Masahiko Sone

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.

Increased plasma urotensin II levels in patients with diabetes mellitus

Urotensin II (UII) is the most potent vasoconstrictor peptide, whereas it acts as a vasodilator on some arteries. We studied plasma levels of UII in diabetic patients with normal serum creatinine levels (<90 micromol/l) and the expression of UII and its receptor in cultured human vascular endothelial cells. Plasma UII levels were significantly elevated by 1.8-fold in diabetic patients without proteinuria (7.8+/-0.6 fmol/ml; P <0.0001) and 1.7-fold in those with overt proteinuria (7.3+/-0.9 fmol/ml; P =0.0018) when compared with healthy subjects (4.4+/-0.2 fmol/ml). No significant correlation was obtained between plasma UII levels and fasting blood sugar (P =0.631 and P =0.853 in non-proteinuric and proteinuric diabetic patients respectively), glycated haemoglobin levels (P =0.376 and P =0.888 respectively) or serum creatinine levels (P =0.301 and P =0.568 respectively). Reverse transcriptase-PCR analysis showed the expression of mRNAs encoding UII precursor and UII-receptor precursors in cultured human coronary artery endothelial cells and umbilical vein endothelial cells, suggesting that vascular endothelial cells are one of the sources of UII in blood. These findings suggest that elevation of plasma UII levels may be an important background factor in diabetic cardiovascular and organ complications in diabetic subjects without renal failure.

Immunocytochemical localization of adrenomedullin-like immunoreactivity in the human hypothalamus and the adrenal gland

Adrenomedullin is a potent vasodilator peptide that was isolated from pheochromocytoma. Localization of adrenomedullin-like immunoreactivity was studied by immunocytochemistry in the human hypothalamus and adrenal gland. Adrenomedullin-immunoreactive cell bodies were found in the paraventricular, supraoptic and infundibular nuclei of the hypothalamus. Both magnocellular and parvocellular cells of the paraventricular nucleus were positively immunostained. Adrenomedullin-like immunoreactivity was localized in the adrenal medulla. No positive immunostaining was observed in the vascular endothelium, vascular smooth muscle cell or adrenal cortex. The preabsorption of the antiserum with synthetic human adrenomedullin (1-52) abolished the immunostaining. These findings indicate that adrenomedullin-like immunoreactivity is localized in the paraventricular, supraoptic and infundibular nuclei as well as in the adrenal medulla, and suggest that adrenomedullin acts as a neurotransmitter, a neuromodulator or a neurohormone in the human hypothalamus.

Human brain natriuretic peptide-like immunoreactivity in human brain

The presence of immunoreactive human brain natriuretic peptide in the human brain was studied with a specific radioimmunoassay for human brain natriuretic peptide-32. This assay showed no significant cross-reaction with human alpha atrial natriuretic peptide, porcine brain natriuretic peptide or rat brain natriuretic peptide. Immunoreactive human brain natriuretic peptide was found in all 5 regions of human brain examined (cerebral cortex, thalamus, cerebellum, pons and hypothalamus) (0.6-6.7 pmol/g wet weight, n = 3). These values were comparable to the concentrations of immunoreactive alpha atrial natriuretic peptide in human brain (0.5-10.1 pmol/g wet weight). However, Sephadex G-50 column chromatography showed that the immunoreactive human brain natriuretic peptide in the human brain eluted earlier than synthetic human brain natriuretic peptide-32. These findings suggest that human brain natriuretic peptide is present in the human brain mainly as larger molecular weight forms.

Elevated plasma levels of immunoreactive urotensin II and its increased urinary excretion in patients with Type 2 diabetes mellitus: association with progress of diabetic nephropathy

Urotensin II (UII) is the most potent vasoconstrictor peptide ever identified. In order to clarify the pathophysiological role of UII in diabetes mellitus, we examined plasma immunoreactive UII levels and urinary excretion of immunoreactive UII in 10 control subjects and 48 patients with Type 2 diabetes mellitus. The patients were divided into three groups according to the renal function: Group I with Ccr > or = 70 ml/min, group II with 30 < or = Ccr <70 ml/min and group III with Ccr <30 ml/min. Plasma immunoreactive UII levels were elevated in the three diabetic groups compared with normal controls (P <0.05). Group III patients had significantly higher plasma immunoreactive UII levels (15.9 +/- 2.2 fmol/ml, mean +/- S.E.M., n=6) by approximately 1.6-fold than did group I (10.9 +/- 0.9 fmol/ml, n=17) and group II (10.8 +/- 0.8 fmol/ml, n=25) (P <0.05). Urinary excretion of immunoreactive UII was significantly increased in group III patients (52.4 +/- 14.8 pmol/day) by more than 1.8-fold compared with control subjects, groups I and II (P <0.005). Fractional excretion of immunoreactive UII significantly increased as renal function decreased. Presence of diabetic retinopathy or neuropathy had negligible effects on plasma immunoreactive UII levels and urinary immunoreactive UII excretion. Reverse phase HPLC analyses showed three immunoreactive peaks in normal plasma extracts and multiple immunoreactive peaks in normal urine extracts. Thus, Type 2 diabetes mellitus itself is a factor to elevate plasma immunoreactive UII levels, and accompanying renal failure is another independent factor for the increased plasma immunoreactive UII levels in Type 2 diabetic patients. Increased urinary immunoreactive UII excretion in Type 2 diabetic patients with advanced diabetic nephropathy may be due not only to the elevated plasma immunoreactive UII levels but also to increased UII production and/or decreased UII degradation in the diseased kidney.

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.

Regional distribution of urocortin-like immunoreactivity and expression of urocortin mRNA in the human brain.

Regional distribution of urocortin-like immunoreactivity (UCN-LI) in the human brain was studied by radioimmunoassay and was compared with that of corticotropin-releasing hormone (CRH). In addition, the expression of UCN mRNA was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) method. UCN-LI was detected in every region of brain examined, including hypothalamus, pons, cerebral cortex, and cerebellum. The concentrations of UCN-LI in the human brain were approximately 3 pmol/g wet weight in any brain region, and no marked regional difference was noted. On the other hand, the highest concentrations of CRH-LI were found in the frontal cortex, temporal cortex, and hypothalamus and the lowest in the pons. Reverse phase high-performance liquid chromatography of the UCN-LI in the human brain extract showed two immunoreactive peaks; one peak eluting earlier and one in the position of synthetic human UCN. RT-PCR showed that UCN mRNA was expressed in every region of brain examined. These findings indicated that UCN and UCN mRNA were widely expressed in the human brain.

PRODUCTION AND SECRETION OF ADRENOMEDULLIN FROM GLIAL CELL TUMORS AND ITS EFFECTS ON CAMP PRODUCTION

The expression of adrenomedullin (ADM) and its mRNA was studied in human glial cell tumors and cultured glioblastoma cell lines, T98G and A172. Northern blot analysis showed that ADM mRNA was expressed in all brain tumors examined (three anaplastic astrocytomas and two glioblastomas multiforme) and in the glioblastoma cell lines. Immunoreactive (IR-) ADM was detectable in these brain tumors by radioimmunoassay (0.31-2.0 pmol/g wet weight), except for one anaplastic astrocytoma. Reverse phase high performance liquid chromatography of the tumor extracts showed a single peak eluting in the position of ADM-(1-52). IR-ADM concentrations in the cultured media of T98G cells were 205.5 +/- 8.4 fmol/10(5) cells/24 h (mean +/- SEM, n = 5). Treatment of T98G cells with interferon gamma or interleukin 1 beta increased the expression levels of ADM mRNA and the IR-ADM concentrations in the cultured media, whereas tumor necrosis factor alpha decreased them in a dose-dependent manner. Treatment with synthetic ADM-(1-52) (10(-8) or 10(-7) mol/l) increased the cAMP concentrations in the cultured media of T98G cells. These findings suggest that ADM is secreted from glial cell tumors and is related to the pathophysiology of these tumors.

Expression of Adrenomedullin mRNA in the Human Brain and Pituitary

We have recently reported the presence of immunoreactive (IR) adrenomedullin (ADM) in the human brain. In the present study, the expression of ADM mRNA was studied by Northern blot analysis in the human brain and pituitary, and the presence of IR-ADM in the human pituitary was studied by radioimmunoassay. ADM mRNA was clearly detected in every region of the brain examined and in the pituitary. High concentrations of IR-ADM were present in the whole pituitary (16.7 +/- 2.0 pmol/g wet weight, mean +/- SEM, n = 4). Reverse phase high performance liquid chromatography of the pituitary showed a peak eluting in the position of human ADM(1-52). These findings suggest that ADM acts as a neuromodulator or a neurotransmitter in the brain, and as an autocrine factor, a paracrine factor, or a neurohormone in the pituitary.

Increased Secretion of Adrenomedullin from Cultured Human Astrocytes by Cytokines

Abstract: Adrenomedullin, originally discovered from pheochromocytoma, is a member of the calcitonin gene‐related peptide family. The production and secretion of adrenomedullin by cultured human astrocytes were studied by northern blot analysis and radioimmunoassay. Northern blot analysis showed the expression of adrenomedullin mRNA in cultured human astrocytes. Immunoreactive adrenomedullin concentrations in the culture medium were 29.6 ± 1.2 fmol/105 cells/24 h (mean ± SEM, n = 4). Treatment with interferon‐γ (100 U/ml), tumor necrosis factor‐α (1 and 10 ng/ml), or interleukin‐1β (1 and 10 ng/ml) for 24 h caused >20‐fold increases in immunoreactive adrenomedullin levels in the culture medium of human astrocytes. On the other hand, northern blot analysis showed only small increases (∼40%) in the adrenomedullin mRNA expression of human astrocytes with either 100 U/ml interferon‐γ or 10 ng/ml interleukin‐1β and no noticeable change with tumor necrosis factor‐α. Reverse phase HPLC of the medium extracts of human astrocytes treated with interferon‐γ, tumor necrosis factor‐α, or interleukin‐1β showed that most of immunoreactive adrenomedullin was eluted in the position of adrenomedullin‐(1‐52). On the other hand, immunoreactive adrenomedullin in the medium of human astrocytes without cytokine treatment was eluted earlier than the adrenomedullin standard, suggesting that this immunoreactive adrenomedullin represents adrenomedullin with some modifications or fragments of the adrenomedullin precursor. The present study has shown the production and secretion of adrenomedullin by human astrocytes and increased secretion of adrenomedullin by cytokines.