Figure 4
Dose response effects of ATP on [Ca2+] c are inhibited by purinergic blockers in malarial parasites. (A and B) Representative traces of Fluo4/AM (green fluorescence calcium indicator) changes over time by addition of ATP (200 μM) in P. berghei and P. yoelii, respectively. (C and D) Bar graph analyses of Ca2+ concentration in P. berghei and P. yoelii Fluo4/AM labelled isolated parasites (108 cells ml-1) after addition of ATP (25, 50, 70, 200 and 250 μM) (1.15 a.u. ± 0.04, n = 8; 1.4 a.u. ± 0.06, n = 14; 1.5 a.u. ± 0.07, n = 11; 1.9 a.u. ± 0.1, n = 15; 1.6 a.u. ± 0.1, n = 11, respectively) in P. berghei or ATP (50, 70 and 200 μM) (2.3 a.u. ± 0.02, n = 3; 1.9 a.u. ± 0.1, n = 6; 2.1 a.u. ± 0.14, n = 12), respectively in P. yoelii. Mobilization of Ca2+ after treatment with ATP (200 μM) was blocked in the presence of purinoreceptor inhibitors PPADS (50 μM) (0.9 a.u. ± 0.03, n = 9, P < 0.0001), TNP-ATP (50 μM) (1.0 a.u. ± 0.01, n = 7, P < 0.0001), Ip5I (10 μM) (1.1 a.u. ± 0.02, n = 12, P < 0.0001) or KN-62 (10 μM) (1.0 a.u. ± 0.02, n = 9, P < 0.0001, respectively) in P. berghei and TNP-ATP (50 μM) (1.5 a.u. ± 0.09, n = 9, P = 0.001) in P. yoelii. P values were calculated by comparison with the ATP (200 or 50 μM) data (2.0 a.u. ± 0.1, n = 12 and 2.3 a.u. ± 0.02, n = 3) in P. berghei and P. yoelii, respectively. Bar graphs represent means with SEM. The fluorescence was measured continuously (acquisition rate: every 0.5 seconds) for 400 seconds.