bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of retinoids on protein kinase C activity and on the binding characteristics of the tri-iodothyronine nuclear receptor
Authors: Pailler-Rodde I. , Garcin H. , Higueret P.
Published: December 2008

Retinoids and thyroid hormones exert profound effects on the development, growth and homeostasis of vertebrates. The receptor proteins which bind retinoic acid, tri-iodothyronine (T3) or steroid hormones and, as a result of this binding, interact with DNA to stimulate expression of specific genes, belong to the same recently discovered superfamily. The functionality of thyroid and steroid hormone receptors is thought to be related to a phosphorylation-dephosphorylation cycle. In the present work, the action of two retinoids (retinol and retinoic acid) was studied on the properties of T3-nuclear receptors and on protein kinase C (PKC) activity in the rat liver (PKC is known to be a phosphorylating enzyme for various proteins). The influence of 12-O-tetradecanoyl phorbol-13-acetate (TPA; known to enhance PKC activity) on the properties of T3-nuclear receptors was also investigated. Measurements of binding characteristics and enzyme activity were performed 4 or 12 h after a single i.p. injection of retinol or retinoic acid (6 mg/kg body weight) or 1 h after a single i.p. injection of TPA (0·7 mg/kg). The activity of PKC was increased 4 h after administration of the retinoids, and the affinity of the T3-nuclear receptor protein was increased markedly after 12 h. The activity of PKC and the affinity of the nuclear T3 receptor were both increased 1 h after administration of TPA. These observations provide indirect evidence that retinoids, particularly retinoic acid, induce an increase in PKC activity and a subsequent increase in the affinity of the T3-nuclear receptor protein.

Journal of Endocrinology (1991) 128, 245–251
Identification of a non-covalent oxytocin/neurophysin-I complex in the bovine ovary
Authors: Shukovski L. , Findlay J. K. , Smith A. I.
Published: December 2008

Acid extracts of bovine preovulatory granulosa cells and corpora lutea (CL) were subjected to high-performance liquid chromatography (HPLC) and found to contain two peaks of immunoreactive (ir) oxytocin (OT), one corresponding to authentic OT and the second eluting 8 min later. The second peak was more abundant than authentic irOT in preovulatory follicles and in the early CL, but became less abundant as the CL matured (mid luteal) and was close to the limit of detection in the late CL. This peak could be detected only by an OT antiserum which recognized both the biologically active form of OT, as well as the post-translational processing intermediate Gly10-extended oxytocin. A second more specific OT antiserum (OT-933) did not recognize the second peak as strongly. Further analysis of the second peak revealed a complex of OT bound to its neurophysin (NP-I) which could be dissociated under denaturing conditions. Furthermore, we were able to create this complex in vitro by combining the two materials together under acid conditions, similar to the pH predicted in secretory granules, but not under neutral conditions. Measuring irNP-I by radioimmunoassay showed a single peak with a similar retention time to the OT/NP-I complex, confirming the identity of the unknown peak. Incubation of CL slices in culture showed a time-related release of both OT and NP-I, with OT having a greater rate of release in the mid luteal CL.

These data suggest the presence of an OT/NP-I complex in the bovine preovulatory granulosa cells and CL, as well as the unbound peptide presumably within the secretory granules. The ratio of OT/NP-I complex and free peptide changes with ageing of the the CL, perhaps indicating regulated differences in the post-translational processing of the prohormone.

Journal of Endocrinology (1991) 128, 305–314
Kallidin stimulates prolactin secretion from individual rat anterior pituitary cells
Authors: Stirland J. A. , Brown B. L. , Dobson P. R. M.
Published: December 2008

Kinins are localized within the adenohypophysis where they have been shown to stimulate the release of pituitary hormones. In the present study we have investigated the effect of [Lys]-bradykinin (kallidin) on prolactin secretion at the single cell level from cultured male rat anterior pituitary cells. This was assessed by use of a reverse haemolytic plaque assay which permits quantitative evaluation of the proportion of all pituitary cells which are secreting prolactin, and the amount of prolactin secreted per lactotroph (plaque area). The rate of plaque development was used as an index of the rate of hormone secretion in time-course studies.

Kallidin induced a dose-dependent increase in both the percentage of plaque-forming cells and the median plaque area during the first 2 and 3 h of incubation respectively. The threshold concentration of kallidin was 10 nmol/l. After 4 h of kallidin stimulation there was no difference between treated and control mono-layers with respect to median plaque area and the total secretion index. Although recruitment of additional cells into the secretory pool cannot be excluded, this seems unlikely since at 3 and 4 h little or no difference was observed in the number of plaque-forming cells. The data suggest that initially kallidin accelerated the rate of prolactin secretion primarily by inducing an increase in the number of cells secreting prolactin, and subsequently by increasing the amount of hormone secreted per lactotroph. The results presented here are consistent with the proposed role of the kallikrein–kinin system in the paracrine or autocrine control of prolactin release from the pituitary gland.

Journal of Endocrinology (1991) 128, 315–320
Insulin binding to Leydig cells and insulin levels in testicular interstitial fluid at different stages of development in the rat
Authors: Grizard G. , Fournet M. , Rigaudière N. , Lombard-Vignon N. , Grizard J.
Published: December 2008

Binding of insulin to purified intact Leydig cells (LC) and LC membranes, and levels of insulin in plasma and testicular interstitial fluid (IF) were quantitatively evaluated in rats at three stages of development. Specific insulin binding to intact LC increased significantly with age. Scatchard analysis of the binding data always gave curvilinear plots; the number of high affinity binding sites/LC were 2590±514, 3977±701 and 8342±2039 at 21, 40 and 70 days respectively. When the results were expressed per μg membrane protein, the maximal specific insulin binding also increased between 21 and 40 days but did not significantly change thereafter. With ageing, insulin levels in testicular IF decreased (3·05±0·30, 2·48±0·22 and 1·66±0·13 μg/l in 21-, 40- and 70-day-old rats) whereas plasma insulin increased. Taken together, these results suggest (1) that the intratesticular environment in this hormone cannot be evaluated by plasma insulin levels—testicular IF insulin concentration is probably a better index and (2) that insulin may play a role in the development of LC function during sexual maturation.

Journal of Endocrinology (1991) 128, 375–381
Evidence for the role of the secretory pattern of growth hormone in the regulation of serum concentrations of cholesterol and apolipoprotein E in rats
Authors: Oscarsson J. , Carlsson L. M. S. , Bick T. , Lidell A. , Olofsson S.-O. , Edén S.
Published: December 2008

Adult male Sprague–Dawley rats were hypophysectomized and connected to an automatic i.v. infusion system. The same daily dose of human GH (hGH) was given either as eight daily pulses (3-h intervals) to mimic the male specific secretory pattern of GH or as a continuous infusion of GH, to mimic the female secretory pattern. Hypophysectomized rats received i.v. replacement therapy with l-thyroxine and cortisol. The rats were treated for 5 days. The serum cholesterol concentration was higher when hGH was given continuously than when hGH was given as eight daily pulses. The concentration of high-density lipoprotein (HDL)-cholesterol was not influenced by intermittent GH treatment, but increased when hGH was given as a continuous infusion. The serum concentration of apolipoprotein (Apo) E increased following treatment with a continuous infusion of hGH, whereas eight daily pulses of hGH had no effect. The serum concentration of ApoA-I was unaffected by hGH treatment. The serum concentration of ApoB decreased to the same degree whether hGH was given as a continuous infusion or as eight daily pulses. The serum concentration of triglycerides was not affected by hGH treatment.

These results indicate that the higher serum HDL-cholesterol and serum ApoE concentrations of female rats may be due to their more continuous secretion of GH. In contrast, the effects of GH on the serum concentration of ApoB, which is not sexually differentiated, may be independent of the mode of GH secretion.

Journal of Endocrinology (1991) 128, 433–438
Developmental changes in growth hormone, insulin-like growth factors (IGF-I and IGF-II) and IGF-binding proteins in plasma of young growing pigs
Authors: Owens P. C. , Conlon M. A. , Campbell R. G. , Johnson R. J. , King R. , Ballard F. J.
Published: December 2008

The relationship between plasma concentrations of normally secreted GH and insulin-like growth factor-I (IGF-I) was investigated in pigs after weaning. Frequent blood sampling for between 12 and 24 h showed that plasma GH was pulsatile in pigs of 10, 20 and 35 kg liveweight. Pulses were brief in duration, low in amplitude and variable in frequency. Basal and average daily plasma concentrations of GH changed significantly with development, increasing by about 50% between 10 and 20 kg liveweight. Concentrations of IGF-I in plasma showed little or no evidence of diurnal periodicity and were not increased by GH pulses. Average daily concentrations of both IGF-I and IGF-II in plasma progressively increased between 10 and 35 kg liveweight, as did the total desaturated IGF-binding protein (IGFBP) activity of plasma. A strong positive correlation was observed between the total concentration of IGFs (IGF-I plus IGF-II) in the circulation and plasma IGFBP activity. The developmental rise in IGFBP activity of plasma was associated with increased labelling with 125I-labelled human IGF-II in ligand blots of binding proteins of apparent molecular masses > 200, 50, 43 and 29 kDa. One class of binding proteins of 34·5 kDa decreased with development. This study of young growing pigs shows that normally secreted endogenous GH exerts no significant immediate control over plasma IGF-I concentrations, and that plasma levels of IGF-I and IGF-II increase with maturation in this species. The close relationship between the concentrations of IGFs and IGFBPs in plasma is consistent with recent evidence indicating that binding proteins partially regulate the levels of IGF-I and IGF-II in blood by controlling their rates of clearance from the circulation.

Journal of Endocrinology (1991) 128, 439–447
Oxygen sensitivity of potassium- and angiotensin IIstimulated aldosterone release by bovine adrenal cells
Authors: Brickner R. C. , Raff H.
Published: December 2008

Angiotensin II (AII) and extracellular K+, acting through different intracellular mechanisms, stimulate aldosterone release in a synergistic fashion. We have previously shown that decreases in oxygen (O2) within the physiological range inhibit AII, cyclic AMP (cAMP) and ACTH-stimulated aldosterone release. The present experiment evaluated the effect of various concentrations of O2 on K+-stimulated aldosterone release in the presence and absence of AII. Dispersed bovine adrenal glomerulosa cells were incubated with different concentrations of K+ (0·9–5·4 mmol/l) without and with AII (10 nmol/l) under different concentrations of O2 (0, 5 or 50%); 21% O2 (pO2 = 19·9±0·5 kPa, n = 9) was used as reference control for comparison.

In all cases, increases in K+ stimulated aldosterone release, an effect augmented by AII. Under 0% O2 (pO2 = 8·1±0·3 kPa, n = 3) and 5% O2 (pO2 = 12·8±0·5 kPa, n= 3), aldosterone release stimulated by K+ or K+/AII was significantly inhibited compared with that under 21% O2. Conversely, under 50% O2 (pO2 = 36·3±2·5 kPa, n= 3), aldosterone release stimulated by K+ or K+/AII was significantly augmented. Cortisol secretion was not significantly affected by 5% or 50% O2 but was significantly decreased under 0% O2.

The effect of O2 on K+/AII stimulation of aldosterone release, as well as previous experiments with cAMP, progesterone and ACTH, suggest a final common post-receptor oxygen-sensitive component of the aldosterone synthetic pathway. It is suggested that one or more enzymes in the aldosterone synthetic pathway is/are exquisitely sensitive to small changes in O2 within the physiological range.

Journal of Endocrinology (1991) 129, 43–48
An inherited non-amyloidogenic transthyretin variant, [Ser6]-TTR, with increased thyroxine-binding affinity, characterized by DNA sequencing
Authors: Fitch N. J. S. , Akbari M. T. , Ramsden D. B.
Published: December 2008

Amplification and sequencing of the four transthyretin (TTR) exons of a subject with a variant TTR with four-fold increased affinity for thyroxine revealed a heterozygous G to A point mutation at base 7 of exon 2. This results in a serine for glycine change at residue 6 of the mature TTR monomer. No other mutations were found in any exon. Amplification and MspI digestion of TTR exon 2 from the leucocyte DNA of eight members of the subject's family revealed that all but one member were also heterozygous for [Ser6]-TTR.

Journal of Endocrinology (1991) 129, 309–313
Interleukin-1α-induced changes in androgen and cyclic adenosine 3′,5′-monophosphate release in adult rat Leydig cells in culture
Authors: Moore C. , Moger W. H.
Published: December 2008

Interleukin-1 (IL-1) has been proposed as a paracrine regulator of testicular function. The effect of this cytokine on adult rat Leydig cells in primary culture was investigated. Interstitial cells were purified on a two-step Percoll gradient and cultured in the presence or absence of recombinant human IL-1α (IL-1α). The presence of IL-1α in the culture media resulted in a dose-dependent increase in 24-h basal androgen release (half-maximal effective concentration = 40–50 U/ml). The stimulatory effect of IL-1α peaked on days 3 and 4, and was often still significant after 6 days of culture. Removal of IL-1α was followed by a return of basal androgen release to control levels within 3 days. In contrast, cells treated with IL-1α (100 U/ml) for 3 days released significantly less androgen (per 4 h) in response to 1–100 ng LH/ml than control cells. A similar inhibitory effect on the response to dibutyryl cAMP and pregnenolone was observed. Under basal conditions, IL-1α-treated cells released significantly more cAMP than did control cells. In contrast, the increase in cAMP release seen with LH-stimulated cells was significantly inhibited by treatment with IL-1α. These results suggest that IL-1α has a dual effect on adult rat Leydig cells in culture. It stimulates basal but inhibits LH-induced androgen release with parallel changes in cAMP levels. An additional inhibitory effect appears to lie at the level of the 17α-hydroxylase/C17–20 lyase enzyme.

Journal of Endocrinology (1991) 129, 381–390
Stimulation of steroidogenesis by forskolin in rat adrenal zona glomerulosa cell preparations
Authors: Purdy S. J. , Whitehouse B. J. , Abayasekara D. R. E.
Published: December 2008

The actions of forskolin have been investigated to determine to what extent its effects on steroidogenesis in rat adrenal preparations are dependent on activation of adenylate cyclase. In zona glomerulosa preparations, stimulation of both aldosterone and corticosterone production was obtained at concentrations of forskolin between 1 and 10 μmol/l. The effects of 10 μmol forskolin/l were additive with those of low doses (1 pmol/l) of corticotrophin (ACTH), but not with those of high doses (1 nmol/l) of ACTH. In contrast, in zona fasciculata/reticularis cells, doses of forskolin up to 10 μmol/l produced no significant stimulation of corticosterone production either alone or in the presence of ACTH (1 pmol/l and 1 nmol/l). The response to 1 nmol ACTH/l was attenuated in the presence of forskolin (10 μmol/l) in both zona glomerulosa and zona fasciculata/reticularis cell preparations. Cyclic AMP production increased progressively with dose up to 100 μmol forskolin/l in zona glomerulosa cells, whereas corticosterone production was maximal between 10 and 30 μmol forskolin/l and decreased at 100 μmol forskolin/l. In zona fasciculata/reticularis cells, cyclic AMP production was also increased by forskolin (1 and 10 μmol/l).

The stimulation of zona glomerulosa steroido-genesis by forskolin (1–10 μmol/l) and ACTH (1–100 pmol/l) were both reduced by the adenylate cyclase inhibitor, N6-phenylisopropyladenosine (100 μmol/l). The calcium channel inhibitor, nifedipine, only reduced the steroidogenic response to forskolin (3 μmol/l) at doses of 300 μmol/l whereas the response to 8·4 mmol K+/l was inhibited at 10 μmol nifedipine/1. Although there is some dissociation between the effects of forskolin on cyclic AMP and steroidogenesis, the results are generally consistent with the view that the effects of forskolin in rat zona glomerulosa cells are mainly dependent on activation of adenylate cyclase. This contrasts with the effects of forskolin in bovine fasciculata cells which are reported to be mediated by activation of voltage-regulated calcium channels.

Journal of Endocrinology (1991) 129, 391–397
κ-Opioid modulation of vasopressin secretion in conscious rats
Authors: Wells T. , Forsling M. L.
Published: December 2008

A series of studies has been performed in the conscious rat to investigate the effect of the intracerebroventricular (i.c.v.) administration of the selective κ-opioid receptor agonist, U50 488H, on arginine vasopressin (AVP) secretion stimulated by i.c.v. administration of hypertonic NaCl. Similarly, the effect of the i.c.v. administration of morphine and the i.v. administration of naloxone on AVP secretion was investigated. The response of AVP to an i.c.v. injection of hypertonic NaCl was potentiated by naloxone at a dose of 0·4 mg/kg, but a higher dose (1·2 mg/kg) was required to increase the basal plasma concentration of AVP. Prior treatment with U50 488H or morphine attenuated the increase in plasma concentrations of AVP stimulated by i.c.v. injection of hypertonic NaCl from 13·92±4·44 to 1·22±0·34 and 1·78±0·74 pmol/l respectively (n = 7; P<0·05). Prior administration of U50 488H also attenuated the potentiating effect of naloxone on AVP secretion stimulated by i.c.v. injection of hypertonic NaCl. These results indicate that basal AVP secretion is under tonic inhibitory control by dynorphin, and that μ-and κ-opioid receptors mediate an inhibitory influence of endogenous opioids on osmoreceptor-mediated AVP secretion.

Journal of Endocrinology (1991) 129, 411–416
Hormone concentrations and ovulatory response in rats treated with antiprogestagens
Authors: Uilenbroek J. Th. J.
Published: December 2008

Administration of antiprogestagens (2 mg/day) to female rats for 21 days induces high serum prolactin levels. These levels stimulate luteal progesterone production and an increase in ovarian weight. Compared with RU486 (mifepristone) the increase in prolactin is less after treatment with ZK299 (onapristone), an antiprogestagen with lower antiglucocorticoid activity. To study whether cyclic ovulations occur in rats treated with antiprogestagens, 5-day cyclic rats were given daily injections of RU486 or ZK299 (2 mg) from metoestrus (day 1) to pro-oestrus. This treatment advanced the forthcoming ovulation by 1 day; however, the ovulation rate was low. Injection of 10 IU human chorionic gonadotrophin on the afternoon of pro-oestrus (day 3) increased the ovulation rate, but not to the level found in oil-treated rats.

Serum LH concentrations measured from metoestrus to oestrus at 10.00 and 17.00 h were higher in antiprogestagen- than in oil-treated rats from day 2 (17.00 h) onwards. A low preovulatory LH surge was found in antiprogestagen-treated rats on the after-noon of pro-oestrus (day 3). Ovarian histology at the day of oestrus (day 4) confirmed the presence of a low LH surge as, besides ruptured follicles, unruptured follicles with dispersion of cumulus cells were present. The pro-oestrous surge of prolactin was also advanced by 24 h. The magnitude, however, was not different from that in oil-treated rats at day 4.

In conclusion, daily administration of antiprogestagens to 5-day cyclic rats results in increased basal levels of serum LH and advancement of the preovulatory surge of prolactin and LH by 1 day. The ovulatory response is low due to the low pre-ovulatory surge of LH and to a reduced ability of preovulatory follicles to respond to LH.

Journal of Endocrinology (1991) 129, 423–429
Effects of insulin-like growth factor-II on growth hormone release from human somatotrophinoma cells in vitro
Authors: Harris P. E. , Daniels M. , James R. A. , Turner S. J. , Dewar J. , Kendall-Taylor P.
Published: December 2008

Insulin-like growth factor-I (IGF-I) and IGF-II receptors have previously been demonstrated on membranes prepared from human somatotrophinomas. IGF-I has been shown to have a variable effect on GH secretion by these tumours in vitro. The effects of purified IGF-II on GH secretion have not been described. We have studied the direct actions of human recombinant IGF-II on GH release from eight somatotrophinomas cultured in vitro.

Somatotrophinoma cells were cultured as monolayers at a density of 105 cells/0·5 ml. Treatment with IGF-II for 4 and 24 h resulted in discrete inhibitory effects on GH release from two tumours (tumour 5:4 h, IGF-II 0·5 nmol/l; tumour 2; 24 h, IGF-II 1 nmol/l). Treatment with IGF-II for 24 h resulted in significant inhibitory effects on GH release from one tumour over a range of concentrations tested (IGF-II 0·5–10 nmol/l). Addition of human GH-releasing factor (hGRF)(1–44) (20 nmol/l) for 4 and 24 h resulted in stimulation of GH release by five tumours. Two tumours demonstrated significant inhibitory effects of IGF-II on GRF-stimulated GH release (tumour 2: 24 h, IGF-II 1–5 nmol/l; tumour 3; 4 h, IGF-II 5 nmol/l; 24 h, IGF-II 0·5–50 nmol/l).

These data emphasize the heterogeneity of somatotrophinomas in terms of their response to modulators of GH secretion. IGF-II does not appear to have a modulatory role on GH release by most somatotrophinomas.

Journal of Endocrinology (1991) 129, 447–451
Endogenous natriuretic factors: atrial natriuretic hormone and digitalis-like substance in Cushing's syndrome
Authors: Soszynski P. , Slowinska-Srzednicka J. , Kasperlik-Zaluska A. , Zgliczynski S.
Published: December 2008

In order to investigate the effect of chronic hypercortisolaemia on endogenous natriuretic factors (atrial natriuretic hormone (ANH) and the Na+/K+ pump inhibitor) digitalis-like substance (DLS), and their relation to hypertension, 28 patients with pituitary-or adrenal-dependent Cushing's syndrome and six patients on high-dose prednisone treatment were studied. Plasma ANH levels were increased in patients with Cushing's syndrome (36·0±1·4 (s.e.m.) ng/l) compared with those in healthy controls (28·6±1·3 ng/l, P <0·01). In prednisone-treated patients, ANH levels (43·8±4·5 ng/l) were higher than those in patients with Cushing's syndrome and in controls (P <0·05 and P <0·01 respectively). DLS measured by radioimmunoassay and binding of [3H]ouabain to erythrocytes was not altered in patients with hypercortisolaemia. Slightly decreased DLS activity in the erythrocyte 86Rb uptake inhibition assay was found in patients with Cushing's syndrome (52·9±2·7%) compared with that in controls (60·9±1·8%, P <0·02). With the exception of cortisol (r = 0·52, P<0·01), none of the other factors determined correlated with the mean arterial pressure in patients with Cushing's syndrome.

Thus, a chronic excess of endogenous and exogenous glucocorticoids increases plasma levels of ANH, but does not substantially influence DLS activity or plasma levels. Neither natriuretic factor is directly related to hypertension in Cushing's syndrome.

Journal of Endocrinology (1991) 129, 453–458
INSULIN-LIKE GROWTH FACTOR TYPE I AND INSULIN-LIKE GROWTH FACTOR TYPE II STIMULATE OESTRADIOL-17β HYDROXYSTEROID DEHYDROGENASE (REDUCTIVE) ACTIVITY IN BREAST CANCER CELLS
Authors: Singh Anita , Reed M.J.
Published: December 2008

Oestradiol-17β hydroxysteroid dehydrogenase (E2DH) is present in normal and malignant breast tissues and also in cultured breast cancer cells. It can act in a reductive direction to convert oestrone to the biologically active oestrogen, oestradiol, or in an oxidative direction to metabolize oestradiol to oestrone and may therefore have a crucial role in regulating breast tissue concentrations of oestradiol. Insulin-like growth factor-type I (IGF-I) and IGF-II are both mitogens for breast cancer cells. In this study we have examined the effect of these growth factors on the reductive and oxidative activities of E2DH in MCF-7 (receptor positive) and MDA-MB-231 (receptor negative) breast cancer cells. Both IGF-I (80 ng/ml) and IGF-II (80 ng/ml) significantly stimulated E2DH reductive activity (up to 138%) in MCF-7 cells but had no effect on oxidative activity. Addition of IGF-II (100 ng/ml) to MDA-MB-231 cells resulted in a small but statistically significant (p<0.05) increase in E2DH reductive activity (18%) but in these cells reductive activity is 25-70 times lower than oxidative activity. If IGF-I and IGF-II act to stimulate E2DH reductive activity in breast tumours then such a mechanism could account for the increased concentrations of oestradiol detected in breast tumours.
DEVELOPMENT OF A TWO-SITE IMMUNORADIOMETRIC ASSAY FOR DIMERIC INHIBIN USING ANTIBODIES AGAINST CHEMICALLY SYNTHESIZED FRAGMENTS OF THE α AND β SUBUNIT
Authors: Knight P.G. , Groome N. , Beard A.J.
Published: December 2008

A two-site (liquid-phase) immunoradiometric assay (IRMA) for dimeric inhibin has been developed using antibodies raised against synthetic peptide sequences corresponding to the N-terminus (1-32) of the α subunit and the C-terminal region (82-114) of the βA subunit of Mr ∼30,000 human inhibin. Highly-purified Mr 32,000 bovine inhibin (standard) gave a dilution curve parallel to those for bovine follicular fluid (bFF), human (h)FF and rat ovary extract. Whilst the assay detected both Mr 56,000 and 32,000 inhibin forms in bFF, little reaction with higher Mr forms was evident. Cross-reaction of 'free' inhibin subunit (Mr 25,000 form) and recombinant human activin A in the IRMA were minimal (< 0.1 and < 2% respectively). Although the detection limit of the IRMA (∼ 50 pg/tube) was similar to that of several reported radioimmunoassays (RIA), the IRMA was unable to detect dimeric inhibin in jugular or utero-ovarian vein plasma of heifers. Similarly, when assayed by IRMA, bFF, hFF and rat ovary extract contained 8-58 times less inhibin than was indicated by RIA. These observations are consistent with earlier evidence that the ovary secretes a substantial excess of 'free' inhibin α subunit and that this material reaches the peripheral circulation. Surprisingly, however, the inhibin contents of bFF, hFF and rat ovary extract determined by in vitro bioassay were 8-23 times greater than the corresponding IRMA values, being similar to those derived by RIA. It is suggested that this quantitative discrepancy between inhibin contents estimated by IRMA and bioassay may be due to (1) loss of bioactivity of the inhibin standard during its purification and/or storage, (2) failure of the IRMA to detect high Mr forms of bioactive inhibin and/or (3) cross reaction of follistatin and other FSH-suppressing substances in the in vitro bioassay.
Studies on the roles of phospholipase A2 and eicosanoids in the regulation of corticotrophin secretion by rat pituitary cells in vitro
Authors: Cowell A. M. , Flower R. J. , Buckingham J. C.
Published: December 2008

Dispersed anterior pituitary cells were used to investigate the possible roles of phospholipid metabolites released by phospholipase A2 (PLA2) in the control of immunoreactive ACTH (ir-ACTH) secretion in vitro. PLA2 (15 600–62 500 U/1), the PLA2 activator melittin (0·5–20 mg/l) and arachidonic acid (1 mmol/l) all produced increases in ir-ACTH release from the cells, whilst platelet-activating factor (PAF), prostaglandin F2α (PGF2α), the prostacyclin analogues iloprost and BW245C, the thromboxane A2 (TXA2) analogue U46619, and the leukotrienes LTB4 and LTC4 were ineffective in this respect. PGF2α (100 nmol/l and 1 μmol/l), iloprost (1 μmol/l) and BW245C (100 nmol/l and 1 μmol/l) depressed corticotrophin-releasing factor-41-induced ir-ACTH secretion, while the PAF antagonist BN52021 (10 and 100 μmol/l) and LTC4 (100 nmol/l and 1 μmol/l) had no discernable effects. The secretory responses of the cells to hypothalamic extracts (0·2 hypothalami/ml) and arachidonic acid (1 mmol/l) were generally unaffected by the cyclooxygenase inhibitors ibuprofen (10 and 100 μmol/l) and indomethacin (10 μmol/l), the TXA2 synthetase inhibitor imidazole (10 μmol/l–1 mmol/l), the lipoxygenase inhibitor nordihydroguaiaretic acid (10 and 100 μmol/l) and the dual cyclo-oxygenase/lipoxygenase inhibitors phenidone (1–100 μmol/l) and BW755C (10 and 100 μmol/l). They were, however, inhibited by the dual cyclo-oxygenase/lipoxygenase inhibitor eicosatetraynoic acid (10 and 100 μmol/l), which also blocks epoxygenase and PLA2 activity and by the cytochrome P450 inhibitor SKF-525A (1 mmol/l). The results suggest that the stimulatory effects of PLA2 and arachidonic acid on ir-ACTH secretion are not effected by products generated by the cyclo-oxygenase or lipoxygenase pathways but may be mediated by metabolites generated by the cytochrome P450 pathway.

Journal of Endocrinology (1991) 130, 21–32
Identification of circulating growth hormone-binding proteins in domestic poultry: an initial characterization
Authors: Vasilatos-Younken R. , Andersen B. J. , Rosebrough R. W. , McMurtry J. P. , Bacon W. L.
Published: December 2008

Multiple growth hormone (GH)-binding proteins (GHBPs) were identified in serum and plasma samples from domestic chickens and turkeys. Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis on 10% acrylamide, 2·7% bis discontinuous gels under reducing conditions and electrotransferred to nitrocellulose paper. Western blots were incubated with 125I-labelled recombinant chicken GH (cGH) or bovine GH and GHBPs visualized by means of autoradiography. In fresh samples (< 2 h from collection to gel electrophoresis), multiple minor high Mr bands were evident between approximately 72 000 and 175 000. Two major bands were observed at approximately 69 500 and 27 500. The latter is consistent with previous reports for the rat and mouse serum GHBPs based on nucleotide sequence analysis. The minor bands were essentially undetectable after storage at −25 °C for several months, and an additional major band at Mr approximately 52 500 appeared. The Mr-69 500 major protein contained N-linked carbohydrate, as determined by a reduction in molecular size by treatment with peptide N-glycosidase F. Binding of 125I-labelled GH was partially inhibited by co-incubation with 50 μg unlabelled pituitary-derived cGH/ml and excess unlabelled porcine GH as well as ovine prolactin, but not by bovine insulin. Non-specific binding of 125I-labelled GH by serum albumin was also observed.

A comparison was made between these GHBPs and the hepatic GH receptor (e.g. molecular weight estimates, affinity for homologous versus heterologous GHs, cross-reactivity with prolactin, presence of N-linked carbohydrate). The origin and relationship among the various molecular weight species of GHBPs identified, and their potential role in regulation of the biological activity of GH in birds, remain to be determined.

Journal of Endocrinology (1991) 130, 115–122
Hormonal regulation of insulin-like growth factor (IGF)-binding proteins secreted by isolated sheep thyroid epithelial cells: relationship with iodine organification
Authors: Wang J. F. , Becks G. P. , Hanada E. , Buckingham K. D. , Phillips I. D. , Hill D. J.
Published: December 2008

Isolated sheep thyroid follicles release specific insulinlike growth factor-binding proteins (IGFBPs). Since IGFBPs can modulate IGF bioactivity, at least in vitro, their presence in thyroid tissues may influence synergistic interactions between TSH and endogenous IGF-I or -II which are known to control both thyroid growth and function. We have examined the hormonal control of IGFBP release in relation to iodine organification. Sheep thyroid follicles were isolated by incubation with collagenase and differential centrifugation, grown in Coon's modified Ham's F12M medium with the addition of transferrin, glycylhistidyl-lysine, somatostatin (3H), TSH, cortisol and insulin (6H), and maintained in OH (hormone-free) or 3H medium with or without further supplements for 48 h. Conditioned culture medium was separated by 8% sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis, transferred to nitrocellulose and incubated with 125I-labelled IGF-II followed by autoradiography (ligand blot). Additionally, the radioactive bands were cut from the filters and quantified by γ-spectrometry. Iodine organification was assessed by incubation of follicles with 106 c.p.m. Na125I for 3 h before washing, solubilization in 0·1 mol NaOH/l and the precipitation of organified radioisotope with 10% (v/v) trichloroacetic acid. Cells conditioned in OH or 3H medium released specific IGFBPs of 46, 34, 28 and 19 kDa on ligand blot analysis. The proteins of 34 and 19 kDa were immunopositive on Western blot analysis using anti-bovine IGFBP-2 antiserum. The 46-kDa IGFBP was retained by Concanavalin A–Sepharose chromatography and demonstrated to be glycoprotein. This is probably ovine IGFBP-3. The addition of TSH, or TSH plus cortisol, to OH or 3H medium significantly decreased the 125I-labelled IGF-II associated with the 34- and 28-kDA IGFBP species. All IGFBP species were substantially reduced in 6H medium, which was predominantly due to the effects of TSH and cortisol. When total 125I-labelled IGF-II associated with IGFBPs was considered, a significant (P < 0·01) inverse correlation existed between IGFBP activity and iodine organification in the same cultures; the latter being greatest in OH or 3H medium supplemented with TSH and cortisol. None of these hormone additions altered the endogenous release of IGF-II by the cells. These results suggest that endogenous IGFs, under hormonal control, may modulate the action of endogenous IGF in the regulation of thyroid function.

Journal of Endocrinology (1991) 130, 129–140
A decline in prolactin levels in amniotic fluid and decidua at term pregnancy after the initiation of labour
Authors: Kinoshita T. , Taketani Y. , Mizuno M.
Published: December 2008

Prolactin is known to inhibit the production of prostaglandins, key substances in the initiation of labour. To explore a possible role of prolactin in human parturition, prolactin levels were therefore measured in amniotic fluid and decidua at full-term pregnancy. Prolactin levels in amniotic fluid before labour were about three times higher than those at labour and they tended to decrease with time during labour. Prolactin levels in decidua before labour were about twice as high as those at labour. These findings suggest a possible involvement of prolactin in human parturition.

Journal of Endocrinology (1991) 130, 151–153
Effects of follicular fluid administration on serum bioactive and immunoactive FSH concentrations and compensatory testicular hypertrophy in hemicastrated prepubertal rats
Authors: Brown J. L. , Dahl K. D. , Chakraborty P. K.
Published: December 2008

In prepubertal rats, removal of one testis (hemicastration) results in increased FSH secretion and a compensatory hypertrophy of the remaining testis. To determine whether these two events are related, testis weight was measured after inhibin-rich porcine follicular fluid (FF) was administered to hemicastrated rats twice daily for 14 days to block the compensatory rise in FSH. After hemicastration, serum immunoreactive FSH concentration approximately doubled and testis weight was increased by ∼60%. A slight but non-significant increase in serum bioactive FSH was also observed. Treatment of hemicastrated rats with FF completely prevented the increase in serum FSH and compensatory growth of the remaining testis, whereas concomitant treatment of these animals with ovine FSH reversed the inhibitory effects of FF. Serum inhibin concentrations were determined using two radioimmunoassay (RIA) systems; one assay utilized an antiserum generated against intact bovine inhibin and the other was directed against an α-inhibin fragment. Both assays showed a decline in inhibin levels following hemicastration. In addition, increases in serum inhibin concentrations were observed following FF administration in both RIAs; however, the relative increase in inhibin over levels in hemicastrated rats was greater using the intact inhibin assay.

In summary, these data suggest that the increase in FSH concentrations after hemicastration is related to a reduction in inhibin levels, and that this FSH rise is a primary signal for initiating compensatory testicular hypertrophy in prepubertal rats. Furthermore, exogenous FSH administration overcame the inhibitory effects of FF on the testes, suggesting that inhibin may not act directly at the gonadal level.

Journal of Endocrinology (1991) 130, 207–212
The effect of uninephrectomy on osteocalcin metabolism in sheep: a direct evaluation of renal osteocalcin clearance
Authors: Farrugia W. , Yates N. A. , Fortune C. L. , McDougall J. G. , Scoggins B. A. , Wark J. D.
Published: December 2008

Indirect evidence has suggested that the kidney is a major organ of clearance for osteocalcin, a circulating marker of osteoblast function. The objectives of the present study were (1) to confirm the role of the kidney in osteocalcin clearance (2) to quantify the contribution of extrarenal sites and (3) to investigate the renal mechanism(s) of osteocalcin clearance. Plasma osteocalcin levels, osteocalcin plasma clearance rate (PCR) and plasma production rate (PPR) were determined in oophorectomized (OX) and uninephrectomized oophorectomized (UOX) sheep. The osteocalcin renal extraction efficiency (REE) and the effective renal plasma flow (ERPF) were measured, and the osteocalcin renal clearance rate (RCR) was calculated.

The osteocalcin PCR was reduced significantly in UOX compared with OX sheep (2·0±0·1 (n = 9) vs 2·5±0·1 litres/h (n = 44); P < 0·0005). In UOX sheep with plasma creatinine levels ≤ 130 μmol/l, the osteocalcin REE was 9±1·3% and the osteocalcin RCR was 50–91% of osteocalcin PCR (n = 4). In UOX sheep with plasma creatinine levels in the range 100–440 μmol/l, there was a linear relationship between osteocalcin PCR and ERPF; the osteocalcin RCR was related to the osteocalcin PCR (RCR = 0·9 × PCR −0·50). Intravenous infusion of the synthetic glucocorticoid triamcinolone acetonide (TA) in UOX sheep led to marked decrements in plasma osteocalcin levels and the osteocalcin PPR, and a significant increase in the osteocalcin PCR. These changes were accompanied by a 44% increase in ERPF. During i.v. infusion of 125I-labelled osteocalcin in three UOX sheep, the urinary excretion of trichloroacetic acid-precipitable radioactivity represented 27% (range 22–31%) of the amount cleared by the kidney. Bio-Gel P6 chromatography of urine suggested the presence of intact 125I-labelled osteocalcin and at least one radiolabelled osteocalcin fragment.

These findings confirm that the kidney is the major site of osteocalcin clearance and show that extrarenal sites also make an appreciable contribution. ERPF is an important determinant of the osteocalcin PCR. Augmentation of the ERPF by TA may mediate the induction of osteocalcin clearance by this glucocorticoid. In the UOX sheep, urinary excretion of intact osteocalcin may account for up to 30% of renal osteocalcin clearance.

Journal of Endocrinology (1991) 130, 213-221
Effects of insulin-like growth factor-I on aromatase cytochrome P450 activity and oestradiol biosynthesis in preimplantation porcine conceptuses in vitro
Authors: Hofig A. , Simmen F. A. , Bazer F. W. , Simmen R. C. M.
Published: December 2008

The effects of insulin-like growth factor-I (IGF-I) on aromatase P450 activity and steroid production in preimplantation pig conceptuses were evaluated in vitro. Conceptuses recovered from gilts on days 10 and 12 of pregnancy were incubated for 6 h in modified Eagle's Minimum Essential Medium (MEM) plus IGF-I (0·1 μg/ml) or insulin (8·5 μg/ml), and conceptuses were monitored for their ability to convert [1,2-3H]β-testosterone into oestrogens. Aromatase activity of day-10 conceptuses was low and unaffected by IGF-I or insulin. In contrast, basal aromatase activity in day-12 conceptuses was about threefold higher and was further increased by IGF-I (P < 0·02), but was unaffected by insulin. To determine whether higher aromatase P450 activity was associated with increased oestradiol production, concentrations of oestradiol were determined by radioimmunoassay in culture medium of day-11 and -12 conceptuses, after incubation in MEM alone or in the presence of dehydroepiandrosterone (DHA, 1 μg/ml) with or without IGF-I (0·1 μg/ml) or insulin (0·1 or 8·5 μg/ml) for 24 h. Conceptuses in MEM plus DHA produced more oestradiol (P < 0·01) than those in MEM alone. Addition of IGF-I or insulin did not increase the effect of DHA. Basal oestradiol production was dependent on conceptus size; however, IGF-I or insulin did not affect basal or DHA-stimulated oestradiol production regardless of conceptus size. These findings demonstrate that IGF-I can modulate aromatase activity in vitro, without affecting overall de-novo steroidogenesis. Thus, the developmental increase in conceptus oestradiol production observed during early pregnancy in the pig may reflect synergistic interactions between IGF-I and other regulatory factors present within the conceptus and/or uterine environment.

Journal of Endocrinology (1991) 130, 245–250
Protein kinase C, an endogenous regulator of hormone-induced cyclic AMP induction in porcine luteal cells
Authors: Rajkumar K. , Chedrese P. J. , Ly H. , Murphy B. D.
Published: December 2008

LH, in addition to increasing cyclic AMP (cAMP) in ovarian cells, stimulates phosphoinositide hydrolysis producing inositol trisphosphate and diacylglycerol (DG). DG activates phospholipid- and calcium-dependent protein kinase (PKC). In the present study, we have used both PKC activators and inhibitors to examine the interactions of the PKC pathway on hormone-induced cAMP production in porcine luteal cells. Phorbol 12-myristate 13-acetate (PMA) enhanced LH- and forskolin-induced cAMP production. A time-course study indicated that the facilitatory effect of PMA was greater when added to incubation tubes following addition LH or forskolin. The non-tumour-promoting phorbol ester 4α-phorbol 12,13-didecanoate, which does not stimulate PKC activation, did not facilitate hormone-induced cAMP induction. PKC inhibitors polymyxin B, sphingosine and 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H7) antagonized the facilitatory effect of PMA on LH-induced cAMP production. The cAMP induction by both LH and forskolin was inhibited in the presence of PKC inhibitors. Polymyxin E, which differs from polymyxin B by a single amino acid and does not inhibit PKC activation, did not inhibit LH- or forskolin-induced cAMP induction.

The results of this study provide evidence for a facilitative action of the PKC effector system on hormonally stimulated cAMP production. Furthermore, PKC may be an important endogenous regulator of adenylate cyclase activity in porcine luteal cells.

Journal of Endocrinology (1991) 130, 273–280
Induction of calcitonin receptor expression by glucocorticoids in T47D human breast cancer cells
Authors: Kurokawa M. , Michelangeli V. P. , Findlay D. M.
Published: December 2008

When T47D cells were maintained long term in medium containing 0·1 μmol cortisol/l, calcitonin receptor (CTR) expression was stimulated compared with the very low levels of binding in untreated cells grown from frozen stocks. The time-course of the appearance of CTR following treatment with cortisol was slow, requiring up to 3 weeks of continuous exposure of the cells to the steroid. Binding capacity of control cells also increased slowly with time in culture, but after 3 months was only 20–30% of that in cells continuously treated with cortisol. Removal of cortisol resulted in rapid loss of CTR so that binding was reduced to ∼ 50% of treated cell levels within 1 week of removal. Scatchard analysis of the binding data showed that the increased binding capacity induced by cortisol was due solely to a change in average receptor number per cell, with no change in receptor affinity. That this induction of CTR was due to a glucocorticoid effect was shown by the more rapid (< 96 h) and more potent (< 1 nmol/l) action of dexamethasone than of cortisol. In addition, induction was inhibited by the glucocorticoid inhibitor RU486. The induced receptors were shown to be functional, since salmon calcitonin-stimulated adenylate cyclase was induced in parallel with CTR. These results indicate that glucocorticoids are potential regulators of the CTR.

Journal of Endocrinology (1991) 130, 321–326
Growth hormone receptors in the liver and osmoregulatory organs of rainbow trout: characterization and dynamics during adaptation to seawater
Authors: Sakamoto T. , Hirano T.
Published: December 2008

Specific binding sites for chum salmon growth hormone (sGH) were identified in the membranes obtained from tissues of rainbow trout. Specific binding of 125I-labelled sGH (% per mg protein) was found in the liver (37%), ovary (6%), brain (6%), gill (4%), intestine (4%) and posterior body kidney (4%). Specific binding was not significant in head kidney, anterior body kidney, spleen, heart, skeletal muscle or skin. Scatchard analyses demonstrated the presence of a single class of high-affinity low-capacity receptors in the liver, gill, intestine and kidney. The association constants for the membranes from liver, gill, intestine and kidney were of the same order (1 litre/nmol). Chum salmon prolactin did not inhibit the binding of 125I-labelled sGH to receptors in the liver, gill, intestine and kidney.

Transfer of rainbow trout from fresh water to 80% seawater evoked a rise in plasma concentration of GH and a significant decrease in the GH binding to the liver membranes after 1 day. Binding in the gill and kidney was not altered significantly. Membranes were treated with 4 mol MgCl2/l to remove bound GH from the receptors, and the results indicated that the reduction in binding in the liver after transfer to sea-water was probably due to receptor occupancy by increased endogenous GH. The occupancy of liver GH-binding sites was maximal 4 days after transfer. Total (MgCl2-treated) binding sites in the liver increased significantly 14 days after transfer. Scatchard analysis indicated that receptors were altered in capacity without changes in binding affinity. Although GH may also directly affect osmoregulatory organs through their GH receptors, the present results indicate the likelihood of at least partial mediation by the liver of the seawater-adapting action of GH in the rainbow trout.

Journal of Endocrinology (1991) 130, 425–433