Regulation of the Hypothalamic GnRH Neuron

下丘脑 GnRH 神经元的调节

基本信息

批准号：
7594115
负责人：
Kevin J Catt
金额：
$ 77.07万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7594115
关键词：
Action Potentials Agonist Amino Acids Angiotensin II Bioluminescence COS-7 Cell Calcium-Activated Potassium Channel Cell Line Cell physiology Cells Couples Coupling Cyclic AMP Data Dose Energy Transfer Exhibits Family Feedback Fire - disasters Frequencies G-Protein-Coupled Receptors GTP-Binding Proteins Glutamine Gonadotropin Hormone Releasing Hormone Gonadotropin Receptors Gonadotropin-Releasing Hormone Receptor Gonadotropins Green Fluorescent Proteins Growth Human Chorionic Gonadotropin Hypothalamic structure Individual KISS1 gene KISS1R gene Label Ligands Mediating Membrane Membrane Potentials Metabolic Mus Mutant Strains Mice Mutate Mutation Nature Neurons Neurosecretion Neurosecretory Systems Pituitary Gland Positioning Attribute Potassium Production Property Proteins RNA analysis Rate Receptor Signaling Regulation Renilla Luciferases Reproduction Research Role Signal Pathway Signal Transduction Structure Study Section System Whole-Cell Recordings Wild Type Mouse autocrine human GNRH1 protein hypothalamic pituitary gonadal axis kisspeptin-10 large-conductance calcium-activated potassium channels markov model millisecond mutant neuroregulation paracrine patch clamp peptide hormone programs receptor receptor coupling receptor function reproductive function response vector

项目摘要

Our previous studies in native hypothalamic GnRH neurons and their immortalized counterparts, GT1-7 cells have shown that the autocrine actions of GnRH on its cells of origin cause both inhibitory and stimulatory responses due to the activation of multiple G proteins, and that these actions are essential for episodic GnRH secretion. Also, GnRH neurons were found to express G protein-gated inwardly rectifying potassium (GIRK) channels that are activated by LH/hCG, leading to inhibition of membrane excitability and pulsatile GnRH secretion. The presence of GIRK channels in GnRH neurons is also highly relevant to the mechanism of pulsatile GnRH release, as a consequence of episodic inhibition of neuronal firing and neurosecretion. In more recent studies, the GPR54 receptor and its endogenous ligand, kisspeptin, both of which are essential for activation and regulation of the hypothalamic-pituitary-gonadal axis, were found to be expressed in hypothalamic GnRH neurons and GT1-7 cells. Analysis of RNA extracts from individually identified hypothalamic GnRH neurons with primers for GnRH, KiSS-1, and GPR54 revealed expression of all three gene products. Furthermore, constitutive and GnRH agonist-induced bioluminescence resonance energy transfer BRET(2) between Renilla luciferase (Rluc)-tagged GnRH-R and GPR54 labeled with green fluorescent protein GFP(2), expressed in HEK-293 cells, revealed hetero-oligomerization of the two receptors. Whole-cell patch-clamp recordings from identified GnRH neurons showed that kisspeptin has an initial depolarizing effect on membrane potential, followed by increased action potential firing and an increase in GnRH peak-amplitude and duration. Kisspeptin production and secretion in hypothalamic GnRH neurons and GT1-7 cells was significantly reduced by treatment with GnRH. These findings suggest that kisspeptins can act as paracrine and/or autocrine regulators of the GnRH neuron. The stimulation of GnRH release by kisspeptin, and the opposing effects of GnRH on kisspeptin secretion, indicate that GnRH receptor/GnRH and GPR54/kisspeptin autoregulatory systems are integrated by negative feedback to control the production and secretion of both GnRH and kisspeptin from GnRH neurons. In perifusion studies, treatment of GT1-7 neuronal cells with kisspeptin-10 increased GnRH peak-amplitude and duration. The production and secretion of kisspeptin in cultured hypothalamic neurons and GT1-7 cells was detected by a specific radioimmunoassy, and was significantly reduced by treatment with GnRH. The expression of kisspeptin and GPR54 mRNAs in identified hypothalamic GnRH neurons, as well as kisspeptin secretion, indicate that kisspeptins may act as paracrine and/or autocrine regulators of the GnRH neuron. The stimulation of GnRH release by kisspeptin, and the opposing effects of GnRH on kisspeptin secretion, show that GnRH receptor/GnRH and GPR54/kisspeptin autoregulatory systems are integrated by negative feedback to control the secretion of GnRH and kisspeptin from GnRH neurons. In spontaneously active GnRH neurons, the firing of individual and/or bursts of action potential (AP) is followed by a hyperpolarization which can last from several milliseconds (ms) up to several seconds (s). Such hyperpolarization is mediated by the activation of two families of Ca2+-activated K+ channels. Big conductance (BK) channels contribute to AP repolarization, and small conductance (SK) channels underlie the afterhyperpolarization (AHP) and mediate firing frequency and spike-frequency adaptation. Under whole-cell recording 85% of identified GnRH neurons exhibited spontaneous AP firing. The majority of recorded GnRH neurons (48%) showed irregular AP firing with transition between narrow, high spike amplitude rhythmic firing, and intervals of broader, lower spike amplitude, burst-like AP firing. Narrow high-amplitude APs were followed by fast AHP (fAHP) with a decay constant of 0.9 ms and medium AHP (mAHP) a with decay constant of 27.4 ms. During treatment with GnRH (10 nM) the decay constants of fAHP and mAHP were not significantly changed. However, mAHP was abolished during treatment with GnRH (1 microM), while fAHP remained unchanged and was followed by subthreshold after-depolarization potential (ADP) and significant reduction of the frequency of AP firing. Broader, lower spike amplitude APs during basal recording had a high rate of firing, lacked fAHP and generated mAHP with a decay constant of 39.5 ms. These data provide evidence that the modes of spontaneous action potential firing can determine the nature of the after-hyperpolarizing current in hypothalamic GnRH neurons. An analysis of the role of the first intracellular loop of the mouse GnRH receptor in G protein coupling performed in transfected COS-7 and HEK-293 cells. Previous experimental data, and prediction of coupling by the Hidden Markov model, revealed that the mouse gonadotropin-releasing hormone receptor (mGnRH-R) can couple to Gq11, Gs, and Gi/o. The predicted coupling suggests that residues in the first intracellular (1i) loop between 50 and 63, and in the 3i loop between 228 and 240, are involved in Gq11 coupling. Coupling to Gs is predicted to be in the 1i loop between residues 75 to 85. Amino acids predicted for Gi/o coupling in the 1i loop are residues 57 to 64; 2i loop, residues 137 to 145; and 3i loop, residues 262 to 270. Hidden Markov prediction of coupling suggests that residues in the 1i loop from 50 through 85 are involved in multiple G protein coupling including Gq11, Gs, and Gi/o. Agonist activation of the wild type mGnRH-R expressed in COS-7 cells in a pcDNA 3.1 vector, and in HEK-293 cells using a pGFP2-N vector, caused a monotonic and dose-dependent increase in cAMP production. The predicted promiscuity of the 1i loop of the mGnRH-R in signal transduction was evaluated by mutating selected residues located in its N-terminus. Double Lys/Gln mutations were performed at residues 59 and 62. In contrast to the wild type mGnRH-R, agonist-induced cAMP production by the Lys/Gln mutant showed a biphasic response. cAMP production increased significantly during treatment with 5 pM GnRH and reached its maximal level at 20 pM GnRH. Maximal cAMP production was not sustained by increased GnRH concentrations, and was significantly decreased with a maximal inhibitory effect at 1 microM GnRH. The cAMP profile induced by activation of the mutant mGnRH-R differed significantly from that induced by GnRH activation of the wild type receptor, where maximal cAMP production was obtained with 1 microM GnRH. In summary, mutation of two Lys residues at position 59 and 62 in the 1i loop of the mGnRH-R promotes receptor coupling to Gs at low picomolar GnRH concentrations and increases cAMP production. Conversely, at high nanomolar and micromolar GnRH concentrations the GnRH-R couples to Gi/o and inhibits cAMP production.

我们先前在天然下丘脑GNRH神经元及其不朽的对应物中进行的研究GT1-7细胞表明，GnRH对其原产性细胞的自分泌作用引起的抑制性和刺激反应，由于多种G蛋白的激活，这些作用对于情节GNRH的发作至关重要。同样，发现GNRH神经元表达GNRH神经元通过LH/HCG激活的GNRH神经元向内门控钾（GIRK）通道，从而抑制膜兴奋性和脉冲GNRH分泌。 GNRH神经元中的GIRK通道的存在也与脉动GNRH释放的机理高度相关，这是由于神经元释放和神经分泌的发作抑制。在最近的研究中，发现在下丘脑GNRH神经元和GT1-7细胞中表达了GPR54受体及其内源性配体，这对于下丘脑 - 垂体 - 基达轴的激活和调节至关重要。分析来自单独鉴定的下丘脑GNRH神经元的RNA提取物，其引物用于GnRH，KISS-1和GPR54，揭示了所有三种基因产物的表达。此外，组成型和GNRH激动剂诱导的生物发光共振能量转移（2）在肾荧光素酶（RLUC）标记的GnRH-R和GPR54之间和GPR54中标记在HEK-293细胞中表达，在HEK-293细胞中表达，揭示了HEK-293细胞，揭示了两个受体 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素 - 元素化。来自已鉴定的GNRH神经元的全细胞斑块钳记录表明，亲肽素对膜电位具有初始的去极化作用，然后增加动作电位射击，而GnRH峰值增强和持续时间增加。通过用GNRH治疗可显着降低下丘脑GNRH神经元和GT1-7细胞中的Kisspeptin和分泌。这些发现表明，吻肽可以充当GNRH神经元的旁分泌和/或自分泌调节剂。 Kisspeptin对GNRH释放的刺激以及GnRH对Kisspeptin分泌的相反影响表明GNRH受体/GNRH和GPR54/KISSPEPTIN自动调节系统由负反馈整合，以控制GNRH和GNRH和Kisspepteptin of Gnrh neurons的生产和分泌。在促进研究中，用Kisspeptin-10治疗GT1-7神经元细胞增加了GnRH峰振幅和持续时间。特定的放射性免疫和GNRH处理可显着降低，在培养的下丘脑神经元和GT1-7细胞中亲吻肽的产生和分泌。在确定的下丘脑GNRH神经元以及Kisspeptin分泌中，Kisspeptin和GPR54 mRNA的表达表明，亲吻蛋白可以充当GnRH神经元的旁分泌和/或自分泌调节剂。 Kisspeptin对GNRH释放的刺激以及GNRH对Kisspeptin分泌的相反影响表明GNRH受体/GNRH和GPR54/KISSPEPTIN自动调节系统由负反馈组成，以控制GNRH和Kiskpeptin from GNRH Neurons的分泌。在自发活跃的GNRH神经元中，个体和/或动作电位（AP）的发射后，超极化可以持续几毫秒（MS），最高为几秒钟（s）。这种超极化是通过两个Ca2+活化的K+通道的激活来介导的。大电导（BK）通道有助于AP复极化，而小电导（SK）通道是余后过时（AHP）的基础，并介导射击频率和尖峰频率适应。在全细胞记录下，有85％已鉴定出的GNRH神经元表现出自发的AP触发。大多数记录的GNRH神经元（48％）显示出不规则的AP解散，狭窄，高尖峰振幅有节奏的放电和较宽，更低的尖峰振幅，爆发状的AP射击之间的过渡。狭窄的高振幅AP之后是快速AHP（FAHP），衰减常数为0.9 ms，中等AHP（MAHP）A，衰减常数为27.4 ms。在用GnRH（10 nm）处理过程中，FAHP和MAHP的衰减常数没有显着改变。然而，在用GnRH（1 microM）治疗期间，MAHP被废除，而FAHP保持不变，其后是阈值后偏度势（ADP）和AP解火频率的显着降低。基础记录期间，更广泛的较低的尖峰振幅AP具有很高的射击速度，缺乏FAHP，并产生了MAHP，衰减常数为39.5 ms。这些数据提供了证据表明，自发作用电位触发的模式可以确定下丘脑GNRH神经元中造后过敏电流的性质。分析小鼠GnRH受体在转染的COS-7和HEK-293细胞中进行的小鼠GnRH受体在G蛋白偶联中的作用。以前的实验数据以及隐藏的马尔可夫模型对耦合的预测表明，促性腺激素释放激素受体（MGNRH-R）可以将其与GQ11，GS和GI/O相对。预测的耦合表明，第一个细胞内（1i）中环中的残基在50到63之间，以及在228至240之间的3i环中的残基参与GQ11耦合。预计与GS耦合在75至85的残基之间的1i环中。预测1I环中GI/O偶联的氨基酸是残基57至64； 2i循环，残基137至145;和3i环，残基262至270。隐藏的马尔可夫预测耦合的预测表明，从50到85的1i环中的残基参与了多个G蛋白偶联，包括GQ11，GS和GI/O。在PCDNA 3.1载体中表达的野生型MGNRH-R的激动剂激活，并使用PGFP2-N载体在HEK-293细胞中激活，导致营地产量的单调和剂量依赖性增加。通过突变位于其N末端的选定残基来评估MGNRH-R在信号转导中的1I环的预测滥交。在59和62的残基上进行了双LYS/GLN突变。与野生型MGNRH-R相反，Lys/Gln突变体会引起激动剂诱导的cAMP产生，显示出双相反应。在5p pm GNRH治疗期间，营地产量显着增加，并在晚上20点GNRH达到其最大水平。 GNRH浓度升高并不能在1 microM GNRH下具有最大的抑制作用来显着降低，从而显着降低了cAMP的产生。突变体MGNRH-R激活引起的cAMP轮廓与野生型受体的GNRH激活引起的cAMP谱图显着不同，在该野生型受体的激活中，使用1 microM GNRH获得了最大的cAMP产生。总而言之，在MGNRH-R的1i环中位置59和62处的两个Lys残基的突变促进了低皮摩尔GNRH浓度下的受体偶联，并增加了营地产量。相反，在高纳摩尔和微摩尔GNRH浓度下，GnRH-R夫妇将其与GI/O偶联并抑制cAMP的产生。