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ABSTRACT
We have investigated the effect of TRH on the accumulation of glycosylated TSH in the rat anterior pituitary gland. Hemipituitaries from adult male rats were incubated in medium containing [3H]glucosamine in the presence of TRH. [3H]Glucosamine-labelled TSH in media and pituitaries was measured by immunoprecipitation and characterized by isoelectric focusing after affinity chromatography. Incorporation of [3H]glucosamine into immunoprecipitable TSH in the media and pituitaries increased progressively with the period of incubation. Although the release of [3H]glucosamine-labelled and unlabelled TSH into media was stimulated by the addition of TRH in a time- and dose-dependent manner, TRH administration did not alter the amounts of labelled or unlabelled TSH in the anterior pituitary lobes. The anterior pituitaries were found, by isoelectric focusing analysis, to be composed of four major component peaks of [3H]glucosamine-labelled TSH. Administration of TRH caused profound changes in the radioactivity of these components and evoked new radioactive peaks, resulting in the appearance of six components in total. The present data provide evidence that TRH significantly changes the heterogeneity of glycosylated TSH in the anterior pituitary without altering the amount of the glycosylated form.
J. Endocr. (1986) 109, 227–231
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The intracellular localization of l-[4,5-3H]leucine in chromaffin cells has been observed using light and electron microscopic autoradiography and the association of the labelled amino acid with particular cell components confirmed by statistical analysis. By making observations at short intervals after a single intravenous pulse of [3H]leucine it has been possible to follow the movement of the isotope from the endoplasmic reticulum through the Golgi complex to the chromaffin granules. No evidence for movement of the label through the Golgi complex was observed in adjacent cortical cells. The time sequence of transport of the amino acid through the various cell organelles was very similar to that observed by previous workers in protein-secreting exocrine cells.
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Search for other papers by S. KOBAYASHI in
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SUMMARY
The fate of l-[2,5,6-3H]DOPA, and the intracellular localization of its metabolic products dopamine, noradrenaline and adrenaline, have been determined by the simultaneous use of assay techniques following separation of amines by chromatography and light and electron microscopic autoradiography.
During the first 24 h after i.v. or i.p. injection of [3H]DOPA, synthesis of the above catecholamines occurred. Throughout this time the labelled amines were associated with chromaffin granules or immediately adjacent cytosol and not with either the Golgi complex or rough endoplasmic reticulum. Labelling of chromaffin granules occurred simultaneously throughout the cell and there was no evidence of regions containing recently labelled granules and others containing previously charged (older) granules. Adrenaline-storing cells took up [3H]DOPA and its products more rapidly and lost recently synthesized adrenaline more rapidly than noradrenaline-storing cells took up and stored their equivalent amines. This was in keeping with a more rapid turnover of catecholamines in adrenaline-storing elements.
Search for other papers by S Asakuma in
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Leptin suppresses food intake and increases energy expenditure in the hypothalamus. Rats consume most of their daily food intake during the dark phase of the diurnal cycle. Lactating rats have increased food intake, but the involvement of leptin in the regulation of food intake in this physiological condition is not well understood. The present experiment was carried out to determine the circadian pattern of leptin concentrations in plasma and cerebrospinal fluid (CSF) in relation to the feeding behavior of non-lactating and lactating rats.Female rats were maintained on a controlled lighting schedule (lights on between 0600 and 1800 h) and the food intake of lactating rats was two- or threefold higher than that of non-lactating rats. In both groups, food intake was three times greater in the dark phase (P<0.01) compared with the light phase. The plasma concentrations of leptin were lower (P<0.01) in lactating rats than non-lactating rats in both light and dark phases, but there were no differences in plasma leptin levels between light and dark phases. In contrast, and in both groups, the leptin concentrations in CSF were lower (P<0.01) in the dark phase than in the light phase. Leptin levels in CSF were lower (P<0.01) in lactating rats than in non-lactating rats. We conclude that a diurnal pattern of leptin levels within the brain (but not in plasma) reflects characteristics of feeding behavior in lactating and non-lactating rats.
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Search for other papers by I. Kobayashi in
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Search for other papers by K. Wakabayashi in
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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
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Search for other papers by A Miyamoto in
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The newly formed corpus luteum (CL) rapidly develops after ovulation and has the features of active vascularisation and mitosis of steroidogenic cells. These stage-specific mechanisms also may contribute to gain the function of prostaglandin F2 alpha (PGF2 alpha)-resistant CL at this stage. Recent studies suggest that the vasoactive peptide angiotensin II (Ang II) regulates luteal function. Thus, this study aimed to investigate (i) the expression of angiotensin-converting enzyme (ACE) mRNA by RT-PCR and the ACE protein expression by immunohistochemistry, (ii) the effects of angiogenic growth factors, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), on the secretion of Ang II, PGF2 alpha, progesterone and oxytocin (OT), and (iii) the effects of luteal vasoactive peptides (Ang II and endothelin-1 (ET-1)) or OT on the secretion of PGF2 alpha, progesterone and OT from bovine early CL (days 3--4 of the oestrous cycle), and evaluate a possible interaction of these substances with PGF2 alpha. The expression of mRNA for ACE was found in theca interna of mature follicle, early CL and endothelial cells from developing CL as well as pituitary and kidney, but granulosa cells of mature follicle were negative. The immunohistochemical analysis revealed that blood capillaries (endothelial cells) were stained for ACE, but luteal cells were negative in early CL. To examine the effects of substances on the secretory function of the CL, an in vitro microdialysis system was used as a model. The infusion of bFGF and VEGF stimulated Ang II and PGF2 alpha secretion as well as progesterone, but not OT secretion in early CL. The infusion of Ang II after PGF2 alpha infusion continued the stimulatory effect on progesterone and OT release within early CL until 3 h thereafter. However, the infusion of ET-1 alone had no effect on progesterone or OT release. The infusion of luteal peptides such as Ang II and OT stimulated PGF2 alpha secretion, whereas the infusion of ET-1 did not. In conclusion, the overall results of this study indicate that a functional angiotensin system exists on the endothelial cells of early CL, and that angiogenic factors bFGF and VEGF upregulate luteal Ang II and PGF2 alpha secretion, which fundamentally supports the mechanism of progesterone secretion in bovine early CL. This idea supports the concept that the local regulatory mechanism involved in active angiogenesis ensures the progesterone secretion in the developing CL in vivo.
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Search for other papers by M. Ohtani in
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ABSTRACT
Prostaglandin F2α (PGF2α) is a primary luteolysin in the cow. Although the mechanisms involved in luteolysis are thought to be a complex of its direct action on luteal cells and indirect effect on luteal blood flow, the detailed mechanisms remain to be elucidated. This study focuses on the possible interaction of endothelial cells-derived endothelin-1 (ET-1) with PGF2α in the rapid suppression of progesterone release from the bovine corpus luteum (CL). In in vitro microdialysis system (MDS) of CL, PGF2α acutely stimulated the release of progesterone and oxytocin during infusion and ET-1 release after infusion. Moreover, PGF2α induced slight decrease of progesterone release during the last period of the experiment (8-11 h after PGF2α exposure). Two 1 h-perfusions of ET-1 at 3 h intervals induced only a slight decrease of progesterone release after the second perfusion. This treatment also affected the oxytocin release; the first ET-1 perfusion produced an acute stimulation, whereas the second ET-1 perfusion inhibited the release to below 50%. When the CL pieces were pre-perfused with PGF2α for 2 h, the two consecutive perfusion of ET-1 at 3 h intervals induced drastic decrease in progesterone and oxytocin release only after the second ET-1 perfusion. Thus, a pre-exposure with PGF2α clearly potentiated the inhibiting activity of ET-1 in the progesterone release. These results suggest a physiological impact of PGF2α and ET-1 in the rapid cascade of functional luteolysis in vivo, and a possible interaction between endothelial cells and luteal cells.
Search for other papers by O. Carnevali in
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Search for other papers by K. Yamamoto in
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Search for other papers by T. Kobayashi in
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Search for other papers by S. Kikuyama in
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Search for other papers by A. M. Polzonetti-Magni in
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ABSTRACT
Male and female Rana esculenta liver was induced in an in-vitro system by homologous and Rana catesbeiana pituitary to synthesize and release vitellogenin, a lipoglycophosphoprotein precursor of yolk proteins, lipovitellins and phosvitins, in oviparous vertebrates.
In the present experiments, the action of prolactin on hepatic vitellogenin synthesis and release was investigated, using ovine prolactin and Rana catesbeiana prolactin. The effects of prolactin on hepatic vitellogenin synthesis displayed different trends related to sex; male liver was found to be more responsive than female liver to both ovine and frog prolactin; moreover, the response to prolactin was dose-related (r = 0·998; P <0·05) in male but not in female liver. In both sexes, a high degree of seasonality in the responsiveness of the liver was found, since the vitellogenin levels induced by prolactin during the winter phase were significantly (P < 0·001) higher than those produced during the summer phase. Thus, there was no significant difference between the action of ovine and frog prolactin on vitellogenin synthesis; in fact, mammalian prolactins are structurally similar with regard to nucleotide and amino acid sequences.
The direct action of prolactin on hepatic vitellogenin synthesis in the frog Rana esculenta is discussed, on the basis of the role played by prolactin as an important growth modulatory hormone in fetal and adult tissues.
Journal of Endocrinology (1993) 137, 383–389
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Ghrelin is a multifunctional peptide that promotes an increase of food intake and stimulates GH secretion. Ghrelin secretion is regulated by nutritional status and nutrients. Although a high-protein (HP) diet increases plasma ghrelin secretion in mammals, the mechanisms and the roles of the elevated ghrelin concentrations due to a HP diet have not been fully established. To clarify the roles of elevated acylated ghrelin upon intake of a HP diet, we investigated the regulation of ghrelin concentrations in plasma and tissues in wethers fed with either the HP diet or the control (CNT) diet for 14 days, and examined the action of the elevated plasma ghrelin by using a ghrelin-receptor antagonist. The HP diet gradually increased the plasma acylated-ghrelin concentrations, but the CNT diet did not. Although the GH concentrations did not vary significantly across the groups, an injection of ghrelin-receptor antagonist enhanced insulin levels in circulation in the HP diet group. In the fundus region of the stomach, the ghrelin levels did not differ between the HP and CNT diet groups, whereas ghrelin O-acyltransferase mRNA levels were higher in the group fed with HP diet than those of the CNT diet group were. These results indicate that the HP diet elevated the plasma ghrelin levels by increasing its synthesis; this elevation strongly suppresses the appearance of insulin in the circulation of wethers, but it is not involved in GH secretion. Overall, our findings indicate a role of endogenous ghrelin action in secretion of insulin, which acts as a regulator after the consumption of a HP diet.
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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
