Properties and regulation of high-affinity pituitary receptors for corticotropin-releasing factor

Specific receptors for corticotropin-releasing factor (CRF) were identified in the rat anterior pituitary gland by binding studies with 125I-Tyr-CRF. Binding of the labeled CRF analog to pituitary particles was rapid and temperature-dependent, and reached steady state within 45 min at 22 degrees C. The CRF binding sites were saturable and of high affinity, with dissociation constant (Kd) of 0.76 X 10(-9) M. Pituitary binding of 125I-Tyr-CRF was inhibited by CRF, Tyr-CRF and the active 15-41 fragment of CRF, but not by the inactive 21-41 CRF fragment and unrelated peptides. The binding-inhibition potencies of the CRF peptides were similar to their activities as stimuli of adrenocorticotropic hormone (ACTH) release. The high-affinity CRF sites were markedly reduced in adrenalectomized rats, and this change was reversed by dexamethasone treatment. These data indicate that the high-affinity CRF sites demonstrated in the anterior pituitary are the functional receptors which mediate the stimulatory action of the peptide on ACTH release, and that CRF receptors are down-regulated during increased secretion of the hypothalamic hormone.     

A reference map of a human pituitary adenoma proteome

In order to compare the proteomes from different cell types of pituitary adenomas for our long-term goal to clarify the molecular mechanisms that participate in the formation of pituitary adenoma, and to detect any tumor-related marker for an "early-stage" diagnosis, the two-dimensional gel electrophoresis (2-DE) reference map of a pituitary adenoma tissue proteome is described here. A vertical, two-dimensional (2-D) polyacrylamide gel electrophoresis system and PDQuest image analysis software have been used to provide a high level of between-gel reproducibility and to accurately array each protein expressed in a pituitary adenoma tissue. Mass spectrometry (matrix-assisted laser desorption/ionization-time of flight MALDI-TOF and liquid chromatography-electrospray ionization-quadrupole-ion trap LC-ESI-Q-IT) and protein databases were used to characterize each protein in the 2-D gel. The results demonstrate that a good reproducibility of the 2-D gel pattern was attained. The position deviation of matched spots among four 2-D gels was 1.95 +/- 0.45 mm in the isoelectric focusing direction, and 1.70 +/- 0.53 mm in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis direction. A total of ca. 1000 protein spots were separated by 2-DE, and 135 protein spots that represent 111 proteins were characterized with mass spectrometry (96 spots for MALDI-TOF, 39 spots for LC-ESI-Q-IT). The characterized proteins include pituitary hormones, cellular signals, enzymes, cellular-defense proteins, cell-structure proteins, transport proteins, etc. Those proteins were located in the cytoplasmic, cellular membrane, mitochondrial, endoplasmic reticulum, nuclear, ribonucleosome, extracellular fractions, or were secreted in plasma, etc. Those identified proteins contribute to a functional profile of the pituitary adenoma proteome. These data will be used to expand the proteome database of the human pituitary, which can be accessed in the website http://www.utmem.edu /proteomics.     

Analysis of the proteome in the human pituitary

The pituitary is the master endocrine gland responsible for the regulation of various physiologic and metabolic processes. Proteomics offers an efficient means for a comprehensive analysis of pituitary protein expression. This paper reports on the application of proteomics for the mapping of major proteins in a normal (control) pituitary. Pituitary proteins were separated by two-dimensional gel electrophoresis with immobilized pH 3-10 gradient strips. Major protein spots that were visualized in the two-dimensional gel by silver staining were excised, and the proteins in these spots were digested with trypsin. The tryptic digests were analyzed by mass spectrometry, and the mass spectrometric data were used to identify the proteins through searches of the SWISS-PROT or NCBInr protein sequence databases. The majority of the proteins were identified on the basis of peptide mass fingerprinting data obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Several proteins were also characterized based on product-ion spectra measured by post-source decay analysis and/or liquid chromatography-electrospray-quadrupole ion trap mass spectrometry. To date, 62 prominent protein spots, corresponding to 38 different proteins, were identified. The identified proteins include important pituitary hormones, structural proteins, enzymes, and other proteins. The protein identification data were used to establish a two-dimensional reference database of the human pituitary, which can be accessed over the Internet (http://www.utmem.edu/proteomics). This database will serve as a tool for further proteomics studies of pituitary protein expression in health and disease.     

EST dataset of pituitary and identification of somatolactin and novel genes in Chinese sturgeon, <Emphasis Type="Italic">Acipenser sinensis</Emphasis>

Chinese sturgeon (Acipenser sinensis) is a rare and endangered species and also an important resource for the sturgeon aquaculture industry, however, a few genes have been identified in this species. We report here construction of a pituitary cDNA library from a 24 years old female Chinese sturgeon just after its spawning, and obtained 2,025 ESTs from the library. 885 unique sequences were identified, which were categorized into 12 functional groups. More than half of the unique sequences (57%) do not match with annotated sequences in the public databases. Three of these novel genes were further identified. Notably, a full-length of cDNA (1,143 bp) encoding somatolactin of 232 amino acids was identified. Phylogenetic analysis showed 97% amino acid identity with White sturgeon somatolactin. RT-PCR analysis indicated that the somatolactin mRNA was only detected in pituitary. Pituitary-specific expression of the somatolactin suggested that the protein may play important physiological functions in pituitary-endocrine system of the Chinese sturgeon.     

Phenotypical and Pharmacological Characterization of Stem-Like Cells in Human Pituitary Adenomas

The presence and functional role of tumor stem cells in benign tumors, and in human pituitary adenomas in particular, is a debated issue that still lacks a definitive formal demonstration. Fifty-six surgical specimens of human pituitary adenomas were processed to establish tumor stem-like cultures by selection and expansion in stem cell-permissive medium or isolating CD133-expressing cells. Phenotypic and functional characterization of these cells was performed (1) ex vivo, by immunohistochemistry analysis on paraffin-embedded tissues; (2) in vitro, attesting marker expression, proliferation, self-renewal, differentiation, and drug sensitivity; and (3) in vivo, using a zebrafish model. Within pituitary adenomas, we identified rare cell populations expressing stem cell markers but not pituitary hormones; we isolated and expanded in vitro these cells, obtaining fibroblast-free, stem-like cultures from 38 pituitary adenoma samples. These cells grow as spheroids, express stem cell markers (Oct4, Sox2, CD133, and nestin), show sustained in vitro proliferation as compared to primary cultures of differentiated pituitary adenoma cells, and are able to differentiate in hormone-expressing pituitary cells. Besides, pituisphere cells, apparently not tumorigenic in mice, engrafted in zebrafish embryos, inducing pro-angiogenic and invasive responses. Finally, pituitary adenoma stem-like cells express regulatory pituitary receptors (D2R, SSTR2, and SSTR5), whose activation by a dopamine/somatostatin chimeric agonist exerts antiproliferative effects. In conclusion, we provide evidence that human pituitary adenomas contain a subpopulation fulfilling biological and phenotypical signatures of tumor stem cells that may represent novel therapeutic targets for therapy-resistant tumors.     

4龄中华鲟垂体的EST分析

表达序列标签 (Expressed sequence tag, EST) 是鉴定基因表达规律和发现新基因的一种有效的分子生物学手段。为了能在中华鲟(Acipenser sinensis Gray) 中发现与生长和生殖内分泌调控相关的基因,我们构建了中华鲟垂体的SMART cDNA质粒文库。垂体是调节生长和生殖内分泌的重要器官。在本研究中,通过测序筛选得到了944个EST克隆,将所得EST 与 GenBank 数据库中的序列进行比对, 结果表明,802 (84.96%) 个克隆可以找到同源序列,共代表461个基因, 其中含132个已知功能基因;而 142 (15.04%) 个克隆不能找到同源序列。研究发现,在所有基因中,阿黑皮素原基因 (Proopiomelanocortin, POMC) 是出现次数最高的基因,占总EST数的10.17%, 显示出其在垂体中的重要地位。我们还发现了7个未知功能的基因并重点研究了其在心脏、肝脏、脾脏、肾脏、肌肉、精巢、卵巢和垂体等组织中的表达特异性。结果发现,4个基因：EG009334、EG009337、EG009338 和 EG009340为垂体特异性表达或垂体和卵巢特异性表达。对这些基因进一步的功能研究将有利于我们更好地了解中华鲟生长和生殖内分泌调控的分子机制。     

A combined opiate agonist and antagonist treatment reduces prolactin secreting pituitary tumor growth

Prolactin secreting pituitary adenomas (prolactinomas) is the most common pituitary tumors in humans. Animal studies have identified aggressive prolactinoma development in fetal alcohol exposed rats. We have recently identified a combination treatment of a μ opioid receptor antagonist naltrexone and a δ opioid receptor agonist D-Ala2-,N-Me-Phe4,Gly-ol Enkephalin (DPDPE) increases innate immune function. In this study, we tested whether naltrexone and DPDPE combination therapy is useful to control pituitary tumor growth. Fetal alcohol exposed and control Fischer 344 female rats at 60 days of age were ovariectomized and received an estrogen implant to induce prolactinomas. Six weeks after the estrogen implant, these animals received treatments of naltrexone and DPDPE or saline. The growth of the pituitary tumor prior to and after opioidergic agent treatments was visualized using magnetic resonance imaging (MRI). At the end of the treatment, pituitary weights, plasma prolactin and splenic levels of cytotoxic factors were determined. Both imaging data and weight data indicated that the volume and the weight of the pituitary were increased more after estrogen treatment in animals exposed to fetal alcohol than control. Naltrexone and DPDPE treatment reduced the weight and volume of the pituitary gland and plasma levels of prolactin in both fetal alcohol exposed and control-fed animals. The treatment of opioidergic agents also increased the levels of cytotoxic factors in the spleen. These data provide a novel possibility in treating pituitary tumors using a combination therapy of naltrexone and DPDPE.     

GnRH相关肽在大鼠垂体前叶的细胞学定位

本研究应用特异性抗GnRH相关肽(GAP)N端11个氨基酸的抗血清和六种垂体前叶激素的抗血清,通过免疫组织化学双重染色技术观察GAP在大鼠垂体前叶细胞的定位。结果发现,GAP样免疫反应性物质存在于LH细胞和FSH细胞,而未见于GH、PRL、TSH和ACTH细胞。本文首次证明GAP存在于正常大鼠垂体促性腺激素细胞,为GAP调节LH和FSH的分泌提供了形态学证据;也支持GAP的功能序列在其分子的N端,或GAP进一步裂解出N端片段而发挥作用。     

GABAergic control of anterior pituitary hormone secretion

Anatomical and biochemical studies have identified a hypothalamic tubero-infundibular GABAergic system, which plays a functional role on anterior pituitary hormone secretion. Experimental and clinical evidence support the presence of a dual component in the action of GABA; one mediated via the central nervous system and the other exerted directly at the anterior pituitary level. The two sites of action may be responsible for the excitatory and inhibitory effects of GABA on pituitary hormone and especially prolactin secretion. The future characterization of this system will provide a better understanding of the involvement of GABA in the physiology of anterior pituitary hormone secretion and will contribute to the development of new pharmacological agents for the therapy of neuroendocrine disorders.     

Nitroproteins from a human pituitary adenoma tissue discovered with a nitrotyrosine affinity column and tandem mass spectrometry

The aim of this study was to characterize endogenous nitroproteins, and those proteins that interact with nitroproteins, in a human pituitary nonfunctional adenoma so as to clarify the role of protein nitration in adenomas. A nitrotyrosine affinity column (NTAC) was used to preferentially enrich and isolate endogenous nitroproteins and nitroprotein-protein complexes from a tissue homogenate that was prepared from a human pituitary nonfunctional pituitary adenoma. The preferentially enriched endogenous nitroproteins and nitroprotein-protein complexes were subjected to trypsin digestion, desalination, and tandem mass spectrometry analysis. Nine nitroproteins (Rho-GTPase-activing protein 5, leukocyte immunoglobulin-like receptor subfamily A member 4 precursor, zinc finger protein 432, cAMP-dependent protein kinase type I-beta regulatory subunit, sphingosine-1-phosphate lyase 1, centaurin beta 1, proteasome subunit alpha type 2, interleukin 1 family member 6, and rhophilin 2) and three proteins (interleukin 1 receptor-associated kinase-like 2, glutamate receptor-interacting protein 2, and ubiquitin) that interacted with nitroproteins were discovered. The nitration site of each nitroprotein was located onto the functional domain where nitration occurred, and each nitroprotein was related to a corresponding functional system. Those data indicate that protein nitration might be an important molecular event in the formation of a human pituitary nonfunctional adenoma.