Figure 5

Bradykinin-induced [Ca²⁺] _i mobilization is partially dependent on the activation of P2Y ₁₂ purinoceptors. Panel A shows the effect of bradykinin (BK, 30 μM) after pretreating human subcutaneous fibroblasts with apyrase (2 U/mL, Ai), which catabolizes ATP/ADP into AMP, and after inhibition of ectonucleotidases, with POM-1 (20 μM, Aii) or after removing Mg²⁺ from the incubation fluid (Aiii). Panel C shows the effects of BK (30 μM) in the absence or presence of selective P2Y₁, P2Y₁₂ and P2Y₁₃ receptor antagonists, respectively MRS 2179 (0.3 μM, Ci), AR-C 66096 (0.1 μM, Cii) and MRS 2211 (10 μM, Ciii). Cells were pre-incubated with the cell-permeant fluorescent calcium indicator, Fluo-4 NW (2.5 μM, see Methods). [Ca²⁺]_i transients were calibrated to the maximal calcium load produced by ionomycin (5 μM, 100% response). Black arrows indicate the time of drugs application. No changes in baseline fluorescence were observed after application of the modulators. Each point 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. Panel B illustrates the time course of the extracellular catabolism of adenine nucleotides in human subcutaneous fibroblasts grown in culture for 11 days. ATP, ADP or AMP (3 μM) were added to the culture medium at time zero; samples (75 μl) were collected at indicated times. Each sample was analyzed by HPLC to separate and quantify ATP (white), ADP (black), AMP (grey), adenosine (ADO, red), inosine (INO, orange) and hypoxanthine (HX, green). Each point represents pooled data from two individuals; 2 replicas were performed for each individual. The calculated half-life time (t_½) for each initial substrate is shown for comparison. Panel D shows immunoreactivity of human subcutaneous fibroblasts against the P2Y₁₂ receptor; shown image is representative of three independent experiments. Image scale bar is 30 μm.