Effect of cAMP agonists and DMSO on ATP release. (A) Two separately prepared concentrated forskolin stock solutions in DMSO (final concentration 30 µM), or equivalent volume of DMSO, were directly applied to cell suspensions at time 0, and ATP release was quantified at different time points, with the controls being nonstimulated cells (representative of n = 4 experiments). (B) Low DMSO concentration (<10%) does not induce RBC ATP release. The same amount of DMSO was added to RBC suspension either as a small aliquot of 100% concentrated stock or after 10-fold dilution (to 10% DMSO in PSS), giving the same final concentration of 0.27%. Black bars (scale on the left) – extracellular ATP, white bars (scale on the right) – hemoglobin released. Each bar represents an average of n = 4 experiments ± SEM. No statistically significant difference was found in either ATP or hemolysis between the controls and samples treated with 10% DMSO. The difference between the controls and samples treated with 100% concentrated stock was statistically significant (P < .05, Mann-Whitney U test). (C) Effect of cAMP stimulation on extracellular ATP. Cells were incubated for 5 minutes at 37°C with forskolin (F, 30 µM), or a mixture of F (30 µM) plus isoproterenol (I, 10 µM) and papaverine (P, 100 µM). Extracellular ATP level (x-axis) was plotted against absorbance at λ = 414 nm (y-axis) measured in the same supernatant samples. Extracellular ATP showed strong linear correlation with hemolysis (P < .0001; R = 0.975). The slope of linear fit (gray line) corresponded to intracellular ATP concentration of 1.48 mM. Open circles, control, nonstimulated cells; closed circles, cells stimulated by F; closed squares, cells stimulated by F+I+P cocktail. Inset: ATP (fmol/10⁶ cells) in RBC supernatants treated with F or F+I+P mixture (average ± SEM, n = 6). Average ATP release was not significantly different for all 3 conditions (P = .23). The data are representative of n = 4 similar experiments.