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


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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H Tanaka, A Wakisaka, H Ogasa, S Kawai, and CT Liang

In order to establish the cellular basis for using growth factors as possible therapeutic agents for the age-dependent deficit in bone formation activity, we examined the individual and combined effects of IGF-I and/or platelet-derived growth factor (PDGF) on the gene expression of osteoblast-related markers in male rats. The expression of osteoblast markers was examined in the femurs of adult and old rats following marrow ablation, which amplifies gene expression activity. The mRNA levels of collagen(alpha1) (I) (COLI), alkaline phosphatase (AP), osteopontin (OP) and osteocalcin (OC) were significantly lower in the old as compared with the adult rats. To determine whether growth factors can abolish the age-related deficits in mRNA expression in old bone, PDGF and/or IGF-I were infused directly into the right femur for 5 days following marrow ablation. The contralateral femur was infused with vehicle only and used as a control. PDGF stimulated the expression of OP mRNA in both adult and old rats, whereas COLI, AP and OC mRNAs were not affected. IGF-I infusion did not have a significant effect on mRNA expression in adult rats. In contrast, treatment with IGF-I significantly enhanced the mRNA levels of COLI, AP and OP in old rats. To examine whether the combination of both factors could affect the expression of osteoblast markers synergistically, PDGF and IGF-I were infused together. In adult bones, the combined treatment with PDGF and IGF-I caused a slight increase in the level of OP gene expression but no change in AP, OC or COLI genes. Although neither IGF-I nor PDGF alone was effective in stimulating the expression of OC, the combined treatment in old bones enhanced OC expression significantly. The expression of COLI, AP and OP was also stimulated, but the stimulation was no different from that of IGF-I alone. In PDGF plus IGF-I treatment with a high dose, no dose-response effects were observed. Within the limits of the present study, it is suggested that IGF-I and, to a much lesser extent, PDGF may partially restore the deficit in the expression of osteoblast markers in old bones, and that the combination of both factors is slightly better than IGF-I alone in stimulating OC expression.

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K Ohtani, H Shimizu, Y Tanaka, N Sato, and M Mori


Pioglitazone hydrochloride (AD-4833), one of the thiazolidinedione analogs, is a new anti-diabetic agent which improves peripheral insulin resistance in diabetic patients. We determined the direct effect of AD-4833 on insulin secretion in HIT-T 15 cells. The effects of AD-4833 (10−7 m to 10−5 m) on insulin secretion were examined in 3 and 7 mm glucose-containing F-12 K media. The addition of 10−5 m AD-4833 significantly increased insulin secretion in both media, but its stimulatory effect was more potent in the medium containing 7 mm glucose. The addition of 10−5 m AD-4833 caused an immediate, significant increase in cytosolic free Ca2+ concentration ([Ca2+]i). Nifedipine at all concentrations from 10 to 1000 nm significantly attenuated insulin secretion by 10−5 m AD-4833. In addition, 10−5 m AD-4833 failed to stimulate insulin secretion in the Ca2+-free Kreb's-Ringer bicarbonate buffer. These data indicated that AD-4833 stimulates in vitro insulin secretion in HIT-T 15 cells, perhaps by inducing Ca2+ influx.

Journal of Endocrinology (1996) 150, 107–111

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T Sugiyama, H Minoura, N Kawabe, M Tanaka, and K Nakashima


The mRNA species for prolactin receptor (PRL-R) isoforms, long and short form PRL-Rs, were estimated by the reverse transcription-polymerase chain reaction method in the rat brain (cerebrum) during the oestrous cycle, pregnancy and lactation. The levels of long form PRL-R mRNA increased at pro-oestrus and oestrus, at the same time as serum prolactin levels increased, whereas the mRNA level of short form PRL-R was relatively unchanged. Long form PRL-R mRNA expression was also markedly increased in the brain at mid- and late gestation, and this elevated mRNA level was maintained during the period of lactation. In contrast, basal levels of short form PRL-R mRNA were also maintained throughout these periods of gestation and lactation. Ovariectomy moderately reduced brain mRNA levels of both long and short form PRL-R from the levels of those in control dioestrous rats, and hypophysectomy further suppressed them to the lowest levels. Administration of oestradiol valerate (E2V) or 17α-hydroxyprogesterone caproate (17OHPC) to ovariectomized rats resulted in dramatic increases in long form PRL-R mRNA levels in the brain, whereas no significant increase in short form PRL-R mRNA was observed. In rats which were ovariectomized and hypophysectomized, the administration of 17OHPC, rat prolactin or rat GH partially restored the brain level of long form PRL-R mRNA but not short form PRL-R mRNA. E2V, on the other hand, had no effect on the expression of brain PRL-R mRNAs in these hypophysectomized rats, suggesting that the stimulatory effect of E2V on long form PRL-R mRNA expression in ovariectomized rats was mediated by an enhanced secretion of a pituitary hormone, prolactin. These results suggest that the expression of long form PRL-R mRNA in the rat brain is directly induced by progesterone, prolactin or GH during the oestrous cycle, pregnancy and lactation.

Journal of Endocrinology (1994) 141, 325–333

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H. Shimizu, Y. Uehara, Y. Tanaka, Y. Shimomura, and I. Kobayashi


Evidence is accumulating that adrenal steroids may be involved in the metabolic effects of cytokines. We evaluated the possible involvement of glucocorticoids in the inhibition of pancreatic insulin secretion by interleukin-1β (IL-1β), one of the cytokines produced by inflammatory cells. In the first group of experiments, adrenalectomized rats showed a significant reduction in basal and glucose (0·5 g/kg, i.v.)-stimulated immunoreactive insulin (IRI) levels after injection of IL-1β (1·0 μg/kg), but intact rats did not. Pretreatment with IL-1β increased plasma glucose levels 2 and 15 min after an i.v. bolus of glucose in adrenalectomized rats. In the second group of experiments, dexamethasone supplement (0·1 mg/kg) given to adrenalectomized rats cancelled the reduction in plasma glucose levels by IL-1β, and rats treated with 1·0 mg dexamethasone/kg showed a significant increase in basal IRI levels and enhanced serum IRI levels after IL-1β injection. However, 1·0 mg deoxycorticosterone/kg given daily for 7 days failed to cancel the effect of IL-1β on the reduction of serum IRI levels, although it attenuated the weight loss after adrenalectomy. The data suggested that withdrawal of glucocorticoids after adrenalectomy potentiates the effect of IL-1β on the reduction of serum IRI levels. Glucocorticoids may have a protective action against the reduction of serum IRI levels by IL-1β.

Journal of Endocrinology (1992) 132, 419–423

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


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 Hayakawa, Y Kawarada, R Mizumoto, H Hibasami, M Tanaka, and K Nakashima


To elucidate whether and how IGF-I is involved in the regeneration of the pancreas after partial pancreatectomy, IGF-I mRNA expression, IGF-I protein synthesis, ornithine decarboxylase (ODC) activity and DNA replication in the remnant pancreas were determined in the dog. After pancreatectomy, IGF-I mRNA expression was remarkably enhanced in the remnant pancreas, showing the maximal value at post-operative day (POD) 1. Subsequently, IGF-I synthesis in the tissue was significantly stimulated at POD 2, and its maximal concentration was observed at POD 3. Following IGF-I synthesis, ODC activity was induced and its maximal activity was also obtained at POD 3. Finally, DNA replication was induced in the remnant pancreas, and its maximal level was observed at POD 5. These responses in the remnant pancreatic tissue to partial pancreatectomy were greatly enhanced as the resection rate was increased up to 95%. Positive correlations were observed between IGF-I concentrations in the remnant pancreas and the activities of ODC and DNA synthesis in the tissue after 95% pancreatectomy. These results suggest that the gene expression of IGF-I is rapidly induced in the remnant pancreas after partial pancreatectomy, and subsequently synthesized endogenous IGF-I peptides may stimulate ODC and other cell growth-related activities in the tissue in paracrine and/or autocrine manners eventually to induce DNA replication and tissue regeneration.

Journal of Endocrinology (1996) 149, 259–267

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K Sakaguchi, T Ohkubo, T Sugiyama, M Tanaka, H Ushiro, and K Nakashima


Prolactin (PRL) exerts a wide variety of physiological effects on mammalian tissues through its receptor (PRL-R) on the target cells. PRL-R in rat tissue consists of two isoforms, the long and the short form, and the regulatory mechanisms of their mRNA expression in tissues are complex and diverse. The present study reports the differential regulation of PRL-R mRNA expression in rat liver and kidney by testosterone and oestradiol. Using Northern blot analysis, short form PRL-R mRNA was clearly detected in female rat liver and male rat kidney, and long form PRL-R mRNA was faintly observed only in female rat liver. However, the reverse transcription-polymerase chain reaction method enabled efficient analysis of mRNA levels in short and long forms of PRL-R in the liver and kidney of both male and female rats. The mRNA levels for the long and short forms of PRL-R were depressed in the liver of male rats but not in that from female rats during sexual maturation. Castration of male rats resulted in the induction of the mRNAs for these two forms of PRL-R in the liver. Testosterone, but not oestradiol, completely blocked the induction by castration of liver PRL-R gene expression. In kidney, in contrast, mRNA levels for both forms of PRL-R were depressed in female rats but not in male rats after sexual maturation. Administration of oestradiol, but not of testosterone, caused marked repression of short form PRL-R mRNA, particularly in the kidney of male rats. The levels of long form PRL-R mRNA in the kidney was less affected by the administration of oestradiol. These results have suggested that the expression of PRL-R mRNAs in rat liver and kidney is differentially regulated by testosterone and oestrogen.

Journal of Endocrinology (1994) 143, 383–392

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T Sugiyama, H Minoura, N Toyoda, K Sakaguchi, M Tanaka, S Sudo, and K Nakashima


Prolactin receptor (PRL-R) mRNA expression levels in the female rat brain (cerebrum) during pup contact stimulation were determined by the reverse transcription-PCR method. The high expression levels of long form PRL-R mRNA found in the brain of lactating rats were markedly reduced by removal of pups, and long form PRL-R mRNA levels were recovered by resumption of pup contact. Interestingly, pup contact stimuli of nulliparous virgin rats also markedly induced long form but not short form PRL-R mRNA expression in the brain in 1·3 days, together with the expression of maternal behaviour. In ovariectomized (OVX) or hypophysectomized (HYPOX) virgin rats, or in OVX plus HYPOX virgin rats, however, brain long form PRL-R mRNA was not significantly induced by pup contact stimuli for as long as 7 days, while maternal behaviour was fully expressed in these rats after 7 days of pup contact. The in situ hybridization experiments revealed that the long form PRL-R mRNA induced in virgin rats in contact with pups or in lactating rats was localized in the epithelial cells of the choroid plexus. No significant increase in mRNA was detected in other regions of the brain, such as the hypothalamus or cortex, in these maternal female rats. These results suggest that pup contact induces the expression of long form PRL-R mRNA in the choroid plexus of the brain in the presence of female sex steroid and pituitary hormones for the rapid expression of maternal behaviour. Our studies also suggested that maternal behaviour can be expressed in OVX or HYPOX rats after exposure to pups for 7 days without any significant increase in brain PRL-R mRNA expression.

Journal of Endocrinology (1996) 149, 335–340

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Y. Nishi, M. Haji, S. Tanaka, T. Yanase, R. Takayanagi, Y. Etoh, and H. Nawata


The effect of human recombinant activin-A on adrenal steroidogenesis was studied in cultured bovine adrenocortical cells. Activin-A significantly reduced cortisol output from ACTH (10nmol/l)-stimulated adrenocortical cells incubated for 24 hours in a dose-dependent manner (10, 100 and 500ng activin-A /ml suppressed cortisol secretion by 19, 33 and 40%), although no significant effect was observed in the case of 3 h incubation. Dehydroepiandrosterone (DHEA) secretion from ACTH-stimulated adrenocortical cells incubated for 24 h was also decreased by the addition of activin-A in a dose-dependent manner. (10, 100 and 500ng activin-A /ml suppressed DHEA secretion by 22, 56 and 58%).

These inhibitory effects of activin-A (100ng/ml) on cortisol and DHEA secretion were partially blocked by the addition of follistatin / FSH-Suppressing Protein (200ng/ml). In contrast, activin-A treatment resulted in no significant decrease in aldosterone secretion. There were no significant effects of activin-A on basal secretions of cortisol, DHEA or aldosterone from adrenocortical cells. These results suggest that activin-A has a direct inhibitory effect on ACTH-stimulated bovine adrenocortical steroidogenesis.