bluuebunny/crossref_metadata_2025 huggingface dataset:
Altered blood pressure profile, autonomic neuropathy and nephropathy in insulin-dependent diabetic patients
Authors: Monteagudo Patricia T , Nóbrega João C , Cezarini Paulo R , Ferreira Sandra RG , Kohlmann Oswaldo , Ribeiro Artur B , Zanella Maria-Teresa
Published: December 2008

Monteagudo PT, Nóbrega JC, Cezarini PR, Ferreira SRG, Kohlmann Jr O, Ribeiro AB, Zanella M-T. Altered blood pressure profile, autonomie neuropathy and nephropathy in insulin-dependent diabetic patients. Eur J Endocrinol 1996;135:683–8. ISSN 0809–4643

To evaluate the relationship between autonomie neuropathy (AN) and nephropathy we measured 24-h blood pressure (BP) and overnight urinary albumin excretion (UAE) in 38 patients with insulin dependent diabetes mellitus (IDDM). Autonomie function was evaluated by the heart rate response to deep breathing. Valsalva maneuver, heart rate at rest and BP variation with posture. Sympathetic cutaneous reflex was also tested in both inferior and superior limbs. Patients with mean day diastolic BP (DDBP) ⩽ 90 mmHg without AN (N = 15) compared to 12 normal controls had similar BP values, but compared to those with DDBP ⩽90 mmHg and AN (N = 12) they had lower night diastolic BP (NDBP) (66 ± 4.8 vs 72 ± 8.8 mmHg; p < 0.05) and UAE (9.8 ± 2.3 vs 107.2 ± 3.5 μg/min; p < 0.001). No difference in DDBP was observed between these two diabetic groups (80 ± 3.9 vs 83 ± 6.1 mmHg). Of the 11 patients with DDBP > 90 mmHg, only three were free of AN and only two of the eight with AN where free of diabetic nephropathy. The percentage day/night changes in systolic BP were lower in patients with AN (13 vs 7.9%; p < 0.05) and were inversely related to autonomie score, used as an index of the degree of autonomie dysfunction (r = −0.48; p < 0.01) and to UAE (r = −0.39; p < 0.05). Furthermore, UAE correlated with autonomie score (r = 0.69; p < 0.0001) and with NDBP (r = 0.44; p < 0.01). Our results show that AN in IDDM patients is associated with a reduced nocturnal fall in BP and suggest a pathogenic role of autonomie dysfunction in the development of diabetic nephropathy, possibly favoring both BP elevation during the night and increases in intraglomerular pressure.

Maria Teresa Zanella, Disciplina de Endocrinologia, Escola Paulista de Medicina, Rua Botucatu 720, Vila dementino, São Paulo, Brazil
Do insulin-like growth factor binding proteins (IGFBPs) modulate the IGF-I growth promoting and differentiating effects in human neuroblastoma cells?
Authors: Cianfarani Stefano , Germani Daniela , Rossi Paola , Spagnoli Anna , Mercanti Delio
Published: December 2008

Cianfarani S, Germani D, Rossi P, Spagnoli A, Mercanti D. Do insulin-like growth factor binding proteins (IGFBPs) modulate the IGF-I growth promoting and differentiating effects in human neuroblastoma cells? Eur J Endocrinol 1996;135:716–23. ISSN 0804–4643

The insulin-like growth factors (IGFs) are known to stimulate both the proliferation and differentiation of neuroblastoma cells, but the role of the IGF binding proteins (IGFBPs) has not yet been established. In this study, human neuroblastoma SH-SY5Y cells have been treated with IGF-I and its potent analogue des (1–3) IGF-I alone or following preincubation with a differentiating agent such as 12-o-tetradecanoylphorbol-13-acetate (TPA). Cell proliferation and differentiation were evaluated. Conditioned medium was tested for the presence of IGFBPs by ligand blotting. The SH-SY5Y cell proliferation was maximally stimulated by des (1–3) IGF-I. The TPA-induced differentiation of SH-SY5Y, evaluated by assessment of cell morphology and GAP-43 expression as a biochemical marker of differentiation, was potentiated by nanomolar concentrations of des (1–3) IGF-I and, to a smaller extent, IGF-I. Conditioned medium showed the presence of a major IGFBP band with an approximate molecular weight of 32.5 kD and a very faint band of approximately 24 kD. The IGFBP immunoblotting results suggest that the predominant band might represent IGFBP-2. Our data represent a first demonstration of the presence of IGFBPs in conditioned medium of human neuroblastoma SH-SY5Y cells. The finding that the potent IGF-I analogue des (1–3) IGF-I with reduced affinity for IGFBPs induce major effects on cell growth and differentiation suggests that the IGFBPs may play an active role in the neuronal response to the proliferative and differentiative effects of IGF-I.

Stefano Cianfarani, Laboratory of Paediatric Endocrinology, Department of Paediatrics, "Tor Vergata" University, via di Tor Vergata 135, 00133 Rome, Italy
Infrequent mutations of p16INK4A and p15INK4B genes in human pituitary adenomas
Authors: Yoshimoto Katsuhiko , Tanaka Chisato , Yamada Shozo , Kimura Takehiko , Iwahana Hiroyuki , Sano Toshiaki , Itakura Mitsuo
Published: December 2008

The p16INK4A and p15INK4B genes on chromosome 9p21 encode the p16 and p15 inhibitors of cyclin D/cyclin-dependent kinase 4 complexes respectively. Mutations and deletions of the p16INK4A gene have been found in melanomas and many other types of tumors. To assess the role of the p16INK4A and p15INK4B genes in tumorigenesis of the pituitary gland, 31 sporadic pituitary adenomas and 2 pituitary adenomas in familial acrogigantism were examined for loss of heterozygosity on 9p21–22 and screened for mutations in the p16INK4A and p15INK4B genes. To identify pituitary adenomas which had lost which had lost 9p21–22, pituitary adenomas were genotyped with markers flanking the p16INK4A and p15INK4B loci. The frequency of mutations in coding regions of the p16INK4A and the p15INK4B genes in pituitary adenomas was determined with polymerase chain reation-single strand conformation polymorphism analysis and sequencing of variants. Of the 33 pituitary adenomas, two revealed loss of 9p21–22 sequences, but none of them had tumor-specific mutations. We conclude that mutations of the p16INK4A and p15INK4B genes are not required for tumorigenesis of the pituitary gland.

European Journal of Endocrinology 136 74–80
The stimulatory effect of endothelin-1 on regenerating adrenal cortex is reversed by nifedipine
Authors: Zieleniewski Wojciech
Published: December 2008

Endothelin-1 (ET-1), a potent vasoconstrictor, was found to act in non-vascular tissues, for example it enhanced aldosterone output from adrenal zona glomerulosa. As the adrenal cortex is capable of regeneration after enucleation, it seemed of interest to study the effects of ET-1 on adrenocortical regeneration. The study was performed on adult rats subjected to left adrenal enucleation combined with contralateral adrenalectomy. Mitotic index was employed to assess the proliferation of regenerating adrenal cortex cells. Plasma corticosterone was measured by a standard RIA kit. ET-1 significantly raised the mitotic index of regenerating rat adrenal cortex by six days after surgery. On the other hand, nifedipine reduced the proliferation ratio and abolished the stimulatory influence of ET-1. Similarly, ET-1 enhanced corticosterone output from the regenerating adrenal cortex, and this could be prevented by the addition of nifedipine.

This study has shown that ET-1 might act as a regulatory factor on the regenerating adrenal cortex via calcium channels.

European Journal of Endocrinology 136 121–122
True hermaphroditism: clinical aspects and molecular studies in 16 cases
Authors: Damiani Durval , Fellous Marc , McElreavey Ken , Barbaux Sandrine , Barreto Elenilde S A , Dichtchekenian Vaê , Setian Nuvarte
Published: December 2008

Although true hermaphroditism (TH) accounts for less than 10% of intersex patients, it stands as a diagnostic challenge and has allowed a better understanding of the mechanisms involved in sexual differentiation. In this paper we review the clinical and laboratory data as well as molecular biology findings on 16 TH patients followed up at the Pediatric Endocrine Unit, Instituto da Criança, Hospital das Clínicas, São Paulo University Medical School. They were of a mean age of 3 years 8 months and nine of them were black. All the patients had ambiguous external genitalia as the main complaint. The 46,XX karyotype accounted for 50% of the cases and the ovotestis was the most frequent gonad found (59%). In the eight TH patients with a 46,XX karyotype, the sex-determining region of the Y chromosome (SRY) was negative, posing an intriguing question about the testicular differentiation mechanisms involved in these cases. In 7/19 ovotestes, the ovarian portion of the gonad has been preserved, keeping open the possibility of fertility. The female sex option was made in 10/16 cases (62·5%) and three patients exhibited spontaneous puberty. The mechanism through which testicular tissue develops without SRY has not yet been completely clarified, suggesting the involvement of the X chromosome as well as autosomal genes in the process.

European Journal of Endocrinology 136 201–204
Mechanism of angiotensin II-induced arachidonic acid metabolite release in aortic smooth muscle cells: involvement of phospholipase D
Authors: Shinoda Junji , Kozawa Osamu , Suzuki Atsushi , Watanabe-Tomita Yasuko , Oiso Yutaka , Uematsu Toshihiko
Published: December 2008

In a previous study, we have shown that angiotensin II (Ang II) activates phosphatidylcholinehydrolyzing phospholipase D due to Ang II-induced Ca2+ influx from extracellular space in subcultured rat aortic smooth muscle cells. In the present study, we have investigated the role of phospholipase D in Ang II-induced arachidonic acid (AA) metabolite release and prostacyclin synthesis in subcultured rat aortic smooth muscle cells. Ang II significantly stimulated AA metabolite release in a concentration-dependent manner in the range between 1 nmol/l and 0·1 μmol/l. d,l-Propranolol hydrochloride (propranolol), an inhibitor of phosphatidic acid phosphohydrolase, significantly inhibited the Ang II-induced release of AA metabolites. The Ang II-induced AA metabolite release was reduced by chelating extracellular Ca2+ with EGTA. Genistein, an inhibitor of protein tyrosine kinases, significantly suppressed the Ang II-induced AA metabolite release. 1,6-Bis-(cyclohexyloximinocarbonylamino)-hexane (RHC-80267), a potent and selective inhibitor of diacylglycerol lipase, significantly inhibited the Ang II-induced AA metabolite release. Both propranolol and RHC-80267 inhibited the Ang II-induced synthesis of 6-keto-prostaglandin F1α, a stable metabolite of prostacyclin. The synthesis was suppressed by genistein. These results strongly suggest that the AA metabolite release induced by Ang II is mediated, at least in part, through phosphatidylcholine hydrolysis by phospholipase D activation in aortic smooth muscle cells.

European Journal of Endocrinology 136 207–212
Influence of prolonged fasting and thyroid hormone on the α2-adrenergic response in isolated epididymal adipocytes of Wistar rats
Authors: Cheikh Rajae Ben , Chomard Philippe , Beltramo Jean Luc , Autissier Nicole , Dumas Paul
Published: December 2008

Fasting and thyroid hormone have been reported to modulate the β-adrenergic pathway of lipolysis in rat, but their effects on the α2-adrenergic response are not well known. The purpose of the present study was to investigate this point. Male Wistar rats, 3 weeks old, were thyroidectomized surgically, kept for 1 month at 25 °C and then fasted or not fasted for 3 days with or without daily intraperitoneal injection of 3,5,3′-tri-iodo-l-thyroni(T3; 4·6 nmol/100 g body weight). Age-matched, sham-operated, fed and fasted euthyroid rats were used as controls. The experiments were carried out using isolated epididymal adipocytes. The α2-adrenergic agonist UK 14304 (UK) inhibited the stimulated lipolysis more in fed than in fasted euthyroid rats whereas it had no effect in hypothyroid or T3-treated hypothyroid rats. The α2-adrenergic antagonist idazoxan reversed the antilipolytic effect of UK more in fasted than in fed euthyroid rats. The α2-adrenergic antagonist RX 82 1002 (RX) did so, but at lower concentrations than those of idazoxan. Idazoxan slightly increased the glycerol release in hypothyroid and especialiy T3-treated hypothyroid rats. RX had practically no effect on the production of glycerol in these animals. The findings suggest that (a) fasting and probably hypothyroidism decrease the α2-adrenergic response in adipocytes from rats, (b) T3 treatment of hypothyroid rats has no effect on the α2 response, and (c) thyroid hormone does not directly modulate the α2-adrenergic response in rat adipocytes.

European Journal of Endocrinology 136 223–229
Decreased serum IGF-I and dehydroepiandrosterone sulphate may be risk factors for the development of reduced bone mass in postmenopausal women with endogenous subclinical hyperthyroidism
Authors: Földes János , Lakatos Péter , Zsadányi Júlia , Horváth Csaba
Published: December 2008

Postmenopausal women with endogenous subclinical hyperthyroidism seem to have reduced bone mass, which does not correlate with serum thyroid hormone levels. Relative insufficiencies of IGF-I and dehydroepiandrosterone sulphate (DHEAS) might be additional risk factors for low bone density in these patients. We measured IGF-I, IGF-binding protein-3 (IGFBP-3) and DHEAS levels together with bone mineral density (BMD) of the femoral neck and lumbar spine in women with an autonomously functioning thyroid nodule. Sixty-three women were classified as subclinical hyperthyroid (31 pre- and 32 postmenopausal) and 39 as overt hyperthyroid (16 pre- and 23 postmenopausal) and results were compared with data obtained from 41 age-matched euthyroid healthy women. In premenopausal women BMD was reduced only in the overt hyperthyroid group, and only in the spine, to 92% (P < 0·05). Serum IGF-I as well as IGFBP-3 were increased in the manifest hyperthyroid group, to 157% (P < 0·001) and 129% (P < 0·05) respectively, whereas DHEAS levels did not change in either premenopausal patient group. In postmenopausal women BMD was significantly reduced both in the subclinical hyperthyroid group (spine to 90% and femoral neck to 88%; P < 0·05), as well as in the hyperthyroid group (spine to 78% and femoral neck to 86%; P < 0·01). In contrast to premenopausal women, serum IGF-I and IGFBP-3 did not change in the two groups who were postmenopausal and serum DHEAS levels were reduced to 58% (P < 0·001) in both postmenopausal groups with subclinical as well as overt hyperthyroidism. In the same two groups of patients, serum IGF-I and DHEAS levels correlated with BMD (femoral neck; both r = 0·50, P < 0·05). In conclusion, women with a solitary autonomous thyroid nodule with subclinical hyperthyroidism have reduced BMD only if they are postmenopausal. This is probably due to the effect of subtle increases in thyroid hormone production together with lack of oestrogen protection of the skeleton. But additional risk factors for the development of enhanced bone loss might be a state of relative IGF-I and DHEAS insufficiency in these patients as well as in postmenopausal women with overt hyperthyroidism.

European Journal of Endocrinology 136 277–281
Trimethoprim/sulfamethoxazole-induced hypoglycemia in a malnourished patient with severe infection
Authors: Hekimsoy Zeliha , Biberoǧlu Sevinç , Çömlekçi Abdurrahman , Tarhan Oktay , Mermut Cem , Biberoǧlu Kadir
Published: December 2008

Hypoglycemia resulting from the combination of sulfonylurea and sulfonamides is a recognized drug interaction. Hypoglycemia induced by sulfonamides alone may be encountered less frequently. Because of their structural similarities to sulfonylureas. sulfonamides are liable to facilitate hypoglycemia by increasing insulin release in susceptible individuals. Sulfonamides can potentiate the hypoglycemic effect of sulfonylurea agents when given in combination. We describe a malnourished patient with severe infection who developed hypoglycemia during high-dose trimethoprim/sulfamethoxazole therapy. Elevated C-peptide concentrations during the hypoglycemic episode indicate that hypoglycemia resulted from increased endogenous insulin secretion. As malnourished patients are prone to hypoglycemia, we suggest that they should be monitored carefully if they are on sulfonamide therapy.

European Journal of Endocrinology 136 304—306
Extrarenal aspects of angiotensin II function
Authors: Csikós Tamás , Gallinat Stefan , Unger Thomas
Published: December 2008

The cloning of angiotensin II (Ang II) receptor genes and the availability of specific receptor ligands allows characterization of Ang II receptor-mediated actions. Most of the well-known Ang II effects such as vasoconstriction, drinking response and cell proliferation are mediated through the AT1 receptor. Little is known about the physiological effect of the AT2 receptor, though there are some reports describing the involvement of the AT2 receptor in blood pressure regulation. Recent data demonstrate that the AT2-mediated actions are inhibitory to AT1- and mitogen-induced growth effects, indicating a balancing mechanism for Ang II-controlled mechanisms. It has also been demonstrated that AT2 receptor inactivation induces endothelial cell proliferation in the presence of Ang II. Additionally, AT2 receptor activation enhances nerve growth factor-induced differentiation of PC12W cells and a role in apoptotic changes has also been reported.

Based on recent findings, this article focuses on the role of Ang II in growth and differentiation processes with respect to the AT2 receptor in these events.

European Journal of Endocrinology 136 349–358
Molecular analysis of the RET proto-oncogene in patients with sporadic medullary thyroid carcinoma: a novel point mutation in the extracellular cysteine-rich domain
Authors: Bugalho Maria João , Frade João Pedro , Santos Jorge Rosa , Limbert Edward , Sobrinho Luis
Published: December 2008

Germline point mutations in the RET proto-oncogene are associated with multiple endocrine neoplasia type 2 (2A and 2B) and familial medullary thyroid carcinoma. On the other hand, somatic point mutations of RET have been described in a subset of sporadic medullary thyroid carcinomas (MTCs).

We examined tumor and blood DNA of thirteen apparently sporadic MTC patients for mutations in RET exons 10, 11, 13, 15 and 16 to determine whether they had true sporadic tumors or either de novo or occult germline mutations.

Three different somatic missense mutations were documented in seven patients. In five patients a mutation in exon 16, codon 918, (ATG→ACG) causing a Met→Thr substitution was found. In the remaining two patients the mutation affected exon 11: codon 630 in one case and codon 634 in the other. In both cases a T→C transversion was identified causing a Cys→Arg substitution.

In conclusion, absence of a germline mutation in RETexons 10, 11, 13 or 16 is evidence against an inherited form in all cases. In seven patients, identification of a somatic mutation supported the previous clinical diagnosis of sporadic medullary thyroid carcinoma; in one of them we identified a hitherto undescribed somatic point mutation at codon 630.

European Journal of Endocrinology 136 42 3–426
Improvement of cytochrome P450c17 catalytic processivity as a novel mechanism to attenuate hormonal consequences of enzyme decay in the testes after a gonadotropin surge
Authors: Kühn-Velten W Nikolaus
Published: December 2008

Objective: The aim of this study was to gain understanding of the apparent discrepancy between the moderate restriction of testosterone synthesizing capacity and the nearly complete decay of the androgen-producing enzyme, cytochrome P450c17 (CYP17; steroid 17α-hydroxylase/17,20-lyase), in rat testes during the desensitization phase induced by a single, high-dose gonadotropin (human chorionic gonadotropin, hCG) injection.

Design and methods: Adult male rats received 25IU hCG i.v., and purified Leydig cells and crude interstitial cell microsomes were prepared 0, 4, 12, 24, 48, 72, 120 and 192 h afterwards. Component CYP17 activities, i.e. simultaneously catalyzed productive androgen formation and abortive 17α-hydroxyprogesterone release, and their ratio (processivity), were compared with CYP17 levels and testosterone secretion rates.

Results: Leydig cells isolated 48 h after the artificial hCG surge produce 62% less testosterone than control cells upon stimulation in vitro, though CYP17 levels are reduced by 97%. Its total activity decreases by 87%, resulting in a 4·5-fold rise in the turnover number; the processivity is additionally improved 5-fold over controls. Parallel changes occur in interstitial cell microsomes; a negative linear correlation exists between the ratio of productive over total CYP17 activities and the actual CYP17 concentrations. CYP17 is partly denatured to P420 during hCG action, but other heme proteins (cytochrome b5) remain unchanged. Animal treatment with estradiol results in CYP17 downregulation without any concomitant effect on enzyme processivity.

Conclusion: Improved CYP17 processivity is suggested to be the consequence of (otherwise ratelimiting) improved electron transfer efficiency towards CYP17. It explains the relatively high testosterone secretion during Leydig cell desensitization and is interpreted to be a protective mechanism to confine adverse consequences of enzyme decay.

European Journal of Endocrinology 136 438–443
The revised 8307 base pair coding sequence of human thyroglobulin transiently expressed in eukaryotic cells
Authors: van de Graaf Simone A R , Pauws Erwin , de Vijlder Jan J M , Ris-Stalpers Carrie
Published: December 2008

We developed a transient transfection system for human thyroglobulin (TG) cDNA in both human thyroid cells and in COS-1 cells.

Four overlapping TG cDNA fragments were amplified by reverse transcription-PCR from RNA of normal thyroid tissue. The most 5′ fragment includes the natural translation initiation site and the sequence encoding the signal peptide (SP). After subcloning, the nucleotide sequence was determined and compared with the published human sequence, resulting in the detection of 30 nucleotide variations. For validation purposes, all variations were screened in 6–12 normal human alleles. Twenty-one were present in all screened alleles and have to be revised in the published nucleotide sequence. Since one variation concerns a triplet insertion, the coding sequence of the mature human thyroglobulin is 8307 nucleotides encoding 2750 amino acids.

The TG cDNA constructs were transiently transfected in HTori 3 and COS-1 cells and protein expression was detected using a polyclonal anti-human-TG on fixed cells and after SDS-PAGE. In both cell-lines all four TG protein fragments were expressed. The mannose structures detected on the proteins by lectins and localization after expression in the cells suggest that only the N-terminal TG fragment (containing the SP) is directed to the endoplasmatic reticulum but is unable to reach the Golgi complex.

The described expression system in human thyrocytes will be a helpful tool in studying the structure–function relationship of human TG in thyroid hormonogenesis.

European Journal of Endocrinology 136 508–515
A novel approach to assess changes in endocrine secretion: analysis of GnRH antagonist (Nal-Glu) suppression of gonadotropin release in ovariectomized ewes
Authors: Normolle Daniel P , Evans Neil P , Dahl Geoffrey E , Padmanabhan Vasantha
Published: December 2008

Circulating hormone levels reflect the outcome of multiple feedback systems. A method to accurately assess the dynamics of hormonal changes in samples collected at infrequent intervals and compare these dynamic processes among treatment groups is presented. In this approach, a smooth curve is fitted to each time series of concentrations produced in an experiment, the curves are summarized by numerical measurements, and the measurements are subjected to statistical analysis. The method is demonstrated on data from an experiment that explores the differential effects of a competitive GnRH receptor antagonist (Nal-Glu) on circulating levels of LH and FSH.

In this experiment, six adult ovariectomized Suffolk ewes were treated with one of three doses of Nal-Glu using a crossover design. LH and FSH concentrations were determined in hourly samples of jugular blood for 24 h after treatment. Applying the analytical approach, we observed differential effects of increasing concentrations of Nal-Glu on circulating LH and FSH concentrations. The magnitude of LH suppression was similar from dose to dose, while the duration of LH suppression was dose-dependent. In contrast, all doses of Nal-Glu elicited similar effects on the amplitude, duration and time to recovery of FSH suppression. Studies conducted in vitro utilizing dispersed ovine pituitary cells in culture demonstrated that the differential effects of Nal-Glu on FSH and LH secretion were not the outcome of differential sensitivity of FSH and LH to GnRH. The differential effects of Nal-Glu on circulating LH and FSH concentrations may be due to a number of factors, including other releasing or release-inhibiting hormones, paracrine modulators involved in selective regulation of FSH, and/or differences in clearances.

European Journal of Endocrinology 136 519–530
Oxytocin-induced changes in single cell K+ currents and smooth muscle contraction of guinea-pig gastric antrum
Authors: Duridanova Dessislava B , Nedelcheva Milena D , Gagov Hristo S
Published: December 2008

To study the effects of oxytocin on both spontaneous phasic contractions and K+ outward currents (IK) of the so-called 'non-target' smooth muscle cells, physiological concentrations of oxytocin ranging between 10−12 mol/l and 10−8 mol/l were applied to smooth muscle preparations and single voltage-clamped cells isolated from the circular layer of the guinea-pig gastric antrum. Oxytocin (10−12mol/l to 10−8 mol/l) suppressed, in a dose-dependent manner, the tetrodotoxin- and atropine-resistant spontaneous phasic contractions and shifted rightward the dose–response curves of 10−7 mol/l charybdotoxin and 10−3mol/l BaCl2. In cells with preloaded intracellular Ca2+ stores, oxytocin (10−12 mol/l to 10−9 mol/l) caused a dose-dependent activation of the charybdotoxin-blockable non-inactivating component of IK (IK(s1)) of single voltage-clamped cells, which was accompanied by hyperpolarization of the cell membranes. 8Lys-vasopressin and 8arg-vasopressin failed to mimic the effects of oxytocin on both contraction and K+ currents. Further, the oxytocin-induced activation of IK(s1) was effectively antagonized by 5× 10−8 mol/l U-73122 or 5× 10−6 mol/l 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (inhibitors of the cell membrane phospholipase C), as well as by intracellularly applied heparin (selective inhibitor of inositol-1,4,5-trisphosphate (IP3)-induced Ca2+ release channels). In cells incubated in the absence of Ca2+ entry throughout the study, oxytocin (10−9 mol/l) caused a slight and transient increase of IK(s1) amplitudes. Neither ryanodine (10−6 mol/l nor cyclopiazonic acid (10−6 mol/l) were able to restore the IK-activating effect of oxytocin in these cells.

The data obtained suggest (i) that selective oxytocin receptors are present on the membranes of guinea-pig antral smooth muscle cells, (ii) that the oxytocin-related relaxation may result from the activation of Ca2+-sensitive K+ conductivity via activation of IP3-induced release of Ca from the submembrane located cisternae of the sarcoplasmic reticulum Ca2+ stores and (iii) in turn, this evokes a non-inactivating component of IK, hyperpolarizing the cell membrane.

European Journal of Endocrinology 136 531–538
Carbamoylcholine regulation of polyphosphoinositide synthesis and hydrolysis in cultured, dispersed, digitonin-permeabilized mouse pancreatic islet cells
Authors: Kardasz Andrew M , Thams Peter , Capito Kirsten , Hedeskov Carl J
Published: December 2008

Continuing formation of inositol phosphates during stimulation of pancreatic β-cells by hormones and neurotransmitters requires the continued synthesis of the polyphosphoinositides phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5 bisphosphate (PIP2) from phosphatidylinositol (PI). In the present study we have investigated how this pathway and the activity of phosphoinositide-specific phospholipase C (PI-PLC) are regulated by carbamoylcholine (CCh), Ca2+, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), GTPγS and NaF in 44-h [3H]inositol-labelled, dispersed and digitonin-permeabilized mouse pancreatic islet cells.

CCh stimulated not only PI-PLC (G-protein-mediated) but also, by an as yet unknown mechanism, significantly enhanced PI 4-kinase activity, estimated as the PIP:PI ratio, by 100%, and further increased the flux from PI to PIP and PIP2. GTPγS and NaF mimicked the effects of CCh on PI-PLC but had no effect on the levels of PIP and PIP2. TPA raised the PIP:PI ratio by 75%. In addition TPA counteracted the CCh stimulation of PI-PLC. There was no effect of 10−6 mol/l Ca2+ on the levels of PIP and PIP2. Experiments with quinacrine and adenosine confirmed that PI-PLC and PI 4-kinase could be regulated independently of each other. In conclusion, these data point to differential regulation of polyphosphoinositide synthesis and breakdown.

European Journal of Endocrinology 136 539–545
Effect of natural menopause on serum levels of IGF-I and IGF-binding proteins: relationship with bone mineral density and lipid metabolism in perimenopausal women
Authors: Nasu Masamichi , Sugimoto Toshitsugu , Chihara Mieko , Hiraumi Mitsuo , Kurimoto Fumihiko , Chihara Kazuo
Published: December 2008

The present study was performed to examine the effect of natural menopause on serum levels of IGF-I, IGF-binding protein (IGFBP-2) and -3 as well as on bone mass and lipid metabolism in perimenopausal women. One hundred and twenty-one healthy Japanese women, who were 45–55 years old, were studied (71 premenopausal and 50 postmenopausal women 1–9 years after menopause). Bone mineral density (BMD) was measured at the middle third of the radius by using dual energy X-ray absorptiometry. Serum levels of IGF-I, but not those of IGFBP-2 or -3, were significantly reduced in the postmenopausal group compared with the premenopausal group. One year after menopause, serum IGF-I levels were significantly lower, and the biochemical markers of bone turnover, such as serum total alkaline phosphatase level and urinary calcium to creatinine ratio, were significantly higher than the premenopausal levels. Serum levels of IGF-I, but not those of IGFBP-2 or-3, were positively correlated with BMD. Serum levels of IGFBP-2, but not those of IGF-I or IGFBP-3, were negatively correlated with body mass index and body weight. Finally, serum levels of IGFBP-3, but not those of IGF-I, were positively correlated with serum levels of total cholesterol and triglyceride. The present findings suggest that a rapid decrease in serum IGF-I levels after menopause might be partly involved in bone loss following gonadal failure and that IGFBP-2 and -3 might be related to the regulation of body mass and lipid metabolism during perimenopause respectively, although the mechanisms remain unknown.

European Journal of Endocrinology 136 608–616
Ovarian 17α-hydroxyprogesterone responses to GnRH analog testing in oligomenorrheic insulin-dependent diabetic adolescents
Authors: Virdis Raffaele , Zampolli Maria , Street Maria E , Vanelli Maurizio , Potau Neus , Terzi Cesare , Ghizzoni Lucia , Ibanez Lourdes
Published: December 2008

Objective: To investigate the pituitary-ovarian function in adolescent girls with insulin-dependent diabetes mellitus (IDDM).

Design: Clinical case-control study.

Methods: The GnRH analog leuprolide acetate was administered subcutaneously to 16 adolescents with IDDM (seven eumenorrheic and nine oligomenorrheic) and 13 controls between 0800 and 0900 h. Blood samples were collected at baseline and 0·5, 3, 6 and 24 h after leuprolide to measure levels of gonadotropins, 17α-hydroxyprogesterone (17-OHP), androgens and estradiol.

Results: Mean baseline serum LH levels were significantly higher in eumenorrheic compared with oligomenorrheic IDDM patients, while peak LH responses to GnRH analog testing were similar in all subjects. Oligomenorrheic IDDM girls showed, as a group, a distinct 17-OHP response to GnRH analog stimulation, which in five out of nine girls was in the range of functional ovarian hyperandrogenism (≥ 8·6 nmol/l). Androgen and estradiol levels were not significantly altered in any group. No correlation was found between steroid levels and HbA1c levels, although the latter were significantly higher in oligomenorrheic than in eumenorrheic patients.

Conclusion: About 50% of the oligomenorrheic IDDM adolescents had an increased ovarian 17-OHP response to GnRH analog stimulation in the range of functional ovarian hyperandrogenism. Factors other than metabolic control, such as stress, may play an etiologic role in IDDM ovarian dysfunction.

European Journal of Endocrinology 136 624–629
Melatonin receptors are present in adult rat Leydig cells and are coupled through a pertussis toxin-sensitive G-protein
Authors: Valenti Sandra , Giusti Massimo , Guido Roberta , Giordano Giulio
Published: December 2008

Previous studies have suggested that melatonin (MLT) acts directly on rat Leydig cells by modulating androgen production. In the present study, the site of action of MLT was investigated. The binding of 2-[125I]iodomelatonin (125I-MLT; 7–240 pmol/l) to Leydig cell membrane fragments was tested in the presence or absence of guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-S; 50 μmol/l). Saturation studies and Scatchard analysis revealed the existence of a high-affinity binding site with a Bmax of 46·70± 3·50 fmol/mg protein and a Kd of 88·70±6·20 pmol/l; treatment with GTP-γ-S reduced the concentration of 125I-MLT binding sites (Bmax 34·03±4·50), while increasing the Kd to 106·5± 2·61 pmol/l.

Pretreatment of the cells with pertussis toxin (PTX; 10 ng/ml for 16 h) resulted in a decreased binding of I-MLT and a lack of effect of GTP-γ-S. Moreover, the effect of MLT on testosterone secretion induced by LH (30 mIU/ml), forskolin (1 μmol/l) and LHRH (100 nmol/l) was studied after 3-h incubation of cells which had been precultured with or without PTX. The inhibition of testosterone secretion due to MLT administration was eliminated by PTX pretreatment during forskolin and LH, but not during LHRH administration. However, 17-hydroxyprogesterone levels were higher in all groups incubated in the presence of MLT, irrespective of PTX pretreatment.

Our data suggest that: (a) MLT receptors are present on the membranes of adult rat Leydig cells; (b) they couple through PTX-sensitive G-protein-coupled binding sites; (c) the mechanism by which MLT blocks 17–20 desmolase enzymatic activity (thus leading to increased 17-hydroxyprogesterone levels), and testosterone secretion during LHRH stimulation is likely to depend on one or more different mechanism(s) of action.

European Journal of Endocrinology 136 633–639
Expression of parathyroid hormone-related peptide (PTHrP) and PTH/PTHrP receptor in newborn human calvaria osteoblastic cells
Authors: Lomri Abderrahim , de Pollak Cindy , Sebag Michael , Goltzman David , Kremer Richard , Marie Pierre J
Published: December 2008

We examined the expression of parathyroid hormone-related peptide (PTHrP) and its receptor in normal newborn human calvaria osteoblastic (NHCO) cells. Northern blot analysis showed that NHCO cells express a single 1·6 kb transcript of PTHrP, which was increased within 1 h (2x) and peaked at 6 h (7x) after serum treatment. In the culture media, the release of PTHrP peptide was maximally increased (4x) 24 h after the addition of serum, as determined by immunoradiometric assay. NHCO cells exhibited a cytoplasmic immunostaining for PTHrP in the presence of serum, and most PTHrP-positive cells were alkaline phosphatase-negative, suggesting that PTHrP was expressed in undifferentiated cells. Furthermore, RT-PCR analysis showed that both PTHrP and PTH/PTHrP receptor were expressed in NHCO cells in basal conditions or after stimulation with serum. The maximal PTHrP expression induced by serum suppressed PTH/PTHrP receptor expression, suggesting that PTHrP down-regulated its receptor in NHCO cells. Treatment with 10 nm human PTH(1–34—which binds to PTH/PTHrP receptors, increased intracellular cAMP levels and alkaline phosphatase activity, and decreased cell growth, indicating that ligand binding to PTH/PTHrP receptors regulates NHCO cell proliferation and differentiation. The expression and synthesis of PTHrP and the presence of functional PTH/PTHrP receptors suggest a possible paracrine mechanism of action of PTHrP in normal human calvaria osteoblastic cells.

European Journal of Endocrinology 136 640–648
Epidermal growth factor receptor expression in patients with ductal breast carcinoma. Comparison with clinicopathological factors and estrogen receptor status
Authors: Harlozinska A , Bar J K , Bebenek M , Kowalski P , Sedlaczek P
Published: December 2008

Epidermal growth factor receptor (EGFR) and estrogen receptor (ER) status were immunohistochemically evaluated in 80 primary ductal breast carcinomas. The comparison between EGFR status and tumor grading, tumor size, lymph node involvement and age of patients was performed. EGFR expression was found in 87.5% of carcinoma samples and 46.3% of tumors were positive for ER staining. The inverse relationship between EGFR and ER expression was confirmed and it was evident that a high concentration of EGFR exceeding 25% of a tissue section may inhibit or significantly limit the ability to express ERs. EGFR presence was significantly associated with poorly differentiated tumors. No correlation was found between EGFR positivity and tumor size, lymph node involvement or age of patients. Our results indicate that EGFR status does not seem to be a valuable independent prognostic indicator, but that the combination of EGFR positivity (above 25% of a tissue section) accompanied by a low or undetectable level of ER expression should be considered as a potential marker of a poor prognosis in patients with ductal breast carcinoma.
Quantitative in-situ hybridization histochemistry studies on growth hormone (GH) gene expression in acromegalic somatotrophs: effects of somatostatin, GH-releasing factor and cortisol
Authors: Levy A. , Lightman S. L.
Published: December 2008

We have examined the effects of human GH-releasing factor (1–44) (GRF), cortisol and somatostatin-(1–14) on GH gene expression in solid tissue and dispersed cells from human pituitary adenomas using quantitative in-situ hybridization histochemistry. Sections cut from tissue obtained at hypophysectomy from three acromegalic patients were hybridized to probes directed against mature α-subunit, GH, prolactin, pro-opiomelanocortin, TSHβ-subunit and LHβ-subunit mRNA. Only one biopsy contained GH mRNA in isolation. A second was found to co-exhibit GH, prolactin and α-subunit mRNA, and a third was found to contain prolactin, TSHβ-subunit, α-subunit and LHβ-subunit mRNA, with GH mRNA below the limit of specific detection, indicating that the sample was composed of normal rather than adenomatous pituitary tissue. GH mRNA in individual dispersed cells derived from the latter declined to barely detectable levels over 287 h, both in cultures containing GRF (10 ng/ml) or GRF (10 ng/ml) plus somatostatin (10 ng/ml) and in controls, but increased fourfold in cultures containing GRF (10 ng/ml) plus cortisol (0·5 μmol/l). GH mRNA remained unchanged in both adenoma samples over 138 and 450 h, irrespective of the addition of GRF or GRF plus hydrocortisone. In these samples, somatostatin plus GRF had no consistent effect. These studies confirm that quantitative in-situ hybridization histochemistry can be used to investigate hormone gene regulation in small samples of human tissue and should enable us to define more clearly the level at which abnormal gene regulation occurs.
Regulation of LH subunit mRNA levels by gonadal hormones in female rats
Authors: Abbot S. D. , Docherty K. , Clayton R. N.
Published: December 2008

To determine the physiological role of the ovaries in regulation of LH subunit gene expression, levels of cytoplasmic mRNA were measured in a cDNA-RNA dot-blot hybridization assay. An increase (twofold) in α mRNA was first detected 8 days after ovariectomy and then remained stable for 4 weeks. In contrast, LH-β mRNA increased by 60–79% within 12 h of removing the ovaries and then rose progressively to six times the intact values at 3 and 4 weeks. Increases in LH-β mRNA were always greater than those of α mRNA. Oestradiol, and oestradiol plus progesterone, but not progesterone alone, prevented the rise in α and LH-β mRNA 10 days after ovariectomy.

Three days after ovariectomy, α mRNA, but not LH-β mRNA, was suppressed to below intact control values by oestradiol and oestradiol plus progesterone, indicating greater sensitivity of α mRNA to oestradiol inhibition at this stage. A single injection of oestradiol (1 μg s.c.) to rats ovariectomized 14 days previously transiently suppressed α and LH-β mRNA levels and serum LH concentrations in parallel for 1–8 h, after which high preinjection values were restored. However, pituitary LH content remained suppressed after LH mRNA levels had returned to the control values of ovariectomized rats.

In most instances there was a qualitative positive correlation between changes in α and LH-β mRNA, pituitary LH content and serum LH concentrations. LH content reflected LH-β mRNA changes more closely than those of α mRNA. However, in oestradiol-treated rats ovariectomized 10 days previously, LH content remained increased despite normalization of the LH-β and α mRNA levels, suggesting differential sensitivity to oestradiol of the gene expression and translational processes. Thus divergence of pre- and post-translational regulation of LH biosynthesis was demonstrated. These results imply an important physiological role for female sex hormones in the control of LH gene expression and LH biosynthesis.

Prolactin mRNA fell by 30–50% for the first 2 weeks after ovariectomy, but by 3 and 4 weeks values were similar to those of intact controls. Serum and pituitary prolactin levels were reduced by 50% or more at all time-points, despite normalization of mRNA. Treatment of ovariectomized rats for 10 days with oestradiol and progesterone, either alone or combined, reversed the fall in prolactin mRNA and serum and pituitary prolactin levels. These changes in prolactin gene expression and synthesis were opposite to those of LH subunits in response to the same in-vivo hormone manipulations.

Growth hormone mRNA levels were unchanged by ovariectomy, oestradiol or progesterone treatment. Levels of TSH-β mRNA increased slightly (maximum up to 50%) after ovariectomy, but were unaltered by oestradiol and progesterone treatment for 10 days. These results support the view that α mRNA changes, resulting from ovariectomy, oestradiol and progesterone treatment, occur in gonadotrophs and not thyrotrophs, which also express the α subunit gene.
Effects of l-leucine, its 2-keto acid metabolite and its nonmetabolized analogue on rat tumoral islet cell function
Authors: Leclercq-Meyer V. , Marchand J. , Sener A. , Blachier F. , Malaisse W. J.
Published: December 2008

l-Leucine and 2-ketoisocaproate stimulated insulin release from perifused rat tumoral islet cells (RINm5F line). The secretory response coincided with an increase in the intracellular ATP/ADP ratio, a stimulation of 45Ca outflow from cells perifused in the presence of extracellular Ca2+, and an increase in 32P efflux from cells prelabelled with radioactive orthophosphate. In contrast to d-glucose, however, l-leucine or 2-ketoisocaproate failed to decrease 86Rb outflow, to inhibit 45Ca outflow from cells perifused in the absence of Ca2+ and to enhance the labelling of inositol-containing phospholipids in cells exposed to myo-[2-3H]inositol. These findings suggest that d-glucose, l-leucine and 2-ketoisocaproate exert dissimilar effects on the subcellular distribution of adenine nucleotides and/or 86Rb. The nonmetabolized analogue of l-leucine, 2-aminobicyclo-[2.2.1]heptane-2-carboxylic acid (BCH), also caused an initial stimulation of insulin release and 32P efflux, but this was soon followed by a severe and irreversible inhibition of insulin output, associated with a permanent enhancement of 86Rb outflow. The dual ionic and secretory response to BCH is interpreted in the light of its dual effect on the catabolism of endogenous amino and fatty acids, and raises the view that BCH could be used to interfere with the function of insulinoma cells.
Oestrogen receptor mRNA and a related RNA transcript in mouse ovaries
Authors: Hillier S.G. , Saunders P.T.K. , White R. , Parker M.G.
Published: December 2008

Oestrogen receptor mRNA expression in mouse ovaries was analysed by Northern blotting of total RNA using 32P-labelled RNA probes complementary to different functional domains of the oestrogen receptor. The ~6·5 kb mouse oestrogen receptor mRNA transcript was present in immature and adult ovaries at extremely low abundance compared with uterus and oviduct. Using a probe complementary to the steroid-binding domain of the oestrogen receptor (probe EF), a novel RNA transcript of ~1·5 kb was also found in the ovaries but was absent from uterus and oviduct. The melting temperature of the hybrid produced by the ~1·5 kb transcript with probe EF was ~10°C lower than that produced by authentic oestrogen receptor mRNA, which demonstrates incomplete sequence homology between the two transcripts and indicates that the ~1·5 kb RNA is not a truncated form of oestrogen receptor mRNA. Furthermore, the ~1·5 kb RNA lacks the DNA-binding domain found in the oestrogen receptor. The ~1·5 kb RNA, but not oestrogen receptor mRNA, was enriched in total RNA from isolated granulosa cells compared with residual ovarian tissue. The encoded product of this novel oestrogen receptor-related RNA could be a steroid-binding protein involved in oestrogen action in the ovaries.
Bradykinin stimulates phosphoinositide metabolism and prolactin secretion in rat anterior pituitary cells
Authors: Jones T.H. , Brown B. L. , Dobson P. R. M.
Published: December 2008

Bradykinin stimulated prolactin secretion from monolayer cultures of rat anterior pituitary cells, the stimulation being greater from the cells of male rats. This stimulated secretion was accompanied by a rise in total inositol phosphate accumulation, suggesting that the action of bradykinin is mediated by phosphoinositide hydrolysis. The increase in inositol phosphate accumulation was biphasic; a further sharp rise occurred when the concentration of bradykinin exceeded 1 μmol/l. This may indicate that bradykinin acts on other cell types in the pituitary gland. Bradykinin had no effect on growth hormone secretion from cells of normal pituitary glands, or on prolactin secretion and phosphoinositide metabolism in GH3 rat pituitary tumour cells. Bradykinin receptor antagonists (both B1 and B2) had no effect on either bradykinin-stimulated inositol phosphate accumulation or prolactin secretion. Kallikreins, the enzymes responsible for the generation of kinins, are known to be present in the adenohypophysis. Therefore, the results presented here would suggest that kinins may have a role as paracrine agents in the pituitary gland.