ApeptidecontainedinthevenomofthepredatorymarinesnailConustulipa,rho-TIA,haspreviouslybeenshowntopossessalpha1-adrenoreceptorantagonistactivity.Here,wefurthercharacterizeitspharmacologicalactivityaswellasitsstructure-activityrelationships.Intheisolatedratvasdeferens,rho-TIAinhibitedalpha1-adrenoreceptor-mediatedincreasesincytosolicCa2+concentrationthatweretriggeredbynorepinephrine,butdidnotaffectpresynapticalpha2-adrenoreceptor-mediatedresponses.InrADIoligandbindingassaysusing[125I]HEAT,rho-TIAdisplayedslightlygreaterpotencyatthealpha1Bthanatthealpha1Aoralpha1Dsubtypes.Moreover,althoughitdidnotaffecttherateofassociationfor[3H]prazosinbindingtothealpha1B-adrenoreceptor,thedissociationratewasincreased,indicatingnon-competitiveantagonismbyrho-TIA.N-terminallytruncatedanalogsofrho-TIAwerelessactivethanthefull-lengthpeptide,withalargedeclineinactivityobserveduponremovalofthefourthresidueofrho-TIA(Arg4).Analaninewalkofrho-TIAconfirmedtheimportanceofArg4foractivityandrevealedanumberofotherresiduesclusteredaroundArg4thatcontributetothepotencyofrho-TIA.Theuniqueallostericantagonismofrho-TIAresultingfromitsinteractionwithreceptorresiduesthatconstituteabindingsitethatisdistinctfromthatoftheclassicalcompetitivealpha1-adrenoreceptorantagonistsmayallowthedevelopmentofinhibitorsthatarehighlysubtypeselective.

SharpeI., etal. (2003) Allostericalpha1-AdrenoreceptorAntagonismbytheConopeptiderho-TIA. JBC.PMID:12824165

The19-aminoacidconopeptide(rho-TIA)wasshownpreviouslytoantagonizenoncompetitivelyalpha(1B)-adrenergicreceptors(ARs).Becausethisisthefirstpeptideligandforthesereceptors,wecompareditsinteractionswiththethreerecombinanthumanalpha(1)-ARsubtypes(alpha(1A),alpha(1B),andalpha(1D)).Radioligandbindingassaysshowedthatrho-TIAwas10-foldselectiveforhumanalpha(1B)-overalpha(1A)-andalpha(1D)-ARs.Asobservedwithhamsteralpha(1B)-ARs,rho-TIAdecreasedthenumberofbindingsites(B(max))forhumanalpha(1B)-ARswithoutchangingaffinity(K(D)),andthisinhibitionwasunaffectedbythelengthofincubationbutwasreversedbywashing.However,rho-TIAhadoppositeeffectsathumanalpha(1A)-ARsandalpha(1D)-ARs,decreasingK(D)withoutchangingB(max),suggestingitactscompetitivelyatthesesubtypes.rho-TIAreducedmaximalNE-stimulated[(3)H]inositolphosphateformationinHEK293cellsexpressinghumanalpha(1B)-ARsbutcompetitivelyinhibitedresponsesincellsexpressingalpha(1A)-oralpha(1D)-ARs.Truncationmutantsshowedthattheamino-terminaldomainsofalpha(1B)-oralpha(1D)-ARsarenotinvolvedininteractionwithrho-TIA.Alanine-scanningmutagenesisofrho-TIAshowedF18Ahadanincreasedselectivityforalpha(1B)-ARs,andF18Nalsoincreasedsubtypeselectivity.I8Ahadaslightlyreducedpotencyatalpha(1B)-ARsandwasfoundtobeacompetitive,ratherthannoncompetitive,inhibitorinbothradioligandandfunctionalassays.Thusrho-TIAnoncompetitivelyinhibitsalpha(1B)-ARsbutcompetitivelyinhibitstheothertwosubtypes,andthisselectivitycanbeincreasedbymutation.Thesedifferentialinteractionsdonotinvolvethereceptoraminoterminiandarenotbecauseofthechargednatureofthepeptide,andisoleucine8iscriticalforitsnoncompetitiveinhibitionatalpha(1B)-ARs.

ChenZ., etal. (2004) Subtype-selectivenoncompetitiveorcompetitiveinhibitionofhuman _α1-Adrenergicreceptorsbyrho-TIA. JBC.PMID:15194691

Therattailarteryhasbeenusedforthestudyofvasoconstrictionmediatedbyalpha(1A)-adrenoceptors(ARs).However,ringsfromproximalsegmentsofthetailartery(withintheinitial4cm,PRTA)wereatleast3-foldmoresensitivetomethoxamineandphenylephrine(n=6-12;p<0.05)thanringsfromdistalparts(betweenthesixthand10thcm,DRTA).Interestingly,theimidazolinesN-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamidehydrobromide(A-61603)andoxymetazoline,whichactivateselectivelyalpha(1A)-ARs,wereequipotentinPRTAandDRTA(n=4-12),whereasbUSPirone,whichactivatesselectivelyalpha(1D)-AR,wasapproximately70-foldmorepotentinPRTAthaninDRTA(n=8;p<0.05).Theselectivealpha(1D)-ARantagonist8-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dionedihydrochloride(BMY-7378)wasapproximately70-foldmorepotentagainstthecontractionsinducedbyphenylephrineinPRTA(pK(B)ofapproximately8.45;n=6)thaninDRTA(pK(B)ofapproximately6.58;n=6),althoughtheantagonismwascomplexinPRTA.5-Methylurapidil,aselectivealpha(1A)-antagonist,wasequipotentinPRTAandDRTA(pK(B)ofapproximately8.4),buttheSchildslopeinDRTAwas0.73+/-0.05(n=5).Thenoncompetitivealpha(1B)-antagonistconotoxinrho-TIAreducedthemaximalcontractioninducedbyphenylephrineinDRTA,butnotinPRTA.Theseresultsindicateapredominantroleforalpha(1A)-ARsinthecontractionsofbothPRTAandDRTAbutwithsignificantcoparticipationsofalpha(1D)-ARsinPRTAandalpha(1B)-ARsinDRTA.Semiquantitativereversetranscription-polymerasechainreactionrevealedthatmRNAencodingalpha(1A)-andalpha(1B)-ARsaresimilarlydistributedinPRTAandDRTA,whereasmRNAforalpha(1D)-ARsistwicemoreabundantinPRTA.Therefore,alpha(1)-ARssubtypesaredifferentiallydistributedalongthetailartery.Itisimportanttoconsiderthesegmentfromwhichthetissuepreparationistakentoavoidmisinterpretationsonreceptormechanismsanddrugselectivities.

KamikiharaSY., etal. (2005) DifferentialDistributionofFunctionalalpha1-AdrenergicReceptorSubtypesalongtheRatTailArtery. JPET.PMID: 15872040