Matching native electrical stimulation by graded chemical stimulation in isolated mouse adrenal chromaffin cells
Matching native electrical stimulation by graded chemical stimulation in isolated mouse adrenal chromaffin cells
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DOI：
10.1016/j.jneumeth.2007.07.004
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发表时间：
2007-11-30
影响因子：
3
通讯作者：
Smith, Corey
中科院分区：
文献类型：
--
作者：
Fulop, Tiberiu;Smith, Corey
肾上腺嗜铬细胞在交感神经刺激下释放多种递质。适度的细胞放电，匹配交感神经的音调，释放少量的可自由溶解的儿茶酚胺。在交感应激下匹配输入的较高的电放电频率会导致儿茶酚胺以及包装在分泌颗粒致密核心内的半溶血管和神经活性多肽的释放。这种依赖活性的差异递质释放已经被证明依赖于胞吐模式的机械性转变，通过调节颗粒和细胞表膜之间的分泌融合孔的扩张。然而，调节融合孔扩张的机制的生化描述仍然难以捉摸。在实验设置中，通过单电池电压钳实现了模拟交感输入的电刺激。这种方法虽然精确，但与生化和蛋白质组学分析不相容，这两种方法都需要大量的样本。我们在当前的研究中解决了这一限制。我们描述了一种批量化学刺激范例，该范例经过校准以匹配定义的电活动。我们利用钙和单细胞安培测量来匹配细胞外钾浓度与交感神经张力和急性应激条件下的生理电刺激。这种方法提供了更大的均匀刺激细胞样本，用于确定肾上腺嗜铬细胞活性依赖的差异递质释放的分子角色。(C)2007 Elsevier B.V.保留所有权利。
Adrenal chromaffin cells release multiple transmitters in response to sympathetic stimulation. Modest cell firing, matching sympathetic tone, releases small freely soluble catecholamines. Elevated electrical firing rates matching input under sympathetic stress results in release of catecholamines as well as semi-soluble vaso- and neuro-active peptides packaged within the dense core of the secretory granule. This activity-dependent differential transmitter release has been shown to rely on a mechanistic shift in the mode of exocytosis through the regulated dilation of the secretory fusion pore between granule and cell surface membranes. However, biochemical description of the mechanism regulating fusion pore dilation remains elusive. In the experimental setting, electrical stimulation designed to mimic sympathetic input, is achieved through single-cell voltage-clamp. While precise, this approach is incompatible with biochemical and proteomic analysis, both of which require large sample sizes. We address this limitation in the current study. We describe a bulk chemical stimulation paradigm calibrated to match defined electrical activity. We utilize calcium and single-cell amperometric measurements to match extracellular potassium concentrations to physiological electrical stimulation under sympathetic tone as well as acute stress conditions. This approach provides larger samples of uniformly stimulated cells for determining molecular players in activity-dependent differential transmitter release from adrenal chromaffin cells. (c) 2007 Elsevier B.V. All rights reserved.