bluuebunny/crossref_metadata_2025 huggingface dataset:
Thyroxine Administration Prevents Streptococcal Cell Wall-Induced Inflammatory Responses*
Authors: Rittenhouse Peter A. , Redei Eva
Published: January 2014

Administration of streptococcal cell wall (SCW) preparation induces an inflammatory response in susceptible animals that is a model frequently used for rheumatoid arthritis. The degree of inflammation produced by SCW is greatly enhanced by low endogenous levels of glucocorticoids due to diminished hypothalamic-pituitary-adrenal activity. Because decreased glucocorticoid production is known to occur in the hypothyroid state, we tested whether hypothyroidism would increase, and conversely, whether hyperthyroidism would decrease, the inflammatory responses to SCW. Adult female Sprague Dawley rats were fed a regular diet (control), L-T4 (T4; hyperthyroid), or 6-propyl-thiouracil (hypothyroid) in drinking water for 7 weeks. Hypothyroidism resulted in elevated plasma levels of TSH and hypothalamic preproTRH messenger RNA (mRNA) while reducing anterior pituitary POMC mRNA and plasma ACTH and corticosterone levels. In contrast, hyperthyroid rats produced opposite results: decreased measures of central thyroid function but increased pituitary-adrenal function. Three days after administration of SCW, macrophage inflammatory protein-1α and interleukin-1β mRNA expression increased dramatically in controls and even further in hypothyroid animals, as measured by Northern blot analysis. In contrast, T4-treated rats showed significant inhibition of these inflammatory markers. Thus, the hyperthyroid state combined with increased endogenous glucocorticoid levels is protective against inflammatory challenges. The inverse relationship between preproTRH expression and pituitary-adrenal function suggests the possibility of a direct inhibitory link connecting the hypothalamic-pituitary-adrenal and thyroid axes, and suggests alternative sites of therapeutic intervention for rheumatoid arthritis and other inflammatory associated disorders.
The Role of Interleukin-6 in the Induction of Hypercalcemia in Renal Cell Carcinoma Transplanted into Nude Mice
Authors: Weissglas Max G. , Schamhart Denis H. J. , Löwik Clemens W. G. M. , Papapoulos Socrates E. , Theuns Harry M. , Kurth Karl-Heinz
Published: January 2014

Hypercalcemia is a well known complication of renal cell carcinoma (RCC). As RCCs can produce IL-6, and IL-6 may stimulate bone resorption and cause mild hypercalcemia, we examined whether IL-6 is involved in renal cancer-associated hypercalcemia in vivo. Three human renal cell carcinoma tumor lines (RC-8, RC-9, and NC-65) growing in nude mice were studied. Tumors were implanted sc, and parameters of bone metabolism and serum human IL-6 levels were determined in relation to tumor volume (TV). All three tumor lines secreted human IL-6, although in different quantities. The maximum level of IL-6 in RC-8 was 434 pg/ml (TV, 200 mm3), that in RC-9 was 81 pg/ml (TV, 1800 mm3), and that in NC-65 was 2368 pg/ml (TV, 1800 mm3). Hypercalcemia developed in RC-8 and RC-9 tumor-bearing animals, but not in NC-65-bearing animals. The hypercalcemia in both RC-8 and RC-9 tumor lines was associated with elevated levels of PTH-related peptide (PTHrP) and loss of trabecular bone volume. Serum calcium and phosphate concentrations showed an almost linear relationship with plasma PTHrP independently of the tumor line and serum IL-6 levels. No hypercalcemia occurred in the NC-65 animals, which had the highest levels of IL-6, but no detectable plasma PTHrP and PTHrP messenger RNA expression in the tumor. Administration of neutralizing antibodies to IL-6 to RC-8 animals normalized serum calcium concentrations and PTHrP values and induced a significant inhibition of tumor growth. No such effect on tumor growth of anti-IL-6 was seen in the other two tumor lines. The normalization of serum calcium in RC-8 mice is most likely attributed to the growth-inhibiting effect of anti-IL-6 on RC-8 tumor. We conclude that IL-6 secreted by RCC does not contribute directly to hypercalcemia, but may enhance hypercalcemia by stimulating the tumor growth of a subpopulation of PTHrP-secreting carcinomas.
Role of Mitogen-Activated Protein Kinase Pathway in Prostaglandin F2α-Induced Rat Puerperal Uterine Contraction
Authors: Ohmichi Masahide , Koike Koji , Kimura Akiko , Masuhara Kanji , Ikegami Hiromasa , Ikebuchi Yoshihide , Kanzaki Tohru , Touhara Kazushige , Sakaue Motoyoshi , Kobayashi Yuzuru , Akabane Masuo , Miyake Akira , Murata Yuji
Published: January 2014

In this study, prostaglandin (PG) F2α was found to activate mitogen-activated protein (MAP) kinase and MAP kinase kinase (MEK) in cultured rat puerperal uterine myometrial cells. PGF2α stimulation also led to an increase in phosphorylation of raf-1, son of sevenless (SOS), and Shc. Furthermore, we examined the mechanism by which PGF2α induced MAP kinase phosphorylation. Both pertussis toxin (10 ng/ml), which inactivates Gi/Go proteins, and expression of a peptide derived from the carboxyl terminus of the β-adrenergic receptor kinase 1 (βARK1), which specifically blocks signaling mediated by the βγ subunits of G proteins, blocked the PGF2α-induced activation of MAP kinase. Ritodrine (1 μm), which is known to relax uterine muscle contraction, attenuated PGF2α-induced tyrosine phosphorylation of MAP kinase. Moreover, to examine the role of MAP kinase pathway in uterine contraction, an inhibitor of MEK activity, PD098059, was used. Although MEK inhibitor had no effect on PGF2α-induced calcium mobilization, this inhibitor partially inhibited PGF2α-induced uterine contraction. These results provide evidence that PGF2α stimulates the MAP kinase signaling pathway in cultured rat puerperal uterine myometrial cells through Gβγ protein, suggesting that this new pathway may play an important role in the biological action of PGF2α on these cells.
Circadian Variation in Basal Plasma Corticosterone and Adrenocorticotropin in the Rat: Sexual Dimorphism and Changes across the Estrous Cycle*
Authors: Atkinson Helen C. , Waddell Brendan J.
Published: January 2014

Sexual dimorphism in the rat hypothalamic-pituitary-adrenal axis was investigated by determination of plasma corticosterone and immunoreactive (I-) ACTH in males and in females at each stage of the estrous cycle. A serial blood-sampling technique enabled assessment of covariation of the two hormones across the full circadian range of their concentrations within individual animals. Distinct diurnal rhythms in plasma corticosterone were evident in all rats, and the degree and timing of this rhythmicity, determined by cosinor analyses, did not vary with gender or cycle stage. There were, however, marked differences in absolute levels of corticosterone across the estrous cycle, with the average daily concentration (mesor) increasing progressively from a minimum at estrus (129 ± 11 ng/ml) to a maximum 3 days later at proestrus (246 ± 14 ng/ml). The mesor corticosterone value in male rats (102 ± 21 ng/ml) was not different from that in estrous females, but was lower than that in females at all other stages of the cycle. In contrast, no gender- or cycle-related differences were detected in absolute levels of I-ACTH, although distinct diurnal rhythms, synchronous with those for corticosterone, were evident in all groups. Accordingly, a strong and positive within-rat relationship between plasma corticosterone and I-ACTH was observed in all groups, but there was a clear shift in the nature of this relationship across the estrous cycle, such that the slope (i.e. concentration of plasma corticosterone per unit concentration of I-ACTH) was minimal in males and estrous females and maximal in proestrous females. In conclusion, this study shows that the extent of sexual dimorphism in resting plasma corticosterone levels is dependent on estrous cycle stage, being absent at estrus and maximal at proestrus. Moreover, this variation in plasma corticosterone was not accompanied by corresponding changes in plasma I-ACTH, suggestive of cycle-related changes in responsiveness of the adrenal cortex to trophic stimulation.
Rapid Membrane Effects of Steroids in Neuroblastoma Cells: Effects of Estrogen on Mitogen Activated Protein Kinase Signalling Cascade and c-fos Immediate Early Gene Transcription
Authors: Watters Jyoti J. , Campbell Jean S. , Cunningham Matthew J. , Krebs Edwin G. , Dorsa Daniel M.
Published: January 2014

Rapid effects of steroid hormones have been observed in neuronal cells for many years. We show here, that in the human neuroblastoma cell line SK-N-SH, the membrane impermeable conjugated 17β-estradiol (E2BSA) activates mitogen activated protein kinase kinase (MAPKK or MEK) and induces the phosphorylation and activation of both ERK-1 and ERK-2 (mitogen activated protein kinase or MAPK). Additionally, E2BSA induces the transcription of a reporter gene construct driven by the promoter of the mouse c-fos proto-oncogene. The effects of this membrane impermeable estrogen on c-fos transcription are not inhibited by the estrogen receptor antagonists. Tamoxifen or ICI 182,780, further excluding the involvement of the intracellular estrogen receptor. This is also illustrated by the observation that E2BSA does not activate estrogen response element (ERE) mediated transcription. This is the first report of rapid membrane effects of 17β-estradiol on growth factor related signalling pathways in neuronal cells, and indicates a potential mechanism by which 17β-estradiol might affect the expression of genes whose promoters do not contain EREs but are responsive to factors acting through other response elements such as AP-1 and SRE sites.
Anorectic Effects of the Cytokine, Ciliary Neurotropic Factor, Are Mediated by Hypothalamic Neuropeptide Y: Comparison with Leptin*
Authors: Xu B. , Dube M. G. , Kalra P. S. , Farmerie W. G. , Kaibara A. , Moldawer L. L. , Martin D. , Kalra S. P.
Published: January 2014

Although ciliary neurotropic factor (CNTF) is a tropic factor in nervous system development and maintenance, peripheral administration of this cytokine also causes severe anorexia and weight loss. The neural mechanism(s) mediating the loss of appetite is not known. As hypothalamic neuropeptide Y (NPY) is a potent orexigenic signal, we tested the hypothesis that CNTF may adversely affect NPYergic signaling in the hypothalamus. Intraperitoneal administration of CNTF (250μ g/kg) daily for 4 days significantly suppressed 24-h food intake in a time-dependent manner and decreased body weight. The loss in body weight was similar to that which occurred in pair-fed (PF) rats. As expected, hypothalamic NPY gene expression, determined by measurement of steady state prepro-NPY messenger RNA by ribonuclease protection assay, significantly increased in PF rats in response to energy imbalance. However, despite a similar loss in body weight, there was no increase in NPY gene expression in CNTF-treated rats. Daily administration of CNTF intracerebroventricularly (0.5 or 5.0 μg/rat) also produced anorexia and body weight loss. In this experiment, negative energy balance produced by both PF and food deprivation augmented hypothalamic NPY gene expression. However, despite reduced intake and loss of body weight, no similar increment in hypothalamic NPY gene expression was observed in CNTF-treated rats. In fact, in rats treated with higher doses of CNTF (5.0 μg/rat), NPY gene expression was reduced below the levels seen in control, freely fed rats. Furthermore, CNTF treatment also markedly decreased NPY-induced feeding. These results suggested that anorexia in CNTF-treated rats may be due to a deficit in NPY supply and possibly in the release and suppression of NPY-induced feeding. The possibility that CNTF-induced anorexia may be caused by increased leptin was next examined. Daily intracerebroventricular injections of leptin (7 μg/rat) decreased food intake, body weight, and hypothalamic NPY gene expression in a manner similar to that seen after CNTF treatment. Leptin administration also suppressed NPY-induced feeding. However, peripheral and central CNTF injections markedly decreased leptin messenger RNA in lipocytes, indicating a deficiency of leptin in these rats; thus, leptin was unlikely to be involved in appetite suppression. Thus, these results show that a two-pronged central action of CNTF, causing diminution in both NPY availability and the NPY-induced feeding response, may underlie the severe anorexia. Further, unlike other members of the cytokine family, suppression of NPYergic signaling in the hypothalamus by CNTF does not involve up-regulation of leptin, but may involve a direct action on hypothalamic NPY neurons or on neural circuits that regulate NPY signaling in the hypothalamus.
KAT45, a Noradrenergic Human Pheochromocytoma Cell Line Producing Corticotropin-Releasing Hormone*
Authors: Venihaki M. , Ain K. , Dermitzaki E. , Gravanis A. , Margioris A. N.
Published: January 2014

KAT45 cells were derived from a human pheochromocytoma, which also caused ectopic Cushing’s syndrome, and developed into a cell line spontaneously after the continuous primary culture of the tumor cells. These human pheochromocytoma cells were compared with the extensively characterized PC12 rat pheochromocytoma cell line. KAT45 cells resembled PC12 cells in morphology, proliferation rate, response to cholinergic stimuli, and the development of dendrite-like projections after exposure to nerve growth factor. They produced norepinephrine and epinephrine in a ratio of 50:1, as opposed to production of dopamine by PC12 cells, in amounts 1 order of magnitude higher compared with PC12. Because of the ectopic Cushing’s syndrome in our patient, her normal ACTH level, and the knowledge that PC12 cells and even normal rat chromaffin cells appear to produce CRH, we examined whether KAT45 cells also produced this neuropeptide. Indeed, KAT45 cells released authentic CRH and contained an apparently intact CRH transcript. Nicotine and KCl depolarization stimulated the secretion of CRH, whereas interleukin-1β, glucocorticoids, and nerve growth factor stimulated its synthesis. In addition to the potential systemic effects of CRH, which in our patient produced ectopic Cushing’s syndrome, CRH can exert paracrine effects within normal or tumoral adrenals. We used KAT45 cells as a model for the study of the local role of CRH. CRH affected several parameters of KAT45 cell metabolism, including their proliferation rate, synthesis of catecholamines, and production of POMC-derived peptides. KAT45 cells, in addition to the data they provided regarding the in vitro profile of a human CRH-producing pheochromocytoma, may prove to be a valuable auxiliary to the PC12 cell line.
Differential Expression of Rat Insulin I and II Messenger Ribonucleic Acid after Prolonged Exposure of Islet β-Cells to Elevated Glucose Levels*
Authors: Ling Zhidong , Heimberg Harry , Foriers André , Schuit Frans , Pipeleers Daniel
Published: January 2014

Prolonged exposure of rat islet β-cells to 10 mmol/liter glucose has been previously shown to activate more cells into a glucose-responsive state (>90%) than has exposure to 6 mmol/liter glucose (50%). The present study demonstrates that this recruitment of more activated cells results in 4- to 6-fold higher levels of proinsulin I and proinsulin II messenger RNA (mRNA). However, only the rate of proinsulin I synthesis is increased. Failure to increase the rate of proinsulin II synthesis in the glucose-activated cells results in cellular depletion of the insulin II isoform, which can be responsible for degranulation of β-cells cultured at 10 mmol/liter glucose. Higher glucose levels (20 mmol/liter) during culture did not correct this dissociation between the stimulated insulin I formation and the nonstimulated insulin II formation. On the contrary, the rise from 10 to 20 mmol/liter glucose resulted in a 2-fold reduction in the levels of proinsulin II mRNA, but not of proinsulin I mRNA; this process further increased the ratio of insulin I over insulin II to 5-fold higher values than those in freshly isolated β-cells. The present data suggest that an elevated insulin I over insulin II ratio in pancreatic tissue is a marker for a prolonged exposure to elevated glucose levels. The increased ratio in this condition results from a transcriptional and/or a posttranscriptional failure in elevating insulin II formation while insulin I production is stimulated in the glucose-activated β-cells.
Corticotropin-Releasing Factor (CRF) Agonists Stimulate Testosterone Production in Mouse Leydig Cells through CRF Receptor-1*
Authors: Heinrich Nadja , Meyer Mike R. , Furkert Jens , Sasse Annette , Beyermann Michael , Bönigk Wolfgang , Berger Hartmut
Published: January 2014

The influence of CRF on testosterone production in primary mouse Leydig cell cultures was studied, and the type of CRF receptor (CRF-R) involved in this activity was determined. CRF directly stimulated testosterone production in mouse Leydig cells, but did not influence the maximum human (h)CG-induced testosterone production. The effect was time- and dose-dependent, saturable with an EC50 of 2.84 nm for hCRF, antagonized by the CRF antagonist α-helical CRF9–41, and accompanied by intracellular cAMP elevation. The rank order of potency of the natural CRF agonists, hCRF, ovine CRF, sauvagine, and urotensin, corresponded to that of their activities on CRF-R1 in rat pituitary cells and also to that reported for this receptor, but not for CRF-R2, when transfected into various cell lines. Furthermore, the difference in response of mouse Leydig cells to[ 11-d-Thr,12-d-Phe]- and[ 13-d-His,14-d-Leu]-ovine CRF corresponded to that measured when COS cells expressing CRF-R1 were activated, but was considerably smaller than that observed for activation of COS cells expressing CRF-R2α or -R2β. The messenger RNA encoding the mouse CRF-R1 was detected by RT-PCR in mouse Leydig cell preparations. In contrast to mouse Leydig cells, CRF agonists had no influence on the basal testosterone and cAMP production by rat Leydig cells, nor did the agonists or antagonist change the hCG-stimulated testosterone and cAMP production by these cells. It is concluded that mouse Leydig cells express CRF-R1, mediating elevation of testosterone production by CRF agonists through cAMP. Because potencies of CRF agonists in activating mouse Leydig cells were more than 10-fold lower compared with their potencies in stimulating rat pituitary cells, it is suggested that the coupling of the CRF-R1 to intracellular signaling in Leydig cells is different from that in corticotropic pituitary cells, at least in quantitative terms.
Expression of Growth Differentiation Factor-9 Messenger Ribonucleic Acid in Ovarian and Nonovarian Rodent and Human Tissues*
Authors: Fitzpatrick Susan L. , Sindoni Deborah M. , Shughrue Paul J. , Lane Malcolm V. , Merchenthaler Istvan J. , Frail Donald E.
Published: January 2014

Growth differentiation factor-9 (GDF-9) is a member of the transforming growth factor-β family that is reported to be expressed exclusively in the ovary, specifically in the oocyte. Female mice deficient in GDF-9 are infertile, suggesting that GDF-9 receptor agonists and antagonists may specifically modulate fertility. We now report that GDF-9 messenger RNA (mRNA) is expressed in nonovarian tissues in mice, rats, and humans. GDF-9 mRNA was detected in mouse and rat ovary, testis, and hypothalamus by Northern blot and RT-PCR analyses. The localization of GDF-9 mRNA specifically in oocytes of the mouse ovary was confirmed by in situ hybridization histochemistry. In mouse testis, although localization in Sertoli cell cytoplasm could not be ruled out, mRNA expression was observed in large pachytene spermatocytes and round spermatids. The expression of GDF-9 mRNA in human tissues was assessed by Northern blot and RT-PCR analyses. GDF-9 mRNA was observed in ovary and testis and, surprisingly, in diverse nongonadal tissues, including pituitary, uterus, and bone marrow. Therefore, GDF-9 mRNA expression in rodents is not exclusive to the ovary, but includes the testis and hypothalamus. Furthermore, human GDF-9 mRNA is expressed not only in the gonads, but also in several extragonadal tissues. The function and relevance of nongonadal GDF-9 mRNA are not known, but may affect strategies for contraception and fertility that are based on GDF-9 activity.
Regional Expression of Transforming Growth Factor-α in Rat Ventral Prostate during Postnatal Development, after Androgen Ablation, and after Androgen Replacement*
Authors: Banerjee Subhadra , Banerjee Partha P. , Zirkin Barry R. , Brown Terry R.
Published: January 2014

The prostate is a highly heterogeneous organ, composed of different types of epithelial and stromal cells organized regionally along the ductal network. Although androgen-stimulated growth and maintenance of the prostate gland primarily involve epithelial cells, it is unclear whether all epithelial cells are androgen dependent. Moreover, the actions of androgens may not be direct; a number of polypeptide growth factors, including transforming growth factor-α (TGFα), are postulated to mediate androgen action in the rat prostate. In this investigation, using an immunohistochemical technique, we examined the cellular and regional expression of TGFα in the rat ventral prostate during postnatal development to adulthood. TGFα-immunopositive cells were located throughout the ductal epithelium from postnatal days 5–20. By day 45 and thereafter, regional variation in TGFα expression became apparent; epithelial cells in the proximal segment exhibited intense staining, whereas those in the distal segment exhibited negligible staining. These observations were coincident with increased serum testosterone concentrations at puberty. To understand the role of androgen in the expression of TGFα in the epithelial cells of the distal and proximal segments of the adult rat ventral prostate, androgen was withdrawn by castration, and testosterone subsequently was administered. Androgen receptor protein expression decreased after castration and reappeared after androgen replacement in both the distal and proximal segments. TGFα staining was negligible in epithelial cells of the distal segment of intact adult rats, became prominent by 7 days after castration, but then diminished after the administration of testosterone. Western blot analyses revealed the presence of a specific 30-kDa immunoreactive form of TGFα in rat ventral prostate, and its quantity reflected the staining intensities observed in the immunohistochemical studies. These results suggest that TGFα expression is negatively regulated by androgen in epithelial cells of the distal segment. In contrast, staining for TGFα in epithelial cells of the proximal segment did not change with castration or testosterone administration, suggesting that TGFα is not regulated by androgen in this region of the ventral prostate. In summary, TGFα expression is differentially regulated among epithelial cells localized in two different regions of the ventral prostate. We hypothesize that TGFα may function as a survival factor for epithelial cells which, as a consequence of its expression, become androgen independent and thus escape apoptotic cell death after androgen ablation.
Adrenal Steroid Regulation of Neurotrophic Factor Expression in the Rat Hippocampus
Authors: Chao Helen M. , Sakai Randall R. , Ma Li Yun , McEwen Bruce S.
Published: January 2014

Adrenal steroids and neurotrophic factors are important modulators of neuronal plasticity, function, and survival in the rat hippocampus. Adrenal steroids act through two receptor subtypes, the glucocorticoid receptor (GR) and the mineralocorticoid receptor, and activation of each receptor subtype has distinct biochemical and physiological consequences. Adrenal steroids may exert their effects on neuronal structure and function through the regulation of expression of neurotrophic and growth-associated factors. We have examined adrenal steroid regulation of the neurotrophins brain-derived neurotrophic factor, neurotrophin-3, and basic fibroblast growth factor, as well as the growth associated protein GAP-43, through activation of GR or mineralocorticoid receptor with selective agonists. Our findings indicated that in CA2 pyramidal cells, adrenalectomy resulted in decreases in the levels of basic fibroblast growth factor and neurotrophin-3 messenger RNA, which were prevented by activation of mineralocorticoid but not glucocorticoid receptors. Adrenalectomy-induced increases in GAP-43 and brain-derived neurotrophic factor messenger RNA levels could be blocked by activation of glucocorticoid receptors in CA1, but not in CA3, pyramidal cells. Thus the extent to which adrenal steroids regulate hippocampal neurotrophic and growth-associated factors, appears to be dependent both on the adrenal steroid receptor subtype activated and on the hippocampal subregion examined.
Prolonged Depletion of Guanosine Triphosphate Induces Death of Insulin-Secreting Cells by Apoptosis*
Authors: Li Guodong , Segu Venkatesh Babu G. , Rabaglia Mary E. , Luo Rui-Hua , Kowluru Anjaneyulu , Metz Stewart A.
Published: January 2014

Inhibitors of IMP dehydrogenase, such as mycophenolic acid (MPA) and mizoribine, which deplete cellular GTP, are used clinically as immunosuppressive drugs. The prolonged effect of such agents on insulin-secreting β-cells (HIT-T15 and INS-1) was investigated. Both MPA and mizoribine inhibited mitogenesis, as reflected by[ 3H]thymidine incorporation. Cell number, DNA and protein contents, and cell (metabolic) viability were decreased by about 30%, 60%, and 80% after treatment of HIT cells with clinically relevant concentrations (e.g. 1 μg/ml) of MPA for 1, 2, and 4 days, respectively. Mizoribine (48 h) similarly induced the death of HIT cells. INS-1 cells also were damaged by prolonged MPA treatment. MPA-treated HIT cells displayed a strong and localized staining with a DNA-binding dye (propidium iodide), suggesting condensation and fragmentation of DNA, which were confirmed by detection of DNA laddering in multiples of about 180 bp. DNA fragmentation was observed after 24-h MPA treatment and was dose dependent (29%, 49%, and 70% of cells were affected after 48-h exposure to 1, 3, and 10 μg/ml MPA, respectively). Examination of MPA-treated cells by electron microscopy revealed typical signs of apoptosis: condensed and marginated chromatin, apoptotic bodies, cytosolic vacuolization, and loss of microvilli. MPA-induced cell death was almost totally prevented by supplementation with guanosine, but not with adenosine or deoxyguanosine, indicating a specific effect of GTP depletion. An inhibitor of protein isoprenylation (lovastatin, 10–100μ m for 2–3 days) induced cell death and DNA degradation similar to those induced by sustained GTP depletion, suggesting a mediatory role of posttranslationally modified GTP-binding proteins. Indeed, impeding the function of G proteins of the Rho family (via glucosylation using Clostridium difficile toxin B), although not itself inducing apoptosis, potentiated cell death induced by MPA or lovastatin. These findings indicate that prolonged depletion of GTP induces β-cell death compatible with apoptosis; this probably involves a direct impairment of GTP-dependent RNA-primed DNA synthesis, but also appears to be modulated by small GTP-binding proteins. Treatment of intact adult rat islets (the β-cells of which replicate slowly) induced a modest, but definite, death by apoptosis over 1- to 3-day periods. Thus, more prolonged use of the new generation of immunosuppressive agents exemplified by MPA might have deleterious effects on the survival of islet or pancreas grafts.
In Vitro Characterization of Somatostatin Receptors in the Human Thymus and Effects of Somatostatin and Octreotide on Cultured Thymic Epithelial Cells
Authors: Ferone Diego , van Hagen P. Martin , van Koetsveld Peter M. , Zuijderwijk Joke , Mooy Diana M. , Lichtenauer-Kaligis Elgin G. R. , Colao Annamaria , Bogers Ad J. J. C. , Lombardi Gaetano , Lamberts Steven W. J. , Hofland Leo J.
Published: January 2014

Somatostatin (SS) and its analogs exert inhibitory effects on secretive and proliferative processes of various cells via high affinity SS receptors (SS-R). SS analogs bind with different affinity to the five cloned SS-R subtypes. Octreotide, an octapeptide SS analog, binds with high affinity to the SS-R subtype 2 (sst2). SS-R have been demonstrated in vivo and in vitro on cells from endocrine and immune systems. Among the lymphatic tissues, the thymus has been shown to contain the highest amount of SS, suggesting a local functional role of the peptide.

We investigated the SS distribution and SS-R expression pattern in the normal human thymus using autoradiography, membrane homogenate binding studies, and RT-PCR. In addition, the effect of SS and octreotide on growth of cultured thymic epithelial cells (TEC) was studied.

By autoradiography, binding of[ 125I-Tyr0]-SS-28 and[ 125I-Tyr3]-octreotide was detected in all seven thymuses studied. Specific[ 125I-Tyr3]-octreotide binding was shown on membrane preparations from thymuses, while not from cultured thymocytes. RT-PCR showed the expression of sst1, sst2A and sst3 messenger RNA (mRNA) in the thymic tissue, whereas sst1 and sst2A mRNAs were found in isolated TEC. SS mRNA was present in thymic tissue and in isolated TEC. SS and octreotide significantly inhibited 3H-thymidine incorporation in 3 of 3 and 6 of 6 TEC cultures, respectively. The percent inhibition ranged from 38.8 to 66.8% for SS and from 19.1 to 59.5% for octreotide.

In conclusion, SS mRNA and sst1, sst2A, and sst3 mRNAs are expressed in the normal human thymus. Cultured TEC selectively express sst1 and sst2A mRNA and respond in vitro to SS and octreotide administration with an inhibition of cell proliferation. These data suggest a paracrine/autocrine role of SS and its receptors in the regulation of cell growth in thymic microenvironment.
Identification of High Affinity Binding Sites for Inhibin on Ovine Pituitary Cells in Culture**This work was supported by consecutive program grants (Regkey 943208 and 983212) from the National Health and Medical Research Council of Australia.
Authors: Hertan Ruth , Farnworth Paul Graham , Fitzsimmons Karen Lesley , Robertson David Mark
Published: January 2014

The aim of this study was to identify and characterize binding sites for inhibin in primary cultures of ovine anterior pituitary cells. Recombinant human 31-kDa inhibin A was iodinated by an optimized lactoperoxidase procedure. Fractionation of the labeled protein by gel filtration chromatography on Sephadex G-100 in 0.1 m HCl yielded two immunoactive peak regions, the second of which was bioactive as assessed by in vitro bioassay, with a ratio of bioactivity/immunoactivity of 0.62–0.77 and an iodine incorporation ratio of 1.7–2.0 mol 125I/mol inhibin. The specific binding of purified [125I]inhibin to cultured ovine pituitary cells varied with time, temperature, and cell number. Displacement of the tracer by unlabeled inhibin, as assessed by Scatchard analysis, revealed two binding sites with average Kd values of 0.28 and 3.9 nm and with approximately 250 and 3100 binding sites/anterior pituitary cell, respectively. There was little cross-reaction between inhibin and activin A (<2%), transforming growth factor-β (<0.2%), or follistatin (≪0.1%). Examination of cell lines that were not expected to have inhibin receptors showed that there was no specific binding of inhibin to human leukemia (Jurkat) cells, whereas the binding to human embryonic kidney (293) cells was displaced by both inhibin and activin with a similar degree of cross-reaction, which suggests binding to an activin receptor. It is concluded that inhibin-binding sites with high affinity and specificity have been identified on ovine pituitary cells, consistent with both inhibin action on the pituitary and the presence of the putative inhibin receptor.
Androgen Receptor, Estrogen Receptor α, and Estrogen Receptorβ Show Distinct Patterns of Expression in Forebrain Song Control Nuclei of European Starlings1
Authors: Bernard Daniel J. , Bentley George E. , Balthazart Jacques , Turek Fred W. , Ball Gregory F.
Published: January 2014

In songbirds, singing behavior is controlled by a discrete network of interconnected brain nuclei known collectively as the song control system. Both the development of this system and the expression of singing behavior in adulthood are strongly influenced by sex steroid hormones. Although both androgenic and estrogenic steroids have effects, androgen receptors (AR) are more abundantly and widely expressed in song nuclei than are estrogen receptors (ERα). The recent cloning of a second form of the estrogen receptor in mammals, ERβ, raises the possibility that a second receptor subtype is present in songbirds and that estrogenic effects in the song system may be mediated via ERβ. We therefore cloned the ERβ complementary DNA (cDNA) from a European starling preoptic area-hypothalamic cDNA library and used in situ hybridization histochemistry to examine its expression in forebrain song nuclei, relative to the expression of AR and ERα messenger RNA (mRNA), in the adjacent brain sections. The starling ERβ cDNA has an open reading frame of 1662-bp, predicted to encode a protein of 554 amino acids. This protein shares greater than 70% sequence identity with ERβ in other species. We report that starling ERβ is expressed in a variety of tissues, including brain, pituitary, skeletal muscle, liver, adrenal, kidney, intestine, and ovary. Similar to reports in other songbird species, we detected AR mRNA-containing cells in several song control nuclei, including the high vocal center (HVc), the medial and lateral portions of the magnocellular nucleus of the anterior neostriatum, and the robust nucleus of the archistriatum. We detected ERα expression in the medial portion of HVc (also called paraHVc) and along the medial border of the caudal neostriatum. ERβ was not expressed in HVc, in the medial and lateral portions of the magnocellular nucleus of the anterior neostriatum, in the robust nucleus of the archistriatum, or in area X. In contrast, ERβ mRNA-containing cells were detected in the caudomedial neostriatum and medial preoptic area in a pattern reminiscent of P450 aromatase expression in the same brain regions in other songbirds. These data suggest that estrogenic effects on the song system are not mediated via ERβ-producing cells within song nuclei. Nonetheless, the overlapping expression of ERβ- and aromatase-producing cells in the caudomedial neostriatum suggests that locally synthesized estrogens may act via ERβ, in addition to ERα, to mediate seasonal or developmental effects on nearby song nuclei (e.g. HVc).
Down-Regulation of Protein Kinase C Inhibits Insulin-Like Growth Factor I-Induced Vascular Smooth Muscle Cell Proliferation, Migration, and Gene Expression1
Authors: Yano Kenji , Bauchat Jeanette R. , Liimatta Marya B. , Clemmons David R. , Duan Cunming
Published: January 2014

Insulin-like growth factor-I (IGF-I) plays an important role in regulating vascular smooth muscle cell (VSMC) proliferation, directed migration, differentiation, and apoptosis. The signaling mechanisms used by IGF-I to elicit these actions, however, are not well defined. In this study, we examined the role(s) of protein kinase C (PKC) in mediating the IGF-I actions in cultured porcine VSMCs. Out of the eleven known members of PKC family, PKC-α, -βI, -ε, -η, -λ,θ , and -ζ, were detectable by Western immunoblot analysis in these cells. Further analysis indicated that the subcellular distribution of several PKC isoforms is regulated by IGF-I. While IGF-I stimulated membrane translocation of PKC-η, -ε, and -ζ and regulated the cytosolic levels of PKC-βI, it had no such effect on PKC-α and -λ. To examine whether PKC activation is required for the IGF-I-regulated biological responses, phorbol myristate acetate (PMA) and GF109203X were used to down-regulate or inhibit PKC activity. Both PMA (1 μm) and GF109203X (20 μm) nearly completely suppressed the total PKC activity after a 30-min incubation (> 90%), and this inhibition lasted for at least 24 h. Down-regulation or inhibition of PKC activity abolished the IGF-I-induced DNA synthesis, migration and IGFBP-5 gene expression. In contrast, the IGFBP-5 expression induced by forskolin was unaffected by PKC down-regulation or inhibition, suggesting that PKC activation is required for the IGF-regulated but not the cAMP-regulated events. Because the actions of IGF-I on DNA synthesis and IGFBP-5 gene expression in VSMCs have been shown to be mediated through the phosphatidylinositol 3-kinase (PI3 kinase) signaling pathway in porcine VSMCs, the potential role of PKC in IGF-I-induced activation of PI3 kinase and PKB/Akt were examined. Treatment with either PMA or GF109203X did not significantly affect the effects of IGF-I on PI3 kinase activation or PKB/Akt phosphorylation. These results indicated that PKC-βI, -η, -ε, and -ζ may play an essential role(s) in IGF-I regulation of VSMC migration, DNA synthesis and gene expression, and that these PKC isoforms may either act independently of the PI3 kinase pathway or act further downstream of PKB/Akt in the IGF signaling network.
Adrenal Capillary Endothelial Cells Stimulate Aldosterone Release through a Protein That Is Distinct from Endothelin1
Authors: Rosolowsky Lori J. , Hanke Craig J. , Campbell William B.
Published: January 2014

We tested the possibility that bovine adrenal capillary endothelial cells (ECs) stimulate aldosterone secretion from bovine zona glomerulosa (ZG) cells by the release of a transferable factor. In coincubations of ZG cells and ECs in serum-free medium, aldosterone release was stimulated approximately 17-fold, and the stimulation was related to the concentration of ECs. The maximal stimulation by ECs was 75% of the maximal response to ACTH. In contrast, adrenal pericytes and fibroblasts were without effect. ECs incubated alone without ZG cells did not produce aldosterone. Conditioned medium from ECs (EC-CM), but not adrenal fibroblasts, stimulated aldosterone release approximately 3-fold. The stimulation increased with the concentration of EC-CM and the duration of conditioning time. Steroidogenic activity in EC-CM was abolished by pronase treatment, indicating that the active factor was a protein. However, the activity in EC-CM was distinct from that of endothelin-1 (ET-1), an endothelial peptide that also stimulates aldosterone secretion, as it was not blocked by the ETB receptor antagonist PD-145065, it did not alter[ 125I]ET-1 binding to ZG cells, and its release occurred before the release of ET-1. Neither ECs nor EC-CM stimulated the production of cortisol from zona fasciculata cells. The activity of EC-CM was not blocked by an angiotensin II AT1 receptor antagonist or a bradykinin B2 receptor antagonist. EC-CM stimulated increased intracellular calcium in fura-2-loaded ZG cells, but did not increase the production of cAMP. Using gel filtration, this peptide had an approximate molecular mass of 3000 Da and migrated earlier than ET-1. This study demonstrates that ECs in vitro alter steroidogenesis through the release of a transferable substance and suggests the existence of an endothelium-derived steroidogenic factor that is produced by adrenal capillary ECs. This endothelium-derived steroidogenic factor may function in the adrenal gland as a paracrine regulator of aldosterone secretion.
Partial Characterization of Circulating Inhibin-B and Pro-αC During Development in the Male Rhesus Monkey1
Authors: Winters Stephen J. , Plant Tony M.
Published: January 2014

Gel filtration chromatography and ELISAs for inhibin-B and pro-αC were used to examine the circulating forms of inhibin in the neonatal (age 2–6 weeks), juvenile (age 1–2 yr), and adult male rhesus monkey. In all samples, isoforms of inhibin-B of 26–36K and 150K were found. Both forms were significantly greater in the adult. Theα -subunit assay detected major peaks at 45–60 and 29–31K, and a minor peak of greater than 100K. As for inhibin-B, the major forms of inhibin pro-αC were highest in adulthood. Inhibin-B and pro-αC were measurable in peripheral plasma at age 1 week, increased with the neonatal rise in plasma FSH, and then decreased but remained detectable through age 1 yr. Values in adult males were higher than at any time during the first year of life. Finally, mean values of plasma inhibin-B and pro-αC in five monkeys, based on multiple blood samples drawn between age 1 week and 1 yr, were rank ordered and were found to be highly positively correlated (r = 0.96), suggesting that inhibin levels in the first year of life may be a marker of Sertoli cell number, and may predict the spermatogenic capacity of the testis in adulthood.
Endotoxin Stimulates Nitric Oxide Production in the Paraventricular Nucleus of the Hypothalamus through Nitric Oxide Synthase I: Correlation with Hypothalamic-Pituitary-Adrenal Axis Activation1
Authors: Uribe Rosa Maria , Lee Soon , Rivier Catherine
Published: January 2014

Nitric oxide (NO) is an unstable gas that is produced in brain tissues involved in the control of the activity of the hypothalamus-pituitary-adrenal (HPA) axis. Transcripts for constitutive neuronal NO synthase (NOS I), one of the enzymes responsible for NO formation in the brain, is up-regulated by systemic endotoxin[ lipopolysaccharide (LPS)] injection. However, this change is delayed compared with LPS induced-ACTH release, which makes it difficult to determine whether it is functionally important for the hormonal response. To obtain a more resolutive time course of the NO response, we first measured NO in microdialysates of the paraventricular (PVN) nucleus of the hypothalamus. The iv injection of 100 μg/kg LPS induced a rapid and short-lived increase in concentrations of this gas, which corresponded to the initiation of the ACTH response. LPS-induced Ca2+-dependent NOS activity in the PVN as well as the number of PVN cells expressing citrulline (a compound produced stoichiometrically with NO) also increased significantly over a time course that corresponded to ACTH and corticosterone release. Finally, blockade of NO production with the arginine derivative Nω-nitro-l-arginine-methylester (L-NAME; 50 mg/kg, sc), which attenuated the ACTH response to LPS, virtually abolished basal NOS activity in the PVN, as well as anterior and neurointermediate lobes of the pituitary, and prevented the appearance of citrulline in the PVN of rats injected with LPS.

Collectively, these results show that LPS-induced activation of the HPA axis correlates with the activation of the PVN NOergic system, and supports a stimulatory role for NO in the modulation of the HPA axis in response to immune challenges.
Central Administration of Leptin to Ovariectomized Ewes Inhibits Food Intake without Affecting the Secretion of Hormones from the Pituitary Gland: Evidence for a Dissociation of Effects on Appetite and Neuroendocrine Function*
Authors: Henry Belinda A. , Goding James W. , Alexander Warren S. , Tilbrook Alan J. , Canny Benedict J. , Dunshea Frank , Rao Alexandra , Mansell Ashley , Clarke Iain J.
Published: January 2014

We have studied the effect of leptin on food intake and neuroendocrine function in ovariectomized ewes. Groups (n = 5) received intracerebroventricular infusions of either vehicle or leptin (20μ g/h) for 3 days and were blood sampled over 6 h on days −1, 2, and for 3 h on day 3 relative to the onset of the infusion. The animals were then killed to measure hypothalamic neuropeptide Y expression by in situ hybridization. Plasma samples were assayed for metabolic parameters and pituitary hormones. Food intake was reduced by leptin, but did not change in controls. Leptin treatment elevated plasma lactate and nonesterified fatty acids, but did not affect glucose or insulin levels, indicating a state of negative energy balance that was met by the mobilization of body stores. Pulse analysis showed that the secretion of LH and GH was not affected by leptin treatment, nor were the mean plasma concentrations of FSH, PRL, or cortisol. Expression of messenger RNA for neuropeptide Y in the arcuate nucleus was reduced by the infusion of leptin, primarily due to reduced expression per cell rather than a reduction in the number of cells observed. Thus, the action of leptin to inhibit food intake is dissociated from neuroendocrine function. These results suggest that the metabolic effects of leptin are mediated via neuronal systems that possess leptin receptors rather than via endocrine effects.
Prostaglandin E2 Cooperates with TRANCE in Osteoclast Induction from Hemopoietic Precursors: Synergistic Activation of Differentiation, Cell Spreading, and Fusion
Authors: Wani Mohan R. , Fuller Karen , Kim Nack Sung , Choi Yongwon , Chambers Tim
Published: January 2014

It was recently found that osteoblastic cells express TRANCE (tumor necrosis factor-related activation-induced cytokine), a newly identified member of the tumor necrosis factor superfamily, and that expression was increased by calciotropic hormones. Furthermore, soluble recombinant TRANCE induces osteoclast formation and resorption in stroma-free populations of hemopoietic precursor cells. However, overexpression of the decoy receptor osteoprotegerin in vivo shows that there are substantial differences in the sensitivity of different sites to resorption-inhibition, suggesting that either alternative ligands exist or the sensitivity of osteoclasts to TRANCE can be modified by cofactors. We therefore tested the possibility that cofactors might enhance osteoclast formation by TRANCE. We found that the number of tartrate-resistant acid phosphatase-positive and calcitonin receptor-positive cells was increased by a factor of 10 by the presence of PGE2 in the absence of stromal cells. Moreover, although the tartrate-resistant acid phosphatase-positive cells that formed in TRANCE alone were typically mononuclear and poorly spread, the addition of PGE2 induced the formation of large, well spread multinuclear cells. There was an increase in bone resorption that corresponded with the increase in osteoclast number. PGE2 did not synergize with TRANCE for resorption-stimulation in mature cells. 8-Bromo-cAMP showed a similar syngergistic effect on osteoclastic differentiation. Thus, PGE2 appears to stimulate bone resorption through a direct effect on hemopoietic precursors, primarily through a synergistic effect on the ability of TRANCE to induce osteoclastic differentiation.
Evidence for Stanniocalcin Gene Expression in Mammalian Bone*
Authors: Yoshiko Yuji , Son Aoi , Maeda Shuji , Igarashi Akira , Takano Shoichi , Hu Jianguo , Maeda Norihiko
Published: January 2014

Stanniocalcin (STC) acts as a regulator of calcium and phosphate homeostasis in an endocrine manner in bony fish. Recently, complementary DNAs encoding human and mouse STC have been characterized, and the messenger RNA (mRNA) expression was identified in various tissues, such as kidney, small intestine, prostate, thyroid, and ovary. Because previous studies concerning the effects of fish STC on mammalian bone have been discussed, there is a good possibility that mammalian STC is a local factor in bone. Here, we demonstrated STC mRNA expression in neonatal mouse calvaria, the primary cultured mouse osteoblast-rich fractions, and human and mouse osteoblastic cell lines. We also mapped the cellular distribution of the STC mRNA in femur and calvaria in developing mice. Several transcripts with a major 4-kb band were detected in all samples. The cellular distribution of the mRNA expression corresponded closely to osteoblasts in both femur and calvaria. Significant labeling of the STC mRNA was also identified in chondrocytes but not in osteoclasts and other bone marrow elements. These results are the first evidence that hormone may be actually expressed in osteoblasts and chondrocytes, and they strongly implicate the involvement of local STC in both endochondral and membrane bone as an autocrine/paracrine factor.