Mitochondrial transmembrane potential is diminished in phorbol myristate acetate-stimulated peritoneal resident macrophages isolated from wild-type mice, but not in those from gp91-phox-deficient mice.
Macrophages produce superoxide (O2-) during phagocytosis or upon stimulation with a variety of agents including phorbol myristate acetate (PMA) through the activation of NADPH oxidase, and the formed O2- is converted to other reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). The aim of the present study was to elucidate the effect of the intracellularly produced ROS on mitochondrial transmembrane potential (MTP) in mouse (C57BL/6) peritoneal resident macrophages stimulated with PMA. Using a fluorescent dye, succinimidyl ester of dichlorodihydrofluorescein (H2DCFDA), O2- was visualized in intracellular compartments in a certain subpopulation of macrophages isolated from wild-type mice. Cells deficient in gp91-phox, one of the membrane components of NADPH oxidase, were negative for the fluorescence. When cells were loaded with both H2DCFDA and MitoCapture, a fluorescent dye for mitochondria, mitochondrial fluorescence was diminished in O2- -producing cells, but not in O2- -deficient cells. Flow cytometry also revealed the decrease of mitochondrial fluorescence in wild-type cells, but not in gp91-phox-deficient cells. The loss of mitochondrial fluorescence was prevented by microinjection of catalase into cells. The present findings demonstrate that MTP is diminished by ROS, including the H2O2 dismutated from O2-, produced intracellularly by activation of the NADPH oxidase in mouse peritoneal resident macrophages stimulated with PMA.
Kobayashi
Toshihiro
T
Department of Anatomy and Cell Biology, Kochi Medical School, Kochi University, Kohasu, Okoh-cho, Nankoku, 783-5305 Kochi, Japan. kobayash@kochi-ms.ac.jp
Ogawa
Yasuhiro
Y
Watanabe
Yoshiya
Y
Furuya
Masato
M
Kataoka
Sayo
S
Garcia del Saz
Eva
E
Tsunawaki
Shohko
S
Dinauer
Mary C
MC
Seguchi
Harumichi
H
D000818:Animals; D002374:Catalase / Q000378:metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D046529:Microscopy, Electron, Transmission; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000633:toxicity*
D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D017382:Reactive Oxygen Species; D002374:Catalase; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D013755:Tetradecanoylphorbol Acetate