C. Duparc

Intraadrenal Corticotropin in Bilateral Macronodular Adrenal Hyperplasia

BACKGROUND Bilateral macronodular adrenal hyperplasia is a rare cause of primary adrenal Cushings syndrome. In this form of hyperplasia, hypersecretion of cortisol suppresses the release of corticotropin by pituitary corticotrophs, which results in low plasma corticotropin levels. Thus, the disease has been termed corticotropin-independent macronodular adrenal hyperplasia. We examined the abnormal production of corticotropin in these hyperplastic adrenal glands. METHODS We obtained specimens of hyperplastic macronodular adrenal tissue from 30 patients with primary adrenal disease. The corticotropin precursor proopiomelanocortin and corticotropin expression were assessed by means of a polymerase-chain-reaction assay and immunohistochemical analysis. The production of corticotropin and cortisol was assessed in 11 specimens with the use of incubated explants and cell cultures coupled with hormone assays. Corticotropin levels were measured in adrenal and peripheral venous blood samples from 2 patients. RESULTS The expression of proopiomelanocortin messenger RNA (mRNA) was detected in all samples of hyperplastic adrenal tissue. Corticotropin was detected in steroidogenic cells arranged in clusters that were disseminated throughout the adrenal specimens. Adrenal corticotropin levels were higher in adrenal venous blood samples than in peripheral venous samples, a finding that was consistent with local production of the peptide within the hyperplastic adrenals. The release of adrenal corticotropin was stimulated by ligands of aberrant membrane receptors but not by corticotropin-releasing hormone or dexamethasone. A semiquantitative score for corticotropin immunostaining in the samples correlated with basal plasma cortisol levels. Corticotropin-receptor antagonists significantly inhibited in vitro cortisol secretion. CONCLUSIONS Cortisol secretion by the adrenals in patients with macronodular hyperplasia and Cushings syndrome appears to be regulated by corticotropin, which is produced by a subpopulation of steroidogenic cells in the hyperplastic adrenals. Thus, the hypercortisolism associated with bilateral macronodular adrenal hyperplasia appears to be corticotropin-dependent. (Funded by the Agence Nationale de la Recherche and others.).

Expression of vasopressin receptors in ACTH-independent macronodular bilateral adrenal hyperplasia causing Cushing's syndrome: molecular, immunohistochemical and pharmacological correlates

Cortisol secretion in ACTH-independent macronodular adrenal hyperplasia (AIMAH) causing Cushings syndrome can be controlled by illegitimate receptors. The aim of the present study was to characterize the molecular, immunohistochemical, and pharmacological profiles of vasopressin receptors in cells derived from three patients with AIMAH (H1-H3), in order to evaluate the role of ectopic vasopressin receptors in the physiopathology of hypercortisolism. Expression of mRNAs encoding the vasopressin receptor types (V(1a), V(1b), and V(2)) were analyzed by RT-PCR in adrenal tissues. The presence of V(1a) and V(2) receptors was studied by immunohistochemistry on adrenal sections. The pharmacological profiles of vasopressin receptors involved in the control of cortisol secretion were investigated using the V(1a) receptor antagonist SR49059 and the V(2) receptor agonist [deamino-Cys(1), Val(4), D-Arg(8)]-vasopressin on cultured cells. The V(1a) receptor protein was present and functional in H1 and H3 tissues, whereas the V(1b) receptor was not expressed in any of the tissues. RT-PCR experiments revealed that V(2) receptor mRNAs were detected in the three tissues. In contrast, immunohistochemical and cell incubation studies showed that the V(2) receptor was involved in the stimulatory effect of AVP on cortisol secretion in H1 and H2, but not in H3 cells. Taken together, these data show that expression of functional ectopic V(2) receptors and repression of eutopic V(1a) receptor can coexist in some hyperplastic corticosteroidogenic tissues. They also reveal that immunohistochemical and incubation studies are essential for the characterization of ectopic receptors actually involved in the control of cortisol secretion by AIMAHs.

Circulating EM66 is a highly sensitive marker for the diagnosis and follow-up of pheochromocytoma

We have previously demonstrated that measurement of tissue concentration of the novel secretogranin II‐derived peptide EM66 may help to discriminate between benign and malignant pheochromocytomas. The aim of the present study was to characterize EM66 in plasma and urine of healthy volunteers and pheochromocytoma patients, in order to further evaluate the usefulness of this peptide as a circulating marker for the management of the tumors. HPLC analysis of plasma and urine samples demonstrated that the EM66‐immunoreactive material coeluted with the recombinant peptide. In healthy volunteers, plasma and urinary EM66 levels were, respectively, 2.6 (1.9–3.7) ng/ml and 2.9 (1.9–4.6) ng/ml. In patients with pheochromocytoma, plasma EM66 levels were 10‐fold higher than those of healthy volunteers (26.9 (7.3–44) ng/ml), and returned to normal values after removal of the tumor. In contrast, urinary EM66 levels were not significantly different from those of healthy volunteers (3.2 (2.2–3.9) ng/ml). Measurement of total or free plasma metanephrines and 24 hr urinary metanephrines in our series of patients revealed that these tests, taken separately, are less sensitive than the EM66 determination. Pheochromocytes in primary culture secreted high levels of EM66, suggesting that the chromaffin tumor was actually responsible for the increased plasma peptide concentrations in the patients. These data indicate that EM66 is secreted in the general circulation and that elevated plasma EM66 levels are correlated with the occurrence of pheochromocytoma. Thus, EM66 is a sensitive plasma marker that should be considered as a complementary tool in the management of pheochromocytoma.

Mast Cell Hyperplasia Is Associated With Aldosterone Hypersecretion in a Subset of Aldosterone-Producing Adenomas

CONTEXT Adrenal mast cells can stimulate aldosterone secretion through the local release of serotonin (5-HT) and activation of the 5-HT4 receptor (5-HT4). In aldosterone-producing adenomas (APAs), 5-HT4 receptor is overexpressed and the administration of 5-HT4 receptor agonists to patients with APA increases plasma aldosterone levels. These data and the well-documented role of mast cells in tumorigenesis suggest that mast cells may be involved in the pathophysiology of APA. OBJECTIVE The study aimed at investigating the occurrence of mast cells in a series of APA tissues and to examine the influence of mast cells on aldosterone secretion. DESIGN The occurrence of mast cells in APAs was investigated by immunohistochemistry. Mast cell densities were compared with clinical data. The influence of mast cells on aldosterone production was studied by using cultures of human mast cell and adrenocortical cell lines. RESULTS In APA tissues, the density of mast cells was found to be increased in comparison with normal adrenals. Mast cells were primarily observed in adrenal cortex adjacent to adenomas or in the adenomas themselves, distinguishing two groups of APAs. A subset of adenomas was found to contain a high density of intratumoral mast cells, which was correlated with aldosterone synthase expression and in vivo aldosterone secretory parameters. Administration of conditioned medium from cultures of human mast cell lines to human adrenocortical cells induced a significant increase in aldosterone synthase (CYP11B2) mRNA expression and aldosterone production. CONCLUSION APA tissues commonly contain numerous mast cells that may influence aldosterone secretion through the local release of regulatory factors.

Evaluation of endocrine testing of Leydig cell function using extractive and recombinant human chorionic gonadotropin and different doses of recombinant human LH in normal men

BACKGROUND The functional testing of endocrine testis uses extractive human chorionic gonadotropin (ehCG). Recombinant human hCG (rhCG), avoiding any contamination, should replace ehCG. Moreover, a functional evaluation with recombinant human LH (rhLH) would be closer to physiology than a pharmacological testing with hCG. METHODS The study was conducted in normal men. We first evaluated the dose-effect of ehCG on plasma testosterone and estradiol levels, before and after injection of either hCG or vehicle. Secondly, the responses to the optimal dose of ehCG were compared with those of rhCG. Thirdly, we investigated the dose-effect of rhLH, on steroid hormone secretion. LH, testosterone, and estradiol plasma levels were measured after the injection of either rhLH or placebo. RESULTS ehCG induced dose-dependent increases in plasma estradiol and testosterone levels. They respectively peaked at 24 and 72 h after the injection. The most potent dose of ehCG (5000 IU) induced results similar to those observed with 250 microg (6500 IU) rhCG. By comparison with placebo, rhLH induced a significant and dose-dependent increase in plasma testosterone levels 4 h after the injection. Peak response of testosterone to rhLH and rhCG was significantly correlated. rhLH did not induce significant change in plasma estradiol level. CONCLUSIONS In normal men, a single i.v. injection of 150 IU rhLH induces a 25% rise in plasma testosterone levels by comparison with placebo. At the moment, the dynamic evaluation using hCG remains the gold standard test to explore the Leydig cell function. The use of 250 microg rhCG avoiding any contamination should be recommended.

Ectopic expression of serotonin7 receptors in an adrenocortical carcinoma co-secreting renin and cortisol.

Abnormal expression of membrane receptors has been previously described in benign adrenocortical neoplasms causing Cushings syndrome. In particular, we have observed that, in some adreno corticotropic hormone (ACTH)-independent macronodular adrenal hyperplasia tissues, cortisol secretion is controlled by ectopic serotonin(7) (5-HT(7)) receptors. The objective of the present study was to investigate in vitro the effect of serotonin (5-hydroxy tryptamine; 5-HT) on cortisol and renin production by a left adrenocortical carcinoma removed from a 48-year-old female patient with severe Cushings syndrome and elevated plasma renin levels. Tumor explants were obtained at surgery and processed for immunohistochemistry, in situ hybridization and cell culture studies. 5-HT-like immunoreactivity was observed in mast cells and steroidogenic cells disseminated in the tissue. 5-HT stimulated cortisol release by cultured cells. The stimulatory effect of 5-HT on cortisol secretion was suppressed by the 5-HT(7) receptor antagonist SB269970. In addition, immunohistochemistry showed the occurrence of 5-HT(7) receptor-like immunoreactivity in carcinoma cells. mRNAs encoding renin as well as renin-like immunoreactivity were detected in endothelial and tumor cells. Cell incubation studies revealed that the adrenocortical tissue also released renin. Renin production was inhibited by 5-HT but was not influenced by ACTH and angiotensin II (Ang II). In conclusion, the present report provides the first demonstration of ectopic serotonin receptors, i.e. 5-HT(7) receptors, in an adrenocortical carcinoma. Our results also indicate that 5-HT can influence the secretory activity of malignant adrenocortical tumors in an autocrine/paracrine manner. The effects of 5-HT on adrenocortical tumor cells included a paradoxical inhibitory action on renin production and a stimulatory action on cortisol secretion involving 5-HT(7) receptors.

Paradoxical inhibitory effect of serotonin on cortisol production from adrenocortical lesions causing Cushing's syndrome.

In the human adrenal gland, serotonin (5‐HT) stimulates cortisol production through a paracrine mechanism involving 5‐HT4 receptors positively‐coupled to adenylyl cyclase. A hyperresponsiveness of adrenocortical tissue to 5‐HT has also been described in several cases of ACTH‐independent bilateral macronodular adrenal hyperplasias (AIMAHs) and adenomas causing Cushings syndrome. In the present study, we report two cases of cortisol‐producing adrenocortical lesions, i.e. one AIMAH (case 1) and one adenoma (case 2), whose secretory activity was inhibited in vitro by 5‐HT. The potencies (pIC50) and efficacies (Emax) of 5‐HT to inhibit cortisol secretion were 8.2 ± 0.4 and − 64.1% ± 7.5% in case 1, and 9.2 ± 0.5 and − 32.3% ± 3.8% in case 2. The specific 5‐HT4 antagonist GR 113808 failed to influence the 5‐HT‐induced decrease in cortisol production by the two tissues, indicating that the paradoxical inhibitory effect of 5‐HT could not be accounted for by activation of eutopic 5‐HT4 receptors. These results suggest that the tissues expressed aberrant 5‐HT receptors. In conclusion, the present study provides the first evidence for an inhibitory effect of 5‐HT on cortisol secretion in adrenocortical lesions causing Cushings syndrome. Our data also suggest that expression of illegitimate membrane receptors by cortisol‐producing adrenal hyperplasias and/or adenomas may convert a paracrine stimulatory factor into an inhibitory signal.

Effect of Serotonin4 (5-HT4) Receptor Agonists on Aldosterone Secretion in Idiopathic Hyperaldosteronism

Serotonin (5-HT) stimulates aldosterone secretion in man through 5-HT4 receptors positively coupled to adenylyl cyclase. In particular, it has been shown that oral administration of a single dose of the 5-HT4 receptor agonist cisapride induces a significant increase in plasma aldosterone levels (PAL) in healthy volunteers. Idiopathic hyperaldosteronism (IH) is a rare disorder characterized by hypertension, hypokalemia and bilateral adrenal hypersecretion of aldosterone. In patients with IH, administration of the 5-HT precursor 5-hydroxytryptophan (5-HTP) is followed by a significant increase in PAL. 5-HTP-induced aldosterone secretion has been attributed to the activation of central serotonergic pathways. The aim of the present study was to evaluate the effect of the oral administration of a single dose of cisapride (10 mg) on aldosterone secretion in 15 patients with IH, in a simple blind fashion versus placebo. Cisapride induced a significant increase in PAL but did not affect renin, cortisol and potassium levels. The present study demonstrates that 5-HT4 receptor agonists are able to stimulate aldosterone secretion in patients with IH. These data also indicate that hyperplastic glomerulosa tissue, like normal glomerulosa cells, expresses a functional 5-HT4 receptor. Therefore, 5-HT4 receptor antagonists may represent a new approach in the treatment of primary hyperaldosteronism.

Paracrine control of steroidogenesis by serotonin in adrenocortical neoplasms.

Serotonin (5-hydroxytryptamine; 5-HT) is able to activate the hypothalamo-pituitary-adrenal axis via multiple actions at different levels. In the human adrenal gland, 5-HT, released by subcapsular mast cells, stimulates corticosteroid production through a paracrine mode of communication which involves 5-HT receptor type 4 (5-HT4) primarily located in zona glomerulosa. As a result, 5-HT is much more efficient to stimulate aldosterone secretion than cortisol release in vitro and administration of 5-HT4 receptor agonists to healthy individuals is followed by an increase in plasma aldosterone levels without any change in plasma cortisol concentrations. Interestingly, adrenocortical hyperplasias and tumors responsible for corticosteroid hypersecretion exhibit various cellular and molecular defects which tend to reinforce the intraadrenal serotonergic tone. These pathophysiological mechanisms, which are summarized in the present review, include an increase in adrenal 5-HT production and overexpression of 5-HT receptors in adrenal neoplastic tissues. Altogether, these data support the concept of adrenal serotonergic paracrinopathy and suggest that 5-HT and its receptors may constitute valuable targets for pharmacological treatments of primary adrenal diseases.

Effects of serotonin and vasopressin on cortisol production from an adrenocortical tumor causing subclinical Cushing's syndrome.

In dexamethasone-suppressed healthy volunteers, the serotonin4 (5-HT4) receptor agonist cisapride and lysine vasopressin [LVP, an analog of arginine vasopressin (AVP)] have no influence on plasma cortisol levels (PCL). In contrast, cisapride and AVP have been shown to stimulate cortisol secretion in patients with adrenal tumor or bilateral adrenal hyperplasia and Cushings syndrome. In this report, we describe a case of adrenocortical adenoma causing subclinical Cushings syndrome. Cisapride and terlipressin, a precursor of LVP, both induced an increase in PCL reaching +88% and +100%, respectively, without any significant variation of plasma ACTH levels. In vitro experiments were conducted to investigate the effects of 5-HT and AVP on cortisol production from cultured tumor cells and normal adrenocortical cells. 5-HT and AVP both induced a dose-dependent increase in cortisol production from cultured tumor cells. Comparison of the data obtained with tumor and normal cells, respectively, showed that 5-HT was more efficient to stimulate steroidogenesis in adenomatous than normal cells. Concurrently, the efficacy and potency of AVP were both higher in tumor than normal cells. Collectively, these results show that the abnormal in vivo responses of the adrenocortical adenoma to cisapride and LVP could be ascribed to an increased sensitivity of the tumor tissue to 5-HT and AVP. The data also suggest that the adrenocortical tumor overexpressed eutopic 5-HT4 and V1 receptors.