1. Hemolysis is a primary ATP-release mechanism in human erythrocytes
by Jacek Sikora, Sergei N. Orlov, Kishio Furuya, and Ryszard Grygorczyk
Blood
Volume 124(13):
September 25, 2014
©2014 by American Society of Hematology

2. Hypotonic shock-induced ATP release tightly correlates with cell lysis.
Hypotonic shock-induced ATP release tightly correlates with cell lysis. (A) ATP content in RBC supernatants exposed for 5 minutes to isotonic (control) or 25%, 30%, or 37% hypotonic solutions in 6-well plate format. The data are average ± SEM (n = 3 determinations), and, for hypotonic conditions, they are significantly different from control values (P < .05, Mann-Whitney U test, indicated by *). (B) Absorbance spectra (λ = nm) of RBC supernatants exposed to different hypotonic solutions from the experiment reported in A. Absorbance of supernatant from cell lysates equivalent to 100, 1000, and 5000 cells/µL is also shown. Note that the absorbance spectra of 30% hypotonic stress supernatants overlap with those of lysates of 1000 cells/µL. From the calibration curve of peak absorbance (at 414 nm) vs cell lysate density, as illustrated in supplemental Figure 1B, we could calculate the number and percentage of lysed cells in each experimental condition. (C) For low hemolysis levels, lysed cell number was evaluated more precisely by plotting the entire absorbance spectra of the supernatant vs the absorbance spectra of the reference lysate. y-axis: Absorbance spectra of RBC supernatants exposed to different hypotonic solutions from the experiments reported in A are plotted against absorbance of reference lysate of 1000 cells/µL (x-axis) for the entire spectrum between 380 and 620 nm. From the slope of linear fit, the exact number of lysed cells in the supernatants was determined for each experimental condition: Iso, Hypo 25%, 30%, and 37%. The corresponding number of lysed cells was 226, 603, 1073, and 6470/µL, respectively. (D) Hypotonic shock-induced ATP release vs number of lysed cells. The number of lysed cells in control (Iso) and hypotonic conditions was calculated by fitting their corresponding absorbance spectra to those of a reference cell lysate of 1000 cells/µL (white circles), shown in C. The solid line is a least square linear fit to the hypotonic shock-induced ATP release data with 95% confidence bands indicated by dashed lines and correlation coefficient R = Red triangles indicate ATP measured in cell lysates prepared from the same batch of blood and adjusted to corresponding numbers of lysed cells found under Iso and hypotonic conditions. The slope of the linear relationship corresponds to an intracellular ATP concentration of 1.42 mM. (E) Correlation between extent of hemolysis (shown as absorbance – left axis, or % hemolysis – right axis) and ATP release induced by hypotonic shock. Paired values of extracellular ATP and free hemoglobin in the supernatant samples were fitted by linear regression with Origin Laboratory 7.5. The data are examples of 3 independent experiments (each indicated by a different symbol) similar to those in A-D but performed in 12-well plate format. In each experiment, RBC suspensions were incubated in either isotonic or hypotonic (20%, 25%, 30%, 35%, or 40%) solution. The slopes of the fitted lines correspond to the intracellular ATP concentration and are indicated on the graph. (F) Effect of CBX (100 μM) on ATP release stimulated by 5-minute exposure to 25% hypotonic shock. Cells were preincubated for 10 minutes with CBX in isotonic PSS, then transferred to a Petri dish containing CBX in hypotonic medium. Average of n = 3 experiments ± SEM.
Jacek Sikora et al. Blood 2014;124:
©2014 by American Society of Hematology

3. Luminescence imaging of ATP release from RBCs
Luminescence imaging of ATP release from RBCs. (A) Image a is an IR-DIC image of RBCs before hypotonic shock application.
Luminescence imaging of ATP release from RBCs. (A) Image a is an IR-DIC image of RBCs before hypotonic shock application. Image b was obtained by subtracting control image a (before hypotonic shock) from that acquired ∼25 minutes after 20% hypotonic shock. The resulting white dots correspond to cells that lysed and were missing in images captured after hypotonic stimulation. c is an overlay of image b and cumulative ATP-dependent luminescence (in red) observed during the entire experiment. Note that regions of ATP release coincide with spots where cells lysed. (B) (a) Time-course of luminescence responses due to lysis of single RBCs in the experiment shown in A. Luminescence responses were measured at individual release sites seen in A and are indicated by traces of different colors. See supplemental Movie 1 for the entire time course of ATP release. (b) ATP release durations for the responses depicted in a measured from the start of release until the luminescence response decayed to 1/e of its peak value (black circles). Average (±SEM) release time was 17.6 ± 2.1 seconds (white square). (C) (a) Two examples (upper and lower row images) illustrating ATP release due to lysis of single RBCs. IR images of RBCs (in green) are overlaid with extracellular ATP-dependent luminescence (in red). 20% hypotonic shock-induced ATP release (center image) occurs shortly before lysis/collapse of a single RBC (indicated by a white arrow; see also supplemental Movie 2). No ATP release from intact RBCs is evident. The elapsed time (minutes:seconds) is shown in the upper-right corner. (b) Average delay time between start of ATP release and cell lysis for the responses shown in B (black circles). Average (±SEM) delay time was 47 ± 10.6 seconds (white square).
Jacek Sikora et al. Blood 2014;124:
©2014 by American Society of Hematology

4. Mechanically stimulated ATP release and hemolysis.
Mechanically stimulated ATP release and hemolysis. Inset: The amount of ATP (fmol/106 cells) detected in supernatants depends on the duration of mechanical shear stress stimulation. Each bar represents the average of 6 measurements ± SEM. The data on stimulated release are significantly different from control values (P < .05, Mann-Whitney U test, indicated by *). The main graph shows tight, linear correlation (R = 0.923; P < .0001) between ATP release induced by shear stress and extent of hemolysis (depicted as absorbance – left axis, or % hemolysis – right axis) for all data points (n = 30) of the experiment reported in the inset. Open symbols refer to the controls and solid symbols to stimulated RBC samples. The slope of linear fit corresponds to intracellular ATP concentration of 0.67 mM. The data are representative of n = 4 similar experiments. The estimated volume averaged shear stress was ∼4 dyne/cm2 (see “Materials and methods”).
Jacek Sikora et al. Blood 2014;124:
©2014 by American Society of Hematology

5. Effect of cAMP agonists and DMSO on ATP release.
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.
Jacek Sikora et al. Blood 2014;124:
©2014 by American Society of Hematology

6. Effect of hypoxia on ATP release and cell lysis.
Effect of hypoxia on ATP release and cell lysis. (A) Average ATP (left) and hemolysis (right) in control and hypoxia-treated RBCs. Each bar represents an average of 12 or 13 samples ± SEM from 4 independent experiments performed with the same blood batch, *Statistically significant difference compared with the controls (P < .05, Mann-Whitney U test). (B) Relationship between extracellular ATP and hemolysis. The graph shows tight, linear correlation (P < .0001) between hemolysis extent (depicted as absorbance – left axis, or % hemolysis – right axis) and ATP release induced by hypoxia for all data points of the 4 independent experiments reported in A. Each experiment is represented by a different symbol, with black symbols referring to hypoxia and open symbols indicating the control (normoxia) condition. The slope of linear fit corresponds to intracellular ATP concentration of 1.98 mM.
Jacek Sikora et al. Blood 2014;124:
©2014 by American Society of Hematology