Difficult-to-control hypertension due to bilateral aldosterone-producing adrenocortical microadenomas associated with a cortisol-producing adrenal macroadenoma.

The patient was a 54-year-old woman who developed a right adrenal tumour, Cushingoid features, elevated levels of cortisol that were not suppressed by 1 nor 8 mg of dexamethasone, and suppression of adrenocorticotropin (ACTH) during treatment for severe hypertension. Computed tomography (CT) revealed a right adrenal tumour and an atrophic left adrenal gland. In addition, elevated plasma aldosterone concentration (PAC) and suppressed plasma renin activity (PRA) with an aldosterone-to-renin ratio of 128 (ng per 100 ml per ng ml⁻¹ h⁻¹) suggested aldosterone excess. Urinary excretion of aldosterone was relatively high, and the captopril and rapid ACTH tests resulted in no response of PRA and exaggerated increase in PAC, respectively. ACTH-loaded adrenal venous sampling showed bilateral excess of aldosterone with right predominance of cortisol. Right laparoscopic partial adrenalectomy (ADX) and immunohistochemical analysis showed both a cortisol-producing adenoma and an aldosterone-producing microadenoma (microAPA) within the attached adrenal, which had not been detected by CT preoperatively. After the right partial ADX, her blood pressure, aldosterone level and suppressed PRA remained unchanged. Subsequently, laparoscopic total left ADX was performed. Two microAPAs with paradoxical hyperplasia were revealed within the apparently atrophic left adrenal gland. Soon after the second surgery, her blood pressure normalized without requiring any anti-hypertensive medication.

Adrenocortical peptides: autocrine or paracrine regulators for the steroid hormone secretion or the cell proliferation?

There is accumulating evidence that indicates the production and secretion of some peptides by adrenal cortex and adrenocortical tumors. These peptides include adrenomedullin, proadrenomedullin N-terminal 20 peptide (PAMP), endothelin-1, cerebellin, urotensin-II etc., and appear to be involved in the regulation of steroid hormone secretion and the proliferation of adrenocortical cells as autocrine and/or paracrine factors. It is noteworthy that expression of adrenomedullin is induced by hypoxia and inflammatory cytokines in various cells, raising the possibility that the adrenocortical production of adrenomedullin (and probably PAMP) may be increased in some inflammatory and/or ischemic conditions, and these two peptides may modulate the states of inflammatory and/or ischemic disorders. In addition to adrenomedullin and PAMP, adrenal cortex and/or some adrenocortical tumors express endothelin-1, cerebellin, urotensin-II, etc. The adrenocortical peptides may form a novel autocrine or paracrine regulatory system for the steroid hormone secretion and the cell proliferation in the adrenal cortex, and involve in the pathophysiology of inflammatory, ischemic or neoplastic diseases of the adrenal cortex

Role of urotensin II in patients on dialysis.

Urotensin II is a potent vasoconstrictor, which also has some vasodilatory properties. We investigated its expression in various tissues and in the plasma of patients with renal dysfunction. Plasma concentrations of urotensin II-like immunoreactivity were 2-fold higher in patients not on dialysis and 3-fold higher in those on haemodialysis thanin healthy individuals. Messenger RNA encoding theurotensin II precursor and the urotensin II receptor precursor were expressed in various human tissues. The peptidemight act as an important regulator in the cardiovascularand renal systems. Urotensin II antagonists could, therefore, be useful in the treatment of diseases affecting theseorgans.

Expression of urotensin II and urotensin II receptor mRNAs in various human tumor cell lines and secretion of urotensin II-like immunoreactivity by SW-13 adrenocortical carcinoma cells.

Urotensin II is the most potent vasoconstrictor peptide identified so far. Expression of urotensin II and urotensin II receptor mRNAs was studied in various human tumor cell lines by reverse transcriptase polymerase chain reaction (PCR) method. Secretion of urotensin II by these tumor cells was studied by radioimmunoassay. The tumor cell lines studied were T98G glioblastoma cells, IMR-32 neuroblastoma cells, NB69 neuroblastoma cells, BeWo choriocarcinoma cells, SW-13 adrenocortical carcinoma cells, DLD-1 colorectal adenocarcinoma cells and HeLa cervical cancer cells. Urotensin II mRNA was expressed in 6 tumor cell lines except for NB69 neuroblastoma cells. Urotensin II receptor mRNA was expressed in all 7 tumor cell lines. A significant amount of urotensin II-like immunoreactivity was detected only in the culture medium of SW-13 adrenocortical carcinoma cells by radioimmunoassay. Sephadex G-50 column chromatography showed that the urotensin II-like immunoreactivity in the culture medium extract was eluted earlier than synthetic human urotensin II, suggesting that SW-13 cells secreted higher molecular weight materials, perhaps partially processed forms of the urotensin II precursor. Reverse phase high-performance liquid chromatography (HPLC) showed three immunoreactive peaks, one of which was eluted in the position of urotensin II. The present study has shown for the first time expression of urotensin II and urotensin II receptor mRNAs in various tumor cell lines and the secretion of urotensin II-like immunoreactivity by SW-13 adrenocortical carcinoma cells.

Expression of melanin-concentrating hormone receptor messenger ribonucleic acid in tumor tissues of pheochromocytoma, ganglioneuroblastoma, and neuroblastoma.

Expression of melanin-concentrating hormone (MCH) receptor messenger ribonucleic acid (mRNA) was studied by RT-PCR and Northern blot analysis in human brain; pituitary; adrenal glands; tumor tissues of adrenal tumors, ganglioneuroblastomas, and neuroblastomas; and various cultured tumor cell lines. RT-PCR analysis showed that MCH receptor mRNA was widely expressed in brain tissues, pituitary, normal portions of adrenal glands (cortex and medulla), tumor tissues of adrenocortical tumors (12 of 13 cases), pheochromocytoma (all 7 cases), ganglioneuroblastoma (1 case), neuroblastoma (all 5 cases), and various cultured tumor cell lines (6 of 7 cell lines), including 2 neuroblastoma cell lines. Northern blot analysis showed the expression of MCH receptor mRNA ( approximately 2.4 kb) only in the tumor tissues of 5 pheochromocytomas, 1 ganglioneuroblastoma, and 4 neuroblastomas, indicating that the expression levels of MCH receptor mRNA are much higher in these tumors than in the other tissues. These findings raised the possibility that MCH or MCH-like peptides may be related to the pathophysiology of these neural crest-derived tumors.

Induction of adrenomedullin by hypoxia in cultured retinal pigment epithelial cells.

PURPOSE: To explore the effects of hypoxia on the production and secretion of adrenomedullin (ADM) and endothelin (ET)-1 in human retinal pigment epithelial (RPE) cells. METHODS: RPE cells were cultured under normoxic or hypoxic (1% O2) conditions. Expression of ADM and ET-1 was examined by Northern blot analysis and radioimmunoassay. Effects of ADM and ET-1 on the number of RPE cells were examined by modified 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: ADM mRNA expression levels and immunoreactive ADM levels in the medium were increased by hypoxia in all three human RPE cell lines (ARPE-19, D407, and F-0202). Immunoreactive ET was detected in the cultured media of D407 cells and ARPE-19 cells and identified as ET-1 by reversed-phase high performance liquid chromatography. Hypoxia treatment for 48 hours increased immunoreactive ET levels approximately 1.3-fold in the cultured media of D407, but not ARPE-19 cells. Hypoxia decreased the number of ARPE-19 cells and F-0202 cells, and the treatment with ADM ameliorated the hypoxia-induced decrease in the cell number. In contrast, exogenously added ET-1 had no significant effects on the number of ARPE-19 cells under normoxia and hypoxia. CONCLUSIONS: Hypoxia increased the expression of ADM in all three human RPE cell lines, whereas the induction of ET-1 by hypoxia was found only in D407 cells. ADM induced by hypoxia may have protective roles against hypoxic cell damage in RPE cells.

Immunoreactive orexin-A in human plasma.

Orexin-A and orexin-B are newly discovered neuropeptides which are implicated in feeding behavior and arousal state. We studied immunoreactive(IR)-orexin-A concentrations in human plasma by radioimmunoassay. IR-orexin-A concentrations in plasma obtained from 17 healthy subjects in the morning were 1.94 +/- 0.24 pmol/liter (mean +/- SEM). IR-orexin-A levels in the plasma obtained at night were not significantly different from those obtained in the morning in 9 female subjects. The HPLC analysis of the plasma extract showed two immunoreactive peaks; one peak eluting in an identical position to synthetic orexin-A, and one eluting earlier. This study has shown for the first time the presence of orexin-A in human plasma.

Differential expression of adrenomedullin and its receptor component, receptor activity modifying protein (RAMP) 2 during hypoxia in cultured human neuroblastoma cells.

Adrenomedullin is a potent vasodilator peptide originally isolated from a pheochromocytoma. Recently, a novel adrenomedullin receptor has been identified as a complex consisting of calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP) 2. To explore possible pathophysiological roles of adrenomedullin and its receptor component RAMP2 in hypoxic tissues, we studied effects of hypoxia on expression of adrenomedullin and RAMP2 in two human neuroblastoma cell lines, IMR-32 and NB69, by radioimmunoassay and Northern blot analysis. Expression levels of adrenomedullin were increased by hypoxia in both cell lines. Treatment with cobalt chloride or desferrioxamine mesylate also increased expression levels of adrenomedullin mRNA. On the other hand, expression levels of RAMP2 mRNA were decreased in IMR-32 cells and were not changed in NB69 cells by hypoxia. Treatment with cobalt chloride or desferrioxamine mesylate decreased expression levels of RAMP2 mRNA in both IMR-32 and NB69 cells. These findings indicate that adrenomedullin expression is induced during hypoxia in IMR-32 and NB69 neuroblastoma cells, but RAMP2 expression is rather suppressed under the same conditions. The decreased expression of RAMP2 and the ADM expression induction under hypoxia may constitute one mechanism of cellular adaptation to hypoxic stress.

Adrenomedullin and its receptor complexes in remnant kidneys of rats with renal mass ablation: decreased expression of calcitonin receptor-like receptor and receptor-activity modifying protein-3.

Adrenomedullin (AM) has vasodilator and diuretic actions, similarly to natriuretic peptides. AM receptor complexes are composed of calcitonin receptor-like receptor (CRLR) and receptor-activity modifying protein-2 (RAMP2), or CRLR and RAMP3. We aimed to know whether gene expression of AM and AM receptor complexes are regulated in kidneys under pathophysiological conditions. Expression of AM, RAMP2, RAMP3 and CRLR mRNA was studied in the remnant kidney of rats with renal mass ablation using competitive quantitative RT-PCR techniques. Partial cloning was performed to determine the rat RAMP3 nucleotide sequence. In normal rat kidneys, expression levels of RAMP2, RAMP3, CRLR and AM mRNAs were 26.5 +/- 1.9 mmol/mole of GAPDH, 7.7 +/- 0.9 mmol/mole of GAPDH, 3.6 +/- 0.2 mmol/mole of GAPDH and 0.57 +/- 0.03 mmol/mole of GAPDH (mean +/- SE, n = 6), respectively. RAMP3 mRNA levels decreased significantly to about 50% and about 70% of control (sham-operated rats) 4 days and 14 days after 5/6 nephrectomy, respectively. CRLR mRNA levels also decreased significantly to about 30% and about 43% of control. Sodium intake restriction had no significant effects on the RAMP3 and CRLR gene expression. On the other hand, RAMP2 mRNA expression in the kidney was suppressed by sodium intake restriction regardless of nephrectomy, while RAMP2 levels in the remnant kidney were not significantly changed by 5/6 nephrectomy. Neither 5/6 nephrectomy or sodium intake restriction had any significant effects on the AM gene expression in the kidney. The present study showed that expression of mRNAs encoding AM, RAMP2, RAMP3 and CRLR were differentially regulated in remnant kidneys of rats with renal mass ablation.

Increased gene expression of adrenomedullin and adrenomedullin-receptor complexes, receptor-activity modifying protein (RAMP)2 and calcitonin-receptor-like receptor (CRLR) in the hearts of rats with congestive heart failure.

Adrenomedullin is a vasodilator peptide produced in various organs, including heart and kidney. A novel adrenomedullin receptor complex has recently been identified, namely the calcitonin receptor-like receptor (CRLR) and receptor-activity modifying protein (RAMP) 2. In the present study, we have examined gene expression of RAMP2, CRLR and adrenomedullin in hearts and kidneys of rats with congestive heart failure caused by coronary artery ligation. Partial cloning was performed to determine the rat RAMP2 nucleotide sequence. Messenger RNA levels were then determined using competitive, quantitative reverse transcription-PCR techniques. Significantly increased expression levels (means+/-S.E.) of RAMP2, CRLR and adrenomedullin mRNA were found in the atrium (1.8+/-0.2-fold, 1. 8+/-0.2-fold and 2.1+/-0.1-fold, respectively, compared with sham operated rats) and in the ventricle (1.4+/-0.1-fold, 1.3+/-0.03-fold and 3.0+/-0.5-fold respectively). On the other hand, expression levels of RAMP2, CRLR and adrenomedullin mRNAs were not significantly changed in the kidney. These findings suggest potential roles of locally-produced and locally-acting adrenomedullin in the failing heart.

Regional distribution of immunoreactive prolactin-releasing peptide in the human brain.

Regional distribution of prolactin-releasing peptide (PrRP) in the human brain was studied by radioimmunoassay. The antiserum raised against human PrRP-31 in a rabbit was used in the assay, which showed 100% cross reaction with PrRP-20 and no significant cross reaction with other peptides. The highest concentrations of immunoreactive-PrRP were found in hypothalamus (912 +/- 519 fmol/g wet weight, n = 6, mean +/- SEM), followed by medulla oblongata (496 +/- 136 fmol/g wet weight) and thalamus (307 +/- 117 fmol/g wet weight). On the other hand, immunoreactive-PrRP was not detected in frontal lobe or temporal lobe (<50 fmol/g wet weight). Sephadex G50 column chromatography of the immunoreactive-PrRP in the hypothalamus and medulla oblongata showed three immunoreactive peaks; one peak eluting in the position of PrRP-20, one eluting in the position of PrRP-31 and one eluting earlier. Reverse phase high-performance liquid chromatography (HPLC) of these brain tissue extracts showed a peak eluting in the position of PrRP-20 and PrRP-31. The present study has shown for the first time the presence of immunoreactive-PrRP in the human brain. The immunoreactive-PrRP levels in the human hypothalamus were, however, lower than the levels of other neuropeptides with prolactin-releasing activity, such as thyrotropin-releasing hormone and vasoactive intestinal polypeptide.

Expression of endothelin-1 and endothelin receptors in cultured human glioblastoma cells.

Production and secretion of endothelin-1 (ET-1) by a human glioblastoma cell line, T98G, were studied by radioimmunoassay and Northern blot analysis. Immunoreactive ET was detected in the culture medium of T98G (17.6 +/- 0.6 fmol/10(5) cells/24 h, mean +/- SEM, n = 5). Reverse-phase high-performance liquid chromatography (HPLC) of immunoreactive ET in the culture medium extract showed a single peak eluting in the position of ET-1. Northern blot analysis showed expression of ET-1 mRNA in T98G cells. Treatment with interferon-gamma decreased the expression of ET-1. Treatment with TNFalpha or interleukin-1beta (IL-1beta) increased the expression of ET-1. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) showed expression of endothelin-A- and -B- (ET(A) and ET(B)) receptor mRNAs in T98G glioblastoma cells. These findings indicate that glioblastoma cells produce and secrete ET-1, and express ET receptor mRNAs. ET-1 secreted by glioblastoma cells may act locally on tumor cells, possibly as a growth modulator.

Secretion of endothelin-1 and adrenomedullin by SW-13 human adrenocortical carcinoma cells.

The adrenal medulla and pheochromocytomas are known to secrete various neuropeptides and vasoactive peptides. On the other hand, the production and secretion of peptides by adrenocortical tumors have not been studied in detail. The study reported here therefore set out to examine these two functions for two vasoactive peptides, endothelin-1 (ET-1) and adrenomedullin (ADM) in SW-13 human adrenocortical carcinoma cells by radioimmunoassay and Northern blot analysis. Both immunoreactive ET (irET) and irADM were detected in the culture medium of SW-13 cells. Northern blot analysis showed the expression of ET-1 and ADM mRNAs in SW-13 cells. On the other hand, no significant amounts of calcironin-gene-related peptide, corricotropin-releasing-hormone, neuropeptide Y or urocorlin were secreted by SW-13 cells. This study has shown that ET-1 and ADM are the two unique vasoactive peptides that are produced and secreted by adrenocortical carcinoma cells.

Induction of adrenomedullin during hypoxia in cultured human glioblastoma cells.

Adrenomedullin is a potent vasodilator peptide originally isolated from pheochromocytoma. Adrenomedullin is produced by various types of cells including neurons and astrocytes. To explore possible pathophysiological roles of adrenomedullin in hypoxic brain, we studied the effects of hypoxia on the expression of adrenomedullin in T98G human glioblastoma cells by radioimmunoassay and northern blot analysis. Expression levels of adrenomedullin mRNA and immunoreactive adrenomedullin levels in the culture medium were increased by hypoxia about six- and about threefold, respectively. Treatment with cobalt chloride increased expression levels of adrenomedullin mRNA about threefold and immunoreactive adrenomedullin levels in the culture medium about threefold in T98G cells. Using actinomycin D, we showed that hypoxia did not cause the stabilization of the adrenomedullin mRNA, suggesting that the increased adrenomedullin mRNA levels in response to hypoxia are caused mainly by increased transcription. Treatment with cycloheximide caused increases in adrenomedullin mRNA levels in both normoxic and hypoxic states, raising the possibility that some protein(s) may act as a suppressor of adrenomedullin gene expression in T98G cells. These findings indicate that adrenomedullin is highly induced during hypoxia in T98G glioblastoma cells and suggest that increased expression of adrenomedullin during hypoxia may be important in the defense against hypoxia or ischemia in the brain.

Chicken ovalbumin upstream promoter transcription factor II in the human adrenal cortex and its disorders.

Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is an orphan member of the steroid/thyroid hormone receptor superfamily. COUP-TFII has been demonstrated to negatively regulate the transcriptional activity of adrenal 4-binding protein, a steroidogenic cell-specific transcription factor that activates the transcription of various steroidogenic P450 genes. We therefore examined immunolocalization of COUP-TFII in the human adrenal cortex and its disorders, including functioning and nonfunctioning cortical tumors, to study its possible correlation with adrenocortical steroidogenesis. In nonpathological adrenal cortex, COUP-TFII immunoreactivity was marked in the nuclei of adrenocortical cells in definitive and fetal zones from 16 gestational weeks to 2 months after birth. Immunoreactivity for COUP-TFII was marked in the zona glomerulosa and weak in the zonae fasciculata and reticularis from 7 months to 8 yr of age, but thereafter markedly decreased in these zones (P < 0.05, between age 7 months to 8 yr and 24-62 yr of age, respectively). In adrenocortical tumors, COUP-TFII immunoreactivity was marked in the nuclei of tumor cells of aldosteroma (H score, 134 +/- 15.9; P < 0.001 vs. Cushing's adenoma and P < 0.05 vs. nonfunctioning adenoma and carcinoma), modest in nonfunctioning adenoma (82.7 +/- 19.8) and adrenocortical carcinoma (79.6 +/- 56.3), and low in Cushing's adenoma (38.2 +/- 24.5). Results from immunoblotting performed in seven cases of adenomas were consistent with those of immunohistochemistry. In the attached nonneoplastic adrenal cortex of the adenomas, immunoreactivity for COUP-TFII was markedly increased compared to that in nonpathological adrenal cortex in adults and was especially marked in the zona glomerulosa in the attached adrenal of aldosteroma (P < 0.001) and the zona fasciculata in that of Cushing's adenoma (P < 0.05). COUP-TFII immunoreactivity was universally detected in stromal cells of the adrenal glands. These results suggest that COUP-TFII plays an important role in the regulation of steroidogenesis in human adrenal cortex and its disorders.

Adrenomedullin in cultured human retinal pigment epithelial cells.

PURPOSE: To determine whether adrenomedullin (ADM), a vasorelaxant peptide is produced and secreted by human retinal pigment epithelial (RPE) cells, whether ADM expression is regulated by inflammatory cytokines and a growth factor, and whether ADM has proliferative effects on these cells. METHODS: Production and secretion of ADM by cultured human RPE cells were examined by Northern blot analysis and radioimmunoassay. Regulation of the ADM expression by basic fibroblast growth factor, interferon (IFN)-gamma, tumor necrosis factor-alpha, interleukin (IL)1beta, or all-trans-retinoic acid was studied. In addition, proliferative effects of ADM on human RPE cells were examined by modified 3-(4,5-dimetylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: ADM mRNA was expressed constitutively in all three human RPE cell lines (F-0202, D407, and ARPE-19) examined. Immunoreactive ADM was detected in the cultured media by radioimmunoassay. Sephadex G-50 column chromatography of the cultured medium showed a single peak eluting in the position of ADM-(1-52). Treatment with IFN-gamma or IL-beta increased ADM mRNA levels and immunoreactive-ADM levels in the medium in dose- and time-dependent manners in ARPE-19 cells. Exogenously added ADM increased the number of F-0202 cells and ARPE-19 cells, and the treatment with ADM antibody or ADM-(22-52) (an ADM antagonist) decreased it. CONCLUSIONS: Human RPE cells produced and secreted ADM. IFN-gamma and IL-1beta induced ADM expression in ARPE-19 cells. Furthermore, ADM stimulated proliferation of RPE cells. These results raise the possibility that ADM is related to the pathophysiology of some inflammatory and proliferative ocular diseases.

Transcriptional control of adrenomedullin induction by phorbol ester in human monocytic leukemia cells.

Adrenomedullin is a potent vasodilator peptide that was originally identified from human pheochromocytoma. In this study, we investigated the induction of adrenomedullin gene expression in THP-1 acute monocytic leukemia cells during differentiation into macrophage-like cells by 12-O-tetradecanoylphorbol-13-acetate (TPA), and identified a cis-regulatory region of the human adrenomedullin gene responsible for TPA-induced adrenomedullin expression. Upon treatment with TPA (100 ng x mL(-1)) for 24 h, immunoreactive adrenomedullin concentrations in the culture medium and adrenomedullin mRNA levels were increased more than 10-fold, concomitant with the differentiation of THP-1 cells into macrophage-like cells. Actinomycin D abolished the TPA-induced adrenomedullin expression, indicating that the induction of ADM gene expression by TPA was regulated at the transcriptional level. Transient transfection assay revealed that a cis-acting region (positions -70 to -30) of human adrenomedullin gene was necessary for TPA-induced reporter gene expression. This region contains multiple copies of activator protein 2 (AP-2) binding sites, which are bound by purified AP-2 protein, as judged by electrophoretic mobility shift assay. The binding activity to this region was undetectable in nuclear extracts prepared from untreated THP-1 cells, but was increased in extracts prepared from TPA-treated cells. The protein binding was abolished by unlabeled oligonucleotides containing the AP-2 consensus sequence. These results indicate that the region (-70 to -30) of the human ADM gene containing multiple AP-2 binding sites is responsible for TPA-induced adrenomedullin expression in THP-1 cells.

Orexin-A in the human brain and tumor tissues of ganglioneuroblastoma and neuroblastoma.

Regional distribution of orexin-A-like immunoreactivity in the human brain and pituitary, and the presence of orexin-A-like immunoreactivity in the tumor tissues of pheochromocytomas, ganglioneuroblastomas and neuroblastomas were studied by radioimmunoassay. Expression of orexin mRNA was studied by reverse transcriptase polymerase chain reaction (PCR) method. Orexin-A-like immunoreactivity was detected in every region of human brain, but not in the pituitary. The highest concentration of orexin-A-like immunoreactivity in the human brain was found in hypothalamus (17.8 +/- 4.3 pmol/g wet weight, mean +/- SEM, n = 7), followed by thalamus, medulla oblongata, and pons. Orexin-A-like immunoreactivity was detected in the tumor tissues of ganglioneuroblastoma and neuroblastoma, but not in the tumor tissues of pheochromocytoma. Reverse phase high performance liquid chromatographic analyses of the orexin-A-like immunoreactivity in the human brain extracts and neuroblastoma extracts showed a single immunoreactive peak, which was eluted in an identical position to synthetic human orexin-A. Orexin mRNA was expressed in the hypothalamus and in the tumor tissues of ganglioneuroblastoma and neuroblastoma. These findings suggest that orexin-A is produced in the hypothalamus and transported to various brain regions via axons. In addition, this study has shown for the first time the production of orexin-A by ganglioneuroblastomas and neuroblastomas.

Binding sites for melanin-concentrating hormone in the human brain.

Binding sites for melanin-concentrating hormone (MCH) in human brain were investigated and characterized by radioligand binding. Specific binding sites for MCH were present in every region of human brain (cerebral cortex, cerebellum, thalamus, hypothalamus, pons, and medulla oblongata) obtained at autopsy. alpha-Melanocyte stimulating hormone or ACTH was a poor inhibitor of (125)I-MCH binding (IC(50) 1 microM) compared with MCH (IC(50) = 0.3 +/- 0.07 nM, mean +/- SEM, n = 3). Scatchard plots of (125)I-MCH binding in human brain (thalamus) gave a dissociation constant of 0.2 +/- 0.06 nM and maximal binding of 5.8 +/- 0.3 fmol/mg protein (n = 3). These findings suggest that specific MCH binding sites that differ from the melanocortin receptors exist in human brain.

Production and secretion of two vasoactive peptides, adrenomedullin and endothelin-1, by cultured human adrenocortical carcinoma cells.

The production and secretion of peptides by adrenocortical tumors have not been well studied. We therefore studied the production and secretion of two vasoactive peptides, adrenomedullin and endothelin-1 in SW-13 human adrenocortical carcinoma cells by radioimmunoassay and Northern blot analysis. Both immunoreactive-adrenomedullin and immunoreactive-endothelin were detected in the culture medium of SW-13 cells (27.7 +/- 1.6 fmol/10 (5) cells/24 h and 11.0 +/- 0.8 fmol/10 (5) cells/24 h, respectively, mean +/- SEM, n = 6). Northern blot analysis showed the expression of adrenomedullin mRNA and endothelin-1 mRNA in SW-13 cells. On the other hand, no significant amount of calcitonin gene-related peptide, corticotropin-releasing hormone, neuropeptide Y, or urocortin was secreted by SW-13 cells. Treatment with ACTH (10(-9)-10(-7) mol/l), angiotensin II (10(-9)-10(-7) mol/l), or dexamethasone (10(-8)-10(-6) mol/l) for 24 h had no significant effects on immunoreactive-adrenomedullin levels and immunoreactive-endothelin levels in the culture medium of SW-13. Treatment with tumor necrosis factor (TNF)-alpha (20 ng/ml) increased significantly both immunoreactive-adrenomedullin levels and immunoreactive-endothelin levels in the culture medium. Interferon-gamma (100 U/ml) increased the immunoreactive-endothelin levels, but not immunoreactive-adrenomedullin levels, whereas interleukin-1 (IL-1)beta (10 ng/ml) increased immunoreactive-adrenomedullin levels, but not immunoreactive-endothelin levels. These findings indicate that SW-13 human adrenocortical carcinoma cells produce and secrete two vasoactive peptides, adrenomedullin, and endothelin-1 and that the secretion of these two peptides is modulated differently by cytokines.