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

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.