Signaling Pathways Regulating GnRH Secretion

调节 GnRH 分泌的信号通路

• 批准号: 6821982
• 负责人: RICHARD Ira WEINER
• 金额: $ 28.62万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6821982
• 关键词: Signaling; Pathways; Regulating; GnRH; Secretion

EXCEED THE SPACE PROVIDED. GnRH pulses are tightly regulated for the maintenance of reproductive cycles. Pulsatile GnRH release is an intrinsic property of GT1 GnRH cells and endogenous GnRH neurons. Based on findings in GT1 cells, we hypothesize that the cAMP signaling pathway participates in the stimulation of GnRH secretion and the timing of GnRH pulses. Our findings show that increases in cAMP stimulate GnRH secretion by opening cAMP-gated cation (CNG) channels leading to increased excitability and depolarization of the neuron. Increased neuron excitability is reflected in increased action potentials, Ca2+ oscillations and GnRH secretion. Increased cAMP levels also activate PKA that appears to initiate negative feedback pathways. We will study the role of these signaling molecules on the regulation of GnRH secretion in vitro in the GT1 GnRH cell lines and in vivo in transgenic rats. We will decrease neuron excitability by lowering cAMP levels by expressing the constitutively active phosphodiesterase, PDE4D1, or inhibiting CNG channel activity by expressing a dominant/negative (D/N) mutant of the CNG2 channel subunit (DMCNG2). We will increase neuron excitability by inhibiting the PKA negative feedback pathway by expression of the D/N mutant of the regulatory subunit of PKA mRAB and by increasing cAMP levels by expressing a constitutively active soluble adenylate cyclase (sAC). In GT1 cells we will use adenovirus vectors to target expression of the genetic probes. We will study changes in GT1 neuron excitability (Ca2+ oscillations) and the frequency and amplitude of GnRH pulses. We have now shown that expression of PDE4D1 in GT1 cells inhibits Ca2+ oscillations and pulsatile GnRH release. Genetic probes shown to be effective in experiments with GT1 cells will be cell specifically targeted to GnRH neurons in transgenic rats using the rat GnRH gene promoter/enhancer. We have now shown that targeted expression of PDE4D1 in a line of transgenic rats decreased the frequency of LH pulses in castrated males and females. Females were infertile and had blunted LH ovulatory surges or polycystic ovaries. In addition to advancing our knowledge of the signaling pathways involved in timing pulsatile GnRH secretion these animals will provide important models for studying the effects of alterations in GnRH pulsatility on reproductive function. Potentailly these findings may be relevant to the understanding of human disorders. PERFORMANCE SITE ========================================Section End===========================================

超出所提供的空间。GnRH脉冲被严格调节以维持生殖周期。脉冲式GnRH释放是GT 1 GnRH细胞和内源性GnRH神经元的固有特性。基于在GT 1细胞中的发现，我们假设cAMP信号通路参与GnRH分泌的刺激和GnRH脉冲的定时。我们的研究结果表明，cAMP增加刺激GnRH分泌的cAMP门控阳离子（CNG）通道开放，导致增加兴奋性和去极化的神经元。增加的神经元兴奋性反映在增加的动作电位，Ca 2+振荡和GnRH分泌。增加的cAMP水平也激活PKA，PKA似乎启动负反馈途径。我们将研究这些信号分子在体外GT 1 GnRH细胞系和体内转基因大鼠中对GnRH分泌的调节作用。我们将通过表达组成型活性磷酸二酯酶PDE 4D 1来降低cAMP水平，或通过表达CNG 2通道亚基（DMCNG 2）的显性/阴性（D/N）突变体来抑制CNG通道活性，从而降低神经元兴奋性。我们将通过表达PKA mRAB调节亚基的D/N突变体来抑制PKA负反馈途径，并通过表达组成型活性可溶性腺苷酸环化酶（sAC）来增加cAMP水平，从而增加神经元兴奋性。在GT 1细胞中，我们将使用腺病毒载体靶向表达基因探针。我们将研究GT 1神经元兴奋性（Ca 2+振荡）和GnRH脉冲的频率和幅度的变化。我们现在已经证明，GT 1细胞中PDE 4D 1的表达抑制了Ca 2+振荡和脉动式GnRH释放。在使用GT 1细胞的实验中显示有效的遗传探针将是使用大鼠GnRH基因启动子/增强子的转基因大鼠中的GnRH神经元的细胞特异性靶向。我们现在已经表明，PDE 4D 1在一系列转基因大鼠中的靶向表达降低了去势雄性和雌性大鼠LH脉冲的频率。雌性不育，LH排卵高峰或多囊卵巢变钝。除了推进我们的知识的信号通路参与定时脉冲式GnRH分泌这些动物将提供重要的模型，研究GnRH脉冲性的改变对生殖功能的影响。这些发现可能与人类疾病的理解有关。性能现场=