PINEALOCYTE GAP JUNCTIONS

松果体细胞间隙连接

• 批准号: 3860981
• 负责人: JUAN SAEZ
• 金额: --
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 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/3860981
• 关键词: Drosophilidae; electrical potential; electrophysiology; fluorescent dye /probe; gap junctions; hypothalamic hormones; laboratory rat; melatonin; monoclonal antibody; neurotransmitters; norepinephrine; northern blottings; pineal body; radioimmunoassay; steroid hormone biosynthesis

Gap junctions between pinealocytes provide a pathway for intercellular communication affecting electrical activity, metabolism, gene expression and cell secretion. We intend to combine techniques of electrophysiology, biochemistry, immunology and molecular biology to characterize these junctions and determine their physiological role. We will study the biophysical properties of junctional communication between pairs of acutely dissociated pinealocytes. We will measure macroscopic junctional conductance (gj), determine its voltage dependence and measure single channel conductances. The presence of other gating processes including closure by H+, Ca2+ and octanol will be tested for. We will study short term modulation of gj by agents that affect melatonin secretion, e.g. norepinephrine (NE) and the effect of environmental factors including normal innervation, light/dark cycles, constant illumination, and constant darkness. Using immunological techniques and Northern blot analysis we found that rat pineals express both connexins 43 and 21 but not connexin 27. Connexin 26 is restricted to pinealocytes and connexin 43 is found only in the astrocytes. Levels of pinealocyte connexin 26 will be measured by Western blotting after treatments found to affect gj. If levels of connexin 21 change, the levels of the mRNA encoding the gap junction protein and its transcription rate will be measured. Turnover of the protein and of the mRNA will be determined in primary cultures of pairs and small groups of pinealocytes or in pineals in organ culture by means of [(35)S]-methionine and 3H-uridine pulse-chase experiments respectively. These measurements should show whether the expression of these gap junction proteins is regulated at the level of mRNA synthesis, mRNA stability, protein synthesis or protein degradation. Since it is thought that gap junctions participate in the regulation of secretion, we propose to study the relationship between pinealocyte coupling, gap junction permeability to Ca2+, and melatonin secretion. Permeability to Ca2+ will be assayed using fura-2 and ratio image processing. The effect of secretion of melatonin will be evaluated indirectly by comparing secretion by isolated cells and cell clusters and by determining the effect on secretion by isolated cells and cell cluster of uncoupling concentration of octanol. We expect to obtain insight into the regulation of gap junctions formed by connexin 21, which could be relevant to function of the pineal gland as well as to regulation of gap junctions in general. The pineal gap junction may also serve as a model of electrical synapses in the central nervous system.

松果体细胞之间的缝隙连接为细胞间的相互作用提供了途径。 影响电活动、代谢、基因表达的通信 和细胞分泌。 我们打算联合收割机电生理学，生物化学， 免疫学和分子生物学来表征这些连接， 决定其生理作用。我们将研究生物物理学 急性分离神经元对之间连接通讯的性质 松果体细胞我们将测量宏观结电导（gj）， 确定其电压依赖性并测量单通道电导。 其他门控过程的存在，包括H+，Ca 2+和 将检测辛醇。我们将研究短期调制的gj， 影响褪黑激素分泌的药剂，例如去甲肾上腺素（NE）和 环境因素的影响，包括正常的神经支配，光/暗 循环，持续的照明，和持续的黑暗。使用免疫学 技术和北方印迹分析，我们发现大鼠松果体表达 连接蛋白43和21，但不连接蛋白27。连接蛋白26仅限于 连接蛋白43仅在星形胶质细胞中发现。水平 松果体细胞连接蛋白26将通过蛋白质印迹法测量， 发现治疗影响GJ。如果连接蛋白21的水平发生变化， 编码差距连接蛋白的mRNA及其转录速率 将被衡量。蛋白质和mRNA的周转将 在成对和小群松果体细胞的原代培养物中测定，或 用[（35）S]-蛋氨酸和~ 3 H-尿苷在器官培养的松果体中 脉冲追踪实验。这些测量应该显示 这些缝隙连接蛋白的表达是否在细胞内受到调节， mRNA合成水平、mRNA稳定性、蛋白质合成或蛋白质 降解由于缝隙连接被认为参与了 调节分泌，我们建议研究 松果体细胞偶联、缝隙连接对Ca ~（2+）的通透性和褪黑素 分泌物。将使用fura-2和比率测定对Ca 2+的渗透性 图像处理将评估褪黑激素分泌的影响 间接地通过比较分离的细胞和细胞簇的分泌， 通过测定分离的细胞和细胞簇对分泌的影响 辛醇的解偶联浓度。我们希望能深入了解 由连接蛋白21形成的间隙连接的调节， 与松果体的功能以及间隙的调节有关 总的来说，连接。松果体间隙连接也可以作为一个模型， 中枢神经系统中的电突触