PINEAL GAP JUNCTION--CHARACTERIZATION AND REGULATION

松果体间隙连接——特征和调节

• 批准号: 3242093
• 负责人: JUAN CARLOS SAEZ
• 金额: $ 12.81万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-01-01 至 1992-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3242093
• 关键词: acidity /alkalinity; alpha antiadrenergic agent; astrocytes; beta antiadrenergic agent; biochemistry; calcium channel; cell type; central nervous system; cyclic AMP; cyclic GMP; cytogenetics; electrophysiology; gap junctions; gene expression; hormone metabolism; hormone regulation /control mechanism; hydrogen transport; immunology; isoproterenol; laboratory rat; melatonin; membrane permeability; membrane potentials; messenger RNA; methionine; molecular biology; neurotransmitters; norepinephrine; northern blottings; organ culture; phenylephrine; pineal body; radiotracer; second messengers; secretion; synapses; tissue /cell culture; tritium; uridine; vasoactive intestinal peptide; voltage gated channel; western blottings

Gap junctions between pinealocytes provide a pathway for intercellular communication affecting electrical activity, metabolism, and possibly gene expression and secretion. We intend to combine techniques of electrophysiology, biochemistry, immunology and molecular biology to characterize these junctions and determine their physiology 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. [Permeability to Ca2+ will be assayed quantitatively as a indicator of permeability to second messengers, e.g. cAMP and cGMP]. 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 26 but not connexin 32. [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 26 change, the levels of the mRNA encoding it and the 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 [35S]-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. The effect on secretion of melatonin will be evaluated indirectly; secretion by isolated cells and by cell clusters in the presence and absence of an uncoupling agent such as octanol will be compared. The effect of octanol on secretion by isolated cells will control for its effect on junctions formed by connexin 26, 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.

松果体之间的间隙连接为细胞间的途径提供了途径 沟通影响电活动，代谢和可能的基因 表达和分泌。 我们打算将 电生理学，生物化学，免疫学和分子生物学 表征这些连接并确定其生理作用。 我们将 研究对之间的连接通信的生物物理特性 急性解离的松果体。 我们将测量宏观 连接电导（GJ），确定其电压依赖性和度量 单通道电导。 其他门控过程的存在 包括H+，Ca2+和Octanol的闭合。 [对Ca2+的渗透性将被定量分析作为指标 对第二使者的渗透性，例如营地和CGMP]。 我们将学习 影响褪黑激素分泌的药物对GJ的短期调制，例如 去甲肾上腺素（NE）和环境因素的影响 正常的神经支配，光/黑色周期，恒定照明和恒定 黑暗。 使用免疫学技术和北印迹分析我们 发现大鼠松果体同时表达连接蛋白43和26，但不能表达连接蛋白 [32。 仅在星形胶质细胞中]。 松果体连接蛋白26的水平为 通过发现影响GJ的治疗后，通过蛋白质印迹测量。 如果 连接素26的水平变化，编码它的mRNA水平及 将测量转录率。 蛋白质和蛋白质的营业额 mRNA将在一对原发性培养物和小组中确定 松果体或在器官培养中的松果体中通过[35s] - 甲硫氨酸 和3H-尿苷的脉搏追踪实验。 这些测量值 应该说明这些间隙连接蛋白的表达是否是 在mRNA合成水平，mRNA稳定性，蛋白质合成水平 或蛋白质降解。 由于认为差距连接参与 在分泌的调节中，我们建议研究这种关系 在松果体耦合之间，间隙连接到Ca2+和 褪黑激素分泌。 对褪黑激素分泌的影响将是 间接评估；分离的细胞和细胞簇分泌 诸如辛醇之类的解偶联剂的存在和不存在将是 比较的。 辛醇对分离细胞分泌的影响将 控制它对连接蛋白26形成的连接的影响，这可能是 与松果体的功能以及间隙调节有关 一般而言。 松果体间隙连接点也可以用作 中枢神经系统中的电突触。