Phorbol12-myristate13-acetate(PMA)isaPKC-activatingphorbolester,increasestheintracellularCa²⁺concentration([Ca²⁺]_i)inadose-dependentmanner,withanEC₅₀of11.7nM.

EC50:11.7nM(PKC)^[1]

InordertoexaminetheroleofPKCinp38MAPKphosphorylation,thecellsarestimulatedwiththePKCactivator,PMA(100nM),whichmimicsthebindingofDAG,thenaturalactivatorofPKC,totheC1regionofthePKCs.p38MAPKphosphorylationbyPMAisobservedinthetwocelltypessimilartothatobservedbyGnRHinαT3-1cells,thatis,aslowsustainedactivation(3.2-foldand3.6-fold,respectivelyat30min).TheparadoxicalfindingsthatPKCsactivatedbyGnRHandPMAplayadifferentialroleinp38MAPKphosphorylationmaybeexplainedbydifferentiallocalizationofthePKCs.Basal,GnRH-andPMA-stimulationofp38MAPKphosphorylationinαT3-1cellsismediatedbyCa²⁺influxviavoltage-gatedCa²⁺channelsandCa²⁺mobilization,whileinthedifferentiatedLβT2gonadotropecellsitismediatedonlybyCa²⁺mobilization^[2].

PMAisaPKCagoNIST,whichreversesthedamageinducedby5-hydroxydecanoicacid(5-HD).Thus,activationofthemitoKATPprotectedmitochondrialfunctioninSODandMDAviathePKCpathway^[3].

• [1].XuF,etal.ProteinkinaseC-mediatedCa2+entryinHEK293cellstransientlyexpressinghumanTRPV4.BrJPharmacol.2003Sep;140(2):413-21.

  [2].MugamiS,etal.DifferentialrolesofPKCisoforms(PKCs)andCa2+inGnRHandphorbol12-myristate13-acetate(PMA)stimulationofp38MAPKphosphorylationinimmortalizedgonadotropecells.MolCellEndocrinol.2017Jan5;439:141-154.

  [3].HouS,etal.MechanismofMitochondrialConnexin43"sProtectionoftheNeurovascularUnitunderAcuteCerebralIschemia-ReperfusionInjury.IntJMolSci.2016May5;17(5).pii:E679.

  [4].ZhangT,etal.MPTP-InducedDopamineDepletioninBasolateralAmygdalaviaDecreaseofD2RActivationSuppressesGABAAReceptorsExpressionandLTDInductionLeADIngtoAnxiety-LikeBehaviors.FrontMolNeurosci.2017Aug7;10:247.

Phorbol12-myristate13-acetate(PMA)isdissolvedinDMSOandstored,andthendilutedwithappropriatemediumbeforeuse^[2].

αT3-1andLβT-2cellsaregrowninmonolayerculturedinDMEMsupplementedwith10%fetalcalfserum(FCS)andL-glutamine2mM,penicillinandstreptomycin(100units/mL)inhumidifiedincubator5%CO₂at37°C.Serumstarvationiswith0.1%FCSinthesamemediumfor16h.GnRHandPMAarethenaddedforthelengthoftimeasindicated.Ingeneral,αT3-1cellsaretransientlytransfectedbyExGen500orbyjetPRIME,whileLβT2cellsonlybyjetPRIMEtransfectionreagent.Forexperimentswithdominant-negative(DN)PKCs,αT3-1cells(in6cmplates)aretransfectedwith1.5μgofp38α-GFPwith3μgofcontrolvector,pCDNA3,orwith3μgoftheDN-PKCsconstructs.ForLβT2cells,transfectionsareperformed(in10cmplates)with4μgofp38α-GFPalongwith9μgofcontrolvector,pCDNA3,orwith9μgoftheDN-PKCsconstructs.Approximately30haftertransfection,thecellsareserumstarved(0.1%FCS)for16handlaterstimulatedwithGnRHorPMA,washedtwicewithice-coldPBS,treatedwiththelysisbuffer,followedbyonefreeze-thawcycle.Cellsareharvested;followingcentrifugation(15,000×g,15min,4°C)supernatantsaretakenforimmunoprecipitationexperiments^[2].MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.

Phorbol12-myristate13-acetate(PMA)sdissolvedinDMSOanddilutedwithPBSorsaline^[3].

Rat^[3]
AllexperimentsqreperformedwithmaleWistarrats(weighing250-280g).Onehundredandthirty-fiveWistarratsarerandomlydividedintosevengroups.(1)Ratsintheshamgroup(n=21)aregivenalateralcerebralventricleinjectionof0.9%normalsaline;(2)RatsintheIRgroup(n=21)aregivenalateralcerebralventricleinjectionof0.9%normalsaline30minbeforemiddlecerebralarteryocclusion(MCAO);(3)RatsintheCarbenoxolone(CBX)group(n=21)aregivenalateralcerebralventricleinjectionofCBX(5μg/mL×10μL)30minbeforeMCAO;(4)RatsintheDiazoxide(DZX)group(n=21)aregivenalateralcerebralventricleinjectionofDZX(2mM×30μL)30minpriortoMCAO;(5)Ratsinthe5-HDgroup(n=21)aregivenalateralcerebralventricleinjectionof5-HD(100mM×10μL),andafter10min,DZXisinjected15minpriortoMCAO;(6)TheratsintheDZX+Rogroup(n=15)aregivenalateralcerebralventricleinjectionofDZX,andafter10min,Ro-31-8425(400μg/kg)isinjected15minpriortoMCAO;(7)Theratsinthe5-HD+PMAgroup(n=15)aregivenanintraperitonealinjectionofPMA(200μg/kg)aftertheinjectionof5-HDandDZX.MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.

• [1].XuF,etal.ProteinkinaseC-mediatedCa2+entryinHEK293cellstransientlyexpressinghumanTRPV4.BrJPharmacol.2003Sep;140(2):413-21.

  [2].MugamiS,etal.DifferentialrolesofPKCisoforms(PKCs)andCa2+inGnRHandphorbol12-myristate13-acetate(PMA)stimulationofp38MAPKphosphorylationinimmortalizedgonadotropecells.MolCellEndocrinol.2017Jan5;439:141-154.

  [3].HouS,etal.MechanismofMitochondrialConnexin43"sProtectionoftheNeurovascularUnitunderAcuteCerebralIschemia-ReperfusionInjury.IntJMolSci.2016May5;17(5).pii:E679.

  [4].ZhangT,etal.MPTP-InducedDopamineDepletioninBasolateralAmygdalaviaDecreaseofD2RActivationSuppressesGABAAReceptorsExpressionandLTDInductionLeadingtoAnxiety-LikeBehaviors.FrontMolNeurosci.2017Aug7;10:247.

616.83

C₃₆H₅₆O₈

16561-29-8

RoomtemperatureincontinentalUS;mayvaryelsewhere

DMSO:≥62mg/mL

Phorbol12-myristate13-acetate(PMA)isdissolvedinDMSOandthendilutedwithsterilesaline^[4].

*"<1 mg/ml"="" means="" slightly="" soluble="" or="" insoluble.="" "≥"="" means="" soluble,="" but="" saturation="">

• [1].XuF,etal.ProteinkinaseC-mediatedCa2+entryinHEK293cellstransientlyexpressinghumanTRPV4.BrJPharmacol.2003Sep;140(2):413-21.

  [2].MugamiS,etal.DifferentialrolesofPKCisoforms(PKCs)andCa2+inGnRHandphorbol12-myristate13-acetate(PMA)stimulationofp38MAPKphosphorylationinimmortalizedgonadotropecells.MolCellEndocrinol.2017Jan5;439:141-154.

  [3].HouS,etal.MechanismofMitochondrialConnexin43"sProtectionoftheNeurovascularUnitunderAcuteCerebralIschemia-ReperfusionInjury.IntJMolSci.2016May5;17(5).pii:E679.

  [4].ZhangT,etal.MPTP-InducedDopamineDepletioninBasolateralAmygdalaviaDecreaseofD2RActivationSuppressesGABAAReceptorsExpressionandLTDInductionLeadingtoAnxiety-LikeBehaviors.FrontMolNeurosci.2017Aug7;10:247.