Normal Human Adrenals Research Articles

Expression of steroidogenic enzyme messenger ribonucleic acids and corticosteroid production in aldosterone-producing and "nonfunctioning" adrenal adenomas.

"Nonfunctioning" adrenal adenomas are often diagnosed in patients without recognizable clinical symptoms of adrenocortical hyperfunction. The objective of this study was to determine directly the steroidogenic activity of such adenomas (n = 12) and compare them histologically and functionally to normal human adrenals (n = 6) and aldosterone-producing adenomas (n = 15). The histological appearances of nonfunctioning and aldosterone-producing adenomas were surprisingly similar. Nonfunctioning adrenal adenomas expressed all mRNAs of P450scc, P450c17, P450c21, adrenodoxin, and adrenodoxin reductase with relative levels comparable to those found in normal adrenals. Consistent with their hormone-producing nature, these adenomas had cortisol and aldosterone contents as high as those in normal adrenal tissues, a significantly (P < 0.05) increased 17-hydroxyprogesterone content, and a disproportionally low expression of P450c21 mRNA compared to aldosterone-producing adenomas. Cells isolated from both aldosterone-producing and nonfunctioning adrenal adenomas exhibited highly ACTH-sensitive cortisol and aldosterone production, suggesting again the presence of both zona glomerulosa-like and zona fasciculata-like steroidogenesis in these adenoma tissues. These results indicate that so-called nonfunctioning adrenal adenomas are not without steroidogenic activity. Therefore, the assumption that adrenal adenomas are entirely nonfunctioning in the absence of recognizable hormonal hyperfunction may not be correct.

Expression of progesterone-binding protein in normal and neoplastic human adrenals

Specific progesterone-binding protein (P4-BP) is demonstrated in adrenocortical nuclei of the guinea pig, but, not in nuclei of other animals. We tried to demonstrate the progesterone-binding activity in nuclei of human normal adrenals and adrenal tumors. Normal adrenals were obtained from six patients with renal cell carcinomas undergoing radical nephrectomy. Seven adrenocortical adenomas were obtained: five tumors from patients with Cushing's syndrome, one tumor from non-functioning adenoma, and one from aldosteronoma. Nuclei were purified from the tissues, and progesterone binding assay was performed. We could not demonstrated progesterone-binding activity in nuclei of six normal human adrenals. However, we demonstrated progesterone-binding activity in nuclei purified from human adrenocortical adenomas associated with Cushing's syndrome. Saturation analysis revealed a Kd of 13.85 +/- 1.99 nM (mean +/- SD, n = 5) and a binding capacity of 1.95 +/- 0.37 pmol/mg DNA (mean +/- SD, n = 5). A Kd of progesterone-binding activity in human adrenocortical adenoma was similar to that of guinea pig P4-BP, and a binding capacity was about one-fifteenth of guinea pig P4-BP. However, nuclei purified from a non-functioning adrenocortical adenoma and an aldosteronoma failed to demonstrate progesterone-binding activity. The binding activity was specific for progesterone. 5 alpha-pregnane-3,20-dione was a modest competitor, while 17 beta-estradiol, testosterone, cortisol, and other related steroids were poor competitors. Thus the progesterone-binding activity in human adrenals was similar to guinea pig P4-BP in the affinity and specificity of binding.(ABSTRACT TRUNCATED AT 250 WORDS)

The modification and expression of 21-hydroxylase gene in normal human adrenal gland and adrenal cancer

Genomic DNA and total RNA from three adrenal cancer tissues were analyzed by hybridization with human 21-hydroxylase gene. Southern blot analysis with restriction enzyme Eco RI revealed major fragments at 18, 13 and 9 kilobase (kb) in normal adrenal glands. In two of three adrenal cancers, however, the band at 18 kb was either absent or decreased and the 9 kb fragment showed an increase in its intensity. Normal liver, kidney and leucocytes has only 18 and 13 kb while lacking the 9 kb fragment. Using Taq I, Bam HI or Bgl II, we found no difference in restriction fragment patterns between adrenal cancer and normal tissues. Cleavage with Hpa II after digestion with Bgl II showed that significantly more DNA was digested into low-molecular weight fragments in adrenal cancer and the normal adrenal gland than those in other normal tissues. By Northern blot analysis, there was difference of signal intensity of hybridizing mRNA between the adrenal cancers and normal adrenal glands. These results suggest that the Eco RI site in the flanking region of the 21-hydroxylase gene may be modified in adrenal cancer tissue, and that inadequate 21-hydroxylase is present in some forms of adrenal cancers.

Ultrastructural morphometry of normal adrenal cortex and of hyperplastic adrenals in gushing's disease.

Surgical specimens of 4 normal adrenal glands and of 5 hyperplastic ones in Cushing's disease were studied ultrastructurally. The ultrastructure of the three zones of each adrenal gland was morphometrically and statistically analyzed. Comparing the statistical data of the three zones of the normal gland, the development of smooth endoplasmic reticuium and rough endoplasmic reticuium showed an increase from the outer zona glomerulosa to the inner zona fasciculata and reticularis. Also, the mitochondria were more numerous in the inner zona reticularis than in the outer zones. In Cushing's disease, the smooth endoplasmic reticuium, the rough endoplasmic reticuium, and the mitochondria were developed to a significantly higher degree than in normal human adrenal glands. The distribution of the cell organelles in the zona fasciculata and zona reticularis is almost alike in normal adrenal glands and in the adrenal cortex in Cushing's disease, except that the smooth endoplasmic reticuium in the zona reticularis was less extensive than in the zona fasciculata. The volume percentages of lipid vacuoies in the hyperplastic zona fasciculata in Cushing's disease was strongly and significantly decreased in comparison to normal adrenal glands. Our ultrastructural findings and the statistical data were in accordance with the results from animal experiments with adrenocorticotropic hormone (ACTH) stimulation and confirmed former qualitative ultrastructural findings concerning human adrenal gland changes in ACTH-dependent Cushing's disease.

Characteristics of atrial natriuretic hormone receptors in human pheochromocytomas

The presence of functional receptors for human atrial natriuretic hormone in human pheochromocytomas was recently reported. The present study reports the binding of hANH as measured by Scatchard analysis in 4 human adrenal glands and in 5 human pheochromocytomas. Binding assays using [3H]ANH revealed a single class of high-affinity binding sites for hANH in both tissues. Human pheochromocytomas present a lower number of binding sites than normal human adrenal gland (Bmax of 7.1 +/- 2.1 vs 33.6 +/- 6.9 fmol/mg protein, respectively). However, the decreased number of ANH receptors was not paralleled by modifications of tissular cyclic GMP (cGMP). Moreover, plasma hANH concentrations in 7 patients with pheochromocytomas (20.2 +/- 2.7 pmol/l) were statistically higher than those obtained in 25 normal control humans (8.1 +/- 0.6 pmol/l, p less than 0.001). We also demonstrated the presence of immunoreactive ANH in the tumour itself.

Alpha-Interferon Induces Cortisol Release by Human Adrenals in vitro

It is at present not clear whether alpha interferon (INF-alpha) can participate in the control of glucocorticoid blood levels through direct action on the adrenal gland. In this study, the possible action of INF-alpha on cortisol release by adrenal tissue was tested in vitro. Slices of normal human adrenals were incubated with INF-alpha for 3 h at 37 degrees C in 95% air and 5% CO2. Cortisol release by adrenal tissue was stimulated by INF-alpha, showing a dose-response curve from 20 IU/ml, the lowest dose that gave a response, to a maximal dose of 60 IU/ml when the response reached a plateau. The effect of INF-alpha on cortisol liberation by adrenal tissue in vitro may be implied in neuroimmune regulatory interactions.

Anti-corticosteroid antibodies in AIDS patients.

Patients suffering from AIDS tend to have symptoms that resemble those encountered in adrenocortical insufficiency. Serum sodium concentrations and blood pressure values were monitored and found to be subnormal, despite the fact that renin activity and aldosterone levels were either normal or elevated. We report the presence of autoantibodies directed against hydrocortisone among such AIDS patients. Indirect immunofluorescence technique using patients' sera and adrenal glands from AIDS patients as antigen showed antibodies to adrenocortical cells in sera from 9 of the 12 AIDS patients and in none from patients with autoimmune diseases and viral diseases or healthy blood donors. No similar reaction was seen in normal human or monkey adrenal glands. An ELISA technique was developed using hydrocortisone as antigen and using this technique 45% of the AIDS patients were found to have antibodies to hydrocortisone. It is possible that anti-corticosteroid antibodies (ACSA) may play a role in the pathophysiology of the Addison's like syndrome seen in terminal phases of AIDS.

Multiple peptide production and presence of general neuroendocrine markers detected in 12 cases of human phaeochromocytoma and in mammalian adrenal glands.

In this study, antibodies to a range of markers of neuroendocrine differentiation were evaluated for their use in the histopathological assessment and characterisation of phaeochromocytomas. Routinely processed wax blocks from eleven adrenal phaeochromocytomas (10 benign and 1 malignant) and one benign phaeochromocytoma of the urinary bladder were investigated. In addition to these tumours, normal human, cat and piglet adrenal glands were examined. In the phaeochromocytomas, immunostaining was obtained with 21 of the 25 antisera used. Of the general neuroendocrine markers, neuron-specific enolase was found in all tumours, and chromogranin and protein gene-product 9.5 in most of the cases. A range of regulatory peptide immunoreactivities could be demonstrated, such as enkephalin, neuropeptide tyrosine (NPY), 7B2, galanin and vasoactive intestinal polypeptide (VIP). In addition, two peptides were found which have not been reported previously in these tumours, peptide histidine methionine (PHM) and the cryptic fragment of the precursor encoding VIP. Co-localisation studies revealed that peptides derived from the same precursor or peptide family were found in the same tumour cells (e.g. VIP and PHM, NPY and its C-flanking peptide CPON). In the normal adrenal medulla, all the peptides previously reported to be present could be demonstrated immunocytochemically. Galanin was present in a subpopulation of cells also immunoreactive for enkephalin. Neuropeptide tyrosine and CPON were demonstrated in another subpopulation. Occasionally, cells were found to contain all four antigen immunoreactivities. Using antisera to enzymes involved in catecholamine synthesis, galanin was found to be present in noradrenaline-containing cells. The study demonstrates the presence of various antigens in chromaffin tissue of the adrenal gland. A range of substances can also be identified immunocytochemically in phaeochromocytoma tissue, using routinely-processed material.

Presence of functional receptors for atrial natriuretic peptide in human pheochromocytoma

Pheochromocytoma, a catecholamine-secreting adrenomedullary tumor, has been shown to contain the functional receptor for human atrial natriuretic peptide(h-ANP). Release of catecholamines from tissue slices of pheochromocytoma was inhibited by h-ANP in a dose-dependent manner. Binding assays using 125I-ANP revealed a single class of high affinity binding sites for ANP. When covalently tagged with 125I-ANP and electrophoresed under non-reducing and reducing conditions, the receptor migrated as a 140-kDa band and a 70-kDa band, respectively, reflecting its disulfide-linked subunit structure. The presence of ANP receptor in pheochromocytoma was further demonstrated by immuno-histochemistry; the tumor was positively stained with an anti-receptor antiserum. The antiserum was also useful to establish the zona glomerulosa localization of ANP receptor in the normal human adrenal gland.

Ontogeny of adrenal steroid hydroxylases: evidence for cAMP-independent gene expression

Total RNA from normal and anencephalic human fetal adrenals was examined by blot analysis for transcripts encoding P-450 scc, P-450 11β P-450 17α P-450 c21 and adrenodoxin using bovine cDNA clones specific for these different enzymes. The specific contents of RNA encoding these components of the adrenocortical steroidogenic pathway were found to be similar in both types of adrenal tissue. Likewise, immunoblot analysis showed comparable concentrations of P-450 scc, P-450 17α and adrenodoxin protein to be present in adrenal tissues from normal and anencephalic human fetuses. Immunoblot analysis of homogenates of fetal sheep adrenals of increasing gestational age (85–145 days) showed constant levels of P-450 scc and P-450 11α, but increasing P-450 17α content, especially near term. Both sheep fetuses prior to 136 days gestational age and human anencephalic fetuses are known to have extremely low circulating levels of immunoreactive ACTH as well as very low adrenal adenylate cyclase activity. Thus, it is concluded that factors other than pituitary ACTH which operate independent of adenylate cyclase activation are required for the initial expression (imprinting) of steroid hydroxylase genes.

Chromogranin from normal human adrenal glands: purification by monoclonal antibody affinity chromatography and partial N-terminal amino acid sequence

A method is presented for the purification of human chromogranin from adrenal glands obtained at autopsy. The procedure involved homogenization of whole glands in aqueous buffer, salt precipitation, affinity chromatography using a highly specific monoclonal antibody (LK2H10) and reverse-phase high-pressure liquid chromatography. Chromogranin purified from autopsy adrenal glands revealed a high degree of polypeptide heterogeneity when analyzed by silver-stained SDS polyacrylamide gels. Greater than 90% of the protein was represented by a cluster of polypeptides with an M r = 70 000 (i.e. chromogranin A), while the remaining protein was highly disperse in molecular weight. That these various polypeptides were in fact chromogranin was shown by Western blotting using monoclonal antibody LK2H10. About 6 nmol of chromogranin were obtained from 97 g of starting adrenals which was estimated to be a 25% yield and a 250-fold enrichment from adrenal homogenates. Critical to achieving reasonable yields of this protein was the need for particular low pH buffers for resuspension of chromogranin after solvent removal steps. Chromogranin purified from human adrenal glands was similar in amino acid composition, and identical in the N-terminal amino acid sequence (24 residues) to bovine chromogranin A. A secondary sequence representing 25% of the total protein and missing the first three residues of the N-terminus suggested the possibility of N-terminal processing of chromogranin in situ. The conservation of the N-terminal amino acid sequence of human and bovine chromogranin contrasts with the strong sequence variability predicted by antisera cross-reactivity and suggests that N-terminus of chromogranin may be critical for its biological activity.

Satellite cells in the normal human adrenal gland and in pheochromocytomas. An immunohistochemical study.

In light of the recent finding of the S-100 antigen in satellite cells of the rat adrenal medulla, we looked for S-100-labelled cells in both the normal human adrenal medulla and in pheochromocytomas. Immunostaining enabled us to detect S-100-labelled satellite cells by both light and electron microscopy in a significant number of pheochromocytomas and, as expected, in the normal human adrenal medulla.

ACTH and prostaglandin receptors in human adrenocortical tumors. Apparent modification of a specific component of the ACTH-binding site.

The failure of certain adrenal tumors to respond to ACTH was investigated in vivo be administration of corticotropin-(1-24)-tetracosapeptide (ACTH1-24) and dexamethasone and in vitro by studying the binding properties of ACTH1-24 and prostaglandin E1 (PGE1) and their effect on adenylate cyclase activity of the tumors' crude membranes; in addition, in five cases the stimulation of cortisol production in isolated adrenal cells by both hormones and dibuttyryl cyclic adenosine 3',5'-monophosphate (cAMP) was also studied. The results obtained in 13 hormone-producing tumors of the human adrenal cortex, i.e. 10 carcinomas and 3 adenomas, were compared with those found in normal human adrenal glands. According to the adenylate cyclase responses to ACTH1-24 and PGE1, the tumors fall into different categories. In the first group are six rumors in which the adenylate cyclase was stimulated by both ACTH1-24 and PGE; in addition specific binding could be demonstrated for the two hormones in all six. The binding affinity for 125I-ACTH1-24 was found to be about 10 times higher than that for 125I-ACTH11-24. In the one tumor in which the experiment was performed, bound 125I-ACTH1-24 was displaced by ACTH1-10. These results are similar to the ones found in normal human adrenal preparations. For two rumors of the group in which ACTH did not increase steroidogenesis in vivo, the biochemical abnormality might be located beyond cAMP formation. A second group encompasses six tumors in which the steroidogenesis in vivo and the adenylate cyclase activity were insensitive to ACTH1-24 but in which the enzyme was stimulated by PGE1 and NaF. However, these preparations bound 125I-ACTH1-24 and 125I-ACTH11-24, the binding affinity being similar for both peptides but 10 times lower than the one found in normal adrenal cortex for 125I-ACTH1-24. In the only case of this group where it was tested, ACTH1-10 did not displace bound 125I-ACTH1-24. This result strongly suggests the possibility of a modification or a loss of the receptor site that binds the N-terminal sequency (1-10) of ACTH, the biologically active part of the molecule. In the last tumor, both PGE1 and ACTH were unable to stimulate adenylate cyclase activity and steroid production in a preparation of isolated adrenal cells, although steroidogenesis was stimulated by dibutyryl though steroidogenesis was stimulated by dibutyryl cAMP. No specific binding for PGE1 could be demonstrated. However, 125I-ACTH1-24 and 125I-ACTH11-24 were found to be bound to the tumor with the same affinity.

ACTH AND PROSTAGLANDINS, RECEPTORS IN HUMAN ADRENOCORTICAL TUMORS

The binding properties of prostaglandin PGE and ACTH, and their action on adenylate cyclase activity (AC) have studied in 12 hormone producing tumors of the human adrenal cortex and compared with those found in the same type of subcellular preparation (20 000 xg) from normal human adrenal glands. A specific binding of PGE and a stimulation of AC by this hormone and by NaF was obtained in all cases. A specific binding of 125I-ACTH was also observed in all cases, but ACTH only stimulated AC in six cases. In the other six cases where AC was insensitive to ACTH, the steroidogenesis was not stimulated “ in vivo ” by this hormone. However these tumors bound specifically 125I-ACTH1–24 and 125I-ACTH11–24, the KD being similar for both peptides but ten times lower than the one found in normal adrenal cortex for 125I-ACTH1–24. This results strongly suggests the possibility of a modification or a loss of the receptor site which binds the N-terminal sequence (1–10) of ACTH known as the biologically active part of the molecule.

Interactions of acth with its adrenal receptors: Specific binding of ACTH 1–24, its O-nitrophenyl sulfenyl derivative and ACTH 11–24

ACTH 1–24, its o-nitrophenyl sulfenyl derivative and ACTH 11–24 were iodinated by the chloramine T method and purified on carboxymethylcellulose. Enzymatic digestion shows that all the iodine was incorporated in the form of monoiodotyrosine. The three ACTHs bound to adrenal crude membranes (pellet 20,000 g) prepared from normal human, rat and sheep adrenals. In the presence of an excess of membrane protein more than 60% of the radioactivity was bound specifically. The binding of 125I-ACTH 1–24 to its adrenal receptor is very rapid; it is temperature dependent, inhibited by ions and by the pancreatic trypsin inhibitor. ACTH 1–24 and its o-nitrophenyl sulfenyl derivative inhibited identically the binding of 125I-ACTH 1–24. ACTH 11–24 and ACTH 1–10 were only, respectively, 15% and 6%, as active as ACTH 1–24 in displacing the bound 125I-ACTH 1–24. The binding of 125I-ACTH 11–24 is more inhibited by ACTH 1–24 than by ACTH 11–24 ACTH 1–10 at high concentrations (10 −4 M) had no effect. The adenyl cyclase of the same adrenal preparations was stimulated by ACTH 1–24; NPS-ACTH 1–24 stimulated adenyl cyclase activity but inhibited the action induced by ACTH 1–24 in the same way as ACTH 11–24 which was without effect on adenyl cyclase. On the other hand, ACTH 1–10 stimulated adenyl cyclase activity and its action was additional to ACTH 1–24. It can be concluded that the 1–10 N-terminal sequence of ACTH, in addition to being the biological active site of the molecule, contributes to binding and increases the affinity of ACTH for its receptor.

Defective steroidal synthesis by adrenal tissue: the control of gene expression causing loss of active steroidogenic enzymes.

ABSTRACT RNA was isolated from normal human adrenal glands and found to cause the formation of Δ5-3β-hydroxysteroid dehydrogenase-isomerase and steroid 21-hydroxylase activities by a Krebs II ascites cell-free protein synthesising system. Although no functional steroid 21-hydroxylase in vivo or in vitro was found in a gland from a patient with virilism due to congenital adrenocortical hyperplasia the RNA would still give steroid 21-hydroxylase-like activity in the protein synthesising system which suggests that the inherited defect was not in the structural gene. Activity could not be induced by RNA from a 'non-functioning' adrenocortical tumour or rat liver.

Short communications tissue culture of adult human adrenocortical cells.

Department of Anatomy, Laboratories of Tissue Culture and Electron Microscopy, University of Padua, Italy (Received 12 March 1974) Attempts to grow adult mammalian adrenocortical cells in vitro are justified by the fact that most of the work reported until now (Kahri, 1966; Milner & Villee, 1970) refers to cultured foetal or newborn rat and human adrenal glands and, furthermore, by the knowledge that the zonation and the metabolic features of adrenocortical cells at these early stages of life differ from those possessed by adult cells (Bloch, 1968). We recently reported a method for tissue culture of the adrenal cortex of adult rats (Armato & Nussdorfer, 1972). In this communication, we describe the successful cultivation of adult human adrenocortical cells. Wedge biopsies of normal human adrenals were obtained from adult patients undergoing urological operations, put into cold (4 °C) Eagle's MEM (Wellcome, London) and were brought to the laboratory. About 20

Metabolism of steroids in normal and pathological human adrenal glands

Metabolism of steroids in normal and pathological human adrenal glands

Micro-angiography of normal and pathologic human adrenals in prenatal and aging course.

Micro-vascular patterns in the human adrenals in the course of development and in adults were visualized by postmortal angiography and examined in relation to adrenocortical lesions. The terminal unit of the arterial blood supply to the adult adrenal cortex consists of the capillary dome in the glomerulosa with brush-like capillaries perforating the fasciculata, while the zona reticularis receives arterial blood from the arteriae medullae. Therefore, the proper fasciculata is situated in the utmost periphery of the arterial circulation. The capillary dome as the terminal unit of the arterial blood supply plays an important role in precipitating various adrenocortical lesions. Capillary dysarrangement was rather indistinct in nodular hyperplasia, but it was remarkable in solitary intracortical nodules receiving several arterioles. In fetuses and newborns, cortical lesions appeared diffusely because they lacked capillary domes. The cortico-medullary vein drained from the glomerulosa into the medullary vein, while the emissary vein was demonstrated as large communications between the capsular and medullary veins. A circulatory blockade between the permanent and fetal cortex proceeding with fetal maturity was found responsible for neonatal hemorrhage involving exclusively the fetal cortex. The similarity of the adrenal vascular pattern of anencephalic newborns to that of normal infancy was ascribed to early involution of the fetal cortex in anencephalus.

泌尿器科領域における Acid Phosphatase Isozyme の研究

The physico-chemical properties of acid phosphatase isozymes were investigated in the normal human prostate, prostatic tumor, adrenal gland, kidney, seminal vesicle, semen and dog kidney, using pevikon zone electrophoretic and DEAF-Sephadex A-50 column chromatographic techniques. The existence of acid phosphatase isozymes (API and APII) were demonstrated in normal human prostate and prostatic tumor (adenoma and carcinoma). The two acid phosphatase active fractions were obtained on DEAE-Sephadex column chromatography of prostate extract while another active fraction (APK) was obtained in kidney tissue, showing these are isozymes each other. The characteristics of acid phosphatase isozymes of prostate were as follows: The optimum pH of AP I was pH 4.0, and that of AP II was pH 5.0. The Km values of AP I and AP II were 0.166×10-6M and 0.250×10-6M, respectively.These two enzyme proteins were not resistant against heat treatment, although AP II was more stable than AP I. The inhibitory effects of various compounds, such as EDTA, magnesium chloride, sodium fluoride, formaldehyde solution, tartrate, diethylstilbesterol (Honvan), testosterone propionate (Enarmone) were tested. All those affected both isozymes to the same extent.The characteristics of kidney acid phosphatase were as follows:The optimum pH was pH 4.25 and the Km value was 0.570×10-6M. The isozyme (APK) was found to be more resistant than AP I and AP II against heat treatment. APK was strongly inhibited by formaldehyde solution more than AP I and AP II were, and less inhibited by sodium fluoride and tartrate than AP I and AP II were.The isozyme patterns of the bladder, seminal vesicles and semen were similar to those of the prostate, and those of the adrenal gland and dog kidney were similar to those of the human kidney.