisone available to bind to the glucocorticoid receptor. Primary human bronchial epithelial cells (PBECs) were isolated from seven autopsy specimens and cultured in F12/Dulbecco's modified Eagle's medium with 5% fetal bovine serum until approximately 80% confluent. Cells were preincubated with 10(-9) M to 10(-5) M hydrocortisone for 24 h in the presence or absence of 10(-6) M of the 11betaHSD inhibitor glycyrrhetinic acid, after which the cells were stimulated with 5 ng/ml interleukin-1beta for 24 h. Granulocyte macrophage colony-stimulating factor (GM-CSF) levels were quantitated in the resulting supernatants by enzyme-linked immunosorbent assay. Hydrocortisone inhibited GM-CSF release in stimulated PBEC with a concentration that produces 50% inhibition of maximum effect (IC(1/2)max) of 5.0 x 10(-8) M. In the presence of glycyrrhetinic acid, the potency of hydrocortisone was increased approximately 33-fold (IC(1/2)max with glycyrrhetinic acid, 1.5 x 10(-9) M). Hydrocortisone activity was maximally enhanced at concentrations between 10(-9) M and 10(-8) M, levels that are comparable to plasma levels of hydrocortisone not bound to plasma proteins. Glycyrrhetinic acid had no effect on the suppression of GM-CSF release by hydrocortisone in the transformed cell line BEAS-2B, which does not express the 11betaHSD enzyme. Glycyrrhetinic acid also had no effect on the inhibition of GM-CSF release in PBECs by the synthetic glucocorticoids budesonide, beclomethasone dipropionate, fluticasone propionate, mometasone furoate, and triamcinolone acetonide, steroids not metabolized by 11betaHSD. Together, these findings suggest that metabolism of hydrocortisone by 11betaHSD may regulate glucocorticoid activity in human airway epithelial cells.

11-beta-Hydroxysteroid Dehydrogenases↗

Cyclical oxidation-reduction of the C3 position on bile acids catalyzed by rat hepatic 3 alpha-hydroxysteroid dehydrogenase. I. Studies with the purified enzyme, isolated rat hepatocytes, and inhibition by indomethacin.

We recently identified that the Y' bile acid binders are 3 alpha-hydroxysteroid dehydrogenases (3 alpha-HSD). In the present studies, purified 3 alpha-HSD catalyzed rapid 3H loss from [3 beta-3H, C24-14C]lithocholic and chenodeoxycholic acids without net conversion to 3-oxo bile acids under physiologic pH and redox conditions. [3 beta-3H]Cholic acid was a poor substrate. The Y' fraction of hepatic cytosol was exclusively responsible for this activity and 3H was transferred selectively to NADP+. Time-dependent 3H loss was also seen in isolated hepatocytes. Further hydroxylation products of lithocholic and chenodeoxycholic acids lost 3H at the same rate, whereas 3H loss from lithocholic acid rapidly ceased, which suggests compartmentation of this bile acid in hepatocytes. Indomethacin inhibited 3H loss from bile acids either in incubations with the pure enzyme or in isolated hepatocytes. Indomethacin did not alter the initial uptake rate of bile acids by hepatocytes, but caused a redistribution of unconjugated bile acids into the medium at early time points (2.5 and 5.0 min) and that of conjugated bile acids at later time intervals (30 min). 3H loss from the 3 beta position therefore can be used to probe the interaction between bile acids and cytosolic 3 alpha-HSD in intact cells, and indomethacin is capable of inhibiting this interaction.

3-Hydroxysteroid Dehydrogenases↗

Cyclical oxidation-reduction of the C3 position on bile acids catalyzed by 3 alpha-hydroxysteroid dehydrogenase. II. Studies in the prograde and retrograde single-pass, perfused rat liver and inhibition by indomethacin.

[3 beta-3H, 24-14C]Lithocholic, chenodeoxycholic, and cholic acids were administered in tracer bolus doses either prograde or retrograde in the isolated perfused rat liver. Little 3H loss from cholic acid was observed, whereas with the other bile acids, 20-40% of the administered 3H was lost in a single pass from perfusate to bile. Most of the 3H loss occurred rapidly (5 min) and was recovered as [3H]water in perfusate. Excretion of bile acids was delayed with retrograde administration, and 3H loss was more extensive. In both prograde and retrograde studies, indomethacin markedly inhibited the excretion of the bolus of bile acid into bile. Indomethacin inhibited the extraction of glycocholate (50 microM) during steady state perfusion without affecting transport maximum for excretion. At lower glycocholate concentration (5 microM), indomethacin inhibited both extraction and excretion. A greater effect was seen on excretion in the latter case, which suggests that displacement of bile acid from the cytosolic protein lead to redistribution in the hepatocyte as well as reflux into the sinusoid. These data suggest that binding of bile acids to cytosolic 3 alpha-hydroxysteroid dehydrogenases occurs extensively during hepatic transit and is important in mediating the translocation of bile acids from the sinusoidal to canalicular pole of the cell.

3-Hydroxysteroid Dehydrogenases↗

Distribution of 11 beta-hydroxysteroid dehydrogenase along the rabbit nephron.

It has been recently proposed that 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) is responsible for aldosterone tissue specificity. A 11 beta-OHSD deficiency has been invoked as a cause of the syndrome of apparent mineralocorticoid excess, and 11 beta-OHSD inhibition by liquorice has been invoked to explain the hypertension induced by this drug. Since the renal tubule is composed of aldosterone-sensitive and insensitive segments, we determined the distribution of 11 beta-OHSD along the rabbit tubule. Pools of tubular segments isolated by microdissection were incubated for 2 h at 37 degrees C in the presence of [3H]corticosterone (3H-B, 8.10(-9) M). Afterwards, the amounts of 3H-B and of the metabolite 11-dehydrocorticosterone (3H-A) were determined using HPLC analysis. In the proximal tubule, in either its convoluted or straight portion, and in the medullary thick ascending limb, the amount of 3H-A was 19.6 +/- 3.8% (n = 12), 17.9 +/- 3.4 (n = 8), and 15.0 +/- 2.2 (n = 4), respectively, of the sum of 3H-A + 3H-B. In the cortical ascending limb and the collecting tubule in its cortical and medullary parts, it was 74.7 +/- 6.8% (n = 4), 74.1 +/- 4.9 (n = 9) and 64.6 +/- 14.1 (n = 3), respectively. In both proximal and cortical collecting tubule, addition of carbenoxolone 8.10(-4) M, an inhibitor of 11 beta-OHSD, almost completely inhibited the conversion of 3H-B to 3H-A. Thus, 11 beta-OHSD activity was high in the aldosterone-sensitive segments, and low in the aldosterone-insensitive segments. These results strongly favor the hypothesis that 11 beta-OHSD is a key enzyme in mineralocorticoid tissue specificity along the rabbit nephron. They reinforce the notion that a defect in 11 beta-OHSD plays a major role in the syndrome of apparent mineralocorticoid excess and liquorice-induced hypertension.

11-beta-Hydroxysteroid Dehydrogenases↗