Mechanism of Cell Volume Regulation in Liver

描述（由申请人提供）：本提案的主要目标是 确定肝细胞对肿胀反应的调节机制 由生理压力产生的。这些机制对于正常的 肝功能，它们的紊乱是气球样变性的基础 这是从急性肝炎 肝移植术后保存损伤。补偿反应 肝细胞肿胀，称为调节性容积减少（RVD），介导 由于钾离子活化引起的液体和电解质流出， 氯离子通道然而，我们对这些类型的理解仍然存在很大的差距 钾离子通道以及它们是如何被激活的。最近的观察 在该实验室中表明：（a）肝细胞肿胀增加细胞溶质 通过细胞内钙库释放和钙浓度 内流;（B）它激活钙依赖性大电导（BK）和小电导 电导（SK）钾通道;和（c）RVD是钙依赖性的， 通过阻断BK和SK通道抑制。因此，工作假设 这一建议是，肝细胞钙的动态增加通过以下途径控制RVD： BK和SK通道的激活。以下每个特定目标将测试 这一假设的关键组成部分，并将提供新的信息， 控制肝脏细胞体积的基本分子机制。具体目标 主要有：（1）确定肝细胞肿胀促进钙离子释放的机制 介导容量调节反应的信号;（2）阐明如何 肿胀诱导的肝细胞钙增加导致BK活化， SK通道;以及（3）定义BK和SK通道对 肝细胞肿胀后体积恢复。具体目标1的研究将 确定肿胀诱导钙储存释放的机制， 发生钙内流。具体目标2中的研究将定义 钙浓度、细胞内钙动员和钙内流 BK和SK频道开通。具体目标3中的研究将评估 改变细胞水平对肝细胞体积调节的影响 我们已经从肝脏克隆了功能性BK和SK通道同种型。拟议 研究将使用互补的最先进的细胞和分子生物学 研究模型肝细胞系中体积调节的方法以及 来自大鼠和条件性SK 3敲除小鼠的原代肝细胞。这些 研究将在一个重要但知之甚少的领域提供新的信息 肝细胞生物学，他们将产生新的见解肝病 与病理性细胞肿胀有关。

DESCRIPTION (provided by applicant): The broad goal of this proposal is to determine regulatory mechanisms by which liver cells respond to swelling produced by physiological stresses. These mechanisms are crucial for normal liver function, and their derangement underlies the ballooning degeneration that is a hallmark of pathological conditions that range from acute hepatitis to preservation injury after liver transplantation. The compensatory responses to liver cell swelling, termed regulatory volume decrease (RVD), are mediated by fluid and electrolyte efflux resulting from the activation of potassium and chloride channels. However, large gaps remain in our understanding of the types of potassium channels involved and how they are activated. Recent observations in this laboratory indicate that: (a) liver cell swelling increases cytosolic calcium concentration through intracellular calcium store release and calcium influx; (b) it activates calcium-dependent large conductance (BK) and small conductance (SK) potassium channels; and (c) RVD is calcium-dependent and inhibited by blockade of BK and SK channels. Thus, the working hypothesis for this proposal is that dynamic increases in liver cell calcium control RVD via activation of BK and SK channels. Each of the following Specific Aims will test critical components of this hypothesis and will provide new information about basic molecular mechanisms that control cell volume in liver. The specific aims are: (1) to determine mechanisms by which liver cell swelling elicits calcium signals that mediate volume regulatory responses; (2) to elucidate how swelling-induced increases in liver cell calcium lead to activation of BK and SK channels; and (3) to define the contributions of BK and SK channels to volume recovery after liver cell swelling. Studies in Specific Aim 1 will determine the mechanisms by which swelling induced calcium store release and calcium influx occur. Studies in Specific Aim 2 will define the influence of calcium concentration, intracellular calcium mobilization, and calcium influx on BK and SK channel opening. Studies in Specific Aim 3 will evaluate the impact on hepatocellular volume regulation of altering cellular levels of functional BK and SK channel isoforms we have cloned from liver. The proposed studies will use complementary state-of-the-art cell and molecular biological approaches to study volume regulation in a model liver cell line as well as primary hepatocytes from rats and from conditional SK3 knockout mice. These studies will provide new information in an important but poorly understood area of liver cell biology, and they will yield new insights into liver diseases associated with pathological cell swelling.