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T. Endo, H. Watanabe, H. Yamamoto, S. Tanaka and M. Hashimoto

ABSTRACT

While prostaglandin F (PGF) has been thought to be a natural luteolysin in non-primates, a luteolytic effect in the human corpus luteum is less evident. We therefore investigated the action of PGF on monolayer cultures of human luteal cells obtained from mid-luteal phase corpora lutea.

PGF increased basal and human chorionic gonadotrophin (hCG)-stimulated progesterone production by human cultured luteal cells. A potent tumour-promoting phorbol ester, phorbol 12-myristate-13-acetate (PMA), also stimulated progesterone production by cultured human luteal cells.

Although human luteal cells were incubated for 24 h with PMA, hCG was still able to stimulate the production of progesterone by PMA-pretreated cells. However, PMA pretreatment blocked the ability of PGF to stimulate progesterone production. It is possible that the luteotrophic effect of PGF may be mediated, in part, by the activation of protein kinase C.

Addition of PGF to suspensions of human luteal cells preincubated with myo-[2-3H]inositol promoted an increase in labelled inositol phosphates. PGF also rapidly increased intracellular free Ca2+ in human luteal cells loaded with the fluorescent Ca2+ probe, fura-2.

We conclude that PGF and PMA stimulate progesterone production and that PGF increases the intracellular free calcium and inositol phosphates of human cultured luteal cells in the mid-luteal phase.

Journal of Endocrinology (1992) 133, 451–458

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

ABSTRACT

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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T. Endo, H. Fukue, M. Kanaya, M. Mizunuma, M. Fujii, H. Yamamoto, S. Tanaka and M. Hashimoto

ABSTRACT

The present studies were carried out to investigate the effect of several growth factors on human endometrial stromal cells.

In human endometrial stromal cells, bombesin and bradykinin provoked an increase in intracellular free Ca2+ and in labelled inositol phosphates when pre-incubated with [3H]myoinositol. Some or possibly all of the initial increase in intracellular free Ca2+ represented a mobilization of Ca2+ from intracellular stores and the second phase of the response depended on Ca2+ influx from the extracellular medium.

[3H]Thymidine was added to human cultured endometrial stromal cells with bombesin, bradykinin, epidermal growth factor (EGF), prostaglandin F, vasopressin and platelet-derived growth factor. Bombesin, bradykinin and EGF stimulated the incorporation of [3H]thymidine into DNA in quiescent cells.

In conclusion, bombesin and bradykinin are growth factors which activate phospholipase C in human endometrial stromal cells, while EGF stimulates DNA synthesis without the activation of phospholipase C.

Journal of Endocrinology (1991) 131, 313–318

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

ABSTRACT

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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H Otsubo, S Hyodo, H Hashimoto, M Kawasaki, H Suzuki, T Saito, T Ohbuchi, T Yokoyama, H Fujihara, T Matsumoto, Y Takei and Y Ueta

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

ABSTRACT

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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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.

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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.