胆囊吸收功能增强是胆囊结石形成的重要原因之一，但其发生机制不清。我们在前期研究工作中发现结石形成过程中胆囊粘膜胆固醇含量增高和NHE3活性增强，由此我们推测，高胆固醇可能通过抑制PKA功能和激活PKC活性，以协同作用的方式调控NHE3在胆囊粘膜上皮细胞中的定位，提高NHE3在顶质膜的表达水平，进而增强其转运活性和促进胆囊吸收。本课题主要拟采用免疫荧光共聚焦技术、免疫共沉淀和非变性胶电泳分析等技术手段研究胆囊结石形成过程中是否出现了PKA和PKC信号通路调控的改变；明确PKA是否抑制NHE3的磷酸化进而抑制其从细胞膜到细胞质的转运，PKC是否活化ezrin进而促进NHE3从细胞质到细胞膜的转运。该研究将明确PKA和PKC信号通路异常上调NHE3活性的机制，为胆囊结石的预防与治疗提供新靶点。

Enhanced absorptive function of the gallbladder is a major cause of gallstone disease, but the pathogenic mechanisms are not clear. Our preliminary data showed that NHE3 activity and cholesterol content of the plasma membrane were up-regulated in the process of gallstone formation. We propose that activation of PKC and inhibition of PKA by cholesterol synergistically stimulates NHE3 activity by enhancing its apical membrane localization. By using modern cellular and molecular techniques, including but not limited to confocal immunofluorescence, co-immunoprecipitation and native-PAGE electrophoresis, we will test whether cholesterol alters PKA and PKC signaling, whether attenuation of PKA activity down-regulates the phosphorylation and internalization of NHE3, and whether PKC promotes NHE3 exocytosis from the intracellular pool through enhanced phosphorylation of ezrin. The proposed study will shed novel light on the underlying mechanisms of NHE3 regulation by high cholesterol content, thereby to provide novel strategies in the prevention and treatment of gallstones.