Background Volatile anesthetics (VAs) may affect varied and complex physiology processes by manipulating Ca2+-calmodulin (CaM).However,the detailed mechanism about the action of VAs on CaM has not been elucidated.This study was undertaken to examine the effects of VAs on the conformational change,hydrophobic site,and downstream signaling pathway of CaM,to explore the possible mechanism of anesthetic action of VAs.Methods Real-time second-harmonic generation (SHG) was performed to monitor the conformational change of CaM in the presence of VAs, each plus 100 μmol/L Ca2+. A hydrophobic fluorescence indicator,8-anilinonaphthalene-1-sulfonate (ANS),was utilized to define whether the VAs would interact with CaM at the hydrophobic site or not.High-performance liquid chromatography (HPLC) was carried out to analyze the activity of CaM-dependent phosphodiesterase (PDE1) in the presence of VAs.The VAs studied were ether,enflurane,isoflurane,and sevoflurane,with their aqueous concentrations 7.6,9.5,11.4 mmol/L; 0.42,0.52,0.62 mmol/L; 0.25,0.31,0.37 mmol/L and 0.47,0.59,0.71 mmol/L respectively,each were equivalent to their 0.8,1.0 and 1.2 concentration for 50％ of maximal effect (EC50) for general anesthesia.Results The second-harmonic radiation of CaM in the presence of Ca2+ was largely inhibited by the VAs.The fluorescence intensity of ANS,generated by binding of Ca2+ to CaM,was reversed by the VAs.HPLC results also showed that AMP,the product of the hydrolysis of cAMP by CaM-dependent PDE1,was reduced by the VAs.Conclusions Our findings demonstrate that the above VAs interact with the hydrophobic core of Ca2+-CaM and the interaction results in the inhibition of the conformational change and activity of CaM.This in vitro study may provide us insight into the possible mechanism of anesthetic action of VAs in vivo.