Fig. 4. Mechanical stimulation, `touch', induces intercellular Ca²⁺ waves. Two images of indo-1 fluorescence (F405 and F480) were superimposed onto a Nomarski image where red represented F405 and green represented F480. So, cells in which Ca²⁺ increased turned red, as shown in color scale bar for Ca²⁺-increases. Arrows and asterisks show touched cells. Magnification scales are 20 µm (A-C are the same magnification). (A) A Slight touch with a blunted thin glass rod induced intercellular Ca²⁺-waves in subepithelial fibroblasts. See also supplementary material Movie 1. (B) MRS2179 (100 µM) inhibited touch-induced Ca²⁺ waves. The washing out of MRS2179 caused the recovery of Ca²⁺ waves. (C) CBX (100 µM), a blocker of gap junctions, is not effective on the initiation and propagation of Ca²⁺ waves. (D) Touch-induced Ca²⁺ waves could propagate to physically non-contacted cells. (E) Ca²⁺ waves were induced and propagated even in Ca²⁺-free solution similarly under normal conditions. (F) Time courses of Ca²⁺ changes in individual cells are shown by increases in fluorescence ratios (F405/F480) after touch stimulation in normal Ringer (left) and in Ca²⁺-free solutions (right). Cells touched for a second time also induced Ca²⁺ waves, although Ca²⁺ increases in touched cells (shown by dark-blue rhomboids) were suppressed in Ca²⁺-free solution.