Fig. 2. Yeast cells synthesize NO upon apoptosis induction, which is dependent on L-arginine. (A) NO production in untreated, H₂O₂-treated and chronologically aged cells was indirectly assessed through measurement of nitrite and nitrate concentrations as described in Materials and Methods. ^*P0.05 versus untreated cells, ^**P0.03 versus 1-day-old cells; t-test, n=3. (B) NO production in untreated and H₂O₂-treated (1.5 and 2.0 mM) cells assessed by flow cytometric quantification of cells stained with the NO indicator DAF-FM diacetate, in the absence (white area under the peak) or presence (shaded area) of the non-metabolized L-arginine analogue L-NAME. The data are presented in the form of frequency histograms displaying relative fluorescence (x axis) against the number of events analyzed (y axis). (C) Direct measurement of L-arginine-dependent NO production upon H₂O₂-induced apoptosis. NO production was recorded with a NO-selective electrode (AmiNO-700) upon addition of 4 mM H₂O₂ to 5x10⁸ wild-type cells (black line) or to wild-type cells pre-incubated with the non-metabolized D-arginine (blue line) or L-NAME (green line). A control experiment without cells was also recorded and is represented as a red line. (D) Rate of NO production is H₂O₂ dependent. 2 mM or 4 mM of H₂O₂ was added to 5x10⁸ wild-type cells and NO production assessed using the NO-selective electrode (AmiNO-700). Data presented correspond to the linear part of the NO production curve. Rate of NO production was calculated from the slope.