Figure 3

Bradykinin elicits ATP release from human fibroblasts by a mechanism depending on intracellular Ca²⁺mobilization. Panels A, B and D represent cells loaded with quinacrine (30 μM, a fluorescent dye that specifically binds ATP), for 60 min at 37°C. ATP release was detected by single-cell confocal microscopy in the time-lapse mode measuring the fluorescence intensity decay 4 min after bradykinin (BK, 30 μM, A and B) application as compared to the control situation, in which only Tyrode’s solution was applied (A). Panel D, shows the effect of BK (30 μM) after pretreatment of the cells with the selective endoplasmic reticulum Ca²⁺-ATPase inhibitor, thapsigargin (2 μM, Di), and after removal of extracellular Ca²⁺ (Ca²⁺-free medium plus EGTA, 100 μM, Dii); the effect of BK (30 μM) in the presence of the selective Panx1 inhibitor, ¹⁰Panx (100 μM, Diii), is also shown. Image scale bars: 30 μm. Graphs show quinacrine fluorescence decay (arbitrary units, a.u.) plotted versus time in the presence of Tyrode’s solution (B), thapsigargin (2 μM, Di) and Ca²⁺-free medium ( Dii). Black arrows indicate the time of drugs application. Each point represents pooled data from an n number of cells. The vertical bars represent S.E.M.. Panel C, shows the ATP content in human subcutaneous fibroblast cultures at given time intervals (0-240 seconds) in the presence of BK (30 μM, open bars) as compared to the control condition where only Tyrode’s solution was applied (closed bars). Relative luminescence units (RLUs) were calibrated using a 4 nM ATP standard (left hand-side black vertical bar). Each bar represents pooled data from an n number of experiments. The vertical bars represent S.E.M.. *p < 0.05 represent significant differences from BK (30 μM) alone.