Bradykinin stimulates the release of intracellular Ca2+stores and Ca2+influx from the extracellular space. Panel A shows immunoreactivity of cells cultured from explants of human subcutaneous tissue against fibroblast-cell markers, vimentin (red, Ai) and type I collagen (green, Ai), and α-smooth muscle actin (SMA-FITC, green, Aii). Negative controls, in which cells were incubated only with secondary antibodies, Alexa Fluor 488 (green) and Alexa Fluor 568 (red), are shown for comparison purposes ( Aiii ); a positive control of SMA-FITC immunoreactivity in rat cardiac myofibroblasts is also shown (green, Aiv). Cell nuclei are stained with DAPI (blue); scale bar 60 μm. Panel B illustrates intracellular Ca2+ ([Ca2+]i) oscillations in cultured human subcutaneous fibroblasts loaded with the fluorescent calcium indicator, Fluo-4 NW (2.5 μM, see Methods) obtained in the absence and in the presence of bradykinin (BK, 30 μM). Changes in fluorescence were detected in the time-lapse mode with a confocal microscope. Calibration to the maximal calcium load produced by ionomycin (5 μM, 100% response) is also shown for comparison. Image scale bars: 30 μm. Panel C shows that the kinetics of BK-induced [Ca2+]i signals differed slightly between cells of a given population. Panel D depicts the concentration-response curve of [Ca2+]i oscillations produced by BK (0.003-100 μM). Panels E, F and G, represent [Ca2+]i oscillations produced by BK (30 μM) applied in the absence (E) and in the presence of the selective endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin (2 μM, F), and after removal of extracellular Ca2+ (Ca2+-free medium plus EGTA, 100 μM, G). Black arrows indicate the time of drugs application. 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.