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  • Author: Toshio Tsushima x
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Kazue Takano, Naomi Hizuka, Kazuo Shizume, Yoko Hasumi and Toshio Tsushima

Abstract.

Serum somatomedin A was significantly reduced after 3 days of fasting in rats with a mean decrease of 23.6 ± 2.4% (N = 18) of initial values. Re-feeding for one day produced a definite increase in somatomedin A, with a rise in body weight.

When re-fed isocalorically for 21 days with diets of different quality, a low protein diet led to smaller increases in both seum somatomedin A and body weight in comparison to those of control-, high-protein- and high fat-diets (P < 0.001). There is a positive correlation between the increase in body weight and serum somatomedin A levels (N = 70, r = 0.71, P< 0.001). The effect of growth hormone on somatomedin generation was abolished in hypophysectomized rats fed with low-protein diet.

Our study suggests that protein in the diet is important for the generation of somatomedin A, which is necessary for normal growth.

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Eiji Ohmura, Toshio Tsushima, Hitomi Murakami, Kae Wakai and Kazuo Shizume

Abstract. The effect of phorbol diester tumour promoters on the release of growth hormone (GH) and prolactin (Prl) was studied in rat pituitary cells cultured in monolayer. 12-O-tetradecanoyl phorbol-13-acetate (TPA), the most potent phorbol ester, stimulated GH accumulation in the cultured medium in a dose-dependent manner. TPA also stimulated Prl accumulation. A time course study indicated that TPA mainly stimulates release of GH. The maximal stimulation of GH release by TPA (100 ng/ml) was 3–4-fold over control. Phorbol-12,13-dibutyrate (PDB), another tumour-promoting phorbol ester, stimulated GH release to an extent similar to that of TPA, while a biologically inactive compound, phorbol-12,13-diacetate (PDA), had no effect. TPA-stimulated GH release was not affected by the presence of indomethacin, an inhibitor of prostaglandin (PG) synthesis, indicating that PG is not involved in the process of TPA-stimulated GH release. Co++, a competitive antagonist of Ca++, at 2.0 mm completely suppressed the GH release induced by TPA, and this inhibition was partially reversed by the addition of 2.0 mm Ca++. Verapamil, a Ca++ channel blocker, reduced TPA-stimulated GH release, and trifluoperazine, an inhibitor of Ca-calmodulin formation, had a similar effect. Somatostatin (SRIF) also inhibited the GH release by TPA. These observations are compatible with the idea that Ca++ may be involved in the process of TPA-stimulated GH release. Since TPA has been reported to activate a Ca++- and phospholipiddependent protein kinase (protein kinase C), it is possible that TPA stimulate GH release by activating the enzyme. Further studies are required to clarify this point.

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Norio Sasaki, Yasuo Imai, Toshio Tsushima and Fukashi Matsuzaki

Abstract.

Both somatotrophic and lactogenic binding sites increase markedly in pregnant mouse liver membranes. The regulatory mechanisms of these binding sites involved in pregnancy are studied. The study of the effects of various hormone administration to female mice reveals that oestradiol increases and testosterone decreases lactogenic binding sites, and that other hormones affect neither somatotrophic nor lactogenic binding sites. Hysterectomy, adrenalectomy or foetectomy of pregnant mice cause a remarkable reduction of somatotrophic binding sites to the level observed in non-pregnant or post-partum animals. In contrast, the lactogenic binding sites are not influenced by these treatments. Replacement with corticosterone after adrenalectomy does not prevent the decrease of somatotrophic binding sites. The contents of placental lactogen from both adrenalectomized and foetectomized pregnant mice are similar to those of the normal pregnant group. These observations indicate that the regulatory mechanisms modulating somatotrophic and lactogenic binding activities are quite different and that increased somatotrophic binding sites during pregnancy are supported by the factor(s) generated in the foeto-placental unit and maternal adrenal gland.

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Tetsuro Okabe, Hiroshi Hidaka, Nakaaki Ohsawa and Toshio Tsushima

Abstract. In an attempt to obtain an in vitro experimental model for aldosteronoma, primary culture was initiated with adenomas from 3 patients with primary aldosteronism. The cells grown in culture retained the morphology and functional properties characteristic of aldosteronoma cells well for periods of up to 200 days. The cells formed monolayer cell colonies and showed an epithelioid morphology with small nuclei containing prominent nucleoli. The cells possessed a clear, eosinophilic cytoplasm resembling that of aldosteronoma cells in vivo. The cultured cells continued to secrete large amounts of aldosterone throughout the culture period. The cells responded to angiotensin II and III by increased release of aldosterone into the culture medium. They also responded to Db-cAMP and ACTH by increased secretion of the hormone.

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Tohru Yashiro, Yoshito Ohba, Hitomi Murakami, Takao Obara, Toshio Tsushima, Yoshihide Fujimoto, Kazuo Shizume and Kunihiko Ito

Abstract.

The presence of IGF-I receptors was demonstrated in normal and neoplastic tissues of human thyroid. Binding of [125I]IGF-I to thyroid membranes was dependent on time and temperature of incubation, and maximal binding was achieved at 4°C and 18 h of incubation. [125I] IGF-I binding was dose-dependently displaced by unlabelled IGF-I; half-maximal inhibition occurred at concentrations of 10–20 μg/l. IGF-II and insulin had relative potencies of 5 and 1% compared with IGF-I. Scatchard analysis of binding data revealed a single class of IGF-I receptors with high affinity (Ka: 1.2–8.6 × 109 1/mol) in normal thyroid tissues. Affinity cross-linking and autoradiography demonstrated the type I IGF receptors. Specific binding of [125I] IGF-I in thyroid cancer tissues (9.69 ± 2.07% per 200 μg protein; mean ± sem, N = 8) was significantly (p <0.05) higher than that in the surrounding normal tissues (3.03 ± 0.35%, N = 8). In contrast, there was no difference in the binding between adenoma tissues (4.19 ± 0.53%, N = 5) and the adjacent normal tissues (2.94 ± 0.24%, N = 5). The higher IGF-I binding in cancer tissues was due to an increase in the binding capacity without any change in the affinity. The presence of IGF-I receptors suggests a possible role of IGF-I and its receptors in the growth of thyroid cancer cells.

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Megumi Kogawa, Kazue Takano, Kumiko Asakawa, Naomi Hizuka, Toshio Tsushima and Kazuo Shizume

Abstract. The effect of insulin on the generation of somatomedin by a monolayer culture of rat hepatocytes was investigated. Release of somatomedin into the medium of cultured hepatocytes was observed for at least three days, as determined by a radioreceptor assay for somatomedin A. The addition of 1–100 μg/ml insulin to the medium caused dose-related increases in somatomedin A in the conditioned medium for up to three days. The conditioned medium also stimulated DNA synthesis in human fibroblasts.

To investigate hormone degradation by hepatocytes, labelled somatomedin A or insulin was added to the medium and the degradation was examined by trichloroacetic acid (TCA) precipitation, and gel chromatography. Although [125I]somatomedin A was slightly degraded even after 24 h of incubation, [125I]insulin was degraded within 6 h and the TCA precipitation at 24 h was 15% only. This degradation of hormones was inhibited by 1 mm bacitracin. Insulin stimulation of somatomedin production was significantly increased to 131.2% as compared with cultures without bacitracin.

The molecular size of the somatomedin released into the medium was greater than that of purified somatomedin, suggesting that this somatomedin is bound to carrier proteins.

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Doo Chol Han, Kanji Sato, Yuko Fujii, Minoru Ozawa, Hidehito Imamura, Toshio Tsushima and Kazuo Shizume

Abstract. To elucidate the effect of rT3 on iodothyronine-5′-deiodinating activity (I-5′-DA) in the liver of neonatal mice, rT3 was injected sc on the 5–8th day after birth and I-5′-DA in the liver was determined. A single injection of rT3 (0.01–1 μg/g) inhibited the ontogenetically developing I-5′-DA in a dose- and time-dependent manner. The inhibitory effect was reversible and specific for I-5′-DA. Lineweaver-Burk analysis revealed that the time- and dose-dependent decrease in the enzyme activity was due to a decrease in Vmax with no alteration in Km values (5 × 10−8 mol/l). The maximal inhibitory effect was observed at a dose of 1 μg rT3/g, whereas the inhibitory effect was diminished at greater doses (4–10 μg/g), probably owing to a contamination with T4 of the rT3 preparation administered. Furthermore, consistent with our previous in vitro findings, rT3 inhibited the I-5′-DA induced by T3 in the liver of neonatal mice. These findings suggest that rT3 inhibited I-5′-DA in the liver of neonatal mice by decreasing the amount of enzyme available to the substrate and that rT3 also elicited an antagonistic effect against T3 in the induction of I-5′-DA in vivo.

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Kanji Sato, Doo Chol Han, Minoru Ozawa, Yuko Fujii, Toshio Tsushima and Kazuo Shizume

Abstract. A highly sensitive bioassay for PTH was developed by using rat osteosarcoma cells (ROS 17/2.8). By limiting dilution, ROS cells were subcloned and the subclonal cell line (ROS 17/2.8–5) most responsive to PTH was selected. When subconfluent ROS 17/2.8–5 cells were treated with hydrocortisone for 3 days and then incubated with PTH, the cAMP response was significant at 10–40 ng/l hPTH (1–34) (4 ∼ 16 × 10−12 mol/l). Osteoclast activating factors such as human interleukin I alpha and beta, and tumour necrosis factor alpha did not stimulate cAMP production, whereas a conditioned medium of oesophageal carcinoma cells established from a patient with humoral hypercalcaemia stimulated cAMP production. By selecting PTH-responsive subclonal cells and treating them with hydrocortisone, the sensitivity for detecting PTH was improved approximately 15 times. This method will be useful in the characterization and purification of PTH-like factors produced by malignant tumours from hypercalcaemic patients.

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Kanji Sato, Hisako Mimura, Shuichi Kato, Osamu Isozaki, Toshio Tsushima and Kazuo Shizume

Abstract. The serum levels of propylthiouracil (PTU) were determined by radioimmunoassay in 10 normal subjects and in 11 patients with Graves' disease after a single 100 or 200 mg oral dose of PTU. The serum half-life of PTU in the normal subjects and in hyperthyroid patients with uneventful clinical course was 75 ± 19 min (mean ± sd, n = 6) and 73 ± 13 min (n = 7), respectively. Maximum serum PTU concentrations were usually attained within 1 h after a single 200 mg oral dose and at 1 h were 5.3 ± 1.4 μg/ml (3.1 ± 0.82 × 10−5 m) in normal subjects (n=6) and 4.8 ± 2.4 μg/ml (2.8 ± 1.4 × 10−5 m) in hyperthyroid patients (n = 7). These betweengroup differences were not significant. Serum PTU concentrations were low in a pregnant hyperthyroid patient with a weak response to PTU treatment. In another patient, who appeared resistant to PTU therapy, the serum PTU level increased as expected at testing, and it was later confirmed that, during treatment, he had not taken the drug as prescribed. In a patient who developed agranulocytosis due to methimazole and subsequently fever due to PTU, the half-life of PTU was prolonged to about 130 min.

These findings suggest that monitoring the serum PTU levels in patients with Graves' disease can be of clinical value in patients who do not respond to treatment. Furthermore, it may provide some clues as to the mechanism by which toxic reaction develops.

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Osamu Isozaki, Toshio Tsushima, Kanji Sato, Motoyasu Saji, Yoshito Ohba, Naoya Emoto, Yuji Sato and Kauzo Shizume

Abstract. A 54 year old man with markedly elevated serum T3, but without an apparent thyroid disease, was found to have a specific antibody to T3. His serum thyroxine, TBG and TSH were in normal range, but T3-RSU was markedly low. Antibodies to thyroglobulin and microsome were negative. He was judged euthyroid because of a normal basal metabolic rate and a normal thyroidal 123I uptake which was suppressed by T3 adminnistration. When serum was extracted with ethanol prior to assay, serum T3 was found to be in the upper border of normal range. Several experiments revealed the presence of an antibody to T3 in his serum with an affinity constant of 3.3 × 109 m −1. The binding capacity of the antibody was 7.6 ng/mg of IgG. The binding of [125I]T3 was almost specific to T3, and potencies of T4 and fT3 in displacing [125I]T3 binding were only 1.0 and 0.3%, respectively, of that of T3. The antibody contained both kappa and lambda chains and was therefore polyclonal.

The T3 metabolic clearance rate, which was determined by disappearance of injected [125I]T3 from serum, was lower in this patient (7.44 I/day) than in normal. The T3-production rate was decreased to 14.9 μg/day, and serum free T3 concentration as well as urinary T3 excretion rate were also reduced. Since both serum total and free T4 concentrations were normal, the supply of T4 to peripheral tissues would be sufficient to keep this patient in a euthyroid state in spite of the anti-T3 antibody.