Signaling Pathways Regulating GnRH Secretion

调节 GnRH 分泌的信号通路

• 批准号: 6986831
• 负责人: RICHARD Ira WEINER
• 金额: $ 27.83万
• 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/6986831
• 关键词: Signaling; Pathways; Regulating; GnRH; Secretion

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：GNRH脉冲受生殖周期的维持。脉冲GNRH释放是GT1 GNRH细胞和内源性GNRH神经元的内在特性。根据GT1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脉冲的时间。 我们的发现表明，cAMP的增加通过打开营地门控阳离子（CNG）通道刺激GNRH分泌，从而导致神经元的兴奋性和去极化增加。神经元兴奋性的增加反映在增加的动作电位，Ca2+振荡和GNRH分泌中。 camp水平的提高还激活了似乎启动负反馈途径的PKA。 我们将研究这些信号分子在GT1 GNRH细胞系中的GNRH分泌和转基因大鼠体内的作用。 我们将通过表达组成型活性磷酸二酯酶，PDE4D1或通过表达CNG2通道亚基CNG2通道亚基的显性/负（D/N）突变体（DMCNG2）来抑制CNG通道活性来降低神经元的兴奋性。 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，并通过表达组成性活跃的溶解腺酸环化酶（SAC）来提高神经元的兴奋性。在GT1细胞中，我们将使用腺病毒载体靶向遗传探针的表达。 我们将研究GT1神经元兴奋性（CA2+振荡）的变化以及GnRH脉冲的频率和振幅。现在，我们已经表明，PDE4D1在GT1细胞中的表达抑制Ca2+振荡和脉动GNRH释放。 使用大鼠GNRH基因启动子/增强子针对转基因大鼠的GNRH神经元专门针对GNRH神经元的细胞，可在使用GT1细胞实验中有效的遗传探针。 现在，我们已经表明，在转基因大鼠系中，PDE4D1的靶向表达降低了cast骨雄性和女性中LH脉冲的频率。雌性不育，lh排卵或多囊卵巢钝化。 除了促进我们对定时脉冲GnRH涉及的信号通路的了解外，这些动物还将为研究GNRH脉动改变对生殖功能的影响的影响提供重要模型。 这些发现可能与对人类疾病的理解有关。