bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunological detection of the oestradiol receptor protein in cell lines derived from the lymphatic system and the haematopoietic system: variability of specific hormone binding in vitro
Authors: Jakob F. , Tony H. P. , Schneider D. , Thole H. H.
Published: December 2008

Extracts of human MCF 7 mammary carcinoma cells, the human lymphoblastoid cell lines AEH 1 and IM 9, T-cell derived CCRF cells, HL 60 myeloic leukaemia cells and murine myeloma cells SP 0 and NS I were analysed for immunoreactivity with polyclonal goat antibodies raised against homogeneous preparations of C-terminal fragments (32 kDa) of porcine uterine oestradiol receptor (ER). Whole cells and low speed cytosols were analysed for specific oestradiol-binding activity. ERs were enriched from cell extracts by either fractionated ethanol precipitation (0–25% (v/v) ethanol) and/or microscale-immunoaffinity chromatography. Immunoreactive proteins of identical molecular weight (approximately 65 kDa) were detected in all cell lines examined. Whole cell binding assays showed specific oestradiol-binding activity in MCF 7, IM 9 and CCRF cells. Borderline binding was found in HL 60 myeloid cells. No specific binding could be detected in AEH 1, NS I and SP 0 cells. Identical results were obtained using agarelectrophoresis after dextran-coated charcoal treatment. Immunoaffinity purified ERs from MCF 7, AEH 1 and HL 60 cells were subjected to limited proteolysis, where identical tryptic fragments were generated. In conclusion, we have confirmed by immunological methods that ERs are expressed in a variety of cell lines derived from the immune system and the haematopoietic system. The lack of specific hormone binding or very low-affinity hormone binding in some of the cells examined may be due to post-translational events or point mutations.

Journal of Endocrinology (1992) 134, 397–404
A simple bioassay for epidermal growth factor using pig thyrocytes
Authors: Metcalfe R. A. , Crosby S. R. , White A. , Hanford L. , Mac Neil S.
Published: December 2008

A bioassay for epidermal growth factor (EGF) is described using an eluted stain assay (ESTA) of dehydrogenase activity in pig thyrocytes. Optimal responsiveness to EGF was obtained in confluent cultures of primary pig thyrocytes cultured with EGF or biological samples containing EGF for either 24 or 48 h. Dehydrogenase activity was determined by measuring the production and then the release of a coloured formazan product produced by reduction of a 3-[4,5-dimethylthiazol-2 -yl]-2,5-diphenyltetrazolium bromide substrate added to the cells.

The assay responded equally to mouse, human and recombinant EGF and was suitable for measuring EGF activity in some but not all biological fluids. Specificity of detection of EGF activity was confirmed using antibody to EGF. The ESTA assay compared favourably with the radioreceptor assay for EGF in terms of sensitivity to EGF with half-maximal activation at 0·24 ±0·06 nmol/l (mean ± s.e.m., n= 22 experiments) for the ESTA assay and 0·60 ±0.13 nmol/l (n=7) experiments) for the radioreceptor assay.

Journal of Endocrinology (1992) 134, 449–457
Origin of aldosterone in trypsin-stimulated rat adrenal zona glomerulosa incubations
Authors: Vinson G. P. , Laird S. M. , Hinson J. P. , Teja R.
Published: December 2008

The time-course for the in-vitro secretion of aldosterone and 18-hydroxycorticosterone (18-OH-B) by rat adrenal whole capsular tissue (largely zona glomerulosa) was studied under control and stimulated conditions. The stimulatory effect of trypsin was relatively delayed, and the steroids were significantly enhanced only after 1 h, in contrast to the actions of ACTH, which produced effects after 15 or 30 min. Tissue-sequestered 18-hydroxydeoxycorticosterone (t-18-OH-DOC), which is not affected by ACTH, was significantly depleted by trypsin, but secreted 18-OH-DOC was not consistently affected by either stimulant. In contrast to the apparent mobilization of t-18-OH-DOC, the conversion of exogenously added [3H]18-OH-DOC to [3H]18-OH-B was inhibited by trypsin, and aldosterone was unaffected. When trilostane was added to inhibit de-novo steroidogenesis, under conditions in which the steroid secretory response to ACTH is completely inhibited, aldosterone and 18-OH-B secretion was still stimulated by trypsin although yields were lower. Compared with controls, trilostane reduced t-18-OH-DOC concentrations, and trypsin caused a further depletion.

In other studies, glomerulosa plasma membrane enriched preparations were homogenized and centrifuged, and the supernatants were dialysed and added to incubations of dispersed zona glomerulosa cells in the presence or absence of stimulators of aldosterone secretion. The addition of the supernatants, which contained high concentrations of sequestered t-18-OH-DOC, stimulated aldosterone and 18-OH-B production by collagenase-dispersed zona glomerulosa cells to a greater extent than the addition of an equivalent amount of free 18-OH-DOC or corticosterone. When trypsin, ACTH, the phorbol ester phorbol myristate acetate or increased potassium were also added, there was a further increase in 18-OH-B production, and final recoveries of 18-OH-DOC were correspondingly decreased.

The results are consistent with the hypothesis that, because of the nature of its disposition in the glomerulosa cell, t-18-OH-DOC may be utilized as a substrate for aldosterone and 18-OH-B production. The plasma membrane location of this stored steroid pool, and the known actions of phorbol ester or trypsin stimulation, suggest that it may be mobilized by protein kinase C activation.

Journal of Endocrinology (1992) 135, 125–133
The induction of specific proteases for insulin-like growth factor-binding proteins following major heart surgery
Authors: Cwyfan Hughes S. C. , Cotterill A. M. , Molloy A. R. , Cassell T. B. , Braude N. , Hinds C. J. , Wass J. A. H. , Holly J. M. P.
Published: December 2008

Insulin-like growth factors (IGF-I and IGF-II) circulate bound to specific high-affinity binding proteins (IGFBPs). Recent evidence has shown that in pregnancy and severe illness, specific proteases modify these binding proteins, reducing their affinity for IGFs. We have studied 12 patients, undergoing elective coronary artery vein-bypass graft surgery, for the appearance of these proteases and have demonstrated the induction of two independent, heat-labile, cationdependent proteases. Proteolytic activity directed against IGFBP-3 was detected in all patients between 24 h and 5 days after surgery; the second IGFBP-4 specific protease was active 1 h after sternotomy. The total IGF-I levels were found to decrease following surgery, with the IGF-I distribution in the plasma being radically altered from that seen prior to the operation. One day after the operation the majority of the IGF-I, instead of being bound in the relatively inert 150 kDa complex, was associated with the smaller binding proteins which are more readily accessible to the tissues. These findings are in contrast to pregnancy where, despite similar proteases, the majority of the IGF-I remains in the 150 kDa complex. The alteration seen in IGF-I distribution after surgery did not appear to be a direct result of the IGFBP-3 proteolytic activity or an effect of the addition of heparin to the circulation. The potential increase in bioavailability of IGFs caused by the alteration in carrier protein may play a pivotal role in countering the catabolic state induced by surgery.

Journal of Endocrinology (1992) 135, 135–145.
Carbachol increases intracellular free calcium concentrations in human granulosa-lutein cells
Authors: Mayerhofer A. , Föhr K. J. , Sterzik K. , Gratzl M.
Published: December 2008

We investigated whether the stimulation of human granulosa-lutein cells with muscarinic and nicotinic receptor agonists can cause increases in intracellular free calcium (Ca2+), using Fura-2 microfluorimetry. The addition of carbachol (a non-selective muscarinic and nicotinic receptor agonist) to cultured human granulosa-lutein cells increased intracellular free Ca2+ levels. Concentrations as low as 10 nmol/l were effective. In contrast, nicotine did not evoke elevations of intracellular free Ca2+. Basal Ca2+ levels ranged around 70–140 nmol/l and maximal, carbacholinduced peaks reached 1·1 μmol/l. The carbachol-induced Ca2+ signal was abolished after preincubation of the cells with the muscarinic receptor antagonists quinuclidinyl benzilate or atropine, but it was not affected by removal of extracellular Ca2+. Further evidence for the involvement of intracellular Ca2+ stores is provided by experiments in the absence of extracellular Ca2+. While thapsigargin (a blocker of ATP-driven Ca2+ uptake by intracellular stores) and ionomycin (an ionophore by which Ca2+ is released from intracellular stores) evoked small Ca2+ transients, cells pretreated with these agents did not respond to carbachol any more. These data suggest the presence of a functional muscarinic receptor on human granulosa-lutein cells and imply the involvement of intracellular Ca2+ stores during the cellular response. These results also suggest the participation of the nervous system, acting through muscarinic receptors, in the control of the function of human granulosa-lutein cells.

Journal of Endocrinology (1992) 135, 153–159
Gonadotrophin surge-attenuating factor attenuates in-vitro LH secretion induced by gonadotrophin-releasing hormone from cultured ovine pituitary cells only during the breeding season
Authors: Fowler P. A. , Townsend C. , Messinis I. E. , Cunningham P. , Templeton A.
Published: December 2008

Primary cultures of ovine pituitaries from adult ewes were used to investigate aspects of gonadotrophin surge-attenuating factor (GnSAF) bioactivity in human follicular fluid (hFF) from superovulated women. During the autumn and first half of the winter, LH secretion induced by gonadotrophinreleasing hormone (GnRH) was markedly reduced (43·5 ± 5·2% of control GnRH-induced LH secretion) by incubation for 48 h with steroid-free hFF. For the rest of the year, treatment with the same batch of steroid-free hFF resulted in non-significant reduction or stimulation of GnRH-induced LH secretion (71·3± 13·2 to 117·8±11·2% of control GnRH-induced LH secretion). Incubation of pituitary cells for 48 h with oestradiol (1 pmol/l to 1 μmol/l), progesterone (1 pmol/l to 1 μmol/l) or oestradiol and progesterone combined (1 pmol/l to 1 μmol/l) in a two-way titration for 48 h had no significant effect on GnRH-induced LH secretion (83·4±7·6 to 110·6±5·0% of control secretion). Separating hFF into fractions of different molecular mass by ultrafiltration demonstrated that GnSAF bioactivity was present in a form 10–30 kDa in size. Incubation for 48 h with these fractions had no significant effect on basal FSH secretion but significantly attenuated GnRH-induced LH secretion during the autumn. The same fractions had little effect on GnRH-induced LH secretion from pituitary cells collected during the summer. We conclude that ovine pituitaries display at least partial reduction in sensitivity to GnSAF outside the breeding season. In addition, neither oestradiol nor progesterone singly or in combination caused the observed attenuation of GnRH-induced LH secretion that is ascribed to GnSAF bioactivity.

Journal of Endocrinology (1992) 135, 221–227
The role of thromboxane in the uterotrophic response in the gravid normotensive and spontaneously hypertensive rat
Authors: Kerr M. B. , Marshall K. , Senior J.
Published: December 2008

The role of thromboxane in the gravid normotensive (CD) and hypertensive (SHR) rat was investigated (by utilizing two thromboxane receptor-blocking drugs, EP092 and AH23848) both at mid-gestation and at term. The parameters examined were uterine blood flow (blood flows were measured by the microsphere technique) and uterine weight and placental blood flow at term, fetal mass and number.

At mid-gestation EP092 significantly (P< 0·005) increased uterine blood flow in both strains whilst the increases seen with AH23848 were not statistically significant. At term (day 22 in the CD and day 23 in the SHR rat) the antagonists increased uterine blood flow in the CD rats alone. However, at this time the antagonists caused an increase in placental blood flow in both strains.

Thromboxane appears to be involved in the regulation of uteroplacental blood flow. The observation that the antagonists were able to potentiate blood flow by mid-gestation may provide a clinical indication with respect to potential prophylactic use of this class of compounds in cases of pregnancy-induced hypertension in women.

Journal of Endocrinology (1992) 135, 257–261
Passive immunization against circulating insulin-like growth factor-I (IGF-I) increases protein catabolism in lambs: evidence for a physiological role for circulating IGF-I
Authors: Koea J. B. , Gallaher B. W. , Breier B. H. , Douglas R. G. , Hodgkinson S. , Shaw J. H. F. , Gluckman P. D.
Published: December 2008

Primed constant infusions of [14C]urea were used to determine the acute effect of passive immunization against circulating free and protein-bound insulin-like growth factor-I (IGF-I) on the rate of net protein catabolism (NPC) in castrated male lambs fasted for 48 h. Following an intravenous bolus of 50 ml IGF-I antiserum, the rate of NPC increased to a peak 30 min after injection of 1·69 ± 0·16 g/kg per day from a baseline value of 1·45±0·22 g/kg per day (P<0·05, n = 4). In three animals given 50 ml equivalents of the purified immunoglobulin fraction, NPC increased from 1·31 ±0·20 to 1·59±0·16 g/kg per day (P<0·05). A similar trend was observed in animals given 25 ml antiserum (n = 4). The rate of NPC did not increase following a bolus of non-immune serum in control animals and the rate of NPC in the treated lambs returned to control levels within 60 min of antibody injection. Plasma insulin and glucose concentrations in both the treated and control groups were unchanged throughout the study. These data suggest that circulating IGF-I has a physiological role in regulating whole body protein turnover during starvation and possibly other catabolic states. The effect of immunoneutralization of circulating IGF-I is transient and this suggests that while IGF-I has an endocrine role in the regulation of protein turnover, other regulatory mechanisms are involved.

Journal of Endocrinology (1992) 135, 279–284
Immunochemical detection of the major vitelline envelope proteins in the plasma and oocytes of the maturing female rainbow trout, Oncorhynchus mykiss
Authors: Hyllner S. J. , Haux C.
Published: December 2008

The major vitelline envelope proteins were detected in the plasma of female rainbow trout maturing under natural conditions by using the Western blot technique. Females were sampled every month from July until ovulation in January. The amount of vitelline envelope proteins in plasma increased markedly as the gonads increased in size from 0·4 to about 15% of the total body weight. The plasma level of oestradiol-17β largely followed the alterations in the amount of vitelline envelope proteins, indicating the endocrine control of vitelline envelope protein synthesis. In addition, plasma vitellogenin changed in a manner that resembled the changes in the amount of plasma vitelline envelope proteins. The appearance and growth of the vitelline envelope during oocyte development was demonstrated using immunohistochemical methods. The vitelline envelopes from oocytes at different stages of development were immunoreactive with the antibodies directed against the major vitelline envelope proteins. No immunoreactivity could be observed in the ooplasm or in the surrounding follicular cells, which indicated that the major vitelline envelope proteins were of extraovarian origin. The present study further supports the hypothesis that the major protein constituents of the vitelline envelope in teleosts are under the endocrine control of oestradiol17β and that the site of synthesis is outside the ovary.

Journal of Endocrinology (1992) 135, 303–309
Activation of phospholipase A2 and phospholipase C by endothelin-1 in human endometrium
Authors: Ahmed A. , Cameron I. T. , Ferriani R. A. , Smith S. K.
Published: December 2008

Human endometrium contains specific binding sites for iodinated endothelin (ET)-1, ET-2 and ET-3, and ET-1 stimulates prostaglandin (PG) F2α synthesis from explants of proliferative endometrium in short-term culture. This study has investigated the cellular responses of normal proliferative endometrium to ET-1. Radioimmunoassay was used to measure PG release and Dowex anion-exchange column chromatography was utilized to assess the accumulation of inositol phosphates. Endothelin-1 induced a significant increase in PGF2α release (basal median: 1465 pg/mg per 60 min (range: 541–3935 pg/mg per 60 min); ET-1-stimulated: 1813 pg/mg per 60 min (1021–5714 pg/mg per 60 min); P < 0·04 using Wilcoxon signed rank test). The effect of ET-1 was attenuated in the presence of the phospholipase A2 inhibitor quinacrine. Endothelin-1 induced a rapid, transient and concentration-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), measured by the accumulation of tritiated inositol phosphates. Following a 1-min stimulation with ET-1 (100 nmol/l), [3H]inositol mono-, bisand trisphosphate fractions increased from median values of 490·0 d.p.m./mg dry wt (range: 348·0–807·0 d.p.m./mg dry wt), 120·0 d.p.m./mg dry wt (93·6–144·1 d.p.m./mg dry wt) and 67·0 d.p.m./mg dry wt (54·2–85·0 d.p.m./mg dry wt) to 939·0 d.p.m./mg dry wt (635·9–1596·0 d.p.m./mg dry wt; P < 0·03), 145·0 d.p.m./mg dry wt (127·0–293·9 d.p.m./mg dry wt; P < 0·05) and 146·0 d.p.m./mg dry wt (77·5–187·0 d.p.m./mg dry wt; P < 0·03) respectively. These results suggest that ET-1 activates the phospholipase A2 and PtdIns(4,5)P2-specific phospholipase C in human proliferative endometrium, resulting in the generation of PGF2α and second messengers respectively which are pivotal to endometrial function.

Journal of Endocrinology (1992) 135, 383–390
Plasma atrial natriuretic peptide in vasopressin deficiency the effects of acute water deprivation in rats
Authors: Burgess W. J. , Balment R. J.
Published: December 2008

Plasma concentrations of atrial natriuretic peptide (ANP) and other renally active hormones were measured in Long–Evans (LE) rats and vasopressin-deficient Brattleboro rats with diabetes insipidus (DI) in conditions of water repletion and deprivation, and in DI rats following chronic vasopressin replacement.

In water-replete rats, vasopressin deficiency was associated with elevated circulating ANP and angiotensin II (AII) concentrations, while plasma adrenal steroid concentrations were depressed by comparison with LE rats. These differences were fully reversed after 7 days of vasopressin replacement in DI rats to restore normal water turnover.

Water deprivation for 4 h had little effect on plasma tonicity or hormone profile in LE rats. In contrast, however, the unreplaced fluid loss during 4-h water deprivation in the DI rat was associated with a marked increase in plasma tonicity evident within 30 min. Plasma ANP concentrations fell substantially to levels below those in LE rats, coincident with a rise in adrenal steroid levels and independent of any clear change in AII. These changes in circulating ANP concentration were directly correlated with changes in plasma Na+ concentration, osmolality and tissue water content in the DI rats, underlining the importance of body fluid status in modulating the secretion of ANP.

These data clearly show that plasma ANP concentration is increased in vasopressin deficiency, but emphasize the sensitivity of circulating hormone levels in vasopressin-deficient animals to acute changes in the state of hydration, underscoring the complex and labile interaction between body fluid and hormonal factors involved in the control of ANP secretion.

Journal of Endocrinology (1992) 135, 431–438
Growth hormone (GH)-binding protein in normal and GH-deficient dwarf rats
Authors: Carmignac D. F. , Wells T. , Carlsson L. M. S. , Clark R. G. , Robinson I. C. A. F.
Published: December 2008

There are GH-binding proteins (GHBPs) present in the blood of many species, and these correspond to the extracellular GH-binding domain of the GH receptor. In the rat, GHBP arises by alternative splicing of the GH receptor mRNA, but little is known of the physiological role of circulating GHBP, or its relationship with episodic GH secretion. We have developed a sensitive radioimmunoassay based on recombinant GHBP, and have measured rat GHBP levels in small samples of plasma from normal and GH-deficient dwarf rats. In normal adult rats, GHBP levels were two- to threefold higher in females than in males (16·6 ± 0·8 vs 6·4 ± 0·4μg/l, P < 0·001), but this sex difference was not seen in dwarf rats. A continuous infusion of human GH in dwarf males raised plasma GHBP to 23·5 ± 3·5 μg/l compared with 6·7 ± 0·5 μg/l in sham-infused animals, whereas suppression of GH by continuous infusion of a long-acting somatostatin analogue in female dwarf rats had no effect on GHBP. In anaesthetized rats, large changes in plasma GH caused by i.v. administration of rat GH, somatostatin or GH-releasing factor did not affect GHBP acutely. Both GH and GHBP were also measured in serial blood samples from conscious normal and dwarf rats. A sexually dimorphic GH secretory pattern was observed in both strains. Males showed peaks and troughs of GH every 3 h varying over a 100-fold range, whereas females exhibited more continuous GH secretion. Despite the large fluctuations in endogenous GH, GHBP levels remained relatively constant in individual normal or dwarf males, as well as in females of both strains, and there was no significant correlation between GH and GHBP either in individual rats or as a group. Our results suggest that GHBP is GH-dependent in the longer term, and that the higher GHBP levels in female rats require their continuous GH secretory pattern. However, plasma GHBP levels remain stable and are not affected by acute changes in endogenous or exogenous GH.

Journal of Endocrinology (1992) 135, 447–457
Immunization with human thyrotrophin receptor peptide induces an increase in thyroid hormone in rabbits
Authors: Ohmori M. , Endo T. , Ikeda M. , Onaya T.
Published: December 2008

Eight rabbits were immunized with a synthetic peptide corresponding to the unique N-terminal region (termed N peptide; amino acid residues 29–57) in the extracellular domain of the human thyrotrophin (TSH) receptor. After 10 weeks, all of the eight rabbits produced anti-N peptide antibodies. Western blot analysis revealed that the antibodies recognized rabbit TSH receptor as an approximately 100 kDa protein. We compared the level of thyroid hormone in serum taken before immunization (preimmune sera) with that of serum taken after immunization (postimmune sera) in these immunized rabbits. Postimmune sera from the eight rabbits had higher mean (± s.d.) levels of tri-iodothyronine (T3) and thyroxine (T4) than did preimmune sera (T3, preimmune 0·82 ± 0·26 μg/l vs postimmune 1·33 ± 0·35, P < 0·01; T4, preimmune 33·7 ± 10·0 μg/l vs postimmune 41·0 ± 6·0, P < 0·05). T3 levels in four rabbits and T4 levels in four rabbits after immunization were over the normal range obtained from six age-matched control rabbits. Seven rabbits exhibited thyroid-stimulating antibody (TSAb) activity with various degrees (241–545%). The concentration of T3 and T4 did not increase over 10 weeks in either non-immunized rabbits (T3, preimmune 0·89 ± 0·34 μg/l vs postimmune 0·82 ± 0·22; T4, preimmune 31·1 ± 7·3 μg/l vs postimmune 30·3 ± 5·1) or other peptide-immunized rabbits (T3, preimmune 0·68 μg/l (n = 2) vs postimmune 0·69; T4, preimmune 33·1 μg/l vs postimmune 26·4). These results indicate that experimentally produced anti-TSH receptor antibody with TSAb activity induces an increase in thyroid hormone in rabbits.

Journal of Endocrinology (1992) 135, 479–484
Effects of FSH on Leydig cell morphology and function in the hypogonadal mouse
Authors: O'Shaughnessy P. J. , Bennett M. K. , Scott I. S. , Charlton H. M.
Published: December 2008

The hypogonadal (hpg) mouse has a congenital deficiency in gonadotrophin-releasing hormone and the gonads consequently lack exposure to endogenous gonadotrophins during development. To determine the effect of FSH on Leydig cell function in these animals adult hpg mice were injected twice daily with FSH (2 μg injections) or LH (40 ng injections, the presumed LH contamination of FSH used). Following FSH treatment there was a clear stimulation of the seminiferous epithelium and in animals injected with FSH plus [3H]thymidine, the incorporation of label was largely confined to the germ cells with no apparent uptake by the Sertoli cells. In FSH-treated testes the Leydig cells contained numerous large lipid droplets, similar to the unstimulated hpg testis. There was no evidence of the interstitial hyperplasia which is observed following injection of high doses of LH (2 pg twice daily).

There was no change in basal androgen content of the testis in vivo following FSH treatment but injection of a maximal dose of human chorionic gonadotrophin (hCG), 1 h before death, markedly increased testicular androgen content only in the FSH-treated group. Testicular androgen production in vitro was significantly increased following FSH treatment both under basal conditions (FSH-treated, 17·4 pmol/testis; control, 1·46 pmol/testis) and during stimulation by hCG (FSH-treated, 940 pmol/testis; control, 81 pmol/testis). Associated with the increased androgen production following FSH treatment there were significant increases in the activities of three steroidogenic enzymes; cholesterol side-chain cleavage (186-fold increase over control), 17α-hydroxylase (103-fold increase) and 17-ketosteroid reductase (177-fold increase). The fourth enzyme involved in androgen synthesis, 3β-hydroxysteroid dehydrogenase, shows relatively high activity in the control hpg testis and was only increased by sixfold following FSH treatment. There was no effect of FSH on 5α-reductase activity.

Results show that FSH causes a marked stimulation of the steroidogenic capacity of the hpg testis. Leydig cells do not contain FSH receptors and it is assumed that FSH acts through paracrine factors released by the Sertoli cells.

Journal of Endocrinology (1992) 135, 517–525
Expansion of plasma volume by intragastric isotonic saline inhibits supraoptic neurones in rats
Authors: Pendlebury S. T. , Dyball R. E. J. , Honda K.
Published: December 2008

To determine whether an increase in plasma volume might directly influence supraoptic neurones, single cell extracellular recordings were made from magnocellular neurones of the supraoptic nucleus in urethane-anaesthetized rats as plasma volume was expanded by intragastric injection of isotonic saline. Continuous ratemeter records taken before, during and after intragastric injections of 10 ml isotonic saline showed that the firing rate of putative vasopressin cells was reduced by 2·21 spikes/s (P < 0·02; n = 9; paired t-test) after 50 min. Putative oxytocin cells, after an initial increase in firing rate which lasted approximately 30 min, showed a decrease of 0·98 spikes/s (P < 0·02; n = 6; paired t-test). A population of 93 control cells of both types had a median firing rate of 4·69 spikes/s, a comparable group of 65 cells recorded 1 h after intragastric injection had a median firing rate of 3·15 spikes/s and another group of 68 cells recorded 1 h after a second injection had a median firing rate of 2·5 spikes/s. These differences were significant (P < 0·04 and P < 0·01; Mann–Whitney U test). The haematocrit of plasma samples taken from five similarly anaesthetized control animals was 49·7%. One hour after one intragastric injection the value was significantly (P < 0·02; paired t-test) reduced to 46·7% and 1 h after a second injection it was further reduced to 42·1% (P < 0·01). Thus a modest increase in plasma volume was associated with a reduced firing rate of both vasopressin and oxytocin cells so that it is probable that an increase in plasma volume inhibits the secretion of both neurohypophysial hormones.

Journal of Endocrinology (1992) 135, 527–533
DIRECT STIMULATION OF BOVINE OVARIAN PROGESTERONE SECRETION BY LOW CONCENTRATIONS OF a-INTERFERON
Authors: Luck M.R. , Shale J.A , Payne J.H.
Published: December 2008

The ruminant conceptus secretes proteins during early pregnancy which maintain the corpus luteum. These trophoblast proteins are related to the αII-interferons and prevent luteolysis indirectly by disrupting the secretion of endometrial prostaglandin. Although trophoblast interferons appear to be largely confined to the uterine lumen, it remains possible that they also act peripherally. This report describes in vitro studies which suggest that interferon may influence hormone secretion by the ovary directly. The study employed i) a well defined serum-free culture model in which bovine granulosa cells secrete the luteal hormones progesterone and oxytocin, and ii) serum-free and serum-supplemented cultures of cells from early CL. Dose-response experiments were performed using bovine recombinant α-interferon (brIFN). Progesterone and oxytocin secretions were measured over 4-5 days of culture and DNA content was also determined.

Low concentrations of brIFN (10−15 mol/l to 10−11 mol/l) stimulated progesterone secretion by granulosa cells by up to three fold, without significantly affecting oxytocin concentrations or culture DNA content. Concentrations of 10−10 mol/l to 10−7 mol/l suppressed progesterone secretion in a log dose-related manner (r=0.97) with evidence of toxicity (lower oxytocin concentrations and significantly reduced DNA compared with controls). Progesterone secretion by luteal cells in serum-free culture was stimulated in the presence of 10−15 mol/l brIFN, whilst high concentrations again caused inhibition. The data show that ovarian cells can respond directly to low concentrations of interferon-like proteins. They also demonstrate an inhibition by high doses which may mask the stimulatory effect in this model. The data suggest that the early corpus luteum may be directly influenced by α-interferon. A stimulation of progesterone, but not of oxytocin, secretion from ovarian cells would be consistent with a role for conceptus proteins in maintaining the corpus luteum of pregnancy.
Oxytocin receptor concentrations, inositol phosphate turnover and prostaglandin release by endometrium from ewes induced to ovulate during the early post-partum period
Authors: Wallace J. M. , Thompson M. G. , Aitken R. P. , Cheyne M. A.
Published: December 2008

Induction of ovulation early post partum in sheep is associated with a high incidence (30–40%) of premature luteolysis. The present study was designed to characterize oxytocin receptor levels, oxytocin-stimulated inositol phosphate (IP) turnover (second messenger) and oxytocin-stimulated prostaglandin F2α (PGF2α) release in the endometrium of post-partum ewes induced to ovulate 21 days after parturition and expected to exhibit a range of corpus luteal functions subsequently.

Ovulation was induced on day 21 post partum using a controlled internal drug release device and pregnant mare serum gonadotrophin, and uterine tissues were collected on days 5, 10 or 15 of the cycle (n = 4/day). A further 12 ewes whose interval from previous parturition exceeded 150 days were similarly treated and acted as controls. Measurement of daily peripheral progesterone concentrations revealed that while all control ewes exhibited normal luteal function, abnormal luteal function was evident in two, two and one post-partum ewes studied on days 5, 10 and 15 of the cycle respectively. Oxytocin receptor binding was detected (by receptor-binding assay and in-vitro autoradiography) in the endometrium and myometrium of post-partum ewes at all three stages of the oestrous cycle but only at day 15 in control ewes. To determine IP turnover, 100 mg caruncular endometrium was incubated in duplicate for 2·5 h with 10 μCi [3H]inositol and treated with 0 or 2 μmol oxytocin/l for 30 min, then [3H]inositol mono-, bis- and trisphosphates were quantified. Oxytocin stimulated total IPs in all day-5 and day-15 post-partum ewes, in three of four day-10 ewes and in all day-15 control ewes. Basal endometrial PGF2α release measured in triplicate (100 mg/well) during a 2 h incubation was higher in post-partum versus control ewes on days 5 and 10 but not on day 15 of the cycle. Similarly, oxytocin stimulated PGF2α release to varying levels at all stages of the cycle in post-partum ewes but only on day 15 in control ewes. Irrespective of the treatment group endometrial oxytocin receptor number was significantly (P < 0·001) correlated with oxytocin-stimulated IP turnover and PGF2α release.

Thus the induction of ovulation and the subsequent luteal phase in post-partum ewes is against a back ground of high oxytocin receptor expression and enhanced PGF2α release which in some ewes may contribute to abnormal luteal function.

Journal of Endocrinology (1993) 136, 17–25
Characterization of the gonadotrophin-releasing hormone receptor in αT3-1 pituitary gonadotroph cells
Authors: Anderson L. , Milligan G. , Eidne K. A.
Published: December 2008

The present study has characterized the gonadotrophin-releasing hormone (GnRH) receptor in immortalized αT3-1 pituitary gonadotroph cells. GnRH and GnRH analogues produced both a dose- and time-dependent increase in total inositol phosphate (IP) accumulation. The rank order of potency of these analogues was the same as that obtained in parallel receptor-binding studies in αT3-1 cells. These responses were abolished following pretreatment with a GnRH antagonist. The use of a specific inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) assay demonstrated a rapid but short-lived rise in Ins(1,4,5)P3 production. Intracellular calcium ([Ca2+]i) was subsequently measured in αT3-1 cells using dual wavelength fluorescence microscopy combined with dynamic video imaging. GnRH produced a biphasic rise in [Ca2+]i. The initial calcium transient was complete within seconds while the smaller secondary plateau phase lasted several minutes. G-protein involvement in the IP response to GnRH in αT3-1 cells was investigated using sodium fluoride (NaF) and pertussis toxin (PTx) which activate and inactivate G-proteins respectively. Like GnRH, NaF produced a dose- and time-dependent increase in IP accumulation. Activation of phospholipase C in these cells by either GnRH or NaF was PTx-insensitive, suggesting that the G-protein involved was neither Gi nor Go but more probably Gq. Immunoblot analysis of αT3-1 cell membranes using antisera raised against the predicted C-terminal decapeptide of the α subunit of Gq demonstrated the presence of Gq in αT3-1 cells. Collectively these results show that the GnRH receptors expressed in αT3-1 cells are coupled to the phosphatidylinositol second messenger pathway via a specific G-protein. αT3-1 therefore represents a convenient model in which to study GnRH-related second messenger pathways.

Journal of Endocrinology (1993) 136, 51–58
Induction of ornithine decarboxylase immunoreactivity in the male mouse kidney following testosterone treatment: an axial heterogeneity in the proximal tubule
Authors: Koibuchi N. , Matsuzaki S. , Ma H.-T. , Sakai M. , Yamaoka S.
Published: December 2008

The effect of testosterone on the activity of ornithine decarboxylase (ODC), its protein level and immunocytochemical distribution were examined in the mouse kidney. Male BALB C mice at 8 weeks of age were used throughout. Fourteen hours before death, they received a subcutaneous injection of testosterone (1 mg/animal) or solvent to measure renal ODC activity or to detect the distribution of ODC immunoreactivity in the kidney. Renal ODC activity and the content of the enzyme were markedly increased after testosterone treatment. Histologically, few cells that were obviously immunoreactive to ODC were observed in the control animals and in the testosterone-treated animals a marked increase in ODC immunoreactivity was observed only in the cortex. ODC immunoreactive cells were located diffusely in the proximal tubule. In the pars recta, cells were stained weakly and homogeneously, while in the pars convoluta, the luminal surface of the cells showed stronger immunoreactivity. Moreover, many granule-like particles that were strongly ODC immunoreactive were observed inside the lumen of the pars convoluta. These results show that testosterone treatment induces an increase in ODC content in certain cells located in the proximal tubule of the cortex.

Journal of Endocrinology (1993) 136, 85–89
Growth hormone deficiency in 'little' mice results in aberrant body composition, reduced insulin-like growth factor-I and insulin-like growth factor-binding protein-3 (IGFBP-3), but does not affect IGFBP-2, -1 or -4
Authors: Donahue L. R. , Beamer W. G.
Published: December 2008

Although GH is known to regulate somatic growth during development, its role in regulating adult body composition is less well defined. The effects of GH on individual body compartments – water, fat, protein and mineral – are achieved both by the action of GH and by a GH-induced hormone, insulin-like growth factor-I (IGF-I). We used a genetic model of GH deficiency, the 'little' (gene symbol lit) mouse, to determine the GH regulation of IGF-I and its insulin-like growth factor-binding proteins (IGFBPs) and to define the interaction between these hormones and each body compartment in adults.

Our results showed that GH-deficient lit/lit mice had reduced levels of serum IGF-I (range 38–130 μg/l) compared with normal lit/+ littermates (range 432–567 μg/l) between 2 and 52 weeks of age. The lit/lit mice did not experience the fivefold increase in IGF-I between 2 and 4 weeks of age that was seen in lit/+ mice. In lit/lit serum, overall binding of 125I-labelled IGF-I to the four IGFBPs was reduced, solely in response to a reduced amount of IGFBP-3. No overall differences were found between lit/lit and lit/+ mice in the binding of 125I-labelled IGF-I to IGFBP-2, -1 or -4. Age-related declines in IGF-I and IGFBPs were seen in lit/lit mice. However, adult levels of IGF-I were maintained in lit/+ mice to at least 52 weeks of age, as were levels of IGFBP-1 and -4, while IGFBP-3 and -2 declined with age.

With respect to body composition, comparison of lit/lit with lit/+ mice showed that the lit/lit mice were characterized by abnormally large adipose tissue stores and reduced body water, protein and mineral from 2 weeks onward. These changes occurred despite normal energy intake in lit/lit mice up to 52 weeks of age, indicating that neither undernutrition nor hyperphagia is characteristic of this GH-induced model of obesity. Furthermore, lit/lit males accrued more body fat beginning at an earlier age than lit/lit females. With advancing age, the per cent body fat increased in both lit/lit and lit/+ mice, while the per cent body water and mineral declined. In lit/lit but not lit/+ mice, per cent protein also declined with age. The changes in body water and fat are attributable to lack of adequate GH in the genetically GH-deficient lit/lit mouse. On the other hand, the changes in body protein are more likely to be effects of IGF-I. Changes in mineral observed in lit/lit mice could be the result of action by GH, IGF-I or both hormones. Therefore, when GH is chronically manipulated by GH deficiency as in lit/lit mice, by GH excess as in acromegaly, or by GH therapy, all four body compartments are affected, suggesting that GH therapy is most valuable when the treatment goal is to alter overall body composition.

Journal of Endocrinology (1993) 136, 91–104
Oestradiol-17β modulates the actions of pharmacologically distinct forms of protein kinase C in rat anterior pituitary cells
Authors: Thomson F. J. , Johnson M. S. , MacEwan D. J. , Mitchell R.
Published: December 2008

Phorbol ester-induced release of LH and GH from rat anterior pituitary tissue in vitro is differentially inhibited by some, but not other, inhibitors of protein kinase C (PKC), suggesting that pharmacologically distinct species of PKC may have different functional roles in these cells. Since stimulus-induced anterior pituitary hormone release can be enhanced by oestradiol-17β (OE2) pretreatment, we investigated the effect of OE2 treatment of long-term (4 weeks) ovariectomized rats on the amount, activity and cellular actions of pharmacologically distinct PKC species in rat anterior pituitary tissue. Here we report that OE2 treatment enhanced phorbol 12,13-dibutyrate (PDBu)-induced LH but not GH release measured in vitro. This effect of OE2 on LH release may involve synthesis of additional PKCs that are not targeted by the synthetic diacylglycerol, 1,2-dioctanoyl-snglycerol (DOG). Measurements of anterior pituitary PKC activity and [3H] phorbol ester-binding studies suggested that the facilitatory action of OE2 on LH release may occur, at least in part, by altering the quantity and activity of PKC(s). Our results also demonstrate that the OE2-induced PKC(s) which facilitate LH release may be of the type that are not dependent upon raised intracellular Ca2+ for their activation and display distinct pharmacological properties (being readily activated by PDBu, but not by DOG, and are staurosporine-sensitive but H7-insensitive). This facilitatory action of OE2 on PKC-induced LH release does not appear to involve OE2-induced changes in the affinity of existing PKC(s) for PDBu, or changes in the amount of releasable LH in the pituitary prior to the stimulus.

Journal of Endocrinology (1993) 136, 105–117
Bradykinin and vasopressin activate phospholipase D in rat Leydig cells by a protein kinase C-dependent mechanism
Authors: Vinggaard A. M. , Hansen H. S.
Published: December 2008

In the present study we report that bradykinin stimulated phospholipase D activity in rat Leydig cells. Bradykinin added for 8 min stimulated choline formation in a dose-dependent manner and, in the presence of ethanol, bradykinin (100 nmol/l) stimulated transphosphatidylation by phospholipase D resulting in the formation of phosphatidylethanol. This stimulation was abolished after down-regulation of protein kinase C by long-term pretreatment for 22 h with phorbol 12-myristate 13-acetate (PMA). The stimulation of phospholipase D by the simultaneous addition for 8 min of maximum concentrations of PMA and vasopressin (AVP), PMA and bradykinin, or AVP and bradykinin produced no additive phosphatidylethanol or choline response, suggesting that AVP, bradykinin and PMA stimulated phospholipase D-catalysed phosphatidylcholine hydrolysis by a similar protein kinase C-dependent mechanism. Furthermore, LH (10 ng/ml), insulin (500 nmol/l), GH (100 ng/ml), interleukin-1β (5 U/ml) and platelet-activating factor (200 nmol/l) were found not to activate phospholipase D, whereas the Ca2+ ionophore A23187 (10 μmol/l) stimulated phosphatidylethanol formation, suggesting that Ca2+ might be a regulator of phospholipase D in Leydig cells.

Journal of Endocrinology (1993) 136, 119–126
Growth hormone (GH) suppression of catecholamine turnover in the chicken hypothalamus: implications for GH autoregulation
Authors: Lea R. W. , Harvey S.
Published: December 2008

GH administered centrally or peripherally inhibits basal or secretagogue-induced GH secretion in domestic fowl. Since the release of pituitary GH is neurally regulated by the hypothalamus, GH autoregulation may be mediated by changes in the content or metabolism of hypothalamic monoamines. When chicken GH (500 μg/kg body weight) was injected i.v. into laying hens, tissue catecholamine (adrenaline, noradrenaline and dopamine) concentrations in the preoptic area (POA) and medial basal hypothalamus (MBH) were depleted for 2–24 h, as were concentrations of dihydroxyphenylacetic acid, a dopamine metabolite. The serotonin (5-HT) content of the POA and MBH was unaffected by i.v. GH administration, although a reduction in MBH 5-hydroxyindoleacetic acid suggested a tissue-specific inhibition of 5-HT turnover. Qualitatively similar results were observed in laying hens 24 h after the intracerebroventricular injection of chicken GH (10 μg/bird). These results therefore demonstrate aminergic actions of GH within the chicken hypothalamus which may mediate GH autoregulation. However, as amine metabolism is not only suppressed when endogenous GH secretion is reduced, but also at times when normal GH secretion is restored, these aminergic effects may also reflect other actions of GH on central function.

Journal of Endocrinology (1993) 136, 245–251
Acute or chronic immunoneutralization of somatostatin does not affect growth hormone or thyroid hormone secretion in sheep
Authors: Van Kessel A. G. , Laarveld B.
Published: December 2008

The effect of acute or chronic immunoneutralization of somatostatin (SRIF) on plasma GH, thyrotrophin (TSH) and thyroid hormones was examined. Acute responses to SRIF immunoneutralization were examined using 30 intact male lambs (19·8 ±0·6 kg) assigned to one of five treatment groups such that control (C) lambs received no anti-SRIF immunoglobulin and SRIF-immunized (SI) lambs received 2 mg (SI2), 10 mg (SI10), 20 mg (SI20) or 100 mg (SI100) anti-SRIF immunoglobulin/kg body weight (BW). Control immunoglobulin was administered such that all lambs received 100 mg total immunoglobulin protein/kg BW. Effects of chronic SRIF immunoneutralization were examined using C and SI100 lambs which received additional (40 mg/kg BW) control and anti-SRIF immunoglobulin respectively, 4 and 8 days following the initial dose. Blood samples were collected from all lambs, at 10-min intervals, for 5 h immediately following initial immunoglobulin infusion and, from C and SI100 lambs, at 10-min intervals, for 5 h at 11 days following initial immunoglobulin infusion. At the end of each 5-h sampling period, pituitary and thyroid function was examined by i.v. challenge with thyrotrophin-releasing hormone (TRH; 0·33 μg/kg BW). Basal plasma GH and thyroxine (T4) and the GH, TSH, T4 and tri-iodothyronine (T3) responses to TRH were not influenced by acute or chronic immunoneutralization of SRIF. Acute, but not chronic, immunoneutralization of SRIF elevated basal plasma T3 in SI100 lambs only. The results suggest that SRIF, under physiological conditions, does not influence GH or thyroid hormone secretion in sheep but may influence thyroid hormone metabolism acutely.

Journal of Endocrinology (1993) 136, 261–269