Amiloride slows down calpain-mediated ABCA1 degradation through inhibition of hypoxia-induced NHE1 expression |
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| Authors: | MO Xian-gang ZHANG Li ZHANG Luo-chao WANG Long XIANG Ning YANG Juan SONG Xiang |
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| Institution: | Department of Geriatric Medicine, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, China |
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| Abstract: | AIM: To examine the effects of hypoxia on sodium-hydrogen exchange 1(NHE1) expression, intracellular Ca2+ concentration (Ca2+]i) and calpain activity, and to explore the effect of amiloride on adenosine triphosphate-binding cassette transporter A1(ABCA1) degradation and its calpain-related mechanism. METHODS: RAW264.7 cells were exposed to hypoxia for 0 h, 12 h, 24 h and 48 h. The cell viability was measured by MTT assay and the expression of NHE1 at mRNA and protein levels was detected by real-time PCR and Western blot. Ca2+]i was analyzed by flow cytometry. Calpain activity was assessed by the method of Suc-LLVY-aminoluciferin. Furthermore, the protein levels of ABCA1 in the RAW264.7 cells exposed to hypoxia for 24 h were determined after 6 h or 12 h treatment with NHE1 inhibitor amiloride in the presence of cycloheximide. ABCA1 protein levels and calpain activity were detected after 12 h incubation with calpain inhibitor ALLN or intracellular calcium-chelating agent BAPTA. RESULTS: Hypoxia inhibited the cell viability in a time-dependent manner. Hypoxia up-regulated the mRNA and protein expression of NHE1, and increased Ca2+]i and calpain activity. Hypoxia increased the degradation of ABCA1 and amiloride slowed down the ABCA1 degradation. ALLN or BAPTA increased ABCA1 protein level and decreased calpain activity. CONCLUSION: NHE1 inhibitor amiloride attenuates the calpain-mediated degradation of ABCA1, indicating that hypoxia-induced NHE1 might, at least in part, participate in the ABCA1 degradation. |
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| Keywords: | Adenosine triphosphate-binding cassette transporter A1 Amiloride Sodium-hydrogen exchanger 1 Calpain |
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