Pretreatment with phenoxybenzamine, guanethidine, or reserpine attenuated vasopressor ac- tivities of these peptides. These data suggested that vasopressor ...
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K i i c h i r oNak句 ima, andShumpeiS a k a k i b a r a Pe p t i d eI n s t i t u t e ,I n c . ,P r o t e i nRe s e a r c hFoun d a t i o n ,M i n o h s h i ,Osa k a5 6 2 ,Japan
R e c e i v e dDecemb巴r 2 0 ,1 9 9 5 A d r e n o m e d u l l i n( A M )i sav a s o r el a x a n tp e p t i d et h a tw a sr e c e n t l yi s o l a t e df r o mh u m a np h e o c h r o m o c y t o m a I nc o n t r a s tt ohum a n( h )AM,w h i c hh a sv a s o d e p r e s s o ra c t i v i t y,as y n t h e t i cNt e r mi n a lf r a g m e n to fhAM, hAMー(l-25)-NH2 s h ow e dvasopr巴ssor a ct i v i t yi nt h ea n e s t h e t i z e dr a t .T h eN t e r m i n a lp e p t i d e shAM( 131 ) NH 2 ,hAM-(1 -25 ）ーOH, h A M ( l -21 ) NH2 ,a c e t yl -hAM ( 1 6 2 1 )NH 2 ,a n da c e t y lh AM( 1 63 6 )OHa ls h o w e d v a s o p r e s s o ra c i t v i t i e s .Th ep o t e n c yo fhAM( I21 )NH2 ,a c e t yl hAM( 1 621 )NH 2w a sg r e a t e rt h a nt h a to f hAM・（！・25)-NH2・ Pr官treatm巴nt w i t hp h e n o x y b e nz a m i ne , g u a n e t h id i n e, o rr e s e r p i n ea t t e n u a t e dv a s o p r e s s o ra cｭ u g g e s t e dt h a tv a s o p r e s s o ra c t i v i t yo fN t e r m in a lf r a g m e n to fh AMi sd u e t i v it i e so ft h e s ep e p t i d e s .Thes巴 data s t oas t i m u la i t o nof 巴ndog巴nous c a t e c h o l ami ner e l e a s e. ｩ 1 9 9 6A c a d e m i cP r e s s , I n c s o l a t e dfroma n A d r e n o m e d u l l i n(AM)i sap o t e n tv a s o r e l a x a n tp e p t i d et h a th a sr e c e n t l ybe巴n i o fhumanpheochromocytoma( 1) . Th巴 amino a c i ds e q u e n c eo fhumanA M, a52-amino a c i dp e p t i d e(hAM( l 5 2 )NH 2 ) ,i sTyr1-Arg-Gln-Ser-Met-Asn-AsrトPhe-Gln-Gly-Leu A r g S e r 13 Phe-GlyCys1 6 A r g P h e G l y T h rCys2 1 Thr ValGlnLys2 5 LeuAlaH is G l n I l e T y r 3 1 G l n P h eｭ Thr-Asp-Lys3 6-Asp-L ys A spAsn-ValAla P r o 43 ArgS e r L y sl leSe rP r oGln-Gly-Tyr 5 2-NH2 ( 1 ) .C u l t u r e dr a tv a s c u l a rsmoot hmusclec elsp o s s e s ss p e c i f i cA Mr e c e p t o r s ,whicha r ef u n c t i o n ｭ a l l yc o u p l e dt oa d e n y l a t ec y c l a s e( 2 , 3 ).I nas t r u c t u r e a c t i v i t yr e l a t i o n s h i ps t udy,w巴 demonstrated t h a ts y n t h e t i c hAM-(1 司52)-NH2 andi t sN t e r m i n a lt r u n c a t e dd e r iva t iv e s [hAM-(13-52) NH 2 , o r m a t i o nw i t hn e a r lye q u a l hAM( 1 6 52)-NH 2 ]i n h i b i t e d1 2 51-hAMb i n d i n gandstimulat巴d cAMPf p o t e n c y .Whileap e p t i d ehAM( 1-51)-NH 2i nwhicht h eCt e r m i n a lTy r 5 2hadbeenremoved show巴d b o t has i g n i f i c a n t l ylowe rr e c e p t o rb i n d i n ga c t i v i t ya ndcAMPr e s p o n s e( 3 ).Wehav巴 o b t a i n e ds i m i l a rr e s u l t sw i t hr eg a r d st ot h estructure』activity r e l a t i o n s h i po ft h ehAMi nme a su r e ｭ 4 ). me n t so ft h ev a s o r e l axa n ta c t i v i t yi na ne st h e t i z e dr a t s( Ast h ehAM( 15 2 )NH 2ha st h r e eLysr e s i d u e s ,wed i g e s t e dhAMwi t hal y s y lendopeptidas巴 (EC3 . 4. 2 1 . 5 0 ) .I napr・eliminary s t u d y ,hAM-(1-25)OHwast h eo n l ym句 or p r o d u c t .Wede t e r ｭ minedt h eb i o l o g i c a la c t i v i t yo ft h isp e p t i d eonb l o odp r e s s u r e.S u r p r is i ngl y ,i nc o n t r a s tt ohAMｭ (l-52)-NH 2wh i c hhasv a s o r e l a x a n ta c t i v i t y,t h eN t e r m i n a lf r a g m e n to fhAM,hAM-(1- 25 ）ーOH showedvasop r e s s o ra c t i v i t y.Toco n f i r mt h i svasopr巴ssor a c t i v i t yi na n e s t h e t iz edr a t ,wes y n t h e ( 12 5 ) -NH 2ando t h e rr e l a t e dp e p t i d e s .Thep r e s e n ts t u d ywasde s i z e dhAM- (1 -25 ）ーOH , hAMs i g n e dt od e t e r m i n ewhichaminoa c i dr e si d ue sa r ee s s e n t i a lf o rt h i sv a s o p r e s s o ra c t i v i t ya ndt o e l u c i d a t et h emecha nismbywhicht h i sa c t i v i t ymightc o mea b o u t . acid 巴xtract
MATERIALSANDMETHODS Pe p t i d e s .A d r e nom e d u l l i na n a l o g u e sw e r esynthesiz巴d a sr e p o r t e dp r e v i o u s l y( 5 ) .B r i e f ly ,p e p t i d e sw e r es yn t h e s i z e db y as o i l dp h a s ep r o c e d u r eu s i nga nA p p l i e dBi o s y s t e msMo d e l4 3 0Ap e p t i d es y n t h e si z e ro naB o cs t r a t e g y.Th ec r u d e p r o d uc t swi t hd i s u l f i d eb o n dwe r ep u r i f ie dt oh o m o g e n e i t yb yr e v e r s ep h a s eh ig hp e r f o r ma n c el i q u i dc h r o m a t o g r a p h y ( H P L C ) .T h ep u r it yo ft hep r o d u c t sw a sc on f i r me db ya n a ly t i c a l HPLCa n dam i n oa c i da n a l y s i s .Th ea m i noa ci dc o mp o s i t i o n sand 出巴 net c o n t e n t so ft h ep e p t i d ew e r ed e t e r m i n e db ya m i n oa ci da n a l y si s .Th ep u r i t yo fe a c hp r e p a r a t i o nw a so v e r 98% 。f t h ef i n a lp u r i f ie dp巴ptid巴
1 T owhomc o r r e s p o n d e n c es h o u l db ea d d r e s s e d . Fax: 8 ト727-29-4 1 24. 5 9 0 0 0 6 2 9 1 X / 9 6$1 8 . 0 0 C o p y r i g h tｩ 1 9 9 6b yA c a d e m i cP r e s s ,I n c . A l l i r g h t so fr e p r o d u c t i o ni na n yf o r mr e s e r v e d
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P r e s s o ra c t i v i t i e s .M aleSpragu巴 Daw ！巴y r a t s( 2 6 0 2 9 0g )w e r ean巴sthetiz巴d w i t hp e n t o b a r b i t a l( 5 0mg/kg,i n t r a p e r i t o ｭ n e a l )a n dat r a c h e o t o m ywasp e r f o r m e d .A r t e r i a lb l o o dp r e s s u r ewasd e t e r m i n e ddir巴ctly t h r o u g haf e m o r a lart巴rial c a n n u l a a sr e p o r t e dpr巴v i ousl y ( 6 ) .P e p t i d esw e r ei n j e c t e da sab o l us( 0 . 5m l / k g )v i aaf e m o r a lv e n o usc a n n u l a .I na n o t h e rseri巴s o f experim巴nts u s i n gac o ns c i o u sr a t, t h ea o r t i cb l o o dp r e ss u r ewasd e t e r m i n e dd i r e c t l yw i t h o u tan巴s thes ia o rres仕aint 2ー7 d a ys f o l l o w i n gt h ei n s e r t i o no fac a n n u l ai n t ot h ea b d o m i n a la o r t av i at h el e f tf e m o r a la r t e r ya sr e p o r t e dp r e v i o us l y( 7 ) .The s y n t h e t i cP巴ptides w e r ei n j e c t e da sab o l us( I mνkg) through 出e o t h e ra o r t i cc a n n u l av i at h er i g h tf e m o r a la r t e r y .Thet i p b o u t5mmp e r i p h e r a lt ot h ec a n n u l af o rb l o o dp r e ss u r edet巴rmination. B l o o dp r e s s u r ewas o ft hi sc a n n ul awasposition巴d a mon it o r e dw i t ha ne l e c t r o n i cs ys t e m( C e n t u r yT e c h n o l o g yC P 0 1,S t a rM e d i c a lP A 0 1 1a n dS a nE i8K21, T o k y o ) .I nthes巴 e x p e r i m e n t s,t h emeanb l o o dp r e s s u r el e v e lwas9 01 0 0mmHgi na n e s t h e t i z e dr a t s,a n d1 0 0 1 2 0mmHgi nc o n s c i o usr a ts . st h ep巴rcentage o fc h a n g ei nb l o o dp r e s su r ec o m p a r e dt ot h a tbefor巴 peptid巴 C h a n g e si nb l o o dp r e s s u r ew e r eexpr巴ssed a a d m i n i s t r a t i o n . S t a t i s t i c a la n a l y s i s . Ther e s ul t sa r eg i v e na st h emeanｱ S . E .w i t ht h enumbero fexperim巴nts i np a r e n t h e s e s .The s t a t i s t i c a ls i g n i f i c a n c eo ft h er e s u l t swase v a l u a t e db yu n p a i r e dStudent’ s tt es . tA v a l u eo fP( 8-37) ( 3 0n m o l / k g / m i n ,i . v . )hadane f f e c t hAM( 2 252 ）司NH2 (100n a t an o tshown).Thesed a t as u g g e s t e dt h a tt h e ont h ep r e s s o re f f e c to fhAM-(1-25)-NH2( n= 3,d fhAMmightbed i f f e r e n tfromt h a to fhAM-(l-52)-NH2 ・ r e c e p t o rf o rt h eN-t巴rminal fragmento I na d d i t i o n,we synthesiz巴d o t h e ra n a l o g so fhAM(1-25)-NH2and d e t e r m i n e dv a s o p r e s s o r ot h a t a c t i v i t y( F i g .4 ) .Thes h o r t e rf r a g m e n t ,hAM-(1-21)-NH2hadag r e a t e rpotencycompar巴d t n l yt h er i n gs t r u c t u r eo fa c e t y l (Ac)-hAM-(16-21)-NH2had o fhAM-(125)-NH2 ・ Int巴restingly, o t e r m i n a lp e p t i d eo ft h i sr i n gm o i e t y ,Ac-hAM-(16s t r o n gvasopr巴ssor 』 activity. Theelongat巴d C r e s s o re f f e c t .AlthoughhAM-(1-31)-NH2wasasp o t e n ta shAM-(1-25) ｭ 3 6 )NH2hadar巴duced p o s ti t sa c t i v i t y.Thesed a t ah i g hｭ NH2,t h ee l o n g a t e dC t e r m i n a lpeptid巴， hAM-(1-43)-NH2 hadl i t h i nt h er i n gstructur巴 for t h ep r e s s o r l i g h t e dt h ei m p o r t a n c eo ft h es i xaminoa c i dresidu巴s w a c t i v i t yo fN t e r m i n a lf r a g m e n to fhAM.Wes e a r c h e di nt h ed a t ab a s ePRF/SEQDBo ft h eP r o t e i n ResearchFoundationf o rp e p t i d e st h a tc o n t a i n sr e g i o n st h a twerea tl e as t83% homologoust othes巴 s i xaminoa c i dr e s i d u e s.Thes e a r c hi d e n t i f i e danumbero fpolyp巴ptides, i n c l u d i n gth巴 epidermal growthf a c t o rp r e c u r s o r( 1 1 ),f i b r i l l i n( 1 2 ) ,andanumbero fo t h e r s . Howev巴r, nohomologous p e p t i d e swerei d e n t i f i e dt h a ta r eknownt ober e l a t e dt ot h ec a r d i o v a s c u l a rs y s t e m . I nc o n c l u s i o n ,th巴 N-terminal fragmento fhAMproducedad o s e d e p e n d e n ti n c r e a s ei nblood p r e s s u r ei nr a t s .T h i sv a s o p r e s s o re f f e c tmightbecausedbyr e l e a s eo fendogenousc a t e c h o l a m i n e. Fmthermore,t h er i n gs t r u c t u r eo fs i xaminoa c i dr e s i d u e swase s s e n t i a lf o rt h ev a s o p r e s s o ra c t i v i t y fendog巴nous o fN t e r m i n a lf r a g m e n to fhAM.Themechanismso fa c t i o n ,i n c l u d i ngth巴 source o cat巴cholamine s h o u l dbeas u b j e c tf o rf u r t h e ri n v e s t i g a t i o n. ACKNOWLEDGMENTS Wethaは Drs. H .M a ts u oa n dK .K a n g a w a ,N a t i o n a lCa r d i o v a s c u l r uC e n t e rR e s e a r c hI ns t i t u t e, f o rh e l p f u ld is c u ss i o n .We a l s ot h a n kD r .C .R巴dhead ( M a xP l a n kI n s t i t u t e,G e r m a n y )f o rk i n d l yc o m m e n t i n go nt hem a n u s c r i p t .
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