Mechanism of Cell Volume Regulation in Liver

描述(申请人提供)：本提案的主要目标是确定肝细胞对肿胀作出反应的调节机制。 由生理压力产生的。这些机制对于正常的 肝功能，而它们的紊乱是气球状退变的基础 这是从急性肝炎到各种病理情况的标志。 对肝移植后保存损伤的影响。补偿性反应 对肝细胞肿胀，称为调节性体积减少(RVD)是介导的 由于钾和钙的活化而产生的液体和电解质外流 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 所涉及的钾通道以及它们如何被激活。最近的观察结果 在这个实验室表明：(A)肝细胞肿胀增加胞浆 细胞内钙库释放的钙浓度和钙 内流；(B)激活钙依赖的大电导(BK)和小电导 电导(SK)钾通道；和(C)RVD是钙依赖的， 被BK和SK通道阻断所抑制。因此，工作假说 这一建议是动态增加的肝细胞钙控制RVD通过 激活BK和SK通道。以下每个具体目标都将进行测试 这一假说的关键组成部分，并将提供关于 控制肝脏细胞体积的基本分子机制。具体目标 是：(1)确定肝细胞肿胀引起钙离子的机制 调节音量调节反应的信号；(2)阐明 肿胀诱导的肝细胞钙升高导致BK和 SK频道；以及(3)定义BK和SK频道对 肝细胞肿胀后体积恢复。对特定目标1的研究将 确定肿胀诱导钙储存释放和 发生钙离子内流。在具体目标2中的研究将确定 钙浓度、细胞内钙动员和钙内流 关于BK和SK频道的开通。特定目标3的研究将评估 细胞水平改变对肝细胞体积调节的影响 我们从肝脏中克隆了具有功能的BK和SK通道亚型。建议数 研究将使用互补的最先进的细胞和分子生物学 研究模型肝细胞系体积调节的方法以及 来自大鼠和条件性SK3基因敲除小鼠的原代肝细胞。这些 研究将在一个重要但鲜为人知的领域提供新的信息 他们将对肝脏疾病产生新的见解 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.