bluuebunny/crossref_metadata_2025 huggingface dataset:
Vasopressin-stimulated insulin secretion and inositol phosphate production: interactions with glucose and phorbol esters
Authors: Richardson S B , Laya T , VanOoy M
Published: December 2008

Arginine vasopressin (VP) activates phospholipase C (PLC) and stimulates insulin secretion and inositol phosphate (insP) production in mouse islets and clonal hamster beta cells (HIT). The insulin response to VP is reportedly dependent on extracellular glucose and there is evidence that glucose also activates PLC. The interactions of VP and glucose have been further examined in HIT cells. Glucose stimulated insulin secretion but not insP production and VP stimulated both insulin secretion and insP production in the absence of extracellular glucose. However, in the presence of glucose the insulin and insP responses to VP were potentiated. The phorbol ester, tetradecanoylphorbol acetate (TPA), which activates protein kinase C (PKC), stimulated basal insulin secretion but inhibited insP production. In the presence of submaximal concentrations of VP, 100 nmol/l TPA inhibited VP-stimulated inositol monophosphate production and there was no additivity of stimulated insulin secretion. With a maximal (10 nmol/l) concentration of VP, TPA caused additive insulin secretion and insP levels were stimulated above baseline. Twenty-four hour preincubation with TPA to down-regulate PKC did not inhibit the insulin response to VP. We conclude that extracellular glucose does not activate PLC and is not required for VP-stimulated insulin secretion although it potentiates VP-stimulated insulin secretion and insP production.

Journal of Endocrinology (1995) 145, 221–226
Opioids modify the release of LH at the pituitary level: in vitro studies with entire rat pituitaries
Authors: Dragatsis I , Papazafiri P , Zioudrou C , Gerozissis K
Published: December 2008

It is currently accepted that opioids modify the secretion of LH by affecting the release of GnRH in the hypothalamus. A direct action of opioids at the pituitary level is not yet fully established. To this end, we tested the effects of opioids on the release of LH by the entire pituitary in adult male rats. Opioid agonists with mu (DAGO), delta (DSLET) and kappa (U-50488H) specificity were tested at 0·01 to 10 μm in static incubations. DAGO inhibited dose-dependently the spontaneous and GnRH-induced release of LH. DSLET inhibited only the GnRH-induced release of LH. On the other hand, U-50488H increased spontaneous LH release dose-dependently. The opioid antagonists naloxone, diallyl-G (delta antagonist) and MR 2266 (kappa antagonist) blocked the effects induced by DAGO, DSLET or U-50488H respectively, implying an opioid receptor-mediated effect. The above results showed that opioids with mu, delta and kappa specificity act on the entire pituitary and modify differentially the release of LH. In this study we also compared spontaneous and GnRH-induced LH release by anterior and entire pituitaries and found that the amount of LH released by the anterior lobe was twofold higher, suggesting that inhibitory factors present in the neurointermediate part may affect the release of LH.

Journal of Endocrinology (1995) 145, 263–270
3,5-Di-iodo-l-thyronine suppresses TSH in rats in vivo and in rat pituitary fragments in vitro
Authors: Horst C , Harneit A , Seitz H J , Rokos H
Published: December 2008

3,5-Di-iodo-l-thyronine (T2) is a naturally occurring metabolite of thyroxine (T4). Contrary to earlier findings, T2 has recently been shown to have rapid effects in rat liver and in mononuclear blood cells. In the experiments described here, T2 was tested to determine whether it has a TSH suppressive effect in rats in vivo and in rat pituitary fragments in vitro.

In experiments over 2 weeks in rats in vivo, low doses of T2 (20–200 μg/100 g body weight per day) had no significant influence on body and organ weights, but significantly decreased TSH and T4 serum concentrations. At 200 μg/100 g per day, T2 suppressed TSH to 43% and T4 to 29% of control levels. At 1–15 μg/100 g per day, 3,5,3′-tri-iodo-l-thyronine (T3), used as a comparison to T2, had significant effects on TSH and T4 levels, and also on body weight. Fifteen μg T3/100 g per day decreased TSH to 44%, T4 to 25%, and body weight to 59% of control levels.

In experiments over 3 months in rats in vivo, a low dose (25 μg/100 g per day) of T2 suppressed TSH to 60% and T4 to 57% of control levels and had no significant influence on other parameters. Conversely, 0·1 μg/100 g per day T3 had significant effects on body and organ weights as well as pellet intake, but a less pronounced TSH suppressive effect: TSH concentrations were unchanged and T4 concentrations were down to 80% of control values.

In rat pituitary fragments in vitro, a clear suppression of TSH secretion after a TRH pulse was demonstrated.

To summarise, T2 is a specific agonist in the negative feedback mechanism on TSH secretion at the pituitary level without other apparent thyromimetic effects.

Journal of Endocrinology (1995) 145, 291–297
Ontogeny of the GH response to phorbol ester and phospholipase C in rat pituitary cells
Authors: Cuttler L , Collins B J , Szabo M
Published: December 2008

GH secretory patterns undergo marked change during early mammalian development. The factors that underlie these changes and the major components of signal transduction in the immature somatotrophs are not fully understood. Increasing evidence suggests that protein kinase C (PKC) plays a central role in perinatal organ differentiation and function. To evaluate the possible role of PKC as a mediator of GH secretion from immature pituitaries, we tested the effects of the PKC activating phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA), alone or together with GH-releasing factor (GRF), somatostatin (SRIF), and Ca2+ modifying agents; an inactive phorbol analogue (4α-12–13-didecanoate; 4α-PDD), and phospholipase C on GH release from pituitary cell cultures from perinatal and mature rats.

Pituitary primary cell cultures were prepared from fetal (day 20 of 21·5 days of gestation), 2-day-old, 12-day-old, and adult male (2- to 4-month-old) rats. Each experiment was performed on at least three separate occasions. The magnitude of TPA (0·15–150 nm)-induced GH release was markedly age-dependent, fractional GH release being greatest from pituitaries of fetal and newborn rats, and least from those of adults (P<0·001). Further, the minimum dose of TPA required to stimulate GH release over basal levels was tenfold higher for adult pituitaries (15 nm) than for perinatal pituitaries (1·5 nm). Phospholipase C (1 and 10 U/ml) also caused greater fractional GH release from neonatal pituitaries than from adult pituitaries (P<0·01). By contrast, the inactive phorbol ester 4αPDD did not stimulate GH release from pituitary cells of 2-day-old, 12-day-old or adult rats.

SRIF (0·1–10 nm) inhibited TPA (150 nm)-induced GH release in a dose-dependent manner, but the degree of inhibition was significantly less in perinatal than in mature pituitaries (P<0·001). Combined treatment with near maximal stimulatory doses of GRF (1 nm) and TPA (150 nm) resulted in a partially additive GH response. Inhibitors of Ca2+ mobilization reduced TPA-induced GH release in all age groups, although GH suppression was least in pituitaries of immature rats.

These findings indicate that PKC activators such as TPA and phospholipase C are potent secretagogues of GH in perinatal pituitaries. The action of phorbol ester was mediated by Ca2+-dependent pathways in both immature and mature pituitaries. Somatotroph responsiveness to TPA and phospholipase C was strikingly age-dependent, with greatest GH response in the perinatal period and declining response with advancing age after birth. These data suggest that immature somatotrophs possess functional PKC-dependent signal transduction pathways whose relative contribution declines during postnatal development.

Journal of Endocrinology (1995) 145, 307–314
IGF-I and serine protease inhibitor 2·1 nuclear transcript abundance in rat liver during protein restriction
Authors: Hayden J M , Straus D S
Published: December 2008

Restriction of dietary protein consumption of young male rats results in decreased growth velocity and a reduction in the abundance of hepatic IGF-I mRNA. It is not known whether the reduction in IGF-I mRNA abundance in the liver of protein-restricted rats results from a decrease in IGF-I gene transcription. In the present study, three experiments were performed with 4-week-old male rats to examine the effect of protein restriction on IGF-I gene transcription in liver. In these experiments, we monitored IGF-I nuclear transcripts (pre-mRNA) within total cellular RNA using a ribonuclease protection assay.

In the first experiment, a consistent decrease in IGF-I mRNA from animals fed isocaloric diets containing 20% (control), 12%, 8% and 4% protein (dietary effect, P<0·001) was not paralleled by a decrease (P>0·50) in IGF-I pre-mRNA. Two additional experiments examining the effect of 4% vs 20% protein diets yielded comparable results. Pooled results from these two studies (n=12/treatment) demonstrated that a 64% reduction (P<0·0001) in IGF-I mRNA abundance was not accompanied by a decrease in IGF-I pre-mRNA (1·17 vs 1·31 ±0·21 image density units for 4% and 20% protein treatments). Unlike IGF-I, the abundance of carbamyl phosphate synthetase-I (CPS-I) pre-mRNA and mRNA was comparably reduced (∼70%, P<0·001), indicating that the decrease in mRNA of this urea cycle enzyme during protein restriction occurs predominantly by a transcriptional mechanism. A common feature of all experiments was a pronounced variability in the expression of hepatic IGF-I pre-mRNA among animals, which was not diet specific. To test whether the variability in IGF-I gene transcription was correlated with variability in the transcription of another gene that is regulated by GH, we quantified the abundance of nuclear transcripts for the serine protease inhibitor 2·1 (SPI 2·1 gene. A positive association (r=0·81, P<0·0001) between SPI 2·1 and IGF-I nuclear transcripts was demonstrated. The correlation between IGF-I and SPI 21 transcripts was specific, because the quantity of IGF-I and CPS-I nuclear transcripts was not correlated in this study. Although transcription of the IGF-I and SPI 2·1 genes was similar, the abundance of SPI 21 mRNA was not altered by protein deprivation.

In summary, these studies indicated that protein restriction does not substantially alter the mean quantity of IGF-I nuclear transcripts, suggesting that the decrease in IGF mRNA occurs predominantly by a post-transcriptional mechanism. In addition, nuclear transcript abundance of the IGF-I and SPI 21 genes varies in a co-ordinate manner, supporting the hypothesis that transcription of these genes responds rapidly to a common variable factor such as plasma GH.

Journal of Endocrinology (1995) 145, 397–407
Consistent GH responses to repeated injections of GH-releasing hexapeptide (GHRP-6) and the non-peptide GH secretagogue, L-692,585
Authors: Fairhall K M , Mynett A , Smith R G , Robinson I C A F
Published: December 2008

GH release is normally stimulated by the naturally occurring GH-releasing factor (GRF). However, smaller GH-releasing peptides (GHRPs) and non-peptide analogues have been described which stimulate GH release in animals and man. Although these compounds release GH in vitro, their in vivo activity in conscious animals has proved more difficult to study since the GH responses are variable, and prone to desensitization. We now compare the GH-releasing properties of GHRP-6 and a novel benzolactam GH secretagogue L-692,585 using chronically cannulated guinea pigs and automated blood microsampling to study the effects of repeated exposure to these secretagogues. L-692,585 was approximately tenfold less potent than GHRP-6 for GH release, but it synergized strongly with GRF. Serial injections of GRF, GHRP-6 or L-692,585 at intervals of 60 or 90 min produced variable GH release which followed a cyclic pattern of responsiveness. Prolonging the pulse interval to 3 h produced more regular responses to both GHRP-6 and L-692,585. Continuous i.v. infusion of low doses of either secretagogue elicited an initial GH release, and amplified the spontaneous GH secretory pattern over the next 6 h. We conclude that L-692,585 and GHRP-6 share similar in vivo properties, and that intermittent responsiveness to frequent injections is similar for all three secretagogues, and is a property of the conscious animal rather than of any secretagogue type. More consistent responses can be obtained with less frequent injections that more closely match the endogenous GH rhythm, whereas continuous exposure to these secretagogues leads to amplified endogenous secretion. Our results show that the interpretation of in vivo effects of these peptide and non-peptide secretagogues will need to take account of their interaction with the endogenous mechanisms governing GH release.

Journal of Endocrinology (1995) 145, 417–426
Characteristics of the binding of 32P-labelled human relaxins to the human fetal membranes
Authors: Garibay-Tupas J L , Maaskant R A , Greenwood F C , Bryant-Greenwood G D
Published: December 2008

The two human relaxin genes termed H1 and H2 are expressed in the choriodecidua and placenta and have been proposed to act via specific receptors as local modulators of collagenolysis in the fetal membranes. Such receptors have been inferred, but not demonstrated, from studies of the effect of adding exogenous relaxin to these tissues. Thus conditions were optimized for the binding of 32P-labelled human relaxin H2 to membrane-enriched particulate fractions of human fetal membranes, amnion and chorion, with adhering decidua. The membrane protein concentration was optimal at 250 μg, when incubated at 27 °C for 60 min, at pH 7·5 with Mn2+ and Mg2+ ion concentrations of 2·0 mm. Incubation of membrane particulate fractions with increasing amounts of labelled relaxin H2 suggested the presence of a single class of binding sites with an affinity constant (Ka) of 2·15 nm. The binding was primarily to the chorion and decidua with very little to the amnion layer. The competition for binding of the 32P-labelled human relaxin H2 with unlabelled relaxin H2 gave an IC50 of 28 pm, while an IC50 of 60 pm and 280 pm was obtained for relaxin H1 and porcine relaxin respectively. In contrast, unlabelled guinea-pig relaxin inhibited this binding by only 10% even at a 1000-fold greater concentration than H2, and human recombinant insulin failed to inhibit even at a million-fold concentration of unlabelled relaxin H2.

Relaxins H2 and H1 can readily displace the binding of either 32P-labelled human relaxins H1 or H2 and gave very similar displacement curves. The binding affinity of relaxin H2, however, was fivefold higher than that of relaxin H1. These data provide evidence for the presence of a relaxin receptor that may preferentially bind relaxin H2.

Journal of Endocrinology (1995) 145, 441–448
Pituitary-adrenal function in the immature ovine foetus
Authors: McFarlane A C , Potocnik S , Towstoless M , Moritz K , Wintour E M
Published: December 2008

Pituitary-adrenal responses to intravenous infusion of ovine corticotrophin-releasing hormone (oCRH) or arginine vasopressin (AVP) and to haemorrhage were examined in the ovine foetus prior to 90 days of gestation (term 145–150 days). In chronically cannulated foetuses (n=8), between 74 and 84 days of gestation, basal ACTH levels were less than 20 pg/ml while cortisol levels were 6·5 ± 1·5 nmol/l (mean±s.e m.). Intravenous infusion of oCRH (1 μg/h for 60 min) or AVP (1 μg/h for 60 min) significantly increased ACTH (P<0·05 for both treatments) and cortisol (P<0·01 for both treatments) levels, although the response to both hormones was modest. In acutely studied foetuses of a similar age (70–90 days of gestation, mean 82·0 ± 1·4 days, n=7), exteriorization and progressive haemorrhage significantly (P<0·05) elevated ACTH levels from 117·4 ± 32·1 pg/ml to a maximal value of 329·2 ± 112·8 pg/ml, the maximal ACTH response corresponding to the removal of a volume of blood equivalent to 6·6 ±1·2% of the pre-haemorrhage body weight. The present study has demonstrated that the ovine foetal pituitary, in vivo, is responsive to exogenous and endogenous stimuli by mid-gestation and, at this age, although basal cortisol levels are low, the foetal adrenal is capable of responding to elevated ACTH levels in the short term.

Journal of Endocrinology (1995) 145, 455–460
Plasma clearance, tissue uptake and expression of pituitary peptide 23/pancreatitis-associated protein in the rat
Authors: Chakraborty C , Sharma S , Katsumata N , Murphy L J , Schroedter I C , Robertson M C , Shiu R P C , Friesen H G
Published: December 2008

The secretion of peptide 23 by rat pituitary cells is stimulated by growth hormone-releasing hormone and inhibited by somatostatin. Recent cloning of the cognate cDNA for peptide 23 revealed that it is identical to pancreatitis-associated protein (PAP). In the present study, the clearance and tissue uptake of recombinant peptide 23/PAP in normal adult male rats was assessed. The plasma half-life of recombinant peptide 23/PAP was 4·8 ±1·4 (s.d.) min. Maximal accumulation of radiolabelled peptide 23/PAP was observed in the kidney, stomach, small intestine and pancreas whereas negligible uptake was seen in the liver, lung or heart. Peptide 23/PAP was detected in a variety of tissue extracts using a radioimmunoassay. Extracts of ileum contained the highest concentrations of peptide 23/PAP. In situ hybridization analysis showed that peptide 23/PAP mRNA was highly expressed in the columnar epithelial cells of ileum, jejunum and duodenum. These observations demonstrate that peptide 23/PAP, a protein previously thought to be of pituitary origin, is widely expressed in the gastrointestinal tract and that it is rapidly removed from the circulation by the kidney and by tissues which express peptide 23/PAP.

Journal of Endocrinology (1995) 145, 461–469
Epidermal growth factor stimulates cell proliferation and inhibits iodide uptake of FRTL-5 cells in vitro
Authors: Asmis L M , Gerber H , Kaempf J , Studer H
Published: December 2008

In accordance with the available data most authors conclude that epidermal growth factor (EGF) has very little or no effect on FRTL-5 cells. This has been viewed as a serious handicap of this cell line. In the present study we cultivated three strains of FRTL-5 cells from different sources and assessed their response to EGF with regard to proliferation, function and differentiation.

Cell proliferation was assessed by counting in a Coulter cell counter after culturing cells at suboptimal conditions in well plates. Cell function was studied by measuring iodide uptake. Cell differentiation was examined immunocytochemically by staining monolayer cultures for thyroglobulin (Tg) and EGF receptor (EGFr) as well as morphologically by microscopical evaluation of monolayer cultures.

All three FRTL-5 cell lines investigated express EGFr. In two wild type FRTL-5 cell lines EGF stimulates growth, an effect that is enhanced by the presence of TSH, and partially inhibits iodide uptake. A third mutated strain of FRTL-5 cells does not respond to EGF. Tg expression can be demonstrated immunocytochemically in EGF-treated cells as well as in controls. Morphologically, in monolayer culture EGF-treated cells cannot be distinguished from controls.

Contrary to previous reports, these studies demonstrate EGF effects on FRTL-5 cells that are consistent with EGF effects established in other thyroid follicular cells.

Journal of Endocrinology (1995) 145, 513–520
Investigation into the potential physiological sources of rat milk IGF-I and IGF-binding proteins
Authors: Donovan S M , Hintz R L , Rosenfeld R G
Published: December 2008

We have previously reported the presence of IGF-I and IGF-binding proteins (IGFBP-2, -3 and -4) in rat milk. Herein, the potential sources of rat milk IGF-I and IGFBPs were investigated. Lactating dams (day 14 postpartum) were separated from their pups and injected intraperitoneally with 0·45 μCi 125I-IGF-I or 125I-IGFBP-3. After 3 h, serum and milk of rats receiving 125I-IGF-I contained 7642 ± 3121 and 14 455 ± 7837 c.p.m./ml respectively. Serum and milk of rats given 125I-IGFBP-3 contained 7232 ± 1366 and 10 371 ± 4091 c.p.m./ml respectively. Sephacryl S-200 gel filtration chromatography demonstrated that the 125I-IGF-I in both serum and milk was primarily in the 150 kDa IGF-binding complex, whereas the distribution of 125I-IGFBP-3 differed between serum and milk. In serum, most of the 125I-IGFBP-3 was in the 150 kDa fraction, while most 125I-IGFBP-3 in milk was in the 40 kDa fraction. Northern analysis of liver showed IGFBP-1 and -3 mRNA expression, with variable expression of IGFBP-2 and -4 mRNA. In contrast, mammary tissue expressed only IGFBP-2 and -4 mRNA, suggesting that these IGFBPs in milk may arise from de novo synthesis within the mammary gland. The lack of detectable IGFBP-3 mRNA in mammary tissue and the translocation of 125I-IGFBP-3 from the serum suggest that milk IGFBP-3 arises from the maternal circulation.

Journal of Endocrinology (1995) 145, 569–578
PROTEINS IN TISSUE EXTRACTS WHICH BIND INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-3 (IGFBP-3)
Authors: Hodgkinson S. , Fowke P. , Somai N. Al , McQuoid M.
Published: December 2008

Membrane associated IGFBP-3 is now known to play a role in the modulation of IGF at the cellular level, but mechanisms involved in cell membrane binding are far from certain. In this study we report the identification and initial structural characterisation of proteins in a range of sheep and rat tissues which specifically bind recombinant human non-glycosylated IGFBP-3. Tissues were homogenised in Tris HCl (0.1 M, pH 7.4), containing proteolytic enzyme inhibitors and the residues re-extracted in buffer containing SDS and Triton X-100 (both 1% w/v) prior to analysis. These were subjected to SDS-PAGE, electro-blotted onto nitrocellulose and subjected to ligand blot analysis (LBA) using radioiodinated IGFBP-3 as ligand. LBA revealed a major band of binding activity migrating at 60 kDa in extracts of rat muscle while sheep muscle contained forms of 52 and 40 kDa as the principal species and ovine pancreatic extracts an abundance of the 40 kDa variant alone. Distribution of the binding activity appears tissue specific. Apart from skeletal muscle, pancreas and a small amount of the 52 kDa form in pituitary, analysis revealed no evidence of IGFBP-3 binding in a range of other sheep tissues including liver, kidney, spleen, intestine, adrenal, brain, mammary, uterus, ovary and plasma. The binding activity is TCA precipitable, trypsin digestible, dose responsive and relatively specific for IGFBP-3 since the related proteins recombinant human IGFBP-2 and purified caprine IGFBP-4 failed to bind. Additionally, IGFBP-3 binding to these species appeared unaffected by presaturation of IGFBP-3 with IGF-I, nor by their transient acidification which together with molecular weight estimates and their pattern of tissue distribution suggests they are not related to the acid labile subunit of the ternary 150 kDa IGFBP complex. IGFBP-3 binding and molecular mobility of the species were unaffected by sample reduction but binding was suppressed in a concentration dependent fashion by heparin. Finally LBA using 125I-Concanavalin A revealed the 40 kDa species to be glycosylated while the 52 kDa form was not. Functions of these novel proteins in targeting or modulating IGF activity at the cellular level remain to be clarified.
Effect of restriction of placental growth on expression of IGFs in fetal sheep: relationship to fetal growth, circulating IGFs and binding proteins
Authors: Kind K L , Owens J A , Robinson J S , Quinn K J , Grant P A , Walton P E , Gilmour R S , Owens P C
Published: December 2008

To determine whether tissue production of the IGFs is altered when fetal growth is retarded, IGF-I and -II mRNAs were measured in tissues of fetal sheep subjected to placental restriction and the relationships between IGF gene expression, circulating IGF protein and fetal growth were examined. The majority of potential placental attachment sites were surgically removed from the uterus of 12 non-pregnant ewes to restrict placental size in a subsequent pregnancy. Blood and tissues were collected at 121 days of gestation (term=150) in 12 fetuses with restricted placental size and eight normal fetuses. IGF-I and IGF-II mRNA was detected by solution hybridization/ribonuclease protection assay in placenta and all fetal tissues studied. IGF-I mRNA was most abundant in skeletal muscle and liver and IGF-II mRNA was highest in kidney and lung. Restriction of placental size reduced fetal weight by 17% and reduced the pO2 (18%) and glucose concentration (23%) of fetal blood. Placental restriction also reduced IGF-I mRNA in fetal muscle (P<0·002), lung (P<0·05) and kidney (P<0·01) but had no significant effect on IGF-II mRNA in any tissue. IGF-I mRNA in fetal liver, kidney and skeletal muscle correlated positively with the concentration of IGF-I protein in fetal blood (P<0·01). There was no relationship between the concentration of IGF-II protein in fetal blood and IGF-II mRNA in any fetal tissue examined. The concentration of IGF-binding protein-3 (IGFBP-3) in fetal arterial blood plasma measured by RIA correlated positively with fetal weight and with plasma IGF-I. This study shows that restriction of placental growth in sheep reduces circulating levels of IGF-I and IGFBP-3 in the sheep fetus and reduces the capacity of the fetus to produce IGF-I at a number of tissue sites. Altered production of IGF-I, but not IGF-II, by fetal tissues may contribute to retarded fetal growth.

Journal of Endocrinology (1995) 146, 23–34
Corticotrophin-releasing hormone (CRH)-binding protein interference with CRH antibody binding: implications for direct CRH immunoassay
Authors: Linton E A , Perkins A V , Hagan P , Poole S , Bristow A F , Tilders F , Corder R , Wolfe C D A
Published: December 2008

Direct immunoassay of plasma corticotrophin-releasing hormone (CRH) is potentially subject to interference from high levels of CRH-binding protein (CRH-BP) that exist in the human circulation. In this study, we tested the effect of CRH-free, native CRH-BP (6·4 nmol/l) purified from human plasma, CRH-BP diluent alone, normal human plasma (containing 5·8 nmol endogenous CRH-BP/1) and normal sheep plasma (containing no CRH-BP) on the binding of 125I-labelled CRH tracer to five N-terminal and four C-terminal CRH antibodies. All anti-(1–20)CRH N-terminal antibody dilution curves displayed marked inhibition of binding in the presence of purified CRH-BP and human plasma in comparison with the curves with the control diluent or sheep plasma. Almost no inhibition of binding was obtained with any of the C-terminal antibodies (all directed against epitopes within the last six amino acids of CRH) and the four dilution curves were nearly superimposable. Liquid-phase CRH IRMAs were then developed with different combinations of two of each of the N- and C-terminal antibodies, using radiolabelled IgG prepared from purified C-terminal antisera as tracer and raw N-terminal antisera as the link antibodies to the separating system. The addition of dilutions of purified CRH-BP over the range 1·25–20 nmol/l to the IRMA standard curve in assay buffer resulted in a dose-dependent reduction in the signal; with 5 nmol CRH-BP/1, a level commonly found in human plasma, the reduction in binding was 67% and 81% in two different IRMAs at a CRH concentration of 631 pmol/l. With endogenous CRH-BP in human plasma, a dose-dependent inhibition of binding similarly resulted, with the plasma containing the most CRH-BP causing the greatest inhibition. Since plasma CRH-BP levels in humans vary widely, direct plasma IRMA using these type of antibodies will give inaccurate results and initial extraction of the CRH is necessary. Methanol extraction of synthetic or endogenous CRH is shown to be both highly efficient and unaffected by variable amounts of endogenous or exogenous CRH-BP; it is therefore suitable as the first step in plasma CRH measurement by IRMA.

Journal of Endocrinology (1995) 146, 45–53
Acute hyperinsulinemia, androgen homeostasis and insulin sensitivity in healthy man
Authors: Ebeling P , Stenman U-H , Seppälä M , Koivisto V A
Published: December 2008

The acute effects of hyperinsulinemia on androgen homeostasis and a possible association of androgens to insulin sensitivity, serum lipids and lipoproteins and to lipid oxidation were examined in 19 healthy males (27 ± 1 yrs, body mass index 24 ± 1 kg/m2). In each subject, a 240 min euglycemic hyperinsulinemic clamp was performed and glucose and lipid oxidation were determined by indirect calorimetry. During hyperinsulinemia serum sex hormone-binding globulin (SHBG) concentration decreased by 5% (P<0·01), insulin-like growth factor binding protein (IGFBP-1) by 88% (P<0·001) and dehydroepiandrosterone sulphate (DHEAS) by 12% (P<0·001), with no change in total or free testosterone concentrations. In the basal state, IGFBP-1 and C-peptide were inversely related (r= −0·54, P<0·05). Fasting concentrations of serum free testosterone (r=0·59, P<0·01) and DHEAS (r=0·47, P<0·05) correlated positively with serum free fatty acid (FFA) concentrations during hyperinsulinemia, but not with fasting FFA level. Lipid oxidation rate in the basal state correlated positively to the decline in SHBG (r=0·61, P<0·01) and DHEAS concentrations (r=0·62, P<0·01) during hyperinsulinemia. While the fasting serum high density lipoprotein cholesterol level correlated positively with the insulin-induced decline in DHEAS level (r=0·58, P<0·01), no associations were found between serum androgens and total cholesterol, low density lipoprotein cholesterol or triglyceride concentrations. Insulin sensitivity was not related to SHBG, IGFBP-1, DHEAS or testosterone concentrations. It is concluded that, in the healthy man with normal androgen homeostasis, (1) acute hyperinsulinemia decreases SHBG, IGFBP-1 and DHEAS concentrations, (2) the relative insulin-induced decline of IGFBP-1 level is 18-fold greater than that of the SHBG level, (3) androgens may maintain lipolysis during hyperinsulinemia and (4) there is no association between physiological androgen concentrations and insulin sensitivity.

Journal of Endocrinology (1995) 146, 63–69
Effect of ovarian steroid hormones and the presence of the fetus on oxytocin gene expression in the uterus
Authors: Higuchi T , Liu C-X , Saito H , Negoro H , Matsukawa S
Published: December 2008

Oxytocin (OT) is a neurohypophysial hormone with potent stimulating activity of the pregnant uterus, but its physiological role in parturition is still unclear. Recently, OT was found to be synthesized in the pregnant uterus, indicating that OT originating from the uterus, not from the posterior pituitary gland, may trigger the onset of labour. In order to define the factors responsible for the induction of uterine OT, the effect of ovarian steroid hormones and conceptus on the induction of OT mRNA in the rat uterus was examined by Northern and dot blot hybridization analysis. OT mRNA in the uterus started to increase on day 14 of pregnancy and showed very high levels at the time of parturition. Uterine OT mRNA was not altered by any steroid treatment, oestradiol-17β (0·2 μg), progesterone (4 mg) or both in combination, for 6 days. The gravid horn of the uterus had 3·6-fold as much OT mRNA as the non-gravid horn on day 21 of pregnancy in hemipregnant rats with one ligated oviduct. The ovarian steroid hormones could not induce accumulation of OT mRNA in the uterus of ovariectomized rats, at least under the conditions used, but the presence of a conceptus may be critical for the very high levels of OT mRNA.

Journal of Endocrinology (1995) 146, 81–85
Suppression of LH secretion in food-restricted lactating females: effects of ovariectomy and bromocryptine treatment
Authors: Walker C-D , Mitchell J B , Woodside B C
Published: December 2008

It has been shown that restricting food in lactating rats for the first 2 weeks postpartum at a level of 60% of the ad-libitum daily ration increases the length of lactational dioestrus by about 7 days but little is known about correlated changes in hormone levels. In the first experiment we report changes in LH, prolactin (PRL) and ACTH secretion in food-restricted and ad-libitum fed lactating rats at various stages of lactation. Our results demonstrate that food restriction during the first 2 weeks of lactation did not affect PRL or ACTH secretion, but decreased plasma LH levels despite comparable GnRH receptor density between food-restricted and ad-libitum fed females. In the second experiments we investigated a possible causal relationship between the increased secretion of progesterone seen in food-restricted females and the suppression of plasma LH levels, by determining the effects of bromocryptine treatment and ovariectomy on LH secretion in both ad-libitum fed and food-restricted lactating females. LH suppression in food-restricted lactating females was not affected by ovariectomy or bromocryptine treatment, although the latter treatment significantly increased GnRH receptor number. These data suggest that factors other than ovarian steroids, PRL or increased adrenocortical activity modulate LH secretion and the length of lactational dioestrus in food-restricted lactating females.

Journal of Endocrinology (1995) 146, 95–104
Type II thyroxine 5′-deiodinase in the rat thymus
Authors: Molinero P , Osuna C , Guerrero J M
Published: December 2008

In the present study we have shown type II thyroxine 5′-deiodination (5′D) in the rat thymus. The enzyme activity was identified in crude extract homogenates by measuring the 125I released from [3′,5′-125I]thyroxine which is used as a substrate of the reaction. The release of 125I is dependent on protein tissue concentration, time, temperature and pH, and is saturable by increasing the substrate concentration, indicating its enzymatic nature. Characteristics of the enzyme activity also include a low Km (9·1 nm), its dependence on dithiothreitol, and its inhibition by iopanoic acid, but not by propylthiouracil. Experiments to investigate the cellular location of the enzyme in the thymic gland showed that the enzyme is present in both stromal cells and thymocytes. At the subcellular level, 5′D activity was associated with cellular membranes. Thyroid status appears to regulate 5′D activity in rat thymus. Hypothyroidism caused an increase in thymus 5′D activity. The Km value remained unchanged (9·1 vs 10·5 nm) during hypothyroidism, but Vmax increased significantly from 17·7 fmol/mg protein per h in euthyroid rats to 53·5 fmol/mg protein per h in hypothyroid rats. 5′D activity was also modulated by catecholamines through β-adrenergic receptors because isoproterenol, but not methoxamine or clonidine, could activate the enzyme. Because these characteristics define the type II iodothyronine-deiodinating pathway in other tissues, we suggest that the rat thymus also shares this pathway.

Journal of Endocrinology (1995) 146, 105–111
Pronounced seasonal rhythms in plasma somatolactin levels in rainbow trout
Authors: Rand-Weaver M , Pottinger T G , Sumpter J P
Published: December 2008

The aim of this study was to establish whether there are seasonal or age-related changes in circulating levels of somatolactin (SL) in rainbow trout (Onchorhynchus mykiss). SL levels were determined in blood sampled at monthly intervals over a 2-year period from a population of rainbow trout maintained under a natural daylength and temperature regime (North-West England, latitude 54°20′ N). SL levels displayed a distinct circannual cycle, with peak levels in summer (17–20 μg/l) and lowest levels occurring in winter (0·2–2 μg/l). This variation in SL levels was closely correlated with water temperature (P<0·001) but was out of phase with changes in daylength. Plasma SL levels were significantly higher in both mature male and female fish compared with immature fish. Plasma prolactin levels were determined to provide information on a hormone structurally related to SL, and also because mammalian prolactin is known to show distinct seasonal cycles. However, trout prolactin, in contrast to SL, did not show a pronounced seasonal pattern, although prolactin levels were significantly, but inversely, correlated with water temperature (P<0·001).

Journal of Endocrinology (1995) 146, 113–119
Differential acetylation of pro-opiomelanocortin-derived peptides in the pituitary gland of Xenopus laevis in relation to background adaptation
Authors: van Strien F J C , Galas L , Jenks B G , Roubos E W
Published: December 2008

Immunocytochemical analysis revealed the presence of acetylated endorphins in both melanotropes and corticotropes of the pituitary gland of Xenopus laevis. Chemical acetylation studies to determine the steady-state level of acetylated versus non-acetylated endorphins showed that virtually all endorphins are acetylated in both melanotropes and corticotropes. Apparently Xenopus is unique among vertebrates as non-acetylated endorphins are major endproducts in the distal lobe of all other vertebrate species studied thus far. The dynamics of endorphin biosynthesis in melanotrope cells using pulse-chase analysis coupled to immunoaffinity chromatography revealed that processing of pro-opiomelanocortin to produce N-terminalacetylated endorphins is very rapid. To determine the effect of long-term background adaptation on acetylation status of endorphins and α-MSH-related peptides, Xenopus laevis were adapted for 3 or 6 weeks to either a black or a white background. In both physiological states the major intracellular form of α-MSH-related peptides in melanotropes was desacetyl α-MSH while the major endorphin-related peptide was α,N-acetyl-β-endorphin[1–8]. In the medium of superfused neurointermediate lobes of black background-adapted animals the major form of secreted melanotropins and endorphins was α-MSH and α,N-acetyl-β-endorphin[1–8] respectively. We conclude that there is a marked spatio-temporal difference in acetylation of melanotropin and endorphins, with rapid intracellular acetylation of endorphins while melanotropin is acetylated at the time of its exocytosis. In the medium of superfused neurointermediate lobes of white background-adapted animals the amount of desacetyl α-MSH was much higher than in the medium of lobes of black-adapted animals. Therefore, the secretory signals from melanotrope cells of black- and white-adapted Xenopus appear to differ with respect to the degree of acetylation of the melanotropins. This difference may underlie the strategy of Xenopus to regulate dermal melanophore activity during physiological background adaptations.

Journal of Endocrinology (1995) 146, 159–167
Uptake of thyroxine in the human choriocarcinoma cell line JAR
Authors: Mitchell A M , Manley S W , Payne E J , Mortimer R H
Published: December 2008

We have studied the uptake of 125I-thyroxine (125I-T4) in the human choriocarcinoma cell line JAR. Uptake of 125I-T4 was time-dependent, stereospecific and reversible, with a saturable component of 33% after 120 min of incubation. Kinetic analysis of the initial specific uptake rates indicated the presence of a single uptake process with a Michaelis constant of 59·4 ± 13·9 nm (n=12) and maximum velocity of 0·29 ± 0·06 pmol/min per mg protein. Uptake was dependent on intracellular energy as, in the presence of 2 mm potassium cyanide, saturable uptake was reduced to 60·6 ± 8·5% (n=4) of control uptake. Uptake was also temperature-dependent. Saturable 125I-T4 uptake after 60 min of incubation was 26·1 ± 3·0% at 25 °C (n=6) and 27·3 ± 5·7% at 4 °C of control uptake at 37 °C. Ouabain did not inhibit 125I-T4 uptake indicating that the uptake was independent of the Na gradient across the cell membrane. Although T4 uptake was stereospecific, as d-T4 failed to inhibit 125I-l-T4 uptake, it was not specific for T4, as tri-iodothyronine (T3) and reverse T3 also inhibited 125I-T4 uptake. We conclude that JAR cells have a saturable, stereospecific and reversible membrane transport mechanism for T4 which is dependent on intracellular energy, but independent of the Na+ gradient across the cell membrane.

Journal of Endocrinology (1995) 146, 233–238
The in vivo effects of fibroblast growth factor and epidermal growth factor on the secretion of oestradiol, androstenedione and progesterone by the autotransplanted ovary in the ewe
Authors: Scaramuzzi R J , Downing J A
Published: December 2008

An experiment was conducted to determine the effects of epidermal growth factor (EGF) and fibroblast growth factor (FGF), infused into the ovarian artery, on the secretion of ovarian steroids during the mid-luteal phase in ewes with an autotransplanted ovary. The infusion of EGF (5 μg/h) for 12 h suppressed the secretion of oestradiol and androstenedione during the infusion and for up to 30 h after the infusion. The secretion of progesterone tended to be lower immediately after the infusion (not significant) but had recovered by 24 h after the end of the infusion and then increased significantly (P<0·05) to rates higher than in control animals. There were no effects of the infusion of EGF on the characteristics of pulsatile LH secretion. FSH concentrations increased 24 h after the end of the infusion probably as an indirect consequence of the changes in oestradiol secretion and not as a consequence of a direct effect of EGF on the hypothalamo-pituitary axis although this latter possibility cannot be unequivocally eliminated. The infusion of FGF (1·5 μg/h) for 12 h also suppressed the secretion of oestradiol and androstenedione during and for up to 30 h after the infusion. The infusion of FGF had no detectable effect on the secretion of progesterone or the characteristics of pulsatile LH secretion. FSH concentrations increased steadily during the infusion but declined rapidly to below pre-infusion concentrations after the end of the infusion. These data provide tentative in vivo evidence for paracrine and autocrine effects of EGF and FGF on follicular and luteal function in sheep.

Journal of Endocrinology (1995) 146, 301–311
Functional and morphological changes of the thyroid gland following 5 days of pulsatile TRH stimulation in male rats
Authors: Hoang-Vu C , Brabant G , Leitolf H , von zur Mühlen A , Dralle H , Schröder S , Sierralta W D
Published: December 2008

The aim of the present study was to evaluate in vivo the selective effects of a small increase in plasma TSH levels on thyroid function, proliferation and morphology. Chronically catheterized male Sprague–Dawley rats were stimulated i.v. over 5 days either with TRH (2 μg TRH in 100 μl 0·9% (w/v) NaCl (TRH-P) or the NaCl carrier alone (P), both given as pulses every 2 h. Control groups were cotreated i.v. with 10 μg thyroxine (T4)/100 g body weight per day (TRH-P+T4) starting 2 days before pulsatile stimulation. TSH plasma levels were approximately doubled by TRH-P (P≤0·001), T4 plasma levels significantly increased (P≤0·001) but tri-iodothyronine plasma levels did not change compared with treatment with P. No significant changes between groups were found in thyroid weight and in intrathyroidal iodine content, but the percentage of 5-bromo-2′-desoxyuridinelabelled thyrocytes as a marker of proliferation in TRH-P-treated animals was significantly increased over P or TRH-P+T4 (P≤0·001). Ultrastructural analysis of the thyroid evaluated by electron microscopy revealed a significant increase in the number of lysosomes (P≤0·001). The size of the endoplasmic reticulum (ER) in relation to the cytoplasm was significantly increased when treated with TRH-P compared with P or TRH-P+T4 (P≤0·001). Post-embedding immunogold staining revealed Tg as a major product within ER cisternae. Immunogold labelling was moderate in controls and higher densities of gold particles were obtained in TRH-P-treated animals (P≤0·001). In conclusion, short-term pulsatile TRH stimulation increasing the plasma levels of immunoreactive TSH only twofold is capable of inducing hypertrophy of the thyrocytes by gross ultrastructural changes which are paralleled by an increase in circulating T4. These data underscore the dominant role of TSH on thyroid ultrastructure within the narrow boundaries of normal physiological regulation.

Journal of Endocrinology (1995) 146, 339–348
Influence of treatment with the anti-oestrogen 3-hydroxytamoxifen (droloxifene) on plasma sex hormone levels in postmenopausal patients with breast cancer
Authors: Geisler J , Haarstad H , Gundersen S , Raabe N , Kvinnsland S , Lønning P E
Published: December 2008

Plasma levels of oestradiol (Oe2), oestrone (Oe1), oestrone sulphate (Oe1S), androstenedione, testosterone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulphate (DHEAS), sex hormone-binding globulin (SHBG) and the gonadotrophins (FSH and LH) were determined in 20 postmenopausal women with breast cancer treated with the anti-oestrogen droloxifene (3-hydroxytamoxifen). Plasma oestrogens were measured before and after 3, 6 and 12 months of therapy. The other hormones were measured before and after 6 months of therapy. Droloxifene treatment had no significant influence on plasma levels of Oe2. Plasma levels of Oe1 and Oe1S increased during treatment (mean increase of 11·9-15·9% and 24·5–69·4% respectively after different time-intervals on treatment). The Oe1S/Oe1 and Oe1S/Oe2 ratios increased by mean values of 13·8–45·2% and 25·9–52·4% respectively. Plasma SHBG increased significantly by a mean value of 73·9%, while FSH and LH fell non-significantly by 19·7% and 20·4% respectively. Plasma levels of testosterone, androstenedione, DHEA and DHEAS all increased during treatment, but none of these alterations were of statistical significance. While the influence of droloxifene on plasma SHBG resembled that which is seen during treatment with tamoxifen, its influence on plasma oestrogens and the gonadotrophins seems to be different. Possible explanations of such differences and the clinical implications of alterations in plasma hormones during treatment with droloxifene are discussed.

Journal of Endocrinology (1995) 146, 359–363
Corticotrophin-releasing hormone-binding protein in human fetal plasma
Authors: Perkins A V , Wolfe C D A , Eben F , Soothill P , Linton E A
Published: December 2008

During pregnancy maternal plasma corticotrophinreleasing hormone (CRH) levels rise 1000-fold whilst fetal plasma levels are often 100-fold higher than the concentrations seen in normal non-pregnant human plasma. Despite these high CRH levels neither the maternal nor fetal pituitary releases excessive amounts of ACTH. A specific CRH-binding protein (CRHBP) exists in the maternal circulation which is able to bind and inactivate the ACTH releasing activity of CRH. In this study we have used a specific CRHBP radioimmunoassay to determine the level of CRHBP in fetal and maternal plasma samples. Fetal samples were collected by cordocentesis between 20 and 33 weeks gestation and matched maternal samples were taken by venepuncture at the same time. In a second study, plasma samples were collected from 8 women at fortnightly intervals from week 20 to term, at labour and post-partum. A fetal sample, taken from the umbilical vein, was collected immediately post-delivery. The mean maternal CRHBP concentration for the samples collected between 20 and 33 weeks (n=23) was 8·12 nmol/l and the fetal level was 8·62 nmol/l. Data from the second study showed that at term the maternal CRHBP concentration decreased significantly (P<0·025) to 6·32 nmol/l. The fetal CRHBP level also decreased significantly (P<0·001) at term to a level of 5·84 nmol/l. The CRHBP in both fetal and maternal plasma was shown to be functional by 125I-CRH binding and gel permeation chromatography. The capacity of maternal and fetal plasma to bind 125I-CRH decreased at term in agreement with the quantitation of plasma CRHBP by radioimmunoassay.

Journal of Endocrinology (1995) 146, 395–401