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Direct detection of intracellular superoxide generation from freshly isolated rat aorta: a novel approach
Author(s) -
Goel Aditya,
Matkar Smita,
Rahimian Roshanak
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.719.5
Subject(s) - superoxide , fluorescence microscope , phorbol , aorta , chemistry , intracellular , nitric oxide , incubation , acetylcholine , biophysics , fluorescence , biochemistry , medicine , biology , protein kinase c , physics , organic chemistry , quantum mechanics , enzyme
Although superoxide (O 2 − ) plays an important role in vascular diseases, direct measurement of O 2 − in viable tissue remains a challenge. The aim of this study was to design and validate a procedure to measure O 2 − generation using freshly isolated aorta. Method: The 8 mm fresh aortic segments from male and female rats were cut longitudinally in Krebs buffer at 37°C and mounted on the silicon elastomer surface with the lumenal surface facing upward. The segments were incubated with the nitric oxide probe 4,5‐diaminofluorescein diacetate (DAF‐2DA, 5 μM) or O 2 − probe dihydroethidium (DHE, 2.5 μM), followed by fluorescent recoding at longitudinal edge using Leica DMIRE2 microscope. Acetylcholine‐induced increased DAF fluorescence was taken as evidence for the endothelial integrity. To examine the effect of O 2 − generator, DHE fluorescence was quantified before and after incubation of segments with phorbol myristate acetate (PMA, 0.1 μM), or PMA in the presence of O 2 − scavenger (tempol, 100 μM) for 30 min. Results: The extent of PMA‐induced O 2 − increase was greater in female tissues (9.2 fold) than that in males (2.8 fold), and this was prevented by the presence of tempol. This data was comparable to our recent report on gender difference in PMA‐induced O 2 − production in rat aorta using isometric tension study. Conclusion: This procedure will allow us to detect O 2 − generation in translucent viable vessels. (Supported by NHLBI)