Figure 3

Bitter tastants increase [Ca²⁺]_i in isolated gallbladder cells. In silico analysis of a mouse gallbladder scRNA data set identified two cell clusters (#3 and 5) with high levels of Acta2 expression (A). Cell cluster #5 represented smooth muscle cells, since it expressed the CCK receptor, Cckar (B), but not Cckbr (C). Based on CCK (1 µM) responsiveness, cells were grouped into CCK⁺ (presumably SMC) and CCK^– (all other) cells. Confocal laser scanning recording of fluorescence intensity of the calcium indicator Fluo-4 was expressed in arbitrary units (AU) over time in response to increasing concentrations of (D) denatonium (Den), (E) quinine, and (F) dextromethorphan (DXM), respectively, after CCK (1 µM) as an initial test stimulus. All drugs were added under continuous flow in the chamber, so that indicated concentrations were reached initially and then washed out. Mean ± SEM, n refers to number of cells/number of gallbladders from which they were taken. (G,I,K) Relative frequencies of responders (> 10% increase in [Ca²⁺]_i; dark column) and non-responders (light column) to increasing concentrations of Den, quinine, and DXM in the experiments depicted in (A–C). Frequencies of responders among CCK⁺ cells were compared to CCK^– cells by chi-square test, p-values indicated above the columns; n as in (A–C), absolute numbers indicated in the columns. (H,J,L) Maximum increase in [Ca²⁺]_i in responders (cells that exhibited > 10% increase in [Ca²⁺]_i to at least one concentration of the respective bitter tastant); extent of reaction in CCK⁺ cells is compared to that in CCK^– cells by Mann–Whitney-U-test, p-values are indicated; n refers to number of cells/number of gallbladders from which they were taken.