CHROMAFFIN CELLS--STIMULUS SECRETION COUPLING

本申请集中于Ca通道和钙通道的机制。 嗜铬细胞中的胞吐作用。在动物体内， 肾上腺髓质分泌儿茶酚胺和几种神经肽 荷尔蒙分泌是由Ca 2+进入细胞触发的， 电压门控钙通道。嗜铬细胞有三种不同的类型 钙通道。一种类型，易化钙通道，不被激活， 单次短暂去极化。这个频道通常是静默的;它不 有助于电活动或Ca 2+内流。 促进案例 通道被许多去极化激活， 电位，或通过去极化到非常正的电位。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钙通道都存在于 嗜铬细胞能够触发分泌;易化Ca 渠道是最有效的。易化Ca产生的分泌 通道更大、更快，并且与 其他通道类型产生的释放，即使当钙电流 更小。通过同时测量Ca电流、[Ca 2 +]和分泌 (with电容），我们希望确定最佳条件， 触发每种钙通道的释放。我们将模拟分泌， 嗜铬细胞中存在每种类型的Ca通道。 嗜铬细胞储存并分泌肾上腺素或去甲肾上腺素 (some细胞可以分泌两者）。在一些初步研究中， 发现含有细胞的细胞没有易化Ca通道。使用 伏安技术来鉴定嗜铬细胞是肾上腺素 或去甲肾上腺素分泌我们会验证这一发现去甲 细胞将被广泛研究，以更好地了解儿茶酚胺 从细胞分泌，推测没有促进钙通道。所有 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 含有肾上腺素的细胞具有易化钙通道。这些 研究可能揭示分泌的改变， 钙通道的存在。 在嗜铬细胞中记录的钙电流使用穿孔补丁整体- 细胞构型不同于用全细胞记录的电流 膜片钳配置与低EGTA的移液管。这是一 重要的一点，因为几乎所有的电容研究分泌都使用 低EGTA的全细胞记录条件。我们将研究分泌 从嗜铬细胞使用穿孔的全细胞记录条件。 鉴于我们的初步结果，我觉得研究分泌物是至关重要的， 细胞内成分完好无损最后，我们将确定 神经递质可以调节分泌，同时保持Ca电流 [Ca 2 +]i常数。 如果发现了调制，我们将试图阐明 机制。

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.