A Primary Study On The Effects Of Volatile Anesthetics On The In Vivo And In Vitro Ca²⁺-cam System

The study on the effects and mechanism of the volatile anesthetics on the intrastore of ParameciumObjective To investigate the effects and mechanism of volatile anesthetics on the intracellular Ca2+of the Paramecium directely. Methods We monitored Ca2+change of the intra-store of Paramecium by dual-wavelength fluorescence method, with Mag-Fura2, AM as indicator. MLCK peptide and its control peptide (MLCK peptide control) were given to the permeabilized Paramecium to define whether it is the CaM inhibition resulted in the Ca2+mobilization from the intra-store of the Paramecium cells. The calculated0.8EC50,1.0EC5,1.2EC50concentrations of ether, enflurane, isoflurane and sevoflurane were introduced to permeabilized Paramecium cells separately, and Ca2+signals were monitored. At last, after pretreatment with5μM MLCK peptide for5min, the permeabilized Paramecium cells were administrated with enflurane at concentration of1.2EC50to see whether the anesthetic would cause Ca2+mobilization as previous. Results Volatile anesthetics and MLCK peptide caused a decrease in the Ca2+fluorescence ratio of Paramecium Ca2+stores, while MLCK peptide control didn't. The application of1.2EC50concentration of enflurane after5-min pretreatment with MLCK peptide didn't cause decrease of Ca2+signal in permeabilized Paramecium Ca2+stores. Conclusion MLCK peptide evoeked Ca2+mobilization from intra-stores by inhibition of CaM. Pretreating Paramecium with5μM MLCK peptide abolished the effects of volatile anesthetics on Ca2+stores, indicating that volatile anesthetics caused Ca2+mobilization from intra-stores by the same mechanism with MLCK peptide. Part ⅡChelation of the raised [Ca2+]i on the effects of volatile anesthetics on the Paramecium cells Objective To evaluate the effects of the elevated [Ca2+]i that induced by volatile anesthetics during the early stage of the anesthetic given period. Methods The harvested Paramecium cells were separated into two groups, control group and BAPTA group. The BAPTA group was incubated with5μM BAPTA-AM for30min to chelate Ca2+before test. The Paramecium cells in the two groups were both introduced to aqueous solutions with different concentrations of volatile anesthetics, and the whole swimming process of those Paramecium cells was recorded by video and then analyzed by Motion Analysis1.2system. The volatile anesthetics studied were ether, enflurane, isoflurane, and sevoflurane, and their aqueous concentrations were7.6,9.5,11.4mM;0.42,0.52,0.62mM;0.25,0.31,0.37mM and0.47,0.59,0.71mM, respectively equivalent to0.8,1.0and1.2EC50for general anesthesia. Results After chelation with Ca2+, the excited effects of the volatile anesthetics on Paramecium cells, behaved in the way of accelerated swimming, were canceled off, and the inhibitory effects of the volatile anesthetics on Paramecium cells, behaved in the way of decreasd swimming speed, were impaired. Conclusion The elevated Ca2+induced by volatile anesthetics in the early stage of the anesthetics exposure was fundamental for the excitatory effects of volatile anesthetics, and was also responsible for the inhibitory effects of volatile anesthetics. PartⅢIn vitro study on the effects of volatile anesthetics on the structure and function of CaMObjective To investigate the effects of volatile anesthetics on the structure and function of CaM in vitro, and thereby explore the possible action site of volatile anesthetics. Methods A hydrophobic fluorescence indicator,8-anilinonaphthalene-1-sulfonate (ANS), was used to define the hydrophobic area of CaM. For fluorescence spectrometry study, samples were divided into three groups:control group, EGTA group and volatile anesthetics group, the fluorescence spectras of ANS which was coexisted with CaM were detected. High-performance liquid chromatography (HPLC) was utilized to analyze the effects of volatile anesthetics on the activity of CaM-dependent phosphodiesterase1(PDE1), which was indicated by the content of the hydrolysis product of cAMP. The volatile anesthetics studied were ether, enflurane, isoflurane, and sevoflurane, and their aqueous concentrations were7.6,9.5,11.4mM;0.42,0.52,0.62mM;0.25,0.31,0.37mM and0.47,0.59,0.71mM, respectively equivalent to0.8,1.0and1.2EC50for general anesthesia. Results Ca2+induced large enhancement of ANS fluorescence intensity, while EGTA abolished this effect. The Ca2+-induced effect was reversed by volatile anesthetics, and the reversed extent was in accordance with their concentrations. HPLC showed that AMP, the hydrolysis product of cAMP, was reduced by volatile anesthetics. Conclusion Our findings demonstrated that volatile anesthetics interacted with the hydrophobic core of CaM in the presence of Ca2+and inhibited the activity of CaM-dependent PDE1. 1Volatile anesthetics could induce Ca2+release from intrastores of Paramecium.2CaM inhibitory peptide-MLCK peptide, in contradiction with its control peptide would cause Ca2+release from intra-stores of Paramecium, indicating that it's the inhibition of CaM resulted the Ca2+release.3The pretreatment of CaM inhibitory peptide-MLCK peptide would cancel the Ca2+release function of volatile anesthetics on Paramecium, denoting that the function was due to CaM inhibition.4Abolishing the Ca2+release that induced by volatile anesthetics would canceled the excitatory effects of volatile anesthetics on Paramecium, indicating that the Ca2+elevation evoked by volatile anesthetics is the fundamental reason of the excitatory effects of volatile anesthetics on Paramecium.5Abolishing the Ca2+release that induced by volatile anesthetics attenuated the inhibitory effects of volatile anesthetics on Paramecium, indicating that the Ca2+release was involved in the inhibitory effects of volatile anesthetics on Paramecium.6Volatile anesthetics reversed the fluorescence enhancement of the ANS, indicating that volatile anesthetics could interfere with the ANS interaction with the hydrophobic area of CaM.7Volatile anesthetics inhibited the hydrolysis ability of CaM-dependent PDE1, indicating that volatile anesthetics could inhibit the CaM activation of downstream enzymes.