Direct intracellular nitric oxide detection in isolated adult cardiomyocytes: Flow cytometric analysis using the fluorescent probe, diaminofluorescein
Date
2004
Authors
Strijdom H.
Muller C.
Lochner A.
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
We assessed the possibility to detect intracellular nitric oxide (NO) with the NO-specific probe 4,5-diaminofluorescein-2/diacetate (DAF-2/DA), by flow cytometry, in fresh adult rat cardiomyocytes, and compared the findings with results obtained from quantitation of cellular nitrate/nitrite (NOx) levels. Methods. - Cardiomyocytes were isolated by collagenase perfusion, followed by incubation in a Krebs-Henseleit/2% bovine serum albumin buffer in the presence of 10 μM DAF-2/DA (∼0.5 × 106 cells/ml). Experimental conditions were: (i) baseline control, (ii) NO donor (2-(N,N-diethylamino)-diazenolate 2-oxide, DEA/NO) administration, and (iii) 120 min simulated ischemia (hypoxia). In addition, control and hypoxic groups were incubated with the NO synthase (NOS) inhibitor, NW-nitro-L-arginine methyl ester (L-NAME). Following incubation and washing, intracellular fluorescence of DAF-triazol (DAF-2T, oxidized form of DAF-2/DA) was analyzed by flow cytometry. NOx levels were determined with an NOx assay. Fluorescence-activated cell sorter (FACS) data were expressed as mean fluorescence intensity (percentage of control) and NOx levels as pmol/106 cells. Results. - Optimal baseline fluorescence was obtained when myocytes were incubated with DAF-2/DA for 3 h at 37 °C. The NO donor DEA/NO (500 μM) and hypoxia significantly increased DAF fluorescence and NOx levels. L-NAME addition significantly reversed these trends in the hypoxia groups. Conclusions. - We have demonstrated that intracellular NO can be detected in fresh isolated adult cardiomyocytes by flow cytometry with 10 μM DAF-2/DA. Furthermore, we demonstrated that hypoxia is an activator of adult cardiomyocyte NOS, as demonstrated by both end-points. Reproducibility observed between results obtained by FACS analysis and NOx assays suggests that DAF-2/DA fluorescence can be regarded as an independent marker for intracellular NO in cardiomyocytes. © 2004 Elsevier Ltd. All rights reserved.
Description
Keywords
2 (n,n diethylamino)diazenolate 2 oxide, bovine serum albumin, collagenase, diaminofluorescein 2 diacetate, fluorescein derivative, n(g) nitroarginine methyl ester, nitric oxide, nitric oxide donor, nitric oxide synthase inhibitor, unclassified drug, animal cell, article, cell culture, cell isolation, controlled study, data analysis, flow cytometry, fluorescence activated cell sorting, fluorescence analysis, heart muscle cell, heart muscle ischemia, heart muscle perfusion, hypoxia, nonhuman, priority journal, rat, reproducibility, response time, statistical significance, urea cycle, Animals, Cell Hypoxia, Cells, Cultured, Flow Cytometry, Fluorescein, Fluorescent Dyes, Myocytes, Cardiac, Nitric Oxide, Nitric Oxide Synthase, Rats, Bovinae
Citation
Journal of Molecular and Cellular Cardiology
37
4
37
4