Molecular Basis For Sasanquasaponin That Induces Increase Of Chloride/bicarbonate Exchange Of Anion Exchanger 3 And Promotes The Intracellular Chloride Efflux In Hypoxia/Reoxygenation Cardiomyocytes

Objective:We investigated 1)whether SQS could promote phosphorylation of Ser67 of AE3 through activating PKCεin H9c2 cells undergoing hypoxia/Reoxygenation（H/R）;2)whether PKCε-dependent phosphorylation of serine 67 on AE3 is responsible for the increase of Cl^-/HCO₃^-exchange of AE3 and intracellular chloride efflux by SQS in H9c2 cells.Method:1.To explore the effects of sasanquasaponin（SQS）on PKCεactivation in H9c2 cells undergoing hypoxia/reoxygenation（H/R）,H9c2 cells were incubated for24 h with SQS（10μM）alone or combination with PKCεinhibitor（εV1-2,1.0μM）,followed by H/R.Level of phosphorylation of PKCεwas analysed by Western blot using an anti-phospho-PKCεspecific antibody.2.To observe the role of effects of sasanquasaponin（SQS）on phosphorylation of AE3 in H9c2 cells undergoing hypoxia/reoxygenation（H/R）,H9c2 cells were incubated for 24 h with SQS（10μM）alone or combination with PKCεinhibitor（εV1-2,1.0μM）,or infected with Ad-AE3-S67A 24 h before SQS pretreatments,followed by H/R.AE3 phosphorylation was measured in AE3 immunoprecipitates with the anti-phosphoserine antibody.3.In order to examine the role of effects of PKCεactivator（DCP-LA）on phosphorylation of AE3 in Ad-AE3 or Ad-AE3-S67A–infected H9c2 cells undergoing hypoxia/reoxygenation（H/R）,H9c2 cells were infected with Ad-AE3 or Ad-AE3-S67A for 24 h and then incubated for 24 h with 1.0μM DCP-LA,followed by H/R.AE3 phosphorylation was detected in AE3 immunoprecipitates with the anti-phosphoserine antibody.4.To examine the effects of sasanquasaponin（SQS）on AE3 activity and intracellularCl^-concentration（[Cl^-]_i）inεV1-2-pretreatedor Ad-AE3-S67A-infected H9c2 cells undergoing hypoxia/reoxygenation（H/R）,H9c2cells were incubated for 24 h with SQS（10μM）alone or combination with PKCεinhibitor（εV1-2,1.0μM）,or infected with Ad-AE3-S67A 24 h before SQS pretreatments,followed by H/R.The anion exchange activity were determined by the fluorescence of BCECF-AM by a spectrofluorometer,the[Cl^-]_i were determined by the fluorescence of MQAE by a flow cytometry.5.To observe the effects of sasanquasaponin（SQS）on ROS generation,Ca²⁺overload and H/R injury inεV1-2-pretreated or Ad-AE3-S67A-infected H9c2 cells undergoing hypoxia/reoxygenation（H/R）,H9c2 cells were incubated for 24 h with SQS（10μM）alone or combination with PKCεinhibitor（εV1-2,1.0μM）,or infected with Ad-AE3-S67A 24 h before SQS pretreatments,followed by H/R.Intracellular Ca²⁺concentration were measured by a flow cytometry.Intracellular ROS level were assessed by a fluorescence microscope.Cell viability were detected by MTT method.LDH and CPK were detected by a colorimetric method.Results:1.In H9c2 cells,SQS pretreatment can obviously increase PKCεphosphorylation,whereas the expression of total PKCεproteins remaine unchanged.In addition,when given 60 min before and during SQS pretreatment,1.0μMεV1-2（PKCεinhibitor）abolished the activation of PKCεinduced by SQS.2.In H9c2 cells,SQS pretreatment can significantly up-regulate AE3expression and increase AE3 phosphorylation in H9c2 cells undergoing H/R.pretreatment with 1.0μMεV1-2 blocked SQS-induced AE3 phosphorylation but did not affected AE3 expression.More intriguingly,in Ad-AE3-S67A–infected H9c2 cells,the S67A mutation completely abrogated the SQS-induced phosphorylation of AE3.3.DCP-LA treatment resulted in phosphorylation of AE3 in Ad-AE3–infected H9c2 cells but not in Ad-AE3-S67A–infected H9c2 cells.4.In H9c2 cells,SQS pretreatment led to a significant increase of AE3activity and a substantial reduction in[Cl^-]_i in H9c2 cells undergoing H/R.However,these effects were attenuated in bothεV1-2 pretreatment and Ad-AE3-S67A infection H9c2 cells.5.In H9c2 cells,SQS attenuated Ca²⁺overload and ROS production that normally follows H/R injury,accompanied by reduction of LDH and CPK release and increase of the viability.However,in the presence ofεV1-2 or Ad-AE3-S67A,the inhibitory effects of SQS on Ca²⁺overload and ROS production were reversed,and the cardioprotective effect of SQS were attenuated.Conclusion:PKCε-dependent phosphorylation of serine 67 on AE3 is a critical molecular basis by which SQS increases activity of Cl^–/HCO₃^–exchange of AE3,promotes the intracellular chloride efflux,and elicites cardioprotection in H9c2 cells undergoing H/R.