bluuebunny/crossref_metadata_2025 huggingface dataset:
The natriuretic actions of vasopressin in the female rat: variations during the 4 days of the oestrous cycle
Authors: Forsling M L , Zhou Y , Windle R J
Published: December 2004

The renal actions of vasopressin were studied in the conscious female rat. Vasopressin caused a dose-dependent increase in sodium excretion when administered at 40–160 pmol/min. The highest dose, which increased sodium excretion from 10·4 ± 0·3 μmol/min (n=32) to 18·3 ± 0·8 μmol/min (n=8, P<0·001), also caused a significant rise in glomerular filtration rate (GFR). The antidiuretic and natriuretic responses to vasopressin varied significantly over the 4 days of the oestrous cycle. Both responses were greatest on pro-oestrus, being −57 ± 3 and 52 ± 3% above the control values with 80 pmol vasopressin/min. Responses of similar magnitude were also seen on dioestrus day 1. On these two cycle days the effects on urine flow and sodium excretion were accompanied by a significant increase in GFR. Smaller antidiuretic and natriuretic responses were seen on oestrus and dioestrus day 2, without concomitant changes in GFR. As the plasma vasopressin concentrations produced by hormone infusion were similar on each day of the cycle, the renal responsiveness to vasopressin appears to vary over the 4 days of the oestrous cycle. This may be important in terms of alteration and possible disturbances of fluid balance which may occur during reproductive cycles and pregnancy.

Journal of Endocrinology (1996) 148, 457–464
Changes in the immunohistochemical localisation of fibroblast growth factor-2, transforming growth factor-β1 and thrombospondin-1 are associated with early angiogenic events in the hyperplastic rat thyroid
Authors: Patel V A , Hill D J , Eggo M C , Sheppard M C , Becks G P , Logan A
Published: December 2004

Administration of a goitrogen (methimazole) and a low iodine diet to rats over a two-week period resulted in hypothyroidism and thyroid hyperplasia compared with controls (control: total serum thyroxine (T4) 66 ± 4 nmol/l, thyroid weight 5±1 mg/100 g body weight; experimental: T4 undetectable, thyroid weight 27 ± 4 mg/100 g body weight after 2 weeks of treatment; mean ± s.d., n=10). Immunohistochemistry carried out using a specific endothelial cell marker, CD31, and morphometric analysis (point counting of immunopositive cells) revealed that the progression of goitre in the rat thyroid is accompanied by an increase in capillary endothelial cell growth (neovascularisation). Fibroblast growth factor-2 (FGF-2) immunohistochemistry revealed widespread staining for the protein in the follicular cells of control glands. Less intense staining was found in the stroma and follicular cell nuclei. During hyperplasia and subsequent neovascularisation there was a progressive increase in the FGF-2 immunoreactivity at all locations during the two-week treatment period. Thrombospondin-1 (TSP1) immunoreactivity in the control rat thyroid was found in the stroma and in the endothelial cells, while weak follicular cell staining was also present. In the goitrous rat thyroid the TSP1 immunoreactivity was present after 1 week of treatment in the endothelial cells and most follicular cells, whilst stromal localisation was weak. After week 2 of treatment the endothelial cell and stromal localisation was no longer apparent, although a follicular localisation was still present. Transforming growth factor-β1 (TGFβ1) immunoreactivity was present in the cytoplasm of a minority of the follicular cells in control rat thyroids, while their nuclei were unstained. In the goitrous rat thyroid an increased intensity of staining for TGFβ1 was seen in all follicular cells, many of which now also demonstrated immunopositive nuclei, within one week of goitrogen administration. These results show that in the hyperplastic thyroid increases in FGF-2 and TGFβ1, and decreases in TSP1, accompany angiogenesis. These factors may interact in an autocrine/paracrine relationship to stimulate the neovascularisation that occurs during goitre formation.

Journal of Endocrinology (1996) 148, 485–499
Effect of corticosterone on protein degradation in isolated rat soleus and extensor digitorum longus muscles
Authors: Bowes S B , Jackson N C , Papachristodoulou D , Umpleby A M , Sönksen P H
Published: December 2004

The net catabolic effect of glucocorticoids on protein metabolism is well documented but the acute and chronic effect of glucocorticoids on protein breakdown remains controversial. In the present studies protein breakdown was measured by the release of tyrosine from the isolated soleus and extensor digitorum longus (EDL) muscles of control rats and rats treated with corticosterone (10 mg/100 g body weight/day) for 5 days.

The effect of corticosterone in arresting growth was confirmed since corticosterone-treated rats weighed significantly less than control rats after 2, 3, 4 and 5 days of treatment (P<0·001). Furthermore, the weights of soleus and EDL muscles from corticosterone-treated rats were significantly reduced (P<0·001, at least P<0·05 respectively) compared with muscles from control rats on days 3–5.

In the EDL muscle tyrosine release was significantly elevated after corticosterone treatment for 2 days (257 ± 21 nmol/g tissue/h, P<0·05), 3 days (205 ± 9 nmol/g tissue/h, P<0·01), 4 days (255 ± 20 nmol/g tissue/h, P<0·005) and 5 days (218 ± 8 nmol/g tissue/h, P<0·05) compared with EDL from control rats (192 ± 13, 171 ± 7, 187 ± 7, 180 ± 12 nmol/g tissue/h respectively). In the soleus muscle, tyrosine release was significantly elevated after corticosterone treatment for 2 days (226 ± 14 nmol/g tissue/h, P<0·001), 3 days (223 ± 16 nmol/g tissue/h, P<0·001) and 4 days (199 ± 10 nmol/g tissue/h, P<0·001) compared with control rats (158 ± 7, 132 ± 6 and 153 ± 7 nmol/g tissue/h respectively). After 5 days there was no significant difference in tyrosine release from soleus muscle between corticosterone-treated (176 ± 15 nmol/g tissue/h) and control rats (157 ± 6 nmol/g tissue/h). Plasma glucose concentrations were not significantly different in rats treated with corticosterone and control rats whilst insulin levels were significantly raised in the corticosterone-treated rats on all days compared with control rats (P<0·05 on day 1; P<0·001 on days 2, 3, 4 and 5). It is suggested that insulin may have prevented hyperglycaemia developing in the corticosterone-treated rats. Results from these studies indicate that the acute effect of glucocorticoids is to increase muscle proteolysis but this is not maintained with longer-term treatment.

Journal of Endocrinology (1996) 148, 501–507
Regional adrenal blood flow responses to adrenocorticotropic hormone after chronic embolization of the fetal placental circulation in sheep
Authors: Carter A M , Challis J R G , Svendsen P
Published: December 2004

To ascertain whether repeated hypoxic stress would alter the response of the adrenal cortex to adrenocorticotropic hormone (ACTH), by premature activation of the hypothalamic–pituitary–adrenal axis, we studied fetal sheep subjected to daily reduction of arterial oxygen content by embolization of the fetal placental circulation with 15 μm microspheres for 8 days from about day 124 of gestation (term ∼147 days) and sham-embolized controls. Starting before the final embolization (or shamembolization) on day 8, and continuing for 24 h, the fetus was given an intravenous infusion of ACTH1–24 (0·5 μg/h) or vehicle. Fetal and maternal blood samples were taken for determination of immunoreactive cortisol, and regional adrenal and fetal placental blood flows were measured by the microsphere technique at three time points: 1 h before infusion, 3 h after the start of the infusion (1 h after embolization), and after 24 h of infusion. Prior to infusion of ACTH or vehicle, fetal placental blood flow was lower in microsphere-embolized fetuses than in sham-embolized controls (199 ± 15 vs 292 ± 25 ml/min per 100 g tissue; mean ± s.e.; P<0·01). However, plasma cortisol and adrenal cortical blood flow did not differ between embolized fetuses and controls. Adrenal vascular responses to the 24-h infusion of ACTH were similar in embolized and shamembolized fetuses. Adrenal cortical blood flow increased 3-fold (P<0·05) due to decreased vascular resistance (P<0·01), with no change in adrenal medullary blood flow. Thus, while daily embolization of the fetal placental circulation caused a sustained decrease in cotyledonary blood flow, no evidence of altered responsiveness of the adrenal cortex to ACTH was found in these experiments.

Journal of Endocrinology (1996) 148, 517–522
Expression of α2- and β-adrenoceptor subtypes in human islets of Langerhans
Authors: Lacey R J , Chan S L F , Cable H C , James R F L , Perret C W , Scarpello J H B , Morgan N G
Published: December 2004

Sequences from cDNA molecules encoding α2-adrenoceptor subtype genes were subcloned into prokaryotic vectors and riboprobes generated to hybridise selectively with each of the human α2C2-, α2C4- and α2C10-adrenoceptor subtype mRNA species. The riboprobes were labelled with either 32P or digoxigenin and used to study the expression of α2-adrenoceptor subtypes in sections of human pancreas, in isolated human islets of Langerhans and in clonal HIT-T15 pancreatic β-cells. Using a ribonuclease protection assay protocol, expression of mRNA species encoding both α2C2 and α2C10 was demonstrated in preparations of isolated human islets of Langerhans. mRNA encoding α2C4 was also detected in human islet RNA, using reverse transcription coupled with the polymerase chain reaction. In situ hybridisation was then employed to examine the distribution of each α2-adrenoceptor subtype in sections of human pancreas. All three subtypes of α2-adrenoceptor mRNA were identified in sections of formalin-fixed, paraffinembedded human pancreas using riboprobes labelled with digoxigenin. Although some labelling of the three α2-adrenoceptor mRNA subtypes was seen in the islets, the labelling was most intense in the exocrine tissue of the pancreas for each receptor subtype. The specificity of the digoxigenin-labelled RNA probes was confirmed in several control tissues and by in situ hybridisation studies using sense probes in the pancreas. The integrity of the pancreas sections was confirmed by in situ hybridisation with an antisense riboprobe derived from human insulin cDNA. The results demonstrate that multiple α2-adrenoceptor subtypes are expressed in human pancreas. Both the exocrine and endocrine cells express more than one receptor subtype, although the islets stain less intensely than the bulk of the tissue suggesting that the islet cells may have lower levels of expression than the acinar tissue. The presence of α2-adrenoceptor subtype mRNA species in pancreatic β-cells was confirmed by Northern blotting of RNA extracted from the clonal β-cell line, HIT-T15. Transcripts encoding each of the three cloned α2-adrenoceptor subtypes were detected in HIT-T15 cells.

Hybridisation of sections of human pancreas with oligodeoxynucleotide probes designed to hybridise with β2-adrenoceptor mRNA revealed expression of this species in islet β-cells but not in the exocrine tissue of the pancreas.

Journal of Endocrinology (1996) 148, 531–543
The effects of N-methyl-d,l-aspartic acid and aspartic acid on the plasma concentration of gonadotrophins, GH and prolactin in the ewe
Authors: Downing J A , Joss J , Scaramuzzi R J
Published: December 2004

Aspartic acid is a neurotransmitter in the central nervous system that acts via the glutamate receptor and the analogue, N-methyl-d,l-aspartic acid (NMA) is an agonist that stimulates GnRH secretion. Under normal dietary conditions, the plasma concentration of aspartic acid in ewes is low and if increased by improved nutrition may increase the brain concentration of aspartic acid leading to increased gonadotrophin secretion. In two experiments we investigated the effects of NMA on pituitary hormone concentrations and the effects of aspartic acid on ovulation rate and pituitary hormone concentrations. The intravenous injection of NMA into cycling ewes resulted in an immediate (within 15 min) release of a pulse of LH and of GH and a prolonged (up to 1 h) suppression of prolactin secretion. There were marked differences in responsiveness to NMA between individual ewes. The intravenous infusion of aspartic acid for 5 days in the late luteal phase of the oestrous cycle did not affect ovulation rate but reduced the mean LH (P<0·05) and FSH (P<0·05) concentrations in plasma. The frequency of LH pulses also tended to be lower (P<0·1) in ewes infused with aspartic acid. It is suggested that the decrease in gonadotrophin secretion in ewes infused with aspartic acid is due to effects on the hypothalamus or the anterior pituitary gland which are not related to increased levels of ovarian feedback. These changes are likely to involve decreased GnRH secretion.

Journal of Endocrinology (1996) 149, 65–72
Inhibin-related proteins in rat prostate
Authors: Risbridger G P , Thomas T , Gurusinghe C J , McFarlane J R
Published: December 2004

Inhibin and activin are members of the transforming growth factor β (TGFβ) family which can regulate cell proliferation in a number of tissues. The presence of inhibins and the related proteins, activins, in the prostate has been implicated by the detection of activin type II receptors. The aim of this study was to determine whether or not immunoactive (ir) inhibin and ir-activin are present in the rat prostate and to study the acute regulation by androgens. The results showed that mRNAs for the α and β inhibin subunits were detected in rat prostate by reverse transcription-PCR together with ir-inhibin and ir-activin in prostate cytosols. The levels of ir-activin in the prostate (223 ± 44 ng/gland) were greater than the levels of ir-inhibin (6·89 ng/gland), and activin immunoreactivity was localised to the epithelial cells. The presence of these proteins and the subunit mRNAs suggests that these proteins are produced in the prostate and may have a role in prostate function. The study of the effect of androgen withdrawal on the levels of ir-activin and ir-inhibin in these tissues showed no change in the content of ir-inhibin or ir-activin (ng/g tissue) after 3 days of castration or following the administration of the cytotoxic drug ethane dimethane sulphonate (EDS), although there was a significant (P<0·01) decline in prostate weight. Fourteen days after EDS treatment, as the prostate weight fell significantly lower, the amount of ir-inhibin and ir-activin per prostate gland was significantly (P<0·01) reduced although the concentration was unaffected. These data demonstrate, for the first time, that inhibin α and β subunit mRNA and ir-inhibin and ir-activin are present in the prostate; the role of these proteins in prostate function remains to be established.

Journal of Endocrinology (1996) 149, 93–99
Functional heterogeneity of human term cytotrophoblasts revealed by differential sensitivity to extracellular Ca2+ and nucleotides
Authors: Bradbury R A , McCall M N , Brown M J , Conigrave A D
Published: December 2004

We have prepared purified cytotrophoblasts from human term placentas and examined the sensitivity of fura-2 loaded cells to the nucleotides ATP and UTP and to changes in extracellular Ca2+ concentration ([Ca2+]o). Purified cytotrophoblasts were obtained by collagenase digestion and separation according to density using selfgenerated Percoll gradients. The cytotrophoblast fraction was free of red cell and largely free of white cell contamination (as assessed by uniformly negative staining for vimentin and the failure of >90% of fura-2 loaded cells to respond to the chemotactic peptide fMet-Leu-Phe). Purified cells secreted progesterone in a linear fashion over several hours in the presence of 25-hydroxycholesterol. The cells ranged in size from approximately 7·5 to 50 μm in diameter as described previously for purified cytotrophoblasts, and an analysis of cells for sensitivity to [Ca2+]o or nucleotides suggested functional heterogeneity within the cytotrophoblast population. Small cells (7·5–10 μm) were negative for cytokeratin-8 and, after loading with fura-2, were insensitive to extracellular nucleotides but sensitive to elevations in [Ca2+]o. Medium-sized cells (12–20 μm) were largely cytokeratin-positive (70% of cells) and sensitive to both ATP and UTP but largely insensitive to [Ca2+]o. Large cells (25–50 μm) were uniformly cytokeratin-positive (100% of cells) and, after fura-2 loading, sensitive to both [Ca2+]o and extracellular ATP or UTP. We examined the likely origin of small, medium and large cytotrophoblasts using an immunomagnetic cell sorting procedure that separates villous cytotrophoblasts (which do not express major histocompatibility class I antigens) from extravillous cytotrophoblasts. This procedure resulted in the selective sedimentation of almost all medium and large cells, leading to the conclusion that the small cells were villous cytotrophoblasts whereas medium and large cells were predominantly extravillous in origin. The data suggest that small, medium and large cytotrophoblasts have distinct roles in the function of the term placenta.

Journal of Endocrinology (1996) 149, 135–144
Fluorescence-activated cell sorted rat islet cells and studies of the insulin secretory process
Authors: Josefsen K , Stenvang J P , Kindmark H , Berggren P-O , Horn T , Kjær T , Buschard K
Published: December 2004

Studies of individual cell types in the islets of Langerhans are complicated by the cells' functional coupling by gap junctions and paracrine interaction. Access to purified alpha and beta cells is therefore desirable. We present a simplified and optimized method for fluorescence-activated cell sorting of endocrine pancreatic rat islets. For dispersion of the islets, dispase was superior to trypsin, as the number of vital single cells was higher (1·1 ± 0·1 × 103 vs 0·6 ± 0·1 × 103/islet, P<0·05). The purity of the sorted cells was 96·7 ± 1·2% for the non-beta cells and 97·8 ± 0·6% for the beta cells (numbers in percentages of endocrine cells). In culture, isolated beta cells, non-beta cells and mixtures of beta and non-beta cells formed aggregates, but not at low temperature (4 °C) and not in medium with low serum content (2%). Finally, in pure beta cell aggregates, glucose stimulated changes in cytoplasmic free Ca2+ concentration although both glucose- and arginine-induced insulin secretion was much reduced. We conclude that alpha cells are necessary for insulin secretion but not for glucose sensing.

Journal of Endocrinology (1996) 149, 145–154
The role of pregnenolone-metabolizing enzymes in the regulation of oestradiol biosynthesis during development of the first wave dominant follicle in the cow
Authors: Carrière P D , Harvey D , Cooke G M
Published: December 2004

During the luteal phase in the cow, a first-wave dominant follicle grows to reach ovulatory size, but then ceases to grow, becomes no longer dominant and enters a phase of slow regression. During this growth transition, the concentration of oestradiol has been shown to decrease in follicular fluid. The objective of this study was to determine if follicular fluid oestradiol concentrations are regulated by the activity of three major steroidogenic enzymes, namely P450-aromatase (P450-arom), 3β-hydroxysteroid dehydrogenase/Δ5–Δ4 isomerase (3β-HSD) and 17α-hydroxylase C-17,20 lyase cytochrome P450 enzyme (P450–17α) measured in granulosa and theca cells isolated from individual first-wave dominant follicles. Follicle growth and state of dominance was assessed by ultrasonography and follicles were classified as growing-dominant (GD, n=6), non-growing-dominant (NGD, n=8) or non-growing-non-dominant (NGND, n=6). Mean follicular fluid concentrations of oestradiol were higher in GD than in NGD or NGND follicles (511 ± 98 versus 136 ± 16 and 20 ± 11 nmol/l respectively). Oestradiol was not correlated with P450-arom in any of the three groups. In GD follicles, oestradiol was positively correlated with pregnenolone concentration but neither was correlated with granulosa or theca 3β-HSD activity or with theca P450–17α activity. In NGD follicles, oestradiol was negatively correlated with theca 3β-HSD activity and pregnenolone was negatively correlated with granulosa 3β-HSD activity. In NGND follicles, oestradiol was positively correlated, and pregnenolone was negatively correlated with theca 3β-HSD and P450–17α activities. These studies demonstrated that pregnenolone supply is the principal regulating factor of oestradiol output during follicle dominance and during the loss of dominance but that the levels of P450–17α and 3β-HSD activity become rate-limiting when the follicle is no longer dominant.

Journal of Endocrinology (1996) 149, 233–242
Immunohistochemical localization of the glucocorticoid receptor in human fetal membranes and decidua at term and preterm delivery
Authors: Sun M , Ramirez M , Challis J R G , Gibb W
Published: December 2004

The human fetal membranes and decidua may be important in the onset and/or progression of human labor by providing prostaglandins for this process. Glucocorticoids have been implicated in the regulation of prostaglandin production by these tissues but to date there is no direct evidence for glucocorticoid receptors (GRs) being present in human intrauterine tissues. The purpose of the present study was to determine, using immunohistochemistry, whether the human fetal membranes and decidua contained GRs; to determine the localization of receptors to the cytoplasm or nuclei, and to examine the content and distribution of the GRs in tissues obtained during pregnancy following preterm labor (<37 weeks) and at term prior to and following term labor. Term tissues were obtained prior to labor by elective Cesarian section (n=9) or following vaginal delivery (n=9). Tissues from 14 patients who delivered preterm but with no clinical evidence of infection were also examined. Cryostat sections were thaw-mounted onto microscope slides. The immunoreactive GRs were visualized with an Elite Vectastain ABC Kit using a polyclonal antibody prepared against a synthetic peptide corresponding to amino acids 346–367 of the human GR. At term, nuclear GRs were found in amnion epithelial cells, mesenchyme and the chorion laeve. GRs were present, but were less defined, in the decidua. A similar distribution was found in the preterm tissues. However, nuclear staining in the amnion epithelial cells, mesenchymal cells, chorion and decidua was more pronounced in tissues obtained following preterm labor. This study provides direct evidence for the presence of GRs in human fetal membranes and decidua, and suggests the possible importance of multiple cell types in the action of glucocorticoids in these tissues.

Journal of Endocrinology (1996) 149, 243–248
Some effects of a low sodium intake on the expression of P450 aldosterone synthase in the hamster adrenal cortex: immunoblotting, immunofluorescent and immuno-gold electron microscopic studies
Authors: LeHoux J G , Lefebvre A , Ducharme L , Lehoux J , Martel D , Brière N
Published: December 2004

In the current work we studied the effects of a low sodium intake on P450 aldosterone synthase (P450aldo) in the adrenal cortex of male hamsters by Western blotting analysis. We also investigated the zonal distribution of P450aldo with a specific antibody using immunofluorescence and immuno-gold electron microscopy.

Western blotting analysis revealed a progressive induction of P450aldo in the adrenals of hamsters kept on a low sodium diet, with two-, four- and eightfold increases after 2, 4 and 21 days on the diet.

Immunofluorescence microscopy showed that P450aldo was confined to the zona glomerulosa (ZG) cells. Electron microscopy showed P450aldo to be located in the mitochondria of ZG cells. When hamsters were maintained on a low sodium intake for 2, 11 and 21 days, P450aldo was still found only in the ZG; the ZG appeared either unchanged or sometimes slightly enlarged. Moreover, at days 11 and 21, the intensity of the immunofluorescent signal was much stronger in the ZG of hamsters on the low sodium intake than in controls. Hence, immunocytochemistry using the colloidal-gold technique showed P450aldo to be more abundant in the mitochondria of the experimental animals than in controls.

To conclude, P450aldo is present only in the ZG of hamster adrenals and sodium restriction appears to induce its expression by stimulating production within individual ZG cells rather than by stimulating a proliferation of the ZG cells.

Journal of Endocrinology (1996) 149, 341–349
Osmotic and volaemic regulation of atrial and ventricular natriuretic peptide secretion in conscious eels
Authors: Kaiya H , Takei Y
Published: December 2004

The effects of acute manipulation of plasma osmolality and blood volume on plasma atrial and ventricular natriuretic peptide (ANP and VNP) levels were examined in conscious freshwater eels, Anguilla japonica. A bolus injection of hypertonic NaCl (0·85 m and 1·7 m, 2·5 ml/kg body weight) through a catheter into the ventral aorta produced increases in plasma Na concentration and osmolality with parallel concentration-dependent, transient increases in plasma ANP and VNP levels. Plasma ANP and VNP levels also increased after injection of 1·7 m mannitol solution which produced an increase in plasma osmolality but a decrease in plasma Na concentration. However, injection of a 2·0 m solution of urea, which does not cause cellular dehydration in mammals, produced only small increases in plasma ANP and VNP levels, although plasma osmolality increased.

A bolus injection of 10 or 25 ml/kg isotonic saline supplemented with 2% dextran for colloidal osmotic pressure, which theoretically increased blood volume by 29% or 71%, produced volume-dependent, transient increases in plasma ANP and VNP levels without changes in plasma Na concentration and osmolality. Similar volume expansion with dialysed eel plasma caused greater increases than with dextran-saline. However, these increases were much smaller than those after osmotic stimuli. These results indicate that secretion of ANP and VNP is regulated by two receptor mechanisms: osmoreceptors activated by cellular dehydration, not specifically by hypernatraemia, and volume or stretch receptors activated by hypervolaemia. The relative importance of the osmoreceptive mechanism is greater in eels than in mammals where volaemic regulation dominates over osmotic regulation for ANP secretion.

Journal of Endocrinology (1996) 149, 441–447
Selective toxic effects of tamoxifen on osteoclasts: comparison with the effects of oestrogen
Authors: Arnett T R , Lindsay R , Kilb J M , Moonga B S , Spowage M , Dempster D W
Published: December 2004

We investigated the actions of the trans- and cis-isomers of tamoxifen on the function of neonatal rat osteoclasts in vitro. Both compounds inhibited resorption pit formation by osteoclast-containing mixed bone cell cultures incubated for 24 h on cortical bone slices. Cell counts revealed that the inhibition was closely related to a cytotoxic effect, to which osteoclasts appeared particularly sensitive. Partial inhibition of resorption was seen in the presence of 2 μm trans-tamoxifen, whereas complete abolition of resorption and osteoclast viability occurred with 10 μm trans-tamoxifen; survival of mononuclear cells was unimpaired at either concentration. Cis-tamoxifen appeared to be slightly more toxic, with significant inhibitions of osteoclast viability and thus resorption pit formation at a concentration of 2 μm, and also of mononuclear cell numbers at 10 μm. Time-lapse video observations indicated that osteoclast death occurred rapidly (within 2–3 h) following exposure to 10 μm of either trans-tamoxifen or cis-tamoxifen. The morphological appearance of the dying cells was consistent with apoptosis. These results may help to explain the anti-resorptive action of tamoxifen seen in vivo in rats and humans. In contrast, oestradiol-17β consistently exerted no significant effects on resorption pit formation by rat osteoclasts over 24 h, even at grossly supraphysiological concentrations (up to 10 μm).

Journal of Endocrinology (1996) 149, 503–508
The effect of ovarian arterial infusion of human recombinant inhibin and bovine follicular fluid on ovarian hormone secretion by ewes with an autotransplanted ovary
Authors: Campbell B K , Scaramuzzi R J
Published: December 2004

Recombinant human inhibin A (rhInh) or steroid-free bovine follicular fluid (bFF) were infused into the ovarian artery of anoestrous ewes with ovarian autotransplants induced to ovulate with a pulsatile regimen of GnRH applied after a 10-day pretreatment with progestagen sponges. In the period 12–24 h after sponge withdrawal ewes received ovarian arterial infusions of saline (n=6), 0·3 μg rhInh/h (n=5), 1·6 μg rhInh/h (n=5) or 25 μl bFF/h (n=4).

Controls had a normal follicular phase with an LH surge 43 ± 3 h after sponge withdrawal which resulted in ovulation (six out of six). Both doses of rhlnh increased ovarian venous inhibin concentrations in a dose-related fashion (P<0·05) but resulted in depressions (P<0·05) in FSH concentrations of similar magnitude. Both doses of rhInh acutely inhibited ovarian oestradiol and androstenedione secretion (P<0·01) but at the end of rhInh infusion oestradiol secretion was quickly re-established without a corresponding increase in FSH. LH surges were detected in five out of five and three out of five ewes infused with low and high doses of rhInh respectively, and progesterone concentrations during the subsequent luteal phase were depressed (P<0·05). Infusion of bFF had no effect on inhibin or FSH concentrations but resulted in acute inhibition (P<0·01) of ovarian oestradiol, androstenedione and inhibin secretion, a delay (P<0·05) in the time to the LH surge and a depression (P<0·05) in luteal-phase progesterone concentrations.

In conclusion, while the depression in FSH induced by rhlnh cannot be excluded as a cause for the inhibitory effects of rhInh treatment on ovarian function, such a mechanism cannot fully explain the ovarian responses obtained to rhInh infusion. These results therefore support a direct ovarian role for inhibin in the modulation of ovarian function in addition to its indirect role in controlling FSH. This conclusion is supported by the demonstration that bFF can induce similar inhibitory effects on ovarian function without changing FSH.

Journal of Endocrinology (1996) 149, 531–540
THE ROLE OF IGFBP-3 IN THE REGULATION OF IGFBP-4 PROTEOLYSIS
Authors: Donnelly M.J. , Holly J.M.P.
Published: December 2004

The biological effects of insulin-like growth factors (IGFs) are mediated by cell surface receptors but their bioavailability is regulated by IGF binding proteins (IGFBPs) which bind IGF with higher affinity than the receptor. Proteolytic cleavage of the binding proteins reduces their affinity for IGF making the IGF more available to the cell receptor. In the current study we have examined the regulation of IGFBP-4 protease produced by cultured human dermal fibroblasts.

IGF-I and the analogs of IGF-I (LR3 and Des[1-3]) induced a dose dependent increase in both proliferation and IGFBP-3 production. Low concentrations of IGF-I induced a marked loss of IGFBP-4 by Western ligand blotting (WLB). This effect was confirmed by the ability of media collected from cells exposed to increasing concentrations of IGF-I to fragment recombinant IGFBP-4, an effect blocked by EDTA.

IGFBP-4 proteolysis was observed when cells were exposed to Des[1-3] (albeit at higher concentrations) but not with LR3. Both analogs bind to the IGF receptor but do not bind to IGFBP-4 and have reduced (Des[1-3]) or no (LR3) affinity for IGFBP-3. This demonstrated that neither receptor activation nor ligand binding directly to IGFBP-4 was necessary for IGF induced proteolysis. Protease activity correlated with affinity for IGFBP-3 suggesting a role for IGFBP-3 in the regulation of IGFBP-4 proteolysis. This was confirmed by the ability of excess recombinant IGFBP-3 to inhibit the IGF-I and Des[1-3] induced proteolysis of IGFBP-4. Addition of IGF-I to media from cells unexposed to IGF induced IGFBP-4 proteolysis but this was not seen with LR3 which does not bind to IGFBP-3. Fragmentation occured at higher concentrations of Des[1-3] consistent with its reduced affinity for IGFBP-3

This data suggests that IGFBP-4 proteolysis is regulated in a novel manner by IGFBP-3 which is dependent on the relative proportions of the different binding proteins and the levels of IGFs
Expression of the steroidogenic acute regulatory protein mRNA in adrenal tumors and cultured adrenal cells
Authors: Liu J , Heikkilä P , Kahri A I , Voutilainen R
Published: December 2004

The steroidogenic acute regulatory protein (StAR) has recently been shown to be a factor necessary for cholesterol transport into adrenal and gonadal mitochondria, which is the regulated, rate-limiting step in steroidogenesis. We show here that StAR mRNA is highly expressed in normal adult adrenals (n=9), adrenocortical adenomas (n=16), adrenal hyperplasias (n=6), adrenocortical carcinomas (n=6) and adrenals adjacent to tumor tissues (n=9). There was a good correlation between the expression of StAR and the cholesterol side-chain cleavage enzyme/20,22-desmolase (P450 scc) mRNAs both in normal (r=0·93; P<0·01) and in tumor (r=0·97; P<0·001) tissues. No StAR mRNA was detected in Northern blots of liver, kidney, breast, parathyroid or phaeochromocytoma RNAs.

In cultured adrenocortical cells, adrenocorticotropin (ACTH), (Bu)2cAMP, and cholera toxin increased StAR and P450 scc mRNA accumulation 6- to 18-fold, dose-and time-dependently. StAR (and P450 scc) mRNA increased relatively slowly in response to ACTH treatment, with the maximal increment at 24 h, while the mRNA of the early response gene c-fos peaked within 2 h. The protein kinase inhibitor H-7 inhibited basal and ACTH-induced StAR mRNA expression. Our results show that StAR mRNA is expressed at high levels in normal human adrenals and adrenocortical neoplasms. It is up-regulated in parallel with P450 scc by ACTH in adult adrenocortical cells, which suggests that ACTH is at least one of the key regulators of adrenal StAR expression.

Journal of Endocrinology (1996) 150, 43–50
In vivo manipulation (depletion versus activation) of testicular macrophages: central and local effects
Authors: Gaytan F , Bellido C , Morales C , García M , van Rooijen N , Aguilar E
Published: December 2004

Testicular macrophages are a relevant cell type for the regulation of Leydig cell steroidogenesis. The availability of liposome technology allows in vivo manipulation of macrophages in order to analyze their role in the regulation of the hypothalamic-pituitary-testicular axis. In this study, adult (70 days of age) and prepubertal (22 days of age) rats were injected intratesticularly with liposomes containing either dichloromethylene diphosphonate (C12MDP) to deplete testicular macrophages or muramyl tripeptide (MTP-PE) to activate them. Control rats were injected with the corresponding volumes of 0·9% NaCl. Animals were killed 10 days after treatment. Adult rats injected bilaterally or unilaterally with C12MDP liposomes showed increased serum LH and testosterone concentrations, as well as increased testosterone concentrations in the testicular interstitial fluid. In unilaterally injected rats, testosterone concentrations in the interstitial fluid were higher in the macrophage-containing testes than in the contralateral, macrophage-depleted testes. Adult rats treated bilaterally with MTP-PE liposomes showed increased numbers of testicular macrophages, whereas the number of Leydig cells was unchanged. Serum LH concentrations were decreased, but no changes were found in testosterone concentrations. Prepubertal rats treated bilaterally with C12MDP liposomes showed decreased numbers of Leydig cells. However, serum LH and testosterone concentrations were increased. Otherwise, prepubertal rats treated bilaterally with MTP-PE liposomes showed increased numbers of macrophages and Leydig cells, as well as increased serum testosterone concentrations. These data suggest that testicular macrophage-derived factors act at two different levels in the pituitary-testicular axis: first, at a central level by inhibiting LH secretion, and secondly, at a local level by stimulating Leydig cell steroidogenesis.

Journal of Endocrinology (1996) 150, 57–65
Inhibitory effects of fumagillin and its analogue TNP-470 on the function, morphology and angiogenesis of an oestrogen-induced prolactinoma in Fischer 344 rats
Authors: Stępień H , Grochal M , Zieliński K W , Mucha S , Kunert-Radek J , Kulig A , Stawowy A , Pisarek H
Published: December 2004

The process of angiogenesis occurs in many physiological states, but it is also essential for the growth of solid tumours and metastasis formation. An abnormal arterial vascularization has been shown in prolactin-secreting pituitary adenomas induced by prolonged treatment with oestrogens in Fischer 344 (F344) rats. It is thought that anti-angiogenic agents might be useful in therapy for these tumours.

Fumagillin and its analogue TNP-470 are known to inhibit endothelial cell proliferation selectively, but their effect on lactotroph cell secretory function and prolactinoma formation has not yet been described. The aim of the present study was to examine the effects of fumagillin and TNP-470 on prolactin secretion, and morphological and vascular changes within the anterior pituitary in long-term oestrogen-treated male F344 rats in vivo and in vitro. As expected, 7 weeks after s.c. implantation of Silastic tubes containing 10 mg diethylstilboestrol (DES), a very high rise in serum prolactin levels was found. Both angiogenesis inhibitors injected s.c. at doses of 10 mg/kg body weight for 24 days attenuated the stimulatory effect of DES on prolactin production and release. They also diminished prolactin cell density and inhibited cell proliferation expressed as the number of anterior pituitary cells labelled with bromodeoxyuridine (BrdU), but the effect of TNP-470 was minor compared with fumagillin. Both angioinhibitors suppressed neovascularization within the anterior pituitary with similar potency but, on the other hand, they did not affect DES-induced increases in prolactin secretion from cultured rat pituitary cells and cell proliferation in vitro.

In conclusion, our results provide strong evidence for the anti-tumour and anti-prolactin activity of angiogenesis inhibitors in the experimentally oestrogen-induced pituitary adenoma; this might be mediated indirectly through the inhibition of angiogenesis.

Journal of Endocrinology (1996) 150, 99–106
Pioglitazone hydrochloride stimulates insulin secretion in HIT-T 15 cells by inducing Ca2+ influx
Authors: Ohtani K , Shimizu H , Tanaka Y , Sato N , Mori M
Published: December 2004

Pioglitazone hydrochloride (AD-4833), one of the thiazolidinedione analogs, is a new anti-diabetic agent which improves peripheral insulin resistance in diabetic patients. We determined the direct effect of AD-4833 on insulin secretion in HIT-T 15 cells. The effects of AD-4833 (10−7m to 10−5 m) on insulin secretion were examined in 3 and 7 mm glucose-containing F-12 K media. The addition of 10−5m AD-4833 significantly increased insulin secretion in both media, but its stimulatory effect was more potent in the medium containing 7 mm glucose. The addition of 10−5 m AD-4833 caused an immediate, significant increase in cytosolic free Ca2+ concentration ([Ca2+]i). Nifedipine at all concentrations from 10 to 1000 nm significantly attenuated insulin secretion by 10−5m AD-4833. In addition, 10−5m AD-4833 failed to stimulate insulin secretion in the Ca2+-free Kreb's-Ringer bicarbonate buffer. These data indicated that AD-4833 stimulates in vitro insulin secretion in HIT-T 15 cells, perhaps by inducing Ca2+ influx.

Journal of Endocrinology (1996) 150, 107–111
Plasma clearance and tissue distribution of labelled chicken and human IGF-I and IGF-II in the chicken
Authors: McMurtry J P , Francis G L , Upton Z , Walton P E , Rosselot G , Caperna T J , Brocht D M
Published: December 2004

The metabolic clearance of chicken IGF-I (cIGF-I), cIGF-II, human IGF-I (hIGF-I), and hIGF-II was examined in the chicken using 125I-labelled growth factors. Superose-12 chromatography of plasma collected at 7·5 min post-infusion revealed peaks of radioactivity corresponding to 150 and 43 kDa and unbound tracer. Statistical analysis of trichloroacetic acid (TCA)-precipitable radioactivity in sequential plasma samples as well as following chromatography of the same samples revealed that clearance of the radiolabelled peptides followed an apparent triphasic pattern. The close similarity of the individual chromatographically defined pools in their clearance rate compared with the three components described by TCA precipitation strongly suggested their identity. Both free 125I-labelled cIGF-II (3·11 min) and hIGF-II (3·01 min) were cleared at a greater rate than their IGF-I counterparts. Unbound hIGF-I was cleared at a greater rate than cIGF-I (4·45 vs 5·66 min respectively). A similar pattern for clearance was evident in the radiolabelled growth factors associated with the 43 kDa component, although at a longer half-life. There was no difference in the apparent clearance of the radiolabelled growth factors associated with the 150 kDa component between IGF-I or -II or between species. Analysis of the chromatographic profiles of radioactive IGF-I peptides complexed to serum proteins versus those bound to labelled IGF-II peptides revealed the presence of a large molecular mass binding protein in vivo. Ligand blotting of chicken serum determined that a binding protein with a mass of 70 kDa was detectable with 125I-IGF-II probes only, and was not present in pig serum. In addition, tissue uptake of 125I-cIGF-I and -II was evaluated. Similar patterns of tissue distribution and uptake were observed for 125I-cIGF-I and -II, except that cIGF-II uptake by the liver exceeded that of 125I-cIGF-I at 15 min post-infusion. The rank order of tissue distribution was as follows: kidney > testis > heart > liver > pancreas > small intestine> cartilage > bursa > gizzard > leg muscle > breast muscle > brain. We conclude from these studies that the clearance of IGFs from the compartments identified in blood and the potential target tissues is dependent on their interactions with IGF-binding proteins and receptors.

Journal of Endocrinology (1996) 150, 149–160
A model of intrauterine growth retardation caused by chronic maternal undernutrition in the rat: effects on the somatotrophic axis and postnatal growth
Authors: Woodall S M , Breier B H , Johnston B M , Gluckman P D
Published: December 2004

While it is well established that severe maternal undernutrition during pregnancy causes intrauterine growth retardation (IUGR), there has been relatively little study of the endocrine consequences and postnatal development of growth-retarded offspring. We have developed a model in the rat of IUGR by nutritional restriction of the mother throughout gestation and have examined the effects of fetal growth retardation on the endocrine and metabolic status during the perinatal period. Timed matings were performed in Wistar rats and dams were randomly assigned to one of two dietary treatment groups. Food was available ad libitum throughout pregnancy to a control group (ad libitum group) and a restricted group was fed 30% of the ad libitum intake (restricted fed group). After birth, food was available ad libitum in both groups and litter size was adjusted to eight pups per litter. Dams lost a significant amount of body weight throughout gestation due to undernutrition but were able to catch up to the ad libitum group by day 10 postnatally. Litter size was not affected by maternal undernutrition. Maternal plasma IGF-I levels were significantly reduced in the restricted fed group throughout gestation (P<0·001) but were not different postnatally. Maternal plasma IGF-binding proteins (IGFBPs)-1, -2 and -3 were significantly (P<0·05) increased in the restricted fed dams. The mean body weights of fetuses in late gestation from the restricted fed dams were significantly lower (P<0·001) in comparison with fetuses from control dams. Placental weights were also significantly (P<0·01) reduced in the restricted fed compared with control dams. Body weights were significantly lower in the offspring of restricted fed dams than control dams from birth (P<0·01) until 90 days of age (P<0·05). Nose–rump length was reduced in the fetuses of the restricted fed group at day 22 of gestation (P<0·001) until weaning (P<0·05). Plasma IGF-I levels were significantly reduced in the pups of restricted fed dams from day 22 of gestation (P<0·01) until postnatal day 9 (P<0·05) but were not significantly different at the later time-points. Plasma insulin levels were significantly reduced in the pups of restricted fed dams at birth (P<0·05) but not at later time-points. Plasma IGFBP-1 and -2 levels were significantly increased in the offspring from restricted fed dams at day 22 of gestation, at birth and at day 9 postnatally (P<0·05). 125I-Bovine GH specific binding to liver membranes was significantly lower (P<0·05) in offspring from restricted fed dams at 21 days of age but not at 90 days of age. These data demonstrate that nutritional deprivation in the pregnant rat leads to IUGR and postnatal growth failure and to changes in allometric growth patterns and endocrine parameters of the somatotrophic axis postnatally.

Journal of Endocrinology (1996) 150, 231–242
Long-term normalization of GLUT-4 protein content in skeletal muscle of diabetic rats following islet transplantation
Authors: Davalli A M , Napoli R , Weitgasser R , Capotorto J V , Hirshman M F , Finegood D T , Bonner-Weir S , Horton E S , Weir G C
Published: December 2004

Skeletal muscle GLUT-4 content is decreased in streptozotocin (STZ)-diabetic rats. This decrease is associated with impairment in glucose transport across the plasma membrane. In this study we investigated whether islet transplantation might normalize GLUT-4 content. Transplantation of syngeneic islets restored long-term near-normoglycemia in STZ-diabetic Lewis rats. Transplanted rats, followed up to 6 months, maintained slightly but significantly higher fasting and fed glucose levels when compared with age-matched normal controls. Although fasting insulin levels of transplanted rats were significantly higher than those of controls, insulin levels did not increase significantly with feeding. Plasma glucose levels following an oral glucose load (2 g/kg) were only slightly higher than in normal controls 2 months after transplantation, whereas after 6 months more severe glucose intolerance was detected. Transplanted rats completely lost the first-phase insulin release in response to i.v. glucose although they showed an increased second phase and preserved response to arginine. Six months after transplantation, endocrine beta cell mass of the grafts was similar to pretransplantation values. GLUT-4 protein content in skeletal muscle homogenates was reduced in untreated diabetic animals whereas it was completely restored by islet transplantation. In conclusion, achievement of long-term near-normoglycemia after islet transplantation was associated with complete normalization of skeletal muscle GLUT-4 content in the diabetic animals, even in the presence of abnormal glucose tolerance and an altered pattern of insulin secretion.

Journal of Endocrinology (1996) 150, 255–263