signals in human subcutaneous fibroblasts involve connexin and pannexin-1 containing hemichannels opening. Panel A shows representative confocal micrographs (Ai) and blots (Aii) of Cx43 and Panx1 hemichannels immunoreactivity in cultured human subcutaneous fibroblasts from 3-4 individuals. Image scale bars: 60 μm. β-tubulin was used as a reference protein to determine the relative density of Cx43 and Panx1 immunoblots (Aiii). Panel B shows the effect of bradykinin (BK, 30 μM) in the presence of inhibitors of connexin and/or Panx1 hemichannels, namely mefloquine (MFQ, 3 μM, Bi), 2-octanol (1 mM, Bii), carbenoxolone (CBX 300 μM, Biii) and 10Panx (100 μM, Biv). The effects of the vesicular transport inhibitor, brefeldin A (BFA, Bv) and of the specific inhibitor of vacuolar H+-ATPases, bafilomycin A1 (Baf A1, Bvi), are also shown for comparison. Cells were pre-incubated with the cell-permeant fluorescent calcium indicator, Fluo-4 NW (2.5 μM, see Methods). Changes in fluorescence were detected using a microplate reader. [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. None of the inhibitors significantly change baseline fluorescence when applied alone. 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 C shows representative traces of single-cell [Ca2+]i oscillations in rat subcutaneous fibroblasts loaded with Fluo-4 NW (Ci) obtained in parallel with the incorporation of the membrane-selective fluorescent dye, FM4-64 (Cii), used to evaluate vesicle endocytosis and exocytosis in living cells. Changes in fluorescence were detected in the time-lapse mode using a laser-scanning confocal microscope. Vertical lines indicate the time of drugs application. Fluorescence confocal micrographs were obtained at the indicated time points (a, b and c). Image scale bars: 30 μm.