Phosphorylation and subcellular localization of Na+/H+exchanger isoform 3 (NHE3) are associated with altered gallbladder absorptive function after formation of cholesterol gallstones.
Na+/H+exchanger isoform 3 (NHE3) dysfunction is thought to contribute to the altered gallbladder absorption that occurs in cholesterol gallstone disease, but the mechanism is unknown. The current study was undertaken to examine the expression, phosphorylation, and subcellular localization of NHE3in gallbladder epithelium cells (GBECs) of male C57BL/6 mice on a control or lithogenic diet. Thirty-six 8-week-old male C57BL/6 mice were randomly assigned to receive a high cholesterol diet or a regular diet for 8 weeks. Gallstone formation was recorded. Gallbladder bile cholesterol, phospholipid, and total bile acids were examined. RT-PCR was used to measure NHE3mRNA expression. NHE3protein expression and subcellular localization were examined by Western blotting and immunofluorescence microscopy, respectively. Gallstones were formed in all mice fed the lithogenic diet. Despite higher NHE3mRNA expression in gallbladders of the mice on the lithogenic diet than in those on the control diet, there was no significant difference in expression of total NHE3protein. However, a higher level of NHE3phosphorylated at serine-552 (P-NHE3) was seen on the lithogenic diet. In immunofluorescence studies, NHE3protein was expressed both on the apical membrane and in the cytoplasm of mouse GBEC. This pattern of subcellular distribution of NHE3strongly corroborates an exchanger trafficking mechanism in NHE3activity regulation in mouse GBEC. We conclude that increased phosphorylation of NHE3following gallstone formation leads to turnover of the exchanger, resulting in decreased gallbladder concentrating function.
Department of General Surgery, Shengjing Hospital of China Medical University, No. 36, San Hao Street, Heping District, Shenyang, Liaoning Province, 110004, China.
Yongsheng
Y
Chen
Department of General Surgery, Shengjing Hospital of China Medical University, No. 36, San Hao Street, Heping District, Shenyang, Liaoning Province, 110004, China. surgeon_cys@163.com.
Shuodong
S
Wu
Department of General Surgery, Shengjing Hospital of China Medical University, No. 36, San Hao Street, Heping District, Shenyang, Liaoning Province, 110004, China.
Yu
Y
Tian
Department of General Surgery, Shengjing Hospital of China Medical University, No. 36, San Hao Street, Heping District, Shenyang, Liaoning Province, 110004, China.
Jing
J
Kong
D065567:Absorption, Physiological; D000818:Animals; D002791:Cholesterol, Dietary / Q000009:adverse effects; D019826:Cholic Acid / Q000009:adverse effects; D059305:Diet, High-Fat / Q000009:adverse effects; D004848:Epithelium / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D005704:Gallbladder / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D042882:Gallstones / Q000209:etiology / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D005786:Gene Expression Regulation; D008297:Male; D008810:Mice, Inbred C57BL; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D011897:Random Allocation; D012694:Serine / Q000378:metabolism; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
D002791:Cholesterol, Dietary; D012333:RNA, Messenger; C000613386:Slc9a3 protein, mouse; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D012694:Serine; D019826:Cholic Acid
Absorption; Cholesterol gallstone disease; Na+/H+ exchanger isoform 3 (NHE3); Phosphorylation