WHOLE CELL CAPACITANCE MEASUREMENTS IN SALIVARY CELLS

描述：这是一项小额拨款提案的重新提交，在该提案中 申请者Kenneth Izutsu博士建议将一种新的方法应用于 胞吐作用的调节及其机制的研究 分泌颗粒，使用膜片钳仪器能够测量 全电池电容。因为分泌颗粒的胞吐作用增加了 膜到细胞的表面膜面积，而且因为整个细胞 电容与表面膜面积成正比，这是 技术可以有效地用作分泌物胞吐作用的一种测量 颗粒。这些研究，类似于之前对其他细胞类型的研究， 将在大鼠下颌下腺腺泡细胞中进行 文化。这些细胞的优点是不是转化的细胞 线条，有许多分泌颗粒，身体状况良好 以生化观点为特征的。初步数据显示， 已经成功地制定了程序，以准备 分散的、单独的腺泡细胞，可以进行常规记录 在全细胞膜片钳模式下从这些细胞中分离出来。为回应这一事件 关于对第一个提交的意见的批评，提供了关于 将颗粒内容物的分泌与细胞相关的方法 膜电容发生变化。具体地说，建议使用CELL 免疫印迹法测量单个细胞分泌的特定蛋白质。 Bertil Hille博士将担任安装和使用 电池电容硬件和软件以及对电池性能的影响 免疫印迹分析和里查多·马丁内斯博士 腺泡细胞制剂的发展，并已进行了许多 对颌下腺泡细胞的生化研究，将作为一种 研究中蛋白质分泌方面的顾问。 经修订的申请的具体目的如下： 具体目标L：获得并安装制造电池所需的设备 使用全细胞膜片钳技术测量电容。 具体目标2：检验电池电容变化是否相关 大鼠颌下腺分泌颗粒内容物的分泌 原代培养中的腺泡细胞。这将主要使用以下工具进行测试 去甲肾上腺素刺激。 特定目的3：检测分泌颗粒膜的内吞作用 如通过电池电容的变化来衡量，则在没有 外源钙离子。 特定目的4：检测分泌颗粒中的离子通道 在颗粒过程中观察到的细胞电导变化与细胞膜有关 胞吐。 申请人解释说，这些目标中的第一个涉及安装 设备，第二个涉及重大实验工作，第三个 第四个目标是使用所开发的方法进行额外的研究 并在前两个阶段使用，目的是进一步验证系统的有效性。这个 申请人建议，如果成功，这种技术应该会打开一个新的 分泌物胞吐作用调节机制的研究进展 颗粒。

DESCRIPTION: This is a resubmission of a small grant proposal in which the applicant, Dr. Kenneth Izutsu, proposes to apply a new methodology to the study of the regulation of, and the mechanisms underlying, the exocytosis of secretory granules, using patch clamp instrumentation capable of measuring whole-cell capacitances. Because exocytosis of secretory granules adds membrane to the surface membrane area of the cell, and because whole-cell capacitance is directly proportional to the surface membrane area, this technique can be used effectively as a measure of exocytosis of secretory granules. These studies, similar to previous studies in other cell types, are to be performed in rat submandibular gland acinar cells in primary culture. These cells have the advantage of not being a transformed cell line, of having numerous secretory granules, and of being fairly well characterized from a biochemical point of view. Preliminary data show that procedures have been successfully developed for the preparation of dispersed, individual acinar cells, and that routine recordings can be made from these cells in the whole-cell patch clamp mode. In response to the critique of the first submission, more details are provided concerning the methods to be used to correlate secretion of granule contents with cell membrane capacitance changes. Specifically, it is proposed to use cell immunoblots to measure secretion of specific proteins from individual cells. Dr. Bertil Hille will serve as the consultant on installing and using the cell capacitance hardware and software as well as on performing the cell immunoblot assay, and Dr. Richardo Martinez, who collaborated on the development of the acinar cell preparation and has performed many biochemical studies on submandibular acinar cells, will serve as a consultant on the protein secretion aspects of the studies. The specific aims in the revised application are: Specific Aim l: To obtain and install the equipment necessary to make cell capacitance measurements using the whole-cell patch clamp technique. Specific Aim 2: To test whether cell capacitance changes are correlated with secretion of secretory granule contents in single rat submandibular gland acinar cells in primary culture. This will primarily be tested using norepinephrine stimulation. Specific Aim 3: To test whether endocytosis of secretory granule membranes as measured by cell capacitance changes, is inhibited in the absence of external Ca2+. Specific Aim 4: To test whether ion channels in the secretory granule membranes contribute to the cell conductance changes observed during granule exocytosis. The applicant explains that the first of these aims involves installing the equipment, the second involves the major experimental work, and the third and fourth aims involve additional studies using the methodology developed and used in the first two aims to further validate the system. The applicant suggests that, if successful, this technique should open a new approach to the study of the regulation of the exocytosis of secretory granules.