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Pituitary hormone gene expression and secretion in human growth hormone-releasing hormone transgenic mice: Focus on lactotroph function

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ENDOCRINOLOGY 2000年第1期141卷 81-90页

作者： Moore, JP Cai, AH Hostettler, ME Arbogast, LA Voogt, JL Hyde, JF Univ Kentucky Coll Med Dept Anat & Neurobiol Lexington KY 40536 USA So Illinois Univ Sch Med Dept Physiol Carbondale IL 62901 USA Univ Kansas Med Ctr Dept Mol & Integrat Physiol Kansas City KS 66160 USA

The human GH-releasing hormone (hGHRH) transgenic mouse has a hyperplastic anterior pituitary gland that eventually develops into an adenoma. We showed previously that the number of lactotrophs in the male hGHRH transgenic mouse is increased 2-fold, yet there is no concomitant increase in plasma levels of PRL. To further elucidate underlying changes in lactotroph function in the hGHRH transgenic mouse, the objectives of this study were to 1) examine the relative differences in PRL gene expression in transgenic mice and their siblings, 2) quantify PRL secretion at the level of the individual cell, 3) determine whether tyrosine hydroxylase gene expression and/or activity are altered in the hypothalamus of transgenic mice, and 4) assess dopamine receptor gene expression and functional sensitivity in lactotrophs of transgenic mice. Total PRL messenger RNA (mRNA levels were increased nearly 5-fold in the hGHRH transgenic mouse, whereas the concentrations of PRL mRNA (PRL mRNA per mu g total RNA) were unchanged. In contrast, total PRL contents were unchanged, whereas the concentrations of PRL (micrograms of PRL per mg total protein) were decreased 3-fold. Hypothalamic tyrosine hydroxylase steady state mRNA levels were not altered in the hGHRH transgenic mice, but hypothalamic tyrosine hydroxylase activity was increased a-fold in transgenic mice. Dopamine D-2 receptor mRNA concentrations in the anterior pituitary were increased 2.5-fold in hGHRH transgenic mice, and total pituitary D-2 receptor mRNA levels were increased nearly 10-fold. Furthermore, the basal secretory capacity of lactotrophs from transgenic mice was increased significantly at the level of the single cell, and dopamine inhibited the secretion of PRL to a greater extent in hGHRH transgenic mice. Thus, although the total number of lactotrophs is increased 2-fold in hGHRH transgenic mice, the present data are consistent with the hypothesis that increased hypothalamic dopamine synthesis and release coupled wit

关键词：色谱法高压液相多巴脱羧酶/拮抗剂和抑制剂酶抑制剂/药理学生长激素释放激素/生物合成生长激素释放激素/遗传学肼类/投药和剂量肼类/药理学下丘脑/酶学下丘脑/代谢免疫印迹法原位杂交小鼠转基因垂体前叶/细胞学垂体前叶/代谢垂体前叶/生理学垂体激素类/生物合成垂体激素类/遗传学垂体激素类/分泌催乳素/生物合成放射免疫测定受体多巴胺D2/生物合成受体多巴胺D2/遗传学酪氨酸单氧化酶/生物合成酪氨酸单氧化酶/遗传学动物女(雌)性人类男(雄)性小鼠

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Somatostatin modulation of excitatory synaptic transmission between periventricular and arcuate hypothalamic nuclei in vitro

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JOURNAL OF NEUROPHYSIOLOGY 2000年第3期84卷 1464-1474页

作者： Lanneau, C Peineau, S Petit, F Epelbaum, J Gardette, R INSERM U159 Ctr Paul Broca F-75014 Paris France

Hypophysiotropic somatostatin (SRIF) and growth hormone-releasing hormone (GHRH) neurons are primarily involved in the neurohormonal control of growth hormone (GH) secretion. They are located in periventricular (PEV) and arcuate (ARC) hypothalamic nuclei, respectively, but their connectivity is not well defined. To better understand the neuronal network involved in the control of GH secretion, connections from PEV to ARC neurons were reconstructed in vitro and neuronal phenotypes assessed by single-cell multiplex RT-PCR. Of 814 stimulated PEV neurons, monosynaptic responses were detected in only 45 ARC neurons. Monosynaptic excitatory currents were detected in 29 ARC neurons and inhibitory currents in 16, indicating a 2/1 ratio for excitatory versus inhibitory connections. Galanin (GAL), NPY, pro-opiomelanocortin (POMC), and SRIF mRNAs were detected in neurons from both nuclei but GHRH mRNA almost exclusively in ARC. Among the five SRIF receptors, only sst1 and sst2 were expressed, in 94% of ARC and 59% of PEV neurons, respectively. Of 128 theoritical combinations between neuropeptides and sst receptors, only 22 were represented in PEV and 25 in ARC. For PEV neurons, neuropeptide phenotypes did not influence excitatory connections. However, the occurrence of presynaptic sst receptors on GAL and SRIF PEV neurons significantly increased their probability of connection to ARC neurons. GHRH ARC neurons expressing sst2, but not sst1, receptors were always connected with PEV neurons. Physiological responses to sst1 (CH-275) or sst2 (Octreotide) agonists were always correlated with the detection of respective sst mRNAs. In conclusion, 1) SRIF-modulated excitatory transmission develops in vitro from PEV to ARC neurons, 2) ARC GHRH neurons bearing sst2 receptors appears directly controlled by fast glutamatergic transmission from PEV neurons simultaneously expressing one to four neuropeptides, 3) GHRH neurons bearing sst1 receptors lack this control, and 4) these results sugg

关键词：细胞分离共同培养技术电刺激兴奋性突触后电位/生理学加兰肽/分析加兰肽/遗传学生长激素/生物合成生长激素释放激素/分析生长激素释放激素/遗传学神经网/代谢神经元/化学神经元/细胞学神经元/代谢神经肽Y/分析神经肽Y/遗传学下丘脑室旁核/细胞学下丘脑室旁核/胚胎学下丘脑室旁核/代谢表型前阿片黑素细胞皮质激素/分析前阿片黑素细胞皮质激素/遗传学 RNA 信使/分析受体生长抑素/激动剂受体生长抑素/分析受体生长抑素/遗传学逆转录聚合酶链反应/方法生长抑素/分析生长抑素/遗传学生长抑素/代谢突触传递/生理学动物小鼠

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Expression of growth hormone-releasing factor, growth hormone, insulin-like growth factor-1 and its binding proteins in human lung

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NEUROPEPTIDES 2000年第2期34卷 98-107页

作者： Allen, JT Bloor, CA Kedia, RK Knight, RA Spiteri, MA N Staffordshire Hosp Dept Resp Med Lung Injury & Inflammat Res Grp Stoke On Trent ST4 6QG Staffs England Natl Heart & Lung Inst Dept Cyst Fibrosis Imperial Coll London England

Reverse transcription PCR showed that mRNA encoding the neurohormones growth hormone-releasing factor (GRF) and GH, and its receptor GH-R, together with IGF-1 splice variants and IGFBPs are expressed by inflammatory cells found in the normal human airway. Unfractionated BALC moderately express GRF, GH and GH-R, IGFBP-2 to IGFBP-6, and IGFBP-rPI. In addition, BALC preferentially express the class 1 IGF-1Ea splice variant of the IGF-1 gene. A similar pattern of expression occurs in purified AM, except they do not appear to express GH-R. In marked contrast, AM precursor peripheral blood monocytes, do not express neuropeptides or IGF-1 and only express IGFBP-1, -4 and -6 and IGFBP-rPI. These data suggest that normal human inflammatory airway cells possess a powerful array of neurohormones and IGFBPs that are available for modulating local IGF-1 bioavailability in the lung. (C) 2000 Harcourt Publishers Ltd.

关键词：选择性剪接支气管肺泡灌洗液/细胞学细胞计数细胞分级分离外显子生长激素释放激素/遗传学生长激素释放激素/代谢人生长激素/遗传学人生长激素/代谢胰岛素样生长因子Ⅰ/遗传学胰岛素样生长因子Ⅰ/代谢巨噬细胞肺泡/代谢单核细胞/代谢 RNA 信使/代谢受体 IGF1型/遗传学受体 IGF1型/代谢受体促生长素/遗传学受体促生长素/代谢逆转录聚合酶链反应成年人女(雌)性人类男(雄)性

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Development of a transgenic mouse that overexpresses a novel product of the growth hormone-releasing hormone gene

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ENDOCRINOLOGY 2000年第4期141卷 1377-1383页

作者： Fang, SJ Steinmetz, R King, DW Zeng, PY Vogelweid, C Cooper, S Hangcoc, G Broxmeyer, HE Pescovitz, OH Wells Ctr Pediat Res Sect Pediat Endocrinol Dept Pediat Indianapolis IN 46202 USA Indiana Univ Sch Med Lab Resource Ctr Indianapolis IN 46202 USA Indiana Univ Sch Med Dept Microbiol & Immunol Indianapolis IN 46202 USA Indiana Univ Sch Med Walther Canc Inst Indianapolis IN 46202 USA

The GH-releasing hormone (GHRH) precursor molecule contains a 30-amino acid C-terminal region that has been designated GHRH-related peptide (GHRH-RP). To begin to understand the physiological role of GHRH-RP, transgenic (Tg) mice that constituitively express this peptide were developed. To generate these mice, a transgene (SS-RP) was constructed by overlap primer extension PCR. This transgene, under the control of the mouse phosphoglycerate kinase gene, selectively expresses GHRH-RP, but not GHRH. Western blot analysis confirmed that the transgene produces GHRH-RP. Animals were evaluated for the effect of excess GHRH-RP on growth, fertility, behavior, stem cell factor (SCF) expression, and hematopoiesis. Northern blot and RT-PCR were used to demonstrate ubiquitous expression of the transgene in tissues from GHRH-RP Tg animals. These tissues also had marked overexpression of SCF messenger RNA compared with controls. Tg animals had significantly increased cell cycling for granulocyte-macrophage, erythroid, and multilineage progenitor cells. Transgenic animals did not differ from control mice in their growth, fertility, or behavior. These findings demonstrate, for the first time, that in vivo the C-terminal peptide of the pro-GHRH molecule is a biologically active peptide that is capable of stimulating the expression of SCF and hematopoiesis in vivo and suggests that GHRH-RP may play a role in normal blood cell development.

关键词：印迹法 RNA 印迹法蛋白质生育力/生理学基因表达生长激素释放激素/遗传学生长激素释放激素/代谢生长激素释放激素/生理学血细胞生成/生理学小鼠近交C3H 小鼠转基因/遗传学小鼠转基因/代谢小鼠转基因/生理学 RNA 信使/代谢逆转录聚合酶链反应干细胞因子/遗传学干细胞因子/代谢动物小鼠

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Posttranslational processing of progrowth hormone-releasing hormone

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ENDOCRINOLOGY 1999年第12期140卷 5817-5827页

作者： Nillni, EA Steinmetz, R Pescovitz, OH Brown Univ Rhode Isl Hosp Sch Med Dept MedDiv Endocrinol Providence RI 02903 USA Indiana Univ James Whitcomb Riley Hosp Children Dept PediatHerman B Wells Ctr Pediat Res Sect Pediat Endocrinol & Physiol Indianapolis IN 46202 USA Indiana Univ James Whitcomb Riley Hosp Children Dept Physiol Indianapolis IN 46202 USA

The prepro-GH-releasing hormone (prepro-GHRH;12.3 kDa) precursor, like other neuropeptide precursors, undergoes proteolytic cleavage to give rise to mature GHRH, which is the primary stimulatory regulator of pituitary GH secretion. In this study we present the first model of in vitro pro-GHRH processing. Using pulse-chase analysis, we demonstrate that at least five peptide forms in addition to GHRH are produced. The pro-GHRH (after removal of its signal peptide, 10.5 kDa) is first processed to an 8.8-kDa intermediate form that is cleaved to yield two products: the 5.2-kDa GHRH and GHRH-related peptide (GHRH-RP;3.6 kDa). GHRH-RP is a recently described peptide derived from proteolytic processing of pro-GHRH that activates stem cell factor, a factor known to be essential for hemo-poiesis;spermatogenesis, and melanocyte function. Further cleavage results in a 3.5-kDa GHRH and a 2.2-kDa product of GHRH-RP. Like GHRH, there is GHRH-RP immunostaining in hypothalamic neurons in the median eminence as detected by immunohistochemistry and immunoelectron microscopy. Based on deduced amino acid sequences of the pro-GHRH processing products, several peptides were synthesized and tested for their ability to stimulate the cAMP second messenger system. GHRH, GHRH-RP, and one of these peptides [prepro-GHRH-(75-92)-NH2] all significantly stimulated the PKA pathway. This work delineates a new model of pro-GHRH processing and demonstrates that novel peptides derived from this processing may have biological action.

关键词：氨基酸序列细胞培养的环AMP依赖性蛋白激酶类/代谢酶激活/药物作用生长激素释放激素/分析生长激素释放激素/化学生长激素释放激素/遗传学下丘脑/化学免疫组织化学免疫吸附技术正中隆起/化学分子序列数据神经元/化学肽碎片/化学合成肽碎片/代谢肽碎片/药理学蛋白质前体/分析蛋白质前体/化学蛋白质前体/遗传学蛋白质加工转译后动物女(雌)性人类妊娠大鼠

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