bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunolocalization of mineralocorticoid receptor in human kidney, pancreas, salivary, mammary and sweat glands: a light and electron microscopic immunohistochemical study
Authors: Sasano H. , Fukushima K. , Sasaki I. , Matsuno S. , Nagura H. , Krozowski Z. S.
Published: December 2008

Using a polyclonal antibody against a synthetic fusion protein corresponding to 167 amino acids of the N-terminal region of the human renal mineralocorticoid receptor (MR), an immunohistochemical study was performed to investigate the intraglandular and intracellular localization of the receptor in human kidney, salivary gland, pancreas, mammary gland and sweat gland both at the light and electron microscopic levels. In the kidney, immunoreactivity was observed in distal convoluted tubules, branches of Henle's loop, and collecting tubules in the renal cortex, and papillary and Henle's loops' ducts in the renal medulla. No significant differences in the distribution of immunoreactivity were observed using different fixatives (10% neutral formalin, 100% methanol, 4% paraformaldehyde, PLP (periodate–lysine–2% paraformaldehyde) solution and Zamboni solution) and processing methods (paraffin embedding and frozen sectioning). Immunoreactivity in the kidney was observed in both the cytoplasm and nucleus, with cytoplasmic staining predominant, regardless of the methods of tissue preparation. Immunoelectron microscopy, employing a pre-embedding method, demonstrated the presence of immunoreaction precipitates in nuclei, endoplasmic reticulum, perinuclear cisternae, free cytoplasm and cell membranes. In nuclei, immunoreactivity was observed in euchromatin but not in heterochromatin, which is consistent with an association of MR with specific DNA regulatory elements located in transcriptionally active euchromatin. In other organs, MR was expressed in cells of the excretory ductal system where mineralocorticoids are known to play a role in electrolyte homeostasis.

Journal of Endocrinology (1992) 132, 305–310
Daily administration of gonadotrophin-releasing hormone increases pituitary gonadotroph number and pituitary gonadotrophin content, but not serum gonadotrophin levels, in female rats on day 1 of dioestrus
Authors: Kotsuji F. , Hosokawa K. , Tominaga T.
Published: December 2008

Gonadotrophin-releasing hormone (GnRH) has been shown to regulate the synthesis and release of gonadotrophins acutely, yet few studies have investigated the chronic effects of this agent on pituitary gonadotrophins. In the present study we determined the effect of chronic administration of GnRH on the female rat pituitary gland. Rats of 8 weeks of age were injected s.c. with various doses of GnRH daily for 30 days. After completion of the GnRH treatment, treated rats and age-matched controls were killed by decapitation at 09.00 h on the first day of dioestrus, as determined from vaginal smears. Treatment with 10 ng–10 μg GnRH/day increased pituitary contents of FSH and LH in a dose-dependent manner. The change in FSH content was much greater than that of LH content. The pituitary FSH content of rats treated with 40 μg GnRH was significantly less than that of rats treated with 10 μg GnRH. There was a marked increase in the number of cells which stained positively for FSH (266%) and LH (28%) in the anterior pituitary of rats given 10 μg GnRH, but there was no demonstrable change in the areas of single cells stained positively for FSH and LH. Serum levels of LH, FSH and oestradiol were not affected by the GnRH treatment. These data indicate that chronic administration of GnRH is capable of increasing the pituitary gonadotrophin content and numbers of FSH and/or LH-stained cells and that FSH cells are affected more than LH cells by the GnRH treatment. The increase in pituitary gonadotrophin content, however, does not necessarily produce an increase in circulating levels of gonadotrophins.

Journal of Endocrinology (1992) 132, 395–400
Circulating maternal immunoreactive inhibin levels during pregnancy in the rat: effects of oophorectomy, hypophysectomy, hemihysterectomy, delayed implantation and pseudopregnancy
Authors: Yohkaichiya T. , Irby D. , de Kretser D. M.
Published: December 2008

This study examines the source of inhibin in the maternal circulation of pregnant rats by measuring serum immunoactive inhibin levels following a range of experimental procedures. Ovariectomy at days 7, 13 or 19 of gestation, with maintenance of pregnancy by supplementation with progesterone and oestradiol dipropionate, led to a profound fall of serum inhibin levels in comparison with controls, demonstrating that the ovary is a major source of circulating inhibin. This conclusion was supported by the inhibition of the late rise (days 16–22) in serum inhibin in pregnant rats which were hypophysectomized on day 15 and maintained with oestrogen and progesterone supplementation. These data support the view that the rise in serum inhibin from days 16 to 22 is due to re-activation of follicular development in preparation for the post-partum oestrus. Reduction of fetal numbers by hemihysterectomy on days 7, 13 or 19 did not alter serum inhibin levels. Induction of delayed implantation by ovariectomy on day 3 and progesterone supplementation together with initiation of reimplantation by the addition of oestradiol dipropionate on day 7 or 11 did not significantly alter inhibin levels. The induction of pseudopregnancy by mating to vasectomized rats did not result in the maintenance of stable serum inhibin levels until oestrous cycles recommenced. Taken together, the studies have identified the ovary as the predominant source of circulating maternal inhibin levels throughout pregnancy in the rat.

Journal of Endocrinology (1992) 132, 469–475
Effects of in-vivo administration of insulin-like growth factor-I on the rate of glucose utilization in the soleus muscle of the rat
Authors: Dimitriadis G. , Parry-Billings M. , Dunger D. , Bevan S. , Colquhoun A. , Taylor A. , Calder P. , Krause U. , Wegener G. , Newsholme E. A.
Published: December 2008

This study investigated the effects of insulin-like growth factor-I (IGF-I) administered to rats in vivo on the soleus muscle isolated from these rats. In order to study the interactions between IGF-I and insulin, the soleus muscles were incubated in the presence of various concentrations of insulin. IGF-I (190–200 μg) was given twice daily; the rats were killed 1 h after one injection of IGF-I (acute administration) or after treatment with IGF-I for 10 days (prolonged administration). The level of IGF-I in plasma was increased by ∼ 100% after acute administration and by around 30% after 10 days of treatment with IGF-I. Acute administration of IGF-I to the rats increased the flux of glucose to hexose monophosphate and the rates of lactate formation and glycogen synthesis in the soleus muscles; however, the responsiveness of these muscles to insulin was lost: the increase in the rate of glucose utilization by IGF-I at physiological concentrations of insulin (10 or 100 mU/l) was similar to that observed at maximal concentrations of insulin (1000 mU/l). Similar results were obtained after prolonged treatment of the rats with IGF-I; however, the increase in the rate of glucose utilization was less pronounced than when IGF-I was given acutely and the muscles were still capable of responding to insulin. These results suggest that: (a) acute or chronic increases in the serum level of IGF-I in the rat in vivo increase the basal rate of glucose utilization in skeletal muscle; this increase is independent of insulin; (b) the effects of insulin on the rate of glucose utilization are not additive to those of IGF-I; however, this may depend on the level of IGF-I in serum.

Journal of Endocrinology (1992) 133, 37–43
Effects of insulin-like growth factor-I (IGF-I) infusion and dietary tri-iodothyronine (T3) supplementation on growth, body composition and plasma hormone levels in sex-linked dwarf mutant and normal chickens
Authors: Tixier-Boichard M. , Huybrechts L. M. , Decuypere E. , Kühn E. R. , Monvoisin J.-L. , Coquerelle G. , Charrier J. , Simon J.
Published: December 2008

This study used a sex-linked dwarf mutant (SLD) chicken to evaluate growth-promoting and metabolic effects of recombinant human insulin-like growth factor-I (rhIGF-I) treatment. The SLD chicken is characterized by a 30% reduction in body weight and by high plasma GH levels, low plasma IGF-I and triiodothyronine (T3) levels and very low GH binding on liver membranes, suggesting reduced functional GH receptors compared with normal chickens. The effects of a continuous s.c. infusion by osmotic mini-pump of 0·1 mg rhIGF-I/kg per day from 4 to 8 weeks of age on body weight, bone growth and body composition were investigated in female SLD and normal chicks. In addition, half of the birds received a dietary supplement of T3 (0·1 parts per million). Plasma levels of IGF-I, GH, T3, thyroxine and insulin were followed during the treatment. In normal chicks, rhIGF-I infusion had no effect on growth and little effect on plasma hormone levels except for a decrease in plasma insulin. In dwarf chicks, rhIGF-I infusion slightly increased body weight but had no effect on longitudinal bone growth. In addition, plasma GH levels were decreased and T3 levels remained lower than in normal chicks. Normal and dwarf chicks showed a decrease in abdominal fat after both IGF-I administration and T3 supplementation, the treatments having additive effects in dwarf chicks only. The combined rhIGF-I and T3 treatment restored a quasi-normal hormonal pattern in dwarf chicks, except for insulin which remained lower than in normal chicks. These results suggest that IGF-I in the chicken has no direct endocrine effect on statural growth.

Journal of Endocrinology (1992) 133, 101–110
Characterization of inhibin and related proteins in bovine fetal testicular and ovarian extracts: evidence for the presence of inhibin subunit products and FSH-suppressing protein
Authors: Torney A. H. , Robertson D. M. , de Kretser D. M.
Published: December 2008

Bovine fetal gonads have been shown previously to contain inhibin bio- and immunoactivity although the ratio of these activities was markedly lower in testicular compared with ovarian extracts throughout gestation. The basis for this difference is examined in this study. Fetal testicular and ovarian high-speed supernatant preparations from bovine fetuses aged 180 to 270 days of gestation were sequentially fractionated by dye affinity chromatography, gel permeation chromatography, reversed phase highperformance liquid chromatography and preparative polyacrylamide gel electrophoresis and monitored by inhibin radioimmunoassay and in-vitro bioassay. Three immunoactive fractions were identified in testicular extracts with molecular masses of 30 kDa (Peak I), 43 kDa (Peak IIa) and 29 kDa (Peak IIb). Peak I material only was bioactive. On the basis of these characteristics, Peak I is probably 31 kDa inhibin as previously described, and Peaks IIa and lib are probably different inhibin α subunit precursor fragments.

In ovarian extracts, two bio- and immunoactive fractions were identified with molecular masses of 30 kDa (Peak I) and 29 kDa (Peak II). On the basis of size, and biological and immunological activities, the ovarian extract Peak I material is probably bovine 31 kDa inhibin, while the Peak II material is probably a novel inhibin-like protein. FSH-suppressing protein (or follistatin) bio- and immunoactivities were also identified in both testicular and ovarian extracts.

It is concluded that the low ratio of inhibin biological/immunological activity in testicular extracts is attributed to the presence of high concentrations of immunoactive α subunit precursor fragments which are low to non-detectable in ovarian extracts. These results support our previous hypothesis that, in contrast to the ovary, the inhibin α subunit is produced in excess in the fetal testis.

Journal of Endocrinology (1992) 133, 111–120
Modulation of differentiation of rat granulosa cells in vitro by interferon-γ
Authors: Xiao S. , Findlay J. K.
Published: December 2008

The effects of recombinant rat interferon-γ (rRaIFN-γ) and rat IFN (RaIFN, a mixture of IFN-γ and -α) on basal and FSH-induced ovarian granulosa cell function were studied. Granulosa cells were harvested from diethylstilboestrol-treated immature rats and cultured (2 × 105 viable cells/well per 0·5 ml) in serumfree medium with or without treatment for 48 h. In the presence of FSH (20 ng/ml), rRaIFN-γ (10–1000 U/ml) significantly inhibited FSH-stimulated aromatase activity (76·4 ± 2·3% maximum inhibition compared with FSH treatment alone), inhibin (40·4 ± 3·7%), progesterone (47·7 ± 8·6%) and 20α-hydroxypregn-4-en-3-one (20α-OHP) (51·8±1·7%) production in a dose-dependent manner. Furthermore, rRaIFN-γ inhibited FSH- and forskolin (FSK; 30 μmol/l)-induced extracellular cAMP accumulation (46·0 ± 6·6% and 29·1 ± 7·3% respectively). The inhibitory effect of rRaIFN-γ on FSK-induced cAMP was accompanied by decreased FSK-induced aromatase activity, inhibin, progesterone and 20α-OHP production. rRaIFN-γ had no detectable effect on aromatase activity, progesterone production and 20α-OHP production in the absence of FSH, but significantly stimulated basal inhibin production by 1·5-fold. rRaIFN-γ alone also caused a small but significant increase in basal levels of cAMP. The timecourse studies showed that FSH-induced aromatase activity and inhibin production were consistently suppressed by rRaIFN-γ, FSH-induced progesterone and 20α-OHP were inhibited at 1 and 2 days and then stimulated on days 3, 4 and 5 relative to FSH alone. There was no difference in DNA content between treatment and non-treatment wells during 5 days of culture. RaIFN had similar effects to rRaIFN-γ. We conclude that IFN-γ can inhibit FSH-induced granulosa cell differentiation and that, in the absence of FSH, IFN-γ stimulated undifferentiated granulosa cells to produce more inhibin. The mechanism of its action is likely to involve changes in cAMP production.

Journal of Endocrinology (1992) 133, 131–139
Chromatographic characterization of insulin-like growth factor-binding proteins in human pregnancy serum
Authors: Wang H. S. , Chard T.
Published: December 2008

Insulin-like growth factor-binding proteins (IGFBPs) in maternal and umbilical cord sera have been analysed by combinations of gel filtration chromatography, affinity cross-linking and electrophoresis. On gel filtration chromatography, the majority of circulating IGF-I in non-pregnant and pregnant women was present in the large molecular mass (150 kDa) binding proteins (IGFBP-3). In umbilical cord serum, by contrast, most IGF-I was present in the 40 kDa binding proteins (consisting of IGFBP-1 and IGFBP-2). Western blots demonstrated an apparent progressive attenuation of IGFBP-3 and IGFBP-2 in serum from pregnant women with an increase in IGFBP-1. After prior cross-linking with disuccinimidyl suberate, the 150 kDa fractions (IGFBP-3) from non-pregnant and pregnant serum showed a similar pattern on SDS-PAGE (several bands at different molecular masses). However, IGFBP-2 (one of the components of the 40 kDa fractions) was undetectable, even after cross-linking, in serum from pregnant women later than 8 weeks of gestational age and in a mixture of maternal serum at term delivery and serum from non-pregnant women. This suggests that serum IGFBP-2 was degraded by specific proteases present in pregnancy serum. Following acid treatment, the 150 kDa fractions from pregnancy serum were split into smaller subunits or fragments while the 40 kDa fractions remained unchanged, suggesting that the 40 kDa binding proteins are acid-stable. The present data demonstrate that IGFBP-3 is the principal IGFBP in pregnancy serum even though there is an apparent reduction in serum IGFBP-3 activity as revealed on Western blots. The absence of IGFBP-2 in serum from pregnant women may be due to degradation by proteases. In the fetal circulation IGFBP-1 and IGFBP-2 appear to be the major binding proteins for IGF-1.

Journal of Endocrinology (1992) 133, 149–159
Characterization and hormonal regulation of 24 kDa protein synthesis by the adult murine epididymis
Authors: Jimenez C. , Lefrancois A. M. , Ghyselinck N. B. , Dufaure J. P.
Published: December 2008

The pattern of labelled proteins synthesized and secreted in vitro by the adult mouse epididymis was studied by one- and two-dimensional polyacrylamide gel electrophoresis. The presence of a major 24 kDa protein synthesized and secreted in a tissue-specific manner by the caput epididymidis was detected. For this molecular weight, two-dimensional analysis indicated several proteins including two polypeptides (pI 8·4 and 8·8) whose expression is under androgenic control. Partial amino acid sequence analysis showed a complete N-terminal identity between these two peptides. A polyclonal monospecific immune serum was raised against the two proteins. Only purified immunoglobulins precipitated them, showing that immunological affinity is restricted to these two proteins in the epididymis. Indirect immunofluorescence assay revealed specific binding of antibodies on the acrosomal region of spermatozoa isolated from the caput, corpus or cauda epididymides. Testicular spermatozoa were not labelled under the same conditions.

To investigate the physiological role of androgens in the synthesis and secretion of the 24 kDa proteins, tissue slices of epididymides from adult mice which had been castrated, or castrated then injected with testosterone were incubated with [35S]methionine. Castration and testosterone replacement kinetics revealed that alterations in 24 kDa protein synthesis follow immediately upon androgenic privation and replacement.

Journal of Endocrinology (1992) 133, 197–203
Eel insulin: isolation, characterization and stimulatory actions on [35S]sulphate and [3H]thymidine uptake in the branchial cartilage of the eel in vitro
Authors: Duan C. , Noso T. , Moriyama S. , Kawauchi H. , Hirano T.
Published: December 2008

Our previous studies have shown that mammalian and salmon insulins stimulate sulphate uptake by cultured eel cartilage, suggesting the possible involvement of insulin in the regulation of cartilage matrix synthesis. In the present study, homologous eel insulin was isolated and characterized, and its effects on cartilage matrix synthesis and DNA synthesis were examined in vitro. Insulin was extracted from eel pancreas with acid–ethanol, and subsequently purified by isoelectric precipitation at pH 5·3, gel filtration on Sephadex G-50, and reversed-phase high-performance liquid chromatography. The amino acid composition and complete sequence (50 residues) of eel insulin revealed high homology to teleostean and mammalian insulins. The isolated eel insulin produced a more pronounced and longer lasting hypoglycaemic effect than bovine insulin in the eel. Homologous eel insulin, like bovine insulin-like growth factor (IGF-I) and insulin, stimulated sulphate uptake by cultured eel cartilage in a dose-dependent manner (16–1000 ng/ml). Combination experiments using maximal concentrations of bovine IGF-I (250 ng/ml) and increasing amounts of eel insulin (10–250 ng/ml) showed no additive effects of insulin on sulphate uptake, suggesting that insulin and IGF-I may share a common mechanism(s) of action. Eel insulin and bovine IGF-I also enhanced thymidine incorporation by eel cartilage in a dose-dependent manner (4–1000 ng/ml); eel insulin was equipotent with bovine IGF-I. These results suggest that insulin, like IGF-I, may exert direct growth-promoting actions in branchial cartilage of the eel.

Journal of Endocrinology (1992) 133, 221–230
Lipopolysaccharide is able to bypass corticotrophin-releasing factor in affecting plasma ACTH and corticosterone levels: evidence from rats with lesions of the paraventricular nucleus
Authors: Elenkov I. J. , Kovács K. , Kiss J. , Bertók L. , Vizi E. S.
Published: December 2008

Stimulation of the immune system or experimental conditions (bacterial lipopolysaccharide (LPS) treatment) provoke a broad spectrum of physiological responses. It was recently shown that one of them is the activation of the hypothalamic-pituitary-adrenal (HPA) axis. The mechanism and the site or sites through which LPS stimulates the HPA axis are not well understood.

To establish whether the effect of bacterial LPS is related in vivo to the presence of hypothalamic hypophysiotrophic peptides (corticotrophin-releasing factor-41, arginine vasopressin, etc.), plasma ACTH and corticosterone levels were monitored in intact and sham-operated rats, and in rats with paraventricular nucleus lesions in order to remove the main source of these neuropeptides. Evidence was obtained that 4 h after treatment, LPS was able to activate the hypophysial-adrenal system in the absence of hypophysiotrophic neuropeptides of paraventricular origin.

It is suggested that, in vivo, LPS could have a direct effect on the pituitary gland or that it acts through an extrapituitary, non-paraventricular pathway to activate the HPA axis.

Journal of Endocrinology (1992) 133, 231–236
The role of calcitonin gene-related peptide in the regulation of anion secretion by the rat and human epididymis
Authors: Leung A. Y. H. , Leung P. Y. , Cheng-Chew S. B. , Wong P. Y. D.
Published: December 2008

A study was carried out to investigate the role of the calcitonin gene-related peptide (CGRP) in the regulation of electrolyte transport in the rat and human epididymis. In monolayer cultures derived from the rat cauda epididymal cells, CGRP stimulated the short-circuit current (SCC) in a dose-dependent manner with the EC50 (concentration required to produce 50% of the response) at 15 nmol/l. This effect of CGRP was seen when the peptide was added to the basolateral aspect of the cells; apical addition having negligible effect. The CGRP-induced rise in the SCC was dependent on the presence of chloride in the bathing solution. Calcitonin had no effect on the SCC and did not affect the CGRP-induced rise in the SCC. The effect of CGRP on secretion was inhibited in a competitive fashion by the CGRP receptor antagonist CGRP(8–37). In contrast to bradykinin, angiotensin II and endothelin I, the effect of CGRP was independent of prostaglandin synthesis. Measurement of intracellular adenosine 3′:5′-cyclic monophosphate showed a time- and dose-dependent increase upon stimulation with CGRP. CGRP also stimulated the SCC in monolayers grown from the human epididymis. The current could be inhibited by apical application of the chloride channel blocker, diphenylamine-2-carboxylate. Immunoreactive CGRP was found in the epithelia of rat and human cauda epididymidis. It is suggested that CGRP may regulate the electrolyte and fluid secretion in the epididymis, thereby providing an optimal microenvironment for the maturation and storage of spermatozoa.

Journal of Endocrinology (1992) 133, 259–268
Effects of dopamine, high potassium concentration and field stimulation on the secretion of aldosterone by the perfused rat adrenal gland
Authors: Porter I. D. , Whitehouse B. J. , Price G. M. , Hinson J. P. , Vinson G. P.
Published: December 2008

The rat adrenal cortex contains quantities of dopamine that are compatible with its function as a neurotransmitter, suggesting that locally released dopamine may act as a neuroregulator within the gland. This possibility has been tested by comparing the effects of dopamine on aldosterone secretion in the perfused adrenal with the effects of stimuli designed to provoke the release of intraglandular dopamine.

Infusion of dopamine (0·1–100 μmol/l for 10-min periods) into the isolated perfused rat adrenal gland resulted in a transient, dose-related reduction of aldosterone secretion to a minimum of approximately 50% of the basal value at 1 μmol dopamine/l (ratio of experimental to control measurements, R = 0·53 ± 0·06 (s.e.m.); n = 5). In contrast, dopamine (1–100 μmol/l) had no effect on aldosterone production by dispersed zona glomerulosa cell preparations incubated in vitro.

The effects of changes in K+ concentration (3·9–52 mmol/l) on aldosterone secretion in the perfused gland and dispersed cell preparations were also compared. A similar bell-shaped dose–response relationship was seen in both preparations between 6 and 32 mmol K+/l, with a maximum at 8·4 mmol K+/l and a return to control values with 16, 24 or 32 mmol K+/l. However, infusion of media with very high K+ concentrations (42 or 52 mmol K+/l) reduced the secretion of aldosterone by the perfused gland to approximately 50% of the basal value (R = 0·51 ± 0·05, n = 9; R = 0·49± 0·08, n = 9; respectively) but produced no change in aldosterone production by zona glomerulosa cells. Electrical field stimulation (pulse width 1 ms, 1 Hz at 60 V for 5 min) of the perfused gland also resulted in a reduction in aldosterone secretion (R = 0·66 ± 0·66, n = 6). In the presence of 1 μmol haloperidol/l, a dopamine antagonist, no effect on aldosterone secretion was seen under control conditions, but the responses to 1 μmol dopamine/l, 52 mmol K+/l and field stimulation were eliminated.

The results are consistent with the view that aldosterone secretion by the perfused adrenal gland is subject to an inhibitory dopaminergic control, which may originate from catecholaminergic neurones within the gland itself.

Journal of Endocrinology (1992) 133, 275–282
The effects of recombinant human interleukin (IL)-1α, IL-1β or IL-6 on hypothalamo-pituitary-adrenal axis activation
Authors: Harbuz M. S. , Stephanou A. , Sarlis N. , Lightman S. L.
Published: December 2008

We have investigated the effects of recombinant human interleukin (IL)-1α, IL-1β and IL-6 on the activation of the hypothalamo-pituitary-adrenal axis. We have determined the effects of a single i.p. injection of cytokine on circulating ACTH and corticosterone levels, corticotrophin-releasing factor (CRF) mRNA in the parvocellular cells of the paraventricular nucleus and pro-opiomelanocortin (POMC) mRNA in the anterior pituitary at both 4 h and 24 h after injection. IL-1α had no effect on any of the parameters measured at either time-point. In contrast, IL-1β increased CRF mRNA in the parvocellular paraventricular nucleus and POMC mRNA in the anterior pituitary 4 h after injection. Plasma ACTH and corticosterone were increased at 4 h and circulating ACTH was still increased at 24 h after treatment with IL-1β. IL-6 had no effect on message levels but did increase circulating ACTH and corticosterone levels both 4 h and 24 h after injection. The mechanism responsible for the increase in circulating ACTH after IL-6 injection is unclear but would appear to be different from that which is activated by IL-1β which also results in increased CRF and POMC gene expression.

Journal of Endocrinology (1992) 133, 349–355
Insulin-like growth factor-I and its N-terminal modified analogues induce marked gut growth in dexamethasone-treated rats
Authors: Read L. C. , Tomas F. M. , Howarth G. S. , Martin A. A. , Edson K. J. , Gillespie C. M. , Owens P. C. , Ballard F. J.
Published: December 2008

The effects of insulin-like growth factor-I (IGF-I) on the gut of 150 g dexamethasone-treated rats were compared with those of two analogues with reduced affinity for IGF-binding proteins, des(1–3)IGF-I and LR3IGF-I, an N-terminal-extended variant. Administration of IGF-I for 7 days to rats made catabolic by co-treatment with dexamethasone induced a dose-dependent increase in total gut weight, with the highest dose of IGF-I (695 μg/day) increasing gut weight by up to 60%, and gut weight as a fraction of body weight by up to 32%. Effects were apparent in all regions of the gut examined, including the stomach, small intestine and colon. Histological and biochemical analyses of the intestine showed that cross-sectional mass, rather than gut length, was increased, and proportional increases in wet weight, protein and DNA content per unit length were measured in both the mucosa and muscularis layers. The rate of duodenal protein synthesis measured on day 7 of treatment was not increased by IGF-I treatment. The IGF-I analogues had qualitatively similar effects to IGF-I, but were consistently severalfold more potent, providing evidence that IGF-binding proteins reduce the biological activity of exogenous IGF-I in the gut. The results indicate that the gut is one of the most sensitive IGF-I target tissues, and that potency in vivo correlates with a reduced interaction with IGF-binding proteins.

Journal of Endocrinology (1992) 133, 421–431
Exogenous gonadotrophin-releasing hormone (GnRH) stimulates LH secretion in male monkeys (Macaca fascicularis) treated chronically with high doses of a GnRH antagonist
Authors: Weinbauer G. F. , Hankel P. , Nieschlag E.
Published: December 2008

We reported previously that after a single injection of a gonadotrophin-releasing hormone (GnRH) antagonist to male monkeys, exogenous GnRH stimulated LH secretion in a time- and dose-dependent manner, indicating that GnRH antagonist-induced blockade of LH secretion resulted from pituitary GnRH receptor occupancy. The present study was performed to investigate whether GnRH can also restore a blockade of LH and testosterone secretion during chronic GnRH antagonist administration. Four adult male cynomolgus monkeys (Macacafascicularis) received daily s.c. injections of the GnRH antagonist [N-Ac-d-pCl-Phe1,2,d-TRP3,d-Arg6,d-Ala10]-GnRH (ORG 30276) at a dose of 1400–1600 μg/kg for 8 weeks. Before the GnRH antagonist was given and during weeks 3 and 8 of treatment, pituitary stimulation tests were performed with 0·5, 5, 50 and 500 μg synthetic GnRH, administered in increasing order at intervals of 24 h. At 8 weeks, a dose of 1000 μg GnRH was also given. All doses of GnRH significantly (P < 0·05) stimulated serum concentrations of bioactive LH (3- to 8-fold) and testosterone (2·6- to 3·8-fold) before the initiation of GnRH antagonist treatment. After 3 weeks of GnRH antagonist treatment, only 50 and 500 μg GnRH doses were able to increase LH and testosterone secretion. Release of LH was significantly (P < 0·05) more elevated with 500 μg compared with 50 μg GnRH. After 8 weeks, only the highest dose of 1000 μg elicited a significant (P < 0·05) rise in LH secretion. Basal hormone levels just before the bolus injection of GnRH were similar (P > 0·10–0·80). This finding eliminated the possibility that the increasing doses of GnRH had primed the pituitary thereby resulting in higher stimulatory effects of the larger doses of GnRH.

In conclusion, the present data indicate that, even after prolonged exposure to a GnRH antagonist, the pituitary retains some degree of responsiveness to GnRH. This observation supports the view that the inhibitory effects of chronic GnRH antagonist treatment are also mediated, at least in part, by occupancy of the pituitary GnRH receptor rather than by receptor down-regulation.

Journal of Endocrinology (1992) 133, 439–445
Release of interleukin-6 by human thyroid epithelial cells immortalized by simian virus 40 DNA transfection
Authors: Kennedy R. L. , Jones T. H. , Davies R. , Justice S. K. , Lemoine N. R.
Published: December 2008

Interleukin-6 (IL-6) has actions on a variety of endocrine tissues. The cytokine is secreted by cells of the anterior pituitary and endocrine pancreas and has recently been shown to be produced by cultures of thyroid epithelial cells. In this study we have examined some of the factors which regulate IL-6 release from an immortalized human thyroid line (HTori3).

IL-6 release over 24 h was stimulated by TSH (5000 μU/ml), by forskolin (0·01 mmol/l), by fetal calf serum (1–20%) and by epidermal growth factor (20 ng/ml). Stimulation was also apparent with γ-interferon and with tumour necrosis factor at concentrations known to enhance class II major histocompatibility antigen expression by thyroid epithelium. The most potent factor tested was interleukin-1 (IL-1), which controls IL-6 release from other cell types. Threefold stimulation was found with 1 U/ml rising to 350-fold with 1000 U/ml. The effect of IL-1 took 2 h to develop and was blocked by cycloheximide (100 μmol/l). Stimulation was not markedly inhibited by pertussis toxin. Many of the actions of IL-1 are mediated by prostaglandin E2 (PGE2). At concentrations as low as 30 nmol/l, PGE2 stimulated IL-6 release but the maximum stimulation obtained with PGE2 was only threefold. The effect of IL-1 was not inhibited by indomethacin.

These data provide further evidence that IL-6 is produced by human thyrocytes. The effect of IL-1 has not been demonstrated previously. Stimulation of IL-6 release by IL-1 did not appear to be mediated by prostaglandin. IL-6 may influence hormone release from the thyroid as it does in other tissues. High concentrations of IL-6 in the thyroid may increase infiltration by, and activation of, lymphocytes in patients with autoimmune thyroid disease.

Journal of Endocrinology (1992) 133, 477–482
Uptake of l-tri-iodothyronine by human cultured trophoblast cells
Authors: Mitchell A. M. , Manley S. W. , Mortimer R. H.
Published: December 2008

We investigated the uptake of l-tri-iodothyronine (T3) by cultured human trophoblast cells. Uptake was time-dependent, initially linear and approaching equilibrium after 60 min with an approximate half-time of 13 ± 4·5 min (mean ± s.e.m., n = 4). It had a non-saturable component accounting for about 50% of total uptake. We demonstrated a single saturable T3 uptake mechanism with a calculated Michaelis constant (Km) of 755 ± 145 nmol/l (n = 11–13) and a corresponding maximum velocity of 28·8 ± 5·3 pmol/min per mg protein (n = 11–13). The Km value was similar to those reported in other tissues.

Journal of Endocrinology (1992) 133, 483–486
A C TYPE NATRIURETIC PEPTIDE IS A VASODILATOR IN VIVO AND IN VITRO IN THE COMMON DOGFISH
Authors: Bjenning C. , Takei Y. , Watanabe T.X. , Nakajima K. , Sakakibara S. , Hazon N.
Published: December 2008

The effects of an elasmbranch cardiac C-type natriuretic peptide (dogfish CNP-22) on arterial blood pressure were investigated in vivo in chronically cannulated dogfish Scyliorhinus canicula and in vitro by a myographic technique using the distal part of the first branchial artery. In-vivo dogfish CNP-22 caused a dose-dependent reduction in mean arterial blood pressure which was much more potent than that of α-human ANP. In-vitro dogfish CNP-22 also caused a dose-dependent relaxation which was independent of the endothelium. These results are in marked contrast to those obtained in similar studies on other vertebrate species in which CNP exhibited only mild hypotensive effects compared to both atrial and brain natriuretic peptides. This study indicates the importance of using homologous peptides in determing the physiological role of natriuretic peptides in non-mammalian vertebrates.
Effects of a novel hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, on pituitary hormone release in rats
Authors: Hart G. R. , Gowing H. , Burrin J. M.
Published: December 2008

We have demonstrated that the novel hypothalamic peptide pituitary adenylate cyclase-activating poly-peptide (PACAP-38; 0·1–100 nmol/l) caused an increase in the release of GH, ACTH, LH and α-subunit and accumulation of intracellular cyclic AMP from dispersed rat anterior pituitary cells in static culture for 24 h. There were no significant effects on TSH or prolactin release over the same time-period. PACAP-38 (10 nmol/l) increased the release of GH by 1·3-fold (P<0·05), ACTH by 1·9-fold (P<0·05), LH by 3·5-fold (P<0·001) and α-subunit by 2·0-fold (P< 0·005) and the accumulation of intracellular cyclic AMP by >2-fold (P<0·001) after 24 h. However, the time-course for the effect of PACAP-38 (1 mmol/l) on hormone release and intracellular cyclic AMP levels showed a temporal dissociation. The effect of PACAP-38 on GH and ACTH levels did not reach significance until 24 h whereas the effect of PACAP-38 on LH and α-subunit release reached significance after 4 h implying a different mechanism of action for their release.

To investigate the PACAP-induced secretion of LH and α-subunit further, we examined the effects of PACAP after down-regulation of protein kinase C (PKC). PACAP-38 at a dose maximal for the stimulation of LH and α-subunit release (10 nmol/l) added together with the PKC activator, 12-0-tetradecanoyl-phorbol-13-acetate (TPA; 0·1 μmol/l) had no greater effect on LH and α-subunit release than TPA alone over a 4 h incubation period. Increasing the pretreatment time with TPA (0–5 h) at a dose (0·1 μmol/l) known to deplete PKC activity substantially, reduced the ability of PACAP-38 to stimulate LH and α-subunit release and intracellular cyclic AMP levels significantly. We conclude that the stimulatory actions of PACAP on LH and α-subunit relies in part on PKC activity.

Journal of Endocrinology (1992) 134, 33–41
Direct effect of endothelin-1 on the granulosa cells of the porcine ovary
Authors: Kamada S. , Kubota T. , Hirata Y. , Taguchi M. , Eguchi S. , Marumo F. , Aso T.
Published: December 2008

Specific binding sites for endothelin-1 (ET-1), a novel potent vasoconstrictor peptide, as well as the effects of ET-1 on cytosolic free Ca2+ concentration ([Ca2+]i), intracellular total inositol phosphate (IP) generation and steroidogenesis were studied in cultured porcine granulosa cells. Scatchard analysis of a binding study using 125I-labelled ET-1 indicated the presence of a single class of high-affinity binding sites with almost equal affinity for ET-1 and ET-3: the apparent dissociation constant was 0·59 nmol/l and the maximal binding capacity was 1·84 pmol/mg protein. Affinitylabelling of 125I-labelled ET-1 to the membranes using disuccinimidyl tartarate as a cross-linker revealed one major and one minor band with the apparent molecular weights of 32 kDa and 49 kDa respectively. ET-1 dose-dependently (1−100 nmol/l) induced rapid and transient increases in [Ca2+]i in fura-2-labelled cells. ET-1 also dose-dependently stimulated total IPs in cells prelabelled with myo-[3H]inositol. ET-1 had a slight stimulatory effect on the secretion of progesterone but not of oestradiol from porcine granulosa cells. The present data clearly demonstrate the presence of a non-selective ET receptor (ETB) in porcine granulosa cells coupled with phosphoinositide hydrolysis and [Ca2+]i mobilization, and suggest that ET-1 may play some role in the production of progesterone by porcine granulosa cells.

Journal of Endocrinology (1992) 134, 59–66
Tissue kallikrein is associated with prolactin-secreting cells within human growth hormone-secreting adenomas
Authors: Jones T. H. , Figueroa C. D. , Smith C. M. , Cullen D. R. , Bhoola K. D.
Published: December 2008

Tissue kallikrein is a serine protease which may be involved in the intracellular processing of prolactin in the anterior pituitary gland. The expression of tissue kallikrein, in the rat, is promoted by oestrogen and inhibited by dopamine. Human and rat prolactinomas contain markedly increased amounts of tissue kallikrein; this is comparatively reduced if patients are pretreated with the dopamine agonist, bromocriptine, before surgery. Some GH-secreting adenomas are mixed and also contain prolactin-secreting cells. We therefore investigated 27 GH-immunostaining human pituitary adenomas for the presence of immunoreactive tissue kallikrein. Sixteen of the adenomas had positive immunostaining for prolactin; eight of these patients had associated clinical hyperprolactinaemia before the tumour was removed. Tissue kallikrein immunoreactivity was found in ten adenomas, all of which also had prolactin immunopositivity. There was a close relationship between the percentage of cells staining for prolactin and tissue kallikrein but not for GH. A further eight adenomas had patchy positivity, i.e. less than 1% of cells immunostained for tissue kallikrein and six of these also had some prolactin-staining cells. Nine out of eleven purely GH-staining adenomas had no tissue kallikrein immunopositivity, the remaining two showing patchy staining. A review of bromocriptine responsiveness, as assessed by mean GH hormone levels during oral glucose tolerance tests before and after therapy was commenced, indicated that patients with adenomas which stained for prolactin and tissue kallikrein were more likely to respond to bromocriptine than those which failed to do so.

Journal of Endocrinology (1992) 134, 149–154
Comparison of extraction methods for insulin-like growth factor-I in rat serum
Authors: Crawford B. A. , Martin J. L. , Howe C. J. , Handelsman D. J. , Baxter R. C.
Published: December 2008

This study was undertaken to compare various extraction methods for insulin-like growth factor-binding proteins (IGFBPs) from insulin-like growth factor-I (IGF-I) in rat serum systematically, before measurement of IGF-I by radioimmunoassay (RIA). The values obtained in the IGF-I RIA following acid–ethanol (AE), acid-ethanol cryoprecipitation (AEC) and formic acid-acetone (FA) extraction methods were compared with the IGF-I values obtained following high-performance liquid chromatography (HPLC), which was the reference method. Radio-ligand blots were used to determine the pattern and degree of IGFBP removal by these methods.

Over a wide range of circulating IGF-I levels, AE and AEC extraction gave IGF-I levels comparable with those obtained following HPLC. FA extraction resulted in IGF-I levels that were consistently higher (P <0·01) than those obtained following HPLC and gave non-parallel displacement curves in comparison with recombinant IGF-I standards (P <0·01). Ligand blots demonstrated a similar pattern of IGFBP removal among the three methods with almost complete removal of IGFBP-3 but only 30–40% removal of the lower molecular weight IGFBPs. These lower molecular weight IGFBPs did not interfere with the RIA measurements of IGF-I from AE and AEC extracts.

Therefore the AE extraction method of Daughaday, originally validated for use in human serum, is also satisfactory for use in rat serum. The complete removal of IGF-binding activity does not appear essential for accurate measurement of IGF-I by RIA, although this may depend on the specific binding characteristics of the IGF-I antiserum.

Journal of Endocrinology (1992) 134, 169–176
Cytotoxic factor in the serum of diabetic rats and its increase during pregnancy
Authors: Conliffe P. R. , Santos G. L. , Varma D. R. , Mulay S.
Published: December 2008

Diabetic pregnancy is associated with a high incidence of fetal growth abnormalities which cannot be solely ascribed to fetal hyperglycaemia and hyperinsulinaemia. We therefore examined the possibility of other contributing factors using rats made diabetic with streptozotocin as the experimental model. Blood serum from virgin diabetic rats and, to a much greater extent, from pregnant diabetic rats inhibited [3H]thymidine incorporation by fetal lung cells in culture in a concentration- and time-dependent manner; cell number was also decreased. The cytotoxic activity of the serum was decreased by treatment of pregnant diabetic rats with insulin. Sera from non-diabetic rats and from rats at 6 h and 24 h after the injection of streptozotocin were not cytotoxic. The cytotoxic activity of diabetic rat serum was retained after dialysis and was not destroyed by heating it for 60 min at 60 °C. Diabetic rat serum antagonized the stimulatory effects of fetal bovine serum, insulin and insulin-like growth factors on thymidine incorporation by lung cells and inhibited corticosterone production by adrenal cells. Ultrafiltration of diabetic rat serum and high-performance gel permeation chromatography in phosphate buffer suggested that the molecular weight of the cytotoxic factor was approximately 40 kDa. However, gel permeation chromatography in 40% acetonitrile of the cytotoxic eluate from reversed-phase high-performance liquid chromatography using a C18 and C4 column revealed that the cytotoxic factor was a low molecular weight substance, which contained no amino acids. The apparent discrepancy in molecular weights using different separation procedures suggests that the cytotoxic factor is bound to serum proteins. The u.v. spectrum of this factor was different from those of ketone bodies but its exact chemical identity could not be established because of the scarcity of the material. It is suggested that the sera of pregnant diabetic rats and their fetuses contain a cytotoxic factor, which may contribute to fetal developmental abnormalities.

Journal of Endocrinology (1992) 134, 205–214
Interaction between thyrotrophin-releasing hormone and the muscarinic cholinergic system in rat brain
Authors: Garcia S. I. , Dabsys S. M. , Santajuliana D. , Delorenzi A. , Finkielman S. , Nahmod V. E. , Pirola C. J.
Published: December 2008

TRH increases the pressor response to acetylcholine through an increment in muscarinic receptors. As chronic atropinization produces a similar effect, we hypothesized that both phenomena may be related. The effect of chronic atropine treatment on the TRH content of several brain areas in Wistar rats was studied. Atropine produced significant increases in TRH content in the preoptic and septal areas, while decreases were observed in the hypothalamus and hypophysis. The concentration of TRH in cerebrospinal fluid rose significantly in atropine-treated rats compared with controls. A similar effect was observed with eserine, an acetylcholinesterase inhibitor. Finally, perfusion of brain preoptic area slices from normal rats with Krebs–Ringer solution in the presence of pilocarpine increased basal TRH release significantly and this effect was blocked by atropine. These results are compatible with a muscarinic control on the activity of the central TRH system.

Journal of Endocrinology (1992) 134, 215–219
Correlation between Ca2+-ATPase activity of rat islet cells and insulin secretion
Authors: Gronda C. M. , Diaz G. B. , Rossi J. P. F. C. , Gagliardino J. J.
Published: December 2008

Using medium with a low ionic strength, a low concentration of Ca2+ and Mg2+ and devoid of K+, we have measured Ca2+-ATPase activity in the homogenates of rat islets preincubated for 3 min with several hormones in the presence of 3·3 mmol glucose/l. Insulin secretion was also measured in islets incubated for 5 min under identical experimental conditions. Islets preincubated with glucose (3·3 mmol/l) and glucagon (1·4 μmol/l) plus theophylline (10 mmol/l), ACTH (0·11 nmol/l), bovine GH (0·46 μmol/l), prolactin (0·2 μmol/l) or tri-iodothyronine (1·0 nmol/l) have significantly lower Ca2+-ATPase activity than those preincubated with only 3·3 mmol glucose/l. All these hormones increased the release of insulin significantly.

Dexamethasone (0·1 μmol/l) and somatostatin (1·2 μmol/l) enhanced the Ca2+-ATPase activity while adrenaline (10 μmol/l) did not produce any significant effect on the activity of the enzyme. These hormones decreased the release of insulin significantly.

These results demonstrated that islet Ca2+-ATPase activity was modulated by the hormones tested. Their inhibitory or enhancing effect seemed to be related to their effect on insulin secretion; i.e. those which stimulated the secretion of insulin inhibited the activity of the enzyme and vice versa. Hence, their effect on insulin secretion may be due, in part, to their effect on enzyme activity and consequently on the concentration of cytosolic Ca2+. These results reinforce the assumption that Ca2+-ATPase activity participates in the physiological regulation of insulin secretion, being one of the cellular targets for several agents which affect this process.

Journal of Endocrinology (1992) 134, 221–225
Preparation and characterization of an antibody specific to the rat dopamine D2 receptor
Authors: Johnston J. M. , Wood D. F. , James J. D. , Johnston D. G.
Published: December 2008

Antibodies specific to the dopamine D2 receptor have been raised in rabbits using synthetic peptides. The resulting antiserum was sensitive to picogram quantities of peptide as measured by enzyme-linked immunoassay and was shown to have a 33% cross-reactivity with partially purified D2 receptor protein. No detectable cross-reactivity with similarly prepared fungal membranes was observed. D2 receptor preparations from normal rat pituitary cells were used in Western blot analysis. Bands of Mr = 95 000 and 34 000 were detected in these preparations with a third faint band at 120 000. These correspond to the pituitary D2 receptor.

Journal of Endocrinology (1992) 134, 227–233