Direct intracellular nitric oxide detection in isolated adult cardiomyocytes: flow cytometric analysis using the fluorescent probe, diaminofluorescein

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.