Figure 5

APAP-challenged primary hepatocytes presented sustained and repeated intracellular calcium signal due to exogenous ATP administration. (A) Primary mouse hepatocytes (PMH) were isolated and loaded with a fluorescent calcium probe (Fluo4-AM). (B) Following APAP incubation (18 h), PMH viability decreased in a dose-dependent manner, reaching 50% of survival when 20 mM of APAP was used. Therefore, this dose was used to subsequent experiments. (C) ATP administration (10 μM) caused calcium signal in naïve hepatocytes, which returned to baseline values after 60 seconds. Six replicates are represented in the graph. (D) However, APAP-treated cells (20 mM; 6 h) developed a hyper-responsive behavior to the same ATP dose, displaying higher and sustained calcium signal, which was also prolonged for longer periods (200 seconds). (E) Treatment with an unspecific P2 antagonist (suramin, 0.1 mM) completely abrogated APAP effects over ATP stimulation. (F) Representative cells of each group were displayed together. (G) Snapshots from live calcium signal recording using confocal microscopy. Videos were rendered in “rainbow pallet” to facilitate fluorescence observation (generated by Fluo4-AM). Following 110 seconds of ATP stimulation, APAP treated cells remaining responsive with increased intracellular calcium signal, while control cells returned to baseline values after 55–70 seconds. Scale = 20 μm. (H) Incubation of primary hepatocytes with exogenous ATP in the dose range found during necrosis (10-100 μm, 18 h) significantly reduced cell viability. * P < 0.05 in comparison to control group. Data are mean ± SEM.