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N Nakao, M Tanaka, Y Higashimoto, and K Nakashima

Insulin receptor (IR) and IGF-I receptor (IGF-IR) are structurally and functionally related and belong to the tyrosine kinase receptor family. In teleosti such as salmonids and turbot, occurrence of multiple IR and IGF-IR members has been reported, but the structures of a complete set of both IR and IGF-IR members in a single teleost species have not yet been characterized. In this study, we cloned and analysed four distinct cDNA clones for IR and IGF-IR members from the liver and kidney of the Japanese flounder (Paralichthys olivaceus). Deduced amino acid sequence analyses and phylogenetic analysis have revealed that two of them (fIR-1 and fIR-2) belong to IR members and the other two (fIGF-IR-1 and fIGF-IR-2) are IGF-IRs. fIR-1 and fIR-2 comprised 1369 and 1368 amino acid residues respectively, and fIGF-IR-1 and fIGF-IR-2 comprised 1412 and 1418 residues respectively. All the receptor proteins contained cysteine-rich domains in their alpha-subunits, and conserved each transmembrane and tyrosine kinase domains in their beta-subunits. The amino acid sequences of fIRs and fIGF-IRs showed more than 90% sequence identity with turbot IR and IGF-IR respectively. When compared with their mammalian homologues, fIGF-IR-1 and fIGF-IR-2 proteins contained large insertions at their C-termini, as was observed in the corresponding region of turbot IGF-IR. Occurrence of multiple species of mRNA for each IR and IGF-IR was suggested by Northern blot analyses. A ribonuclease protection assay revealed diverse expressions of four receptor mRNAs in a wide range of tissues including heart, liver, ovary, testis, brain, gill arch, kidney, skeletal muscle, intestine, stomach, spleen and eye of the flounder.

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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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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. Kurabayashi, T. Fujimaki, M. Yasuda, Y. Yamamoto, and K. Tanaka


This study was carried out (1) to compare the time-course of the change in bone metabolism in rats administered gonadotrophin-releasing hormone agonist (GnRHa) and bilaterally ovariectomized (OVX) rats and (2) to investigate the changes in bone metabolism after discontinuance of GnRHa.

Seventy female Sprague–Dawley rats, aged 90 days, were divided into four groups. Group 1 underwent a sham operation, group 2 was surgically ovariectomized and group 3 was given a GnRHa (leuprorelin acetate for depot suspension) s.c. injection every 30 days. Group 3 was further divided into three subgroups: rats were administered GnRHa for 12 months (GnRHa 12M), 6 months (GnRHa 6M) or 3 months (GnRHa 3M). Group 4 served as a basal control. The bone mineral density (BMD) of lumbar vertebrae and femoral bone, measured by dual-energy X-ray absorptiometry, and the serum bone metabolic parameters were determined every 45–90 days. The bone histomorphometry of lumbar vertebra was measured on days 180 and 360 after surgery.

GnRHa 12M rats showed significantly lower BMD of vertebrae and femoral bone, lower bone volume and higher bone turnover compared with sham-operated rats and those with secondary hyperparathyroidism on days 180 and 360. Their time-course for changes in bone metabolism was almost the same as that of OVX rats. GnRHa-discontinued rats showed a recovery of bone turnover. The recovery of BMD in GnRHa 6M rats was smaller than that of GnRHa 3M rats after GnRHa discontinuance. The bone volume for GnRHa 6M rats was significantly lower than that for GnRHa 3M on day 360.

Journal of Endocrinology (1993) 138, 115–125

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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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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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K Horiguchi, S Yagi, K Ono, Y Nishiura, M Tanaka, M Ishida, and T Harigaya

Prolactin (PRL) is a single-chain polypeptide hormone that is generally secreted from prolactin cells of the anterior pituitary gland into the blood circulation. However, recent studies indicate that the gene expression of prolactin is ectopic in several tissues across several species. These studies found that lymphocytes also produce PRL, which is involved in the immunoregulatory system. Here, we searched for PRL messenger ribonucleic acid (mRNA), using the reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting in the spleens of mice at various growth stages. We also localized mouse prolactin (mPRL) and its mRNA in the spleens of 30- and 60-day-old mice by immunohistochemistry and in situ hybridization respectively. The mPRL gene was expressed in all spleen samples at 0–60 days postpartum. We localized mPRL mRNA in the sheathed artery, periarterial lymphatic sheath and the marginal zone of the spleen. Moreover, we detected mPRL in essentially the same area as its mRNA. Furthermore, we performed double-fluorescence immunohistochemical staining for mPRL and mouse CD4 that is specifically produced in helper T cells, or for mPRL and mouse CD19 or CD40 specified B cells. We colocalized mPRL immunoreactivity only in some CD4-immunopositive cells. These results clearly suggest that T cells synthesize mPRL in the mouse spleen.

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I Sakata, T Tanaka, M Matsubara, M Yamazaki, S Tani, Y Hayashi, K Kangawa, and T Sakai

Ghrelin was recently isolated from the rat stomach as an endogenous ligand for the GH secretagogue receptor. Although it is well known that a large amount of ghrelin is produced in the gastrointestinal tract, developmental changes in ghrelin mRNA expression and differentiation of ghrelin-immunopositive (ghrelin-ip) and mRNA-expressing (ghrelin-ex) cells in the stomach have not been elucidated. In this study, we therefore investigated the changes in ghrelin mRNA expression levels and in the numbers of ghrelin-ip and -ex cells in the stomachs of 1- to 8-week-old male and female rats by Northern blot analysis, immunohistochemistry and in situ hybridization. Northern blot analysis showed that the level of weak ghrelin mRNA expression was low in the postnatal period but then increased in a dimorphic pattern, i.e. transient stagnation at 4 weeks in the male rats and at 5 weeks in the female rats. The number of ghrelin-ip and ghrelin-ex cells also increased after birth, and more numerous ghrelin cells were found in female rats than in male rats, and this finding was confirmed by Northern blot analysis. Ghrelin-ip and -ex cells first appeared in the glandular base of the fundic gland and then they were found in the glandular base and the glandular neck at 3 weeks of age, suggesting that the distribution of ghrelin cells is extended from the glandular base to the glandular neck during the postneonatal development period. This is the first report on detailed changes in postneonatal ghrelin expression level and in the number of ghrelin cells in the rat stomach. The sexual dimorphism of ghrelin expression and ghrelin cell differentiation suggest that ghrelin plays an important physiological role in the stomach.

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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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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