Abstract | UNLABELLED: 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 (NO(x)) 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 microM DAF-2/DA (approximately 0.5 x 10(6) 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, N(W)-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. NO(x) levels were determined with an NO(x) assay. Fluorescence-activated cell sorter (FACS) data were expressed as mean fluorescence intensity (percentage of control) and NO(x) levels as pmol/10(6) cells. RESULTS: Optimal baseline fluorescence was obtained when myocytes were incubated with DAF-2/DA for 3 h at 37 degrees C. The NO donor DEA/NO (500 microM) and hypoxia significantly increased DAF fluorescence and NO(x) 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 microM 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 NO(x) assays suggests that DAF-2/DA fluorescence can be regarded as an independent marker for intracellular NO in cardiomyocytes.
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Authors | Hans Strijdom, Christo Muller, Amanda Lochner |
Journal | Journal of molecular and cellular cardiology
(J Mol Cell Cardiol)
Vol. 37
Issue 4
Pg. 897-902
(Oct 2004)
ISSN: 0022-2828 [Print] England |
PMID | 15380680
(Publication Type: Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't)
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Chemical References |
- 4,5-diaminofluorescein diacetate
- Fluorescent Dyes
- Nitric Oxide
- Nitric Oxide Synthase
- Fluorescein
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Topics |
- Animals
- Cell Hypoxia
- Cells, Cultured
- Flow Cytometry
- Fluorescein
(chemistry)
- Fluorescent Dyes
- Myocytes, Cardiac
(chemistry, enzymology, metabolism)
- Nitric Oxide
(analysis, metabolism)
- Nitric Oxide Synthase
(metabolism)
- Rats
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