bluuebunny/crossref_metadata_2025 huggingface dataset:
Response of insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) and IGFBP-3 to IGF-I treatment in severe insulin resistance
Authors: Quin J D , Checkley A , Gallagher A , Jones J , MacCuish A C , Miell J P
Published: December 2008

It has been suggested that recombinant human IGF-I (rhIGF-I) is a potential therapeutic agent in diabetes mellitus. It is known to have glucose-lowering effects in normal individuals, in patients with non-insulindependent diabetes (NIDDM) and in extreme insulin-resistant states. IGF-binding proteins (IGFBPs) have the potential to affect the biological activity of rhIGF-I. We have studied the effect of infused rhIGF-I on IGFBP-1 and IGFBP-3 in a patient with Mendenhall's syndrome, a rare insulin-resistant state. During an infusion of 20 mg rhIGF-I, glucose concentrations fell from 44·1 ±7·2 to 31·5 ±7·2 (s.e.m.) mmol/l (P=0·001), and insulin and C-peptide levels fell from 920 ±62 to 542±45 mU/l (P=0·008) and 5466 ±633 to 3071 ±297 pmol/l (P= 0·02) respectively. Significant lowering of phosphate, magnesium and alkaline phosphatase concentrations was also noted. IGF-I levels rose from 48 ±10·2 to 410 ±50·1 μg/l (P=0·001), and those of IGF-II fell from 279·8± 8·3 to 104·3 ±7·9 μg/l (P=0·001). IGFBP-1 concentrations did not significantly change during the infusion but those of IGFBP-3 increased from 1655 ± 127 to 2197 ± 334 μg/l (P=0·002), despite a significant fall in GH concentrations from 10·7±2·6 to 4·1±1·1 mU/l (P= 0·007), suggesting that IGFBP-3 regulation is also IGF-I-dependent. The lack of response of IGFBP-1 to a significant fall in insulin levels suggests that impaired insulin receptor function alters the usual regulation of IGFBP-1 by insulin. These findings are of importance, given the potential use of rhIGF-I in NIDDM.

Journal of Endocrinology (1994) 141, 177–182
Transforming growth factor-β1 synthesis in human thyroid follicular cells: differential effects of iodide and plasminogen on the production of latent and active peptide forms
Authors: Cowin A J , Bidey S P
Published: December 2008

The present study has investigated the relative levels and interconversion of latent and active forms of transforming growth factor-β1 (TGF-β1) in human thyroid follicular cell cultures derived from sporadic non-toxic goitres. Northern blotting of RNA extracted from 72-h cultures revealed a 2·5 kb mRNA transcript hybridizing with a cDNA probe for latent TGF-β1, the intensity of which was doubled in cells exposed to Nal (10 μmol/l). Radioreceptor assay of follicular cell-conditioned medium for TGF-β1 content confirmed a similar enhancing effect of iodide. The endogenous active component of TGF-β1 present in conditioned medium represented only a minor fraction of the total TGF-β1 content, this fraction was not enhanced by exposure of follicular cells to iodide. The low level of endogenous active TGF-β1 in medium conditioned by either control or iodide-treated cells was confirmed by immunoadsorption with a precipitating antiserum against active TGF-β1, when such cells failed to show a reversal of the iodide-induced decrease in [methyl-3H]thymidine incorporation into trichloroacetic acid-precipitable material. In contrast to the inhibitory effect of iodide on de novo DNA synthesis, which appeared not to reflect an increase in active TGF-β1, the inhibitory effects of plasminogen (100 mg/l) or plasmin (2000 U/l) on [methyl-3H]thymidine incorporation into thyroid cells were reversible by TGF-β1 immunoadsorption. This provides evidence that the plasmin-mediated inhibition of DNA synthesis in thyroid follicular cells may be attributed to the growth-regulating action of an increased level of activated TGF-β1. The findings of this study therefore provide evidence that (i) human thyroid follicular cells are potentially capable of activating locally derived latent TGF-β1, (ii) an increase in thyroidal TGF-β1 mRNA and latent TGF-β1 peptide availability, following exposure of cells to iodide, is not accompanied by a corresponding increase in active TGF-β1, and (iii) within the thyroid gland, as in other epithelial tissues, activation of endogenous TGF-β1 may be dependent upon the proteolytic actions of plasmin.

Journal of Endocrinology (1994) 141, 183–190
Spermine-induced precocious intestinal maturation in suckling rats: possible involvement of glucocorticoids
Authors: Kaouass M , Sulon J , Deloyer P , Dandrifosse G
Published: December 2008

The mechanism(s) involved in the spermine-induced precocious postnatal maturation of the intestine in the unweaned rat was examined. Spermine given orally to 11-day-old rats stimulated ACTH and corticosterone secretion. Maximum serum levels of ACTH and corticosterone were observed between 4 and 6 h after spermine ingestion and were five- and sevenfold greater respectively than those of control rats receiving saline alone. Intraperitoneal injection of the polyamine had no effect on corticosterone production. Repeated intraperitoneal administration of gastrin, cholecystokinin, glucagon(1–37) and secretin to 11-day-old rats had no effect on the specific activity of intestinal disaccharidases.

These data indicate that (1) the hypophysial-adrenal axis is implicated in the postnatal development of the gastro-intestinal tract induced by spermine and (2) spermine affects ACTH and corticosterone secretion indirectly, probably by stimulating the release of gastrointestinal hormone(s).

Journal of Endocrinology (1994) 141, 279–283
Protein kinase C signal transduction pathway in ACTH-induced growth effect of rat adrenocortical cells in primary culture
Authors: Arola J , Heikkilä P , Voutilainen R , Kahri A I
Published: December 2008

ACTH exerts a biphasic effect on the growth of fetal rat adrenocortical cells in primary culture when bromodeoxyuridine (BrdU) incorporation is used as an indicator of proliferation. The immediate inhibitory effect during the first 24 h of ACTH stimulation is not dependent on cyclic AMP (cAMP). Protein kinase C (PKC) inhibitors H-7 and staurosporine blocked this inhibitory effect of ACTH, whereas 12-0-tetradecanoyl phorbol-13-acetate (TPA; a PKC activator) mimicked the ACTH-induced antimitogenic effect. The stimulatory growth effect of ACTH appears after 72 h of treatment. A similar mitogenic effect is also achieved with cAMP derivative 8-bromo cAMP (8-Br cAMP). However, both ACTH- and 8-Br cAMP-induced proliferations could be reduced with H-7.

ACTH-induced corticosterone secretion was inhibited 50% with H-7 after 24 h, but 8-Br cAMP-induced secretion was unaffected. However, if the treatments were continued for 72 h, H-7 no longer reduced the steroid secretions. Reduction (50–75%) of cholesterol side-chain cleavage enzyme (P450scc) mRNA expression was also noted with H-7 in ACTH-treated cultures after 6 and 24 h. In contrast, TPA doubled the corticosterone secretion induced by 8-Br cAMP, but did not further increase the ACTH-induced secretion after 24 h. TPA alone, however, was not able to induce steroid secretion or P450scc mRNA expression. The morphological differentiation of fetal rat adrenocortical cells with ACTH or 8-Br cAMP from zona glomerulosa-like cells into zona fasciculata-like cells was not disturbed by H-7 nor was it induced by TPA alone.

These results therefore suggest that PKC- and cAMP-dependent signal transductions are involved in the ACTH-induced biphasic growth effect of fetal rat adrenocortical cells. PKC plays a role in the inhibitory growth effect, and both PKC and cAMP are involved in the stimulatory growth phase of ACTH. Both PKC and cAMP are also involved in the steroid secretion of zona glomerulosa-type cells, but differentiation into zona fasciculata-type cells and their steroid production is transduced through cAMP.

Journal of Endocrinology (1994) 141, 285–293
Co-ordinate actions of FSH and insulin-like growth factor-I on LH receptor expression in rat granulosa cells
Authors: Kanzaki M , Hattori M-A , Horiuchi R , Kojima I
Published: December 2008

The actions of FSH and Insulin-like growth factor-I (IGF-I) were studied in cultured rat ovarian granulosa cells. Cells became differentiated and expressed LH receptors when they were incubated for 72 h with 200 μg FSH/l (high FSH) but not 20 μg FSH/l (low FSH). Treatment with high but not low FSH increased the release of both immunoreactive and bioactive IGF-I into the medium. A combination of low FSH and IGF-I reproduced the effect of high FSH on LH receptor expression. We then examined the critical time when low FSH and IGF-I exerted their effects. In the presence of continuous low FSH, IGF-I was capable of inducing LH receptor expression even when added 24 h after the addition of low FSH. However, when IGF-I was added at 36 h, LH receptor expression measured at 72 h was greatly reduced. In contrast to the action of IGF-I, continuous exposure to low FSH was required for LH receptor expression, and IGF-I had no effect when FSH was not included for the entire 72 h of culture.

DNA synthesis as assessed by both [3H]thymidine incorporation and nuclear bromodeoxyuridine labelling was moderate at the beginning of culture and markedly reduced at 24 h both in the presence and absence of either high FSH or low FSH plus IGF-I. In the presence of either high FSH or a combination of low FSH plus IGF-I, DNA synthesis remained decreased for up to 72 h whereas it began to increase in the absence of either high FSH or a combination of low FSH plus IGF-I. A similar increase in DNA synthesis was observed after 48 h when granulosa cells were treated with low FSH alone, which did not induce LH receptor expression. These results indicate that 1) growth and differentiation of granulosa cells are regulated inversely; 2) FSH and IGF-I act together to induce LH receptor expression; and 3) action of IGF-I is dependent on the presence of FSH.

Journal of Endocrinology (1994) 141, 301–308
Effects of a non-steroidal pure antioestrogen, ZM 189,154, on oestrogen target organs of the rat including bones
Authors: Dukes M , Chester R , Yarwood L , Wakeling A E
Published: December 2008

ZM 189,154 ([1RS,2RS]-2-(4-hydroxyphenyl)-2-methyl -1-[9-(4,4,5,5,5-penta-fluoropentyl)sulphinylnonyl]-1,2, 3,4-tetrahydronaphth-6-ol) is a non-steroidal pure anti-oestrogen. It has a high relative affinity for the oestrogen receptor, completely blocks the trophic action of oestradiol (OE2) on the uterus in immature and ovariectomized (OVX) adult rats and, in the latter, also completely blocks the trophic action of OE2 on vagina, bone and growth rate. ZM 189,154 displays no intrinsic oestrogen-agonist activity on uterus, vagina, bone, LH secretion or growth rate in OVX rats. Differential sensitivity of OE2-regulated processes was more apparent in intact rats. Daily doses of 0·6 mg/kg per day of ZM 189,154 blocked ovulation; 2 mg/kg per day achieved maximal uterine atrophy but did not affect bone density or growth rate; 10 mg/kg per day produced a broader spectrum of effects (reduced bone density, increased basal LH, slightly increased growth rate), but the magnitude of these was smaller than after ovariectomy; the 10 mg/kg dose also produced multiple ovarian follicular cysts. The failure of ZM 189,154 to achieve complete ovariectomy-like effects in intact rats may be due to the action of ovarian factors other than OE2, or to the circulating OE2 levels resulting from the disturbance to ovarian function posing too strong a challenge to the antagonist.

Journal of Endocrinology (1994) 141, 335–341
Expression of IGFBP-1 in normal and cirrhotic human livers
Authors: Ross R J M , Rodriguez-Arnao J , Donaghy A , Bentham J , Clark A , Holly J , Williams R , Gimson A
Published: December 2008

Cirrhosis of the liver, a condition characterised by hepatocyte regeneration, is also associated with elevated insulin levels and insulin resistance. In animal models hepatic regeneration is associated with increased IGFBP-1 gene expression. Insulin is known to be an inhibitor of IGFBP-1 gene expression and circulating insulin levels in man demonstrate a negative correlation with IGFBP-1 levels. To further our understanding of the regulation of IGFBP-1 in cirrhosis we have studied steady state levels of IGFBP-1 mRNA in human liver from three groups of patients: Group 1, tissue obtained at the time of harvesting donor liver for orthotopic liver transplantation (n=4); group 2, patients undergoing major liver resection with no histological evidence of chronic liver disease (n=4); and group 3, patients undergoing orthotopic transplantation for chronic liver failure (n=9). Simultaneous samples of serum were taken at the time of surgery in some patients and in these patients IGFBP-1 mRNA levels were related to circulating levels of IGFBP-1 and insulin.

IGFBP-1 mRNA was detectable in all the human liver samples with the greatest levels seen from the normal livers of group 2 patients. Insulin levels were elevated in the cirrhotic group 3 patients compared to a normal range as were IGFBP-1 levels. There was no relationship between circulating levels of IGFBP-1 and IGFBP-1 gene expression.

In conclusion, IGFBP-1 mRNA is present in human adult liver at the time of surgery and also in cirrhotic liver despite high levels of insulin suggesting that there are factors other than insulin regulating IGFBP-1 gene expression.

Journal of Endocrinology (1994) 141, 377–382
Vasopressin-binding sites in the pig pituitary gland: competition by novel vasopressin antagonists suggests the existence of an unusual receptor subtype in the anterior lobe
Authors: Arsenijevic Y , Dubois-Dauphin M , Tribollet E , Manning M , Sawyer W H , Dreifuss J J
Published: December 2008

Arginine vasopressin (AVP) acts in the pituitary gland, in synergy with corticotrophin-releasing factor, to induce ACTH release in response to stressful stimuli. Pituitary AVP receptors in the rat are coupled to phospholipase C, as are the so-called V1-type AVP receptors. The present study examined [3H]AVP binding in membranes prepared from the anterior lobe of the pituitary gland of the pig. [3H]AVP, alone or in competition with analogues, bound to sites in the pig anterior lobe which are pharmacologically similar to those described previously by others in the rat pituitary gland. For comparison, the same competition studies were performed on membrane preparations from the rat liver which contain the classic V1-type AVP receptor. Pituitary and liver AVP-binding sites were dissimilar; both cyclic and linear V1 antagonists had, in general, a much lower affinity for pituitary AVP-binding sites than for those in the liver. Thus, Phaa-d-Tyr(Et)-Phe-Gln-Asn-Lys-Pro-Arg-NH2 (Phaa=phenylacetyl) has a 2500-fold greater affinity for the latter (negative logarithm of inhibition constant (pKi)=9·64) than for the former (pKi=6·22). One linear antagonist, Pa-d-Tyr-Phe-Val-Asn-Arg-Pro-Arg-Arg-NH2 (Pa=propionyl) had about equal affinities for liver and pituitary membranes (pKi=6·39 and 6·53 respectively). Another compound, Phaa-d-Tyr-Phe-Val-Asn-Arg-Pro-Arg-Arg-NH2 had the highest affinity found to date for binding to AVP sites in the pituitary (pKi=7·43). These findings suggest some ideas for the design of more potent and/or selective AVP analogues acting in the pituitary gland.

Journal of Endocrinology (1994) 141, 383–391
Measurement of dimeric inhibin using a modified two-site immunoradiometric assay specific for oxidized (Met O) inhibin
Authors: Knight P G , Muttukrishna S
Published: December 2008

Several years ago we developed a novel two-site immunoradiometric assay (IRMA) for dimeric inhibin. However, relative to the purified 32 kDa bovine inhibin standard used at that time, the immunopotencies of crude inhibin-containing samples were much less than their biopotencies estimated by pituitary cell bioassay. In attempting to improve assay performance and resolve this discrepancy we recently discovered that introduction of a preassay oxidation step to the IRMA results in a dramatic increase in the immunopotencies of inhibin-containing test samples (e.g.: bovine, human, porcine follicular fluid (FF)) and of a new (purified in 1993) 32 kDa bovine inhibin standard. However, the oxidation step did not affect the immunopotency of our original standard (purified in 1987), indicating that this material had undergone spontaneous oxidation during long-term storage, thus accounting for its higher immunopotency in our original IRMA and providing an explanation for the discrepancy between immunoactivity and bioactivity referred to above.

These findings, together with other observations on the behaviour of oxidized and non-oxidized samples of inhibin, related peptide fragments and inhibin-containing samples in the IRMA and α subunit radioimmunoassay (RIA), indicate that the anti-βA 82–114 monoclonal antibody (E4) used as tracer in the IRMA binds selectively to the oxidized (Met O89,91,108) form of the peptide. This property of the antibody can be exploited to advantage by incorporating simple modifications to existing inhibin/activin immunoassays to ensure that all samples and standards are fully oxidized before antibody addition.

Dimeric inhibin levels in individual bovine FF samples (n=105) estimated by the 'oxidized' IRMA (9·66 ± 0·50 ng/μl) were highly correlated (r=0·90; P<0·001) with values derived using the original IRMA (0·75 ± 0·05 ng/μl) but were 13-fold higher. Levels of total immunoreactive (ir)-β (estimated by RIA) and αβ dimer in bovine FF were highly correlated (r=0·89; P<0·001) whereas no correlation existed between levels of total ir-α (estimated by RIA) and either total ir-β or αβ dimer. This observation indicates that availability of β rather than α subunit regulates the amount of dimeric inhibin produced by each follicle. Preliminary observations indicate that the modified IRMA can detect serum levels of dimeric inhibin in rats and women undergoing ovarian hyperstimulation with exogenous gonadotrophin but not in normal women or cattle.

Journal of Endocrinology (1994) 141, 417–425
Effects of recombinant human FSH in immature hypophysectomized male rats: evidence for Leydig cell-mediated action on spermatogenesis
Authors: Matikainen T , Toppari J , Vihko K K , Huhtaniemi I
Published: December 2008

The mode of FSH actions within the testis was studied in immature hypophysectomized male rats by treatment with recombinant human FSH (recFSH, Org 32489). To elucidate the involvement of Leydig cells and androgens in the maintenance of spermatogenesis in FSH-treated hypophysectomized rats further, the recFSH treatment was given both alone and after destruction of Leydig cells with ethane-1,2-dimethane sulphonate (EDS). Three days after hypophysectomy (at 31 days of age) the rats were given one i.p. injection of vehicle or EDS and, 4 days later, they were implanted with osmotic minipumps releasing either 0·9% (w/v) NaCl or 1 IU recFSH/day.

Recombinant FSH alone increased testicular weights 2·5-fold in 7 days (P<0·01). The effect of FSH was similar in EDS-pretreated rats (P<0·01). Testicular testosterone increased from 6·5 ± 1·6 to 16·9 ± 5·3 (s.e.m.) pmol/g tissue (P<0·05) and serum testosterone from 0·12 ± 0·02 to 0·22 ± 0·03 nmol/l (P<0·05) when the rats were treated with recFSH. EDS alone did not affect testicular testosterone but, when combined with recFSH, it totally abolished the stimulatory effect of FSH on testosterone. Testicular binding of 125I-labelled iodo human chorionic gonadotrophin (hCG) and 125I-labelled iodo recFSH was increased 2·5- and 2·1-fold respectively with recFSH treatment (P<0·01). EDS, either alone or with FSH, abolished specific testicular hCG binding (P<0·01), but had no effect on that of recFSH. However, FSH increased its own receptors only in animals not treated with EDS.

Histological analysis of the testes revealed that the diameters of the seminiferous tubules increased from 115 ± 6·1 to 160 ± 7·2 μm (P<0·05) with recFSH, and a comparable increase was observed when EDS treatment preceded that of recFSH (143 ± 1·5 μm, P<0·05 vs. controls). Quantification of the spermatogenic cells indicated that recFSH supported the progression of spermatogenesis, as shown by increased number of meiotic and haploid spermatogenic cells (P<0·05). In all EDS-treated animals, spermatogenesis was severely disturbed and only a few spermatids were seen.

In conclusion: (1) these results further support the suggestion that FSH has indirect stimulatory effects on Leydig cell function, (2) the completion of meiosis and spermiogenesis are supported by FSH, the effect of which is enhanced by the presence of Leydig cells, suggesting its dependence on androgens, and (3) we show for the first time that FSH is able to stimulate its own receptors only in the presence of Leydig cell-derived factors, probably androgens.

Journal of Endocrinology (1994) 141, 449–457
Cortisol inhibition of growth hormone-releasing hormone-stimulated growth hormone release from cultured sheep pituitary cells
Authors: Sartin J L , Kemppainen R J , Coleman E S , Steele B , Williams J C
Published: December 2008

Cortisol inhibits growth hormone (GH) release in short-term culture and is stimulatory in long-term cultures of rat and human pituitary cells. This study sought to determine the in vitro effects of cortisol on GH release and the signal transduction pathways mediating the effects of cortisol on GH release from cultured ovine somatotrophs. Pituitary cells were dispersed with collagenase and placed in culture medium for 4 days. The data indicate that cortisol inhibited growth hormone-releasing hormone (GHRH)-stimulated GH release by at least 2 h. In short-term culture GHRH-, forskolin- and dibutyryl cyclic AMP-stimulated GH release were inhibited by cortisol, suggesting an effect distal to the membrane and involving a protein kinase A (PKA)-dependent pathway. GH release initiated by KCl was inhibited by cortisol, but GH release caused by the calcium ionophore A23187 was unaffected. This suggests a possible action of cortisol on the calcium channels. The inhibition by cortisol of the calcium-dependent secretion of GH release appeared to play a smaller role in mediating cortisol inhibition of GH release than that seen with PKA. Attempts to overcome cortisol inhibition of GH release using puromycin, arachidonic acid or pertussis toxin were unsuccessful. Since cortisol inhibition of GH release does not occur via the mechanisms found in other cell types, cortisol inhibition of pituitary cell secretions appears to be cell-specific rather than utilizing a single inhibitory mechanism. The majority of cortisol actions on the somatotroph appear to act at a site distal to the production of cyclic AMP. In contrast to man and the rat, the sheep somatotroph does not appear to increase GH release when treated with cortisol for 24 h, perhaps related to the lack of effect of cortisol on somatotroph content of GH.

Journal of Endocrinology (1994) 141, 517–525
Human pituitary somatotropes express transforming growth factor-α and its receptor
Authors: Finley E L , King J S , Ramsdell J S
Published: December 2008

Transforming growth factor-α (TGF-α) is a growth-regulatory peptide produced by a variety of transformed and non-transformed cells. Among non-transformed cells, TGF-α has been identified in the prolactin (PRL)- and GH-secreting cells of the bovine anterior pituitary gland.

In this report, we have examined the expression of TGF-α in human anterior pituitary glands by Western analysis and immunohistochemistry. For the Western analysis, human pituitary glands were extracted in acid/ethanol, an acetic acid-soluble fraction was ether-precipitated and dialysed, and TGF-α was partially purified by C18 chromatography. TGF-α was then identified by immunostaining of Western transfers. Anterior pituitary extracts exhibited a major band(s) migrating at 19 kDa that was immunoreactive with a monoclonal antibody directed against the mature TGF-α. However, no evidence of the fully processed 6 kDa TGF-α was observed.

We next identified TGF-α by immunohistochemistry. Using both monoclonal and polyclonal antibodies, specific immunoreactivity was identified in a population of secretory cells in the anterior pituitary gland. Using antibodies specific for the COOH and NH3 terminals of the TGF-α precursor, a comparable number of TGF-α-positive cells were found to contain TGF-α precursor sequences. These results indicate that the 19 kDa form of TGF-α expressed in the human pituitary gland may exist as the transmembrane form.

We next sought to determine which cells express TGF-α in a human male pituitary gland. On frontal sections, TGF-α-immunopositive cells were evenly distributed in a manner and number indistinguishable from GH-immunopositive cells. By contrast, PRL-immunopositive cells in midfrontal sections were largely restricted to the lateral wings and extended dorsally to the neural lobe. TGF-α was positively co-localized to GH-immunopositive cells but not in PRL-immunopositive cells by immunostaining of consecutive sections. TGF-α-immunopositive cells were also immunopositive for the epidermal growth factor receptor, indicating that TGF-α has the capacity for autocrine action in the human pituitary gland.

These results indicate that TGF-α is expressed in the human anterior pituitary gland and it is not proteolytically processed into the mature 6 kDa form. In addition, immunohistochemistry of an adult male human pituitary gland indicates that TGF-α is expressed in somatotropes and has the capacity for autocrine action.

Journal of Endocrinology (1994) 141, 547–554
Effects of a new synthetic vitamin D analogue, EB1089, on the oestrogen-responsive growth of human breast cancer cells
Authors: James S Y , Mackay A G , Binderup L , Colston K W
Published: December 2008

The anti-proliferative effects of the novel vitamin D analogue, EB1089, were assessed in the hormone-dependent breast cancer cell line, MCF-7, in vitro. In the present study, EB1089 was shown to be at least an order of magnitude more potent at inhibiting MCF-7 cell proliferation than the native hormone, lα,25-dihydroxyvitamin D3 (1,25(OH)2D3). Treatment of MCF-7 cell cultures with combinations of oestradiol and EB1089 ranging from 5 × 10−11 m to 5 × 10−9 m revealed the ability of EB1089 to suppress the mitogenic effects of oestradiol in these cells dose-dependently, as determined by [3H]thymidine incorporation and cell counts. EB1089 also exhibited a significant time- and dose-dependent decrease in MCF-7 oestrogen receptor (ER) concentration, as assessed by ligand binding assay. A fourfold reduction of ER levels by 5 × 10−9 m EB1089 relative to control ER levels was observed, whilst 5 × 10−9 m 1,25(OH)2D3 produced a significant but less dramatic decrease in ER levels. In addition, reduction of ER protein in EB1089-treated cell cultures was also demonstrated using an oestrogen receptor enzyme immunoassay.

The interaction of EB1089 and anti-oestrogens on the oestradiol-stimulated growth of MCF-7 cells was investigated. The treatment of cell cultures with 5 × 10−10 m EB1089 in combination with the pure anti-oestrogen, ICI 182,780 (5 × 10−8 m), and in the presence of between 5 × 10−10 m and 5 × 10−9 m oestradiol, produced an augmented inhibition of MCF-7 cell proliferation compared with the actions of either compound alone.

This study demonstrates that EB1089 is a potent anti-proliferative agent of breast cancer cells in vitro, and that part of its mechanism to inhibit cell growth may involve the modulation of ER expression, such that the responsiveness of cells to the growth-stimulatory effects of oestradiol is diminished. This new vitamin D analogue has potential in the treatment of breast cancer.

Journal of Endocrinology (1994) 141, 555–563
Expression of Messenger Ribonucleic Acid Encoding Matrix Metalloproteinases and Their Tissue Inhibitors is Related to Menstruation
Authors: Hampton Anne L. , Salamonsen Lois A.
Published: December 2008

Matrix metalloproteinases (MMP) degrade components of the extracellular matrix and the balance between enzyme production and that of their specific tissue inhibitors (TIMPs) is likely to be crucial for menstruation. The temporal expression of messenger (m) RNA for proMMP-1 (interstitial collagenase),proMMP-3(stromelysin 1), TIMP-1 and TIMP-2 has been examined by Northern analysis in 73 individually-dated endometrial tissue samples from normal women. Thirteen tissues expressed the mRNA for proMMP-3 and mRNA for proMMP-1 was detected in eleven of the same tissues. All of these tissues were from the menstrual (11) or perimenstrual (2) phases. No expression of mRNA for proMMP-1 or -3 was detected between cycle days 4 and 26. By contrast, mRNA for both TIMP-1 and TIMP-2 was detected in all tissue samples. The abundance of mRNA for TIMP-1 was significantly elevated in menstrual tissue compared with tissue from the rest of the cycle. TIMP-2 mRNA expression displayed considerable variability between individual tissues but mean abundance was also higher in menstrual tissue. Menstruation is therefore associated with a perturbation of the balance between the expression of MMPs and their tissue inhibitors which could lead to tissue degradation.
Glucocorticoids increase insulin-like growth factor-II mRNA accumulation in cultured human phaeochromocytoma cells
Authors: Liu J , Kahri A I , Heikkilä P , Blum W F , Voutilainen R
Published: December 2008

Human phaeochromocytomas abundantly express insulin-like growth factor-II (IGF-II), but its regulation and biological role in these neoplasms is not known at present. To clarify the regulation of IGF-II gene expression in phaeochromocytomas, we studied the effects of glucocorticoids, nerve growth factor (NGF), and protein kinase A and C regulators in primary cultures of human phaeochromocytoma cells. Cytoplasmic RNA was extracted and analysed by Northern and dot blotting with a 32P-labelled cDNA probe for IGF-II. Dexamethasone treatment (500 ng/ml) for 3 and 7 days resulted in a 260% and 515% increase in the accumulation of IGF-II mRNA respectively. The stimulatory effect of dexamethasone was time-and dose-dependent. The increases in the 6·0 and 2·2 kb species of IGF-II mRNAs were the most apparent. Cortisol (1 μg/ml) increased the amount of IGF-II mRNA by threefold compared with the control. NGF (200 ng/ml), dibutyryl cyclic AMP (1 mm) and 12-O-tetradecanoyl phorbol-13-acetate (a protein kinase C activator; 100 ng/ml) had no significant effect on IGF-II mRNA levels. These data suggest that IGF-II gene expression in human phaeochromocytomas may be regulated by microenvironmental glucocorticoids.

Journal of Endocrinology (1994) 142, 29–35
Seasonal variations in the ultrastructure and production of androgen-dependent proteins in the seminal vesicles of a Saharian rodent (Psammomys obesus)
Authors: Gernigon Th , Berger M , Lécher P
Published: December 2008

The effects of seasonal variations and experimental deprivation and substitution of androgen in the seminal vesicles of the Saharian rodent Psammomys obesus were investigated. Cytological studies showed that, during the breeding season, epithelial cells had large amounts of rough endoplasmic reticulum (RER) and substantial apocrine secretion. During the non-breeding season, RER cisternae were no longer expanded and apocrine secretion was rare. Castration during the breeding season was followed by regression of the RER and the disappearance of apocrine secretion. Treating castrated animals and animals in the non-breeding season with testosterone for 15 days caused reactivation. Eight major proteins with molecular weights of 120, 78, 67, 41, 37, 34, 21 and 14·4 kDa were present in homogenates during the breeding season; six (92, 41, 36, 35, 21 and 14·4 kDa) were found in seminal vesicle secretions. During the non-breeding season, the very large amounts of the 21 kDa protein were greatly reduced; conversely the 45 kDa protein increased. Electrophoretic patterns of homogenates from animals castrated during the breeding season showed eight proteins differentially induced (37, 34, 21 and 18 kDa) or repressed (67, 45, 38 and 35 kDa) by testosterone, of which the 21 kDa protein decreased most dramatically after castration. The effects of castration were reversed by the administration of testosterone. During the non-breeding season, the synthesis of the four induced proteins was stimulated by testosterone treatment; conversely that of the 45 kDa protein was suppressed.

Journal of Endocrinology (1994) 142, 37–46
Regulation of rat thyroxine-binding globulin and transthyretin: studies in thyroidectomized and hypophysectomized rats given tri-iodothyronine or/and growth hormone
Authors: Vranckx R , Rouaze-Romet M , Savu L , Mechighel P , Maya M , Nunez E A
Published: December 2008

We have investigated the role of the thyroid compared with the hypophysis in the regulation of the two saturable thyroid hormone carriers of rat serum, thyroxine-binding globulin (TBG) and transthyretin (TTR). We examined, at serum and hepatic mRNA level, the responses of TBG and TTR to thyroidectomy (Tx), hypophysectomy (Hx) and replacement treatments with tri-iodothyronine (T3) or/and GH, both hormones which are depleted when the thyroid or hypophysis are removed. The studies were performed on male rats at the age of 8 weeks, when the developmentally regulated TBG becomes undetectable after its transient postnatal rise, while the non-developmentally regulated TTR presents its normal, age-independent level of expression.

Tx-induced TBG re-expression was completely reversed by T3 replacement and unresponsive to GH replacement. TTR in the serum, on the other hand, was not affected by Tx or T3 replacement, moderately reduced by Tx in terms of the amount of mRNA, and markedly reduced by GH replacement. GH treatment, moreover, inhibited the expression of TTR in euthyroid controls. Hx, like Tx, induced TBG re-expression, an effect efficiently antagonized by T3 replacement. However, TBG synthesis was higher in Hx than in Tx rats and less effectively antagonized by T3 replacement. Most unexpectedly, GH induced a dramatic further increase in TBG synthesis, and the TBG synthesized in the GH-replaced Hx rats was entirely resistant to down-regulation by T3 replacement. TTR was markedly decreased at both serum and hepatic levels by Hx, unaffected by T3 and further decreased by GH replacement.

Our evidence is consistent with two distinct regulatory pathways for TBG, one under direct negative control by the thyroid hormones, without GH mediation, and the other independent of the thyroid, but involving GH, possibly for its role in the control of carbohydrate metabolism. We have shown that TTR depends little on the thyroid and is regulated by pituitary factors in a complex way, since it is inhibited by Hx but also by treatment with GH. The divergent regulatory pathways of TBG and TTR may be important in the homeostasis of thyroid hormone bioavailability.

Journal of Endocrinology (1994) 142, 77–84
Specific binding sites for progesterone in subcellular fractions of the porcine corpus luteum
Authors: Menzies G S , Bramley T A
Published: December 2008

Subcellular fractionation of porcine corpus luteum (CL) homogenates on continuous sucrose gradients has previously demonstrated that most of the endogenous progesterone of the CL was associated with a unique particulate fraction. Exogenous radiolabelled steroids were also sequestered with some specificity by this fraction. We now report that this particulate fraction is capable of binding high levels of exogenous 3H-labelled progesterone (and pregnenolone) in vitro, but only in the presence of the saponin, digitonin. Binding was dependent on the pH, temperature and duration of incubation, and showed specificity and high affinity for progesterone (Kd, 79 nm). Androgens, oestrogens and pregnenolone competed for porcine luteal [3H] progesterone binding sites, but only at much higher concentrations, whereas cholesterol, a number of progesterone receptor agonist and antagonist analogues and inhibitors of 3β-hydroxysteroid dehydrogenase and C17-hydroxylase/C17,20-lyase did not compete.

Analysis of profiles for a number of luteal cell-surface membrane and intracellular organelle markers confirmed previous studies showing the association of an NADH-cytochrome C reductase with this fraction. Moreover, the content of endogenous progesterone associated with particulate subcellular fractions isolated from porcine granulosa cell (GC) and CL homogenates at different stages of the luteal phase and early pregnancy waxed and waned with the stage of the luteal phase (and the secretory activity of the CL). Binding of [3H]progesterone in vitro equilibrated at the same buoyant density as endogenous progesterone: levels of both were highest during the mid-luteal phase and during early pregnancy, lower in early and late luteal CL, and undetectable in corpora albicantia. In contrast, relaxin secretory granules were readily resolved from progesterone binding sites. We propose that these particulate progesterone binding sites may be involved in the sequestration and/or packaging of newly-synthesized steroid for secretion by the luteal cell, or may mediate actions of progesterone within the luteal cell.

Journal of Endocrinology (1994) 142, 101–110
The influence of thyroid hormones on the growth of the atria and ventricles of the heart in immature rats
Authors: Canavan J P , Holt J , Goldspink D F
Published: December 2008

The normal plasma concentrations of tri-iodothyronine (T3) and thyroxine (T4) increase approximately six- and fourfold respectively between the end of gestation and weaning in the rat. This early postnatal surge of thyroid hormones was experimentally modified to produce either a state of hypo- or hyperthyroidism. The growth and rates of protein turnover in the atria and ventricles of the heart were studied, 12 and 20 days postpartum, both as a function of age and of changing thyroid status.

Neonatal hypothyroidism was induced by adding propylthiouracil to the mothers' drinking water late in gestation and throughout lactation. Hyperthyroidism was achieved by giving the suckling pups daily injections of T4 from day 3 postpartum onwards.

Between 12 and 20 days the weight and protein mass of the combined ventricles of the euthyroid animals approximately doubled, along with substantial increases (50%) in the RNA and DNA contents. Over this same 8 days, growth in the combined atria was much slower. During the same period, hypothyroidism significantly retarded the growth of these immature rats and their atria and ventricles. Both the rates of protein synthesis and protein degradation were decreased in the atria and ventricles. In contrast, hyperthyroidism significantly increased growth in both types of cardiac tissue, this being more pronounced in the atria than in the ventricles between 12 and 20 days. The rates of protein synthesis were increased accordingly, principally by increases in the ribosomal activities.

In conclusion, thyroid hormones clearly influence the early postnatal growth of the atria and ventricles of the heart in the rat.

Journal of Endocrinology (1994) 142, 171–179
The effects of ovarian transplantation on bone loss in ovariectomized rats
Authors: Tobias J H , Chambers T J , Gallagher A
Published: December 2008

Although hormone replacement therapy can prevent postmenopausal bone loss, it does not restore bone mass to normal in patients with established osteoporosis. This might reflect a failure to reproduce certain aspects of gonadal function. One method of investigating this possibility would be to examine the effect of ovarian transplantation on the skeleton of osteopaenic ovariectomized rats. However, ovarian transplantation may not fully restore ovarian function to normal, and it is not known whether transplanted ovaries reproduce the action of native ovaries on the skeleton. Therefore, we investigated whether renal capsular or subcutaneous ovarian transplants prevent the effects of ovariectomy on histomorphometric indices of rat tibiae over 44 days. Daily vaginal smears showed that oestrous cycles returned in all but two of 25 animals receiving ovarian transplants. We found that ovarian transplantation prevented the reduction in cancellous bone volume following ovariectomy. While trabecular number was reduced in ovariectomized animals receiving renal capsular ovarian transplants compared to intact animals, trabecular thickness was increased in both transplant groups. Ovarian transplantation also prevented the increase in cancellous and cortical bone formation, cancellous bone resorption and longitudinal growth rate caused by ovariectomy. We conclude that restoration of ovarian function by ovarian transplantation largely prevents the effects of ovariectomy on histomorphometric indices of rat tibiae, suggesting that transplanted ovaries can substitute for the action of native ovaries on the skeleton.

Journal of Endocrinology (1994) 142, 187–192
Effects of ovariectomy and oestradiol replacement on hypothalamic serotonergic and monoamine oxidase activity in the catfish, Heteropneustes fossilis: a study correlating plasma oestradiol and gonadotrophin levels
Authors: Senthilkumaran B , Joy K P
Published: December 2008

Hypothalamic serotonin (5-HT; content and turnover) and monoamine oxidase (MAO) activity were measured in female catfish, Heteropneustes fossilis, after ovariectomy and supplementation with oestradiol-17β (OE2) in the recrudescent and quiescent phases. These factors were correlated with changes in plasma levels of OE2 and gonadotrophin. In the quiescent phase (December), neither ovariectomy nor OE2 supplementation had any significant effect on 5-HT content and MAO activity. Plasma levels of OE2 and gonadotrophin were undetectable in both control and treated fish, indicating that there was no feedback effect. In the recrudescent phase (prespawning, May), ovariectomy caused biphasic responses of MAO activity and 5-HT content. The enzyme activity decreased significantly after 2, 3, 4 and 5 weeks but increased significantly 6 weeks after ovariectomy. The 5-HT content varied in a biphasic manner with a significant increase at 2, 3 or 4 weeks and a significant decrease in week 6; there being no effect in week 5. 5-HT turnover was inhibited significantly only in week 4 after ovariectomy and did not show a biphasic pattern. In the ovariectomized groups, the OE2 level decreased significantly in a progressive manner with a maximum reduction in week 6. The plasma level of gonadotrophin showed a significant bimodal pattern of increase with the peak in week 4 after ovariectomy, indicating a strong negative feedback effect of OE2. The bimodal pattern of pituitary gonadotrophin release could be correlated with a similar pattern of increase in 5-HT content. OE2 treatment of fish which had been ovariectomized 3 weeks previously had dose-dependent effects on the enzyme; the low dose (0·1 μg/g body weight) was stimulatory and the higher doses (0·5, 1·0 and 5·0 μg/g body weight) were inhibitory. The reverse was true for 5-HT content. Serotonergic turnover increased significantly only in the groups given high doses (1·0 and 5·0 μg/g body weight). The low dose of OE2 (0·1 μg/g body weight) restored the gonadotrophin and OE2 levels to those of the sham-ovariectomized vehicle-treated control group, whereas the high doses (0·5, 1·0 and 5·0 μg/g body weight) decreased the release of gonadotrophin in a dose-dependent manner. Our results suggest that OE2 modulates MAO activity to alter hypothalamic 5-HT in a seasonally dependent manner. The ovariectomy-induced changes in plasma levels of gonadotrophin appear to be mediated, at least partly, by the feedback action of OE2 on 5-HT metabolism.

Journal of Endocrinology (1994) 142, 193–203
Tissue distribution and regulation of 5′-deiodinase processes in lactating rats
Authors: Jack L J W , Kahl S , Germain D L St , Capuco A V
Published: December 2008

Thyroxine 5′-deiodinase (5′D) catalyses deiodination of the prohormone thyroxine (T4) to the metabolically active hormone 3,5,3′-tri-iodothyronine (T3). Previously, it has been demonstrated that rat mammary gland expresses a 5′D with enzymatic properties equivalent to those of the type I enzyme (5′D-I) found in rat liver and kidney. Using complementary DNA (cDNA) for rat hepatic 5′D-I, we have examined expression of 5′D-I messenger RNA (mRNA) in liver, and mammary gland from virgin and lactating rats, and in seven other tissues from virgin rats. 5′D-I mRNA could not be detected in mammary gland either by Northern blotting or by the more sensitive technique of reverse transcribing mRNA and then amplifying the cDNA by polymerase chain reaction (RTPCR). Analysis of the seven tissues from virgin rats by RT-PCR showed 5′D-I amplicons in liver, kidney and thyroid. No amplicons were detected in adrenal gland, cardiac muscle, skeletal muscle or spleen. In addition, the effect of lactation intensity on circulating thyroid hormones, hepatic and mammary gland 5′D activity, and hepatic 5′D-I mRNA levels was examined. A strong inverse relationship was noted between increased lactation intensity (suckling burden) and circulating T4 and T3, hepatic 5′D-I activity and hepatic 5′D-I mRNA levels. Mammary gland 5′D activity was positively correlated to lactation intensity. The data presented strongly suggest that the 5′D activity expressed in lactating mammary gland is encoded by a mRNA different from the 5′D-I message found in rat liver, kidney and thyroid gland, and may help explain the differential regulation of 5′D-I activity in these organs during lactation. In addition, hepatic 5′D-I activity was found to be correlated with the concentration of 5′D-I mRNA, suggesting that regulation is pretranslational. Results are consistent with a previously suggested involvement of 5′D in establishing metabolic adaptations to support lactation.

Journal of Endocrinology (1994) 142, 205–215
Parathyroid hormone-related protein in human term placenta and membranes
Authors: Bowden S J , Emly J F , Hughes S V , Powell G , Ahmed A , Whittle M J , Ratcliffe J G , Ratcliffe W A
Published: December 2008

Parathyroid hormone-related protein (PTHrP), the hypercalcaemia of malignacy factor, is expressed in the tissues of the human uteroplacental unit, including the placenta, amnion and chorion. We have used three region-specific immunoassays to quantitate and compare the distribution of PTHrP in tissues obtained at term following spontaneous labour and vaginal delivery or elective Caesarean section. In non-labouring women highest PTHrP(1–86) and (37–67) immunoreactivity was found in amnion covering the placenta, rather than the decidua parietalis of the uterus (reflected amnion) (median 1020 vs 451 fmol/g; 2181 vs 1444 fmol/g respectively). In labouring women, the PTHrP(1–86) concentration in reflected amnion was inversely correlated with the interval between rupture of the membranes and delivery. Tissue PTHrP(1–86) concentrations were lower in placenta than in chorion and amnion (medians 12, 109 and 664 fmol/g respectively) and, in all tissues, PTHrP(1–34) and (37–67) concentrations were significantly higher than that of PTHrP(1–86). Bioactive PTHrP(1–34) was detected in placenta, chorion and amnion using the ROS cell bioassay. The PTHrP(1–86) concentration (mean ± s.e.m.=41·4 ± 4·5 pmol/l) was high in amniotic fluid at term, although in maternal and cord plasma levels were only modestly increased. The molecular forms of PTHrP present in tissues and amniotic fluid were investigated by column chromatography which confirmed its molecular heterogeneity and suggested that processing is tissue-specific and occurs at both amino- and carboxy-terminals of the peptide.

Journal of Endocrinology (1994) 142, 217–224