bluuebunny/crossref_metadata_2025 huggingface dataset:
Beta-cell markers and autoantigen expression by a human insulinoma cell line: similarities to native beta cells
Authors: Cavallo M G , Dotta F , Monetini L , Dionisi S , Previti M , Valente L , Toto A , Di Mario U , Pozzilli P
Published: December 2004

In the present study we have evaluated the expression of different beta-cell markers, islet molecules and autoantigens relevant in diabetes autoimmunity by a human insulinoma cell line (CM) in order to define its similarities with native beta cells and to discover whether it could be considered as a model for studies on immunological aspects of Type 1 diabetes.

First, the positivity of the CM cell line for known markers of neuroendocrine derivation was determined by means of immunocytochemical analysis using different anti-islet monoclonal antibodies including A2B5 and 3G5 reacting with islet gangliosides, and HISL19 binding to an islet glycoprotein. Secondly, the expression and characteristics of glutamic acid decarboxylase (GAD) and of GM2-1 ganglioside, both known to be islet autoantigens in diabetes autoimmunity and expressed by human native beta cells, were investigated in the CM cell line. The pattern of ganglioside expression in comparison to that of native beta cells was also evaluated. Thirdly, the binding of diabetic sera to CM cells reacting with islet cytoplasmic antigens (ICA) was studied by immunohistochemistry. The results of this study showed that beta cell markers identified by anti-islet monoclonal antibodies A2B5, 3G5 and HISL-19 are expressed by CM cells; similarly, islet molecules such as GAD and GM2-1 ganglioside are present and possess similar characteristics to those found in native beta cells; the pattern of expression of other gangliosides by CM cells is also identical to human pancreatic islets; beta cell autoantigen(s) reacting with antibodies present in islet cell antibodies (ICA) positive diabetic sera identified by ICA binding are also detectable in this insulinoma cell line.

We conclude that CM cells show close similarities to native beta cells with respect to the expression of neuroendocrine markers, relevant beta cell autoantigens in Type 1 diabetes (GAD, GM2-1, ICA antigen), and other gangliosides. Therefore, this insulinoma cell line may be considered as an ideal model for studies aimed at investigating autoimmune phenomena occurring in Type 1 diabetes.

Journal of Endocrinology (1996) 150, 113–120
Influence of insulin-like growth factor-binding protein-2 on plasma clearance and transfer of insulin-like growth factors-I and -II from plasma into mammary-derived lymph and milk of goats
Authors: Prosser C G , Schwander J
Published: December 2004

Plasma clearance of insulin-like growth factors-I and -II (IGF-I and -II) and insulin-like growth factor-binding protein-2 (IGFBP-2) from lactating goats (n=4) was determined following a single intravenous injection of the corresponding 125I-labelled human protein. Transfer of these proteins out of the vascular space was monitored by their subsequent appearance in mammary-derived lymph and milk. Clearance of 125I-IGFBP-2 from circulation was 0·37 ± 0·06 ml/min/kg, which is markedly greater than that of 125I-IGF-I or -II (0·11 ± and 0·12 ± 0·01 ml/min/kg respectively). This was also reflected in longer elimination half-lives for IGF-I (353 ± 6 min) and -II (254 ± 8 min) compared with IGFBP-2 (110 ± 9 min). Three hours after injection of the 125I-labelled protein, the plasma:lymph ratio of trichloroacetic acid-precipitable radioactivity was 1·54 ±0·04, 3·3 ±0·6 and 4·1 ±0·4 for IGFBP-2, IGF-I and -II respectively. The form of 125I-IGFBP-2 in lymph was not different from that of plasma. Elevation of plasma concentrations of IGFBP-2 by its intravenous infusion significantly decreased plasma half-life of both IGF-I and -II (251 ± 8 and 198 ±7 min respectively). Although the amount and rate of transfer of IGF into mammary-derived lymph was decreased slightly by IGFBP-2, concentrations eventually obtained were not different from control. However, secretion of IGFs into milk was significantly reduced by IGFBP-2, particularly in the case of IGF-I. These results are consistent with the ability of all three compounds to cross the vascular endothelium intact and of IGFBP-2 to decrease the uptake of IGF by mammary epithelium and subsequent secretion into milk. IGFBP-2 may well have acted to target plasma IGF towards non-mammary tissues, thus explaining the more rapid plasma clearance of IGFs in the presence of elevated IGFBP-2.

Journal of Endocrinology (1996) 150, 121–127
Cytochrome P450 17α-hydroxylase gene expression in differentiating rat trophoblast cells
Authors: Yamamoto T , Chapman B M , Johnson D C , Givens C R , Mellon S H , Soares M J
Published: December 2004

Trophoblast giant cells of the rat placenta express cytochrome P450 17α-hydroxylase (P450c17) and synthesize androgens. The purpose of this study was to investigate androgen production and expression of P450c17 in the Rcho-1 trophoblast cell line. These cells are capable of differentiating along the trophoblast giant cell lineage. Androstenedione production increased approximately 70-fold as Rcho-1 trophoblast cells progressed from the proliferation to the differentiation state. P450c17 enzyme activity and mRNA also showed significant increases associated with trophoblast giant cell differentiation. To study the transcriptional regulation of the P450c17 gene, the activities of a series of P450c17 promoter–luciferase reporter constructs were evaluated following transient transfection into Rcho-1 trophoblast cells. A DNA region located – 98 bp upstream of the P450c17 gene transcriptional start site was the shortest promoter DNA construct consistently possessing activity in Rcho-1 trophoblast cells. Activities of longer constructs (−156 to −1560 bp) in this population of cells were significantly greater than the −98 bp promoter–reporter construct. The − 476 bp P450c17 construct showed maximal promoter activity in transiently transfected Rcho-1 trophoblast cells and was developmentally activated in stably transfected Rcho-1 trophoblast cells. Activation of the cyclic AMP/protein kinase A pathway did not significantly affect P450c17 promoter activity in Rcho-1 trophoblast cells, in contrast to its effects in mouse MA-10 Leydig cells. In summary, Rcho-1 trophoblast cells are capable of endocrine differentiation and are a useful in vitro system for studying the regulation of trophoblast androgen production and P450c17 gene expression.

Journal of Endocrinology (1996) 150, 161–168
Effects of long-term food reduction on the hypothalamus-pituitary-thyroid axis in male and female rats
Authors: van Haasteren G A C , Linkels E , van Toor H , Klootwijk W , Kaptein E , de Jong F H , Reymond M J , Visser T J , de Greef W J
Published: December 2004

The reduced thyroid activity during short-term starvation is associated with a lowered hypothalamic synthesis and secretion of TRH. However, little is known about the cause of the reduced thyroid function during prolonged malnutrition. We have therefore studied the effects of food reduction to one-third of normal (FR33) on the hypothalamus-pituitary-thyroid axis of male and female Wistar rats. After 3 weeks body weights of FR33 rats were almost 50% lower than those of controls. In both sexes, FR33 caused marked increases in serum corticosterone, and decreases in serum TSH, thyroxine (T4), free T4, tri-iodothyronine (T3) and free T3. While the free T3 fraction (FFT3) in serum decreased, the free T4 fraction (FFT4) tended to increase. Electrophoretic analysis indicated that decreased FFT3 was correlated with an increased thyroxine-binding globulin, while the increase in FFT4 seemed due to a decreased thyroxine-binding prealbumin binding capacity. Total RNA and proTRH mRNA in the hypothalamus were not affected by FR33. Median eminence and posterior pituitary TRH content tended to increase in FR33 rats, suggesting that hypothalamic TRH release is reduced in FR33 rats. Anterior pituitary TSH content was decreased by FR33 in both sexes, but pituitary TSHβ mRNA and TRH receptor status were not affected except for increased pituitary TSHβ mRNA in female FR33 rats. Although FR33 had no effect on pituitary weight, pituitary RNA and membrane protein content in FR33 rats were 50–70% lower than values in controls.

In conclusion, prolonged food reduction suppresses the pituitary-thyroid axis in rats. In contrast to short-term food deprivation, the mechanism whereby serum TSH is suppressed does not appear to involve decreases in proTRH gene expression, but may include effects on pituitary mRNA translation. Our results further support the hypothesis that TSH release may be lowered by increased corticosterone secretion, although the mechanism of this effect may differ between acute starvation and prolonged food reduction.

Journal of Endocrinology (1996) 150, 169–178
Role of phosphoprotein phosphatases in the corpus luteum: II Control of progesterone secretion by isolated rat luteal cells
Authors: Abayasekara D R E , Ford S L , Persaud S J , Jones P M
Published: December 2004

The key role of protein kinases and protein phosphorylation in the regulation of luteal steroidogenesis is well documented. However the role of phosphoprotein phosphatases (PP) and dephosphorylation in the regulation of luteal cell progesterone secretion is as yet unknown. We have recently demonstrated the presence and activity of PP1 and PP2A in rat luteal cells and the present study was undertaken to determine the consequences of inhibiting PP activity in terms of progesterone secretion. Three structurally dissimilar inhibitors of PP1/2A, okadaic acid, calyculin A and cantharidin each caused a dose-dependent inhibition of LH-induced progesterone secretion without affecting cyclic AMP accumulation. The less potent derivative of okadaic acid, norokadaone, had no effect on either parameter, suggesting that the inhibitory actions on progesterone secretion are due to their specific actions on PP activity and that this inhibition occurs principally at a locus which is distal to the generation of cyclic AMP. In contrast to the inhibitory effects of PP1/2A inhibitors on progesterone biosynthesis, a PP2B inhibitor, cypermethrin, had no effect on LH-stimulated steroidogenesis. The three PP1/2A inhibitors also caused a concentration-dependent inhibition of dibutyryl cyclic AMP-stimulated progesterone secretion. However, none of the inhibitors affected 22R-hydroxycholesterol-supported steroidogenesis, clearly demonstrating that the inhibitors did not interfere with the activity of steroidogenic enzymes. These results suggest that cycles of phosphorylation/dephosphorylation of specific proteins are required for the sustained production of progesterone. Whilst the precise location and function of putative PP substrates is uncertain, the present results indicate that they are involved in regulating the availability of free cholesterol to steroidogenic enzymes within mitochondria.

Journal of Endocrinology (1996) 150, 213–221
Attenuation of human chorionic gonadotropin release by nitric oxide in choriocarcinoma cell lines
Authors:   Myat-Thanda, Histumoto Y , Saheki S , Kitagawa H , Yano J , Matsuura S
Published: December 2004

Nitric oxide (NO) is involved in the regulation of endocrine functions, but only a few studies have been reported about its role in placental hormone secretion. We investigated whether NO has any function in the release of human chorionic gonadotropin (hCG) in two different choriocarcinoma cell lines, JEG-3 and BeWo. First, nitric oxide synthase (NOS) was characterized in the choriocarcinoma cells. NOS activity was localized mainly in the particulate fraction and depended on calcium/calmodulin. Activity was inhibited by the presence of the l-arginine analog, NG-monomethyl-l-arginine (L-NMMA; 1 × 10−4 m). Western blot analysis showed that the choriocarcinoma cells contained an endothelial isoform of NOS.

The NO donor, sodium nitroprusside (SNP; 1 × 10−5 and 1 × 10−4 m), significantly inhibited hCG secretion in both choriocarcinoma cell lines. The suppression of hCG release by SNP (1 × 10−5 m) was blocked by the addition of an NO scavenger, hemoglobin (1 × 10−6 m). l-Arginine (1 × 10−2 m), a NOS substrate, inhibited basal hCG secretion in JEG-3 cells. Incubation of the cells with L-NMMA (1 × 10−4 and 1 × 10−3 m) significantly increased hCG release. Exposure of both cell lines to increasing concentrations of a cyclic GMP analog (8-bromo-cyclic GMP; 1 × 10−4 to 1 × 10−2 m) caused a dose-dependent inhibition of hCG release. Cyclic GMP accumulation in response to SNP (1 × 10−4 m), however, was not detected in either JEG-3 or BeWo cells. These data demonstrated that the endothelial isoform of NOS and a functional l-arginine-NO pathway are present in the choriocarcinoma cell lines. In addition, these findings support the hypothesis that NO produced in these cell lines is involved in the regulation of hCG secretion. We assume that although cyclic GMP is likely to play a role as a second messenger, a cyclic GMP-independent pathway cannot be excluded as a possible physiological mechanism in the attenuation of hCG release by NO.

Journal of Endocrinology (1996) 150, 243–253
Stimulation of creatine kinase specific activity in human osteoblast and endometrial cells by estrogens and anti-estrogens and its modulation by calciotropic hormones
Authors: Fournier B , Häring S , Kaye A M , Sömjen D
Published: December 2004

We have previously demonstrated sex-specific stimulation of creatine kinase specific activity (CK) in bone cells both in vivo and in vitro, in primary culture cells derived from rat and human bone and in established human bone-derived cell lines. We found that the female-derived cell line, SaOS-2, responded to 17β-estradiol (E2) by increased CK specific activity. The effects of E2 on the CK activity in SaOS-2 cells was inhibited by 100-fold excess of 4-hydroxytamoxifen (Tam) as well as by the other anti-estrogen, ICI 164,384. Tam by itself had some stimulatory effect whereas ICI 164,384 showed no estrogenic activity. We also demonstrated the estrogenic-like effect of another anti-estrogen, raloxifene (Ral), which is agonist only in the SaOS-2 osteoblast-like cells but not in the human endometrial, Ishikawa cell line. Ishikawa cells respond to E2 and to Tam by increased CK activity. In both osteoblasts and endometrial cell lines, Ral and Tam were inhibitory in the presence of E2. The effects of E2 on SaOS-2 cells are at least partially mediated by the estrogen receptor (ER) at the level of transcription as demonstrated by transient transfection experiments using the human creatine kinase promoter chloramphenicol acetyltransferase in these cells.

Pretreatment of SaOS-2 with calcitropic hormones, either 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) or human parathyroid hormone (1–34) (hPTH(1–34)) increased the stimulation of CK by E2 by 40–60% relative to E2 alone and significantly increased the sensitivity of the cells to E2 by lowering the effective hormonal dose needed for stimulation of CK by E2 by 100-fold. This stimulatory effect of pretreatment of the cells with 1,25(OH)2D3 was due to a 2·5-fold increase in the level of ER expression as measured directly by enzyme immunoassay in the SaOS-2/1 subline. The increase in the responsiveness to E2 by hPTH(1–34) was not due to an increase in ER level in the cells. We can conclude that in cell cultures as in vivo, Ral shows different effects depending on the cell type, namely estrogenic-like activity in skeletal cells but not in uterine cells. We can also conclude that as with rat-derived cells, in bone cells derived from human bone 1,25(OH)2D3 increased the sensitivity to E2 due to an increase in the number of ER in the cells, whereas PTH(1–34) augmented the response to E2 without increasing ER, by another, as yet unknown, mechanism. These studies suggest that the treatment of pathological bone disorders may be improved by combined hormone therapy.

Journal of Endocrinology (1996) 150, 275–285
Evolution of growth hormone neurosecretory disturbance after cranial irradiation for childhood brain tumours: a prospective study
Authors: Spoudeas H A , Hindmarsh P C , Matthews D R , Brook C G D
Published: December 2004

To determine the aetiopathology of post-irradiation growth hormone (GH) deficiency, we performed a mixed longitudinal analysis of 56 24 h serum GH concentration profiles and 45 paired insulin-induced hypoglycaemia tests (ITT) in 35 prepubertal children, aged 1·5–11·8 years, with brain tumours in the posterior fossa (n=25) or cerebral hemispheres (n = 10). Assessments were made before (n = 16), 1 year (n = 25) and 2 to 5 years (n = 15) after a cranial irradiation (DXR) dose of at least 30 Gy. Fourier transforms, occupancy percentage, first-order derivatives (FOD) and mean concentrations were determined from the GH profiles taken after neurosurgery but before radiotherapy (n = 16) and in three treatment groups: Group 1: neurosurgery only without DXR (n = 9); Group 2: ≥30 Gy DXR only (n = 22); Group 3: ≥30 Gy DXR with additional chemotherapy (n = 9). Results were compared with those from 26 short normally growing (SN) children.

Compared with SN children, children with brain tumours had faster GH pulse periodicities (200 min vs 140 min) and attenuated peak GH responses to ITT (24·55 (19·50–30·20) vs 8·32 (4·57–15·14) mU/I) after neurosurgery, before radiotherapy. However, spontaneous GH peaks (19·05 (15·49–23·44) vs 14·13 (9·12–21·38) mU/l), 24 h mean GH (5·01 (4·37–5·62) vs 3·98 (2·63–5·89) mU/l) and FODs (1·43 (1·17–1·69) vs 1·22 (0·88–1·56) mU/l per min) were similar. The abnormalities present before radiotherapy persisted in group 1 children at 1 year when 24 h mean GH (2·45 (1·17– 5·01) mU/l) and FODs (0·73 (0·26–1·20) mU/l per min) were additionally suppressed, although partial recovery was evident by 2 years.

With time from radiotherapy, there was a progressive increase in GH pulse periodicity (Group 2: 200 min at 1 year, 240 min at ≥2 years; Group 3: 140 min at 1 year, 280 min at ≥2 years) and a decrease in 24 h mean GH (Group 2 vs Group 3 at ≥2 years: 2·45 (1·70–3·47) vs 1·86 (1·32–2·69) mU/l) and FODs (Group 2 vs Group 3 at ≥2 years; 0·56 (0·44–0·69) vs 0·44 (0·27–0·61) mU/l per min). Initial discrepancies between measures of spontaneous and stimulated (ITT) GH peaks were lost by 2 or more years (spontaneous vs ITT; Group 2: 7·76 (5·89–9·77) vs 3·80 (0·91–15·84) mU/l; Group 3: 6·03 (4·27–8·32) vs 3·80 (0·31–46·77) mU/l).

After cranial irradiation, a number of changes evolved within the GH axis: faster GH pulse periodicities and discordance between physiological and pharmacological tests of GH secretion before irradiation gave way to a slow GH pulse periodicity, decreased GH pulse amplitude and rate of GH change (FOD) and, with time, eventual concordance between physiological and pharmacological measures. The evolution of these disturbances may well reflect differential pathology affecting hypothalamic GH-releasing hormone and somatostatin.

Journal of Endocrinology (1996) 150, 329–342
Acute inhibitory effect of excess iodide on ornithine decarboxylase in the thyroid of propylthiouracil-treated rats
Authors: Ma H-T , Reuse S , Koibuchi N , Matsuzaki S
Published: December 2004

Polyamines such as putrescine, spermidine and spermine have been thought to play an important role in thyroid growth induced by goitrogens. Reduced biosynthesis of these polyamines might play a role in the antigoitrogenic effects of excess iodide. This study was designed to examine the effect of potassium iodide (KI) on ornithine decarboxylase (ODC), a rate-limiting enzyme in the biosynthesis of polyamines. Thyroidal ODC activity, protein content and mRNA were increased in rats made hypothyroid by 10 days of propylthiouracil treatment. The increase in ODC activity was suppressed after subcutaneous injection of KI (13 mg/kg body weight); the apparent half-life of ODC activity after the treatment was estimated to be 19 min and the maximum suppression (90%) was seen 60 min after the treatment. On the other hand, administration of iodine-containing compounds including l-thyroxine, l-di-iodotyrosine, amiodarone, iopanoic acid and erythrosine showed no significant effect on ODC activity. The inhibitory effect of excess iodide was not reversed by pretreatment with dibutyryl cAMP and theophylline. The amount of immunoreactive ODC protein was reduced by iodide treatment (40%). However, the decrease was not as great as the decrease in ODC activity (90%). No significant change in thyroidal ODC mRNA content was seen 1 and 3 h following KI treatment. These results suggest that excess iodide reduces ODC activity in the rat thyroid gland by a post-transcriptional mechanism.

Journal of Endocrinology (1996) 150, 369–376
Comparison between urinary pyridinium cross-links and hydroxylysine glycosides in monitoring the effects of ovariectomy and 17β-estradiol replacement in aged rats
Authors: Pecile A , Netti C , Sibilia V , Villa I , Calori G , Tenni R , Coluzzi M , Moro G L , Rubinacci A
Published: December 2004

This study was undertaken to assess the sensitivity of hydroxylysylpyridinoline (HP), lysylpyridinoline (LP), galactosylhydroxylysine (GHyl) and glucosylgalactosylhydroxylysine (GGHyl) to monitor bone response to estrogen deficiency and replacement by comparing their excretory patterns in ovariectomized aged (11–14 months old) rats. The ovariectomized (OVX) rats were randomized into two groups: (1) OVX plus vehicle; (2) OVX plus 17β-estradiol (17-βE, 10 μg/kg, s.c., 4 days/week). Treatment with 17-βE started immediately after OVX and continued for 60 days. The collagen catabolites were measured in urine for 1 month before OVX and thereafter for 60 days. In temporal coincidence with urine collection, bone area and bone mineral density (BMD) of lumbar vertebrae, femoral diaphysis and distal metaphysis were measured by dual-energy X-ray absorptiometry. In the untreated rats, BMD of the femoral metaphysis and lumbar vertebrae decreased significantly and the urinary excretion of LP, HP, GHyl and GGHyl increased with different patterns. In the treated rats, 17-βE replacement prevented the increment in LP excretion, partially prevented the increase in HP excretion, but had no effect on the excretion of GHyl and GGHyl. In conclusion pyridinolines and glycosides have different sensitivities to the bone response to OVX. Glycoside excretion after OVX also reflects metabolic processes not strictly related to bone loss and, in contrast with LP, is not sensitive to estrogen replacement.

Journal of Endocrinology (1996) 150, 383–390
The effects of steroidal and non-steroidal ovarian hormones on pituitary responsiveness to gonadotrophin surge-attenuating factor
Authors: Byrne B , Fowler P A , Templeton A
Published: December 2004

Primary pituitary cultures from adult female rats were used to investigate the effects of steroidal (oestradiol and progesterone) and non-steroidal (inhibin, follistatin) ovarian hormones on the suppressive actions of the ovarian factor gonadotrophin surge-attenuating factor (GnSAF) in the control of gonadotrophin secretion. The source of GnSAF was a chromatographic preparation from follicular fluid containing four distinct protein bands as resolved on SDS–PAGE. Oestradiol and progesterone added alone had no effect on gonadotrophin secretion but had a wide range of effects on the suppression of both LH and FSH secretion caused by the non-steroidal factors. Oestradiol, progesterone and oestradiol+progesterone enhanced the suppressive actions of GnSAF on GnRH-induced LH secretion (causing 19·3 ± 5·2% (P<0·05), 41·9 ± 3·4% (P<0·001) and 32·2 ± 5·3% (P<0·001) greater suppression than GnSAF alone). Progesterone and oestradiol+progesterone completely abolished the suppression of basal FSH secretion caused by inhibin (causing 157·1 ± 22·2%, P<0·001, and 160·9 ± 11·3%, P<0·001, stimulation compared with inhibin alone). Separately the steroids had no effect on the suppression of gonadotrophin secretion caused by follistatin. However, in combination, oestradiol+ progesterone potentiated the suppressive actions of follistatin on GnRH-induced LH secretion causing 29·9 ± 5·3% (P<0·05) greater suppression than follistatin alone. In combination, high-dose follistatin and GnSAF caused 31·1 ±6·5% (P<0·01) greater suppression than GnSAF alone. Thus in combination high-dose follistatin and GnSAF have additive effects on the suppression of GnRH-induced LH secretion. Recombinant human inhibin and GnSAF added in combination had little further effect compared with either alone suggesting that they may have a similar mechanism of action at the pituitary level. These results demonstrate that while FSH secretion in vitro is mainly controlled by inhibin and follistatin, LH secretion is affected by the presence of a whole range of factors. We have demonstrated that oestradiol and progesterone potentiate the suppressive actions of GnSAF in vitro. These data are compatible with the suggestion that in the late follicular phase it is falling levels of GnSAF that allow positive feedback of the steroids on the pituitary to elicit the LH surge, rather than increases in the stimulatory effects of the ovarian steroids overcoming GnSAF. The actions of GnSAF on the pituitary may be modulated by follistatin but it is unlikely that inhibin has any modulatory effects on the GnSAF-induced suppression of LH secretion.

Journal of Endocrinology (1996) 150, 413–422
Luteolytic effect of LH: inhibition of 3β-hydroxysteroid dehydrogenase and stimulation of 20α-hydroxysteroid dehydrogenase luteal activities in late pregnant rats
Authors: Stocco C O , Deis R P
Published: December 2004

The mechanisms associated with the onset of luteolysis in the pregnant rat are not well known. The effect of a specific rat LH antiserum (AS-rLH) and of ovine LH (oLH) on luteal steroidogenesis on day 19 of pregnancy was examined. Rat LH antiserum administered intrabursally at 1000–1100 h on day 19 of pregnancy prevented the physiological decrease in 3β-hydroxysteroid dehydrogenase (3β-HSD) activity, the increase in 20α-hydroxysteroid dehydrogenase (20α-HSD) activity and the fall in serum progesterone (P4) level observed at 1800 h on day 21 of pregnancy. To see if oLH has a direct effect on luteal steroidogenesis, the gonadotrophin was injected into the periovarian bursa. The intrabursa treatment with 1 μg oLH on day 19 of pregnancy at 0800–0900 h did not modify corpus luteal function 36 h after treatment, but treatment with 4 μg oLH per ovary induced a significant progressive decrease in luteal 3β-HSD activity starting 12 h after treatment, while a significant increase in 20α-HSD activity, concomitant with a decrease in serum P4 level, occurred 48 h after treatment. Luteal P4 content decreased with respect to control groups 36 and 48 h after intrabursal treatment with 4 μg oLH. The intrabursal administration of 8 μg oLH induced an increase in 20α-HSD activity and a decrease in 3β-HSD activity 36 h after treatment. Administration of 4 μg oLH per ovary on day 8 of pregnancy induced a significant increase in serum P4 levels without modifying 3β-HSD activity. In rats treated with oLH on day 19 of pregnancy the decrease in 3β-HSD activity occurred 36 h before the significant increase in 20α-HSD activity and serum P4 level. In conclusion, the luteal enzymatic activity changes and the significant decrease in the intraluteal P4 concentration induced by the intrabursal administration of oLH and the clear effect of AS-rLH preventing the physiological luteal changes preceding parturition provide good evidence of an intraovarian action of LH during the normal progression of luteolysis in late pregnant rats.

Journal of Endocrinology (1996) 150, 423–429
Growth hormone binds to a single high affinity receptor site on mouse osteoblasts: modulation by retinoic acid and cell differentiation
Authors: Slootweg M C , Salles J P , Ohlsson C , de Vries C P , Engelbregt M J E , Netelenbos J C
Published: December 2004

Growth hormone (GH) exerts direct differentiative and proliferative effects on osteoblasts. We studied 125I-labeled human (h) GH binding to primary mouse osteoblasts derived from collagenase-treated 18-day fetal mouse calvaria. Scatchard analysis of the data revealed a single class of high affinity GH receptors (apparent Ka= 5·74 × 109 m−1) with 2200 sites per cell. Affinity cross-linking and SDS-PAGE electrophoresis showed two bands with apparent molecular masses of 120 and 70 kDa. Mouse osteoblasts express GH receptor mRNA with gene transcripts of 4·2 and 1·2 kb, at levels which reach approximately 1/6 of those in mouse liver and 1/3 of those in mouse muscle. Two populations of undifferentiated and diffentiated osteoblasts, obtained by sequential collagenase digestion of mouse calvaria, were used to study the relationship between osteoblastic phenotype and GH receptor expression. Although the affinity of the receptors in undifferentiated and differentiated cells was the same, the capacity was significantly higher (1·45 ± 1·0% vs 2·39 ± 0·9%, P=0·03) in differentiated cells. This stresses the specific importance of the osteoblast as a target cell for GH. The differentiating potential of the vitamin A derivative retinoic acid was subsequently used experimentally to induce differentiation in the cells. Retinoic acid increased 125I-hGH binding to preosteoblasts (153%, P=0·02). Together, these data demonstrate the presence of a high affinity GH receptor in mouse osteoblasts which is related to differentiation.

Journal of Endocrinology (1996) 150, 465–472
Relaxin stimulates atrial natriuretic peptide secretion in perfused rat heart
Authors: Toth M , Taskinen P , Ruskoaho H
Published: December 2004

Relaxin, a reproductive hormone of the insulin-like growth factor family, increases heart rate in experimental animals but its other actions on cardiac function and cellular mechanisms responsible for the positive chronotrophic effect remain unknown. We have studied the actions of human recombinant gene-2 relaxin on the release of atrial natriuretic peptide (ANP) and cardiac function (heart rate, contractile force, perfusion pressure) as well as the underlying signal transduction mechanisms by using the isolated perfused spontaneously beating rat heart preparation. The administration of relaxin into the perfusion fluid at concentrations of 1·5, 3 or 10 nm for 30 min caused a dose-dependent sustained increase in heart rate, while contractile force and perfusion pressure remained unchanged. In addition, infusion of relaxin at a concentration of 10 nm into the perfusate produced a gradual 1·5-fold increase in immunoreactive ANP (IR-ANP) secretion (from 456 ± 76 to 701 ± 124 pg/ml, F=4·5, P<0·001). The ANP secretory and chronotrophic effects of relaxin appear to involve the activation of protein kinase C, since administration of a protein kinase C inhibitor staurosporine at a concentration of 30 nm completely blocked the effect of relaxin (10 nm) on IR-ANP secretion P<0·001) and heart rate (P<0·001). A cAMP-dependent protein kinase inhibitor, H-89 (100 nm), also substantially reduced the ANP secretory effect of relaxin and attenuated the increase in heart rate during the sustained phase of the relaxin infusion (P<0·001). KN-62 (3 μm), a Ca2+/calmodulin-dependent protein kinase inhibitor, decreased the positive chronotrophic effect of relaxin (P<0·001) but did not influence significantly the effect of relaxin on IR-ANP release in isolated perfused rat heart preparation. These results provide the first evidence that relaxin stimulates the secretion of ANP from isolated perfused rat hearts. Our results also suggest that relaxin modulates ANP secretion by activation of protein kinase C and cAMP-dependent protein kinase pathways.

Journal of Endocrinology (1996) 150, 487–495
Initiation of the oestradiol-induced inhibition of pulsatile LH secretion in ewes under long days: comparison of peripheral versus central treatment and neurochemical correlates
Authors: Gallegos-Sanchez J , Picard S , Delaleu B , Malpaux B , Thiéry J C
Published: December 2004

In the ewe, the inhibition of pulsatile LH secretion by oestradiol during long days depends on dopaminergic activity and could involve amino acid transmitters. In the first experiment of the present study we observed the changes in LH secretion in ovariectomised ewes under long days immediately after subcutaneous implantation of oestradiol (peripheral treatment). In the second experiment, in order to identify the site of action of oestradiol, we observed the LH changes following intracerebral infusion of oestradiol through a microdialysis membrane (central treatment) within the preoptic area, the mediobasal hypothalamus (MBH) or the retrochiasmatic area (RCh) and measured amino acids and catecholaminergic transmitters and metabolites within the dialysates. With peripheral treatment, the amplitude, the nadir and the area under the LH pulse curve decreased within 4 to 8 h of the insertion of a subcutaneous oestradiol implant. After 18 h, the amplitude and the area under the pulses increased, as well as the intervals between pulses (from 49·9 ± 1·4 min to 75·6 ± 5·9 min). With central oestradiol treatment, LH changes were similar whatever the site of oestradiol infusion, suggesting either multiple sites of action or diffusion between structures. Twenty hours after the beginning of intracerebral oestradiol treatment, the amplitude and the area under the pulses increased, as did the interval between LH pulses (from 49·5 ± 4·1 min to 73·2 ± 14·2 min). Comparison of peripheral with central oestradiol treatment suggested that the long-lasting decrease in the nadir, as well as the transitory decrease in the amplitude and area, before 18 h in experiment 1 are reflections of hypophysial effects. In contrast, the increases in amplitude and area under the LH pulse curve seen 18–20 h after oestradiol in the two experiments could be due to the higher amplitude of LHRH pulses, as a result of an early stimulatory effect of oestradiol. After central oestradiol infusion, there was a decline in the concentration in the dialysate of two metabolites of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid in the RCh, suggesting an early inhibition of monoamine oxidase by the steroid. During the inhibition of LH pulsatility the concentration of γ-aminobutyric acid in the dialysate from the RCh and the MBH increased, suggesting the participation of this transmitter in the changes induced by oestradiol under long days.

Journal of Endocrinology (1996) 151, 19–28
Role of volume status in vasopressin-induced natriuresis: studies in Brattleboro rats
Authors: Walter S J , Tennakoon V , McClune J A , Shirley D G
Published: December 2004

The influence of volume status on the effect of physiological doses of vasopressin on sodium excretion was assessed in anaesthetized Brattleboro rats. Following a 1 h control period, animals were divided into four groups. Group 1 (control) rats were kept in water balance throughout (by adjustment of the rate of i.v. glucose infusion) and received no vasopressin. In group 2 rats, vasopressin (20 μU/min) was infused i.v. for 2 h, then withdrawn during the following 2 h; the vasopressin-induced antidiuresis and subsequent return to water diuresis were matched by appropriate changes in the i.v. infusion, thus maintaining water balance. In this group, vasopressin had no effect on sodium excretion. Group 3 rats received the same dose of vasopressin, but the infusion rate of the glucose solution was not reduced; consequently these rats became water-loaded. In this group, sodium excretion increased significantly during vasopressin infusion, and rapidly returned to baseline values when the vasopressin was discontinued. Group 4 rats were treated in the same way as group 3 animals except that the vasopressin infusion was maintained (but without additional water loading) for a further 2 h; this did not prevent the fall in sodium excretion during the final 2 h of the experiment. We conclude that the natriuretic effect of physiological levels of vasopressin reported elsewhere may be dependent on an accompanying acute volume expansion during infusion of the hormone.

Journal of Endocrinology (1996) 151, 49–54
Induction of superovulation by immunoneutralization of endogenous inhibin through the increase in the secretion of follicle-stimulating hormone in the cyclic golden hamster
Authors: Kishi H , Okada T , Otsuka M , Watanabe G , Taya K , Sasamoto S
Published: December 2004

The present study was conducted to study the effect of immunoneutralization against endogenous inhibin on FSH, LH, oestradiol-17β and progesterone secretion and to investigate the effect of removal of endogenous inhibin on subsequent follicular development in the hamster. After treatment with anti-inhibin serum (inhibin-AS) at 1100 h on day 2 of the oestrous cycle (day 1=day of ovulation), a marked increase in plasma levels of FSH and a slight increase in plasma levels of LH were noted and pituitary contents of FSH, but not LH, were also increased. In the group treated with inhibin-AS, superovulation occurred on day 1 of the following cycle. Plasma levels of oestradiol-17β markedly increased with the increase in the number of ovulations induced by human chorionic gonadotrophin (hCG) as compared with those in control animals. In the second cycle, plasma concentrations and pituitary contents of FSH in the animals given 200 μl inhibin-AS still showed high values as compared with those in the animals treated with control serum, although superovulation did not occur on day 1 of the third cycle. Plasma concentrations and pituitary contents of LH in the hamster given 200 μl inhibin-AS tended to decrease as compared with those in control animals during the second cycle. Plasma concentrations of oestradiol-17β in the animals treated with 200 μl inhibin-AS changed in a similar way to controls. A marked increase in plasma concentrations of progesterone was noted on days 1 and 2 of the second cycle in the group receiving inhibin-AS. The twice daily injection of 1 IU hCG during the second cycle to the animals given 200 μl inhibin-AS induced superovulation on day 1 of the third cycle.

These results indicate that circulating inhibin may be an important indicator of the number of developing follicles and may maintain the species-specific number of developing follicles through suppression of FSH secretion in the cyclic hamster. They also suggest that high levels of inhibin slightly suppress plasma levels of LH, indicating that plasma LH may also regulate follicular development in the cyclic hamster.

Journal of Endocrinology (1996) 151, 65–75
Transforming growth factor β in bovine milk: concentration, stability and molecular mass forms
Authors: Rogers M-L , Goddard C , Regester G O , Ballard F J , Belford D A
Published: December 2004

Transforming growth factor β (TGF-β) is one of the predominant growth factors present in milk. The concentration, molecular mass forms and stability of TGF-β in bovine milk were investigated using a standard bioassay measuring the growth inhibition of a mink lung epithelial cell line. Most of the TGF-β bioactivity in milk was found to be in a latent form, which was also retained in the whey fraction. After acid activation, the total TGF-β concentration was 4·3 ± 0·8 ng and 3·7 ± 0·7 ng TGF-β per ml of milk and cheese whey respectively. Cation-exchange chromatography at pH 6·5 was used to concentrate latent whey-derived TGF-β, which could be activated by transient exposure to extremes of pH, urea or heat. Heparin did not significantly activate milk-derived TGF-β. Neutral gel filtration of the cationic whey fraction revealed a major peak of latent TGF-β with a molecular mass of 80 kDa and a smaller peak at 600 kDa. Transient acidification of the cationic whey fraction prior to neutral gel filtration, or gel filtration under acidic conditions, released low molecular mass TGF-β from both high molecular mass peaks. Whey-derived TGF-β was purified using a five-step chromatographic procedure. An N-terminal sequence was obtained for TGF-β2, which accounted for over 85% of the TGF-β bioactivity in whey. All TGF-β activity in whey could be neutralised by a monoclonal antibody directed against TGF-β1, -β2 and -β3. The results suggest that the majority of TGF-β in bovine milk is present in a small latent complex.

Journal of Endocrinology (1996) 151, 77–86
Effects of somatostatin-28 on circulating concentrations of insulin and gut hormones in sheep
Authors: Martin P A , Faulkner A
Published: December 2004

The effects of intravenous somatostatin-28 (S28) infusion on glucose-stimulated and glucagon-like peptide-1(7–36)amide (GLP-1)-augmented insulin secretion were studied in sheep. S28 was infused via a jugular catheter for 15 min at a rate of 1·1 pmol/kg/min either alone or together with GLP-1 and/or glucose. S28 infusion did not significantly lower circulating basal insulin concentrations in fed sheep. Glucose-stimulated insulin secretion was significantly inhibited by S28 infusion, serum concentrations decreasing from about 200 to 150 pmol/l. GLP-1 significantly augmented glucose-stimulated insulin secretion, serum concentrations increasing from about 230 to 280 pmol/l. S28 completely counteracted this effect of GLP-1. S28 infusion also significantly decreased the circulating concentrations of glucose-dependent insulinotrophic polypeptide (GIP) and GLP-1 in fed sheep (from about 110 to 45 pmol/l for GIP and from about 25 to 15 pmol/l for GLP-1). The physiological implications of these observations are discussed with particular reference to the ruminant. It is concluded that S28 may have an important endocrine role in the control of insulin secretion and regulation of nutrient partitioning.

Journal of Endocrinology (1996) 151, 107–112
Cellular receptors for sex steroids in human pituitary adenomas
Authors: Jaffrain-Rea M L , Petrangeli E , Ortolani F , Fraioli B , Lise A , Esposito V , Spagnoli L G , Tamburrano G , Frati L , Gulino A
Published: December 2004

Cellular receptors for sex steroids (SSRs) were studied in an unselected series of 55 human pituitary tumors. Cytosolic receptors for estrogen (ERcs) and progesterone (PgRcs) were determined in all cases and cytosolic androgen receptors (ARcs) in 47 cases. Nuclear receptors (ERns, PgRns, ARns) were also studied in 33 cases. ERs and PgRs were determined by an ELISA and ARs by [3H]methyltrienolone binding. Where both cytosolic and nuclear receptors were studied (n=33), ERs, PgRs and ARs were found in at least one subcellular fraction in 66·7, 60·6 and 81·8% of cases respectively, ERs and ARs being mainly recovered from the cytosol and PgRs from the nucleus. No linear correlation was found between preoperative plasma steroid hormones and their specific cellular receptors. Nonetheless, the differential expression of SSRs according to sex and gonadal status at the time of surgery strongly supports their regulation by the steroid environment in vivo: PgRcs were more frequent in tumors found in women (41·4 vs 15·4%, P<0·05), whereas a high expression of ERcs and ARcs (>15 fmol/mg protein) was more common in tumors found in men (34·5 vs 10·3%, P<0·05 and 54·5 vs 24·0% respectively). PgRs were positively correlated with ERns, indicating the possibility of estrogen priming of their expression, and negatively correlated with ARs in nuclear fractions. SSRs appeared to be widely distributed among pituitary tumors, although, compared with other hormone-secreting groups, prolactinomas displayed a higher ERc expression (34·8 ± 11·3 vs 4·8 ± 5·1 fmol/mg protein, P=0·007) and gonadotroph cell adenomas lower ARc values (1·3 ± 0·8 vs 38·2 ± 10·6 fmol/mg protein, P=0·048). Microadenomas were characterized by a higher PgR expression than macroadenomas, whereas hemorrhagic (macro)adenomas were characterized by a high ER expression (>90%). The present results indicate that most pituitary tumors are targets for sex steroids, SSR expression being partially triggered by the steroid environment itself. Possible physiopathological and therapeutic implications of these findings are discussed.

Journal of Endocrinology (1996) 151, 175–184
Inhibitory effect of dihydrotestosterone on human thyroid cell growth
Authors: Rossi R , Zatelli M C , Franceschetti P , Maestri I , Magri E , Aguiari G , Cavazzini P , degli Uberti E C , del Senno L
Published: December 2004

Sex steroid-binding activities have been identified by several authors in normal and pathological thyroids and the expression of the canonic androgen receptor (AR) has recently been demonstrated in human thyroid follicular cells.

In order to assess what influence, if any, androgen exposure has on thyroid cell growth, the effect of dihydrotestosterone (DHT) on [3H]thymidine (thy) incorporation and cell proliferation was investigated in thyroid follicular cells in vitro. In a primary culture of goitrous cells, DHT induced a significant reduction of [3H]thy incorporation at concentrations ranging from 10−12 to 10−8 m, with a more pronounced effect at 10−9 m. At this concentration, the inhibitory effect was evident after both 24 and 48 h of treatment and in various types of primary thyroid cell cultures. In goitrous cells, the DHT-induced decrease of [3H]thy was associated with a reduction of expression of the proliferation-associated nuclear Ki-67 antigen, a protein commonly used to assess cell growth fraction. In TPC cells, an AR-positive thyroid papillary carcinoma cell line, DHT at concentrations between 10−12 and 10−8 m significantly decreased the growth rate. DHT (10−9 m) produced an approximately 50–60% inhibition of cell proliferation and the antiandrogen cyproterone acetate was capable of reversing such effects. The DHT-induced reduction of TPC cell proliferation was associated with a significant reduction of c-myc RNA levels. Thyroperoxidase mRNA levels and thyroglobulin production were not reduced by androgen in primary cultures of goitrous cells.

In conclusion, our results indicated that androgens may have a role in this gland by reducing the proliferation, but not the function, of follicular cells.

Journal of Endocrinology (1996) 151, 185–194
Role of the neuronal histaminergic system in the regulation of somatotropic function: comparison between the neonatal and the adult rat
Authors: Grilli R , Sibilia V , Torsello A , Pagani F , Guidi M , Luoni M , Netti C , Müller E E
Published: December 2004

To study possible age-related differences in the role of neuronal histaminergic pathways in the control of GH secretion, the effects of α-fluoromethylhistidine (α-FMH), an irreversible inhibitor of histamine (HA) synthesis, were examined on basal and opioid-induced GH release in neonatal and adult rats. The mechanisms involved in such effects were evaluated by measuring pituitary GH mRNA levels and hypothalamic levels of GH-releasing hormone (GHRH) and somatostatin (SRIF) mRNAs. Daily injection of α-FMH (20 mg/kg, s.c.) in pups of either sex, from birth until 10 days of age, caused a significant increase in baseline plasma GH and potentiated the GH response to the [Met5]-enkephalin analog FK 33–824 (1 mg/kg, s.c.) administered 3 h after the last α-FMH injection. GH and SRIF mRNA levels were significantly higher in α-FMH-treated pups than in controls, whereas no difference was observed in GHRH mRNA levels. In young adult male rats, acute administration of α-FMH (100 mg/kg, s.c., 3 h before) did not change significantly basal GH levels but potentiated FK 33–824 (0·3 mg/kg, intracarotid)-induced stimulation of GH secretion. Repeated administration of α-FMH (200 μg/rat, i.c.v., for 3 days) failed to modify basal and FK 33–824-induced GH secretion, caused a significant reduction in hypothalamic GHRH mRNA levels and left SRIF and GH mRNAs unchanged.

These findings indicate that HA exerts an inhibitory effect on GH secretion in both neonatal and adult rats. The different effects of short-term HA depletion on hypothalamic and pituitary indices of somatotropic function observed at the two age periods may be ascribed to the immaturity of the HA system in early postnatal life and to a different functional role of GH-regulatory factors during ontogeny.

Journal of Endocrinology (1996) 151, 195–201