Fig. 4.

CCh evoked a Ca²⁺ wave, and global uncaging of InsP₃ caused [Ca²⁺]_c to rise simultaneously throughout the cell. (A) Carbachol evoked a Ca²⁺ wave that initiated from a single site (region 2) and propagated from there as a wave. Low and high levels of InsP₃ uncaging, by varying the voltage used for photolysis (80 V, 90 V and 200 V; see Materials and Methods), resulted in [Ca²⁺]_c increasing almost simultaneously throughout the cell. Regions i–v correspond to those in brightfield image (B). (C) Whereas the amplitude of the transient increased with voltage, the time of activation in each region was approximately similar for InsP₃ as revealed by the rise times (t_1/2 to peak). On the other hand, for CCh, the t_1/2 to peak increased with distance from the initiation site. (D) Example frames showing the Ca²⁺ increase (‘Initial’) during the CCh evoked wave (i) and submaximal (90 V; ii) and maximal (200 V; iii) InsP₃. A sequential subtraction was also performed on the data stacks (‘Subtract’), where the pixel intensity values for each frame were subtracted from the values of the image two frames ahead to enable clear visualisation of the regions where the first change in [Ca²⁺]_c occurred. The CCh-evoked wave (i) initiation site is shown with an arrow on the subtracted data set. With subthresold voltage (90 V; ii), InsP₃ release began at three separate areas (arrows). As it is a significantly longer event, the images in the carbachol wave sequence (i) are each three frames apart, whereas the images for the InsP₃ events (ii, iii) are a single frame apart. We found similar results in four additional cells.