title: Amiloride attenuates lipopolysaccharide-accelerated atherosclerosis via inhibition of NHE1-dependent endothelial cell apoptosis.
abstract: OBJECTIVE
  To investigate the effects of the potassium-sparing diuretic amiloride on endothelial cell apoptosis during lipopolysaccharide (LPS)-accelerated atherosclerosis.
  
  
  METHODS
  Human umbilical vein endothelial cells (HUVECs) were exposed to LPS (100 ng/mL) in the presence of drugs tested. The activity of Na(+)/H(+) exchanger 1 (NHE1) and calpain, intracellular free Ca(2+)level ([Ca(2+)](i)), as well as the expression of apoptosis-related proteins in the cells were measured. For in vivo study, ApoE-deficient (ApoE(-/-)) mice were fed high-fat diets with 0.5% (w/w) amiloride for 4 weeks and LPS (10 μg/mouse) infusion into caudal veins. Afterwards, atherosclerotic lesions, NHE1 activity and Bcl-2 expression in the aortic tissues were evaluated.
  
  
  RESULTS
  LPS treatment increased NHE1 activity and [Ca(2+)](i) in HUVECs in a time-dependent manner, which was associated with increased activity of the Ca(2+)-dependent protease calpain. Amiloride (1-10 μmol/L) significantly suppressed LPS-induced increases in NHE1 activity, [Ca(2+)](i). and calpain activity. In the presence of the Ca(2+) chelator BAPTA (0.5 mmol/L), LPS-induced increase of calpain activity was also abolished. In LPS-treated HUVECs, the expression of Bcl-2 protein was significantly decreased without altering its mRNA level. In the presence of amiloride (10 μmol/L) or the calpain inhibitor ZLLal (50 μmol/L), the down-regulation of Bcl-2 protein by LPS was blocked. LPS treatment did not alter the expression of Bax and Bak proteins in HUVECs. In the presence of amiloride, BAPTA or ZLLal, LPS-induced HUVEC apoptosis was significantly attenuated. In ApoE(-/-) mice, administration of amiloride significantly suppressed LPS-accelerated atherosclerosis and LPS-induced increase of NHE1 activity, and reversed LPS-induced down-regulation of Bcl-2 expression.
  
  
  CONCLUSIONS
  LPS stimulates NHE1 activity, increases [Ca(2+)](i), and activates calpain, which leads to endothelial cell apoptosis related to decreased Bcl-2 expression. Amiloride inhibits NHE1 activity, thus attenuates LPS-accelerated atherosclerosis in mice.
authors.affiliation: Key Laboratory for Zoonosis Research, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun, China.
authors.forename: Gui-mei
authors.initials: GM
authors.lastname: Cui
authors.forename: Yu-xi
authors.initials: YX
authors.lastname: Zhao
authors.forename: Na-na
authors.initials: NN
authors.lastname: Zhang
authors.forename: Zeng-shan
authors.initials: ZS
authors.lastname: Liu
authors.forename: Wan-chun
authors.initials: WC
authors.lastname: Sun
authors.forename: Qi-sheng
authors.initials: QS
authors.lastname: Peng
mesh_terms: D000584:Amiloride / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D050197:Atherosclerosis / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000378:metabolism; D027682:Cation Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D062686:Epithelial Sodium Channel Blockers / Q000494:pharmacology*; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D008070:Lipopolysaccharides / Q000378:metabolism; D008297:Male; D051379:Mice; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D027682:Cation Transport Proteins; D062686:Epithelial Sodium Channel Blockers; D008070:Lipopolysaccharides; D019253:Proto-Oncogene Proteins c-bcl-2; C490998:SLC9A1 protein, human; C490997:Slc9a1 protein, mouse; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D002154:Calpain; D002118:Calcium