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  • Author: K Hashimoto x
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On day 20 of gestation, foetal rats received a subcutaneous injection of 0·01 mg oestradiol benzoate dissolved in 0·05 ml sesame oil; foetuses in other litters were given sesame oil alone. Autopsy was performed on day 22 of gestation, at delivery or at various times after birth. Gravimetric and histological observations of the adrenals from oestradiol-treated, oil-treated and intact litter-mate control foetal and neonatal rats were performed together with determination of plasma corticosterone concentrations. Activity of 3β-ol dehydrogenase was also examined histochemically in the adrenals from these animals.

The results indicated that oestradiol benzoate when given prenatally prevented the neonatal decline of adrenal weight and adrenocortical cell size. In normal or oil-treated rats, plasma corticosterone concentration was greatly increased during delivery and 2 h after birth, declining up to 12 h after birth. Oestradiol benzoate prevented this perinatal increase and suppressed the activity of 3β-ol dehydrogenase in the perinatal adrenal cortices.

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M. Nonomura, K. Hoshino, T. Harigaya, H. Hashimoto and O. Yoshida


Hyperprolactinaemia induced by pituitary isografts in male host mice was confirmed by radioimmunoassay, but plasma testosterone levels determined by radioimmunoassay in these mice showed no changes. Immunoenzyme electron microscopic observations revealed large spherical-shaped immunoreactive prolactin granules in pituitary grafts in male hosts, regardless of the sex of the donor mice, indicating the disappearance of sexual dimorphism in prolactin-producing cells in hyperprolactinaemic mice. In hyperprolactinaemic host mice the male accessory sex glands, particularly the seminal vesicle and the ventral prostate, exhibited considerable proliferation and significant increase in weight. These phenomena do not seem to be mediated by the increased action of testosterone. Such biological effects in host mice were much greater when the donor was female rather than male, and were more noticeable in C57BL mice than in C3H mice.

J. Endocr. (1985) 107, 71–76

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Departments of Veterinary Anatomy and Surgery, College of Agriculture, University of Osaka Prefecture, Sakai City, Osaka 591, Japan

(Received 20 July 1976)

Plasma corticosterone concentration in foetal rats greatly increases during delivery and for 2 h after birth, and then declines up to 12 h after birth (Eguchi, Arishima, Morikawa & Hashimoto, 1976). Although this perinatal increase of plasma corticosterone seems to originate mainly from the foetal adrenal gland, transplacental maternal hormone cannot be completely ignored. Maternal corticosterone can cross the placenta to reach the foetus during late gestation in the rat. After an injection of [14C] corticosterone into a pregnant rat on day 21 of gestation, radioactivity is found 30 min later in the foetal plasma (Zarrow, Philpott & Denenberg, 1970). Foetal corticosterone also seems to reach the mother, again demonstrated by the use of a radioactive hormone (Milković, Paunović, Kniewald & Milković, 1973). However, the placental transfer of

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H. Shimura, T. Endo, G. Tsujimoto, K. Watanabe, K. Hashimoto and T. Onaya


We have characterized α1-adrenergic receptor subtypes in functional rat thyroid cells, FRTL, with relation to iodide efflux, and have also examined the effect of TSH on α1 receptor subtypes. FRTL cells grown in a medium containing 5 mU TSH/ml (6H cells) had five times the number of α1 receptors of those maintained in TSH-free medium (5H cells) (11·2 fmol/106 cells compared with 2·0 fmol/106 cells). Pretreatment with chlorethylclonidine (CEC; 10 μmol/l), which inactivates only α1b receptors, caused 98·8% and 97·0% decreases in the density of specific [3H]prazosin-binding sites in 5H and 6H cells respectively. LIGAND computer program analysis of the displacement curves for 2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4 benzodioxane (WB4101) showed that FRTL cells contained mostly low-affinity WB4101 sites. Using the phenoxybenzamine inactivation method, we found a linear relationship between α1 receptor density and the cytosolic free Ca2+ concentration response in FRTL cells. Pre-exposure of intact FRTL cells to CEC caused a 98·7% decrease in noradrenaline-stimulated maximal increase in cytosolic free Ca2+. Also, CEC and 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8), but not nicardipine, inhibited noradrenaline-stimulated iodine efflux. The results suggest that FRTL cells contain mostly the α1b-adrenergic receptor subtype; that the α1b receptors mediate cytosolic free Ca2+ and iodide efflux responses, and that TSH enhances these responses by increasing the α1b receptor density without affecting the post-receptor mechanism.

Journal of Endocrinology (1990) 124, 433–441

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T Taguchi, T Takao, Y Iwasaki, M Nishiyama, K Asaba and K Hashimoto

Dehydroepiandrosterone (DHEA) is believed to have an anti-tumor effect, as well as anti-inflammatory, antioxidant, and anti-aging effects. To clarify the possible inhibitory action of DHEA on pituitary tumor cells, we tested the effects of DHEA, alone or in combination with the nuclear factor-κB (NF-κB) inhibitor parthenolide (PRT), on AtT20 corticotroph cell growth and function both in vitro and in vivo. We found that, in vitro, DHEA and PRT had potent inhibitory effects on pro-opiomelanocortin and NF-κB-dependent gene expression. They also suppressed the transcription activity of survivin, a representative anti-apoptotic factor, and induced apoptosis in this cell line. Furthermore, using BALB/C nude mice with xenografts of AtT20 cells in vivo, we found that the combined administration of DHEA and PRT significantly attenuated tumor growth and survivin expression. The treatment also decreased the elevated plasma corticosterone levels and ameliorated the malnutrition induced by tumor growth. Altogether, these results suggested that combined treatments of DHEA and PRT potently inhibit the growth and function of corticotroph tumor cells both in vitro and in vivo. This effect may, at least partly, be caused by the suppressive effects of these compounds, such as survivin and other inhibitor of apoptosis proteins, on NF-κB-mediated gene transcription.

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C-X Liu, S Takahashi, T Murata, K Hashimoto, T Agatsuma, S Matsukawa and T Higuchi


Large changes in the responsiveness of target organs to oxytocin are thought to originate from alteration of the number of oxytocin receptors (OTR). To elucidate the molecular mechanisms regulating the synthesis of the OTR, we developed a competitive reverse transcription-PCR protocol to measure OTR mRNA. We synthesized cRNA comprising a small stuffer introduced into the target mRNA. Using this cRNA as an internal standard, we made a quantitative estimation of OTR mRNA. Application of this method to the rat uterus revealed that the mean levels of OTR mRNA remained unchanged until 1030–1100 h on day 21 of pregnancy, increased significantly after 2200–2230 h on the same day and declined rapidly after parturition. A similar rapid increase in uterine OTR mRNA content was observed in rats given prostaglandin on day 18, inducing premature delivery on day 19 of pregnancy. All parturient rats had higher OTR mRNA levels regardless of whether parturition was spontaneous or prostaglandin induced. However, in a few rats, OTR mRNA remained as low as that observed during mid pregnancy even on day 22 of gestation, the expected day of parturition in about 70% of the rats in our colony. A similar increase in uterine OTR mRNA content to that observed at parturition was induced by oestrogen treatment for 3 days in ovariectomized virgin rats, but concomitant injection of progesterone did not influence the effect of oestrogen. The present results revealed that the large increase of uterine OTR at the peripartum period is accompanied by an increase in OTR mRNA content that may be brought about, at least in part, by increased oestrogen secretion following luteolysis.

Journal of Endocrinology (1996) 150, 479–486

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K. Tamura, M. Kobayashi, S. Suzuki, Y. Ishii, S. Koyama, H. Yamada, K. Hashimoto, M. Niwa and F. Shibayama


Monoclonal antibodies (McAb) and polyclonal antibodies (PcAb) against human insulin-like growth factor-I (somatomedin C; hIGF-I) were produced. Using these two antibodies, an enzyme-linked immunosorbent assay (ELISA) system for hIGF-I was established. The ELISA system was able to detect hIGF-I at a range of 1–25 μg/l, compared with the range of 1–50 μg/l detected by radioimmunoassay (RIA). Human IGF-II and human insulin could not be recognized in this system. The plasma concentrations of IGF-I found using the ELISA agreed well with those found using RIA after conventional Sep-Pak C18 cartridge pretreatment. Epitopes of hIGF-I to McAb and PcAb were investigated by enzymatic digestion of hIGF-I followed by comparing the affinity of the antibodies to the peptides obtained proteolytically. The epitope to McAb was found to be a peptide containing Leu10-Val11-Asp12 (epitope 2). Five epitopes to PcAb containing the following key fragments were identified: a conformational structure formed by the disulphide bonds between Cys6 and Cys48, and between Cys47 and Cys52 (epitope 1), Leu10-Val11-Asp12 (epitope 2), Val17-Cys18-Gly19-Asp20 (epitope 3), Arg21-Gly22-Phe23-Tyr24 (epitope 4) and Lys68-Ser69-Ala70 (epitope 5). Of these, the peptide containing epitope 5 showed the highest affinity to PcAb. The results indicated that our ELISA system combined recognition by epitope 2 of McAb and recognition by epitope 5 of PcAb to obtain its good specificity.

Journal of Endocrinology (1990) 125, 327–335

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J Ogino, K Sakurai, K Yoshiwara, Yoichi Suzuki, N Ishizuka, N Seki, Yoshifumi Suzuki, H Koseki, T Shirasawa, N Hashimoto, K Yagui and Y Saito

Several mutations of the tyrosine kinase domain of insulin receptor (IR) have been clinically reported to lead insulin resistance and insulin hypersecretion in humans. However, it has not been completely clarified how insulin resistance and pancreatic β-cell function affect each other under the expression of mutant IR. We investigated the response of pancreatic β-cells in mice carrying a mutation (P1195L) in the tyrosine kinase domain of IR β-subunit. Homozygous (Ir P1195L/P1195L) mice showed severe ketoacidosis and died within 2 days after birth, and heterozygous (Ir P1195L/wt) mice showed normal levels of plasma glucose, but high levels of plasma insulin in the fasted state and after glucose loading, and a reduced response of plasma glucose lowering effect to exogenously administered insulin compared with wild type (Ir wt/wt) mice. There were no differences in the insulin receptor substrate (IRS)-2 expression and its phosphorylation levels in the liver between Ir P1195L/wt and Ir wt/wt mice, both before and after insulin injection. This result may indicate that IRS-2 signaling is not changed in Ir P1195L/wt mice. The β-cell mass increased due to the increased numbers of β-cells in Ir P1195L/wt mice. More proliferative β-cells were observed in Ir P1195L/wt mice, but the number of apoptotic β-cells was almost the same as that in Ir wt/wt mice, even after streptozotocin treatment. These data suggest that, in Ir P1195L/wt mice, normal levels of plasma glucose were maintained due to high levels of plasma insulin resulting from increased numbers of β-cells, which in turn was due to increased β-cell proliferation rather than decreased β-cell apoptosis.

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Y Takei, H Hashimoto, K Inoue, T Osaki, K Yoshizawa-Kumagaye, M Tsunemi, T X Watanabe, M Ogoshi, N Minamino and Y Ueta

Adrenomedullin 5 (AM5) is a new member of the calcitonin gene-related peptide (CGRP) family identified in teleost fish. Although its presence was suggested in the genome database of mammals, molecular identity and biological function of AM5 have not been examined yet. In this study, we cloned a cDNA encoding AM5 in the pig and examined its cardiovascular and renal effects. Putative mature AM5 was localized in the middle of prohormone and had potential signals for intermolecular ring formation and C-terminal amidation. The AM5 gene was expressed most abundantly in the spleen and thymus. Several AM5 genes were newly identified in the database of mammals, which revealed that the AM5 gene exists in primates, carnivores, and undulates but could not be identified in rodents. In primates, nucleotide deletion occurred in the mature AM5 sequence in anthropoids (human and chimp) during transition from the rhesus monkey. Synthetic mature AM5 injected intravenously into rats induced dose-dependent decreases in arterial pressure at 0.1–1 nmol/kg without apparent changes in heart rate. The decrease was maximal in 1 min and AM5 was approximately half as potent as AM. AM5 did not cause significant changes in urine flow and urine Na+ concentration at any dose. In contrast to the peripheral vasodepressor action, AM5 injected into the cerebral ventricle dose-dependently increased arterial pressure and heart rate at 0.1–1 nmol. The increase reached maximum more quickly after AM5 (5 min) than AM (15–20 min). AM5 added to the culture cells expressing calcitonin receptor-like receptor (CLR) or calcitonin receptor (CTR) together with one of the receptor activity-modifying proteins (RAMPs), the combination of which forms major receptors for the CGRP family, did not induce appreciable increases in cAMP production in any combination, although AM increased it at 10 10–10 9 M when added to the CLR and RAMP2/3 combination. These data indicate that AM5 seems to act on as yet unknown receptor(s) for AM5, other than CLR/CTR+RAMP, to exert central and peripheral cardiovascular actions in mammals.