Exp Physiol 95.11 pp 1049–1060 Ca2+ events in living cells 1057 agonist lead to an increase in the frequency of Ca2+ oscillations, and that the resulting contractions can be plotted as a function of frequency rather than ‘mean’ [Ca2+]i (Fig. 10; Perez & Sanderson, 2005a,b). Although high agonist concentrations lead to persistent elevation of global [Ca2+]i in most cells, it is reasonable to argue that Ca2+ imaging has revealed a major ‘digital’, ‘frequency- modulated’ aspect to smooth muscle Ca2+ signalling previously unsuspected from global measurements of average [Ca2+]i (Sanderson et al. 2010). Calcium ion spikes and oscillations had been demonstrated in a range of cell types, both excitable and non-excitable, prior to the widespread use of Ca2+ imaging (Thorn et al. 1993; Friel, 1995), but the ability to observe Ca2+ changes within individual myocytes embedded in their parent tissue using confocal techniques has demonstrated their importance in smooth muscle in a way not previously appreciated from Ca2+ microﬂuorimetry records. It remains to be seen what functional beneﬁt results from the use of complexly patterned Ca2+ signals rather than simple variations in steady-state [Ca2+]i, although oscillations may allow for intermittent activation of Ca2+-dependent processes with a relatively high Ca2+ threshold while reducing the risk of cell damage (Berridge et al. 2000). The temporal patterning of Ca2+ signals may also contribute to signal targeting within the cell just as much as their spatial distribution. This may even play a role in the regulation of protein expression in smooth muscle by Ca2+ signals, a process referred to as ‘excitation–transcription coupling’ (Wamhoff et al. 2006). Figure 9. Calcium imaging reveals cell behaviour not apparent in ‘average’ recordings A, a retinal arteriole (Aa) was loaded with fura-2 AM and changes in mean [Ca2+]i and vessel diameter recorded (Ab). This technique records changes in smooth muscle but not endothelial [Ca2+] (Scholﬁeld & Curtis, 2000). Addition of endothelin-1 (Et-1; 10 nM) leads to an increase in [Ca2+]i, followed by constriction. There is an initial transient Ca2+ peak, followed by a sustained rise but at a lower [Ca2+]i. Images courtesy of Drs C. N. Scholﬁeld and T. M. Curtis. B, confocal Ca2+ imaging of retinal arteriole myocytes in situ within the vessel wall (Ba). Fluo-4-loaded cells have been imaged in a plane near the bottom of the organ bath, revealing an array of adjacent myocytes. These were then scanned along the indicated line to generate a linescan (Bb). Addition of endothelin stimulates asynchronous Ca2+ oscillations in adjacent cells. The temporal pattern of the cell response (Bc) does not mirror that of the global signal seen in A (note the faster time base in B). C 2010 The Author. Journal compilation C 2010 The Physiological Society