PINEALOCYTE GAP JUNCTIONS

松果体细胞之间的缝隙连接为细胞间提供了一条途径 通讯影响电活动、新陈代谢、基因表达 和细胞分泌物。 我们打算将电生理学、生物化学、 免疫学和分子生物学来描述这些连接和 确定它们的生理作用。我们将研究生物物理 急性分离对之间的连接通信的性质 松果体细胞。我们将测量宏观结电导(GJ)， 确定其电压相关性并测量单通道电导。 存在其他门控过程，包括通过H+、Ca~(2+)和 辛醇将被检测。我们将研究GJ的短期调制，通过 影响褪黑激素分泌的药物，如去甲肾上腺素和 神经正常、明暗等环境因素的影响 循环、恒定的照明和恒定的黑暗。使用免疫学 技术和Northern印迹分析发现，大鼠松果体表达 连接蛋白43和21，但不包括连接蛋白27。缝隙连接蛋白26仅限于 松果体细胞和连接蛋白43仅见于星形胶质细胞。级别 免疫印迹法检测松果体细胞间隙连接蛋白26 被发现影响GJ的治疗方法。如果缝隙连接蛋白21水平发生变化， 编码缝隙连接蛋白的mRNA及其转录速率 将会被测量。蛋白质和信使核糖核酸的周转 在成对和小群的松果体细胞的原代培养中确定 [(35)S]-蛋氨酸和~3H-尿苷在松果体器官培养中的应用 分别进行脉冲追逐实验。这些测量结果应该显示 这些缝隙连接蛋白的表达是否在 信使核糖核酸合成水平、信使核糖核酸稳定性、蛋白质合成或蛋白质 退化。因为人们认为缝隙连接参与了 调节分泌，我们建议研究两者之间的关系 松果体细胞偶联、缝隙连接对钙离子的通透性与褪黑素 分泌物。用Fura-2和Ratio测定对钙离子的通透性 图像处理。褪黑素的分泌效果将被评估 间接通过比较分离的细胞和细胞团的分泌和 通过测定分离的细胞和细胞团对分泌的影响 辛醇的解偶联浓度。我们希望能够深入了解 连接蛋白21形成的缝隙连接的调节，这可能是 与松果体的功能以及缝隙的调节有关 大体上是交界处。松果体缝隙连接也可以作为 中枢神经系统中的电子突触。

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.