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InternationalJournalof
MolecularSciences
Article
Roleof 2-AdrenoceptorSubtypesinSuppressionofL-Type
2+
CaCurrentinMouseCardiacMyocytes
,RyounghoonJeon2,SungjoPark2,,2,*
1InstituteofTheoreticalandExperimentalBiophysics,RussianAcademyofScience,Institutskaya3,
142290Pushchino,Russia;******@(.);******@(.)
2DepartmentofCardiovascularMedicine,CenterforRegenerativeMedicine,MayoClinic,Stabile5,Mayo
Clinic,2001stStreetSW,Rochester,MN55905,USA;jeon.******@(.);
park.******@(.)
*Correspondence:alekseev.******@;Tel.:+1-507-284-9501
Abstract:Sarcolemmal 2adrenoceptors( 2-AR),representedby 2A, 2Band 2Cisoforms,
cansafeguardcardiacmuscleundersympathoadrenergicsurgebygoverningCa2+handlingand
-speciflctargetingof 2-ARwouldprovidecardiac
muscle-,
littleisknownaboutthespeciflccontributionofthe 2-ARsubtypesinmodulatingcardiomyocyte
,weanalyzedtheexpressionproflleof 2A, 2Band 2Csubtypesinmouse
ventricleandconductedelectrophysiologicalantagonistassayevaluatingthecontributionofthese
isoformstothesuppressionofL-typeCa2+current(ICaL).Patch-clampelectro-pharmacological
studiesrevealedthatthe 2-agonist-inducedsuppressionofICaLinvolvesmainlythe 2C,toalesser
extentthe 2B,andnotthe -qPCRevaluationrevealedthepresenceofadra2band
Citation:Evdokimovskii,.;Jeon,adra2c( 2Band 2Cisoformgenes,respectively),butwasunabletoidentifytheexpressionofadra2a
R.;Park,S.;Pimenov,.;Alekseev,( 2Aisoformgene)
2-Adrenoceptor 2Bandthe 2-ARisoform-linkedregulationofICaLin
SubtypesinSuppressionofL-Typethemouseventricleprovidesanimportantmolecularsubstrateforthecardioprotectivetargeting.
Ca2+CurrentinMouseCardiac
,22,Keywords:G-proteincoupledreceptors;leftventricle;cellsignaling;guanabenz;BRL44408;ARC
:///239;JP1302
ijms22084135
AcademicEditor:
Received:8February2021
Accepted:14April2021Previously,thecatalogofmyocellularmembranereceptorshasbeenexpandedto
Published:16April2021include 2-adrenoceptors( 2-ARs)thatinlinewithotheradrenergicreceptors( 1-and
-)controlthestress-reactiveresponseofcardiomyocytes[1,2].Wehaveidentifledthat,
Publisher’sNote:MDPIstaysneutralinadditiontotheestablished 2-AR-mediatedfeedbacksuppressionofsympatheticand
adrenalcatecholaminerelease, 2-ARsincardiacmyocytesimproveintracellularCa2+
withregardtojurisdictionalclaimsin
publishedmapsandinstitutionalaffll-handlingandsupportmyocardialcontractility[2,3].Theevidenceindicatesthatprotective
2-ARincardiomyocytescanbemobilizednotonlyagainstthedeleterious
effectsofchronicstimulationbyexcessivecatecholaminebutalsoagainstangiotensinergic
loadstomitigatethedevelopmentofcardiacdysfunctions[1,4].Inthisregard,future
therapeuticdirectionsaimedatcardiacspeciflcrestorationorenhancementof 2-AR
Copyright:© 2-ARisoformsinventricularmyocytes,whichmediate
LicenseeMDPI,Basel,.
ThisarticleisanopenaccessarticleTodate,fourdifferent 2-
distributedunderthetermsandexpress 2A, 2Band 2Creceptorisoformsencodedbytheadra2A,adra2Bandadra2C
conditionsoftheCreativeCommonsgenes,respectively[5–7].Othervertebrates,exceptcrocodiles,alsoexpress 2Disoforms
Attribution(CCBY)license(https://encodedbythegenesadra2Daandadra2Db[8–10].Inmammals,primarilythe 2A-and
/ 2C-receptorsubtypesarepresentinthecentralneuralsystem,whereasallthreereceptor
/).
,22,:///ijms22084135:.
,22,41352of11
exhibitdifferentpotencytonorepinephrine,whichishigherforthe 2C-ARcomparedto
the 2A-AR,aswellasdistinctresponsivenesstoneuronalstimulationfrequencies[11–13].
Geneticablationsofeither 2Aor 2Csubtypeshavealloweddiscriminatingbetween
the 2A-AR-dependentcontrolofplasmanorepinephrineandthepredominant 2C-AR-
2B-
ARmediatesinitialphaseofperipheralhypertensiveresponsefollowedbyhypotension
thatismediatedby 2A-, 2B-ARsinlinewithotherreceptorisoformsalso
mediatetheantinociceptiveresponseto 2-ARagonists[14].
Mechanistically,activationofthepresynaptic 2-ARsresultsinsuppressionofcAMP
levels,openingofK+channelsandinhibitionofvoltage-gatedCa2+channelsdirectlyaf-
fectingtheexocytoticmachinery[15,16].Thus, 2-ARshavebeenrecognizedasshort-loop
feedbacksuppressorsofsympatheticandadrenalcatecholaminereleaseand,thereby,gen-
erallyhaveaninhibitoryinfluenceonsympathoadrenergicdrive[11,17–19].Therangeof
pharmacologyeffectsofthesereceptorisoformsinneuronsalsoreliesonregulationofother
thannorepinephrineneurotransmitterreleaseinthecentralandperipheralnervous,which
contributestoanti-depressivepotentialsofthe 2-ARantagonists[20].Concomitantly,
2-ARagonistsareclinicallyusedasanadjuvantforpremedication,especiallyinpatients
susceptibletopreoperativeandperioperativestressbecauseofitssedative,anxiolytic,
analgesicandsympatholyticproflles[21,22].
Theexpressionof 2A, 2Band 2Cinratheartswasfoundnegligiblecompared
tolevelsofthesereceptorsinneuronal,kidney,liveroraortictissues[23],whichledto
theconventionalbeliefthatdirect 2-AR-mediatedregulationofcardiacexcitationor
-
pressionofall 2-ARisoformsinratventricularmyocytes[24,25]demonstratedthatNO
andcGMPwerecentralintracellularmessengersmediating 2-AR-signaling[2,25].Key
cardiomyocyteresponsestoactivationof 2-ARincludestimulationofendothelialNO
synthase(eNOS),reductionofintracellularCa2+levelsandsuppressionofspontaneous
intracellularCa2+oscillations(presumablythroughtheregulationofSERCA/RyRactivi-
ties)andinhibitionofmembraneinwardCa2+currentsviaL-typeCa2+channels[2,25,26].
Furthermore,bypromotingphosphorylationofErk1/2,AktandeNOSinleftventric-
ularmyocytes,the 2-ARagonistdexmedetomidineimprovedcardiacrecoveryafter
ischemia/reperfusion[24,27].Atthesametime,themaladaptivecardiacremodelingasso-
ciatedwithdevelopmentofcardiachypertrophyandheartfailureisaccompaniedbythe
functionaldesensitization/internalizationof 2-AR[2,28].Comparisonoftheaminoacid
sequencesinthethirdintracellularloopof 2-ARisoforms,theregionresponsibleforphos-
phorylationofmultipleserineorthreonineresidues,revealedlittlesequencehomology
suggestingthesubtypeselectivedesensitizationmechanisms[29,30].Thus,cardiomyocyte-
speciflctargetingof 2-ARsaimedatimprovingcardiacmuscle-delimitedstresscontrol
demandsinformationaboutalinkbetween 2-ARsubtypesandspeciflccell-signaling
pathwaysencompassingcardioprotectivemechanisms.
Whilecurrentevidenceunderlinesacardioprotectivepotentialof 2-ARsincardiomy-
ocytes,littleisknownaboutthespeciflccontributionof 2-ARisoformsincardiacmuscle
2-ARisoformsinthemouseventricle
andanalyzedthe 2-ARagonist-inducedsuppressionofL-typeCa2+currentinisolated
cardiomyocytes,usingselectiveantagonistsof 2A, 2Band 2Creceptorisoforms.
-ARsInhibitsL-TypeCa2+Current
Inisolatedmousecardiomyocytesguanabenz,anon-selectiveagonistofthe 2-
ARisoforms,signiflcantly,butreversibly,reducedinwardtransientcurrentsmeasured
throughoutarangeofdepolarizingmembranepotentials(Figure1a–c).Attheapplied
holdingpotential(40mV)nifedipine(5M),aselectiveinhibitorofL-typeCa2+channels,
eliminatedthemeasuredinwardcurrents(Figure1c).Thisindicatesthatthemeasured
whole-cellcurrentsrepresentonlythelow-thresholdvoltage-gatedL-typeCa2+current:.
-40-30-20-1010203040
mV
controlguanabenz40mM
-30mV
-30mV
-2
-20mV
+30mV
-20mV-4
+20mV
+30mV
+10mV
-10mV
5-6
abc
0mV
2pA/pF
+20mV40msguanabenz:
control
440ms
5-100mMguanabenz40mM
-8
0wash-out
4-40mM
3
+10mV
,22,xFORPEERREVIEW3of11-1
-10mV
nifedipine5mM
3-20mMyohim50mM
yohim50mM
,22,4135holdingpotential(−40mV)nifedipine(5µM),aselectiveinhibitorofL-typeCa2+channels,3of11
-2
eliminatedthemeasuredinwardcurrents(Figure1c).Thisindicatesthatthemeasured
whole-cellcurrentsrepresentonlythelow-thresholdvoltage-gatedL-typeCa2+current
0mV-10
((IICaL)amenabletoregulationby)--responserelationshipconstructedforguanabenzguan20pA/pFmMguan20mMguan50mM
CaLRelativecurrent
theeffectofguanabenzonpeakvaluesoftheeffectofguanabenzonpeakvaluesofIICaL,,measuredat+10mVofmembranepotential,measuredat+10mVofmembranepotential,density-3
-5mM
andfittedwithHill’sequationrevealedICandflttedwithHill’sequationrevealedIC50==±µMandMandhh==±((nn=3=3–5;–5;
502pA/pF
Figure1Figure1d,e).Thetime-courseofguanabenz-inducedinhibitionofthepeakd,e).Thetime-courseofguanabenz-inducedinhibitionofthepeakIICaLCaLvaluesvalues
demonstratedthatthesteadydemonstratedthatthesteady-stateblockingeffectwaseffectivelysuppressedby50-stateblockingeffectwaseffectivelysuppressedby50µMM
dofyohimbine,anon-specificantagonistofα2-ARisoforms(Figure1f).Ofnote,50µMofef
ofyohimbine,anon-speciflcantagonistof 2-ARisoforms(Figure1f).Ofnote,50Mofguan+yohim50Ca-4mM
±%%inhibitionofthepeakIICaLCaLvalues(values(nn=4;Figure1f).=4;Figure1f).
SuppressionofSuppressionofIICaLCaLbyguanabenzmeasuredinthepresenceofyohimbine(rebyguanabenzmeasuredinthepresenceofyohimbine(relativetothelativetothe
1-control
effectofthisantagonistalone)andfittedbyHill’sequationdemonstratedarighteffectofthisantagonistalone)andflttedbyHill’sequationdemonstratedaright-shiftofthe-shiftof
theguanabenzguanabenz-dependentdose-responsecurvetoIC-dependentdose-responsecurvetoIC50==±µMandMandhh==±-5
((nn=3=3–4;Figure1e).Thus,inmousecardiomyocytes,theactivationofsarcolemmal–4;Figure1e).Thus,inmousecardiomyocytes,theactivationofsarcolemmalα2 2-AR-AR
isoformsbyguanabenzresultsinisoformsbyguanabenzresultsinthesuppressionofthesuppressionofIICaLCaL..
-6
2
-7
@+10mV,pA/pF
02004006008001000
1-6-5-4
Time,s
Log[Guanabenz],M
,anagonistofα2Guanabenz,anagonistof -2-AR,suppressedL-typeCaAR,suppressedL-typeCa2+currents(2+currents(ICaLI)inisolatedmousecardiacmyocytes.(CaL),b)
(Wholea,b)Whole-cellcurrentsweremeasuredinresponsetodepolarizationfrom-cellcurrentsweremeasuredinresponsetodepolarizationfrom−40mVtomembranepotentialsindicatednearby40mVtomembranepotentialsindicated
nearbythecurrenttraces.(thecurrenttraces.(c)Voltagec)Voltage–currentrelationshipsdemonstratedreversibleguanabenz-inducedinhibitionof–currentrelationshipsdemonstratedreversibleguanabenz-inducedinhibitionofmembrane
membranecurrentsthat,undertheexperimentalconditions,entirelybelongtothedihydropyridine(nifedipine)-sensitivecurrentsthat,undertheexperimentalconditions,entirelybelongtothedihydropyridine(nifedipine)-sensitiveICaL(n=3–
I5).(d()Representativedosen=3–5).(d)Representativedose-dependentsuppressionof-dependentsuppressionofICaLbyguanabenz.(Ibyguanabenz.(e)Dose-dependentrelationshipsoftheguana-e)Dose-dependentrelationships
CaLCaL
oftheguanabenz-inducedsuppressionofbenz-inducedsuppressionofICaLconstructedbasedonthepeakIconstructedbasedonthepeakICaLvalue
Role of α2-Adrenoceptor Subtypes in Suppression of L-Type Ca2+ Current in Mouse Cardiac Myocytes 2021 Edward V. Evdokimovskii 来自淘豆网www.taodocs.com转载请标明出处.