Potentiation of peptide histidine methionine (PHM) on corticotropin-releasing hormone (CRH) induced ACTH secretion in humans.

The effects of rat corticotropin-releasing hormone (CRH) and of synthetic peptide histidine methionine (PHM) on ACTH release were studied in vivo in humans. PHM alone did not alter the basal plasma ACTH or cortisol levels, but it strongly potentiated CRH-induced ACTH secretion. These results, together with our previous observation that PHM immunoreactivity is present in human hypothalamus and pituitary stalk, suggest that PHM may play a physiological role in the regulation of ACTH secretion in humans.

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.

Peptide histidine methionine may be a prolactin-releasing hormone in humans.

Peptide histidine isoleucine (PHI) was initially isolated from the porcine gastrointestinal tract and may be present in the brain. It has been suggested that PHI may be PRL-releasing hormone (PRH) because of its potent PRL-releasing activity and its existence in hypophysial portal plasma in rats. Vasoactive intestinal peptide and PHI are coded by the same gene, and human PHI has a C-terminal methionine instead of isoleucine [peptide histidine methionine (PHM)]. To investigate the possibility that PHM is a physiological PRH in humans, we measured the immunoreactive PHM concentration in human hypothalamic tissue and cerebrospinal fluid (CSF) using a specific RIA. We also examined in vivo the PRH activity of synthetic PHM. The human hypothalamus contained 19.3 +/- 6.2 (+/- SD; n = 5) pmol/hypothalamus, very similar to the content of GHRH or CRH. Immunoreactive PHM was also present in CSF; its levels in CSF were significantly lower in patients with prolactinomas than in control subjects. The CSF PHM levels in such patients increased after correction of hyperprolactinemia by long term bromocriptine therapy. The CSF PHM levels also were low in pregnant women. There was a significant negative correlation between plasma PRL and CSF PHM levels in all of these subjects. Gel filtration profiles of CSF extracts from normal subjects revealed two peaks of immunoreactive PHM: a high mol wt peak and one at the elution position of synthetic PHM. This profile resembled that of hyppothalamic extract. In contrast, only high mol wt material was detected in CSF from hyperprolactinemic subjects. Intravenous administration of synthetic PHM elicited a significant increase in plasma PRL in normal subjects; the responses to PHM were higher in women than in men. The presence of large amounts of immunoreactive PHM in the human hypothalamus suggests that PHM may participate in the regulation of anterior pituitary hormone secretion. Its specific PRL-releasing activity in vivo and the low CSF PHM levels of hyperprolactinemic subjects suggest that PHM may be a physiological PRH in humans.

Immunoreactive corticotropin-releasing hormone in human plasma during pregnancy, labor, and delivery.

We previously reported that immunoreactive corticotropin-releasing hormone (CRH) is present in human placenta and third trimester maternal plasma, and that such material is very similar to rat CRH and the predicted structure of human CRH. We suggested that maternal plasma immunoreactive CRH may be of placental origin. To further investigate this possibility, we measured plasma immunoreactive CRH in women during pregnancy, labor, and delivery and 1 and 2 h postpartum, and in nonpregnant women. Umbilical cord plasma and placental CRH concentrations were also measured. In the first trimester of pregnancy, the mean maternal plasma level was 5.9 +/- 1.0 pg (+/- SEM)/ml (n = 24), not significantly different from that in 10 nonpregnant women (5.8 +/- 0.8 pg/ml). Plasma CRH concentrations progressively increased during pregnancy (second trimester, 35.4 +/- 5.9 pg/ml (n = 39); early third trimester (28-34 weeks), 263 +/- 41 pg/ml (n = 14); late third trimester (35-40 weeks), 800 +/- 163 pg/ml (n = 20)]. There was a significant correlation between maternal plasma CRH levels and weeks of pregnancy. Plasma CRH concentrations were further elevated (2215 +/- 329 pg/ml; n = 9). During early labor, peaked at delivery (4409 +/- 591 pg/ml; n = 28), and declined rapidly after delivery [1 h postpartum, 1042 +/- (353 pg/ml (n = 13); 2 h postpartum, 346 +/- 91 pg/ml (n = 13)]. There was a significant correlation (r = 0.562; P less than 0.01) between matched maternal plasma and placental CRH concentrations. The mean umbilical cord plasma CRH level (50.6 +/- 6.1 pg/ml; n = 28) was much lower than that in the mother at the time of delivery. Umbilical venous plasma CRH levels were significantly greater than those in simultaneously obtained umbilical arterial plasma (70.8 +/- 11.3 and 41.8 +/- 4.9 pg/ml, respectively; n = 11). There was a significant correlation (r = 0.384; P less than 0.05) between maternal and fetal CRH concentrations. Gel filtration of plasma obtained from women during the third trimester, at delivery, and early postpartum and placental extracts revealed two major peaks of immunoreactive CRH: a high mol wt peak and one at the elution position of rat CRH. In contrast, only rat CRH-sized material was detected in plasma from nonpregnant women and umbilical cord plasma. Maternal plasma immunoreactive CRH-sized material stimulated ACTH release from anterior pituitary tissue in a dose-dependent manner and was equipotent with rat CRH.(ABSTRACT TRUNCATED AT 400 WORDS)

Altered circadian blood pressure rhythm in patients with Cushing's syndrome.

The circadian blood pressure rhythm was compared in patients with Cushing's syndrome, essential hypertension, and primary aldosteronism. In patients with essential hypertension or primary aldosteronism, a clear nocturnal fall in systolic and diastolic blood pressure and heart rate was observed. This fall was seen in untreated subjects as well as in patients receiving combined treatment with a calcium antagonist, diuretic, converting enzyme inhibitor, alpha-blocker and beta-blocker, or sympatholytic drug. In these groups, there was a positive correlation between heart rate and systolic or diastolic blood pressure. On the other hand, in patients with Cushing's syndrome, there was no nocturnal fall in blood pressure but in some patients a rise was observed. In all patients there was a nocturnal fall in heart rate. Thus, there was no significant correlation between heart rate and blood pressure in these patients. Exogenous glucocorticoid eliminated the normal nocturnal fall of blood pressure in patients with chronic glomerulonephritis or systemic lupus erythematosus. These results suggest that the changed circadian blood pressure pattern in patients with Cushing's syndrome is not due to antihypertensive treatment or to the mineralocorticoid excess accompanying this disease, but it is attributable to excess glucocorticoid or the associated disturbance in the adrenocorticotropic hormone-glucocorticoid system (or both). This conclusion also implies that the normal circadian rhythm of blood pressure may be regulated at least in part by the adrenocorticotropic hormone-glucocorticoid system.

Adrenomedullin in human brain, adrenal glands and tumor tissues of pheochromocytoma, ganglioneuroblastoma and neuroblastoma.

Adrenomedullin is a potent vasodilator peptide that was isolated from human pheochromocytoma. But the presence of adrenomedullin in the brain has not been clarified. We studied the presence of adrenomedullin in the human brain obtained at autopsy from 6 subjects by radioimmunoassay, as well as in the human adrenal glands and tumor tissues of pheochromocytoma, ganglioneuroblastoma and neuroblastoma. Immunoreactive adrenomedullin was detected in every region of human brain examined (0.26-1.4 pmol/g wet weight) with the highest concentrations found in thalamus (1.40 +/- 0.39 pmol/g wet weight, mean +/- SEM) and hypothalamus (1.28 +/- 0.48 pmol/g wet weight). Reverse phase high performance liquid chromatography showed that the immunoreactive adrenomedullin in the human brain was eluted in the position of synthetic human adrenomedullin 1-52. High concentrations of immunoreactive adrenomedullin were found in human adrenal glands (12.6 +/- 1.0 pmol/g wet weight, n = 7), pheochromocytoma (4.5 +/- 1.5 pmol/g wet weight, n = 11), ganglioneuroblastoma (2.0 +/- 1.3 pmol/g wet weight, n = 4) and neuroblastoma (0.55 +/- 0.21 pmol/g wet weight, n = 3). The present study has shown that adrenomedullin is present in the human brain in high concentrations, suggesting that adrenomedullin acts as a neurotransmitter, neuromodulator or neurohormone in man.

Distribution, plasma concentration, and in vivo prolactin-releasing activity of peptide histidine methionine in humans.

The distribution of immunoreactive peptide histidine methionine (PHM) in human tissues and its plasma concentrations were examined using a specific RIA and gel filtration chromatography. The effects of synthetic PHM on anterior pituitary hormone secretion also were studied. Immunoreactive PHM was found in all tissues studied; high concentrations were found in the gastrointestinal tract, lung, and parotid gland. Subsequent but smaller amounts of PHM were found in the hypothalamus, pituitary stalk, olfactory lobe, and cerebral cortex. The distribution of immunoreactive PHM in human tissues was very similar to that of vasoactive intestinal polypeptide (VIP), and PHM and VIP were in equimolar concentrations. Immunoreactive PHM was also detectable in plasma of normal subjects, and similar plasma concentrations were found in patients with prolactinomas. Molecular sieve chromatography of extracts of nonneural tissues and plasma extracts revealed only one peak, eluting in the position of synthetic PHM. Two peaks of immunoreactive PHM were found in brain tissue; one coeluted with synthetic PHM, and the other eluted in the high mol wt region. Bolus injections of synthetic PHM significantly increased plasma PRL levels in a dose-dependent manner. However, PHM did not alter plasma GH, TSH, ACTH, LH, or FSH levels. These results indicate that PHM is distributed widely in human tissues, and posttranslational processing of the VIP-PHM precursor molecule may be different in different tissues. The finding of equimolar distributions of PHM and VIP is consistent with the notion that these two peptides are synthesized from a common precursor. The presence of immunoreactive PHM in human hypothalamic and pituitary stalk tissue and its specific in vivo PRL-releasing activity suggest that PHM may play an important role in the regulation of PRL secretion.

Immunoreactive corticotropin-releasing hormone present in human plasma may be derived from both hypothalamic and extrahypothalamic sources.

Immunoreactive CRH concentrations were determined in human plasma using an immunoaffinity chromatographic extraction procedure and sensitive RIA. Immunoreactive CRH was detectable in the plasma of all normal subjects (mean +/- SD, 6.2 +/- 2.4 pg/mL; n = 15). Basal (0800-1000 h) plasma immunoreactive CRH levels were significantly lower in patients with Cushing's syndrome due to adrenal (2.8 +/- 1.1 pg/mL; n = 4) or pituitary adenomas (2.9 +/- 0.8 pg/mL; n = 5), in patients with hypothalamic hypopituitarism (3.2 +/- 0.9 pg/mL; n = 5), and in glucocorticoid-treated patients (3.9 +/- 1.9 pg/mL, n = 8). Basal plasma CRH levels were also low in patients with acromegaly (2.8 +/- 0.8 pg/mL; n = 14) and insulin-treated diabetic patients whose pituitary-adrenal function was normal (3.6 +/- 1.0 pg/mL; n = 12). In normal subjects plasma CRH levels increased after insulin-induced hypoglycemia; this response was abolished by the prior administration of dexamethasone. In contrast, basal plasma CRH levels were not affected by prior administration of metyrapone or dexamethasone. No evidence for diurnal variation in plasma immunoreactive CRH was found in normal subjects. In addition, in normal subjects oral glucose administration elicited a significant increase in plasma CRH (basal, 7.3 +/- 0.9 pg/mL; peak 30 min after glucose, 16.7 +/- 5.8 pg/mL; n = 5; P less than 0.05) without concomitant changes in ACTH. Gel filtration of extracts of pooled plasma from normal subjects revealed a major component of immunoreactive CRH in the position of synthetic rat CRH. Immunoreactive CRH-sized material had the same retention time as authentic rat CRH in a reverse phase high pressure liquid chromatography system. The content of immunoreactive CRH in human placenta, pancreas, and adrenal gland was much larger than that in hypothalamus. These findings suggest that immunoreactive CRH is present in peripheral plasma; the increase in plasma immunoreactive CRH after insulin-induced hypoglycemia may reflect stimulation of hypothalamic CRH release; the increase in plasma immunoreactive CRH after glucose administration may reflect extrahypothalamic CRH release; and the lack of diurnal variation in plasma immunoreactive CRH together with the lack of suppression of CRH by dexamethasone suggest that basal plasma CRH is of extrahypothalamic origin.

Natriuretic peptides in the human kidney.

We studied the presence of three natriuretic peptides--atrial natriuretic peptide (ANP), human brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP)--in the human kidney by radioimmunoassay and immunocytochemistry. Immunoreactive ANP, immunoreactive human BNP, and immunoreactive CNP concentrations in six kidneys were 0.12 +/- 0.07 (mean +/- SD), 0.23 +/- 0.08, and 0.37 +/- 0.07 pmol/g wet wt, respectively. Sephadex G-50 superfine column chromatography and reversed-phase high-performance liquid chromatography of kidney extracts revealed a broad peak of immunoreactive ANP comigrating with ANP-28 and urodilatin. Renal immunoreactive human BNP consisted of three components; the major component comigrated with human BNP-32. Renal immunoreactive CNP consisted of at least two components; the major component comigrated with CNP-22, and the minor component eluted in a position similar to that of authentic human CNP-53. Immunocytochemistry showed that immunoreactive human BNP was colocalized with immunoreactive ANP in the segments of distal tubules, whereas immunoreactive CNP was found predominantly in the proximal tubules. These findings indicate that these three natriuretic peptides are present in the human kidney and raise the possibility that they form a renal natriuretic peptide system that participates in the local regulation of sodium and water transport and renal circulation in the human kidney.

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.

Increased plasma immunoreactive neuropeptide Y concentrations in phaeochromocytoma and chronic renal failure.

To investigate the clinical usefulness of radio-immunoassay of neuropeptide Y (NPY), we measured plasma immunoreactive neuropeptide Y (IR-NPY) concentrations in normal subjects (n = 21), essential hypertensive patients (n = 33), patients with phaeochromocytoma (n = 7), patients with chronic renal disease with serum creatinine levels of less than 1.9 mg/dl (n = 5) and patients with chronic renal failure whose serum creatinine levels were greater than or equal to 1.9 mg/dl (n = 18, eight without haemodialysis and 10 undergoing maintenance haemodialysis), by radio-immunoassay. Plasma IR-NPY concentrations in patients with phaeochromocytoma (577 +/- 256 pg/ml, mean +/- s.d.) were significantly higher (P less than 0.001) than those in normal subjects (151 +/- 28 pg/ml), essential hypertensive patients (177 +/- 49 pg/ml) and patients with chronic renal disease with serum creatinine levels less than 1.9 mg/dl (198 +/- 71 pg/ml). Plasma IR-NPY concentrations in patients with chronic renal failure (without haemodialysis: 330 +/- 63 pg/ml; undergoing maintenance haemodialysis: 374 +/- 80 pg/ml) were also high. These results suggest that NPY is useful as one of the tumour markers of phaeochromocytomas. However, this study revealed that patients with chronic renal failure, without phaeochromocytoma also have increased plasma IR-NPY concentrations.

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.

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.

Cerebellin and cerebellin mRNA in the human brain, adrenal glands and the tumour tissues of adrenal tumour, ganglioneuroblastoma and neuroblastoma.

The expression of cerebellin and cerebellin mRNA was studied by radioimmunoassay and Northern blot analysis in the human brain, adrenal gland and the tumour tissues of adrenal tumour, ganglioneuroblastoma and neuroblastoma. Immunoreactive cerebellin was detected in every region of brain studied, with the highest concentrations found in the hemisphere of the cerebellum (424.2 +/- 12.6 pmol/g wet weight, n = 6, mean +/- S.E.M.) and the vermis of the cerebellum (256.8 +/- 30.5 pmol/g wet weight). Immunoreactive cerebellin was also detected in the pituitary (8.2 +/- 1.8 pmol/g wet weight), the spinal cord (3.3 +/- 0.3 pmol/g wet weight) and the normal parts of adrenal glands (2.98 +/- 0.37 pmol/g wet weight, n = 9) and some tumour tissues, such as phaeochromocytomas, cortisol-producing adrenocortical adenomas, ganglioneuroblastomas and neuroblastomas. Northern blot analysis showed that cerebellin mRNA was highly expressed in the hemisphere and vermis of the cerebellum. Cerebellin mRNA was also expressed in other regions of the brain and the tumour tissues of phaeochromocytoma, cortisol-producing adrenocortical adenoma, ganglioneuroblastoma and neuroblastoma. Immunocytochemistry of the normal adrenal gland showed that immunoreactive cerebellin was localized in the adrenal medulla. The present study has shown the expression of cerebellin and cerebellin mRNA, not only in the cerebellum but also in other regions of the brain and some tumours, such as cortisol-producing adrenocortical adenoma, phaeochromocytoma and neuroblastoma. These findings suggest possible pathophysiological roles of cerebellin peptides, not only in the cerebellum, but also in the extra-cerebellar tissues.

Expression of adrenomedullin and adrenomedullin mRNA in ectopic ACTH-secreting tumors.

OBJECTIVE: To study the expression of adrenomedullin, a potent vasodilator peptide originally isolated from a pheochromocytoma, in ectopic ACTH-secreting tumors. METHODS: Tumor tissue concentrations of adrenomedullin, calcitonin gene-related peptide, neuropeptide Y, endothelin-1, corticotropin-releasing hormone and ACTH were measured in three ectopic ACTH-secreting tumors by RIA. The expression of adrenomedullin mRNA was examined by northern blot analysis of tissue from one of the tumors. RESULTS: Immunoreactive adrenomedullin was detected in tumor tissues of three ectopic ACTH-secreting tumors (0.60-18.5 pmol/g wet weight). Calcitonin gene-related peptide, neuropeptide Y, endothelin-1 and corticotropin-releasing hormone were also detected in the tumor tissues. The tumor tissue concentrations of immunoreactive adrenomedullin were comparable to those of these four peptides, but much lower than those of ACTH. Northern blot analysis showed the expression of adrenomedullin mRNA in one tumor from which sufficient tissue was available for such study. The plasma concentration of immunoreactive adrenomedullin was increased in one patient (41.3 pmol/l, control 13.5 +/- 3.6 pmol/l, mean +/- S.D., n = 12). CONCLUSIONS: These results suggest that adrenomedullin is produced by ectopic ACTH-secreting tumors, together with other neuropeptides, and raise the possibility that adrenomedullin is related to the pathophysiology of these tumors.

Immunoreactive brain natriuretic peptide in human adrenal glands and adrenal tumors.

The presence of brain natriuretic peptide (BNP) in tissues of human adrenal glands and adrenal tumors was investigated by radioimmunoassay. Immunoreactive BNP concentrations were 0.203 +/- 0.061 pmol/g wet tissue (mean +/- SEM) in normal parts of adrenal glands (cortex and medulla, N = 8), 0.205 +/- 0.037 pmol/g wet tissue in pheochromocytomas (N = 8), 0.230 +/- 0.062 pmol/g wet tissue in aldosteronomas (N = 11) and 0.180 +/- 0.054 pmol/g wet tissue in adrenocortical adenomas with Cushing's syndrome (N = 4). Sephadex G-50 superfine column chromatography and reverse-phase high-performance liquid chromatography showed that most (> 70%) of the immunoreactive BNP in the normal part of adrenal glands was eluted in the position of human BNP-32. Sephadex G-50 superfine column chromatography of immunoreactive BNP in the pheochromocytoma and aldosteronoma showed four peaks: one in the position of gamma-BNP, one in the position of BNP-32, one between gamma-BNP and BNP-32 and one in the smaller molecular weight region. The present study has shown that immunoreactive BNP is present both in normal human adrenal glands and in adrenal tumors. Multiple molecular forms of BNP were found to be present in the tumor tissues of pheochromocytoma and aldosteronoma.

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.

Induction of adrenomedullin by hypoxia in cultured human coronary artery endothelial cells.

To explore the role of adrenomedullin (ADM) in pathophysiology of ischemic heart disease, we investigated the effects of hypoxia on the production and secretion of ADM in cultured human coronary artery endothelial cells. Treatment with hypoxia (5% CO2/94% N2/1% O2) for 6 and 12 h increased expression levels of ADM mRNA 2.2-fold and fivefold compared with the normoxia control, respectively. The levels of immunoreactive ADM in the media were increased by 12-h hypoxia about fivefold compared with the control (39.0+/-1.1 fmol/10(5) cells per 12 h under hypoxia and 7.9+/-0.4 fmol/10(5) cells per 12 h under normoxia; P<0.01, n = 4, mean +/- SEM). Reverse-phase high-performance liquid chromatography of the extracts of culture media under normoxia and hypoxia showed one major peak eluting in the position of human ADM standard. The production and secretion of ADM were increased in cultured human coronary artery endothelial cells under hypoxia. ADM may therefore play an important pathophysiological role in ischemic heart disease.

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.

Increases of neuropeptide Y-like immunoreactivity in plasma during insulin-induced hypoglycemia in man.

Neuropeptide Y-like immunoreactivity (NPY-LI) in plasma during insulin-induced hypoglycemia was measured in 4 healthy male volunteers. Plasma NPY-LI increased from 167 +/- 11 pg/ml to 247 +/- 25 pg/ml 30 min after the administration of insulin (0.1 U/kg body weight IV), reached the maximum (296 +/- 6 pg/ml) 45 min after the insulin, and then decreased. These results suggest that NPY is released into the systemic circulation during insulin-induced hypoglycemia in man.