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T. Higuchi, K. Honda, T. Fukuoka, H. Negoro, and K. Wakabayashi

ABSTRACT

A highly sensitive and specific radioimmunoassay (RIA) for oxytocin was developed and used to measure oxytocin concentrations during both suckling and parturition in individual rats. In urethane-anaesthetized rats, the suckling stimuli, provided by ten pups, induced intermittent increases in intramammary pressure of about 10 mmHg. This was associated with a significant (P < 0·01) increase in serum oxytocin levels from 19·5 ± 4·5 (s.e.m., n = 9) to 49·1 ± 7·4 pmol/l (n = 9) in the samples taken within 30 s from the time of the peak in the pressure. These rises in serum oxytocin returned rapidly to the basal levels as expected from the short half-life (1·46 min) of oxytocin in general circulation.

On day 22 or 23 of gestation, serum oxytocin levels remained stable until 0–0·5 h before the first fetus was expelled. They then increased significantly (P < 0·01) from 27·6± 4·6 pmol/l (n = 19) in samples taken 0–0·5 h before to 45·1 ± 5·6 pmol/l in samples taken after the expulsion of the first fetus and gradually increased until the last fetus was expelled. Serum oxytocin concentrations then declined but remained higher than those observed before the first fetus had been born until at least 1–1·5 h after the expulsion of the last fetus. Thus, this oxytocin RIA revealed increased concentrations of the hormone in blood during both suckling and parturition in the rat.

J. Endocr. (1985) 105, 339–346

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S. Wakabayashi, S. Kagawa, K. Nakao, K. Mimura, and A. Matsuoka

ABSTRACT

Monolayer cultures of the pancreas of the neonatal rat were maintained for 7 days in glucose-depleted TCM 199 medium, supplemented with 5·5 mmol galactose/l, with or without 0·1 mmol 2-deoxyglucose/l. Under culture conditions without 2-deoxyglucose, the responsiveness of B cells supported on galactose was totally abolished by day 7 of culture, and islets degenerated. In contrast, the addition of 2-deoxyglucose to the galactose-supplemented medium promoted the survival and the function of B cells even in a glucose-depleted environment and, in addition, yielded the monolayers mostly consisting of endocrine cells by destroying fibroblasts selectively. On day 7 the recovery of insulin in the cells was higher than that of the cells grown in medium with galactose alone (10·7-fold), and than the initial level at day 0 (twofold). Furthermore, the response to an acute challenge with 16·7 mmol glucose/1 was 3·3-fold, to 10 mmol leucine/1 it was 8·5-fold, and to 10 mmol 2-ketoisocaproate/l it was 10·9-fold above each value observed on day 1. In summary, the above data indicate that morphologically intact, functionally competent endocrine B cells can be grown in medium free from glucose.

J. Endocr. (1984) 103, 377–381

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S. Wakabayashi, S. Kagawa, K. Yamashita, and A. Matsuoka

ABSTRACT

The effect of two concentrations of glucose on the maturation of the response of B cells was studied in pancreatic monolayer cultures of the neonatal rat using a perifusion system. After exposure for the initial 3 days to a medium with 5·5 mmol glucose/l and 10 μmol iodoacetic acid/l (day 3), in order to delete fibroblasts selectively, monolayer cultures were kept for a total of 12 days in medium with either 5·5 or 16·7 mmol glucose/l alone. On day 3, B cells responded in a monophasic fashion, with no significant second phase, to acute challenge with 16·7 mmol glucose/1. At a concentration of 10 mmol/l, leucine and 2-ketoisocaproate both produced only minimal increases in the second phase of secretion above the basal level. In contrast, B cells on day 7 cultured in 5·5 mmol glucose/l showed a biphasic response to glucose, leucine and 2-ketoisocaproate. The magnitude in response to glucose was well preserved at day 15 of culture, whereas the stimulatory effects of leucine and 2-ketoisocaproate decreased to 24–57% of that of B cells on day 7. Moreover, B cells on day 7 cultured in 16·7 mmol glucose/l responded in a biphasic manner to glucose, the response being 65% of that of B cells in 5·5 mmol glucose/l. Additionally, the response to leucine and 2-ketoisocaproate still appeared to be monophasic. At day 15 of culture, however, the response of B cells in 16·7 mmol glucose/l to glucose was 105%, to leucine 245% and to 2-ketoisocaproate 127% of that of B cells in 5·5 mmol glucose/l. In conclusion, these results suggest that cultivation in medium with 5·5 mmol glucose/l could induce precocious development of an adult pattern of insulin secretion compared with that in 16·7 mmol glucose/l and, in addition, that the medium with 16·7 mmol glucose/l is superior for long-term culture to the medium with 5·5 mmol glucose/l.

J. Endocr. (1988) 118, 303–310

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S. Wakabayashi, S. Kagawa, K. Nakao, and A. Matsuoka

ABSTRACT

Two groups of monolayer cultures of pancreatic cells from the neonatal rat were maintained in glucosedepleted TCM 199 medium, supplemented with 5·5 mmol galactose/l, with or without 0·1 mmol 2-deoxyglucose/1. Another group was kept in medium with 5·5 mmol galactose/l alone, following exposure for 2 days to a medium with 5·5 mmol galactose/1 and 10 μmol iodoacetic acid/l to kill fibroblasts selectively. Each of these monolayers was cultured in a perifusion system for a total of 7 days so that phasic insulin secretion could be compared. On day 0, B cells responded in a monophasic fashion to acute challenge with 16·7 mmol glucose/l whereas, in the presence of 10 μmol forskolin/1 and 1 mmol 3-isobutyl-1-methyl-xanthine/l, the same dose of glucose stimulated a biphasic response of approximately the same magnitude. At a concentration of 10 mmol/l, leucine and 2-ketoisocaproate both produced only minimal increases in the second phase of secretion above the basal level. No response to secretagogues was seen under culture conditions without 2-deoxyglucose. In contrast, addition of 2-deoxyglucose to the galactose-supplemented medium stimulated B cells to secrete insulin in a biphasic fashion in response to a single dose of glucose, and the stimulatory effects of leucine and 2-ketoisocaproate were also remarkably increased. Moreover, when exposed to a linear concentration gradient of glucose, leucine or 2-ketoisocaproate, these B cells responded to secretagogues in a dose-dependent fashion. On the other hand, B cells which had been exposed briefly to iodoacetic acid and then cultured in medium with galactose alone, showed a biphasic response to glucose, leucine and 2-ketoisocaproate; however, quantitative relationships differed. Thus the response (total insulin secreted during a 30-min stimulation) of B cells grown in 2-deoxyglucose to glucose was 610%, to leucine 504% and to 2-ketoisocaproate 810% of that of cells exposed to iodoacetic acid. The present data suggest that cultivation of neonatal B cells in a glucose-depleted medium containing 2-deoxyglucose may cause functional maturation.

J. Endocr. (1987) 115,169–175

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M. Mori, M. Murakami, T. Iriuchijima, H. Ishihara, I. Kobayashi, S. Kobayashi, and K. Wakabayashi

ABSTRACT

An influence of thyrotrophin-releasing hormone (TRH) on TSH heterogeneity in close association with de-novo biosynthesis was studied in rat anterior pituitary glands. Hemipituitary glands from adult male rats were incubated in Krebs–Henseleit–glucose media containing [3H]glucosamine and [14C]alanine for 3 and 6 h in the presence or absence of 10 ng TRH per ml. Fractions of TSH in the pituitary extracts were obtained using affinity chromatography coupled with an anti-rat TSH globulin. These TSH fractions were analysed by isoelectric focusing. The control pituitary glands were composed of four component peaks (isoelectric point (pI) 8·7, 7·8, 5·3 and 2·5) of [3H]glucosamine and [14C]alanine incorporated into TSH, and the amounts of radioactivity of these components were increased with the incubation time. Of these peaks, radioactive components of pI 8·7 and 7·8 coincided with the non-radioactive TSH components measured by radioimmunoassay. Addition of TRH increased incorporation of [14C]alanine into TSH in each of the components to a greater extent than that of [3H]glucosamine. In addition, new components with pI 7·2, 6·5 and 6·2, each component corresponding to each unlabelled TSH component, were demonstrated in the presence of TRH. Because addition of TRH did not change the amounts of [14C]alanine-labelled TSH in the media, the newly formed components were assumed to be connected with protein synthesis occurring in the anterior pituitary gland, which may be specific substances in response to TRH administration. These results indicate that TRH principally elicits an increase in protein synthesis in TSH at the anterior pituitary level, resulting in an alteration of TSH heterogeneity.

J. Endocr. (1984) 103, 165–171