bluuebunny/crossref_metadata_2025 huggingface dataset:
Serum thyrotropin (TSH) heterogeneity in euthyroid subjects and patients with subclinical hypothyroidism: the core fucose content of TSH-releasing hormone-released TSH is altered, but not the net charge of TSH
Authors: Schaaf L , Trojan J , Helton T E , Usadel K H , Magner J A
Published: December 2008

The aims of the present study were to determine the influence of brief subclinical hypothyroidism on the isoforms of serum thyrotropin (TSH) and to examine the net charge of TSH in different metabolic states. Sera were obtained from euthyroid subjects (n=7) and from patients with subclinical hypothyroidism (n=8) before and 30 min after the intravenous administration of 200 μg thyrotropin-releasing hormone (TRH). The TSH from human pituitary extracts (IRP 68/38), basal and TRH-stimulated serum TSH was immunoconcentrated and further analysed by isoelectric focusing (IEF) and lentil lectin affinity chromatography. TSH immunoreactivity was determined in each specimen or fraction with an automated highly sensitive chemiluminometric TSH assay. We found that basal TSH in subclinical hypothyroidism, and TRH-released TSH in euthyroidism and in subclinical hypothyroidism is distributed in a similar neutral to acidic pattern, which significantly differs from the more alkaline to neutral isoform pattern of intrapituitary TSH (P<0·05). IEF analysis of pituitary standard TSH revealed 3 major peaks (pI values 7·5; 6·6; 5·8) whereas in most euthyroid or subclinically hypothyroid subjects 5 peaks were found. Lentil lectin affinity chromatography revealed that TRH-released TSH in euthyroid subjects has more core fucose residues than TSH from patients with subclinical hypothyroidism (64·6 ±6·7 vs 12·5 ±2·7%, P<0·0001).

Thus pituitary standard TSH seems to be less mature material than circulating TSH. Perhaps no alteration in the IEF pattern of TSH was detected during early hypothyroidism because sialylation of TSH was increasing as sulfatation was decreasing. Nevertheless, a change in the core fucose content of TSH was detectable by lentil analysis.

Journal of Endocrinology (1995) 144, 561–567
The reduction of circulating growth hormone and prolactin in streptozocin-induced diabetic male rats is possibly caused by hypothalamic rather than pituitary changes
Authors: Boujon C E , Bestetti G E , Abramo F , Locatelli V , Rossi G L
Published: December 2008

To gain further information on diabetes-related disorders in the somatotrophic and lactotrophic axes, we undertook a functional, morphometrical and densitometrical study of the arcuate nucleus (AN), median eminence (ME) and anterior pituitary gland of adult male rats one month after streptozocin-induced diabetes (STZ-D). The basal secretory activity of somatotrophs and lactotrophs was tested by the reverse haemolytic plaque assay (RHPA) and plasma GH and prolactin (PRL) levels were determined by RIA. The number of GH-releasing factor (GRF)-labelled axons and the amount of axonal tyrosine hydroxylase (TH)-immunoreactivity increased in STZ-D. There were no significant differences in any of the other densitometrical measurements performed on GRF-, somatostatin-, thyrotropin-releasing hormone- and TH-labelled ME axon cross-sections as well as those on tuberoinfundibular-dopaminergic neurones of the AN in STZ-D compared with control rats. Plasma GH and PRL levels and measurements on anterior pituitary GH- and PRL-labelled structures were decreased in STZ-D. However, the GH and PRL plaque areas were increased after RHPA implying that the secretory capacity of somatotrophs and lactotrophs was not impaired. Taken together, these results suggest that the accumulated GRF in the ME is due to reduced GRF release. This could account for the reduced amplitude and/or frequency of GH secretory pulses. The increased axonal TH-immunoreactivity may indicate an increased dopamine synthesis. If coupled to increased release this could, in turn, be partly responsible for the reduced plasma and anterior pituitary PRL concentration. Although a direct effect of diabetes on the anterior pituitary cannot be ruled out, the reduction of circulating GH and PRL in STZ-D male rats seems to be caused by hypothalamic rather than anterior pituitary changes.

Journal of Endocrinology (1995) 145, 19–26
Inhibitory and stimulatory effect of oestrogens upon ovarian 17α-hydroxylase/C17,20-lyase in immature hypophysectomized rats treated with gonadotrophin
Authors: Johnson D C , Crane L H
Published: December 2008

A study was designed to compare the effects of exogenous and endogenous oestrogens upon the expression of steroid 17α-hydroxylase/C17,20-lyase (CYP17) in the immature hypophysectomized rat ovary. C17,20-lyase activity was measured ex vivo using [21-14C]progesterone, while serum concentrations of androstenedione indicated in vivo activity. Immunocytochemistry was used to localize the enzyme within the ovary. Activity of CYP17 increased dramatically and remained high in thecal and interstitial cells after injection of equine chorionic gonadotrophin, even though serum oestradiol (OE2) levels exceeded 2 nmol/l. Production of comparable serum levels by the use of Silastic capsules containing OE2 greatly suppressed but did not stop, expression of the enzyme induced by repeated doses of human chorionic gonadotrophin (hCG). Subcutaneous implantation of Silastic capsules (1 cm) containing diethylstilboestrol (DES) stimulated follicular growth and increased ovarian weight by 67%, 5 days later. Injection of 50 IU hCG at various times after removal of the implant produced time-dependent changes in CYP17 activity and serum androstenedione levels, when measured 30 h later. Although the initial effect of removal of DES was an increase in CYP17 activity, delaying injection of hCG resulted in a reduced response. The results indicated that: (1) endogenous oestrogens do not inhibit CYP17 expression; (2) exogenous oestrogens only reduce the number of thecal/interstitial cells expressing CYP17 when they are exposed to hCG; (3) pretreatment with oestrogen removes the ovarian interstitial but not the thecal cell expression of CYP17 in response to hCG; and (4) oestrogens can be stimulatory for CYP17 expression in thecal cells.

Journal of Endocrinology (1995) 145, 59–67
Mechanism of phospholipase D activation induced by extracellular ATP in osteoblast-like cells
Authors: Suzuki A , Shinoda J , Oiso Y , Kozawa O
Published: December 2008

We have previously reported that extracellular ATP stimulates Ca2+ influx from extracellular space, resulting in the production of prostaglandin E2 which mediates, at least in part, its proliferative effect on osteoblast-like MC3T3-E1 cells, and that the activation of protein kinase C (PKC) stimulates phospholipase D in these cells. In the present study, we examined the effect of extracellular ATP on phosphatidylcholine-hydrolysing phospholipase D activity in MC3T3-E1 cells. ATP stimulated the formation of both choline and inositol phosphates dose-dependently in the range between 0·1 and 1 mm. The formation of choline by a combination of ATP and NaF, an activator of GTP-binding protein, was synergistic, whereas that of inositol phosphates was not. A combination of ATP and 12-O-tetradecanoylphorbol-13-acetate, a PKC activating phorbol ester, additively stimulated the formation of choline. Staurosporine, an inhibitor of PKC, had little effect on ATP-stimulated formation of choline. Choline formation was significantly reduced by chelating extracellular Ca2+ with EGTA, while being inhibited by W-7, an antagonist of calmodulin. These results suggest that extracellular ATP stimulates phospholipase D in a Ca2+/calmodulin-dependent manner in osteoblast-like cells, and that neither PKC activation nor GTP-binding protein is involved in this mechanism.

Journal of Endocrinology (1995) 145, 81–86
Effect of sodium selenite on growth, insulin-like growth factor-binding proteins and insulin-like growth factor-I in rats
Authors: Grønbæk H , Frystyk J , Ørskov H , Flyvbjerg A
Published: December 2008

Selenium is an essential trace element although at higher doses it is also known to be a toxic agent causing a wide range of symptoms including growth retardation. In order to investigate the effect of sodium selenite on growth, insulin-like growth factor-binding proteins (IGFBPs) and insulin-like growth factor-I (IGF-I), 30 male Wistar rats were randomized into three groups. Group A was treated with sodium selenite in the drinking water (3·3 mg selenium/l). Group B was ad libitum fed with free access to standard fodder and tap water and group C was pair fed relative to the selenium-treated rats. Serum IGF-I and IGFBPs were determined on days 0, 14 and at the end of the study on day 35. Selenium-treated rats had significantly lower body weights compared with group B rats on day 9 and group C rats on day 14 (P<0·05). Tibia length was measured at the end of the study and no difference was observed between groups B and C (3·77 ± 0·04 cm vs 3·60±0·02 cm); however, selenium-treated rats had significantly shorter tibia lengths (3·46±0·03 cm) compared with rats in groups B (P<0·001) and C (P<0·05). Selenium treatment induced a significant reduction in circulating IGF-I by the end of the study compared with ad libitum and pair fed rats (P<0·05). Serum subjected to Western ligand blots showed four distinct IGFBP bands with apparent relative molecular weights of 38–47 kDa (doublet) (IGFBP-3), 30 kDa (IGFBP-1 and/or IGFBP-2) and 24 kDa (IGFBP-4). At the end of the study a significant reduction in IGFBP-3 was observed in group A compared with groups B and C (P<0·05). Selenium treatment also caused a reduction in IGFBP-1 and/or IGFBP-2 compared with ad libitum fed rats; in addition, a reduction was observed in pair fed controls. In conclusion, sodium selenite treatment leads to growth retardation accompanied by reduced circulating levels of IGF-I, IGFBP-3, and IGFBP-1 and/or IGFBP-2. The reduction in IGF-I and IGFBP-3 could not be attributed to reduced caloric intake but seems to be a specific action of selenium.

Journal of Endocrinology (1995) 145, 105–112
Oxytocin modulates the luteinizing hormone response of the rat anterior pituitary to gonadotrophin-releasing hormone in vitro
Authors: Evans J J , Hurd S J , Mason D R
Published: December 2008

Although GnRH is believed to be the primary secretagogue for LH, oxytocin has also been shown to stimulate LH release from the anterior pituitary. We investigated the possibility that the two secretagogues interact in the modulation of LH release. Anterior pituitaries were removed from adult female rats at pro-oestrus, and tissue pieces were pre-incubated in oxytocin for 3 h prior to being stimulated with 15 min pulses of GnRH. LH output over the 1 h period from the beginning of the GnRH pulse was determined. Control incubations were carried out in the absence of oxytocin, and background secretory activities without GnRH stimulation were also determined.

Tissue which was pre-exposed to oxytocin (0·012–1·25 μm) had an increased LH response to GnRH (1·25 nm). The increase was larger than the sum of the LH outputs obtained with oxytocin and GnRH separately, revealing that oxytocin synergistically enhanced LH secretion elicited by GnRH (P<0·05; ANOVA). If stimulation by GnRH was delayed for 2 h after incubation with oxytocin, an increase in LH secretion was still observed, indicating that oxytocin-induced modulation did not rapidly disappear. Oxytocin pre-incubation was observed to result in an increase of maximal GnRH-induced LH output (P<0·001; t-test), as well as an increase of intermediate responses.

The LH response of the anterior pituitary to subsequent pulses of GnRH was modified by the self-priming process. The effect of oxytocin pretreatment on the response of primed tissue to GnRH was also investigated. It was found that pre-incubation in oxytocin also enhanced the LH response of primed tissue to GnRH.

The study has revealed that oxytocin increases the LH output of anterior pituitary tissue in response to GnRH. The effect occurs on both GnRH-primed and unprimed tissues. The results suggest that oxytocin has the potential to regulate the dynamics of the pro-oestrous LH surge.

Journal of Endocrinology (1995) 145, 113–119
Augmentation of the effectiveness of porcine GH with swine antibodies induced by a synthetic GH peptide
Authors: Wang B S , Corbett M J , Shieh H-M , Sadeghi H
Published: December 2008

An effort was made to evaluate the potential usefulness of a peptide vaccine in improving the growth performance of farm animals. It was previously reported that a murine PS-7·6 monoclonal antibody (mAb) which was specific to porcine GH (pGH) enhanced the growth-promoting activity of pGH in an experimental hypophysectomized rat model. Additional data from a epitope mapping study suggested that PS-7·6 mAb recognized a pGH fragment corresponding to an amino acid sequence 54–95. In this report, therefore, a peptide pGH(54–95) was synthesized in an attempt to induce PS-7·6-like antibodies in swine. It was demonstrated that the peptide pGH(54–95) competed with PS-7·6 mAb for the binding to radioactive pGH in a competition radioimmunoassay and also caused pigs to elicit polyclonal antibodies immunoreactive to pGH protein. The association and dissociation rate constants of the swine antibody to pGH were 1·9 × 102 m−1 s−1 and 3·2×10−4 s−1 respectively, thus producing an overall binding affinity of Kd=1·6 × 10−6 m. The swine anti-body partially competed with murine PS-7·6 mAb for the binding to pGH, suggesting that the pGH-recognizing sites for both antibodies might be closely related. The biological effect of the swine antibody was examined in hypophysectomized rats and shown to significantly augment pGH activity in promoting the growth of these GH-deficient animals. The present findings suggest that a synthetic peptide may be developed as a potential growth vaccine for swine to generate antibodies capable of enhancing the effectiveness of endogenous pGH.

Journal of Endocrinology (1995) 145, 163–167
Effect of the level of oestradiol on oxytocin-induced prostaglandin F2α release in the cow
Authors: Mann G E , Lamming G E
Published: December 2008

We have investigated the effects of oestradiol-17β on the development of the luteolytic signal (prostaglandin F2α (PGF2α) release to an oxytocin challenge) using a steroid-treated ovariectomised cow model. Long-term ovariectomised cows were pre-treated with progesterone for 14 days followed by oestradiol-17β for 2 days to induce oestrus (day 0), and were then treated for a further 16 days with physiological doses of progesterone and oestradiol-17β to simulate a luteal phase. On day 11 oestradiol was either withdrawn (low group; n=4), maintained (normal group; n=4) or increased (high group; n=4), generating mean (± s.e.m.) plasma concentrations of oestradiol from days 12–16 of 0·9±0·1, 2·3±0·4 and 3·7±0·3 pg/ml respectively. Basal and oxytocin-stimulated PGF2α secretion was monitored by measuring concentrations of the principal metabolite of PGF2α, 13,14-dihydro-15-keto PGF2α (PGFM), in plasma samples collected before and after daily i.v. injection of 50 IU oxytocin from days 12–16. Basal PGFM was unaffected by the level of oestradiol treatment. In the normal group significant rises in plasma PGFM were seen following oxytocin on days 12, 13 and 14 (P<0·05) as well as days 15 and 16 (P<0·01). In the low group significant elevations in PGFM were only seen on days 15 and 16 (P<0·05), and were smaller (P<0·05) than those seen in the normal group. In the high group two cows showed no elevation in PGFM on any day while in two cows a rise was seen on all days which by day 16 was 84% greater than that seen in the normal group. The results demonstrate that a low level of oestradiol over the second half of the luteal phase leads to a weaker luteolytic signal, while a high level can be stimulatory or inhibitory.

Journal of Endocrinology (1995) 145, 175–180
Amphibian oocyte: a model of a possible regulatory mechanism for prostaglandin E2 and prostaglandin F2α synthesis
Authors: Gobbetti A , Zerani M
Published: December 2008

To clarify the possible mechanisms regulating prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) synthesis, the effects of gonadotropin-releasing hormone (GnRH) and substance P (SP) on the release of these two prostaglandins were studied in the oocytes of the crested newt, Triturus carnifex. Full-grown oocytes of T. carnifex, freed from follicular cells, were incubated in the presence of GnRH or SP and of the inhibitors of several enzymes involved in the release of arachidonic acid (AA) and in the conversion of AA into PGE2 and PGF2α. In parallel, the same experiments were performed on oocytes with membrane phospholipids labelled with [3H] AA. In addition, the PGE2-9-ketoreductase activity was evaluated through the conversion of [3H]PGE2 into [3H]PGF2α. The results showed that GnRH and SP could regulate prostaglandin synthesis through the activation of phospholipase C and diacylglycerol lipase, and through the modulation of PGE2-9-ketoreductase in the oocytes of T. carnifex. In particular, GnRH enhances the activty of PGE2-9-ketoreductase with a consequent increase in PGF2α, while SP inhibits the enzyme which leads to an increase in PGE2. A similar mechanism could also be hypothesized for other vertebrate species.

Journal of Endocrinology (1995) 145, 235–241
Androgen negative feedback during the early rise in LH secretion in bull calves
Authors: Rawlings N C , Evans A C O
Published: December 2008

A transient elevation in mean circulating concentrations of LH and FSH occurs in the young bull calf prior to 24 weeks of age. The functional significance of this is not clear. To see if changes in the ability of androgens to suppress gonadotrophin secretion were involved in the start of this early rise in LH secretion or the cessation of the early rise in LH and FSH secretion, bull calves were treated with flutamide (androgen receptor blocker; n=5; 9 mg flutamide/kg body weight in propylene glycol (i.m./s.c.) in three equal portions at 12-h intervals) at 8, 16 and 24 weeks of age and bled every 15 min for 12 h beginning after the third flutamide treatment; control bulls received vehicle at these times. Control bulls (n=5) were bled every 15 min for 12 h at 4, 8, 12, 16 and 24 weeks of age, and all bulls were bled weekly. Serum samples were assayed for concentrations of LH, FSH and testosterone. Based on weekly and intensive bleedings for control and flutamide-treated bulls, an early rise in LH (8–18 weeks of age) and FSH (4–24 weeks of age) secretion was seen in all bull calves (P<0·05). At 8 weeks of age flutamide treatment resulted in increased mean serum LH concentrations (P<0·05); at 16 weeks of age it resulted in increased basal and mean LH concentrations and increased LH pulse frequency (P<0·05); and at 24 weeks of age in increased mean LH concentrations, LH pulse frequency and amplitude (P<0·05) in comparison with control bulls. Flutamide treatment resulted in decreased FSH pulse amplitude at 8 weeks of age and increased mean serum concentrations of FSH and FSH pulse frequency at 24 weeks of age (P<0·05). In flutamide-treated bull calves testicular growth was greater and sexual maturity was reached earlier than in control bull calves (P<0·05). We conclude that a reduced suppression of LH secretion by androgens does not appear to be a major contributing factor to the onset of the early increase in LH secretion, but increased suppression may be involved in the termination of the early rise of both LH and FSH secretion in the bull calf. The early increase in LH secretion may be a critical initial step in postnatal reproductive development, since flutamide treatment increased early LH secretion and resulted in earlier attainment of sexual maturity.

Journal of Endocrinology (1995) 145, 243–249
Deprivation of phosphate increases IGF-II mRNA in MDCK cells but IGFs are not involved in phosphate transport adaptation to phosphate deprivation
Authors: Ernest S , Coureau C , Escoubet B
Published: December 2008

Phosphate (Pi) deprivation and IGFs stimulate renal Pi reabsorption. We studied the involvement of IGFs in the adaptation of Pi transport to Pi deprivation in MDCK cells.

Deprivation of Pi for 15 h increased the steady-state content of IGF-II mRNA (77 ± 12% whereas IGF-I mRNA was not detectable in MDCK cells in either control or Pi-deprived cells. IGF-II (10 −7 m) and IGF-I 10−8m) stimulated the Na-dependent Pi uptake (1·23and 1·3-fold increase at 15 h respectively). The effect of IGF-I appeared after 15 h and increased up to 40 h of treatment (2·15-fold increase). In contrast, Pi uptake was increased by Pi deprivation as early as 8 h (1·5-fold) and up to 40 h of Pi deprivation (1·9-fold increase). IGF-II mRNA was not increased before 15 h of Pi deprivation and returned to control at 40 h. The combination of IGF-I and Pi deprivation had a more than additive effect on Pi transport (fivefold increase) (P<0·001).

At variance with Pi deprivation, high concentrations of insulin stimulated Na-coupled alanine transport (6 ± 2% and 16 ± 4% in Pi-treated and Pi-depleted cells respectively). Pi deprivation and high concentrations of insulin decreased Na,K-ATPase activity (−48 and −64% respectively) and these effects were not additive.

We concluded that: (1) long-term Pi deprivation increased the content of IGF-II mRNA in MDCK cells but that the paracrine/autocrine effects of IGFs could not account for the effects of Pi deprivation on Pi transport in MDCK cells and (2) IGFs could be involved in other changes occurring during Pi deprivation such as the decrease in Na,K-ATPase activity.

Journal of Endocrinology (1995) 145, 325–331
Growth hormone and longitudinal bone growth in vivo: short-term effect of a growth hormone antiserum
Authors: Loveridge N , Farquharson C , Palmer R , Lobley G E , Flint D J
Published: December 2008

The control of longitudinal growth is poorly understood but GH is considered to be one of the major hormones regulating postnatal growth. However, there is dispute as to whether it has a direct or indirect action. To study the role of GH we used a polyclonal antiserum to rat GH and investigated changes in cell proliferation and enzyme activities associated with bone formation and resorption during longitudinal growth. IGF-I levels were measured by two independent RIAs, DNA synthesis by bromodeoxyuridine incorporation followed by immunocytochemistry and enzyme activities were quantified in situ by microdensitometry.

After 1 day the percentage of chondrocytes undergoing DNA synthesis within the proliferative zone was reduced but no other parameters were affected. By day 4 the labelling index was the same as in pair-fed animals but the number of chondrocytes synthesising DNA was reduced as was the total width of the growth plate and that of the proliferative zone. Alkaline phosphatase (associated with mineralisation) was unchanged but glucose 6-phosphate dehydrogenase activity (associated with cell proliferation) was decreased. Osteoclastic tartrate-resistant acid phosphatase activity (associated with bone resorption) was also significantly reduced. Similar changes were apparent after 10 days. At no time was the circulating level of IGF-I decreased.

These data suggest that, during longitudinal growth, GH affects the number of proliferating chondrocytes but not the percentage of cells undergoing DNA synthesis, indicating that its primary role may be on the commitment of prechondrocytes to a proliferative state. Furthermore, while GH does not seem to have any effect on skeletal mineralisation it may stimulate osteoclastic resorption of the primary spongiosa.

Journal of Endocrinology (1995) 146, 55–62
Follicular dynamics and secretion of inhibin and oestradiol-17β during the oestrous cycle of the hamster
Authors: Kishi H , Taya K , Watanabe G , Sasamoto S
Published: December 2008

Plasma and ovarian levels of inhibin were determined by a radioimmunoassay (RIA) at 3-h intervals throughout the 4-day oestrous cycle of hamsters. Plasma concentrations of FSH, LH, progesterone, testosterone and oestradiol-17β were also determined by RIAs. In addition, hamsters were injected at various times with human chorionic gonadotrophin (hCG) to determine the follicular development. The changes in plasma concentrations of FSH after injection of antisera to oestradiol-17β (oestradiol-AS) and inhibin (inhibin-AS) on the morning of day 2 (day 1=day of ovulation) were also determined.

Plasma concentrations of inhibin showed a marked increase on the afternoon of day 1, remained at plateau levels until the morning of day 4, then increased abruptly on the afternoon of day 4 when preovulatory LH and FSH surges were initiated. A marked decrease in plasma concentrations of inhibin occurred during the process of ovulation after the preovulatory gonadotrophin surges. An inverse relationship between plasma levels of FSH and inhibin was observed when the secondary surge of FSH was in progress during the periovulatory period. Plasma concentrations of oestradiol-17β showed three increase phases and these changes differed from those of inhibin. Changes in plasma concentrations of oestradiol-17β correlated well with the maturation and regression of large antral follicles. Follicles capable of ovulating following hCG administration were first noted at 2300 h on day 1. The number of follicles capable of ovulating reached a maximum on the morning of day 3 (24·8± 0·6), and decreased by 0500 h on day 4 (15·0 ± 1·1), corresponding to the number of normal spontaneous ovulations. Plasma concentrations of FSH were dramatically increased within 6 h after inhibin-AS, though no increase in FSH levels was observed after oestradiol-AS. These findings suggest that changes in the plasma levels of inhibin during the oestrous cycle provide a precise indicator of follicular recruitment, and that the changes in plasma concentrations of oestradiol-17β are associated with follicular maturation. These findings also suggest that inhibin may play a major role in the inhibition of FSH secretion during the oestrous cycle of the hamster.

Journal of Endocrinology (1995) 146, 169–176
Degradation of IGF-I in the adult rat gastrointestinal tract is limited by a specific antiserum or the dietary protein casein
Authors: Xian C J , Shoubridge C A , Read L C
Published: December 2008

To investigate the potential of IGF-I peptides as therapeutics in the gut, the survival profiles of a bolus of 125I-labelled IGF-I (8·6 ng) in vivo in various ligated gut segments of fasted adult rats have been examined. The intactness of IGF-I tracer in the flushed luminal contents was estimated by trichloroacetic acid precipitation, antibody and receptor binding assays. It was found that IGF-I was degraded very rapidly in duodenum and ileum segments with a half-life (t1/2) of 2 min by all three methods. IGF-I was slightly more stable in the stomach (t1/2=8, 5 and 2·5 min by the above three methods), and considerably more stable in the colon (t1/2=38, 33 and 16 min as judged by the three methods). Rates of degradation in gut flushings in vitro were similar to the in vivo rates except for the colon, where IGF-I was proteolysed more rapidly in vivo.

As a means of developing gut-stable and active forms of IGF-I, several approaches were examined for their effectiveness in prolonging IGF-I survival in the upper gut. It was found that the extension peptide on the analogue, LR3IGF-I did not protect IGF-I, nor did association with IGF-binding protein-3. However, an IGF-I antiserum was effective in prolonging IGF-I half-life in duodenum fluid by 28-fold. Charge interaction between IGF-I and heparin could also protect IGF-I in the stomach but not in duodenum flushings. Furthermore, casein (a non-specific dietary protein) and to a lesser extent, BSA and lactoferrin, were effective in preserving IGF-I structural integrity and receptor binding activity in both stomach and duodenum fluids. It can be concluded that IGF-I cannot be expected to retain bioactivity if delivered orally because of rapid proteolysis in the upper gut, but the use of IGF antibodies and casein could represent useful approaches for IGF-I protection in oral formulae.

Journal of Endocrinology (1995) 146, 215–225
Expression of the prolactin receptor gene during the breeding and non-breeding seasons in red deer (Cervus elaphus): evidence for the expression of two forms in the testis
Authors: Clarke L A , Edery M , Loudon A S I , Randall V A , Postel-Vinay M-C , Kelly P A , Jabbour H N
Published: December 2008

The red deer is a seasonally breeding mammal with a circannual cycle of prolactin secretion which reaches its peak during the non-breeding season. This study investigated expression of the prolactin receptor gene in red deer tissues collected in the breeding and non-breeding seasons. A 562 bp fragment of the extracellular domain of the red deer prolactin receptor cDNA was amplified from red deer liver poly(A)+ RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) using primers designed from the human sequence. Northern blots were prepared using 10–20 μg poly(A)+ RNA. The blots were hybridized to the 562 bp cDNA labelled by random priming with α32P-dCTP. A main transcript of 3·5 kb was expressed in liver, heart, kidney and testis throughout the year and in epididymis during the breeding season only. In the testis an additional major transcript of 1·7 kb was present during the breeding and non-breeding seasons. Competitive binding assays using 125I-ovine prolactin (125I-oPRL) were performed on microsomal membrane fractions prepared from liver. Scatchard analyses confirmed the presence of a single class of lactogen-binding receptor with a mean Ka of 0·87 ± 0·12 × 109 m−1 and a Bmax of 73·6 ± 9·8 fmol/mg protein (n=5). Cross-linking of 125I-oPRL to liver microsomes with 0·5 mm disuccinimidyl suberate followed by SDS-PAGE revealed a major band of molecular mass 56 kDa which was displaced by ovine prolactin, suggesting a specific lactogen-binding entity of 33 kDa. This study confirms the expression of the red deer prolactin receptor gene throughout the year, characterizes the prevalent form of receptor in the liver and demonstrates the expression of a separate, short form in the testis.

Journal of Endocrinology (1995) 146, 313–321
Follicular development, steroidogenesis and gonadotrophin secretion in response to long-term treatment with a gonadotrophin-releasing hormone agonist in the rat
Authors: Srivastava R K , Krishna A , Sridaran R
Published: December 2008

Gonadotrophin-releasing hormone (GnRH) and its agonists are implicated in the local control of rat ovarian function. We have evaluated the effects of long-term administration of different doses of GnRH agonist (GnRH-Ag) in vivo (a) on reproductive cyclicity and follicular development, (b) on peripheral gonadotrophin and steroid concentrations and (c) on in vitro cAMP and progesterone production by the follicles in response to stimulatory doses of FSH or LH (1 μg/ml). GnRH-Ag (0·2, 1 or 5 μg/day) administration for 28 days had a profound impact on the oestrous cycle of rats as revealed by vaginal cytology. GnRH-Ag treatment caused a decrease in ovarian and uterine weights, which correlated very well with the decrease in the number of follicles present in the ovary. GnRH-Ag (5 μg/day) reduced the number of early preantral follicles and there was complete disappearance of early as well as late antral follicles. However, a dose of 1 μg GnRH-Ag/day was effective in the complete demise of only late antral follicles with a significant attenuation in the number of early antral follicles. There was an enhancement in serum LH concentrations in response to the highest dose of GnRH-Ag administration with serum FSH concentrations declining in rats treated with the two higher doses. However, serum prolactin concentrations were attenuated only in rats treated with the highest dose of GnRH-Ag. GnRH-Ag treatment decreased serum progesterone and oestradiol concentrations. Preantral follicles obtained from the rats treated with 0·2 or 1 μg GnRH-Ag/day resulted in an attenuated response to LH-or FSH-stimulated progesterone production, whereas antral follicles showed an exaggerated response to the stimulatory doses of FSH in vitro. Antral follicles obtained from the rats treated with 0·2 μg/day showed a robust decrease in cAMP accumulation in response to LH with a slight decrease only with FSH. In contrast, preantral follicles obtained from GnRH-Ag-treated rats did not show any significant attenuation in cAMP production. These data suggest that GnRH-Ag exerts a direct inhibitory effect on follicular development and steroidogenesis and as a result it interferes with the normal oestrous cyclicity in the rat.

Journal of Endocrinology (1995) 146, 349–357
Kaliuretic peptide and long acting natriuretic peptide as well as atrial natriuretic factor inhibit aldosterone secretion
Authors: Vesely D L , Chiou S , Douglass M A , McCormick M T , Rodriguez-Paz G , Schocken D D
Published: December 2008

The present investigation was designed to determine whether atrial natriuretic peptides consisting of amino acids 1–30 (long acting natriuretic peptide), 31–67 (vessel dilator) and 79–98 (kaliuretic peptide) as well as 99–126 (atrial natriuretic factor (ANF)) of the 126 amino acid ANF prohormone inhibit aldosterone secretion. Thirty healthy human subjects were studied following infusion of 100 ng/kg body weight/min for 60 min of each of the respective peptides. Kaliuretic peptide decreased plasma aldosterone concentration by the greatest amount (6-fold) and plasma aldosterone was still significantly decreased (P<0·001) three hours after stopping the infusion. In contrast, within 30 min of cessation of the ANF infusion, plasma aldosterone levels had returned to pre-infusion values. Long acting natriuretic peptide also significantly (P<0·01) decreased plasma aldosterone levels which remained significantly (P<0·001) decreased 3 h after cessation of infusion. Vessel dilator did not decrease plasma aldosterone levels. Kaliuretic peptide, ANF and long acting natriuretic peptide also decreased (P<0·01) urinary aldosterone concentrations. None of these peptides changed the plasma potassium concentration. We conclude that two new peptide hormones (long acting natriuretic peptide and kaliuretic peptide) inhibit aldosterone secretion. The length of time that aldosterone secretion is inhibited following kaliuretic peptide and long acting natriuretic peptide infusion is significantly longer (P<0·001) than following ANF infusion.

Journal of Endocrinology (1995) 146, 373–380
Blood prolactin concentrations affect prolactin transfer into goat milk: implications for maintenance of lactation
Authors: Forsyth I A , Taylor J A , Gabai G , Fleet I R
Published: December 2008

125I-Labelled ovine prolactin was infused for 15 min into a pudic artery supplying one mammary gland of lactating goats (n=17). Between 0 and 4·25 h significandy more total (P<0·01) and trichloroacetic acid (TCA)-precipitable (P<0·001) radioactivity appeared in the milk of the infused compared with the non-infused gland. Gel chromatography and antibody precipitation indicated the presence of undegraded 125I-labelled prolactin in milk whey. Maximum transfer occurred 60–80 min after the end of infusion suggesting passage via a transcellular route. High plasma prolactin concentrations, resulting from infusion of cold prolactin with labelled prolactin in late lactation or from seasonally elevated prolactin at peak lactation, reduced the specific activity of infused prolactin and depressed the difference in secretion of 125I-labelled prolactin into milk of infused and non-infused glands. This suggests the operation of a competitive and saturable mechanism. Together with the increase in the milk to blood ratio of prolactin in goats given long-term (3 week) bromocriptine treatment, the results suggest that the goat mammary gland has a high avidity for prolactin especially when circulating prolactin is low. There was also evidence from TCA precipitation that prolactin may be protected from degradation in these circumstances. These mechanisms may contribute to the resistance of ruminant lactation to reduction in plasma prolactin and protect lactation from seasonal prolactin fluctuations.

Journal of Endocrinology (1995) 146, 411–420
Stimulation of tyrosinase in human melanocytes by pro-opiomelanocortin-derived peptides
Authors: McLeod S D , Smith C , Mason R S
Published: December 2008

Human melanocytes, maintained on bovine corneal endothelium-derived extracellular matrix for at least 4 days in the absence of phorbol 12-myristate 13-acetate (PMA) and cholera toxin (CT), displayed increased tyrosinase activity when exposed to several pro-opiomelanocortinderived (POMC) peptides. Melanocytes from 9 of 14 donors showed significantly increased tyrosinase activity after treatment with adrenocorticotropic hormone (ACTH; mean increase 320±107 (s.e.m.)% of control, P<0·005), while melanocytes from 8 of 13 donors increased tyrosinase in the presence of diacetyl-melanocyte stimulating hormone (di-MSH; mean increase 223±31 (s.e.m.)% of control, P<0·005). Maximal increases in tyrosinase were seen after treatment with 10−10 m ACTH and with 10−6 m di-MSH. In two cell cultures which showed tyrosinase stimulation, melanin synthesis was similarly increased in the presence of added POMC peptides. PMA but not CT increased tyrosinase activity in melanocytes cultured under these conditions. In the presence of staurosporine, an inhibitor of protein kinase C (PKC), the magnitude of the increase in tyrosinase due to PMA, ACTH and di-MSH was significantly reduced. These results indicate that tyrosinase activity in melanocytes from most human donors, under appropriate conditions, is susceptible to the stimulatory effects of POMC peptides, that ACTH is considerably more potent than di-MSH in this test system and that in human cells the PKC pathway may be important in modulating melanogenesis.

Journal of Endocrinology (1995) 146, 439–447
Potassium chloride effects on the hormonal signal transduction mechanisms underlying phasic myometrial contractions
Authors: Phillippe M , Chien E K
Published: December 2008

These studies sought to test the hypothesis that potassium-stimulated phasic myometrial contractions utilize cytosolic calcium oscillation-like mechanisms comparable to those activated in response to oxytocin. Uterine tissue was obtained from pro-oestrus/oestrus Sprague-Dawley rats. In vitro isometric contraction studies were performed using longitudinal myometrial strips; computer digitalized contraction data were analyzed for contraction area, and normalized for tissue cross-section area. Dose–response studies were performed using potassium chloride with and without inhibitors of cytosolic calcium oscillation mechanisms. Qualitative inositol-phosphate production studies were performed after preloading uterine tissue with [3H]inositol; subsequently, the individual inositol-phosphates produced in response to stimulation were isolated by anion exchange chromatography. Potassium chloride over a concentration of 10 to 30 mm produced a dose-related increase in phasic contractile activity. The potassium-stimulated phasic contractions were significantly suppressed in response to inhibition of phospholipase C, stimulation of protein kinase C, inhibition of calcium-induced calcium release, and prevention of extracellular calcium influx. The qualitative inositol-phosphate production studies confirmed activation of phospholipase C in response to 20 mm potassium. These studies have provided support for the hypothesis that potassium-stimulated phasic myometrial contractions activate intracellular signal transduction mechanisms comparable to those activated in response to hormonal uterotonic agonists.

Journal of Endocrinology (1995) 146, 485–493
Estrogen increases the release of epidermal growth factor from individual pituitary cells in female rats
Authors: Mouihate A , Lestage J
Published: December 2008

Rat mixed anterior pituitary cells from cycling females were microscopically demonstrated to produce epidermal growth factor (EGF), in culture, using a reverse hemolytic plaque assay. Anterior pituitary cells of proestrous female rats secrete more EGF than those of metestrous rats, as evaluated by the mean area and the percentage of EGF plaque-forming cells. Estradiol-17β (10 nm) treatment of metestrous culture for 24 h increased significantly the size of EGF plaque-forming cells (from 1290±58 to 2566 ± 57 μm2, P<0·01) and the percentage of EGF plaque-forming cells (from 20·57 to 28·19%; P<0·01) to a level as high as observed in proestrous cultures (basal mean area 2171 ± 157 μm2, and basal percentage of EGF plaque-forming cells 22·71%). These results suggest that the hormonal status of female rats influences EGF secretion in the anterior pituitary gland and that estradiol is implicated in this phenomenon.

Journal of Endocrinology (1995) 146, 495–500
Impact of uraemia on food efficiency and the pulsatile mode of growth hormone secretion in rats
Authors: Krieg R J , Latta K , Niimi K , Veldhuis J D , Chan J C M
Published: December 2008

In view of the profound growth failure resulting from renal insufficiency and uraemia, the present studies were designed to specifically investigate food efficiency and the pattern of GH secretion under these conditions. Animals were made uraemic by 5/6 nephrectomy (Nx) and feeding a high-protein diet. Three groups of animals were studied: uraemic (Ur); sham-operated, fed ad libitum (Sh); and sham-operated pair-fed with the uraemics (PF). Food intake per 100 g body weight and food efficiency (g weight gained per g food consumed) were calculated. Fourteen days after 5/6 Nx, blood samples were taken via intra-atrial catheters at 10-min intervals over a period of 6 h. GH was measured in plasma by radioimmunoassay. GH pulsatility was analysed by multiple parameter deconvolution. The growth rate of Ur animals was significantly lower than that of Sh. The body weights of the Ur animals were also lower than PF due to an initial period of weight loss. Both Sh and PF animals showed the typical negative slope of food intake as body weight increased. In contrast, the Ur animals showed a positive slope of food intake. The lower rate of growth and the elevated food intake corresponded to a decreased food efficiency for the Ur group. Deconvolution analysis of pulsatile GH release demonstrated a significant increase in GH half-life in the Ur animals. The amplitude and mass of GH secretory pulses were decreased, whereas the number of detectable secretory bursts was increased. These changes were specific to uraemia with respect to half-life and number of pulses. We conclude that uraemia results in a decrease in the conversion of food to body mass, which is accompanied by significant changes in the neuroregulation of pulsatile GH secretion.

Journal of Endocrinology (1995) 146, 509–517
Characterization of protein production by caprine placental membranes: identification and immunolocalization of retinol-binding protein
Authors: Liu K H , Huang J C , Godkin J D
Published: December 2008

Caprine chorion, allantois and amnion from days 23, 28, 35, 39 and 45, and yolk sac from day 23 of pregnancy were isolated by dissection and cultured for 24 h in modified minimum essential medium in the presence of [35S] methionine. De novo-synthesized proteins released into the culture medium were analyzed by two-dimensional PAGE and fluorography. Patterns of protein production by these isolated extraembryonic membranes remained relatively unchanged from days 23 to 45 of pregnancy. Electrophoretic profiles of proteins synthesized by allantois and amnion were identical but distinct from that produced by chorion. Yolk sac was the major source of serum-like proteins. An acidic (pI 5·3–6·3) 22 kDa protein, which consisted of four isoelectric variants, was produced by all extraembryonic membranes and demonstrated to immunoreact with antiserum produced against bovine placental retinol-binding protein (RBP). Limited N-terminal sequence analysis of one major isoform indicated that the protein had complete homology with bovine RBP over the first 15 amino acids. Immunoreactive RBP was localized in epithelial cells lining the chorion, allantois and amnion. In this study, we have characterized and compared protein production by isolated extraembryonic membranes through days 23 to 45 of pregnancy and identified the 22 kDa protein as caprine RBP of placental origin.

Journal of Endocrinology (1995) 146, 527–534
Regulation of glucagon-like peptide-1 secretion from rat ileum by neurotransmitters and peptides
Authors: Herrmann-Rinke C , Vöge A , Hess M , Göke B
Published: December 2008

Food ingestion induces a rapid increase in the insulinotropic glucagon-like peptide-1 (GLP-1) in plasma. Paradoxically, GLP-1 originates from the lower intestines and therefore a complex regulation of postprandial GLP-1 secretion must exist. This was addressed in the present study by utilizing an isolated vascularly perfused rat ileum preparation. Peptides and neurotransmitters thought to be candidate mediators triggering GLP-1 secretion were arterially infused and GLP-1 was measured in the venous effluent. Arterial infusion of cholinergic agonists strongly enhanced GLP-1 secretion which was counteracted by the addition of atropine. Histamine, dopamine, 5-hydoxytryptamine, γ-aminobutyric acid, and norepinephrine had no effect. Peptides of the bombesin family were strong stimulants whereas tachykinins, enkephalins, dynorphin, TRH, calcitonin-gene-related peptide and members of the secretin family, vasoactive intestinal peptide, peptide histidine isoleucine and neuropeptide Y, were less effective. The second incretin hormone, gastric inhibitory polypeptide (GIP), was the most potent stimulant of GLP-1 secretion in our study. It enhanced GLP-1 release up to sixfold above basal during the early phase followed by a sustained secretion at 400% above basal. This stimulation remained unaffected by atropine. In conclusion, in addition to luminal stimulation of nutrients, a cholinergic impulse as well as peptidergic mediators (among them possibly GIP and GRP) may have an impact on postprandial GLP-1 secretion from the rat ileum.

Journal of Endocrinology (1995) 147, 25–31
Glucose activation of acetyl-CoA carboxylase in association with insulin secretion in a pancreatic β-cell line
Authors: Zhang S , Kim K-H
Published: December 2008

Malonyl-CoA, which is the unique product of acetyl-CoA carboxylase (ACC), may serve as a metabolic coupler in glucose-stimulated insulin secretion by pancreatic β-cells. Therefore we examined if and how ACC is affected by glucose in association with insulin secretion. Glucose induces a rapid increase in ACC activity which is closely related to insulin secretion in a dose- and time-dependent manner. The acute effect of glucose in increasing ACC activity is caused by dephosphorylation of existing ACC molecules, without the production of new enzyme. Inhibition of ACC dephosphorylation and activation by the use of okadaic acid led to diminished glucose-mediated insulin secretion. Likewise, when ACC phosphorylation and inactivation were induced by the use of 5-amino 4-imidazole-carboxamide ribotide, an AMP analog and activator of 5′-AMP protein kinase, the glucose-induced insulin secretion was virtually nil. In the long term, glucose induced ACC and increased insulin secretion. In β-cells, ACC gene expression is controlled by promoter II and glucose activated promoter II expression. ACC promoter I is not expressed in β-cells. Maximum activation of ACC and insulin secretion by glucose in the short term occurred at 5 mm glucose. On the other hand, activation of the expression of ACC promoter II occurred when the cells were exposed to high glucose concentrations for a long period of time. Thus, we have shown that ACC, the only enzyme that produces malonyl-CoA, is activated by glucose; activation of ACC is accomplished by dephosphorylation in the short term and by induction of ACC by gene activation in the long term.

Journal of Endocrinology (1995) 147, 33–41