Nitric oxide measurements in hTERT-RPE cells and subcellular fractions exposed to low levels of red light

Jeffrey C. Wigle; Cherry C. Castellanos; Michael L. Denton; Eric A. Holwitt

doi:10.1117/12.2036993

18 February 2014 Nitric oxide measurements in hTERT-RPE cells and subcellular fractions exposed to low levels of red light

Jeffrey C. Wigle, Cherry C. Castellanos, Michael L. Denton, Eric A. Holwitt

Author Affiliations +

Proceedings Volume 8932, Mechanisms for Low-Light Therapy IX; 89320D (2014) https://doi.org/10.1117/12.2036993
Event: SPIE BiOS, 2014, San Francisco, California, United States

Abstract

Cells in a tissue culture model for laser eye injury exhibit increased resistance to a lethal pulse of 2.0-μm laser radiation if the cells are first exposed to 2.88 J/cm² of red light 24 hr prior to the lethal laser exposure. Changes in expression of various genes associated with apoptosis have been observed, but the biochemical link between light absorption and gene expression remains unknown. Cytochome c oxidase (CCOX), in the electron transport chain, is the currentlyhypothesized absorber. Absorption of the red light by CCOX is thought to facilitate displacement of nitric oxide (NO) by O² in the active site, increasing cellular respiration and intracellular ATP. However, NO is also an important regulator and mediator of numerous physiological processes in a variety of cell and tissue types that is synthesized from l-arginine by NO synthases. In an effort to determine the relative NO contributions from these competing pathways, we measured NO levels in whole cells and subcellular fractions, with and without exposure to red light, using DAF-FM, a fluorescent dye that stoichiometrically reacts with NO. Red light induced a small, but consistently reproducible, increase in fluorescence intensity in whole cells and some subcellular fractions. Whole cells exhibited the highest overall fluorescence intensity followed by (in order) cytosolic proteins, microsomes, then nuclei and mitochondria.

Citation Download Citation

Jeffrey C. Wigle, Cherry C. Castellanos, Michael L. Denton, and Eric A. Holwitt "Nitric oxide measurements in hTERT-RPE cells and subcellular fractions exposed to low levels of red light", Proc. SPIE 8932, Mechanisms for Low-Light Therapy IX, 89320D (18 February 2014); https://doi.org/10.1117/12.2036993

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