Mechanism of Cell Volume Regulation in Liver

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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.