SEROTONERGIC VESICLES--ACIDIFICATION AND SECRETION

血清素能囊泡——酸化和分泌

• 批准号: 2905955
• 负责人: HADASSAH TAMIR
• 金额: $ 20.83万
• 依托单位: NEW YORK STATE PSYCHIATRIC INSTITUTE DBA RESEARCH FOUNDATION FOR MENTAL HYGIENE, INC
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2905955
• 关键词: G protein; biological signal transduction; calcium channel; electrochemistry; electrophysiology; high performance liquid chromatography; membrane channels; neurons; phosphorylation; protein kinase; serotonin; serotonin transporter; synaptic vesicles; thyroid gland; voltage /patch clamp; voltage gated channel

DESCRIPTION (Adapted from applicant's abstract): Serotonergic neurotransmission plays a critical role in many physiological functions. Nevertheless, little is known about the properties of serotonergic synaptic vesicles. Since it is difficult to purify either serotonergic neurons or their synaptic vesicles from the brain, we have studied the 5-HT-containing secretory vesicles of parafollicular (PF) cells of the thyroid as a model. PF vesicles are unique in that they resemble both the trans-Golgi network-derived vesicles of regulated secretion (in containing peptides) and the endosome-derived synaptic vesicles (in containing a recycling membrane and a small molecular transmitter). Unlike other secretory vesicles, PF vesicles are not constitutively acidic, but acidify when PF cells are exposed to a secretagogue, i.e., increased (Ca2+)e. Acidification results from the stimulus-coupled gating of a vesicular Cl- channel that permits influx or H+. Gating of the Cl- channel is induced by its phosphorylation. The resulting delta pH maximizes the vesicular uptake of 5-HT and thus contributes to the level of secretion. Activation of a G protein-coupled Ca2+ receptor (CaR) on the plasmalemma of PF cells regulates downstream effectors (including: phospholipase C, NO synthase and guanylyl cyclase) that lead to vesicle acidification. We now propose to determine the extent to which this transduction pathway is shared by the secretion process and the degree to which the secretory vesicles of PF cells are similar to the synaptic vesicles of serotonergic neurons. Aim 1 is to determine the mechanism underlying CaR-induced gating of the vesicles of PF cells, by focusing on the relationship between vesicle acidification and secretion as measured by quantal release of 5-HT from PF cells. The interaction of CaR with the voltage-gated Ca2+ channels present in PF cells will be studied using patch-clamp recordings, accompanied by Ca2+ imaging. Aim 2 will be to compare properties of PF vesicles with those of synaptic vesicles of serotonergic neurons in situ and immortalized neuronal serotonergic cell line RN46A. We will examine whether these synaptic vesicles exhibit stimulus-induced acidification, and whether CaR participates in regulating the acidity of serotonergic synaptic vesicles. Secretory and synaptic vesicles that regulate internal pH in response to plasmalemmal stimulation are novel and, when understood, are likely to provide insight into serotonergic neurotransmission, as well as the drugs that affect it.

描述（改编自申请人的摘要）：血清素能 神经传递在许多生理功能中起着至关重要的作用。 然而，关于血清素能突触的特性知之甚少 囊泡。 由于很难纯化5-羟色胺能神经元或 它们的突触囊泡来自大脑，我们研究了含5-HT的囊泡 甲状腺的分泌囊泡作为模型。 PF囊泡是独一无二的，因为它们都类似于反式高尔基 网络衍生的受调节分泌的囊泡（含有肽）和 内体衍生的突触囊泡（包含回收膜 和一个小分子发射器）。 与其他分泌囊泡不同，PF 囊泡不是组成型酸性的，而是酸化PF细胞是酸性的 暴露于秘密的秘密，即增加（Ca2+）e。 酸化结果 从允许 涌入或H+。 Cl-通道的门控由其磷酸化诱导。 所得的三角pH最大化5-HT的囊泡摄取，因此 有助于分泌水平。 激活G蛋白耦合 PF细胞的浆体上的Ca2+受体（CAR）调节下游 效应子（包括：磷脂酶C，无合酶和鸟叶叶尼环酶） 导致囊泡酸化。 我们现在建议确定程度 分泌过程和 PF细胞的分泌囊泡与 血清素能神经元的突触囊泡。 目标1是确定 通过汽车诱导的PF细胞囊泡门控的机理，通过 专注于囊泡酸化与分泌之间的关系 通过从PF细胞中量化5-HT的数量释放来测量。 汽车的相互作用 将研究PF细胞中存在的电压门控Ca2+通道 使用贴片钳记录，并附有CA2+成像。 目标2是 比较PF囊泡的性质与突触囊泡的特性 血清素能神经元原位和永生的神经元血清素能细胞 线RN46A。 我们将检查这些突触囊泡是否显示 刺激引起的酸化，以及汽车是否参与调节 血清素能突触囊泡的酸度。 分泌和突触 响应浆膜刺激的囊泡的囊泡 是新颖的，如果理解，很可能会提供有关 血清素能神经传递以及影响它的药物。