Koh Duk-Su
Department of Physiology and Biophysics, University of Washington, Seattle, USA.
Methods Mol Biol. 2006;337:139-53. doi: 10.1385/1-59745-095-2:139.
Activation of Ca2+ channels in the plasma membrane or on internal Ca2+ stores raises cytosolic Ca2+ concentration ([Ca2+]c). Among diverse functions of Ca2+ signals, the induction of exocytosis-the process in which the contents of secretory vesicles are released by their fusion to the plasma membrane-is one of the most important. For example, in neurons and endocrine cells, it allows intercellular communication and secretion of biomolecules. Exocytosis can be detected by several physical and chemical means. By chemically oxidizing the released secretory products at a fixed electrode potential, carbon fiber amperometry provides excellent temporal and spatial resolution in detecting exocytosis. This method, together with other biophysical techniques such as patch clamp and Ca2+ microphotometry, has greatly contributed to our understanding of the molecular mechanisms involved in the stimulus-secretion coupling. However, amperometry can be performed only on cells that secrete oxidizable molecules. To overcome this limit, we have developed a protocol of loading cells with exogenous neurotransmitters that readily oxidize on a carbon electrode. Several cell types have been successfully loaded, exocytosis of secretory vesicles has been demonstrated, and in pancreatic duct epithelial cells, the modulatory signals of exocytosis have been studied in detail.
质膜或细胞内钙库上的钙离子通道激活会提高胞质钙离子浓度（[Ca2+]c）。在钙离子信号的多种功能中，诱导胞吐作用（即分泌囊泡内容物通过与质膜融合而释放的过程）是最重要的功能之一。例如，在神经元和内分泌细胞中，它允许细胞间通讯和生物分子的分泌。胞吐作用可以通过多种物理和化学方法检测。通过在固定电极电位下化学氧化释放的分泌产物，碳纤维安培法在检测胞吐作用时具有出色的时间和空间分辨率。该方法与其他生物物理技术如膜片钳和钙离子显微光度法一起，极大地促进了我们对刺激-分泌偶联中涉及的分子机制的理解。然而，安培法只能在分泌可氧化分子的细胞上进行。为了克服这一限制，我们开发了一种用外源神经递质加载细胞的方案，这些神经递质在碳电极上容易氧化。已经成功加载了几种细胞类型，证明了分泌囊泡的胞吐作用，并且在胰腺导管上皮细胞中，对胞吐作用的调节信号进行了详细研究。
