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Affinity of the mineralocorticoid receptor (MR) is similar for aldosterone and the glucocorticoids (GC) cortisol and corticosterone, which circulate at concentrations far exceeding those of aldosterone. 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) inactivation of GC within the immediate vicinity of the MR is credited with prereceptor specificity for aldosterone in cells coexpressing MR and 11βHSD2. 11βHSD2 efficacy is also critical to other recently described 11βHSD2 substrates. The aim of this work was to address doubts that low levels of expression of 11βHSD2 in aldosterone target tissues suffice to prevent the initiation of gene transcription by the MR activated by physiological concentrations of corticosterone. Cell models stably expressing an MR/Gaussia luciferase reporter and various levels of constitutive or induced 11βHSD2 at concentrations lower than those in rat kidney homogenates and microsomes were produced. Aldosterone and corticosterone were equipotent transactivators of the MR reporter gene in cells without 11βHSD2. Rate of conversion of tritiated corticosterone to 11-dehydrocorticosterone increased and corticosterone-induced nuclear translocation of MR decreased, as 11βHSD2 expression increased. The 50% maximal MR activation for the reporter gene stimulation by corticosterone rose with increasing 11βHSD2 expression, shifting the steroid dose-response curve for corticosterone-induced MR transactivation to the right. Several stable cell lines expressing an easily and reproducibly measured MR reporter system and consistent incremental amounts of 11βHSD2 protein were produced and used to document that 11βHSD2 within low physiological levels inactivates relevant concentrations of GC and decreases MR transactivation by GC in a dose-dependent fashion, laying to rest doubts of the efficacy of this enzyme.

CYP11B1 and CYP11B2 play pivotal roles in adrenocorticosteroids synthesis. We performed semi-quantitative immunohistochemical analysis of these proteins in adrenals from patients with primary aldosteronism using novel monoclonal antibodies. Clusters of cortical cells positive for CYP11B2 were detected in the zona glomerulosa (ZG) of normal adrenal gland (NA), idiopathic hyperaldosteronism (IHA) and the adjacent adrenal of aldosterone-producing adenoma (APA). In APA, heterogenous immunolocalization of CYP11B2 and diffuse immunoreactivity of CYP11B1 were detected in tumor cells, respectively. The relative immunoreactivity of CYP11B2 in the ZG of adjacent adrenal of APA was significantly lower than that of NA, IHA and APA tumor cells, suggestive of suppressed aldosterone biosynthesis in these cells. These findings did indicate the regulatory mechanisms of aldosterone biosynthesis were different between normal/hyperplastic and neoplastic aldosterone-producing cells in human adrenals. CYP11B2 immunoreactivity in the ZG could also serve as a potential immunohistochemical marker differentiating morphologically hyperplastic ZG of IHA and APA adjacent adrenal.

The G-protein-activated inwardly rectifying potassium channel Kir3.4 is expressed in the zona glomerulosa cell membrane and transports potassium out of the cell. Angiotensin II stimulation of aldosterone secretion is mediated, in part, by suppression of the transcription of KCNJ5, the gene coding for Kir3.4, and blocking channel activity. This results in membrane depolarization, mobilization of intracellular calcium, activation of the calcium-calmodulin pathway and increasing gene transcription of steroidogenic enzymes required for aldosterone secretion. In 40-60% of aldosterone-producing adenomas there is a somatic mutation in the region of the KCNJ5 gene that codes for the selectivity filter that decreases potassium selectivity, allowing sodium to leak into the cells, thus depolarizing the membrane and initiating events that result in increased aldosterone synthesis. The mechanism by which mutated KCNJ5 induces cell proliferation and adenoma formation remains unclear.

Background: Primary Aldosteronism (PA) is commonly due to an Aldosterone-Producing Adenoma (APA), but until now the lack of a specific antibody for human aldosterone synthase (hCYP11B2) has prevented the identification of APA in the excised adrenal gland. Objectives: To investigate the usefulness of a novel monoclonal antibody for hCYP11B2 for the immuno-phenotypic characterization of a large series of consecutive adrenal glands from APA patients diagnosed by the “four corners criteria” fully characterized for KCNJ5 mutations. Methods: To generate the antibodies, spleen cells from mice immunized with peptides against hCYP11B2 were fused to mouse myeloma cells. CYP11B2 and CYP11B1 were detected with immunohistochemistry in the adrenals from 49 consecutive APA patients. Based on results of a pilot study the following classification was developed: Pattern1: adenoma with uniform CYP11B2 staining + few areas of CYP11B1 staining; Pattern2: adenoma with diffuse CYP11B2 staining + few areas of CYP11B1 staining + some cells co-expressing CYP11B2/CYP11B1; Pattern3: adenoma expressing CYP11B1 + clusters of cells CYP11B2; Pattern4: no adenoma + multiple clusters of sub-capsular CYP11B2 cells; Pattern5: multi-nodular adrenal cortex + clusters of sub-capsular CYP11B2 cells. Results: KCNJ5 mutations were found in 38% of the cases (19/49). With CYP11B2/CYP11B1 immunostaining the following results were found: pattern 1 16.3% (8/49); pattern2 32.7% (15/49); pattern 3 16.3% (7/49); pattern 4 22.4% (10/49); pattern 5 12.2% (5/49). Conclusions: The highly variable steroidogenic enzymes (CYP11B2, CYP11B1) expression in a series of consecutive APA show the existence of at least 5 different patterns, thus challenging the classical notion that PA entails only APA or bilateral hyperplasia.

1. The final enzymes in the biosynthesis of aldosterone and cortisol are by the cytochrome P450 CYP11B2 and CYP11B1, respectively. The enzymes are 93% homologous at the amino acid level and specific antibodies have been difficult to generate. 2. Mice and rats were immunized with multiple peptides conjugated to various immunogenic proteins and monoclonal antibodies were generated. The only peptide sequences that generated specific antibodies were amino acids 41-52 for the CYP11B2 and amino acids 80-90 for the CYP11B1 enzyme. 3. The mouse monoclonal CYP11B2-41 was specific and sensitive for use in western blots and produced specific staining of the zona glomerulosa of normal adrenal glands. The rat monoclonal CYP11B1-80 also detected a single band by western blot and detected only the zona fasciculata. Triple immunofluorescence of the adrenal demonstrated that the CYP11B1 and the CYP11B2 did not co-localize, while as expected the CYP11B1 co-localized with the 17α-hydroxylase.

Objectives: Activating autoantibodies (AA) to the AT1R second extracellular loop (ECL2) are present in patients with primary aldosteronism (PA). We investigated whether AA from PA and from AT1R-ECL2 immunized rabbits activate AT1R and thereby contribute to hypertension by a direct contractile effect on the vasculature and stimulate adrenal aldosterone production. Methods: We studied 5 normal controls (NC) and 15 subjects with biochemically confirmed PA [4 APA, 4 IAH non- and 7 indeterminate (IPA)]. AT1R-AA activity in serum or IgG was analyzed in AT1R transfected CHO cells. Contractile effects were assayed in isolated perfused rat cremaster arteries. We determined the effects of an ARB and the L-aa AT1R-ECL2 target epitope on in vitro AT1R cremaster artery responsiveness to patient sera and to MAP-L-aa (AFHYESQ) peptide immunized rabbit serum. We examined the effect of IgG from 7 PA subjects on aldosterone production in HAC15 cells in vitro w/wo 1.0 and 10 nM Ang II; and w/wo ARB. Results: Serum from PA significantly (pnd ECL target reduced contractility to 8%; not different from baseline. Conclusions: AA from PA and AT1R-immunized rabbits activates AT1R transfected CHO cells, increases in vitro arterial contractility and stimulates basal and Ang II induced HAC15 aldosterone secretion. ARBs and decoy peptides were effective in blocking AA effects in vitro . These AA have important etiological and therapeutic implications.