Fig. 2. Ca²⁺ puff activity during local and global signalling. All of the cell types analysed in the present study displayed Ca²⁺ puffs when treated with appropriate agonist concentrations. The aim of this figure is to show some of the different modes in which Ca²⁺ puff activity can be observed in these cells. (A) Rapid Ca²⁺ puff activity in a 16HBE14o- cell stimulated with 5 µM ATP. Several Ca²⁺ puff sites were active before and during the onset of the Ca²⁺ wave. White circles on the inset cell image show the locations of the active puff sites, and the Ca²⁺ signals recorded at these regions are depicted by the correspondingly numbered traces. (B) A HeLa cell stimulated with 1 µM histamine and in which a single Ca²⁺ puff sites was responsible for triggering a regenerative Ca²⁺ wave. The black trace represents the Ca²⁺ signal observed at the puff site and the red trace depicts the global Ca²⁺ signal observed by averaging fluo-3 fluorescence across the whole cell. (C) Ca²⁺ puffs firing at three different sites within a single carbachol-stimulated SH-SY5Y cell (1 µM carbachol). Coloured circles on the inset cell image show the locations of the active puff sites, and the Ca²⁺ signals recorded at these regions are depicted by the correspondingly coloured traces. This example illustrates the observation that, sometimes, the Ca²⁺ puff sites appear to fire in synchrony, whereas, at other times, they do not. (D) Ca²⁺ puffs in a NIH-3T3 cell stimulated with 1 µM ATP. Ca²⁺ puffs were observed at the regions marked by the blue, green and red circles on the inset cell image, and the correspondingly coloured traces indicate the Ca²⁺ changes observed at these sites. Ca²⁺ puffs were observed during the initial latency before the first Ca²⁺ oscillation and also on the rising phases of subsequent Ca²⁺ oscillations.