Receptor binding and biological activity of 18-hydroxycortisol.

Recently, 18-hydroxycortisol (11 beta,17 alpha,18,21-tetrahydroxy-4-pregnene-3,20-dione) was isolated and identified from extracts of urine and adrenal incubates of patients with primary aldosteronism. The receptor-binding activity to the renal gluco- and mineralocorticoid receptors and its biological activity as a glucocorticoid and mineralocorticoid were investigated using synthetic 18-hydroxycortisol. The ability of 18-hydroxycortisol to compete with [3H]aldosterone for renal binding to the receptor was 0.13% that of unlabeled aldosterone. The addition of a specific glucocorticoid, RU-26988 (11 beta,17-dihydroxy-21-methyl-17 alpha-pregna-1,4,6-triene-20-yn-3-one) decreased the competing ability to 0.02%, indicating significant binding to the glucocorticoid receptor. The ability to compete with [3H]dexamethasone for the renal cytoplasmic glucocorticoid receptor was 0.1% that of unlabeled dexamethasone. The mineralocorticoid activity of 18-hydroxycortisol was undetectable. Its glucocorticoid activity using an in vitro bioassay based on the induction of tyrosine aminotransferase in the HTC cell was detectable at 10(-5) M, but was too low for adequate quantification. In a second in vitro glucocorticoid bioassay, inhibition of cell growth of the L929 fibroblast, 18-hydroxycortisol also showed minimal activity. In summary, it is unlikely that 18-hydroxycortisol plays a role in the metabolic syndrome in those patients who produce it in excess due to its inactivity as a gluco- or mineralocorticoid.

Renal receptor-binding activity of reduced metabolites of aldosterone: evidence for a mineralocorticoid effect outside of the classic aldosterone receptor system.

Reduced metabolites of aldosterone have been shown to have antinatriuretic and kaliuretic effects. We have studied the ability of four reduced metabolites of aldosterone to compete with [3H]aldosterone and [3H]dexamethasone for binding to the mineralocorticoid and glucocorticoid receptors of the kidney using adrenalectomized rat renal slices and cytosol, respectively, as sources of the binding proteins. 5 alpha-Dihydroaldosterone had 18.9% the ability to compete with [3H]aldosterone for binding to the cytoplasmic receptor of adrenalectomized rat renal slices in comparison to unlabeled aldosterone. Its antinatriuretic potency varied between 7-17%. Its ability to compete with [3H]dexamethasone for binding to the renal glucocorticoid receptor was only 1.9% in comparison to unlabeled dexamethasone. The relative competitive activities of 3 beta,5 alpha-tetrahydroaldosterone and 3 beta,5 beta-tetrahydroaldosterone with [3H]aldosterone to adrenalectomized rat renal slices cytosol were 1.26% and 0.05%, respectively, in comparison to unlabeled aldosterone. Their reported mineralocorticoid activities using the adrenalectomized rat bioassay (antinatriuresis) were 0.1-0.4% and 0.15%, respectively, in comparison to aldosterone. The most important aldosterone metabolite 3 alpha,5 beta-tetrahydroaldosterone showed negligible competitive activity with [3H]aldosterone or [3H]dexamethasone for the renal corticoid type I or type II receptors, respectively. However, this compound has been reported and confirmed to have weak but clear-cut mineralocorticoid activity (approximately 1/100th that of aldosterone). The mineralocorticoid activity of 3 alpha,5 beta-tetrahydroaldosterone cannot be explained by a mechanism involving the classic renal mineralocorticoid receptor. The mechanism could involve an alternative receptor system, a nonreceptor-mediated renal mechanism, or the conversion to a metabolite that would interact with classic receptors.

Receptor binding and biological activity of 18-oxocortisol.

It has been recently demonstrated that cortisol can be metabolized, producing 18-hydroxycortisol and 18-oxocortisol, following the same pathway by which corticosterone is transformed into 18-hydroxycorticosterone and aldosterone. The influence of a hydroxy group in the 17 alpha position of aldosterone or an aldehyde (actually 11-18 hemiacetal) in the 13-methyl of cortisol on the mineralocorticoid and glucocorticoid activities were studied and compared with the parent steroids. The ability of 18-oxocortisol to complete with [3H]aldosterone for binding to the cytosol receptor of rat renal slices was 8.1% in comparison to unlabeled aldosterone. The addition of a specific glucocorticoid 11 beta, 17 beta-dihydroxy-17 alpha-pregnane-1,4,6- trien-20-yn-21-methyl-3-one decreased this binding to 5.6%. The ability of 18-oxocortisol to compete with [3H]dexamethasone for binding to the renal cytosol receptor was 0.2% that of unlabeled dexamethasone and in the HTC whole cell assay was 1.06% and 3.8% that of unlabeled dexamethasone and cortisol, respectively. The mineralocorticoid activity of 18-oxocortisol in the adrenalectomized rat bioassay was 0.6% that of aldosterone. The glucocorticoid activity in in vitro bioassays was 3.1% compared with that of a cortisol when the induction of tyrosine aminotransferase in HTC cells was measured and 4% when the inhibition of fibroblast L-929 growth was measured. The significance of 18-oxocortisol in the pathogenesis of the hypertension in patients with primary aldosteronism is still unclear.

Endothelin receptor subtypes and stimulation of aldosterone secretion.

Endothelins (ETs) are 21-amino acid peptides with two disulfide bonds that have powerful vasoactive properties. We have previously shown the presence of a specific, high-affinity, saturable receptor for porcine or human endothelin (ET-1) in cultured calf zona glomerulosa cells. ET-1 was a stimulator of aldosterone secretion although not as powerful as angiotensin II. Incubations of cultured calf zona glomerulosa cells with Sarafotoxin S6b (S6b), a snake venom that has a structure highly homologous to ET-1, stimulated aldosterone secretion with a potency similar to that of ET-1. Binding of [125I]ET-1 to the adrenal receptor gave a Kd of 0.17 +/- 0.05 nM and a Bmax of 36 +/- 8.5 fmol/well (n = 4). Displacement of [125I]ET-1 by unlabeled ETs and S6b showed that the concentrations needed to displace 50% of the tracer were 0.3 nM for ET-1, 0.3 nM for ET-2, 10 nM for S6b, and 100 nM for ET-3. Binding of [125I]S6b to cultured adrenal cells revealed a receptor with a Kd of 0.05 +/- 0.01 nM and a Bmax of 8 +/- 2 fmol/well (n = 4). Displacement of [125I]S6b by unlabeled ETs and S6b showed that the concentrations needed to displace 50% of the tracer were 0.03 nM for S6b, 0.06 nM for ET-1, 0.04 nM for ET-2, and 0.05 nM for ET-3. Unlabeled ET-1 and ET-2 preferentially down-regulated the binding of [125I]ET-1, and S6b preferentially down-regulated the binding of [125I]S6b.(ABSTRACT TRUNCATED AT 250 WORDS)

An analysis of acute changes in interleukin-6 levels after treatment of hepatitis C with consensus interferon.

Cytokine production has been implicated in the antiviral response to interferon-alpha (IFN-alpha) in hepatitis C and in the development of IFN-alpha-related side effects. We characterized acute changes in serum cytokine levels following administration of a single dose of consensus IFN (IFN-con1) and during continuous treatment of chronic hepatitis C patients. Serum samples were collected at baseline, at multiple times early after IFN administration, and weekly thereafter. Viral RNA titers were assessed by RT-PCR, and viral kinetics were followed. ELISA assays were used to measure IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), IL-4, IL-6, and IL-16. Serum cytokine levels were low at baseline. IL-6 was detected in patients with hepatitis C but not in healthy control subjects by either ELISA or RT-PCR, indicating that low levels of circulating IL-6 were associated with hepatitis C infection. None of the cytokines measured increased significantly after IFN administration except for IL-6. IL-6 levels rose rapidly, peaked at 6-15 h in a dose-dependent manner, and returned to baseline by 48 h in both patients receiving a single dose of IFN and those receiving continuous treatment. This was confirmed by RT-PCR. Pretreatment IL-6 levels were directly correlated with area under the curve (AUC) for IL-6 during the 24 h after IFN dosing (r = 0.611, p = 0.007). Viral titers decreased within 24-48 h after a single dose of IFN-con1. Changes in hepatitis C RNA titers were not significantly associated with pretreatment IL-6 levels or with changes in IL-6 levels. In conclusion, (1) baseline serum cytokine levels, except for IL-6, were low or within the normal range in patients with hepatitis C, (2) IL-6 levels were detected in some patients with hepatitis C before treatment but not in healthy controls, (3) IL-6 levels increased acutely after a single dose of IFN-alpha, and IL-6 induction was related to baseline IL-6 level, and (4) changes in IL-6 levels did not correlate with the early virologic response to IFN.

Rat mesenteric artery endothelial cells in culture secrete ET-1.

Endothelial cells were harvested by the collagenase perfusion of isolated mesenteric arteries of rats and cultured. An endothelin peptide was detected in the supernatant of these cells by an antibody which recognizes ET-1 but not "rat" endothelin (ET-3). Culture media was extracted using a C-8 solid phase column and subjected to reverse phase HPLC using a system that separates all known endothelins and immunoreactive endothelins measured using another antibody which recognizes all endothelins. The main immunoreactive peak co-eluted with ET-1. We could not detect any ET-2, ET-3 or Vasoactive Intestinal Contractor. A smaller immunoreactive peak of unknown structure that eluted earlier than ET-1 was also detected. In conclusion, rat endothelial cells secrete a peptide of similar chromatographic and immunoreactive properties as ET-1.

The production of a monoclonal antibody to 18-hydroxycortisol and other 18-hydroxylated steroids.

Four strains of mice were immunized with 6 different conjugates of 3-O (carboxymethyl-oximino)-18-hydroxycortisol to bovine serum albumin (3 preparations), turkey serum albumin, porcine thyroglobulin, and keyhole limpet hemocyanin. Spleens from 7 of 48 mice immunized were fused with Fox/NY and/or HL-1 Friendly myeloma cell lines, yielding many positive clones for antibody formation. Short cross-reactivities were done in 293 culture supernatants and were found to have low cross-reactivity (less than 0.001%) to cortisol, but very high cross-reactivity to 18-hydroxy-11-deoxycortisol (70 to 140%). One clone showed over 100% cross-reactivity with all the 18-hydroxylated steroids studied. The major problem encountered in the generation of monoclonal antibodies was the low antibody response in the vast majority of mice injected. Half the mice developed no measurable titer, and the clones evaluated from those that did produce antibodies cross-reacted with other 18-hydroxylated steroids. Nevertheless, the antibody developed could serve in radioimmunoassay for 18-hydroxy-11-deoxycortisol separated chromatographically from other cross-reacting steroids. This is important as no synthetic 18-hydroxy-11-deoxycortisol is available.

The production of monoclonal antibodies against aldosterone.

We have prepared several monoclonal antibodies against aldosterone-3-carboxy-methyloxime-BSA conjugate by fusing spleen lymphocytes from an immunized mouse with the mouse myeloma line HL-1 Friendly. A total of 6 different clones were isolated and expanded. All of the antibodies exhibited low cross-reactivities against most of the compounds tested. Antibodies A5A3, A2E11, and C1E2 exhibited low cross-reactivity with 18-hydroxycorticosterone and 18-hydroxydeoxycorticosterone and showed no detectable displacement of tritiated aldosterone from the antibodies with cortisol, corticosterone, and related steroids. The only steroid that showed moderate cross-reactivity was 3 alpha,5 beta-tetrahydroaldosterone (around 3%). Clone A5H12 antibodies exhibited high cross-reactivity with tetrahydroaldosterone (19.3%) but otherwise was very similar to the above clones. Antibody of clone C1E4 showed high cross-reactivity to tetrahydroaldosterone (41.2%) and 18-hydroxyDOC (2%) with relatively low cross-reactivity to DOC (0.078%). Clone A2G9 antibodies were the only ones for which cortisol and corticosterone displaced tritiated aldosterone with cross-reactivities of 0.0042% and 0.125%, making them unsuitable for a direct radioimmunoassay of plasma aldosterone. The monoclonal antibodies were very sensitive to freezing and thawing. The cross-reactivities of the first three clones' antibodies compare favorably with those polyclonal antibodies that have been described to be suitable for use in direct radioimmunoassays of plasma aldosterone. Their advantage is the reliable supply of an antibody with consistent, predictable properties.

Synthesis of 18-hydroxycortisol and 18-oxocortisol in bovine adrenal slices.

The adrenal cortex shows a histological and functional zonation which allows it to function as two distinct glands. Aldosterone is produced only in the outer area, or zona glomerulosa and cortisol is produced in the zona fasciculata. Adrenal slices (200 mu) were prepared from cores of beef adrenals using a McIlwain Tissue Slicer and incubated. Cortisol, corticosterone, aldosterone, 18-hydroxycorticosterone, 18-hydroxycortisol and 18-oxocortisol were measured by radioimmunoassay. Aldosterone and 18-hydroxycorticosterone were produced primarily in the outer slices of the adrenal. Cortisol and corticosterone were produced throughout the adrenal with the production of cortisol in the first layer probably due to contamination with fasciculata. 18-Hydroxycortisol and 18-oxocortisol were produced primarily in the outer slices. The coexistence of cytochrome P-450 corticosterone methyl oxidase I and II and the 17 alpha-hydroxylase in the outer slices leads to the synthesis of these two hybrid steroids.

Glucocorticoid activity of 11 beta,17 beta-dihydroxy-21-methyl-17 alpha-pregna-1,4,6-trien-20-yn-3-one (RU-26988).

Natural and most synthetic glucocorticoids have either a 17 alpha-21-dihydroxy-20-keto or 21-hydroxy-20-keto side chain. Recently a group of 17 alpha-alkynyl steroids which lacks this side chain has been described to have glucocorticoid activity. We have studied the ability of RU-26988 (11 beta,17 beta-dihydroxy-21-methyl-17 alpha-pregna-1, 4,6, trien-20-yn-3-one) to interact with the glucocorticoid receptor and its biological activity in both in vitro and in vivo bioassays. RU-26988 was as active as dexamethasone in competing with [3H] dexamethasone for the liver cytosol receptor, while it had 120% the ability of dexamethasone to compete with [3H] dexamethasone for the HTC cell receptors. RU-26988 had 20% and dexamethasone 14% of the competing ability of triamcinolone acetonide for [3H] triamcinolone acetonide binding to the L-929 fibroblast receptor system. The in vitro glucocorticoid activity as measured by the induction of tyrosine aminotransferase (TAT) in HTC cells by RU-26988 was only 30% that of dexamethasone and the maximal activity was also reduced, suggesting that it behaves as a suboptimal inducer in this system. In the L-929 fibroblast it had 32% the activity of dexamethasone but the maximal inhibition by the two was similar. RU-26988 had only 3.2% the activity of dexamethasone in in vivo rat bioassays (induction of TAT or glycogen deposition in the liver). These data supplement other reports that RU-26988 might be a promising glucocorticoid for topical use where it behaves as a powerful agonist, but not for systemic use, where it is considerably weaker.