Guipi decoction effects on brain somatostatin levels and receptor mRNA expression in rats with spleen deficiency*☆

BACKGROUND： Somatostatin is abundant in the hypothalamus, cerebral cortex, limbic system, and mesencephalon. Somatostatin mRNA expression in the brain of rats with spleen deficiency is noticeably reduced, as well as attenuation of cognitive function. OBJECTIVE： To observe the interventional effect of Guipi decoction on somatostatin level and somatostatin receptor 1 （SSTRl） mRNA expression in different encephalic regions of rats with spleen deficiency, and to compare the interventional effects of Guipi decoction, Chaihu Shugan powder, and Tianwang Buxin pellet. DESIGN： A randomized controlled observation.
SETTING： Basic Medical College, Beijing University of Traditional Chinese Medicine.
MATERIALS： Fifty adult Wistar male rats, of clean grade, weighing （160 ± 10） g, were provided by Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. The protocol was performed in accordance with ethical guidelines for the use and care of animals. Somatostatin 1 polyclonal anti-rabbit antibody and SSTRl in situ hybridization kit were provided by Department of Neuroanatomy, Shanghai Second Military Medical University of Chinese PLA. The drug for developing rat models of spleen deficiency was composed of Dahuang, Houpu and Zhishi, and prepared at 2：1：1. Guipi decoction, Chaihu Shugan powder, and Tianwang Buxin pellet recipes were made according to previous studies. METHODS： This study was performed at the Basic Medical College, Beijing University of Traditional Chinese Medicine from March 2002 to March 2005. The rats were randomly divided into 5 groups, with 10 rats in each group： normal, model, Guipi decoction, Chaihu Shugan powd.er, and Tianwang Buxin pellet groups. Rat models of the latter 4 groups were developed by methods of purgation with bitter and cold nature drugs, improper diet, and overstrain. The rats received 7.5 g/kg of the drugs each morning and were fasted every other day, but were allowed free access to water at all times. The rats were forced to swim in 25 ℃ water until fati     

Anatomically specific changes in the expression of somatostatin, growth hormone-releasing hormone and growth hormone receptor mRNA in diabetic rats

Growth hormone (GH) secretion is altered in poorly controlled diabetic animals. However, modifications in the hypothalamic neuropeptides that control GH secretion, somatostatin and GH-releasing hormone (GHRH), as well as changes in the sensitivity of the hypothalamus and pituitary to the feedback effects of GH, are less clear. We have used RNase protection assays and in-situ hybridization to address whether the mRNA expression of GH, somatostatin and GHRH, as well as of the GH receptor (GHR) in the hypothalamus and anterior pituitary, are altered in streptozotocin-induced diabetic rats. After induction of diabetes, rats were treated with insulin twice daily for 3 weeks to obtain either poorly controlled (mean plasma glucose >300 mg/dl) or well-controlled diabetic rats. Although no significant change in pituitary GH mRNA expression was found, the hypothalamic expression of GHRH and somatostatin mRNA was reduced in poorly-controlled diabetic rats and returned to control values with normalisation of plasma glucose concentrations (P<0.0001 and P<0.002, respectively). Somatostatin mRNA expression was reduced only in the central portion of the periventricular nucleus, with no change being seen in the other areas of the periventricular nucleus or in the arcuate, suprachiasmatic or paraventricular nuclei. A significant decline in GHRH mRNA expression was observed in both the arcuate nucleus and ventromedial hypothalamus. Anterior pituitary GHR mRNA expression was significantly reduced in both well and poorly-controlled diabetic rats, while there was no change in the hypothalamus. To examine whether the evolution time of the diabetes influences these parameters, in a subsequent experiment, diabetic rats received no insulin for 2 months. A significant decline in GHRH and somatostatin mRNA expression was also observed in these rats. In addition, pituitary GH mRNA expression declined significantly in long-term diabetic rats. These results demonstrate that: (1) the expression of both GHRH and somatostatin declines specifically in anatomical areas involved in anterior pituitary hormone control; (2) GHR mRNA expression is decreased in the pituitary of diabetic rats, but not in the hypothalamus, and does not return to control values with normalisation of mean blood glucose concentrations; and (3) the evolution time of the diabetes is important for detecting some changes, including the decrease in pituitary GH mRNA expression.     

Somatostatin dramatically stimulates growth hormone release from primate somatotrophs acting at low doses via somatostatin receptor 5 and cyclic AMP

Somatostatin and cortistatin have been shown to act directly on pituitary somatotrophs to inhibit growth hormone (GH) release. However, previous results from nonprimate species indicate that these peptides can also directly stimulate GH secretion, at low concentrations. The relevance of this phenomenon in a nonhuman primate model was investigated in the present study by testing the impact of somatostatin/cortistatin on GH release in primary pituitary cell cultures from baboons. High doses (> 10(-10) m) of somatostatin/cortistatin did not alter basal GH secretion but blocked GH-releasing hormone (GHRH)- and ghrelin-induced GH release. However, at low concentrations (10(-17)-10(-13) m), somatostatin/cortistatin dramatically stimulated GH release to levels comparable to those evoked by GHRH or ghrelin. Use of somatostatin receptor (sst) specific agonists/antagonists, and signal transduction blockers indicated that sst2 and sst1 activation via intact adenylate cylcase and mitogen-activated protein kinase systems mediated the inhibitory actions of high-concentration somatostatin. By contrast, the stimulatory actions of low-dose somatostatin on GH release were mediated by sst5 signalling through adenylate cylcase/cAMP/protein kinase A and intracellular Ca(2+) pathways, and were additive with ghrelin (not GHRH). Notably, low-concentrations of somatostatin, similar to sst5-agonists, inhibited prolactin release. These results clearly demonstrate that the ultimate impact of somatostatin/cortistatin on hormone release is dose-dependent, cell type-selective and receptor-specific, where the stimulatory effects of low-concentration somatostatin/cortistatin on GH release extend to primates, thereby supporting the notion that this action is relevant in regulating GH secretion in humans.     

Somatostatin and its receptor family.

Somatostatin (SST), a regulatory peptide, is produced by neuroendocrine, inflammatory, and immune cells in response to ions, nutrients, neuropeptides, neurotransmitters, thyroid and steroid hormones, growth factors, and cytokines. The peptide is released in large amounts from storage pools of secretory cells, or in small amounts from activated immune and inflammatory cells, and acts as an endogenous inhibitory regulator of the secretory and proliferative responses of target cells that are widely distributed in the brain and periphery. These actions are mediated by a family of seven transmembrane (TM) domain G-protein-coupled receptors that comprise five distinct subtypes (termed SSTR1-5) that are endoded by separate genes segregated on different chromosomes. The five receptor subtypes bind the natural SST peptides, SST-14 and SST-28, with low nanomolar affinity. Short synthetic octapeptide and hexapeptide analogs bind well to only three of the subtypes, 2, 3, and 5. Selective nonpeptide agonists with nanomolar affinity have been developed for four of the subtypes (SSTR1, 2, 3, and 4) and putative peptide antagonists for SSTR2 and SSTR5 have been identified. The ligand binding domain for SST ligands is made up of residues in TMs III-VII with a potential contribution by the second extracellular loop. SSTRs are widely expressed in many tissues, frequently as multiple subtypes that coexist in the same cell. The five receptors share common signaling pathways such as the inhibition of adenylyl cyclase, activation of phosphotyrosine phosphatase (PTP), and modulation of mitogen-activated protein kinase (MAPK) through G-protein-dependent mechanisms. Some of the subtypes are also coupled to inward rectifying K(+) channels (SSTR2, 3, 4, 5), to voltage-dependent Ca(2+) channels (SSTR1, 2), a Na(+)/H(+) exchanger (SSTR1), AMPA/kainate glutamate channels (SSTR1, 2), phospholipase C (SSTR2, 5), and phospholipase A(2) (SSTR4). SSTRs block cell secretion by inhibiting intracellular cAMP and Ca(2+) and by a receptor-linked distal effect on exocytosis. Four of the receptors (SSTR1, 2, 4, and 5) induce cell cycle arrest via PTP-dependent modulation of MAPK, associated with induction of the retinoblastoma tumor suppressor protein and p21. In contrast, SSTR3 uniquely triggers PTP-dependent apoptosis accompanied by activation of p53 and the pro-apoptotic protein Bax. SSTR1, 2, 3, and 5 display acute desensitization of adenylyl cyclase coupling. Four of the subtypes (SSTR2, 3, 4, and 5) undergo rapid agonist-dependent endocytosis. SSTR1 fails to be internalized but is instead upregulated at the membrane in response to continued agonist exposure. Among the wide spectrum of SST effects, several biological responses have been identified that display absolute or relative subtype selectivity. These include GH secretion (SSTR2 and 5), insulin secretion (SSTR5), glucagon secretion (SSTR2), and immune responses (SSTR2).     

Differential expression of somatostatin receptor subtypes in brain.

The tetradecapeptide somatostatin has been implicated as an important regulator of neuronal and neuroendocrine function in the CNS. The cellular actions of somatostatin are mediated by specific receptors. The genes encoding two different somatostatin receptors (SSTRs) have been isolated and characterized, and RNA blotting studies have shown that both SSTR1 and SSTR2 are expressed in the brain. In order to gain a better understanding of the functions of somatostatin in the CNS, the distribution of SSTR1 and SSTR2 mRNAs was determined using the technique of in situ hybridization. SSTR1 mRNA was present throughout the mouse brain, particularly in the supra- and infragranular layers of the cortex, the amygdala, hippocampus, bed nucleus of the stria terminalis, substantia innominata, hypothalamus, pretectum, substantia nigra, parabrachial nucleus, and nucleus of the solitary tract. SSTR2 mRNA was primarily observed in the infragranular layers of the cortex, the amygdala, claustrum, endopiriform nucleus, arcuate and paraventricular nuclei of the hypothalamus, and medial habenular nucleus. Several regions of the brain reported to contain dense somatostatin-like immunoreactive terminal fields and receptor binding sites were devoid of both SSTR1 and SSTR2 mRNA, suggesting the existence of additional SSTR subtypes.     

Inhibitory effect of pasireotide and octreotide on lymphocyte activation

Somatostatin (SST) regulates the function of the central and peripheral nervous system, the endocrine and exocrine organs, as well as the vascular and immune system. These actions are mediated by five specific membrane somatostatin receptors. This study compares the effects on human lymphocytes of two long-acting somatostatin analogues that have different receptor affinity: octreotide and pasireotide. Both analogues have an antiproliferative effect on human lymphocyte proliferation, but they act at different concentration and, while octreotide enhances IL10 and inhibits gamma IFN pasireotide inhibits IL2 and gamma IFN. In both sets of experiment the different behaviour of the two analogues could be due to their different affinity to the SSTR subtypes. Finally this study suggest that the growth inhibitory action of somatostatin analogues is an apoptotic phenomenon and it can be mediated by SSTR2a, in the case of octreotide, and by SSTR3 when pasireotide is used or it can be mediated by the heterodimerization of the two receptor.     

Somatostatin receptor 2 knockout/lacZ knockin mice show impaired motor coordination and reveal sites of somatostatin action within the striatum

The peptide somatostatin can modulate the functional output of the basal ganglia. The exact sites and mechanisms of this action, however, are poorly understood, and the physiological context in which somatostatin acts is unknown. Somatostatin acts as a neuromodulator via a family of five 7-transmembrane G protein-coupled receptors, SSTR1-5, one of which, SSTR2, is known to be functional in the striatum. We have investigated the role of SSTR2 in basal ganglia function using mice in which Sstr2 has been inactivated and replaced by the lacZ reporter gene. Analysis of Sstr2lacZ expression in the brain by beta-galactosidase histochemistry demonstrated a widespread pattern of expression. By comparison to previously published in situ hybridization and immunohistochemical data, Sstr2lacZ expression was shown to accurately recapitulate that of Sstr2 and thus provided a highly sensitive model to investigate cell-type-specific expression of Sstr2. In the striatum, Sstr2 expression was identified in medium spiny projection neurons restricted to the matrix compartment and in cholinergic interneurons. Sstr2 expression was not detected in any other nuclei of the basal ganglia except for a sparse number of nondopaminergic neurons in the substantia nigra. Microdialysis in the striatum showed Sstr2-null mice were selectively refractory to somatostatin-induced dopamine and glutamate release. In behavioural tests, Sstr2-null mice showed normal levels of locomotor activity and normal coordination in undemanding tasks. However, in beam-walking, a test of fine motor control, Sstr2-null mice were severely impaired. Together these data implicate an important neuromodulatory role for SSTR2 in the striatum.     

Inhibition of tissue factor by ixolaris reduces primary tumor growth and experimental metastasis in a murine model of melanoma

Melanoma is a highly metastatic cancer and there is strong evidence that the clotting initiator protein, tissue factor (TF), contributes to its aggressive pattern. TF inhibitors may attenuate primary tumor growth and metastasis. In this study, we evaluated the effect of ixolaris, a TF inhibitor, on a murine model of melanoma B16F10 cells. Enzymatic assays performed with B16F10 and human U87-MG tumor cells as the TF source showed that ixolaris inhibits the generation of FX in either murine, human or hybrid FVIIa/TF complexes. The effect of ixolaris on the metastatic potential was further estimated by intravenous injection of B16F10 cells in C57BL/6 mice. Ixolaris (250 μg/kg) dramatically decreased the number of pulmonary tumor nodules (4 ± 1 compared to 47 ± 10 in the control group). Furthermore, a significant decrease in tumor weights was observed in primary tumor growth assays in animals treated with ixolaris (250 μg/kg) from days 3 to 18 after a subcutaneous inoculation of melanoma cells. Remarkably, immunohistochemical analyses showed that inhibition of melanoma growth by ixolaris is accompanied by a significant downregulation of both vascular endothelial growth factor (VEGF) expression and microvascular density in the tumor mass. Our data demonstrate that ixolaris targets B16F10 cell-derived TF, resulting in the reduction of both the primary tumor growth and the metastatic potential of melanoma, as well as the inhibition of tumor angiogenesis. Therefore TF may be a potential target for the treatment of this aggressive malignancy.     

Effects of Chronic Octreotide Treatment on GH Secretory Dynamics and Tumor Growth in Rats Bearing an Ectopic Somatotroph (GC) Tumor

The effects of octreotide, a long-acting somatostatin agonist selective of the sstr2/sstr3/sstr5 receptor subtypes, on ectopic GH secretion and tumor growth were investigated in Wistar-Furth female rats implanted with GH secreting (GC) cells which express mostly somatostatin receptors of the sstr1 and sstr2 subtypes. Octreotide dose dependently inhibited thymidine incorporation (?57%) and GH secretion (?41%) from GC cells in culture. In vivo, 6 weeks after GC cell implantation, plasma GH, IGF-1 and insulin levels were highly elevated. Cluster analysis of GH secretory dynamics revealed that GH secretion was less pulsatile in GC-implanted than in control animals. Furthermore, in GC-implanted animals, passive immunization either with SRIH or GHRH antisera did not affect GH plasma levels. Three weeks after GC cell implantation, when tumors became palpable, octreotide (1 μg/h/kg BW) or saline was infused constantly for three weeks by osmotic minipumps. In octreotide treated rats, GH, IGF-1 and insulin levels were not different from sham-implanted animals and tumors weight were reduced by 80%. High affinity somatostatin binding sites were found in equivalent amounts on tumors from octreotide-treated or saline-treated animals. These findings indicate that GH secretion in GC-rats is mainly derived from the tumors and independent of hypothalamic control and that octreotide reduces both GH secretion and tumor growth. We conclude that the GC-implanted rat represents a good animal model to test the antisecretory and antitrophic properties of somatostatin analogs in vivo.     

Identification of cells expressing somatostatin receptor 2 in the gastrointestinal tract of Sstr2 knockout/lacZ knockin mice

Somatostatin is found in neurons and endocrine cells in the gastrointestinal tract. The actions of somatostatin are mediated by a family of G-protein-coupled receptors that compose five subtypes (SSTR1-5), each of which is encoded by a separate gene. lacZ "knockin" mice, in which the reporter gene lacZ was engineered into the genomic locus of Sstr2 by gene targeting, were used to examine the expression pattern of Sstr2 and identify potential targets for neurally released and hormonal somatostatin in the gastrointestinal tract. In the body of the stomach, a large proportion of epithelial cells and subpopulations of myenteric neurons expressed Sstr2. Double- or triple-labeling with antisera to H(+)K(+)ATPase (to identify parietal cells) and/or histidine decarboxylase (to identify enterochromaffin-like [ECL] cells) combined with beta-galactosidase staining revealed that both parietal cells and ECL cells expressed Sstr2, and these two cell types accounted for almost all of the Sstr2-expressing epithelial cells. Somatostatin inhibits gastric acid secretion. The presence of SSTR2 on both parietal and ECL cells suggests that somatostatin acting on SSTR2 may reduce acid secretion by both acting directly on parietal cells and by reducing histamine release from ECL cells. In the small and large intestine, subpopulations of neurons in the myenteric and submucosal plexuses expressed Sstr2, and many of the Sstr2-expressing myenteric neurons also showed SSTR2(a) immunostaining. Most of Sstr2-expressing neurons in the myenteric plexus showed nitric oxide synthase (NOS) immunoreactivity. Previous studies have shown that NOS neurons are descending interneurons and anally projecting, inhibitory motor neurons. Thus, somatostatin acting at SSTR2 receptors on NOS neurons might modulate descending relaxation.     

Growth hormone releasing factor increases growth hormone release from MtTW15 pituitary tumors

The MtTW15 pituitary tumor secretes growth hormone and prolactin. Perifusion of these dispersed MtTW15 tumor cells with 10 nM growth hormone releasing factor (GRF) increases growth hormone release without affecting prolactin release. This effect is dose-dependent between 0.001 and 0.1 nM and is blocked by 100 nM somatostatin. These findings suggest that this tumor and clones derived from it may be valuable tools in studying the cellular mechanisms of action of GRF and somatostatin.     

Ontogenesis of N-acetyl-aspartate and N-acetyl-aspartyl-glutamate in rat brain

The MtTW15 pituitary tumor secretes growth hormone and prolactin. Perfusion of these dispersed MtTW15 tumor cells with 10 nM growth hormone releasing factor (GRF) increases growth hormone release without affecting prolactin release. This effect is dose-dependent between 0.001 and 0.1 nM and is blocked by 100 nM somatostatin. These findings suggest that this tumor and clones derived from it may be valuable tools in studying the cellular mechanisms of action of GRF and somatostatin.     

Desensitization of somatostatin-induced inhibition of low extracellular magnesium concentration-induced calcium spikes in cultured rat hippocampal neurons

Neuronal excitability is inhibited by somatostatin, which might play important roles in seizure and neuroprotection. The possibility of whether the effect of somatostatin on neurotransmission is susceptible to desensitization was investigated. We tested the effects of prolonged exposure to somatostatin on 0.1 mM extracellular Mg(2+) concentration ([Mg(2+)](o))-induced intracellular free Ca(2+) concentration ([Ca(2+)](i)) spikes in cultured rat hippocampal neurons using fura-2-based microfluorimetry. Reducing [Mg(2+)](o) to 0.1 mM elicited repetitive [Ca(2+)](i) spikes. These [Ca(2+)](i) spikes were inhibited by exposure to somatostatin-14. The inhibitory effects of somatostatin were blocked by pretreatment with pertussis toxin (PTX, 100 ng/ml) for 18-24 h. Prolonged exposure to somatostatin induced a desensitization of the somatostatin-induced inhibition of [Ca(2+)](i) spikes in a concentration-dependent manner. The somatostatin-induced desensitization was retarded by the nonspecific protein kinase C (PKC) inhibitor staurosporin (100 nM) or chronic treatment with phorbol dibutyrate (1 microM) for 24 h, but not by the protein kinase A inhibitor KT5720. The desensitization was significantly retarded by the novel PKCepsilon translocation inhibitor peptide (1 microM). In addition, suramin (3 microM), an inhibitor of G-protein-coupled receptor kinase 2 (GRK2), caused a reduction in the desensitization. After tetrodotoxin (TTX, 1 microM) completely blocked the low [Mg(2+)](o)-induced [Ca(2+)](i) spikes, glutamate-induced [Ca(2+)](i) transients were slightly inhibited by somatostatin and the inhibition was desensitized by prolonged exposure to somatostatin. These results indicate that the prolonged activation of somatostatin receptors induces the desensitization of somatostatin-induced inhibition on low [Mg(2+)](o)-induced [Ca(2+)](i) spikes through the activation of GRK2 and partly a novel PKCepsilon in cultured rat hippocampal neurons.     

Somatostatin receptor subtypes 2 and 5 mediate inhibition of egg yolk‐induced gall bladder emptying in mice

Background Somatostatin inhibits gall bladder contraction. Impaired gall bladder emptying is associated with gall bladder stone formation. The incidence of cholecystolithiasis is high in patients treated with a somatostatin agonist octreotide, which predominantly interacts with somatostatin receptor subtype 2 (SSTR2). Therefore, it is believed that SSTR2 regulates gall bladder contraction; however, evidence has not been provided. Here, we evaluate the effects of SSTR1‐SSTR5‐selective agonists on egg yolk‐induced gall bladder contraction in mice. Methods Homozygous deletion of SSTR2 and SSTR5 was generated by cross‐mating of SSTR2^?/? with SSTR5^?/? mice. Mice of different genotypes were injected with SSTR1‐5‐selective agonists or octreotide 15 min before induction of gall bladder emptying by egg yolk. One hour later, gall bladders were removed and weighed. Key Results Egg yolk‐reduced gall bladder weights in all mice, irrespective of their genotype. Octreotide was the most potent inhibitor of gall bladder emptying in wild‐type mice. In contrast, agonists with high selectivity for SSTR2 or SSTR5 inhibited gall bladder emptying by approximately 50–60%, whereas SSTR1‐, SSTR3‐ and SSTR4‐selective agonists failed to influence gall bladder contraction. In SSTR2^?/? mice, octreotide and an SSTR5‐selective agonist inhibited gall bladder emptying by approximately 50%, whereas SSTR2‐selective agonists were inactive. Octreotide inhibited gall bladder emptying in SSTR5^?/? mice by approximately 50%, without any effect in SSTR2^?/?/SSTR5^?/? mice. Conclusions & Inferences Our study provides evidence for the role of SSTR2 and SSTR5 in regulating gall bladder emptying in mice.     

Activation of multiple growth factor signalling pathways is frequent in meningiomas

A minority of meningiomas are difficult to treat with surgery or radiotherapy, and chemotherapeutic alternatives are limited. This study aims to better understand pathways that are active in meningiomas, in order to direct future treatment strategies. We investigated the expression and activation of multiple growth factor receptors, their ligands and downstream signalling pathways in 30 meningiomas using immunohistochemistry. Expression was correlated with chromosome 22q loss. Membrane expression of VEGF receptor (VEGFR) and platelet‐derived growth factor receptor (PDGFR)β was seen in 83% of tumors, Axl in 70%, EGFR in 50% and insulin‐like growth factor receptor in 47%. Expression was similar in low‐ and high‐grade tumors, but membrane EGFR expression was not seen in tumors showing chromosome 22q loss (P < 0.05). Expression of ligands (IGF, NRG, VEGF, Gas 6), and signalling proteins (Mek, Erk, Jnk, Akt) and pS6RP, was widespread. Western blot confirmed widespread Axl expression and supported selective expression of EGFR in NF2‐intact meningiomas. The majority of meningiomas express and show activation of multiple growth factor receptors and their signalling pathways, irrespective of tumor grade. In addition to previously reported receptors, Axl offers a new therapeutic target. The findings also suggest that anti‐EGFR based therapies may be less effective in meningiomas with 22q loss.     

Somatostatin, an inhibitor of ACTH secretion, decreases cytosolic free calcium and voltage-dependent calcium current in a pituitary cell line

Somatostatin is a neurohormone peptide that inhibits a variety of secretory responses in different cell types. We have investigated the effects of somatostatin on calcium current and intracellular free calcium in AtT-20 cells, a pituitary tumor line in which the inhibitory actions of this peptide have been well characterized. At concentrations similar to those that inhibit adrenocorticotropic hormone (ACTH) release, somatostatin and its analogs reduced the levels of intracellular free calcium (as measured by the Quin-2 technique). Nifedipine and other blockers of voltage-dependent calcium channels also reduced cytosolic calcium levels. The effects of somatostatin and nifedipine were not additive, suggesting that somatostatin might inhibit calcium channels. Experiments using the whole-cell patch-clamp technique showed that somatostatin reduces voltage-dependent calcium current. The effects of somatostatin on cytosolic calcium and calcium current appear to be independent of its ability to reduce secretagogue-stimulated cAMP accumulation in these cells. We propose that the somatostatin-induced decrease in cytosolic calcium concentrations and the voltage-dependent calcium current are one of the mechanisms by which somatostatin suppresses ACTH release in AtT-20 cells.     

Gonadotropin-releasing hormone (GnRH) and its receptor in human meningiomas

Objective

Meningiomas are the most common neoplasms of the central nervous system and are more frequent in women than in men. Many studies have been conducted to determine whether the progesterone receptor (PR) and estrogen receptor (ER) are present or absent in meningiomas. No previous studies, however, have investigated the status (presence or absence) of gonadotropin-releasing hormone (GnRH) and its receptor (GnRH-R), two major factors related to PR and ER, in meningiomas. This study aims to determine the status of GnRH and GnRH-R and to elucidate the correlations of GnRH and GnRH-R with PR, ER, and clinical features in meningiomas.

Methods

Eighty-two specimens of human meningiomas were obtained for immunohistochemical analysis with anti-GnRH, anti-GnRH-R, anti-PR, anti-ER, and anti-Ki-67 (MIB-1) antibodies, and for RT-PCR analysis of the mRNA expressions of GnRH and GnRH-R. Correlations of GnRH and GnRH-R with PR, ER, Ki-67, and clinical features such as age, sex, tumor grade, and tumor histology were assessed.

Results

Seventy-eight (95.1%) of the 82 meningiomas reacted positively in the cytoplasm for the GnRH-R. Forty-nine (59.8%) of the 82 cases reacted positively in the cytoplasm for the GnRH. The positive immunoreactivity for GnRH-R and GnRH was confirmed by the RT-PCR analyses of mRNA. Forty-seven (96%) of the 49 cases with positive immunoreactivity for GnRH-R also had positive immunoreactivity for GnRH. PR expression was higher in the tumors positive for GnRH-R (p = 0.002), and a significantly higher proportion of tumors from male patients exhibited positive immunoreactivity for GnRH (p = 0.02). No significant correlations were found between the status of GnRH-R or GnRH with other clinicopathological features.

Conclusion

Over half of meningiomas may be regulated by GnRH–GnRH-R expression in an autocrine fashion. This unique expression profile of GnRH and GnRH-R may open the way to the development of GnRH analogs as a treatment tool in the future.     

Nocturnal light pulses selectively induce Egr-1/NGFI-A protein in periventricular hypophysiotrophic somatostatinergic neurons

Recent reports showing that circadian light cues may induce gene-specific patterns of expression in periventricular hypothalamic neurons has extended the functional correlates of light-pulse stimuli, which are conventionally restricted to circadian phase-setting actions within the suprachiasmatic nucleus (ScN). The aims of the present study were, first, to broaden these observations to the protein level using an Egr-1/NGFI-A antibody, and second to investigate the cell-type specificity of induction with respect to associated hypophysiotrophic neurons. In order to co-localize Egr-1 with neuroendocrine peptides, a same-species, double immunofluorescence protocol has been used. The Egr-1 antibody used for immunohistochemistry was first characterized by Western analysis, and we have shown that the C19 antisera detects a single 75kDa protein species, which has a primarily nuclear subcellular localization. Immunohistochemical analysis has shown that Egr-1 protein is markedly induced by a 1h nocturnal light pulse both in the body of the suprachiasmatic nucleus and in a dorsal ScN zone, which extends up into the periventricular nucleus (PeN). In contrast, induction of Egr-1 was not observed within the arcuate region of the hypothalamus. Double immunofluorescence histochemistry has shown that Egr-1 is extensively, although not exclusively, co-localized with somatostatin in PeN neurons. The selective induction of the 75kDa protein product of the egr-1 gene in PeN somatostatinergic neurons, as contrasted with hypophysiotrophic (somatostatin and growth hormone-releasing hormone [GH-RH]) neurons of the arcuate nucleus is indicative of a cell-and stimulus-specific neuroendocrine paradigm, which may be used to address temporal characteristics of somatostatin function that determine pulsatile growth-hormone secretion.