CHROMAFFIN CELLS--STIMULUS SECRETION COUPLING

嗜铬细胞--刺激分泌耦合

• 批准号: 2182069
• 负责人: AARON P. FOX
• 金额: $ 19.48万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2182069
• 关键词: action potentials; animal poison; calcium channel; calcium channel blockers; calcium flux; calcium indicator; catecholamines; chelating agents; chromaffin cells; dihydropyridines; dopamine; epinephrine; exocytosis; neurotoxins; neurotransmitter transport; norepinephrine; second messengers; secretion; tissue /cell culture; voltage /patch clamp

This application focusses on the Ca channels and on mechanisms of exocytosis found in chromaffin cells. In the animal, chromaffin cells in the adrenal medulla secrete catecholamines and several neuropeptide hormones. Secretion is triggered by Ca2+ entering the cells through voltage-gated Ca channels. Chromaffin cells possess three different types of Ca channels. One type, the facilitation Ca channel, is not activated by single brief depolarizations. This channel is normally silent; it does not contribute to electrical activity or to Ca2+ influx. Facilitation Ca channels are activated by many depolarizations to physiological potentials, or by depolarizations to very positive potentials. Recruitment of these channels may involve a novel form of channel regulation, voltage- dependent phosphorylation. All three types of Ca channels found in chromaffin cells are capable of triggering secretion; facilitation Ca channels are most efficient. Secretion produced by facilitation Ca channels is larger, faster, and has a shorter latency when compared to release produced by the other channels types, even when the Ca current is smaller. By simultaneously measuring Ca currents, [Ca2+] and secretion (with capacitance), we hope to determine the optimal conditions for triggering release by each type of Ca channel. We will model secretion for each type of Ca channel present in chromaffin cells. Chromaffin cells store and secrete either epinephrine or norepinephrine (some cells may secrete both). In a few preliminary studies norepinephrine containing cells were found to have no facilitation Ca channels. Using voltammetric techniques to identify chromaffin cells as either epinephrine or norepinephrine secreting, we will verify this finding. Norepinephrine cells will be studied extensively to better understand catecholamine secretion from cells that presumably have no facilitation Ca channels. All the Ca channels in norepinephrine cells will be tested for efficacy in promoting secretion. These data will be compared to secretion data from epinephrine containing cells that have facilitation Ca channels. These studies may expose alterations in secretion resulting from differences in the Ca channels present. Ca currents recorded in chromaffin cells using the perforated patch whole- cell configuration are different than currents recorded with whole-cell patch clamp configuration with low EGTA in the pipette. This is an important point as almost all capacitance studies secretion have used whole-cell recording conditions with low EGTA. We will study secretion from chromaffin cells using perforated whole-cell recording conditions. Given our preliminary results I feel it's crucial to study secretion with the intracellular constituents intact. Finally, we will determine whether secretion can be modulated by neurotransmitters, while keeping Ca currents and [Ca2+]i constant. If modulation is found, we will try and elucidate the mechanism.

该应用重点关注 Ca 通道和机制 嗜铬细胞中发现胞吐作用。在动物中，嗜铬细胞 肾上腺髓质分泌儿茶酚胺和多种神经肽 荷尔蒙。 Ca2+ 进入细胞后触发分泌 电压门控 Ca 通道。嗜铬细胞具有三种不同类型 Ca 通道。一种类型，即促进 Ca 通道，不会被激活 单次短暂去极化。该通道通常是静默的；它没有 有助于电活动或 Ca2+ 流入。 便利化钙 通道被许多生理去极化激活 电位，或通过去极化至非常正的电位。招聘 这些通道可能涉及一种新形式的通道调节，电压- 依赖性磷酸化。所有三种类型的 Ca 通道都存在于 嗜铬细胞能够触发分泌；促进钙 渠道是最有效率的。促进 Ca 产生的分泌 与相比，通道更大、更快、延迟更短 即使 Ca 电流是由其他通道类型产生的释放 较小。通过同时测量 Ca 电流、[Ca2+] 和分泌 （用电容），我们希望确定最佳条件 触发每种类型的 Ca 通道的释放。我们将模拟分泌 嗜铬细胞中存在的每种类型的 Ca 通道。 嗜铬细胞储存和分泌肾上腺素或去甲肾上腺素 （有些细胞可能两者都分泌）。在一些初步研究中，去甲肾上腺素 发现含有细胞没有易化 Ca 通道。使用 伏安技术鉴定嗜铬细胞是否为肾上腺素 或去甲肾上腺素分泌，我们将验证这一发现。去甲肾上腺素 将广泛研究细胞以更好地了解儿茶酚胺 可能没有促进 Ca 通道的细胞的分泌。全部 将测试去甲肾上腺素细胞中的 Ca 通道的功效 促进分泌。这些数据将与来自的分泌数据进行比较 含有肾上腺素的细胞具有促进钙离子通道。这些 研究可能会揭示由于差异而导致的分泌变化 存在 Ca 通道。 使用穿孔贴片全记录嗜铬细胞中的 Ca 电流 细胞配置与全细胞记录的电流不同 移液器中具有低 EGTA 的膜片钳配置。这是一个 重要的一点，因为几乎所有的电容研究分泌都使用了 低 EGTA 的全细胞记录条件。我们将研究分泌 使用穿孔全细胞记录条件从嗜铬细胞中提取。 鉴于我们的初步结果，我认为研究分泌物至关重要 细胞内成分完好无损。最后，我们将确定是否 分泌可以通过神经递质调节，同时保持 Ca 电流 和 [Ca2+]i 常数。 如果发现调制，我们将尝试阐明 的机制。