Dopamine receptor expression and function in human normal adrenal gland and adrenal tumors

Dopamine is known to play a role in the modulation of aldosterone and catecholamine secretion from the adrenal gland, where dopamine receptors (DR), in particular the DR type 2 (D(2)), have been found to be expressed. DR expression has also been demonstrated in some types of benign adrenal tumors. The aims of the current study were to evaluate DR expression and D(2) localization in the normal adrenal gland and in different types of benign and malignant adrenal tumors, as well as to evaluate the in vitro effects of the dopamine agonists bromocriptine and cabergoline on hormone secretion in nontumoral adrenal cells. Adrenal tissues from 25 patients, subjected to adrenal surgery for different diseases, were studied. These included three normal adrenals; five adrenal hyperplasias; four aldosterone-secreting, two cortisol-secreting, and two clinically nonfunctioning adrenal adenomas; two aldosterone-secreting, two cortisol-secreting, and two androgen-secreting adrenal carcinomas; and three pheochromocytomas. In all tissues, DR and D(2) isoform (D(2long) and D(2short)) expression was evaluated by RT-PCR. D(2) localization was also evaluated by immunohistochemistry using a specific polyclonal antibody, whereas D(2)-like receptor expression was evaluated by receptor-ligand binding study, using the radiolabeled D(2) analog (125)I-epidepride. The effects of bromocriptine and cabergoline on baseline and ACTH and/or angiotensin II-stimulated aldosterone, cortisol, and androstenedione secretion were evaluated in cell cultures derived from five different adrenal hyperplasia.At RT-PCR, both D(1)-like and D(2)-like receptors were expressed in all normal and hyperplastic adrenals. D(2) and D(4) were expressed in aldosterone- and cortisol-secreting adenomas, cortisol-secreting carcinomas, and clinically nonfunctioning adenomas, whereas no DR was expressed in aldosterone- and androgen-secreting carcinomas. D(2), D(4), and D(5) were expressed in pheochromocytomas. In all D(2)-positive tissues, both D(2) isoforms were expressed, with the exception of one case of aldosterone-secreting adenoma and the cortisol-secreting carcinomas, in which only the D(2long) isoform was expressed. D(2)-like receptor expression was confirmed at receptor-ligand binding study. At immunohistochemistry, D(2) was mainly localized in the zona glomerulosa and reticularis of the adrenal cortex and, to a lesser extent, in the zona fasciculata and medulla of normal and hyperplastic adrenal tissue. In the positive tumors, D(2) was localized in the tumoral cells. At the in vitro study, a significant inhibition of both baseline and ACTH-stimulated aldosterone secretion was found after high-dose cabergoline, but not bromocriptine, administration; and a significant inhibition of angiotensin-II-stimulated aldosterone secretion was found after both bromocriptine and cabergoline administration in the adrenal hyperplasias. In conclusion, the current study demonstrated that both D(1)-like and D(2)-like receptors are expressed in the normal adrenal gland and in a percentage of adrenal adenomas or carcinomas. Bromocriptine and cabergoline induce only a minor inhibition of the secretion of adrenal hormones in the nontumoral adrenal gland in vitro, not excluding, however, the possible effective use of dopamine agonists in vivo in the treatment of adrenal tumors.     

Screening for mutations of 21-hydroxylase gene in Hungarian patients with congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders, causing impaired secretion of cortisol and aldosterone from the adrenal cortex, with subsequent overproduction of adrenal androgens. The most common enzyme defect causing CAH is steroid 21-hydroxylase deficiency. To determine the mutational spectrum in the Hungarian CAH population, the CYP21 active gene was analyzed using PCR. A total of 297 Hungarian patients with 21-hydroxylase deficiency are registered in the 2nd Department of Pediatrics, Budapest, Hungary, and their clinical status was evaluated. Blood samples for CYP21 genotype determination could be obtained from 167 patients (representing 306 unrelated chromosomes and 56.2% of the total group of patients). Eight of the most common mutations were screened [In2 (intron 2 splice mutation), I172N, Del (Del: apparents large gene conversion), Q318X, R356W, 1761Tins, ClusterE6, V281L] using allele-specific amplification. The most frequent mutation in the Hungarian CAH population was found to be In2. Our results have shown a good genotype/phenotype correlation in case of most mutations; the In2 mutation is associated mostly with the severe form of the disease, whereas I172N was expressed in a wide spectrum of phenotypes. 1999)     

Congenital adrenal hyperplasia owing to 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) owing to 21-hydroxylase deficiency is the most common cause of genital ambiguity in the newborn and is present in about 1 in 15,000 live births worldwide. The disease is further characterized in its classic salt-wasting form (approximately 75% of cases) by potentially lethal adrenal insufficiency. A non-salt-wasting form of classic CAH with 21-hydroxylase deficiency is also recognized by genital ambiguity in affected females and by signs of androgen excess in later childhood in males. Nonclassic CAH with 21-hydroxylase deficiency may be detected in 1% to 3% of populations and is often mistaken for idiopathic precocious pubarche in children or polycystic ovary syndrome in young women. This article presents an overview of clinical and genetic aspects of the various forms of CAH with 21-hydroxylase deficiency.     

Genetics of adrenal steroid 21-hydroxylase deficiency

Impairment of 21-hydroxylation is the most common enzymatic deficiency resulting in the syndrome of CAH, which may present either in the classical form in infants or in the nonclassical form in older individuals. Variable signs and symptoms of androgen excess are common to both types of the disorder, which are transmitted as autosomal recessive traits linked to HLA. Virilization begins in the second month of gestational life in classical 21-OHD, but postnatally in the nonclassical form. Salt wasting is a feature of the disease in a large number of classical patients; in the simple virilizing form aldosterone biosynthesis, a function of the adrenal zona glomerulosa, is intact. Additionally, no patient with nonclassical 21-OHD has been found to have salt wasting. Levels of precursor hormones are less markedly elevated in nonclassical 21-OHD, reflecting a less severe enzyme deficiency; coordinates of basal and stimulated 17-OHP are plotted on a nomogram to ascertain diagnostic category within a family. Confirmatory evidence of heterozygosity within the family of an affected proband is found by performing HLA typing. Specific linkage disequilibria exist for the classical and nonclassical forms of 21-OHD. Frequency of the classical disease is 1/5,000-1/15,000 in Caucasians, whereas the nonclassical disease is found in approximately 1/100 individuals in the Caucasian population, placing the latter disorder among the most common autosomal recessive disorders in man. A deletion of the active 21-hydroxylase gene has been detected in some classical patients; further investigations are in progress to elucidate the molecular genetics of this disease.     

The prevalence of 21-hydroxylase deficiency in adrenal incidentalomas - hormonal and mutation screening.

The aim of the present study was to evaluate and compare the response of 17 OHP to ACTH stimulation in patients with various types of adrenal incidentalomas and to examine the occurence of germline CYP21 mutation in these patients. SUBJECTS AND METHODS: 40 patients (27 females, 13 males) with unilateral and bilateral masses were screened for fi ve most common mutations of the CYP21 in peripheral blood DNA samples. A hormonal evaluation, i.e. baseline plasma values of 17OHP, DHEAS as well as plasma 17OHP and DHEA after ACTH stimulation, was performed in all patients. 21 of them had unilateral adrenal adenoma, 13 patients had adrenal hyperplasia (six of them unilateral) and 6 patients had CT characteristics of other tumors (myelolipomas, cysts, adrenocortical carcinoma). RESULTS: There were no significant differences in plasma 17OHP, DHEAS and plasma cortisol between all three groups. Stimulated plasma values of DHEA and 17OHP after ACTH administration were significantly higher in patients with adenomas (p < 0.05 and p < 0.01) and with hyperplasia (p < 0.05 and p < 0.05) compared with those with other tumors. An exaggerated response of 17 OHP was found in 5 (12 % ) patients. However, mutation screening in peripheral blood samples revealed no CYP21 mutation in all examined groups. SUMMARY: Although 12 % of patients with adrenal incidentalomas had an exaggerated response of 17 OHP after ACTH administration indicating a possible 21-hydroxylase deficiency, these findings are not associated with CYP21 mutation estimated in peripheral blood samples. There was found no germline CYP21 mutation in all patients with various adrenal incidentalomas.     

Adrenal myelolipoma associated with congenital adrenal 21-hydroxylase deficiency.

The occurrence of adrenal myelolipomas is reported in an untreated patient with congenital adrenal 21-hydroxylase deficiency. Laparotomy demonstrated the presence of two lesions, a large tumor which arose from an ectopic adrenal cortex and a smaller tumor in the left adrenal gland. Six cases of adrenal myelolipomas and congenital adrenal hyperplasia have been reported in the literature. All patients were associated with excessive ACTH secretion for a long period of time. The relative frequency of this association, coupled with the observation by Selye and Stone (Am J Pathol 26:211, 1950) that anterior pituitary extracts cause myelolipomatous changes in rats, may indicate a possible role for ACTH in the development of myelolipomas.     

Testosterone-secreting virilizing adrenal adenoma with human chorionic gonadotrophin receptors and 21-hydroxylase deficiency

A 60-year-old woman was evaluated for persistently elevated serum testosterone concentrations after bilateral ovariectomy. Her serum cortisol, androstenedione, dehydroepiandrosterone sulphate and 17-hydroxyprogesterone levels were normal, and decreased after dexamethasone administration. Those of testosterone (17.8-18.4 nmol/l) were remarkably high (normal range 0.7-2.8 nmol/l), were not suppressed by dexamethasone, but clearly increased after hCG administration (up to 128 nmol/l). Computed tomography revealed an adenoma in the right adrenal gland and adrenal scintigraphy under dexamethasone suppression visualized this adenoma. A right adrenalectomy was performed. (1) The tumour was histologically and ultrastructurally adrenocortical adenoma of zona reticularis cell type. (2) The adenoma tissue contained hCG receptors (198 fmol/g). (3) During tissue culture both ACTH and hCG were capable of maintaining its testosterone production, which was attenuated with time without stimulation. (4) The adenoma tissue did not elaborate 21-hydroxylated steroids in contrast to normal adrenal tissue. Thus the aberrant endocrine behaviour of this gonadotrophin-responsive testosterone-secreting adenoma of adrenal zona reticularis cell origin can be explained by ectopic functional hCG receptors and the lack of 21-hydroxylase activity.     

Haplotypes of the steroid 21-hydroxylase gene region encoding mild steroid 21-hydroxylase deficiency.

Haplotypes of the complement 4 (C4) and steroid 21-hydroxylase [21-OHase; steroid hydrogen-donor: oxygen oxidoreductase (21-hydroxylating), EC 1.14.99.10] repeated gene complex were studied in nine families with at least one member affected with a mild form of 21-OHase deficiency. DNA probes from different parts of the repeated C4/21-OHase unit were used to follow the segregation of hybridization patterns in the families. Ten structurally distinct haplotypes of the C4/21-OHase gene region were identified, and the encoded phenotype was assigned to 34 of the 36 C4/21-OHase haplotypes. Four structurally different haplotypes with three C4/21-OHase repeat units were found. Eight of the nine haplotypes found with triplications of the C4/21-OHase repeat unit encoded the mild form of 21-OHase deficiency, whereas one particular triplicated haplotype encoded a severe form of the disease. In one case the mild form of 21-OHase deficiency was encoded by a haplotype with a single C4/21-OHase repeat unit. Mild 21-OHase deficiency was predicted in a patient by the presence of a triplicated haplotype. The finding of deranged 21-OHase genes on all triplicated C4/21-OHase haplotypes indicate that most of these common haplotypes carry mutated 21-OHase genes, and thus may cause functional polymorphism of general importance in the population.     

Heterogeneity of the bovine adrenal steroid 21-hydroxylase

The results presented indicate that purified cytochrome P-45021 which migrated upon SDS gel electrophoresis essentially as a single band, is further separable into different species by ion-exchange chromatography. The P-450 eluted from the CM-Sephadex column at different points along the buffer concentration gradient, exhibited significant differences in (1) the 21-hydroxylation of 17 alpha-OH-progesterone compared to progesterone and (2) the Type I spectral change produced by 17 alpha-OH-progesterone compared to that due to delta 4-androstenedione. These results indicate that the purified P-450 which appeared homogeneous contains different species differing in net charge and steroid preferences for 21-hydroxylation and binding. The ratio, 21-hydroxylation of 17 alpha-OH-progesterone/progesterone ranged between 2.6 and 0.86 suggesting that the purified preparation is a mixture of 17 alpha-OH-progesterone preferring and progesterone preferring species. Possible molecular bases for the heterogeneity of the 21-hydroxylase are discussed.     

正常人肾上腺组织基因表达谱的研究

建立正常人肾上腺组织基因表达目录。方法肾上腺文库构建、大规模表达序列标签（ＥＳＴｓ）测序及生物信息学分析。结果基因／蛋白表达类的基因在肾上腺组织表达最高,代谢类基因次之。前３位高表达基因均为类固醇激素合成酶类及相关蛋白。肾上腺表达ＣＲＨ、尿皮素（ＣＲＨ相关蛋白）黑色素浓集素（ＭＣＨ）、可卡因和安菲他明调节转录物（ＣＡＲＴ）及垂体腺苷酸环化酶活化多态（ＰＡＣＡＰ）等激素及各类受体,包括细胞因子,神经     

The 21-hydroxylase activity in the glomerulosa and fasciculata of the adrenal cortex in congenital adrenal hyperplasia

In the two clinical syndromes of congenital adrenal hyperplasia due to a 21-hydroxylation defect of adrenal steroidogenesis, the simple virilizing and the salt-wasting forms, the 21-hydroxylase activity was studied considering the zona fasciculata and the zona glomerulosa of the adrenal cortex as two separate glands under different regulation. To test this hypothesis, we stimulated adrenal steroidogenesis by ACTH infusion or dietary sodium restriction in eight patients with congenital adrenal hyperplasia (four patients with the simple virilizing form and four with the salt-wasting form of congenital adrenal hyperplasia) and in six normal children. Both the 17-hydroxy and 17-deoxy pathways of adrenocortical steroid biosynthesis were examined by measuring serum concentrations of 17-hydroxyprogesterone, cortisol, progesterone, deoxycorticosterone, corticosterone, and aldosterone and the excretion of free deoxycorticosterone, 18-hydroxydeoxycorticosterone, corticosterone, 18-hydroxycorticosterone, cortisol, and aldosterone. We considered the steroids 18-hydroxycorticosterone and aldosterone to be primarily of zona glomerulosa origin. These studies indicated that the zona fasciculata of both the salt-wasting and the simple virilizing forms is defective in 21-hydroxylation of 17-hydroxy and 17-deoxy steroids. The zona glomerulosa demonstrated deficient 21-hydroxylation only in the salt-wasting form, whereas in the simple virilizing form, the glomerulosa was spared this defect.     

Structure of human steroid 21-hydroxylase genes.

We have determined the structure of cDNA and two genomic genes encoding steroid 21-hydroxylase [21-OHase; steroid 21-monooxygenase; steroid, hydrogen-donor:oxygen oxidoreductase (21-hydroxylating); EC 1.14.99.10]. If this cytochrome P-450 enzyme is defective, cortisol cannot be synthesized, resulting in congenital adrenal hyperplasia. The cDNA encoding this enzyme is 2.0 kilobases long, and the encoded protein is predicted to contain 494 amino acid residues with a molecular weight of 55,000. This enzyme is at most 28% homologous to other P-450 enzymes that have been studied. The 21-OHase genomic genes, which are located in the HLA major histocompatibility complex on chromosome 6, each contain 10 exons. This structure is distinct from other characterized P-450 genes, which contain 7 or 9 exons. Studies of individuals with homozygous deletions of the 21-OHase A or B genes suggest that only the B gene encodes an active enzyme. This is confirmed by the finding that the A gene has an 8-base deletion within codons 110-112, resulting in a frameshift that brings a stop codon into the reading frame at codon 130. A second frameshift and a nonsense mutation occur downstream. In contrast, the sequence of the exons of the B gene is identical to the cDNA sequence. The 21-OHase A gene is, therefore, a pseudogene.     

Nucleotide sequence analysis of murine 21-hydroxylase genes: mutations affecting gene expression.

Steroid 21-hydroxylase [21-OHase; steroid 21-monooxygenase; steroid, hydrogen-donor:oxygen oxidoreductase (21-hydroxylating); EC 1.14.99.10] is a cytochrome P-450 enzyme required for the adrenal synthesis of mineralocorticoids and glucocorticoids. The gene encoding this protein is present in two copies (21-OHase A and B) in the S region of the murine major histocompatibility complex. Previous studies utilizing gene-specific oligonucleotide probes and gene transfer showed that only the 21-OHase A gene is expressed in the BALB/c mouse. Here, we present the complete primary structures of both BALB/c 21-OHase encoding genes. Comparison of the nucleotide sequences defines a deletion of 215 nucleotides spanning the second exon of the 21-OHase B gene; other nucleotide changes in the 21-OHase B gene introduce frame shifts and premature termination codons. Southern blot analysis of C57BL/6 and DBA/2J mice indicates that a similar deletion is present in these strains; however the C3H/HeJ strain is a structural variant. A hybrid gene composed of the 21-OHase B promoter placed 5' of the 21-OHase A structural sequences was efficiently transcribed following transfection into Y1 adrenocortical tumor cells. These findings demonstrate that the 21-OHase B gene promoter is functional and suggest that mutations within the 21-OHase B structural gene are responsible for its lack of expression.     

Late diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

Congenital adrenal hyperplasia results from 21-hydroxylase deficiency in more than ninety percent of cases. The classical form of 21-hydroxylase deficiency presents in the neonatal period with virilization or adrenal insufficiency, with or without concurrent salt wasting. We report on a rare case of classic 21-hydroxylase deficiency diagnosed in late adulthood. A 39-year-old male patient presented for workup of infertility. Urologic investigation revealed small testes, bilateral testicular masses, and asthenozoospermia. The patient's steroid metabolism showed markedly increased levels of adrenal androgens, in particular of 17-hydroxyprogesterone amd 21-deoxycortisol. The gas chromatographic-mass spectrometric (GC-MS) urinary steroid profile was dominated by metabolites of 17-hydroxyprogesterone, while the endogenous glucocorticoid production was subnormally low. ACTH levels in plasma were elevated. These hormonal findings were consistent with 21-hydroxylase deficiency. Therapy with dexamethasone was initiated. The CTP21A2 gene analysis revealed the mutation I172N (ATC --> AAC) in exon 4 of allele 1 and a large gene deletion in allele 2. Cases of 21-hydroxylase deficiency diagnosed in late adulthood are rare; however, clinicians should be alert of this possibility.