A second enzyme protecting mineralocorticoid receptors from glucocorticoid occupancy.

We have confirmed that A6 cells (derived from kidney of Xenopus laevis), which contain both mineralocorticoid and glucocorticoid receptors, do not normally possess 11 beta-hydroxysteroid dehydroxgenase (11 beta-HSD1 or 11 beta-HSD2) enzymatic activity and so are without apparent "protective" enzymes. A6 cells do not convert the glucocorticoid corticosterone to 11-dehydrocorticosterone but do, however, possess steroid 6 beta-hydroxylase that transforms corticosterone to 6 beta-hydroxycorticosterone. This hydroxylase is cytochrome P-450 3A (CYP3A). We have now determined the effects of 3 alpha,5 beta-tetrahydroprogesterone and chenodeoxycholic acid (both inhibitors of 11 beta-HSD1) and 11-dehydrocorticosterone and 11 beta-hydroxy-3 alpha,5 beta-tetrahydroprogesterone (inhibitors of 11 beta-HSD2) and carbenoxalone, which inhibits both 11 beta-HSD1 and 11 beta-HSD2, on the actions and metabolism of corticosterone and active Na+ transport [short-circuit current (Isc)] in A6 cells. All of these 11 beta-HSD inhibitory substances induced a significant increment in corticosterone-induced Isc, which was detectable within 2 h. However, none of these agents caused an increase in Isc when incubated by themselves with A6 cells. In all cases, the additional Isc was inhibited by the mineralocorticoid receptor (MR) antagonist, RU-28318, whereas the original Isc elicited by corticosterone alone was inhibited by the glucocorticoid receptor antagonist, RU-38486. In separate experiments, each agent was shown to significantly inhibit metabolism of corticosterone to 6 beta-hydroxycorticosterone in A6 cells, and a linear relationship existed between 6 beta-hydroxylase inhibition and the MR-mediated increase in Isc in the one inhibitor tested. Troleandomycin, a selective inhibitor of CYP3A, inhibited 6 beta-hydroxylase and also significantly enhanced corticosterone-induced Isc at 2 h. These experiments indicate that the enhanced MR-mediated Isc in A6 cells may be related to inhibition of 6 beta-hydroxylase activity in these cells and that this 6 beta-hydroxylase (CYP3A) may be protecting the expression of corticosterone-induced active Na+ transport in A6 cells by MR-mediated mechanism(s).

Down-regulation of the mineralocorticoid receptor by dexamethasone in an amphibian kidney cell line (A6).

The A6 cell line, derived from Xenopus kidney, is an in vitro model of cortico-steroid mediated transepitheial Na+ transport stimulation. We report the apparent down-regulation of mineralocorticoid receptor levels in A6 cells, in response to the presence of the synthetic glucorticoid dexamethasone in the culture medium. Mineralocorticoid receptor binding was suppressed to approximately 25% of control following 24-hour exposure to 10nM dexamethasone. Scatchard analysis of concentration-binding experiments show down-regulation of maximum binding capacity by Dex exposure with no alteration of MR affinity, i.e., alteration of MR number only. The effect is dose-responsive with half-maximal down regulation at 1nM. Maximal inhibition of binding occurred after 24-hours exposure to dexamethasone. The inhibitory effect of dexamethasone on MR binding was unique for the glucocorticoid, with no effect exhibited following similar treatment with an androgen, an estrogen, or a mineralocorticoid.

Renal and hepatic family 3A cytochromes P450 (CYP3A) in spontaneously hypertensive rats.

Troleandomycin (TAO), a selective family 3A cytochromes P450 (CYP3A) inhibitor, decreases enhanced in vivo corticosterone 6 beta-hydroxylation and blood pressure in spontaneously hypertensive rats (SHR). Corticosterone 6 beta-hydroxylation was measured in liver and kidney microsomes, to determine ontogeny and the effect of TAO on CYP3A activity at the organ level. SHR kidney CYP3A activity increased from 4 to 8 weeks, stabilized at 11 and 16 weeks, and was much higher than in control (Wistar-Kyoto, WKY) rats at all ages. Hepatic activity showed less consistency in strain difference. TAO produced a relatively large decrease in renal CYP3A activity compared with liver. Although renal CYP3A mRNA was not present in sufficient quantity for detection by northern blot analysis of total RNA, its presence was demonstrated in SHR by reverse transcriptase-polymerase chain reaction amplification. Correlations between renal CYP3A activity and systolic blood pressure in SHR and WKY rats with variations in age, strain and drug treatment are consistent with the role of the enzyme in the pathogenesis of blood pressure elevation in SHR.

Maternal environment defines blood pressure and its response to troleandomycin in spontaneously hypertensive rats.

Relationship between family-3A cytochrome P-450-dependent (troleandomycin inhibitable) and maternal environmental-dependent systolic blood pressure (SBP) was investigated in spontaneously hypertensive rats (SHR). Adult SHR nursed by foster or natural SHR mothers had indistinguishable SBP. Troleandomycin reduced 50% of Wistar-Kyoto (WKY)-SHR strain difference in SBP. SHR having WKY foster mothers had SBP similar to troleandomycin-reduced SHR levels, which was unaffected by troleandomycin. The two components of SBP elevation appear identical. Because observations of others demonstrated that WKY fostered to SHR show no SBP increase, the maternally dependent/troleandomycin-sensitive component of SBP elevation may reflect epistatic interaction between genes determining maternal differences and offspring sensitivity, respectively.

Augmented arterial pressure responses to cyclosporine in spontaneously hypertensive rats. Role of cytochrome P-450 3A.

Evidence to support a hypertensinogenic role of family 3A cytochrome P-450 (CYP3A) activity is that troleandomycin, a selective inhibitor of CYP3A, decreases both blood pressure and in vivo corticosterone 6 beta-hydroxylation in spontaneously hypertensive rats (SHR). Renal CYP3A activity is markedly increased in SHR compared with Wistar-Kyoto (WKY) rats. Cyclosporine acutely increases both systolic blood pressure and renal total cytochrome P-450 in SHR. We tested the hypothesis that the augmentation of blood pressure by cyclosporine is mediated by a further increase in renal CYP3A activity. Accordingly, we assessed the effect of troleandomycin administration on cyclosporine-induced systolic blood pressure increase and renal and hepatic microsomal CYP3A activity in SHR. Cyclosporine (5 mg/kg SC) given daily in 11-week-old SHR resulted in substantial augmentation of blood pressure after 6 days. This blood pressure increase was attenuated by troleandomycin (40 mg/kg) given either during or after development of hypertension. Cyclosporine increased renal (60%) but decreased hepatic (25%) microsomal CYP3A activity in SHR. In contrast, cyclosporine failed to produce any detectable increase in either blood pressure or renal CYP3A activity in WKY rats. Troleandomycin completely inhibited renal CYP3A activity measured after cyclosporine treatment of SHR, which correlated with its attenuation of the cyclosporine-induced blood pressure increase. These findings suggest that renal CYP3A could play an important role in acute cyclosporine-induced hypertension.

Effects of corticosteroids on urinary ammonium excretion in humans.

This study was designed to examine the selective effects of glucocorticoid and mineralocorticoid classes of steroid hormones on urinary ammonium excretion in humans. In 22 10-day studies, normal male volunteers received either 9 alpha-fludrohydrocortisone or hydrocortisone, alone or with the receptor antagonist spironolactone or mifepristone. The small but significant increase in ammonium excretion noted with the administration of 9 alpha-fludrohydrocortisone was associated with a significant decrease in serum potassium. In contrast, a significantly larger increase in ammonium excretion was noted with hydrocortisone, without concomitant electrolyte changes. Spironolactone did not alter the effect on ammonium excretion by either corticosteroid, whereas mifepristone markedly blunted the hydrocortisone-induced increase in urinary ammonium excretion. It was concluded that glucocorticoids increase urinary ammonium excretion in humans and that this effect occurs through binding to the Type II (glucocorticoid) receptor rather than by cross-occupancy of the Type I (mineralocorticoid) receptor.

Renal corticosterone 6 beta-hydroxylase in the spontaneously hypertensive rat.

Excess 6 beta-OH-corticosterone production by family 3A cytochromes P-450 may play a role in genesis of hypertension in the spontaneously hypertensive rat (SHR), by producing a renal defect in Na+ excretion. Renal cytochromes P-450 may be a causal factor in this genetic model. Since family 3A P-450 is present in rat kidney (collecting duct), the renal family 3A catalytic (6 beta-OHase) and immunoreactive activities were compared in SHR and normotensive control (Wistar-Kyoto; WKY) rats. Corticosterone 6 beta-hydroxylation is markedly higher in SHR than in WKY renal microsomal preparations. Western blot analysis with antibodies to rat and rabbit liver family 3A isoforms demonstrated related proteins. Densitometry revealed greater relative intensity of staining in SHR compared to WKY with both antibodies. Both antibodies inhibited corticosterone 6 beta-hydroxylation by SHR renal microsomes. Increased renal 6 beta-OH-corticosterone production by increased renal family 3A cytochromes P-450 may play a role in the blood pressure elevation in SHR.

When is cortisol a mineralocorticoid?

(1) Decreased 11 beta-OHSD activity permits binding of cortisol to the Type I (mineralocorticoid) receptor in humans, thereby producing spironolactone-inhibitable Na+ retention, hypokalemia and hypertension, the syndrome of apparent mineralocorticoid excess (AME). (2) Blockade of either the Type I receptor with spironolactone or the Type II (glucocorticoid) receptor with RU-486 does not consistently abolish the effects of stress level cortisol on Na+ retention and hypertension in acute studies in normal humans, suggesting the existence of an additional glucocorticoid receptor. (3) Enhanced glucocorticoid 6 beta-hydroxylation could play an etiologic role in certain hypertensive syndromes. (4) Both decreased 11 beta-OHSD and increased 6 beta-OHase are candidates as intermediate phenotypes for the remote phenotype essential hypertension.

Corticosterone 6 beta-hydroxylation correlates with blood pressure in spontaneously hypertensive rats.

Evidence for increased glucocorticoid 6 beta-hydroxylation (enhanced family 3A cytochrome P-450 activity) is found in certain reversible forms of human hypertension. This association was investigated in the spontaneously hypertensive rat (SHR). The proportion of injected [3H]corticosterone excreted in urine as 6 beta-[3H]OH-corticosterone was four- to fivefold higher in SHR than in control Wistar-Kyoto rats, before and after development of overt hypertension. Both hypertension and 6 beta-hydroxylation were inhibited by troleandomycin (a selective inhibitor of family 3A cytochromes P-450), consistent with a role for increased steroid 6 beta-hydroxylation in the genesis of hypertension in the SHR.

Expression of cytochrome P450 3A in amphibian, rat, and human kidney.

Family 3A mammalian liver cytochromes P450 (3A1, rat; 3A3/4, human) catalyze the 6 beta-hydroxylation of endogenous steroids and are steroid inducible. Our recent finding that A6 cells (a toad kidney epithelial cell line) contain corticosterone 6 beta-hydroxylase activity as a steroid-inducible microsomal cytochrome P450 raised the possibility that corticosterone 6 beta-hydroxylase activity in the A6 cells is catalyzed by a member of the 3A family. We found that incubation of A6 cell microsomes from dexamethasone-induced cells with antibodies against family 3A proteins specifically inhibited corticosterone 6 beta-hydroxylase activity. Microsomes from A6 cells analyzed on immunoblots developed with family 3A specific antibodies revealed immunoreactive proteins and treatment of A6 with corticosterone or dexamethasone increased the amounts of 3A immunoreactive protein(s). Furthermore, A6 RNA hybridized with 3A cDNAs on Northern blots and genomic DNA from A6 cells hybridized with a 3A cDNA on a Southern blot. Thus, toad kidney A6 cells express a family 3A P450 that is immunochemically, functionally, and genetically related to the mammalian liver 3A proteins. Prompted by these findings in amphibian kidney, we examined mammalian kidney for evidence of family 3A proteins. Immunocytochemical studies of frozen cryostat sections of normal adult rat kidney incubated with 3A1 antibody showed immunoreactivity only with collecting duct. Immunoblot analysis of human kidney microsomes found three protein bands representing 3A3/4, 3A5, and a 53-kDa Mr protein immunoreactive with human 3A antibody. An unexpected finding was the polymorphic expression of 3A3/4 in human kidney with only one of seven (14%) adult human kidneys tested expressing this protein while 3A5, a protein which is polymorphically expressed in adult human livers, was routinely present in the adult human kidney samples tested. Since human fetal liver contains a family 3A P450 we examined human fetal kidney microsomes by immunoblot analysis with human liver 3A antibody and found expression of a protein tentatively identified as 3A7. Thus, like A6 amphibian cells, family 3A P450 proteins and mRNAs are prominent, functional components in the kidney of mammals, including man.

Heterogeneity of CYP3A isoforms metabolizing erythromycin and cortisol.

The N-demethylation of erythromycin and 6 beta-hydroxylation of cortisol are both functions of the glucocorticoid-inducible CYP3A in human liver microsomes. To determine whether 6 beta-hydroxylation and erythromycin N-demethylation are catalyzed by similar or distinct CYP3A isoforms, erythromycin N-demethylase activity, as reflected by the recently described 14[C]-erythromycin breath test, was compared with urinary 6 beta-hydroxycortisol/cortisol ratios, a measure of cortisol 6 beta-hydroxylase activity, in nine patients. Erythromycin N-demethylation varied fourfold and 6 beta-hydroxycortisol/cortisol ratios varied sevenfold among the subjects; no correlation was found between these activities (r2 = 0.065). New noninvasive tests of CYP3A strongly suggest cortisol 6 beta-hydroxylation and erythromycin N-demethylation are performed by distinct CYP3A isoforms.

11 beta-Hydroxysteroid dehydrogenase activity in the renal target cells of aldosterone.

Aldosterone selectivity in mineralocorticoid target tissues has been suggested to be due to 11 beta-hydroxysteroid dehydrogenase (11-OHSD), which, by inactivating the endogenous glucocorticoids cortisol and corticosterone (CS), would allow aldosterone to bind to the mineralocorticoid receptor that has equal affinity for aldosterone and natural glucocorticoids. However, a recent immunohistochemical study failed to colocalize 11-OHSD and mineralocorticoid receptors in the kidney. The goal of this study was to determine 1) whether metabolism of CS occurs in the renal target cells of aldosterone, i.e. in cortical collecting duct cells, and 2) if it does so, whether this activity is sufficient to reduce intracellular CS levels to allow binding of aldosterone to the mineralocorticoid receptor. Cortical collecting duct cells were isolated by solid phase immunoadsorption, with a cell purity of approximately 98%. Metabolism of CS was studied in both freshly isolated cells and primary cultures grown as monolayers on permeable supports. Freshly isolated cells rapidly converted CS to 11-dehydro-CS, which was the only major metabolite detected. In intact collecting duct cells 11-OHSD had an apparent Km for CS of approximately 60 nM, a value more than 100-fold lower than the Km of 11-OHSD in the rat liver, and a maximum velocity of approximately 1.7 x 10(-14) mol/min.1000 cells. In cultured cells, when [3H]CS was applied to one side of the monolayer, almost all radioactivity on the opposite side was 11-dehydro-CS. The cells were able to almost completely metabolize CS passing through them for up to a concentration of 2.5 x 10(-7) M. Carbenoxolone, an inhibitor of 11-OHSD, reduced CS degradation by 88%. Neither freshly isolated nor cultured collecting duct cells converted [3H]11-dehydro-CS back to CS in a significant amount (less than 1%). These data provide functional evidence for 11-OHSD activity in renal aldosterone target cells and indicate that this enzyme might be a collecting duct-specific isoform of 11-OHSD which can sufficiently reduce intracellular CS concentrations to contribute to the apparent mineralocorticoid selectivity of the collecting duct.

Evidence that high dose cortisol-induced Na+ retention in man is not mediated by the mineralocorticoid receptor.

We have previously shown that high dose cortisol (F; 240 mg/day)-induced Na+ retention and systolic blood pressure (BP) increases are not inhibited by the glucocorticoid (type II) receptor antagonist RU486. Adequacy of type II receptor blockade with RU486 was clearly demonstrated, indicating that the Na+ retention was not mediated through the glucocorticoid receptor. Spironolactone (Sp: 400 mg/day), in a preliminary assessment, also did not inhibit F-induced Na+ retention. The purpose of this study was to determine whether the Na+ retention produced by F administration is mediated by the type I receptor by comparing the effects of F to a potent type I agonist [9 alpha-fludrohydrocortisone (9 alpha FF)] with and without Sp administration. The effects of the two agonists and Sp on urinary K excretion and BP were also compared. Normal male volunteers, on a constant daily diet for 10 days, received either F (240 mg/day) or 9 alpha FF (3.0 mg/day) with or without Sp (400 mg/day) for the last 5 days. The mean cumulative reductions in Na+ excretion during the 5 days compared to baseline values before hormone administration were 255 +/- 38 and 494 +/- 81 mmol/5 days for F (n = 9) and 9 alpha FF (n = 5), respectively (P = 0.01). Sp (n = 5) completely inhibited 9 alpha FF-induced Na+ retention (494 +/- 81 vs. -37 +/- 130 mmol/5 days; P less than 0.01), but had no effect (n = 5) on F-induced Na+ retention (255 +/- 38 vs. 193 +/- 50 mmol/5 days; P = NS). After the expected first day kaliuresis, the effects of both steroids on net cumulative urinary K+ excretion were minimal. Systolic BP was increased by F, but not 9 alpha FF, and Sp did not inhibit this increase. A 2-fold greater Sp-inhibitable Na(+)-retaining effect of the mineralocorticoid demonstrates that the failure of Sp to block F-induced Na+ retention is not due to inadequate type I receptor blockade. Based on these findings and earlier studies, we conclude that high dose (stress level) F-induced Na+ retention and systolic BP increase are not mediated by either the mineralo- or glucocorticoid receptor in normal man.

Synthesis of 6 beta-hydroxyaldosterone by A6 (toad kidney) cells in culture.

Incubation of aldosterone with confluent layers of A6 (toad kidney) cells leads to its hydroxylation at the 6 beta-position. 6 beta-Hydroxyaldosterone is the major metabolite when the incubation is carried out at pH 6.8, whereas the product comprises 6 beta-hydroxy-17-isoaldosterone accompanied by some 6 beta-hydroxyapoaldosterone at pH 7.4. All products were identified by high-field 1H nuclear magnetic resonance spectroscopy. Control experiments indicated that the side-chain isomerization to form the 17-iso and apo derivatives occurs after the cytochrome P 450-dependent synthesis of 6 beta-hydroxyaldosterone.

Corticosterone 6 beta-hydroxylase in A6 epithelia: a steroid-inducible cytochrome P-450.

We found microsomal corticosterone 6 beta-hydroxylase (6 beta-OHase) from cultured A6 kidney epithelial cells to be a cytochrome P-450 enzyme with both similarities to and differences from the rat liver steroid 6 beta-OHase P-450p. Enzyme activity was inhibited by CO, alpha-naphthoflavone, metyrapone, and clotrimazole, well-known inhibitors of P-450 enzymes, and increased by known inducers of P-450 enzymes, including dilantin, phenobarbital sodium, and corticosteroids. Moreover, some additional, relatively specific inducers of P-450p (troleandomycin and pregnenolone-16 alpha-carbonitrile) also induced the A6 6 beta-OHase, whereas inducers of other forms of P-450 (aroclor, spironolactone, and isosafrole) appeared to repress the A6 enzyme. The time course of increase in enzyme activity and increased cellular cytochrome P-450 content were consistent with increased levels of enzyme protein. Induction of 6 beta-OHase by the substrate (corticosterone), the metabolite (6 beta-OH-corticosterone), dexamethasone, and aldosterone was biphasic as a function of inducer concentration, with approximate 50% effective concentration (EC50) values of 10(-8)-10(-9) M and 10(-5)-10(-6) M for the respective components of induction. Cortisol also induced the enzyme at 10(-8)-10(-6) M; however, its metabolite 6 beta-OH-cortisol was ineffective or decreased activity at higher concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Corticosterone is converted to 6 beta-hydroxycorticosterone in rat: effects of the metabolite on urinary electrolyte excretion.

This study was performed to determine whether corticosterone (B), the major glucocorticoid of rat, is metabolized to its 6 beta-OH derivative (6 beta-OH-B) and whether this derivatives has any effects on Na+ or K+ transport in rat kidney. Normal and adrenalectomized (adx) rats were injected with [3H]B and urine was collected for 5 h and examined for metabolites of B. Metabolites were collected by solid phase extraction on mu Bondapak C18 cartridges and fractionated by reversed phase high performance liquid chromatography. Fractions coeluting with 6 beta-OH-B were rechromatographed by normal phase thin layer chromatography. Approximately 5% of the radioactivity recovered from the urine of both intact and adx rats cochromatographed with 6 beta-OH-B on the two systems. Mass spectra of this fraction were virtually identical to those of authentic 6 beta-OH-B, demonstrating that rats do metabolize B to its 6 beta-OH derivative. To evaluate the biological activity of this metabolite, adx rats were injected with NaCl and KCl and with varying dosage of either 6 beta-OH-B or reference steroids (aldosterone, B, 6 beta-OH-F). 6 beta-OH-B produced a significant antinatriuresis at all doses. Kaliuresis occurred only at the highest dose and creatinine excretion increased, suggesting increased glomerular filtration from a glucocorticoid effect. Although 6 beta-OH-B may simply be exerting mineralo- and glucocorticoid actions there are two unexplained findings. First, 6 beta-OH-B (10 micrograms/100 g) significantly decreased urinary K excretion with associated antinatriuresis, an effect which has not been seen with Aldo administration. Second, neither a kaliuretic nor antinatriuretic effect of 6 beta-OH-B could be demonstrated in experiments using a method to enhance mineralocorticoid induced K+ excretion (K+ deprivation and NaCl loading only). Yet, the dose used was clearly antinatriuretic in the initial bioassay. It is concluded that the rat is capable of metabolizing B to its 6 beta-OH-B derivative which appears in substantial quantity in the urine. This metabolite produces antinatriuresis in the adrenalectomized rat.

Adrenocorticotropin and cortisol-induced changes in urinary sodium and potassium excretion in man: effects of spironolactone and RU486.

The role of the glucocorticoid (type II) receptor in the Na+ retention induced by cortisol is not known. The relative contribution of mineralocorticoid (type I) and type II receptor activation to changes in urinary Na+ and K+ excretion in man was studied using spironolactone and RU486 to inhibit type I and II receptors, respectively. Normal men eating a constant daily diet received either ACTH or cortisol for 5 days. Spironolactone (400 mg/day) inhibited ACTH (80 U/day)-induced kaliuresis, but not the Na+ retention produced by ACTH or cortisol (240 mg/day) and only blunted the modest Na+ retention induced by cortisol (120 mg/day). RU486 (1200 mg/day for the first 2 day) inhibited the first day kaliuresis and carbohydrate intolerance produced by cortisol, but did not affect the Na+ retention. Thus, the kaliuresis produced by cortisol and ACTH can be attributed to type II and type I receptor activation, respectively. The failure of RU486 to inhibit the Na+ retention induced by cortisol with evidence of adequate blockade of type II receptors indicates that the Na+ retention produced by cortisol is not mediated by type II receptor activation, but is, at least in part, mediated by the type I receptor.