Makoto Ohneda

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.

Specific adrenomedullin binding sites in the human brain.

Binding sites for adrenomedullin in human brain were investigated and characterized by radioligand binding. Specific binding sites for adrenomedullin were present in every region of human brain (cerebral cortex, cerebellum, thalamus, hypothalamus, pons and medulla oblongata) obtained at autopsy. Despite the homology with calcitonin gene-related peptide (CGRP), CGRP was a poor inhibitor of [125I]adrenomedullin binding (IC50 > 1 microM) compared with adrenomedullin(1-52) (IC50 = 1.2 +/- 0.5 nM, mean +/- SEM, n = 3). Three adrenomedullin fragments, adrenomedullin(1-12), adrenomedullin(22-52), and adrenomedullin(13-52), were also poor inhibitors of the binding (IC50 = 0.3 microM), suggesting that the whole molecule of adrenomedullin(1-52) is required for binding to the receptor. Scatchard plots of [125I]adrenomedullin binding in human brain (cerebral cortex) gave a dissociation constant of 0.17 +/- 0.03 nM and maximal binding of 99.3 +/- 1.9 fmol/mg protein (n = 5). These findings suggest that specific adrenomedullin binding sites that differ from the CGRP receptors exist in human brain. This indicates a possible novel neurotransmitter/neuromodulator role for adrenomedullin in human brain.

Melanin-concentrating hormone in the human brain

The presence of human melanin-concentrating hormone (MCH) was studied in the human brain by radioimmunoassay and immunocytochemistry. Immunoreactive MCH concentrations in the human brain ranged from 0.07 to 19.7 pmol/g wet weight. High performance liquid chromatography of the hypothalamus showed a large immunoreactive peak in the position of human/rat MCH, which was eluted 9 min later than that of salmon MCH. Free-floating sections (40 microns) of the hypothalamus were immunostained. Positive MCH immunostaining was found in perifornical, tuberomammillary, and posterior nuclei. Numerous MCH-immunoreactive nerve fibers were observed throughout the hypothalamus. The presence of high concentrations of MCH in the human brain, in particular in the hypothalamus, suggests that MCH is a neurotransmitter, a neuromodulator, or a neurohormone in man.

C-type natriuretic peptide in the human central nervous system: Distribution and molecular form

The distribution and molecular form of C-type natriuretic peptide (CNP) in the human central nervous system were studied with a specific radioimmunoassay for CNP-22. Immunoreactive (IR-) CNP was detectable in all regions of the brain examined (cerebral cortex, thalamus, hypothalamus, pons, and cerebellum) (0.21-0.81 pmol/g wet tissue, n = 4). The highest concentration of IR-CNP was found in the spinal cord at 1.83 +/- 0.13 pmol/g wet tissue (mean +/- SD, n = 3). Reversed-phase high performance liquid chromatography revealed a major peak migrating at the position corresponding to synthetic human CNP-53 and minor peaks comigrating with synthetic CNP-22 and the methionine-oxidized form of CNP-22, respectively. These findings suggest that IR-CNP is widely present in the human central nervous system mainly in a high molecular weight form as the major component and in the molecular form of CNP-22 as the minor component.

Urinary immunoreactive brain natriuretic peptide in patients with renal disease

Urinary immunoreactive brain natriuretic peptide (BNP) was studied by radioimmunoassay in patients with renal disease. Urinary immunoreactive human BNP excretion measured in 11 normal subjects was 3.82 +/- 0.62 pmol/day (mean +/- SEM). Significantly increased 24-h urinary secretion of immunoreactive human BNP was noted in patients with chronic renal failure (11.07 +/- 1.73 pmol/day, n = 9, P < 0.05 to normal subjects). A significant correlation was noted between 24-h urinary excretion of immunoreactive human BNP and creatinine clearance in patients with various renal diseases (r = -0.43, P < 0.01, n = 45). Gel chromatography of the urine extracts obtained from normal subjects and patients with chronic renal failure showed multiple immunoreactive peaks; two eluting earlier, one in the position of human BNP-32 and others eluting later. Reverse-phase high-performance liquid chromatography of the urine extracts showed a peak in the position of human BNP-32 and a peak eluting earlier. These findings indicate that: (1) immunoreactive human BNP is present in human urine; (2) urinary immunoreactive human BNP consists of multiple components, i.e., human BNP-32 itself or a substance very similar to it, smaller molecular forms which are probably metabolic products of human BNP-32, and larger molecular forms; and (3) 24-h urinary excretion of immunoreactive human BNP is increased in patients with renal dysfunction.

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.

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.

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP)-like immunoreactivity in human hypothalamus: co-localization with arginine vasopressin

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel hypothalamic peptide consisting of 38 amino acids (PACAP1-38) with a potent stimulatory action on adenylate-cyclase in rat pituitary. The presence of PACAP-like immunoreactivity in human brain was studied by radioimmunoassay. Co-localization of PACAP with arginine vasopressin and oxytocin was investigated by immunocytochemistry in the human hypothalamus. Immunoreactive PACAP was detected in all regions of human brain (cortex, thalamus, hypothalamus, pons and hemisphere of cerebellum) with the highest levels found in the hypothalamus (8.5 +/- 1.9 pmol/g wet weight, n = 4, mean +/- S.E.M.). High performance liquid chromatography of the human hypothalamic extract showed that approximately 50% of the immunoreactive PACAP was eluted in the position of PACAP1-38. Immunocytochemical studies showed the presence of PACAP immunoreactive neurons in the paraventricular and supraoptic nuclei of human hypothalamus. PACAP co-localized with arginine vasopressin in magnocellular cells of these nuclei. These findings suggest that PACAP1-38 plays important physiological roles in the human hypothalamus.

Calcitonin gene-related peptide-like immunoreactivities in pheochromocytomas

Calcitonin gene-related peptide (CGRP) is reported to exist in high concentrations in plasma and tumor tissues of medullary thyroid carcinomas. CGRP-like immunoreactivity (LI) in tumor tissues of pheochromocytomas was investigated by radioimmunoassay. CGRP-LI in 9 pheochromocytomas ranged from 0.50 to 1240 ng/g wet tissue. Sephadex G-50 column chromatography revealed that most of CGRP-LI in tumor extracts was eluted in an identical position to synthetic human CGRP. Reverse-phase high pressure liquid chromatography revealed that CGRP-LI in tumor extracts was eluted in an identical position to synthetic human CGRP and in a more hydrophobic position. These results indicate that high concentrations of CGRP-LI also exist in tumor tissues of pheochromocytomas.