Fluorescence-based measurement of nitric oxide synthase activity in activated rat macrophages using...

We investigated the utility of the oxidant-reactive probe dichlorofluorescin (DCFH) for measurement of NO synthase (NOS) activity in rat alveolar macrophages (AMs) activated by culture for 18 h with interferon-gamma (IFN-gamma, 25 U/ml). Using both microplate-based fluorometry and flow cytometric analysis, AMs treated with l (but not d)-arginine showed a dose- and time-dependent increase in DCFH oxidation above buffer control (e.g., DCF production (nM): control, d-arginine 100 microM, l-arginine 100 microM respectively; 91+/-9, 76+/-11, 396+/-45, mean +/- SE, n = 6, 120 min). Furthermore, the NOS inhibitor (nitro-l-arginine) showed complete inhibition of the l-arginine-dependent DCF production. Parallel assays showed a strong correlation in DCFH oxidation with nitrite production in the same samples (e.g., DCF production (nM): 143, 222, 409; nitrite (microM): 2.2, 3.6, 7.1; for 5, 10, 50 microM l-arginine, respectively). In contrast, the alternate oxidant-reactive probes hydroethidine (HE) and dihydrorhodamine (DHR) did not report increased oxidant production in activated AMs incubated with l-arginine, despite their ability to easily detect intracellular superoxide anion production in cells treated with menadione (100 microM). We conclude that DCFH is a useful probe for quantitation of NOS-2 activity in activated rat lung macrophages.