Whereas the vasopressor activity of [Aspl] ANG I is higher than [Asn'] type in the rat (Watanabe et al.,. 1977), the opposite is true in the eel (Takei, 1987). Since.
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The r e n i n a n g i o t e n s i n system has been i d e n t i f i e di n v a r i o u sv e r t e b r a t e s ,fromelasmobranchst omammals. T e t r a p o d( a m p h i b i a n st omammals)a n g i o t e n s i n(ANG) hasAspa tt h eN t e r m i n u s ,butAspi sr e p l a c e dbyAsni n elasmobranchandt e l e o s tf i s h .ANGIhasbeeni s o l a t e d f romi n c u b a t e so fp l a s m aandk i d n e ye x t r a c t so ft h ebowｭ 釦1 Amiac a l v a ,ah o l o s t e a nf i s h ,u s i n gt h ee e lv a s o p r e s s o r a c t i v i t ya sana s s a ys y s t e m ;i t ss e q u e n c ewasf o u n dt obe H-Asp“Arg-Val-Tyr-Val-His-Pro-Phe-Asn四Leu-OH a f t e rs e ｭ q u e n c ea n a l y s i s ,massspectromet可， and c o m p a r i s o nw i t h t h esyn血etic p e p t i d e .T h i ss e q u e n c ei si d e n t i c a lt ob u l l f r o g ANGI .[ A s n 1 ]ANGIwasn o td e t e c t e d .Thust h eb o w f i ni s 由E f i r s tf i s hsped白 which c o n t a i n so n l y[ A s p 1 ]ANGI .The b o w f i nANGIandI Iwerenomorev a s o p r e s s o rt h a ne e l p e p t i d e si nt h eb o w f i n ,i n d i c a t i n gt h a tb o w f i nANG I I A s p 1 ] and [ A s n 1 ] r e c e p t o r sdon o tdis出1guish between [ p e p t i d e s .I nt h er a t ,b o w f i nANGIandr a t[ I l e 5 ,H i s 9 ]ANGI h a v ee q u i p r e s s o ra c t i v i t i e s when examined i nd i f f e r e n t a n i n l a l s ,b u tt h ev a s o p r e s s o ra c t i v i t yo fb o w f i nANGId e ｭ c r e a s e df o l l o w i n gr a tANGIi nt h esamea n i m a l s ,a l t h o u g h t h ea c t i v i t yo fr a t ANG Iwas u n a f f e c t e da f t e rb o w f i n ANG I . The p r e s e n ts t u d yd i r e c t l yd e m o n s t r a t e st h e p r e s e n c eo ft h er e n i n a n g i o t e n s i nsystemi nah o l o s t e a n f i s hand showed t h a ti t sANG I Ir e c e p t o r shaven o t
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 da tO c e a nR e ｭ s e a r c hI n s t i t u t e ,U n i v e r s i t yo fT o k y o ,1 1 5 1M i n a m i d a i ,N a k a n o , T o k y o1 6 4 8 6 3 9 ,J a p a n .F a x:+ 8 1 3 5 3 5 1 6 4 6 3 .E m a i l :t a k e i @ o r i ut o k y o . a c . j p .
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y e tf u l l y coevolved with t h e homologous [ A s p 1 ] peptide.
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The r e n i n a n g i o t e n s i n system was considered t o have a r i s e ni np r i m i t i v e bony f i s h (Bentle予 1976). Howeve乙 a newt ypep e p t i d e ,[ A s n 1 ,P r o 3 ,I l e 5 ]ANGI , hasbeeni d e n t i f i e di nanelasmobranch,T r i a k i ss c y l l i a ( T a k e ie ta l . ,1 9 9 3 ) .Ther e n i n a n g i o t e n s i nsystemmay beamorep h y l o g e n e t i c a l l ya n c i e n thormonalsystem involvedi np r e s s u r eandvolumehomeostasisa sw e l l a sc e l lgrowthandp r o l i f e r a t i o n( W o l f ,1 9 9 3 ;Rosenｭ d o r f f ,1 9 9 6 ) . ANG I Im o l e c u l e s , sequenced t od a t e , may be dividedi n t ot h r e egroupsaccordingt ot h ef i r s tand t h i r daminoa c i dr e s i d u e s :[ A s p 1 ,V a l 3 ]ANGI Ii d e n t i ｭ f i e di n amphibians, r e p t i l e s ,b i r d s , and mammals; [ A s n 1 ,V a l 3 ]ANGI Ii ne e l s ,chumsalmon,andg o o s e ｭ f i s h ;and[ A s n 1 ,P r o 3 ]ANGI Ii nd o g f i s h(Kobayashi andT a k e i ,1 9 9 6 ) .I tappearst h a ta l lf i s hA NGshavean Asnr e s i d u ea tt h e i rN-terminus.Thef i f t haminoa c i d r e s i d u ev a r i e s between V a l and I l ei n mammals. However,t h i sv a r i a t i o nappearst obeaf e a t u r ecomｭ mont oa l lANGs,because[ A s n 1 ,I l e 5 ]ANGIo c c u r si n t h ef l o u n d e r ,P l a t i c h t h y sf l e s u s( R .. JBalmentandY . T a k e iunpublishedd a t a ) .Furthermore,[ V a l 5 ]and[ I l e 5 ] v a r i a n t se x h i b i ts i m i l a rvasopressora c t i v i t yi nt h er a t (Watanabee talリ 1977) andt h eq u a i l( T a k e iandHaｭ segawa,1 9 9 0 ) . 0 0 1 66 4 8 0J9 8$ 2 5. 0 0 C o p y r i g h t ｩ1 9 9 8b yA c a d e m i cP r es n yf o r mr e s e r v e d . A l lr i g h t so fr e p r o d u c t i o nina
A n g i o t e n s i nIi nH o l o s t e a nF i s h I na c c o r dwitht h ehomologousN t e r m i n a lamino a c i dr e s i d u e ,t h ev a s o p r e s s o ra c t i v i t yo f[ A s p 1 ]ANGI was g r e a t e rt h a nt h a to f[ A s n 1 ] ANG Ii nt h er a t (Watanabee ta l . ,1 9 7 7 ) ,butano p p o s i t er e l a t i o n s h i p waso b s e r v e di nt h ee e l( T a k e i ,1 9 8 7 ) .T h i si sa l s ot r u e f o relasmobranchANGIwhiche x h i b i t sg r e a t e ra c t i v ｭ i t yi nt h ed o g f i s hbutl e s s e ra c t i v i t yi nt h er a tcompared withr a tANGI( T a k e ie talリ 1993). T h e r e f o r e ,ANGI I r e c e p t o r sseemt ohaveundergonep a r a l l e le v o l u t i o n witht h e i rl i g a n d ( s ) . ANGIh a sbeeni s o l a t e dfromt e l e o s t e i ,b u tn o tfrom c h o n d r o s t e iandh o l o s t e i .G r a n u l a re p i t h e l i o i dQ u x t a ｭ g l o m e r u l a r l i k e )c e l l so c c u ri nc h o n d r o s t e a nandh o l o ｭ s t e a nf i s h e s ,however( S o k a b eandOgawa,1 9 7 4 ) ,and i n c u b a t e so ft h e i rplasmaandkidneye x t r a c t sp r o ｭ duceds u b s t a n c e swhicha r ev a s o p r e s s o ri nt h er a t (Nishimurae talリ 1970). Theseo b s e r v a t i o n ss u g g e s t e d t h a tt h er e n i n a n g i o t e n s i nsystemmaybep r e s e n ti n t h e s egroupso ff i s h .T h e r e f o r e ,ANGIh a sbeens o u g h t i nt h eb o w f i n ,ah o l o s t e a nf i s h ,byi n c u b a t i n ghomoloｭ gousplasma and kidney e x t r a c t s . The v a s o p r e s s o r a c t i v i t yo fbowfinANGIwasexaminedi nt h ebowfin andr a tt oa s s e s st h ee v o l u t i o no ft h er e n i n a n g i o t e n s i n systemandi t sr e c e p t o r .
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A n i m a l s . Bowfin( A m i ac a l v aL . )o fe i t h e rs e xw e i g h ｭ i n g0 . 8 4 . 0kgwerec o l l e c t e di nLakeO n t a r i obyl o c a l f i s h e r m e n .Theywereh e l di nf l o w i n gd e c h l o r i n a t e d t a pw a t e r(Na+ ,0 . 4 5 ;o,0.95;K+ ,0 . 0 2 ;Ca2+ ,0 . 9 8 ; Mg2+,1 . 5 9mmol/L)a t1 2 -14ｰCundera12L:120l i g h t c y c l ef o ra tl e a s t2weeksb e f o r es t u d y . Bloodandk i d n e yc o l l e c t i o n . Bowfinwerea n e s t h e ｭ t i z e di n 0.03% (wIv) t r i c a i n e methanesulphonate ( S i g m a ,S t .L o u i s ,MO)andbloodwasc o l l e c t e dby c a u d a lvenousp u n c t u r ei n t oac h i l l e ds y r i n g ec o n t a i n ｭ i n g1 0ｵ l /mlbloodo f15%ammoniumEDTA.A f t e r c e n t r i f u g a t i o n ,plasmawasc o l l e c t e dandf r o z e nondry i c e .Thekidneywast h e nremovedandf r o z e nondry i c e .Frozensampleswereshippedt ot h eOceanR e ｭ s e a r c hI n s t i t u t e ,U n i v e r s i t yo fT o k y o ,w i t h i n1weeko f c o l l e c t i o n . ANGIf o r m a t i o n . Frozenkidney t i s s u e s( 3 5g ) weremincedwithar a z o rb l a d e ,homogenizedi nt h e
141
samevolumeo f5 .8m MdisodiumEDTA ,andc e n t r i ｭ 8 , 0 0 0 gf o r3 0mina t4°C. 官官 supernatant and f u g e da t1 plasmawered i a l y z e dag泊nst 5.8 ロホ,1 EDTAf o r2 4ha t 40c. 官官 dialyzed ki也1ey e x t r a c t( 3 0m l ) ,p lasma( 1 0 5m l ) , and2 . 5 %p h e n y l m e t h y l s u l p h o n y lf l u o r i d e( 0 . 8ml)we r e i n c u b a t e dt o g e t h e rwith30mlDowex50W-X2r e s i n (pH7 . 4 ,ammoniumf o r m )f o r6ha t20ｰ C .M a t e r i a l s adsorbedt ot h er e s i nweree l u t e dwith30mlo f0 . 1M d i e t h y l a m i n e( f o rmored e t a i l , s e eT a k e ie ta l .,1 9 9 3 ). Thee l u a t ewast h e ns u b j e c t e dt oc a t i o n e x c h a n g e h i g h p e r f o r m a n c el i q u i dchromatography(HPLC)on an SP-2SW column ( 4 . 6ﾗ 2 5 0 mm, T o s o h ,T o k y o , J a p a n )withal i n e a rg r a d i e n tammoniuma c e t a t ec o n ｭ c e n t r a t i o n( f o rd e t a i l s ,s e el e g e n dt oe a c hf i g u r e ).The f r a c t i o n se x h i b i t i n gv a s o p r e s s o ra c t i v i t yi nt h ee e l( s e e below)weret h e ns u b j e c t e dt og e l p e r m e a t i o nHPLC u s i n gSuperdexP e p t i d eHR1 0I 30column( b e dv o l ｭ ume= 24m l ;PharmaciaB i o t e c h . ,T o k y o ) .Theb i o a c ｭ t i v ef r a c t i o n sweref u r t h e rs u b j e c t e dt or e v e r s e p h a s e HPLConanODS-120Tcolumn( 4. 6ﾗ2 5 0mm,T o s o h ) . Thef r a c t i o nwasc o l l e c t e dbyeachpeakr e f l e c t i o na t 2 2 0nmanda s s a y e d .Thev a s o p r e s s o rm a t e r i a lwas f i n a l l yp u r i f i e dbyr e v e r s e p h a s eHPLCona219T54 phenylcolumn( 4 . 6ﾗ 2 5 0m m ,Vydac ,H e s p e r i a ,CA). A minuteamounto ft h ep u r i f i e dm a t e r i a lwass u b ｭ j e c t e dt o a Kompact Maldi I I I mass s p e c t r o m e t e r (Shimadzu ,Tokyo)t oa s s e s si t sm o l e c u l a rmass ,and h a l fo ft h eremainderwasusedf o raminoa c i ds e q u e n c ｭ i n gi nag a s p h a s ep r o t e i ns e q u e n c e r(Model4 7 6 A , AppliedB i o s y s t e m s , SanF r a n c i s c o ,CA).S i n c et h e s e q u e n c e was i d e n t i c a l with b u l l f r o gANG I( H a ｭ segawadαlリ 1983b), i twaspurchasedfromP e p t i d e I n s t i t u t eI n c .(Osaka ,J a p a n ) ,andi t si d e n t i t ywitht h e p u r i f i e dp e p t i d ewasexaminedu s i n gt h er e m a i n i n g h a l fwithODS-120Tand219T54phenylcolumnswith al i n e a rg r a d i e n to f1 5t o3 5% CH 3 CNf o r40m i n . B i o a s s a y . Thep r e s e n c eo fANGIi ne a c hf r a c t i o n wasexaminedbyt h ev a s o p r e s s o rb i o a s s a yi nt h ee e l . C u l t u r e dimmaturee e l s ,A n g u i l l aj a p o n i c a( c a .2 0 0g ) werea c c l i m a t e dt oal a b o r a t o r yf r e s h w a t e rt a n ka t 1 8ｰ Cf o r more t h a n 1weekb e f o r eu s e.E e l s were a n e s t h e t i z e di n0 . 1% (w/ v )t r i c a i n emethanesulphoｭ n a t e ,andc a t h e t e r swerei n s e r t e di n t ot h ev e n t r a la o r t a f o rbloodp r e s s u r emeasurementandd o r s a la o r t af o r i吋ections ( T a k e i ,1 9 8 8 ). A f t e r2 4hr e c o v e r y ,t h esamples o rs t a n d a r d( e e lANGI )d i s s o l v e di n0 . 9% s a l i n ewas i吋ected i navolumeo f0 . 2 5ml/kgf o l l o w e dbya自由h o ft h ec a t h e t e rwith5 0ｵ ls a l i n e .
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V a s o p r e s s o rA c t i v i t yo fB o w f i nANGs Bou；戸n. B owfino fboths e x e sweighing2 . 1ｱ 0 . 3 kg(n= 1 0 )weret a k e nfroms t o c k( s e ea b o v e )and a c c l i m a t e di nap l e x i g l a s so b s e r v a t i o nchambers u p ｭ p l i e dwitha e r a t e dd e c h l o r i n a t e dt a pwater( 1 2 1 4ｰ C) f o r1weekb e f o r es u r g e r y .Ont h edayo fs u r g e r y ,t h e y werea n e s t h e t i z e d ,andtwoc a t h e t e r swerei n s e r t e d i n t ot h ec o e l o m i ca r t e r y and abdominal v e i nf o r measuremento fa r t e r i a lp r e s s u r eandi吋ections, r e s p e c ｭ tivelテ The f i s hr e c e i v e dd a i l yimi吋ections o f50mgo f a m p i c i l l i n( P e n b r i t i n 5 0 0 ,A y h e r s tL a b s . ,M o n t r e a l , Canada)f o r4daysi n c l u d i n gar e c o v e r yp e r i o do f2 d a y s .Bloodp r e s s u r eandh e a r tr a t ewerea l l o w e dt o s t a b i l i z ef o r3 0 6 0minb e f o r et h ef i r s ti吋ection. P e p ｭ t i d e swerei吋ected i n0 . 2ml/kg,andt h ec a t h e t e rwas i m m e d i a t e l yf l u s h e d with 0 . 1 ml 0.9% s a l i n e . The v e h i c l ei吋ection witht h i svolumed i dn o tc a u s eany changei nt h ebloodp r e s s u r e .A t o t a lo f1 2i吋ections wasmadet oe a c ha n i m a lo v e ra2 d a yp e r i o d .E e land bowfinANGIwerei n j e c t e da t1 0 ,5 0 ,2 0 0 ,and1 0 0 0 pmolI kgi nt h i so r d e rande a c hp a i ro fi吋ections was e r i o dl a s t e df o ra t randomized. The interi吋ection p l e a s t3 0m i n .Ona n o t h e rda予 bowfin ANGI ,e e land bowfinANGI I ,andt h e i rANGI I I( 1 0 0 0pmol/kg) wererandomlyi吋ected. ANGswerepurchasedfrom P e n i n s u l aL a b o r a t o r y( B e l m o n t ,C A ) . R a t s . MaleSprague-Dawleyr a t sweighing2 7 8 . 3ｱ 1 . 6g( n= 1 3 )werea n e s t h e t i z e dbyani pi吋ectionof 5 0 mg/kgo fp e n t o b a r b i t a lsodium( A b b o t t ,NorthC h i ｭ c a g o ,I L )f o l l o w e d by an i v i吋ection o f 5 mgI kg p e n t o l i n i u mt a r t r a t e(DainipponP h a r m a c e u t i c a lC o . , O s a k a ) .Thef e m o r a la r t e r ywasc a n n u l a t e df o rm e a s u r e ｭ mento fa r t e r i a lp r e s s u r e , andt h ef e m o r a lv e i nf o r i吋ections. R a tandbowfinANGIweredissolv ~d 1,in 0.9%NaClc o n t a i n i n g0 . 0 1% T r i t o nX 3 0 5 (N司foryti, K y o t o ,J a p a n )andi吋ected a t1 ,3 ,1 0 ,3 0 ,1 0 0 ,3 0 0 ,and 1 0 0 0pmol/kg.Thei吋ection volumewas0 . 5ml/kg f o l l o w e dbyaf l u s ho ft h ec a t h e t e rwith0 . 1mls a l i n e . S i n c et h ev a s o p r e s s o ra c t i v i t yo fbowfinANGI became lowerwheni twasexamineda f t e ri吋ection o fr a tANG Ii nt h e randomized i吋ection p r o t o c o l ,r a t s were ) ,andd o s e ｭ d i v i d e di n t otwogroups（η ＝ 6andη ＝ 7 r e s p o n s er e l a t i o n s h i p sexaminedi nt h eo r d e ro fbowｭ f i nANGIandr a tANGIi nonegroupandi nt h e r e v e r s eo r d e ri nt h eo t h e r . Thep o t e n c yr a t i owasc a l c u l a t e dbyt h e2ﾗ 3p o i n t a n a l y s i sa td o s e swheregoodp a r a l l e l i s mo ft h ed o s e -
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