Figure 6. Endothelial [Ca²⁺]_i responses to acetylcholine stimulation are unaffected by intracellular alkalinisation.

A, bar chart showing the fraction of ECs responding with a given magnitude of Ca²⁺_i response relative to the average CPA response. All ECs loaded with fluorescent dye within the field of view were analysed. No significant effect of CO₂/HCO₃⁻ on the frequency distribution was observed (n= 6; P= 0.90; two-way ANOVA). B, mean traces (n= 6) of the changes in EC [Ca²⁺]_i-dependent fluorescence during acetylcholine stimulation. The traces represent paired analyses of the same ECs investigated both in the presence and in the absence of CO₂/HCO₃⁻. Cells that responded during the first stimulation to acetylcholine were analysed. The Ca²⁺_i response in the absence of CO₂/HCO₃⁻ was not significantly different from the Ca²⁺_i response in the presence of CO₂/HCO₃⁻ (P= 0.70; paired two-tailed Student's t test). The effect of omitting or restoring CO₂/HCO₃⁻ was investigated 30 min after the buffer change to allow a new steady-state pH_i to be reached (Supplementary Fig. 2A and B).