Development and application of low-cost T-ARMS-PCR assay for AGT and CYP11B1 gene polymorphisms.

Angiotensin II (Ang II: a truncated octapeptide of angiotensinogen, AGT) and 11-ß-hydroxylase influence regulation of blood pressure. Dysregulation of Ang II and 11-ß-hydroxylase can lead to hypertension and elevate aldosterone levels. Polymorphisms in AGT (encodes AGT) and CYP11B1 (encodes 11-ß-hydroxylase) shift the paradigm from physiological to pathological. Currently, various high-throughput techniques are used to genotype these polymorphisms. These techniques require expensive infrastructure and reagents. However, in developing countries, where cost is the main limiting factor, it is not feasible to use expensive techniques. So, the aim of current study was to develop efficient low-cost method for genotyping of cardiovascular disease and hypertension associated polymorphisms of AGT (rs4762, rs5051) and CYP11B1 (rs6410). For this, tetra amplification-refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method was developed and optimized for aforementioned AGT and CYP11B1 gene polymorphisms. Efficiency of T-ARMS-PCR was tested by genotyping 776 human samples. These T-ARMS-PCR assays were also validated by Sanger DNA sequencing, where 100% concordance was found, allowing the efficient use of these T-ARMS-PCR assays for polymorphism genotyping in AGT and CYP11B1 in resource limited settings. T-ARMS-PCR is low-cost, efficient and reliable assay for genotyping of AGT and CYP11B1 gene polymorphisms.

Two novel CYP11B1 mutations in congenital adrenal hyperplasia due to steroid 11ß hydroxylase deficiency in a Tunisian family.

Steroid 11ß hydroxylase deficiency (11ß-OHD) (OMIM # 202010) is the second most common form of congenital adrenal hyperplasia (CAH), accounting for 5-8% of all cases. It is an autosomal recessive enzyme defect impairing the biosynthesis of cortisol. The CYP11B1 gene encoding this enzyme is located on chromosome 8q22, approximately 40kb from the highly homologous CYP11B2 gene encoding for the aldosterone synthase. Virilization and hypertension are the main clinical characteristics of this disease. In Tunisia, the incidence of 11ß-OHD appears higher due to a high rate of consanguinity (17.5% of congenital adrenal hyperplasia). The identical presentation of genital ambiguity (females) and pseudo-precocious puberty (males) can lead to misdiagnosis with 21 hydroxylase deficiency. The clinical hallmark of 11ß hydroxylase deficiency is variable, and biochemical identification of elevated precursor metabolites is not usually available. In order to clarify the underlying mechanism causing 11ß-OHD, we performed the molecular genetic analysis of the CYP11B1 gene in a female patient diagnosed as classical 11ß-OHD. The nucleotide sequence of the patient's CYP11B1 revealed two novel mutations in exon 4: a missense mutation that converts codon AGT (serine) to ATT (isoleucine) (c.650G>T; p.S217I) combined with an insertion of a thymine at the c.652-653 position (c.652_653insT). This insertion leads to a reading frame shift, multiple incorrect codons, and a premature stop in codon 258, that drastically affects normal protein function leading to a severe phenotype with ambiguous genitalia of congenital adrenal hyperplasia due to 11ß hydroxylase deficiency.

Mass Spectrometry Imaging Establishes 2 Distinct Metabolic Phenotypes of Aldosterone-Producing Cell Clusters in Primary Aldosteronism.

Aldosterone-producing adenomas (APAs) are one of the main causes of primary aldosteronism and the most prevalent surgically correctable form of hypertension. Aldosterone-producing cell clusters (APCCs) comprise tight nests of zona glomerulosa cells, strongly positive for CYP11B2 (aldosterone synthase) in immunohistochemistry. APCCs have been suggested as possible precursors of APAs because they frequently carry driver mutations for constitutive aldosterone production, and a few adrenal lesions with histopathologic features of both APCCs and APAs have been identified. Our objective was to investigate the metabolic phenotypes of APCCs (n=27) compared with APAs (n=6) using in situ matrix-assisted laser desorption/ionization mass spectrometry imaging of formalin-fixed paraffin-embedded adrenals from patients with unilateral primary aldosteronism. Specific distribution patterns of metabolites were associated with APCCs and classified 2 separate APCC subgroups (subgroups 1 and 2) indistinguishable by CYP11B2 immunohistochemistry. Metabolic profiles of APCCs in subgroup 1 were tightly clustered and distinct from subgroup 2 and APAs. Multiple APCCs from the same adrenal displayed metabolic profiles of the same subgroup. Metabolites of APCC subgroup 2 were highly similar to the APA group and indicated enhanced metabolic pathways favoring cell proliferation compared with APCC subgroup 1. In conclusion, we demonstrate specific subgroups of APCCs with strikingly divergent distribution patterns of metabolites. One subgroup displays a metabolic phenotype convergent with APAs and may represent the progression of APCCs to APAs.

Primary aldosteronism concurrent with subclinical Cushing's syndrome: a case report and review of the literature.

BACKGROUND: The prevalence of primary aldosteronism concurrent with subclinical Cushing's syndrome was higher than previously thought. Through analyzing a rare clinical case, we summarized the diagnosis and management of primary aldosteronism with subclinical Cushing's syndrome. CASE PRESENTATION: A 54-year-old Chinese man of Han nationality was diagnosed as having primary aldosteronism with subclinical Cushing's syndrome. An abdominal computed tomography scan revealed a mass in his left adrenal gland and a mass in his right adrenal gland. After finishing sequential adrenal venous sampling without adrenocorticotropic hormone, the result reminded us that the left and right nodules were responsible for hypercortisolism and aldosterone hypersecretion, respectively. Right and left adrenalectomy were performed successively. The pathological diagnosis was adrenocortical adenoma for both. Histological findings revealed that the right one had positive immunostaining for CYP11B2 and the left one had positive immunostaining for CYP11B1. The immunohistochemistry result helped us to confirm the diagnosis. Somatic KCNJ5 mutation (Leu168Arg) was found in the right tumor; there was no KCNJ5 mutation in the left adrenal tumor. CONCLUSIONS: We suggest that patients with primary aldosteronism should have a low-dose overnight dexamethasone suppression test to screen for hypercortisolism. It can help avoid misdiagnoses and contribute to proper understanding of the adrenal vein sampling result. Making sure of the nidus of aldosterone and cortisol secretion is crucial for the therapy of patients with primary aldosteronism and subclinical Cushing's syndrome.

[123 I]Iodometomidate for molecular imaging of adrenocortical cytochrome P450 family 11B enzymes.

BACKGROUND: Due to advances in conventional imaging, adrenal tumors are detected with increasing frequency. However, conventional imaging provides only limited information on the origin of these lesions, which represent a wide range of different pathological entities. New specific imaging methods would therefore be of great clinical value. We, therefore, studied the potential of iodometomidate (IMTO) as tracer for molecular imaging of cytochrome P450 family 11B (Cyp11B) enzymes. METHODS: Inhibition of Cyp11B1 and Cyp11B2 by IMTO, etomidate, metomidate, and fluoroetomidate was investigated in NCI-h295 cells and in Y1 cells stably expressing hsCyp11B1 or hsCyp11B2. Pharmacokinetics and biodistribution after iv injection of [(123/125)I]IMTO were analyzed in mice in biodistribution experiments and by small-animal single-photon emission computed tomography (SPECT). Furthermore, four patients with known adrenal tumors (two metastatic adrenal adenocarcinomas, one bilateral adrenocortical adenoma, and one melanoma metastasis) were investigated with [(123)I]iodometomidate-SPECT. RESULTS: In cell culture experiments, all compounds potently inhibited both Cyp11B1 and Cyp11B2. Adrenals showed high and specific uptake of [(123/125)I]IMTO and were excellently visualized in mice. In patients, adrenocortical tissue showed high and specific tracer uptake in both primary tumor and metastases with short investigation time and low radiation exposure, whereas the non-adrenocortical tumor did not exhibit any tracer uptake. CONCLUSION: We have successfully completed the development of an in vivo detection system of adrenal Cyp11B enzymes by [(123)I]IMTO scintigraphy in both experimental animals and humans. Our findings suggest that [(123)I]IMTO is a highly specific radiotracer for imaging of adrenocortical tissue. Due to the general availability of SPECT technology, we anticipate that [(123)I]IMTO scintigraphy may become a widely used tool to characterize adrenal lesions.

Serum 21-Deoxycortisol, 17-Hydroxyprogesterone, and 11-deoxycortisol in classic congenital adrenal hyperplasia: clinical and hormonal correlations and identification of patients with 11beta-hydroxylase deficiency among a large group with alleged 21-hydroxylase deficiency.

INTRODUCTION: 21-Hydroxylase deficiency (21OHD) is the most common cause of congenital adrenal hyperplasia, followed in frequency by 11beta-hydroxylase deficiency (11betaOHD). Although the relative frequency of 11betaOHD is reported as between 3 and 5% of the cases, these numbers may have been somewhat underestimated. MATERIALS AND METHODS: In 133 patients (89 females/44 males; 10 d-20.9 yr) with alleged classic 21OHD and five (three females/two males; 7.3-21 yr) with documented 11betaOHD, we measured serum 21-deoxycortisol (21DF), 17-hydroxyprogesterone (17OHP), and 11-deoxycortisol (S), 48 h after glucocorticoid withdrawal. We also studied 20 sex- and age-matched control subjects. Serum steroid levels were determined by RIA after HPLC purification. OBJECTIVES: The objectives of this study were to: 1) quantify 21DF in patients with congenital adrenal hyperplasia, 2) correlate hormonal with clinical data, and 3) identify possible misdiagnosed patients with 11betaOHD among those with 21OHD. RESULTS: In 21OHD, 17OHP (217-100,472 ng/dl) and 21DF (<39-14,105 ng/dl) were mostly elevated and positively correlated (r = 0.7202; P < 0.001). Except for higher 17OHP in pubertal patients, 17OHP and 21DF values were similar according to sex, disease severity, or prevailing glucocorticoid dose. One additional patient with 11betaOHD was detected (1%) and also one with apparent combined 11beta- and 21OHD. S levels were elevated in 11betaOHD and normal but significantly higher in 21OHD than in controls. CONCLUSION: To recognize patients with 21- and/or 11betaOHD, we recommend evaluation of 17OHP or 21DF and S. Also, 21DF may be useful to follow up pubertal patients with 21OHD. Because 1% of patients with alleged 21OHD may have 11betaOHD, its frequency seems underestimated, as per our experience in a Brazilian population.

A Novel CYP11B2-Specific Imaging Agent for Detection of Unilateral Subtypes of Primary Aldosteronism.

CONTEXT: Although adrenal vein sampling is the standard method to distinguish unilateral from bilateral forms of primary aldosteronism, it is an invasive and technically difficult procedure. (11)C-metomidate (MTO)-positron emission tomography was reported as a potential replacement for adrenal vein sampling. However, MTO has low selectivity for CYP11B2 over CYP11B1. OBJECTIVE: This study aimed to determine the selectivity of (18)F-CDP2230, a new imaging agent, for CYP11B2 over CYP11B1 and determine whether the biodistribution profile of (18)F-CDP2230 is favorable for imaging CYP11B2. METHODS: The IC50 of CDP2230 for the enzymatic activities of CYP11B2 and CYP11B1 was determined using cells with stable expression of either enzyme. In vitro autoradiography of human adrenal sections with aldosterone-producing adenomas was performed to confirm the specific binding ability of (18)F-CDP2230 to CYP11B2-expressing regions. Furthermore, positron emission tomography and magnetic resonance imaging were performed to evaluate the biodistribution of (18)F-CDP2230 in rats. RESULTS: Although CDP2230 showed a significantly lower affinity for CYP11B2 and CYP11B1 than did MTO analogues, its selectivity for CYP11B2 over CYP11B1 was higher than that of MTO analogues. In vitro autoradiography revealed that the binding of (18)F-CDP2230 to CYP11B2-expressing regions in the adrenal gland was more specific than that of (123)I-IMTO. Moreover, the biodistribution study showed that (18)F-CDP2230 accumulated in adrenal glands with low background uptake. CONCLUSIONS: Our study showed a high selectivity of (18)F-CDP2230 for CYP11B2 over CYP11B1 with a favorable biodistribution for imaging CYP11B2. (18)F-CDP2230 is a promising imaging agent for detecting unilateral subtypes of primary aldosteronism.

Synthesis of corticosterone in the vascular wall.

Extra-adrenal steroid 21-hydroxylation and 11 beta-hydroxylation occur in a variety of human tissues. This study was undertaken to determine whether the rat mesenteric artery produces corticosterone and to demonstrate the CYP11B1 mRNA in the vascular tissue. Isolated rat mesenteric arteries were perfused with Krebs-Ringer solution for 4 h. The perfusate was collected and chromatographed in a reverse-phase HPLC system. The fraction corresponding to synthetic corticosterone was collected and analyzed by mass spectrometry. The concentration of corticosterone in the perfusate from the adrenalectomized rats was measured using radioimmunoassay after separation with the HPLC system. The mass spectra of synthetic corticosterone was identical with corticosterone isolated from the perfusate of the rat mesenteric arteries. The radioactive peak of corticosterone was detected in the perfusate after perfusing the mesenteric artery with Krebs-Ringer solution containing [14C]-pregnenolone. The expression of CYP11B1, 11B2, and 11A mRNA was detected in the mesenteric artery using a RT-PCR. The production of corticosterone in the vascular wall was increased in the adrenalectomized rats compared with that of the controls. This study shows that the rat mesenteric artery produces corticosterone, and the corticosterone synthase is existed in the vasculature.

Zona glomerulosa function after life-long suppression in two siblings with the hypertensive virilizing form of congenital adrenal hyperplasia.

The function of the adrenal zona glomerulosa was studied in two pubertal siblings with the hypertensive virilizing form of congenital adrenal hyperplasia who had never been treated. Initially, their plasma 11-deoxycortisol and 11-deoxycorticosterone (DOC) levels were very high, PRA was suppressed, and plasma aldosterone and 18-hydroxycorticosterone (18-OHB) were undetectable. To selectively study zona glomerulosa function, the patients and five normal subjects were given dexamethasone (2 mg/day; thus suppressing zona fasciculata function), and their sodium intake was restricted to 10 mmol/day. After 3-5 days, the zona glomerulosa was stimulated with either angiotensin II or potassium chloride. The same protocol was repeated in the patients at various intervals up to 39 months after beginning maintenance therapy with dexamethasone (0.25 mg twice daily). PRA, plasma aldosterone, and 18-OHB remained low during the first 6 months of treatment. After the first year, PRA recovered, and the zona glomerulosa began to respond. Plasma aldosterone and 18-OHB levels reached normal basal and stimulated values in one of the patients after 2 yr of treatment, but remained subnormal after 39 months of treatment in the other patient. Both patients, however, had persistently elevated plasma DOC concentrations, suggesting slight but definite impairment of 11 beta-hydroxylation in the zona glomerulosa. We conclude that in spite of a severe and persistent 11 beta-/18-hydroxylation deficiency in the zona fasciculata, the zona glomerulosa can recover almost completely after prolonged treatment. Appropriate stimulation, however, discloses a minor 11 beta-hydroxylation impairment also in the zona glomerulosa. In addition, the lack of parallelism in zona glomerulosa 11 beta- and 18-hydroxylation of DOC provides evidence for the concept of different 18-hydroxylating systems in the adrenal cortex.

Mapping of phenytoin-inducible cytochrome P450 immunoreactivity in the mouse central nervous system.

The distribution of phenytoin-inducible cytochrome P450 in non-treated mouse brain and spinal cord was analysed immunohistochemically using polyclonal antibodies against phenytoin-induced mouse cerebral microsomal P450. This P450 protein was proved in Ouchterlony [Volk B. et al. (1988) Neurosci. Lett. 84, 219-224], Western blot, and immunohistochemical analyses to be reactive to the specific antibodies and an IgG fraction raised against phenobarbital-induced rat liver microsomal P450IIB1. The phenytoin-induced P450 is designated P450IIB1* because immunologically it is comparable with P450IIB1; however, it has not yet been analysed for other characteristics of this enzyme. Immunocytochemistry was performed on acetone-fixed serial cryosections of the whole brain using the avidin-biotin-peroxidase detection system. Negative controls included incubations with preimmune serum of the immunized animal instead of the primary antibody and preabsorption of the antibody with the corresponding immunogen. The pattern of immunoreactive sites indicates that P450IIB1* is not distributed evenly throughout the CNS. It was found to be restricted to only some cellular populations. The most striking aspect of immunostaining was a predominant reactivity in the evolutionary old brain parts. Neuropil and neuronal staining was found in the spinal cord (motor neurons of the ventral horn), medulla oblongata (hypoglossal nuclei, magnocellular part of the lateral reticular nuclei), pons (trigeminal, facial, cochlear and pontine nuclei), cerebellum (granule cells), midbrain (dorsal raphe nucleus) and limbic lobe (hippocampal pyramidal cells). Neuropil reactivity alone appeared in cerebellar nuclei, midbrain, thalamus, basal ganglia, neopallium and olfactory brain. Generally, pia mater/arachnoid, ependyma, choroid plexus, vascular system and some astrocytic populations were found to be strongly P450IIB1* immunoreactive. In comparison with astroglia, which is characterized by glial fibrillary acidic protein-positiveness, the astrocytes, which are also P450IIB1* reactive, occurred only in subpial and subependymal layers, and in large fiber tracts of the spinal cord and brainstem, where they were attached to the vascular system. Otherwise, the glial fibrillary acidic protein-positive astrocytes were not P450IIB1* immunoreactive in the cerebellar molecular layer (fibers of Bergmann glia), in remaining neuropils and in white matter areas.

PET imaging of adrenal cortical tumors with the 11beta-hydroxylase tracer 11C-metomidate.

UNLABELLED: The purpose of the study was to evaluate PET with the tracer 11C-metomidate as a method to identify adrenal cortical lesions. METHODS: PET with 11C-metomidate was performed in 15 patients with unilateral adrenal mass confirmed by CT. All patients subsequently underwent surgery, except 2 who underwent biopsy only. The lesions were histopathologically examined and diagnosed as adrenal cortical adenoma (n = 6; 3 nonfunctioning), adrenocortical carcinoma (n = 2), and nodular hyperplasia (n = 1). The remaining were noncortical lesions, including 1 pheochromocytoma, 1 myelolipoma, 2 adrenal cysts, and 2 metastases. RESULTS: All cortical lesions were easily identified because of exceedingly high uptake of 11C-metomidate, whereas the noncortical lesions showed very low uptake. High uptake was also seen in normal adrenal glands and in the stomach. The uptake was intermediate in the liver and low in other abdominal organs. Images obtained immediately after tracer injection displayed high uptake in the renal cortex and spleen. The tracer uptake in the cortical lesions increased throughout the examination. For quantitative evaluation of tracer binding in individual lesions, a model with the splenic radioactivity concentration assigned to represent nonspecific uptake was applied. Values derived with this method, however, did show the same specificity as the simpler standardized uptake value concept, with similar difference observed for cortical versus noncortical lesions. CONCLUSION: PET with 11C-metomidate has the potential to be an attractive method for the characterization of adrenal masses with the ability to discriminate lesions of adrenal cortical origin from noncortical lesions.

Some effects of a low sodium intake on the expression of P450 aldosterone synthase in the hamster adrenal cortex: immunoblotting, immunofluorescent and immuno-gold electron microscopic studies.

In the current work we studied the effects of a low sodium intake on P450 aldosterone synthase (P450aldo) in the adrenal cortex of male hamsters by Western blotting analysis. We also investigated the zonal distribution of P450aldo with a specific antibody using immunofluorescence and immuno-gold electron microscopy. Western blotting analysis revealed a progressive induction of P450aldo in the adrenals of hamsters kept on a low sodium diet, with two-, four- and eightfold increases after 2, 4 and 21 days on the diet. Immunofluorescence microscopy showed that P450aldo was confined to the zona glomerulosa (ZG) cells. Electron microscopy showed P450aldo to be located in the mitochondria of ZG cells. When hamsters were maintained on a low sodium intake for 2, 11 and 21 days, P450aldo was still found only in the ZG; the ZG appeared either unchanged or sometimes slightly enlarged. Moreover, at days 11 and 21, the intensity of the immunofluorescent signal was much stronger in the ZG of hamsters on the low sodium intake than in controls. Hence, immunocytochemistry using the colloidal-gold technique showed P450aldo to be more abundant in the mitochondria of the experimental animals than in controls. To conclude, P450aldo is present only in the ZG of hamster adrenals and sodium restriction appears to induce its expression by stimulating production within individual ZG cells rather than by stimulating a proliferation of the ZG cells.

Mode of action of butylated hydroxyanisole (BHA) and other phenols in preventing loss of 11 beta-hydroxylase activity in cultured bovine adrenocortical cells.

When cultured bovine adrenocortical cells are incubated with cortisol, or other steroids that are pseudosubstrates for 11 beta-hydroxylase (cytochrome P-45011 beta), the activity of the enzyme decreases. In previous experiments, three substances were shown to protect 11 beta-hydroxylase against loss of enzymatic activity in the presence of pseudosubstrates:BHA (butylated hydroxyanisole,2(3)-tert-butyl-4-methoxyphenol), dimethyl sulfoxide (DMSO), and metyrapone. The present experiments examine the protective effects of several phenolic analogs of BHA in this system, and compare their activities to that of DMSO and metyrapone. When a variety of analogs of BHA were tested for their abilities to prevent loss of 11 beta-hydroxylase activity in cultured adrenocortical cells incubated with 50 microM cortisol for 24 hr, phenol itself was found to be about equipotent with BHA. Addition of methyl, methoxy and benzyl groups to phenol did not diminish protective activity of the compound, but addition of one and particularly two tert-butyl groups greatly diminished activity. Thus, BHT(2,6-di-t-butyl-4-methylphenol) was inactive, in contrast to BHA. The hydroxy group of phenol was essential since benzene and fluorobenzene were inactive. Compounds with multiple hydroxyl groups were not as active as phenol itself, with the exception of catechol. No products of phenol formed during incubations of cells with cortisol were detected by high performance liquid chromatography. Estimated EC50 values for protection of 11 beta-hydroxylase by phenols were about 100 microM, whereas the EC50 values for dimethyl sulfoxide and metyrapone were 10 mM and 300 nM respectively. On a semilogarithmic plot, the dose-response curves for all these compounds were approximately parallel. To aid in determining the mechanism of protection of 11 beta-hydroxylase, phenols and DMSO were tested for prevention of loss of 11 beta-hydroxylase activity at three different oxygen concentrations (2, 5, and 19% O2). Lowering the oxygen concentration itself resulted in a small diminution of the loss of 11 beta-hydroxylase. Phenols and dimethyl sulfoxide were more effective at low oxygen and less effective in air. Because the cytochrome P-450 inhibitor metyrapone was found previously to be very effective in protecting 11 beta-hydroxylase against loss of activity, we examined whether phenols and dimethyl sulfoxide may act by directly inhibiting 11 beta-hydroxylase activity. In a 1-hr incubation with cells, BHA, phenol, and dimethyl sulfoxide all inhibited 11 beta-hydroxylase, but at concentrations that ranged from 4- to greater than 100-fold higher than those required for protection.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulatory effect of copper on rat adrenal cytochrome P-450 and steroid metabolism.

Accumulation of Cu2+ in rat adrenal glands produced a biphasic response in concentrations of the mitochondrial cytochrome P-450 and heme which were, in turn, reflected in abnormal steroid biosynthesis and output. In the mitochondria, 1 day after Cu2+ treatment, when the concentration of the metal ion was increased by 2- to 3-fold over the control value, a significant increase in cytochrome P-450-dependent steroid 11 beta-hydroxylase activity was observed. These effects were accompanied by a nearly 85% increase in concentrations of cytochrome P-450 and heme. In addition, the activity of delta-aminolevulinate synthetase was increased by 3-fold. In those animals lipid peroxidation, assessed by measuring concentrations of conjugated dienes, was reduced to approximately 50% of the control value. However, after 7 days of Cu2+ treatment (via a mini-osmotic pump), a significantly lowered rate of 11 beta-hydroxylase activity was noted, and the plasma concentration of corticosterone was also reduced significantly. Also, in the mitochondria, the concentrations of cytochrome P-450 and heme were decreased in comparison with the control values. These decreases were accompanied by elevated levels of the mitochondrial lipid peroxidation and a further increase in adrenal Cu2+ content (5-fold). At this time, delta-aminolevulinate synthetase activity remained elevated but to a lower extent than that observed after 1 day of Cu2+ treatment. In contrast to 11 beta-hydroxylase activity, the reduction in cytochrome P-450 content was not reflected in a decrease in the rate of cholesterol side-chain cleavage; rather this activity was increased in Cu2+-treated animals. Adrenal heme oxygenase activity was unaffected by either Cu2+ treatment, as was the specific content of cytochrome P-450 in the microsomal fraction. The present findings suggest that the Cu2+-mediated regulation of cytochrome P-450-dependent steroidogenic activity in the adrenal mitochondria is predominantly a reflection of the metal ion affecting heme biosynthesis and lipid peroxidation in this organ. Moreover, these actions appear to differentially affect the mitochondrial cytochrome P-450 species catalyzing different hydroxylation reactions in the adrenal steroidogenesis pathway.

Rat adrenal transplants are reinnervated: an invalid model of denervated adrenal cortical tissue.

Adrenal autotransplantation is a widely used approach to investigate the potential for neural modulation of adrenal cortical function. It is believed that regenerating adrenal transplants are not reinnervated, thereby providing a model to investigate adrenal function in the absence of neural modulation. However, the hypothesis that adrenal transplants become reinnervated has not been directly tested. The purpose of the present study was to characterize the time course, extent, and nature of the reinnervation of the regenerating adrenal transplant and to assess whether the recovery of steroidogenic function and enzyme expression correlates temporally with the presence of innervation. Using immunohistofluorescent detection of tyrosine hydroxylase (TH), neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP), and vasoactive intestinal peptide (VIP), the innervation of regenerating adrenals was assessed 14-30 days after transplantation of adrenal capsules beneath the kidney capsule in rats. Extensive reinnervation by TH-, NPY-, and VIP-positive fibres was present by 14 days after transplantation including regions of the adrenal capsule and cortex, with only minimal reinnervation by CGRP-positive fibres up to 30 days. TH- and NPY-positive chromaffin cells were also observed in the regenerating transplants. In addition, there was marked recovery of steroidogenic function and steroidogenic enzyme expression up to 30 days. The finding that nerve fibres are present in the transplants during the re-establishment of steroidogenic function and enzyme expression suggests that innervation may modulate the regeneration and functional recovery of adrenal transplants. In an attempt to prevent reinnervation of transplants, adrenal capsules were autotransplanted to denervated kidneys. Immunohistochemical analysis showed that, despite extensive denervation of the kidney tissue, the reinnervation and regeneration of the adrenal transplants still occurred. These data demonstrate the marked capacity of the regenerating adrenal to become reinnervated and reinforces the conclusion that adrenal transplants are an invalid model of denervated adrenal cortical tissue.

Isolation of two distinct cytochromes P-45011 beta with aldosterone synthase activity from bovine adrenocortical mitochondria.

Two distinct forms of cytochrome P-45011 beta, with apparent molecular weights of 48,500 (48.5K) and 49,500 (49.5K), have been isolated from bovine adrenocortical mitochondria. Their amino acid sequences up to the 19th position from the N-terminus were only different at the 6th position (Val and Ala for the 48.5K and 49.5K enzymes, respectively). Each sequence was assignable to a distinct cDNA clone for cytochrome P-450(11) beta (Kirita, S., et al. [1988] J. Biochem. 104, 683-686), indicating that the two proteins originate from different genes in bovine adrenocortical cells. Both forms of cytochrome P-450(11) beta were capable of catalyzing aldosterone synthesis as well as the 11 beta- and 18-hydroxylation of 11-deoxycorticosterone. Thus, at least two distinct cytochrome P-450(11) beta species exist in the adrenal cortex and participate in steroidogenesis.

Light and electron microscopic immunohistochemistry of the localization of adrenal steroidogenic enzymes.

Recent immunohistochemical studies have revealed the precise localization of the enzymes involved in adrenal steroidogenesis. Light microscopical investigations showed that cytochromes P450 of cholesterol side-chain cleavage enzyme (P450scc) and of 11 beta-hydroxylase (P45011 beta), 3 beta-hydroxysteroid dehydrogenase/ delta 5-4 isomerase (3 beta HSD), and 21-hydroxylase (P450C21) are localized in all the adrenocortical cells, especially in those of the zona fasciculata-reticularis. 17 alpha-Hydroxylase/C17-C20 lyase (P45017 alpha,lyase) is present in the zona fasciculata-reticularis cells of human, bovine, pig, and guinea-pig adrenals, but absent in the adrenals of some rodents such as rat, hamster, and mouse. Aldosterone synthase (P450aldo) is contained only in the zona glomerulosa cells. In the rat adrenal, P45011 beta, which catalyzes the conversion of deoxycorticosterone to corticosterone, is localized in the zona fasciculata-reticularis cells. Electron microscopic investigations demonstrated that P450scc and P45011 beta are colocalized in the matrix side of inner mitochondrial membrane including cristae, while 3 beta HSD, P450C21, and P45017 alpha, lyase are present in the membranes of smooth endoplasmic reticulum (SER). These results clearly indicate that aldosterone, the most potent mineralocorticoid, is synthesized in the zona glomerulosa cells, and glucocorticoids, such as corticosterone and cortisol, are produced in the zona fasciculata-reticularis cells. The conversion of cholesterol to pregnenolone and the final steps of corticosteroid synthesis occur in the mitochondria, while the intermediate steps, leading to the synthesis of deoxycorticosterone or deoxycortisol from pregnenolone, take place in the SER membranes.

Immunohistochemistry improves histopathologic diagnosis in primary aldosteronism.

BACKGROUND: In primary aldosteronism (PA) the main source of aldosterone hypersecretion is an aldosterone-producing adenoma (APA) or a bilateral hyperplasia. Histopathology of the adrenal gland from patients with PA has been difficult, as there are no morphological criteria to ascertain which are the cells that produce aldosterone. We therefore applied new specific antibodies to explore which cells in the adrenal contain the enzymes for aldosterone and cortisol production, respectively. METHODS: Adrenals from 24 patients with PA were studied. After routine preparation, consecutive sections were stained with antibodies for CYP11B1 (cortisol) and CYP11B2 (aldosterone) enzymes. RESULTS: APA had a strong immunoreactivity for CYB11B2. In adrenals from seven patients, we found no APA, but several nodules with strong CYB11B2 immunoreactivity, indicating aldosterone-producing nodular hyperplasia. CONCLUSIONS: Immunohistochemistry of adrenal steroidogenic enzymes provides novel diagnostic information. This may become an important part of routine histopathology, and contribute to improved clinical management in PA.

Expression of Cyp21a1 and Cyp11b1 in the fetal mouse testis.

Fetal Leydig cells and fetal adrenocortical cells may share a common progenitor cell. Both cell types show several similarities, particularly in relation to their primary steroidogenic function. Differences in steroid secretion are largely due to the expression of 21-hydroxylase (CYP21A1) and 11beta-hydroxylase (CYP11B1) activity in the adrenal. To determine whether expression of these enzymes defines a clear difference between adrenocortical and Leydig cells, or is further evidence of a link between the cell types, we have measured Cyp21a1 and Cyp11b1 expression and related enzyme activity in the fetal testis. Expression of both Cyp21a1 and Cyp11b1 was clearly detectable in the fetal testis by RT-PCR and Southern blotting. Real-time PCR studies showed that Cyp11b1 was expressed only in the fetal/neonatal testis with no expression in the pubertal or post-pubertal animal. Cyp21a1 was also predominantly expressed in the fetal testis although some lower expression was also seen in the adult. Expression of Cyp21a1 and Cyp11b1 in neonatal testicular cells was unaffected by incubation in vitro with human chorionic gonadotrophin or ACTH. Using immunohistochemistry, CYP21A1 was localised to a subset of interstitial steroidogenic cells in the fetal testis although CYP11B1 was not detectable. Incubation studies showed that 21-hydroxylase activity was present in the tissue although 11beta-hydroxylase activity could not be detected. Results indicate that a subpopulation of steroidogenic cells in the fetal testis express Cyp21a1 and show 21-hydroxylase activity. This may provide further evidence of a link between fetal Leydig cells and adrenocortical cells but does not discount the possibility that these steroidogenic cells represent 'ectopic' adrenal cells.

P450aldo in hamster adrenal cortex: immunofluorescent and immuno-gold electron microscopic studies.

The zonal distribution of aldosterone synthase cytochrome P450 (P450aldo) in the adrenal cortex of male hamsters was investigated by immunofluorescence and electron microscopy, using an anti-P450aldo peptide antibody. On cryostat sections the immunolocalization of P450aldo was confined to the zona glomerulosa cells. On semi-thin plastic sections, P450aldo was shown to be located in mitochondria. Studies in electron microscopy, using the colloidal gold technique, confirmed that P450aldo was located in mitochondria.