Measurement of cytosolic and mitochondrial pH in living cells during reversible metabolic inhibition

C. Balut; M. Vandeven; S. Despa; I. Lambrichts; M. Ameloot; P. Steels; I. Smets

doi:10.1038/sj.ki.5002632

Measurement of cytosolic and mitochondrial pH in living cells during reversible metabolic inhibition

C. Balut, M. Vandeven, S. Despa, I. Lambrichts, M. Ameloot, P. Steels, I. Smets

Research output: Contribution to journal › Article › peer-review

64 Scopus citations

Abstract

Renal ischemia and subsequent reperfusion lead to changes in the regulation of hydrogen ions across the mitochondrial membrane. This study was designed to monitor pH changes in the cytosol and mitochondria of Madin-Darby Canine Kidney cells exposed to metabolic inhibition and subsequent recovery. A classical one-photon confocal imaging approach using the pH-sensitive fluorophore carboxy SNARF-1 was used to define specific loading, calibration, and correction procedures to obtain reliable cytosolic and mitochondrial pH values in living cells. Metabolic inhibition resulted in both cytosolic and mitochondrial acidification, with a more pronounced decrease of mitochondrial pH as compared to the cytosolic pH. Shortly after removing the metabolic inhibition, cytosolic pH did not recover, whereas mitochondrial pH slowly increased. Our method is applicable to other cell types provided that the mitochondria can be loaded with SNARF-1 and that the cells possess a mitochondria-free region to measure SNARF-1 in the cytosol.

Original language	English
Pages (from-to)	226-232
Number of pages	7
Journal	Kidney International
Volume	73
Issue number	2
DOIs	https://doi.org/10.1038/sj.ki.5002632
State	Published - Jan 10 2008

Bibliographical note

Funding Information:
We acknowledge Mr J Janssen for his excellent technical assistance with the cell culture. We also thank Mr M Jans, P Pirotte, W Leyssens, R Van Werde, and Mrs R Beenaerts for their technical help and Mrs M Ieven for art work. We gratefully acknowledge using ImageJ freeware (NIH, Bethesda, MD, USA; Dr W Rasband: http://rsb.info.nih.gov/ij/plugins/lsm-reader.html ) and plugins like LSM Reader and LSM Toolbox (University of Strasbourg, Strasbourg, France; Drs J Mutterer, Y Krempp and P Pirrotte) and Dr G Hamel, SVI BV. This work was supported by a bilateral research collaboration program between Flanders and Romania (BOF 05 B03), by the tUL impulse financing and by the Research Foundation—Flanders (FWO, GO270.07).

Keywords

2-deoxyglucose
Confluent non-permeabilized renal cells
Cyanide
Ischemia/reperfusion
SNARF-1

ASJC Scopus subject areas

Nephrology

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10.1038/sj.ki.5002632

Cite this

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title = "Measurement of cytosolic and mitochondrial pH in living cells during reversible metabolic inhibition",

abstract = "Renal ischemia and subsequent reperfusion lead to changes in the regulation of hydrogen ions across the mitochondrial membrane. This study was designed to monitor pH changes in the cytosol and mitochondria of Madin-Darby Canine Kidney cells exposed to metabolic inhibition and subsequent recovery. A classical one-photon confocal imaging approach using the pH-sensitive fluorophore carboxy SNARF-1 was used to define specific loading, calibration, and correction procedures to obtain reliable cytosolic and mitochondrial pH values in living cells. Metabolic inhibition resulted in both cytosolic and mitochondrial acidification, with a more pronounced decrease of mitochondrial pH as compared to the cytosolic pH. Shortly after removing the metabolic inhibition, cytosolic pH did not recover, whereas mitochondrial pH slowly increased. Our method is applicable to other cell types provided that the mitochondria can be loaded with SNARF-1 and that the cells possess a mitochondria-free region to measure SNARF-1 in the cytosol.",

keywords = "2-deoxyglucose, Confluent non-permeabilized renal cells, Cyanide, Ischemia/reperfusion, SNARF-1",

author = "C. Balut and M. Vandeven and S. Despa and I. Lambrichts and M. Ameloot and P. Steels and I. Smets",

note = "Funding Information: We acknowledge Mr J Janssen for his excellent technical assistance with the cell culture. We also thank Mr M Jans, P Pirotte, W Leyssens, R Van Werde, and Mrs R Beenaerts for their technical help and Mrs M Ieven for art work. We gratefully acknowledge using ImageJ freeware (NIH, Bethesda, MD, USA; Dr W Rasband: http://rsb.info.nih.gov/ij/plugins/lsm-reader.html ) and plugins like LSM Reader and LSM Toolbox (University of Strasbourg, Strasbourg, France; Drs J Mutterer, Y Krempp and P Pirrotte) and Dr G Hamel, SVI BV. This work was supported by a bilateral research collaboration program between Flanders and Romania (BOF 05 B03), by the tUL impulse financing and by the Research Foundation—Flanders (FWO, GO270.07).",

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AU - Balut, C.

AU - Vandeven, M.

AU - Despa, S.

AU - Lambrichts, I.

AU - Ameloot, M.

AU - Steels, P.

AU - Smets, I.

N1 - Funding Information: We acknowledge Mr J Janssen for his excellent technical assistance with the cell culture. We also thank Mr M Jans, P Pirotte, W Leyssens, R Van Werde, and Mrs R Beenaerts for their technical help and Mrs M Ieven for art work. We gratefully acknowledge using ImageJ freeware (NIH, Bethesda, MD, USA; Dr W Rasband: http://rsb.info.nih.gov/ij/plugins/lsm-reader.html ) and plugins like LSM Reader and LSM Toolbox (University of Strasbourg, Strasbourg, France; Drs J Mutterer, Y Krempp and P Pirrotte) and Dr G Hamel, SVI BV. This work was supported by a bilateral research collaboration program between Flanders and Romania (BOF 05 B03), by the tUL impulse financing and by the Research Foundation—Flanders (FWO, GO270.07).

PY - 2008/1/10

Y1 - 2008/1/10

N2 - Renal ischemia and subsequent reperfusion lead to changes in the regulation of hydrogen ions across the mitochondrial membrane. This study was designed to monitor pH changes in the cytosol and mitochondria of Madin-Darby Canine Kidney cells exposed to metabolic inhibition and subsequent recovery. A classical one-photon confocal imaging approach using the pH-sensitive fluorophore carboxy SNARF-1 was used to define specific loading, calibration, and correction procedures to obtain reliable cytosolic and mitochondrial pH values in living cells. Metabolic inhibition resulted in both cytosolic and mitochondrial acidification, with a more pronounced decrease of mitochondrial pH as compared to the cytosolic pH. Shortly after removing the metabolic inhibition, cytosolic pH did not recover, whereas mitochondrial pH slowly increased. Our method is applicable to other cell types provided that the mitochondria can be loaded with SNARF-1 and that the cells possess a mitochondria-free region to measure SNARF-1 in the cytosol.

AB - Renal ischemia and subsequent reperfusion lead to changes in the regulation of hydrogen ions across the mitochondrial membrane. This study was designed to monitor pH changes in the cytosol and mitochondria of Madin-Darby Canine Kidney cells exposed to metabolic inhibition and subsequent recovery. A classical one-photon confocal imaging approach using the pH-sensitive fluorophore carboxy SNARF-1 was used to define specific loading, calibration, and correction procedures to obtain reliable cytosolic and mitochondrial pH values in living cells. Metabolic inhibition resulted in both cytosolic and mitochondrial acidification, with a more pronounced decrease of mitochondrial pH as compared to the cytosolic pH. Shortly after removing the metabolic inhibition, cytosolic pH did not recover, whereas mitochondrial pH slowly increased. Our method is applicable to other cell types provided that the mitochondria can be loaded with SNARF-1 and that the cells possess a mitochondria-free region to measure SNARF-1 in the cytosol.

KW - 2-deoxyglucose

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KW - Cyanide

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Measurement of cytosolic and mitochondrial pH in living cells during reversible metabolic inhibition

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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