Toraichi Mouri

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.

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.

Circulating plasma kinin in patients with bronchial asthma

Bei 33 Patienten mit Asthma bronchiale von verschiedenem Erkrankungsgrade wurde der Kiningehalt im zirkulierenden Blut bestimmt. Erhöhte Blutkininwerte wurden in den Patienten gefunden, und zwar im allgemeinen mit der Schwere der Krankheit korrelierbar. Daraus folgt, dass das «Kinin» aetiologisch mit Asthma bronchiale verknüpft ist.

Colocalization of Corticotropin-Releasing Factor and Vasopressin in the Paraventricular Nucleus of the Human Hypothalamus

The anatomical relationship between corticotropin-releasing factor (CRF)-containing cells and arginine vasopressin (AVP)-containing cells in the human hypothalamus was investigated by immunocytochemistry. In the paraventricular nucleus of the hypothalamus (PVH), CRF-like immunoreactivity (CRF-LI) was present exclusively in parvocellular cells, while AVP-like immunoreactivity (AVP-LI) was present in both parvocellular and magnocellular cells. No CRF-immunoreactive neurons were observed in the supraoptic nucleus. All CRF-immunoreactive parvocellular cells in the PVH were also AVP immunoreactive. We confirmed the presence of AVP in the CRF-immunoreactive cells by using two kinds of anti-AVP antisera, one of which recognized the side chain of AVP while the other recognized the ring structure of AVP. Colocalization of CRF-LI and AVP-LI was observed not only in the same perikarya but also in the same nerve fibers of parvocellular cells. The present results raise the possibility that AVP and CRF may be secreted together into the human portal circulation.

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.

Detection of immunoreactive endothelin in plasma of hemodialysis patients

Two types of radioimmunoassay (RIA) methods for measuring endothelin (ET) in human plasma were developed. One was an extraction procedure using a Sep‐Pak C18 cartridge, the other being a direct method. By the extraction method, plasma ET levels were lower than the detectable limit (7 pg/ml) in normal subjects and elevated in hemodialysis patients. The absolute values obtained via the direct method were 20‐times higher than those from extraction. Gel‐filtration experiments revealed that this discrepancy was mainly due to immunoreactive (IR‐) endothelin‐like substances of high molecular mass near 11.6 kDa (large IR‐ET). Extraction of the peptide by the C18 cartridge could eliminate interference by large IR‐ET and is important in the accurate measurement of ET concentrations in plasma.

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.

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.