Tetsuo Tsuji

The arcuate nucleus as a primary site of satiety effect of leptin in rats

The obese (ob) gene encodes a fat cell-derived circulating satiety factor (leptin) that is involved in the regulation of energy homeostasis. In the present study, we examined effects of i.c.v. injection of recombinant human leptin on food intake and body weight gain in rats. We also studied effects of direct microinjections of leptin into the arcuate nucleus (Arc), ventromedial hypothalamus (VMH), and lateral hypothalamus (LH). A single i.c.v. injection of recombinant human leptin (0.25-2.0 micrograms/rat) reduced significantly and dose-dependently food intake and body weight gain in rats. Microinjections (0.125-0.5 microgram/site) into the bilateral Arc, VMH, and LH caused dose-related decreases in food intake and body weight gain as compared with vehicle-treated groups with a rank order of potency; Arc > VMH = LH. The present study provides the first direct evidence that the Arc is a primary site of satiety effect of leptin.

Adrenomedullin: a possible autocrine or paracrine inhibitor of hypertrophy of cardiomyocytes.

Adrenomedullin (AM), a potent vasodilator peptide, exists in the cardiac ventricle; however, the role of AM in the ventricular tissue remains unknown. In the present study, we investigated the production and secretion of AM in cultured neonatal rat cardiomyocytes, and we examined the effect of AM on de novo protein synthesis in these cells by measuring [14C]phenylalanine incorporation. The cardiomyocytes cultured with serum-free media secreted AM into the media in a time-dependent manner at the rate of 12.2+/-0.5 fmol/10(5) cells/48 hours (mean+/-SEM). Angiotensin II (1 micromol/L) or 10% fetal bovine serum significantly (P<.01) increased the AM secretion by 115% and 305%, respectively. In addition, Northern blot analysis of total RNA extracted from the myocytes disclosed the expression of prepro-AM mRNA of 1.6 kb. Synthetic AM at 1 micromol/L significantly reduced the 10(-6) mol/L angiotensin II- and 10% fetal bovine serum-stimulated [14C]phenylalanine incorporation into the cells, by 16% (P<.05) and 20% (P<.01), respectively. The inhibitory effect of AM on the angiotensin II-stimulated [14C]phenylalanine incorporation was abolished dose-dependently by a calcitonin gene-related peptide receptor antagonist, CGRP(8-37). Furthermore, blockade of the action of endogenous AM by either 10(-6) mol/L CGRP(8-37) or anti-AM monoclonal antibody significantly enhanced the basal and 10(-6) mol/L angiotensin II-stimulated [14C]phenylalanine incorporation. In summary, cultured neonatal rat cardiomyocytes produce and secrete AM, and the secreted AM inhibits the protein synthesis of these cells. Thus, AM may act on cardiomyocytes as an autocrine or a paracrine factor modulating the cardiac growth.

An autocrine or a paracrine role of adrenomedullin in modulating cardiac fibroblast growth.

Objective: The aim of the present study was to determine the role of adrenomedullin (AM) in cardiac fibroblasts. Methods: The production and secretion of AM were examined in cultured neonatal rat cardiac fibroblasts, and the effects of AM on proliferation and protein synthesis of these cells were assessed by [3H]thymidine and [3H]phenylalanine incorporation, respectively. Results: Cultured cardiac fibroblasts secreted AM into the medium time-dependently at a rate of 20.3±3.0 fmol/5×104 cells/48 h, mean±S.D. Northern blot analysis showed expression of preproAM mRNA of 1.6 kb in these cells. In addition, 10−6 mol/l of angiotensin II (Ang II) and endothelin-1 (ET-1) significantly increased the AM secretion by 55 and 48%, respectively. Synthetic AM significantly reduced 10−6 mol/l Ang II- or 10−7 mol/l ET-1-stimulated [3H]thymidine and [3H]phenylalanine incorporation in a dose-dependent manner, and these effects were attenuated by a calcitonin gene-related peptide (CGRP) type 1 receptor antagonist, CGRP(8-37). Synthetic AM also had a dose-dependent stimulatory effect on cAMP accumulation in these cells, which was significantly attenuated by CGRP(8-37). A cAMP analogue, 8-bromo-cAMP, mimicked the AM effects, inhibiting the Ang II-stimulated [3H]thymidine and [3H]phenylalanine incorporation. Blockage of the effect of endogenous AM by anti-AM monoclonal antibody not only significantly reduced the basal level of intracellular cAMP, but also enhanced the [3H]thymidine and [3H]phenylalanine incorporation into the cells. Conclusions: Cultured neonatal rat cardiac fibroblasts produce and secrete AM, and the secreted AM may inhibit proliferation and protein synthesis of these cells. AM may exert these inhibitory effects partly by elevating intracellular cAMP. It is suggested that AM has an important role in modulating the growth of cardiac fibroblasts in an autocrine or a paracrine manner.

A simple immunoradiometric assay for measuring the entire molecules of adrenomedullin in human plasma

We developed a one-step two-site immunoradiometric assay (IRMA) using two kinds of monoclonal antibodies, which enables us to directly measure the entire molecules of adrenomedullin (AM) (the sum of mature-type AM (abbreviated, m-AM) amidated at the C-terminus and Gly-extended non-amidated AM) in human plasma using a small amount of sample (100 microl) without prior extraction. The detection limit of this assay was 0.5 pmol/l for a 100-microl sample. Intra- and inter-assay precisions were 3.4-7.3% and 5.8-7.6%, respectively. The dilution curves of plasma samples showed good linearity and analytical recovery was 89-118%. The mean total AM in plasma of healthy subjects was 9.00+/-2.13 pmol/l, whereas m-AM was 1.05+/-0.24 pmol/l. This method, together with our previously reported simplified method to specifically measure m-AM (Ohta et al., Clin Chem 1999;45:244-251), allows facile estimation of the plasma concentration of AM-Gly by subtracting m-AM from the total AM measured by the procedure described in this paper. We were able to show that the concentration of total AM in patients with sepsis was markedly higher than that in the healthy controls and that the ratios of m-AM/total AM were significantly different between the controls and patients.

Novel distribution of adrenomedullin-immunoreactive cells in human tissues

Abstract Adrenomedullin (AM) is a novel hypotensive and vasodilator peptide. We previously examined the localization of AM in human, rat, and porcine tissues using a polyclonal antibody against synthetic human AM[40–52]. We demonstrated that AM is widely distributed in the endocrine and neuroendocrine systems, but not in the heart, kidney, or blood vessels, although high levels of AM mRNA were detected in the latter tissues. In this study, we further investigated the distribution of AM by using two newly developed monoclonal antibodies against synthetic human AM peptides, [12–25] and [46–52]. AM immunoreactivity was observed in cardiac myocytes, vascular smooth muscle cells, endothelial cells, and renal distal and collecting tubules. In addition, AM-immunoreactive (IR) cells were found in mucosal and glandular epithelia of the digestive, respiratory, and reproductive systems, as well as the endocrine and neuroendocrine systems. These findings indicate that AM-IR cells are more widely distributed in human tissues and suggest that AM might play multiple biological roles in humans.

Relationship between plasma level of cardiotrophin-1 and left ventricular mass index in patients with dilated cardiomyopathy.

OBJECTIVES The study evaluated the relationship between plasma cardiotrophin-1 (CT-1) concentration and left ventricular (LV) mass in dilated cardiomyopathy (DCM) patients with congestive heart failure (CHF). BACKGROUND Cardiotrophin-1 is a newly identified member of the interleukin-6 (IL-6) family of cytokines and one of the endogenous ligands for gp130 signaling pathways in the heart, and it has potent hypertrophic and survival effects on cardiac myocytes. However, the clinical significance of CT-1 is poorly understood. METHODS We measured the plasma CT-1 level in 51 consecutive patients with DCM. Patients were classified into two groups: small LV mass index group and large LV mass index group, based on the median level of LV mass index. RESULTS The plasma CT-1 level was increased in DCM patients with the severity of CHF and was significantly higher in the large LV mass group than in the small LV mass group, despite the absence of a difference in LV ejection fraction between the two groups. In addition, there was a significant positive correlation between the plasma CT-1 level and the LV mass index (r = 0.627, p < 0.0001). According to stepwise multivariate analyses among hemodynamic and neurohumoral factors, a high plasma CT-1 level showed an independent and significant positive relationship with a large LV mass index in patients with DCM. CONCLUSIONS These results indicate that the plasma CT-1 level is increased in patients with DCM and is significantly correlated with the LV mass index, suggesting that CT-1 plays an important role in structural LV remodeling in patients with DCM.

A new method for histamine release from purified peripheral blood basophils using monoclonal antibody-coated magnetic beads

A new method for evaluating histamine release from purified basophils was developed. Basophil-containing leukocytes were directly purified from a small amount of peripheral blood using monoclonal antibody BA312-coated magnetic beads. The purified basophils still rosetted to magnetic beads maintained a normal response to anti-IgE and to dust mite allergen in comparison with the conventional method using washed leukocytes. This methodology facilitates the purification of basophils, anti-IgE- and allergen-induced histamine release, and subsequent histamine determination within only 3 h. The released histamine was analyzed by an enzyme-linked immunosorbent assay (ELISA) with a characteristic detection profile. Since all steps were performed in 96-well microplates, many clinical samples could be analyzed at the same time, permitting easy applications in routine laboratories.

Development of a sensitive enzyme-linked immunosorbent assay for eotaxin and measurement of its levels in human blood

The CC chemokine eotaxin is potent eosinophil-selective chemoattractant, and it is thought that the function of eotaxin is closely related to the recruitment of eosinophils in certain inflammatory reactions. In order to learn more about the biological role of this molecule, we have developed a new sandwich ELISA method to measure human eotaxin using two monoclonal anitibodies and purified recombinant eotaxin as a standard. The minimal detectable concentration of eotaxin in this assay was 1.5 pg/ml, and the working range was 3.1--200 pg/ml with low CVs (< 10%). Both within- and between-run precision levels were less than 6.7% of the CVs. The dilution curves of two serum and two spiked plasma samples showed good linearity and the recovery range was 92.8--103.3%. No cross-reactivity was found with other similar chemokines. MCP-1, MCP-2, MCP-3, MCP-4, eotaxin-2 and RANTES. This assay was sensitive enough to measure the circulating eotaxin levels of healthy volunteers. However, the eotaxin levels in serum samples (mean+/-SD; 68.6+/-13.4 pg/ml, n=15) were significantly higher than those in matched plasma samples (19.2+/-5.4 pg/ml) separated from blood collected in tubes containing EDTA. Kinetic studies revealed that the eotaxin levels in serum markedly increased depending on the elapsed time before separation from blood cells, but such changes in EDTA-plasma were negligible up to 4 h at 25 degrees C. Our new ELISA is an accurate and useful method for quantifying human eotaxin in blood and demonstrates that the process of preparing blood samples affects the measurement of the eotaxin levels.

Increased adrenomedullin in cerebrospinal fluid after traumatic brain injury in infants and children.

Adrenomedullin is a recently discovered 52-amino acid peptide that is a potent vasodilator and is produced in the brain in experimental models of cerebral ischemia. Infusion of adrenomedullin increases regional cerebral blood flow and reduces infarct volume after vascular occlusion in rats, and thus may represent an endogenous neuroprotectant. Disturbances in cerebral blood flow (CBF), including hypoperfusion and hyperemia, frequently occur after severe traumatic brain injury (TBI) in infants and children. We hypothesized that cerebrospinal fluid (CSF) adrenomedullin concentration would be increased after severe TBI in infants and children, and that increases in adrenomedullin would be associated with alterations in CBF. We also investigated whether posttraumatic CSF adrenomedullin concentration was associated with relevant clinical variables (CBF, age, Glasgow Coma Scale [GCS] score, mechanism of injury, and outcome). Total adrenomedullin concentration was measured using a radioimmunometric assay. Sixty-six samples of ventricular CSF from 21 pediatric patients were collected during the first 10 days after severe TBI (GCS score < 8). Control CSF was obtained from children (n = 10) undergoing lumbar puncture without TBI or meningitis. Patients received standard neurointensive care, including CSF drainage. CBF was measured using Xenon computed tomography (CT) in 11 of 21 patients. Adrenomedullin concentration was markedly increased in CSF of infants and children after severe TBI vs control (median 4.5 versus 1.0 fmol/mL, p < 0.05). Sixty-two of 66 CSF samples (93.9%) from head-injured infants and children had a total adrenomedullin concentration that was greater than the median value for controls. Increases in CSF adrenomedullin were most commonly observed early after TBI. CBF was positively correlated with CSF adrenomedullin concentration (p < 0.001), but this relationship was not significant when controlling for the effect of time. CSF adrenomedullin was not significantly associated with other selected clinical variables. We conclude adrenomedullin is markedly increased in the CSF of infants and children early after severe TBI. We speculate that adrenomedullin participates in the regulation of CBF after severe TBI.

Adrenomedullin (11-26): a novel endogenous hypertensive peptide isolated from bovine adrenal medulla.

Adrenomedullin (AM) is a potent hypotensive peptide originally isolated from pheochromocytoma tissue. Both the ring structure and the C-terminal amide structure of AM are essential for its hypotensive activity. We have developed an RIA which recognizes the ring structure of human AM. Using this RIA, we have characterized the molecular form of AM in bovine adrenal medulla. Gel filtration chromatography revealed that three major peaks of immunoreactive AM existed in the adrenal medulla. The peptide corresponding to Mr 1500 Da was further purified to homogeneity. The peptide was determined to be AM (11-26) which has one intramolecular disulfide bond. Amino acid sequences of bovine AM and its precursor were deduced from the analyses of cDNA encoding bovine AM precursor. The synthetic AM (11-26) produced dose-dependent strong pressor responses in unanesthetized rats in vivo. The hypertensive activity lasted about one minute, and a dose dependent increase in heart rate was also observed. The present data indicate that AM (11-26) is a major component of immunoreactive AM in bovine adrenal medulla and shows pressor activity.