CustomerValidation
- •Diabetes.2017Aug;66(8):2201-2212.
- •PLoSOne.2015Nov5;10(11):e0142087.
Description | AVE0991isanonpeptideandorallyactiveAng-(1-7)agonist.AVE0991competesforhigh-affinitybindingof[125I]-Ang-(1-7)tobovineaorticendothelialcellmembraneswithIC50of21±35nM. |
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IC50&Target | IC50:21±35nM(Ang-(1-7)receptor)[1] |
InVitro | AVE0991isanonpeptidecompoundthatevokeseffectssimilartoAng-(1-7)ontheendothelium.AVE0991andunlabeledAng-(1-7)competeforhigh-affinitybindingof[125I]-Ang-(1-7)tobovineaorticendothelialcellmembraneswithIC50sof21±35and220±280nM,respectively.PeakconcentrationsofNOandO2-releasebyAVE0991sodiumsaltandAng-(1-7)(both10μM)arenotsignificantlydifferent(NO:295±20and270±25nM;O2-:18±2and20±4nM).However,thereleasedamountofbioactiveNOis≈5timeshigherforAVE0991incomparisontoAng-(1-7)[1]. |
InVivo | AVE0991(0.58nmol/g)producesasignificantdecreaseofwaterdiuresisinWTmicecomparedwithvehicle-treatedanimals(0.06±0.03mLversus0.27±0.05;n=9foreachgroup;P<0.01). the="" antidiuretic="" effect="" of="" ave="" 0991="" (ave)="" is="" associated="" with="" an="" increase="" in="" urine="" osmolality="" (1669±231.0="">0.01).>2Oversus681.1±165.8mOsm/KgH2Oinvehicle-treatedmice;P<0.01). the="" genetic="" deletion="" of="">0.01).>MasabolishestheantidiureticeffectofAVE0991duringwaterloADIng(0.37±0.10mL[n=9]versus0.27±0.03mL[n=11]inAVE0991-treatedmice).AsobservedwithC57BL/6mice,administrationofAVE0991(0.58nmol/g)inwater-loadedSwissmicealsoproducesasignificantdecreaseoftheurinaryvolumecomparedwithvehicle-treatedanimals(0.13±0.05mL[n=16]versus0.51±0.04mL[n=40];P<>[2].OneweekoftreatmentwithAVE-0991producesasignificantdecreaseinperfusionpressure(56.55±0.86vs.68.73±0.69mmHginvehicle-treatedrats)andanincreaseinsystolictension(11.40±0.05vs.9.84±0.15ginvehicle-treatedrats),rateoftensionrise(+dT/dt;184.30±0.50vs.155.20±1.97g/sinvehicle-treatedrats),rateoftensionfall(−dT/dt;179.60±1.39vs.150.80±2.42g/sinvehicle-treatedrats).Aslightincreaseinheartrate(HR)isalsoobserved(220.40±0.71vs.214.20±0.74beats/mininvehicle-treatedrats[3]. |
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KinaseAssay [1] | Bindingof[125I]-Ang-(1-7)isperformed.Briefly,100μgofmembranesfromprimaryculturedbovineaorticendothelialcells(BAECs,passage1)areincubatedinatotalvolumeof200μLfor45minutesat25°CinHEPES-bufferedsaline(10mMHEPES,0.1MNaCl,5mMMgCl2)containing0.2%BSAandproteaseinhibitorcocktailComplete(BoehringerMannheim).Saturablebindingof[125I]-Ang-(1-7)iscalculatedbysubtractingnonspecificbinding(40%to50%),determinedinthepresenceof10μMunlabeledAng-(1-7)fromtotalbinding.CompetitionexperimentswithincreasingconcentrationsofAVE0991andunlabeledAng-(1-7)areperformedinthepresenceof10nM[125I]-Ang-(1-7).Assaysareterminatedbyvacuumfiltration(≤15mmHg)overDuraporefilters(0.65μm,Opak96-wellplates)presoakedwith1%BSA.Thefiltersarewashed3timeswitheach100μLofPBS(50mM,NaHPO4and0.15MNaCl,pH7.2).Radioactivityondriedfiltersisquantifiedwithagammacounter[1].MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly. | ||||||||||||||||||||||||||||
CellAssay [1] | Monkeykidney(COS)cellsandChinesehamsterovary(CHO)cellsarestablytransfectedwithrat Mas CDNAdrivenbyacytomegaloviruspromoterandselectedbyneomycin.125I-Ang-(1-7)(0.5×10-9 mol/L)isincubatedin24-wellplatesfor60minutesat4°Cin0.3mLofserum-freemedium(DMEM)supplementedwith0.2%BSA,0.005%bacitracin,0.1mol/LPMSF,and0.5mol/Lorthophenanthrolinewith Mas-transfectedCOScellsinthepresenceorabsenceofAVE0991(AVE,10-10 to-5 mol/L).After2isheswithice-coldserum-freeDMEM,cellsaredisruptedwith0.1%TritonX-100.Boundradioactivityismeasuredinagammacounter.Bindingofrhodamine-Ang-(1-7)in Mas-transfectedCHOcellsisperformedundersimilarconditionsusing2×10-9 mol/Lrhodamine-labeled-Ang-(1-7)inthepresenceorabsenceofAVE(10-6 mol/L),CV11974(10-6 mol/L),orPD123319(10-6 mol/L).NSBisdeterminedinthepresenceof10-6mol/LAng-(1-7)[1].MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly. AnimalAdministration | [2][3] AVE0991(AVE)ispreparedin10μMKOH(Mice)[2]. Mice[2] References |
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