RRC ID 43759
著者 Ogata M, Awaji T, Iwasaki N, Fujimaki R, Takizawa M, Maruyama K, Iwamoto Y, Uchigata Y.
タイトル A new mitochondrial pH biosensor for quantitative assessment of pancreatic β-cell function.
ジャーナル Biochem Biophys Res Commun
Abstract Mitochondrial pH is a key determinant of mitochondrial energy metabolism. We have developed a new fluorescence-based ratiometric pH biosensor using a chloride-insensitive and hydrogen-sensitive probe for direct, quantitative and bleaching-free measurement in a living cell. Fusing this biosensor with a mitochondrial localization signal (MTpHGV) allowed us to determine mitochondrial pH. This new system was applied to measure mitochondrial pH in pancreatic β-cells, in which mitochondrial function plays a pivotal role in insulin secretion. Rat INS1 cells and mouse MIN6 cells are transfected with MTpHGV stably to monitor mitochondrial pH. While carbonyl cyanide 3-chlorophenylhydrazone (CCCP) treatment rapidly decreased mitochondrial pH in cultured rat MTpHGV-INS-1 cells, MTpHGV-MIN6 cells showed a rapid increase. These data suggest that MTpHGV probe exist in matrix side in INS-1 cells, but on the outer side of mitochondrial inner membrane in MIN6 cells. Moreover, while MTpHGV-INS-1 cells showed a rapid increase of pH by glucose stimulation, mitochondrial pH decreased quickly by glucose stimulation in all MTpHGV-MIN6 cells examined and recovered smoothly. Perfusion study of glucose load in MTpHGV-MIN6 cells under aminooxyacetate (AOA) or 100μM diazoxide showed that this mitochondrial pH acidification was dependent on nicotinamide adenine dinucleotide (NADH) shuttle, but independent from KATP channel. This new system for measuring mitochondrial pH is sensitive across the range of physiologic conditions and may be a useful tool for evaluating mitochondrial function in living cells.
巻・号 421(1)
ページ 20-6
公開日 2012-4-27
DOI 10.1016/j.bbrc.2012.03.093
PII S0006-291X(12)00553-0
PMID 22465007
MeSH Aminooxyacetic Acid / pharmacology Animals Biosensing Techniques* Calcium / metabolism Carbonyl Cyanide m-Chlorophenyl Hydrazone / pharmacology Cell Line Diazoxide / pharmacology Glucose / pharmacology Hydrogen-Ion Concentration Insulin / metabolism Insulin Secretion Insulin-Secreting Cells / chemistry* Insulin-Secreting Cells / drug effects Insulin-Secreting Cells / metabolism Mice Mitochondria / chemistry* Proton Ionophores / pharmacology Rats
IF 2.985
引用数 3
WOS 分野 BIOPHYSICS BIOCHEMISTRY & MOLECULAR BIOLOGY
リソース情報
ヒト・動物細胞 COS-7(RCB0539)