Altered absorptive function in the gall bladder during cholesterol gallstone formation is associated with abnormal NHE3 complex formation.

Dysfunction of the Na⁺/H⁺ exchanger 3 (NHE3) contributes to the formation of cholesterol gallstones. We aimed to investigate whether NHE3 dysfunction is associated with abnormalities in NHE3 complex formation. We fed C57BL/6 mice with control or lithogenic diet and study the expression of NHE3, ezrin, and Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) in the gallbladder (GB) using RT-PCR and western blot. Immunofluorescence and immunoprecipitation were performed to investigate the interactions of NHE3 with ezrin or NHERF1. To explore the initiating factor that leads to NHE3 dysfunction, we stimulated cholangiocarcinoma cells with taurochenodeoxycholate (TCDC) and/or forskolin. The effects of TCDC on the expression of NHE3 regulatory proteins, as well as their bindings to NHE3, were detected by western blot and immunoprecipitation. Enzyme-linked immunosorbent assay was used to study the regulation of cAMP production by TCDC. The expression of NHERF1 and ezrin phosphorylation level were increased in the gallbladder epithelial cells (GBECs) of C57BL/6 mice with cholesterol gallstones. Immunofluorescence studies demonstrated that the subcellular localization of ezrin and NHERF1 were similar to that of NHE3 in GBECs. Immunoprecipitation revealed that ezrin formed macrocomplex with NHE3, which were enhanced after gallstone formation. TCDC increased forskolin-induced cAMP accumulation, and NHERF1 and expression in cholangiocarcinoma cells. Under the synergistic effect of forskolin, TCDC stimulated ezrin phosphorylation, with enhanced interaction between ezrin and NHE3. The formation of cholesterol gallstones is associated with abnormal formation of NHE3 complexes. Decreased biliary TCDC may be an initiating factor that leads to abnormal GB absorption.

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