(A) In the presence of ACh and Ca²⁺-free solutions (0 mM Ca²⁺ + 0.5 mM EGTA), ACh induced a rapid, transient contraction. Following the restoration of 2 mM Ca²⁺, a strong, sustained contraction occurred, which was fully relaxed by coptisine (n=7/7 mice). (B) In the presence of coptisine, a Ca²⁺ restoration-induced contraction by ACh was not observed (n=6/6 mice). (C) In the presence of DMSO, the solvent of coptisine, and Ca²⁺-free conditions (0 mM Ca²⁺ + 0.5 mM EGTA), ACh induced a rapid, transient contraction. Following the restoration of 2 mM Ca²⁺, a strong, steady contraction occurred (n=6/6 mice). (D) In the presence of nifedipine and Ca²⁺-free medium (0 mM Ca²⁺ + 0.5 mM EGTA), ACh induced a rapid, transient contraction. Following the restoration of 2 mM Ca²⁺, a strong, steady contraction occurred, which was fully relaxed by coptisine (n=7/7 mice). (E) In the presence of nifedipine and Ca²⁺-free solutions (0 mM Ca²⁺ + 0.5 mM EGTA), ACh induced a rapid, transient contraction. Following the restoration of 2 mM Ca²⁺, a strong, sustained contraction occurred, which was relaxed by Pyr3, gadolinium and coptisine, sequentially. (F) The bar graph showed the average reduction in Pyr3, gadolinium and coptisine, respectively, from six experiments. NS, not significant; *, P<0.05, **, P<0.01.