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.

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.

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.

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.

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.

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.

[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.

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.

Déficit de 11Beta -hidroxilasa: mejoría de talla final con hormona de crecimiento y análogos de LHRH Déficit de 11Beta -hidroxilasa: mejoría de talla final con hormona de crecimiento y análogos de LHRH / 11Beta-hydroxylase deficiency: improvement of final height with growthhormone and gonadotropin-releasing hormone analog

El déficit de 11Beta-hidroxilasa es la segunda causa más frecuente de hiperplasia suprarrenal congénita. La 11Beta-hidroxilasa interviene en la síntesis de cortisol y su déficit conlleva acumulación de andrógenos, produciendo virilización prenatal e hiperandrogenismo posterior, y 11-desoxicorticosterona, causante de hipertensión arterial. Se presenta una paciente de 7 años remitida por pubarquia y maduración ósea acelerada, debidas a déficit de 11Beta-hidroxilasa. Debido a que su pronóstico de talla final era muy inferior a su talla genética, se asoció la combinación de análogos de la hormona liberadora de hormona luteinizante (LHRH) y hormona de crecimiento al tratamiento con corticoides orales. Con dicha combinación terapéutica, su pronóstico de talla final mejoró significativamente, alcanzando a los 13 años y 6 meses una talla acorde a su talla

Steroid 11Beta-hydroxylase deficiency is the second most common cause of congenital adrenal hyperplasia. 11Beta-hydroxylase intervenes in cortisol synthesis and its deficiency leads to accumulation of adrenal androgens producing prenatal virilization and, subsequently, hyperandrogenism as well as 11-deoxycorticosterone, leading to the development of hypertension. We describe a 7-year-old girl who was referred for pubarche and accelerated skeletal maturation due to 11Beta-hydroxylase deficiency. Because the patient's predicted height was below her target height, the combination of gonadotropin-releasing hormone analog and growth hormone was added to oral glucocorticoid therapy. With this therapeutic strategy, the patient's predicted height improved significantly and the girl reached a final height in agreement with her target height at the age of 13 years and 6 months

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.

Transformation of Curvularia lunata IM 2901 with pAN7-1 influences selected physiological properties of the fungus.

Genetic analysis of Curvularia lunata IM 2901 transformants, previously obtained by electroporation with plasmid pAN7-1, was carried out. Isolates displayed several differences in hygromycin B resistance and their physiology. It was shown that plasmid pAN7-1 was integrated in different copy numbers and at different positions in the genome of the strains studied. Both the wild type and pAN7-1 isolates, when growing in liquid media, produced an extracellular emulsifying agent. The transformants differed in their growth kinetics, intensity of surfactant production and in the efficiency of cortexolone 11beta-hydroxylation, in comparison with the wild type. The micro-organisms varied in susceptibility to the lytic enzyme complex (Novozyme 234), which indicated the presence of differences in their cell wall composition and/or in architecture caused by an integrated plasmid pAN7-1.

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.

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.

Adrenal adenoma with bilateral adrenocortical nodular change in a patient with Cushing's syndrome.

We report a 57-year-old male patient with adrenocorticotrophin (ACTH)-independent Cushing's syndrome and long-standing hypertension. Both abdominal computed tomographic scan and magnetic resonance imaging revealed bilateral adrenal enlargement with the presence of a tumour in the left adrenal. Marked uptake of 131I-6 beta-iodomethyl-19-norcholesterol was observed only in the left adrenal gland. Left adrenalectomy and biopsy of the right adrenal gland were subsequently performed. Histological examination revealed the presence of an adrenocortical adenoma in the left adrenal with multiple adrenocortical nodules in both left and right non-neoplastic adrenals. Peri- and intraadrenal arteries and arterioles demonstrated marked arteriosclerotic vascular changes. Immunoreactivity for several steroidogenic enzymes was present in the tumour and markedly diminished in the non-neoplastic adrenals. This patient with Cushing's adenoma is considered to have developed adrenocortical nodules in the nonneoplastic adrenal possibly as a result of localized compensatory overgrowth of adrenocortical cells in response to ischaemic changes due to arteriopathy. When examining patients with Cushing's syndrome and bilateral adrenal enlargement, the possibility of concomitant adenoma and adrenocortical nodule formation should also be considered in the differential diagnosis

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.

Phenotype of proliferating cells stimulated during compensatory adrenal growth.

The phenotype of the proliferating cells during adrenocortical growth has remained controversial although glomerulosa, fasciculata and intermediate zone cells have all been considered possible candidates. This was due in part to the inability to identify specific adrenocortical cell types in comparing different types of growth. In the present studies, using immunocytochemical localization of cytochrome P450 aldosterone synthase (P450aldo) and cytochrome P450 11 beta-hydroxylase (P45011 beta) to identify adrenocortical cell phenotypes as well as Ki-67 to label proliferating cells, we have investigated the phenotype of the proliferating cells in the compensatory adrenal growth response to unilateral adrenalectomy. Between 24 and 96 hrs after unilateral adrenalectomy, most Ki-67(+) nuclei were found in the outermost region of the fasciculata, as defined by P45011 beta immunoreactive cells. Few Ki-67(+) nuclei were found in the glomerulosa, defined by P450aldo cells or in the z intermedia, identified by the absence of both P450aldo and P45011 beta. To test which cell type is activated by unilateral adrenalectomy, we altered the phenotypic configuration of the adrenal cortex; rats were placed on a low Na+ diet for three weeks, resulting in a marked expansion of the number of P450aldo(+) cells. An abundance of proliferating cells was identified primarily in the expanded glomerulosa, but not in the intermedia or fasciculata. In contrast, the proliferation associated with compensatory growth in these low Na+ rats, was localized primarily in the outer P45011 beta(+) zone. These findings suggest that the phenotype of the proliferating cell is specific to the growth promoting stimulus.

Phenotypic changes and proliferation of adrenocortical cells during adrenal regeneration in rats.

Adrenal regeneration after enucleation includes both cell proliferation and differentiation, but the phenotype of the proliferating cell remains controversial. Immunoperoxidase localization of cytochrome P450 aldosterone synthase (P450aldo) and cytochrome P450 11 beta-hydroxylase (P45011 beta) and of Ki-67 was used to identify adrenocortical cell phenotypes and proliferating cells, respectively. Comparisons were made between regenerating and intact adrenals collected from rats on low or normal Na+ diets. During the first week after enucleation, P45011 beta was expressed reflecting the presence of fasciculata cells; however, P450aldo was detected only in adrenals from low Na+ rats. On normal and low Na+, glomerulosa cells were replaced by intermedia cells, whereas on low Na+, glomerulosa cells were replaced by fasciculata cells. Proliferation was observed only in glomerulosa and fasciculata, but not intermedia cells. These findings suggest that the expression of the glomerulosa cell phenotype is decreased in the early stages of adrenal regeneration, that differentiation from a glomerulosa to an intermediate or fasciculata cell phenotype is influenced by low Na+ and that glomerulosa and fasciculata cells proliferate in response to enucleation.

Effects of long term stimulation of ACTH and angiotensin II-secretions on the rat adrenal cortex.

In the rat adrenal cortex, aldosterone synthase cytochrome P450 (P450aldo), a mineralocorticoid synthesizing enzyme, localizes in the zona glomerulosa (zG), while cytochrome P45011 beta (P45011 beta), a glucocorticoid synthesizing enzyme, localizes in the zonae fasciculata-reticularis (zFR). In between zG and zF, a cell-layer which contains neither P450aldo nor P45011 beta is present, where replicating cells were abundant as judged by the incorporation of bromodeoxyuridine (BrdU) and/or by detecting PCNA in their nuclei. When plasma ACTH level of the rat was raised 3-fold for 2-3 weeks by the administration of metyrapone, a potent inhibitor of glucocorticoid formation, most of zG cells containing P450aldo disappeared, while zF cells with P45011 beta increased. Under the conditions, the cell-layer without P450aldo and P45011 beta became very thin, and replicating cells were mainly in the outermost portion of zF. When angiotensin II secretion was also stimulated for 2-3 weeks by feeding the rats on Na-deficient diet, the P450aldo-containing cells proliferated to form a thicker zG (7-8 cells-thick from 1-2), while the width of zF containing P45011 beta decreased slightly. Coincidently the cell-layer devoid of P450aldo and P45011 beta became thin, though slightly, and numbers of replicating cells significantly increased in and around the inner edge of the proliferated zG. When both ACTH and angiotensin II secretions were stimulated simultaneously, the cell-layer without P450aldo and P45011 beta almost disappeared and replicating cells were around the boundary of zG and zF. Based on these results we propose that the cell-layer between zG and zF devoid of P450aldo and P45011 beta is the stem cell layer of rat adrenal cortex.

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.

Clara cell heterogeneity in differentiation: correlation with proliferation, ultrastructural composition, and cell position in the rat bronchiole.

Postnatal differentiation of nonciliated bronchiolar epithelial (Clara) cells occurs in a wave-like pattern beginning in the upper airways and ending in the terminal bronchiole. The heterogeneity of Clara cell differentiation observed during postnatal development in rats may be due to both cell turnover rate and cell position in the airways. To test the importance of these two factors in Clara cell differentiation, terminal bronchioles were examined in rats from gestational day 21 through postnatal day 100. The volume fraction of smooth endoplasmic reticulum (SER), a marker of differentiation, was seen to increase with age, while the epithelial cell labeling index of terminal bronchioles decreased over the same period. This represented a significant inverse correlation between SER volume density and cell proliferation rates (r2 = 0.80, P < 0.02). To evaluate the importance of cell position as a factor in cellular differentiation, the abundance of SER and secretory granules and the expression of cytochrome P450 isozyme 2B in Clara cells were examined along the entire length of the terminal bronchiole in animals 1, 21, and 100 days of age. For all three characteristics, Clara cells showed a similar degree of maturation from the proximal bronchiolar bifurcation to the bronchiole-alveolar duct junction (BADJ) (a span of approximately 35 cells). We conclude that during prenatal and postnatal bronchiolar development in rats: (1) the Clara cell is the most actively dividing cell type for the lower airways; (2) the stage of Clara cell differentiation is inversely related to Clara cell mitotic activity; and (3) the heterogeneity of Clara cell maturation and mitotic activity is not influenced by position within the terminal bronchiole.