Force–pCa relationships and time course of relaxation in Triton X-100–skinned E18.5 urinary bladder strips . (A) Original force tracings of the experimental protocol. Following a test relaxation-contraction-relaxation cycle, isometrically mounted longitudinal strips were stimulated with cumulatively increasing pCa = −log[Ca²⁺] for 5 min at each pCa. (B) Normalized force–pCa from n = 23 WT (Cald1^+/+) strips from 16 fetuses and 16 strips from 10 KO (Cald1^−/−) fetuses from a total of 13 litters; the results from both bladder halves of one fetus were averaged, when applicable, and the mean was taken for further calculation. Solid and broken lines represent nonlinear fit with a sigmoidal function and 95% confidence intervals of the force-pCa relations, respectively. Force at pCa 4.3 (F_max) was taken as 100%, and force at pCa >8 was taken as 0 (symbols represent mean ± SEM). (C) Summary of pCa₅₀ values (pCa for 50% activation) taken from sigmoidal fits of individual experiments. (D) Maximal force at pCa 4.3 was significantly lower (P < 0.001) in KO than in WT. (E) Time required for 50% (t₅₀) and 80% (t₈₀) relaxation from F_max. Symbols represent values from individual fetuses, and bars are means ± SD. **, P < 0.01; ***, P < 0.001.