Measuring the rate of NADPH consumption by glutathione reductase in the cytosol and mitochondria.

Autor: Ting KKY; Department of Immunology, University of Toronto, Toronto, ON, Canada.; Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada., Floro E; Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada.; Institute of Biomedical Engineering, University of Toronto, Toronto, ON, Canada., Dow R; Department of Immunology, University of Toronto, Toronto, ON, Canada.; Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada., Jongstra-Bilen J; Department of Immunology, University of Toronto, Toronto, ON, Canada.; Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada.; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada., Cybulsky MI; Department of Immunology, University of Toronto, Toronto, ON, Canada.; Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada.; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.; Peter Munk Cardiac Centre, University Health Network, Toronto, ON, Canada., Rocheleau JV; Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada.; Institute of Biomedical Engineering, University of Toronto, Toronto, ON, Canada.; Department of Physiology, University of Toronto, Toronto, ON, Canada.; Banting and Best Diabetes Centre, University of Toronto, Toronto, ON, Canada.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2024 Dec 05; Vol. 19 (12), pp. e0309886. Date of Electronic Publication: 2024 Dec 05 (Print Publication: 2024).
DOI: 10.1371/journal.pone.0309886
Abstrakt: Background: NADPH is an essential co-factor supporting the function of enzymes that participate in both inflammatory and anti-inflammatory pathways in myeloid cells, particularly macrophages. Although individual NADPH-dependent pathways are well characterized, how these opposing pathways are co-regulated to orchestrate an optimized inflammatory response is not well understood. To investigate this, techniques to track the consumption of NADPH need to be applied. Deuterium tracing of NADPH remains the gold standard in the field, yet this setup of mass-spectrometry is technically challenging and not readily available to most research groups. Furthermore, NADPH pools are compartmentalized in various organelles with no known membrane transporters, suggesting that NADPH-dependent pathways are regulated in an organelle-specific manner. Conventional methods such as commercial kits are limited to quantifying NADPH in whole cells and not at the resolution of specific organelles. These limitations reflect the need for a novel assay that can readily measure the consumption rate of NADPH in different organelles.
Methods: We devised an assay that measures the consumption rate of NADPH by glutathione-disulfide reductase (GSR) in the mitochondria and the cytosol of RAW264.7 macrophage cell lines. RAW264.7 cells were transfected with Apollo-NADP+ sensors targeted to the mitochondria or the cytosol, followed by the treatment of 2-deoxyglucose and diamide. Intravital imaging over time then determined GSR-dependent NADPH consumption in an organelle-specific manner.
Discussion: In lipopolysaccharide (LPS)-stimulated RAW264.7 cells, cytosolic and mitochondrial NADPH was consumed by GSR in a time-dependent manner. This finding was cross validated with a commercially available NADPH kit that detects NADPH in whole cells. Loading of RAW264.7 cells with oxidized low-density lipoprotein followed by LPS stimulation elevated GSR expression, and this correlated with a more rapid drop in cytosolic and mitochondrial NADPH in our assay. The current limitation of our assay is applicability to transfectable cell lines, and higher expression of plasmid-encoded sensors relative to endogenous glucose-6-phosphate dehydrogenase.
Competing Interests: The authors have declared that no competing interests exist.
(Copyright: © 2024 Ting et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
Databáze: MEDLINE
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