RAS, to know whether lungfish angiotensin is of the fish or tetrapod type. Studies by Sawyer et al. (1976) on conscious Australian lungfish, Neoceratodus forsteri,.
G e n e r a la n dC o m p a r a t i v eE n d o c r i n o l og y1 1 4 , 2 062 1 2(1999 ）品 7 24 6av叫
Article 町cen.1999.
I sPrεsεnt i nA u s t r a l i a n Neoceratodus f o r s t e r i
Tεlεost-TypεAngiotεnsin
Lungfish ヲ
] .M.P .Joss,* , 1Y .Itahara,tT .X.Watanabe,t K .Nakajima,tandY .Takei干 汚chool o fB i o l o g i c a lS c i e n c e s ,MacqumieU n i ve r s i t y , S y d n e y , NSW2 1 0 9, Aus t r a l i a ;t Pe p t i deI n s t i t u t eI n c ., P r o t e i n 6 2 8 6 8 6 ,J a p a n ;and 干 Ocean Re s e a r c hIns titute，刀1 e U n i ve r s i t yo fT o k yo , R e s e a r c hF o u n d a t i o n ,M i n o h,Os a k a5 1 1 5 1M i n a m i d a i,Nak日no, T o k yo1 6 4 -86 3 9 , ] a p a n
A c c e p t e dN o v e m b e r1 9 ,1 9 9 8
)wasp roducedfromt h ei n c u b a t i o n A n g i o t e n s i nI(ANGI o fl u n g f i s hplasmawithhomologous k i d n e ye x t r a c t s . Thep u r i f i e dp e p t i d ewasfoundt oh a v et h es e q u e n c eo f H A s n A r g V a l T y r V a l H i s P r o P h e T h r L e u O H ,whichi s homologousf o rt h ef i r s te i g h tr e s i d u e sw i t ha l lt e l e o s t a n g i o t e n s i n ss of a rs e q u e n c e d ,a l t h o u g hl u n g f i s hg e n e r ｭ a l l yp o s s e s stetrapoιtype hormones.Thel u n g f i s hd e c a ｭ )i nducedd o s e d e p e n d e n ti n c r e a s e si n p e p t i d e(ANGI a r t e r i a lp r e s s u r ei nt h er a t . Thel u n g f i s ho c t a p e p t i d e (ANGI I )r e l e a s e da l d o s t e r o n efromk i d n e y a d r e n a lt i s ｭ nv i t r oi nad o s e d e p e n d e n tmannerandinduced s u ei d o s e d e p e n d e n ti n c r e a s e si na r t e r i a lp r e s s u r eo ft h e l u n g f i s h. S u b s t i t u t i o no fa s p a r a g i n ew i t ha s p a r t i ca c i di n t h ef i r s tp o s i t i o n( t e t r a p o d t y p eANGI I )d i dn o ta l t e rt h e b l o o dp r e s s u r er e s p o n s es i g n i f i c a n t l y ,b u t a second s u b s t i t u t i o no ft h ev a l i n ei nt h e（ヨ）－position w i t hi s o l e u ｭ c i n e(ANGI Iformfoundi nhumanandr a t )a b o l i s h e dt h e r i s ei na r t e r i a lp r e s s u r ei nl u n g f i s ho v e rt h esamed o s e Pi四 r a n g e . ｩ1 9 9 9A c a d e m ic KeyWords:A n g i o t e n s i n ;L u n g f i s h ;Neoceratodus;Vaso” p r e s s o r ;A l d o s t e r o n e
Ani n t a c tr e n i n a n g i o t e n s i nsystem(RAS)hasbeen demonstrated f o ra l lc l a s s e so f gnathostome v e r t e ｭ b r a t e s .Withint h i ss y s t e m ,a n g i o t e n s i nI(ANGI )i s c l e a v e dfroma n g i o t e n s i n o g e ni nt h eb l o o d ,byr e n i n , anenzymer e l e a s e dfromt h ekidneysi nr e s p o n s et o loweredbloodp r e s s u r eo rsodiumd e p l e t i o n .Decapep-
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t i d eANGI ,g e n e r a t e dbyi n c u b a t i o no ft h ehomoloｭ gous plasmawithkidneys fromv a r i o u sv e r t e b r a t e s p e c i e s ,v a r i e si naminoa c i dcompositioni np o s i t i o n s 1 ,3 ,5 ,and9(KobayashiandT a k e i ,1 9 9 6 ) .V a r i a t i o n si n p o s i t i o n s1and5a r es i n g l eaminoa c i ds u b s t i t u t i o n s t h a tcanbee x p l a i n e di nt u r nbyas i n g l ebasechangei n t h e i rg e n e t i cc o d o n s , whereas amino a c i d sa t( 9 ) ｭ p o s i t i o na r emuchmorev a r i a b l e . Onlyt h eelasmoｭ r i a k i ss c y l l i a ,showsav a r i a t i o nfromv a l i n et o b r a n c h ,T p r a l i n ei n( 3 ) p o s i t i o n .S i n c et h ea c t i v eformo fa n g i o ｭ t e n s i ni st h eo c t a p e p t i d e ,ANGI I ,i nwhicht h el a s ttwo aminoa c i d shavebeenremovedbyana n g i o t e n s i n ｭ c o n v e r t i n genzyme,t h ev a r i a t i o ni n( 9 ) p o s i t i o nmay beo fl i t t l es i g n i f i c a n c e.Fromt h esequencest h a thave s of a rbeen d e s c r i b e d ,t h ev a r i a t i o ni n( 1) p o s i t i o n s e p a r a t e smostf i s h ,wherei ti sa s p a r a g i n e ,fromt h e t e t r a p o d s ,wherei ti sa s p a r t i ca c i d .Ther e c e n td e s c r i p ｭ t i o no fa n g i o t e n s i nfromt h en o n t e l e o s tbonyf i s h ,Amia c a l v a ,p r o v i d e st h eonlyexampleo faf i s hi nwhicht h e aminoa c i di nt h ef i r s tp o s i t i o ni sa s p a r t i ca c i d( T a k e i e talリ 1998).
S i n c emostr e c e n tmoleculars t u d i e s( M e y e r ,1 9 9 5 ) morec l o s e l ya l i g ndipnoanbonyf i s hwitht e t r a p o d s thanwitht e l e o s t s ,i ti so fi n t e r e s t ,i ne v o l u t i o no ft h e RAS,t oknowwhetherl u n g f i s ha n g i o t e n s i ni so ft h e f i s ho rt e t r a p o dt y p e .S t u d i e sbySawyeretal .( 1 9 7 6 )on s t e r i , c o n s c i o u sA u s t r a l i a n lung白sh, ふたoceratodus Jor usingt h ef i s h t y p e[ A s n 1 ,V a l 5 ]ANGI I ,foundt h a ti t i n c r e a s e dbloodp r e s s u r eandcausedmildt omoderate g l o m e r u l a rd i u r e s i sandn a t r i u r e s i s .Anotherstudyon
2 0 6
日日 1 6・ 6480 / 99 $3 0 日日
C o p y i r g h tｩ1 9 9 9b y A c a d e m i c P r e s s A l l i r g h s to f r e p r o d u c i t o ni na n yf or mr e s e r v e d
L u n g f i s hA n g i o t e n s m t h es t e r o i d o g e n i cr e s p o n s et of i s h ,t e t r a p o d ,andmamｭ o r s t e r ifoundt h a tt h ef i s ht y p e malianANGI Ii nN.f s i g n i f i c a n t l ye l e v a t e dplasmaa l d o s t e r o n el e v e l sw i t h ｭ o u ta f f e c t i n gc o r t i c o s t e r o n eo rc o r t i s o l ,whereast h e t e t r a p o d t y p e[ A s p 1 ,V a l 5 ]d i dn o ts t i m u l a t ea l d o s t e ｭ r o n eb u td i ds i g n i f i c a n t l yr a i s ec o r t i c o s t e r o n el e v e l si n t h eblood( J o s se ta l . ,1 9 9 4 ) .Witht h ed i s c o v e r yo ft h i s d i f f e r e n c ei ns t e r o i d o g e n i cr e s p o n s et ot h ed i f f e r e n t formso fANGI I ,i tbecamei m p o r t a n tt oa s c e r t a i nt h e s e q u e n c eo flung白sh a n g i o t e n s i n .Theworkr e p o r t e d h e r e was undertaken t oa s c e r t a i nt h es e q u e n c eo f l u n g f i s ha n g i o t e n s i ni no r d e rt ob e t t e rc h a r a c t e r i z et h e s t e r o i d o g e n i cr e s p o n s et oRASi nl u n g f i s handt ob e t t e r understandt h ee v o l u t i o no fRASi ng e n e r a l .
METHODS
Animals Adultl u n g f i s h ,N.f o r s t e r i( 3 . 5 1 0k g ) ,werec o l l e c t e d byg i l ln e t t i n gfromT i n a n aC r e e k ,at r i b u t a r yo ft h e 恥1ary R i v e ri ns o u t h e a s t e r nQ u e e n s l a n d .At 恥1acquarie U n i v e r s i t y ,t h e ywerem a i n t a i n e di nl a r g ef i b e r g l a s s t a n k s ,sometimesf o rs e v e r a lm o n t h s ,f e donad i e to f commercialf i s hp e l l e t s ,waterweeds,andl i v ef r e s h w a ｭ t e rs n a i l sandc r a y f i s h .P r i o rt os a c r i f i c e ,bloodsamples o f2 0 6 0mlwerec o l l e c t e dv i ah e a r tp u n c t u r ei n t o c h i l l e ds y r i n g e sc o n t a i n i n g15%ammoniumEDTA( 1 0 ｵ I /mlb l o o d ) ,immediatelyc e n t r i f u g e da t2500gf o r1 5 m i n ,andt h eplasmas t o r e da t 8 0 ｰF o l l o w i n gblood c o l l e c t i o n ,t h ef i s hwerea n e s t h e t i z e di n0.5%(w/ v ) t r i c a i n emethanesulphonate(MS2 2 2 ,S i g m a ,S tL o u i s , MO)andt h e nk i l l e dbys p i n a ls e v e r a n c e .Thek i d n e y s werer a p i d l yd i s s e c t e do u tande i t h e rf r o z e ni nl i q u i d n i t r o g e nandl a t e rt r a n s f e r r e dt o-80ｰo rmincedf r e s h withf i n es c i s s o r si n as m a l lq u a n t i t yo fL e i b o v i t z medium( F l o wL a b o r a t o r i e s ,S c o t l a n d ) .Bloodsamples and k i d n e y s were c o l l e c t e d from s e v e r a ll u n g f i s h r e p r e s e n t i n gboths e x e s .Thoset h a thadbeens t o r e da t 8 0 ｰwereshippedondryi c et ot h eOceanR e s e a r c h I n s t i t u t e ,U n i v e r s i t yo fT o k y o .F r e s hmincedt i s s u ewas usedf o ra s s a y i n ga l d o s t e r o n e s t i m u l a t i n ga c t i v i t y .
A n g i o t e n s i nIS y n t h e s i s Kidneyt i s s u e s( 3 6g )weret h a w e d ,mincedwitha r a z o rb l a d e ,homogenizedi nt h esamevolumeo f5 . 8
207
m MdisodiumEDTA,andc e n t r i f u g e da t1 8 , 0 0 0 gf o r30 mina t4 ｰ .Thed i a l y z e dkidneye x t r a c t( 4 0m l ) ,plasma ( 1 2 0m l ) , and2.5%phenylmethylsulphonylf l u o r i d e ( 1 . 2m l )werei n c u b a t e dt o g e t h e rwith40mlDowex 50W-X2r e s i n(pH7 . 4 ,ammoniumf o r m )f o r6h a t 2 0 ｰ C .Them a t e r i a l sadsorbedt ot h er e s i nweree l u t e d with4 0 0mlo f0 . 1M d i e t h y l a m i n e( f o rmored e t a i l s , s e eT a k e ie talリ 1993). 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 nfrom1 0m Mt o1M i n10%CH3CNf o r1 2 0 mina taf l o wr a t eo f1mlI m i n .Thef r a c t i o n se x h i b i t i n g v a s o p r e s s o ra c t i v i t yi nt h ee e l( s e ebelow)weret h e n s u b j e c t e dt og e lpermeationHPLCu s i n gSuperdex P e p t i d eHR1 0I 3 0column( b e dvolume= 2 4m l ;P h a r ｭ maciaB i o t e c h n o l o g y ,T o k y o ,J a p a n ) .Thecolumnwas e l u t e dwith30%CH3CNi n0 . 1% t r i f l u o r o a c e t i ca c i d ( T F A )a t0 . 2 5ml/min.Theb i o a c t i v ef r a c t i o n swere f 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 eHPLConanODS120Tcolumn( 4 . 6ﾗ2 5 0mm,T o s o h )ande l u t e dwitha l i n e a rg r a d i e n to fCH3CNc o n c e n t r a t i o nfrom1 0t o 60%i n0 . 1% TFAf o r6 0mina t1ml/m i n .Thef r a c t i o n wasc o l l e c t e dbye a c hpeakr e f l e c t i o na t2 2 0nmand a s s a y e d .Thev a s o p r e s s o rm a t e r i a lwasf i n a l l yp u r i f i e d byr e v e r s e p h a s eHPLCona219T54phenylcolumn ( 4 . 6ﾗ 2 5 0mm,V y d a c ,H e s p e r i a ,CA)withal i n e a r g r a d i e n to fCH 3 CNfrom1 5t o35%i n0 . 1% TFAf o r40 mina t1ml/m i n .A minuteamounto ft h epuri白ed m a t e r i a lwass u b j e c t e dt o aKOMPACTMALDII I I masss p e c t r o m e t e r( S h i m a d z u ,T o k y o ,J a p a n )t oa s s e s s i t sm o l e c u l a rm a s s ,andh a l fo ft h eremainderwasused f o r amino a c i ds e q u e n c i n gi nag a s p h a s ep r o t e i n s 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 ｭ c i s c o ,C A ) .Thesequencedp e p t i d ewass y n t h e s i z e di n ap r o t e i ns y n t h e s i z e r(Model4 3 0 A ,AppliedB i o s y s ｭ tems) ,andi t si d e n t i t ywitht h ep u r i f i e dp e p t i d ewas examinedu s i n gt h er e m a i n i n gh a l fwithaODS-120T anda219T54phenylcolumnswithal i n e a rg r a d i e n to f 1 5t o35%CH3CNi n0.1%TFAfor40m 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 nwasa s s a y e d byv a s o p r e s s o ra c t i v i t yi nt h ee e l .C u l t u r e dimmature e 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 di nt h e l a b o r a t o r yi nf r e s h w a t e rt a n k sa t1 8ｰf o rmoret h a n1
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b k y o ,J a p a n ) , andt oapen ( P A 0 1 1 ,S t a rM e d i c a l ,T r e c o r d e r( 8 K 2 1 ,NEC-Sa ne i ,T o k y o ,J a p a n ) .Ratand l u n g f i s hANGIwered i s s o l v e di n0.9%NaClc o n t a i n ｭ i n g0 . 0 1% T r i t o nX 3 0 5( N a : k a r a i )andi n j e c t e da t1 ,3 , 1 0 ,3 0 , 1 0 0 , 3 0 0 ,and1 0 0 0pmol/kg. Thepotencyr a t i owasc a l c u l a t e dbyt h e2ﾗ3 p 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 edoseｭ r e s p o n s ec u r v ewasd e m o n s t r a t e d .Thepotencyr a t i o s weree x p r e s s e da smeansand95%c o n f i d e n c ei n t e r ｭ v a l s .A l lo t h e rv a l u e sweree x p r e s s e da smeans: t :SEo f t h emean.Thed i f f e r e n c eo ft h epotencywasexamined byANOVA.Thesigni白cance wasdetermineda tP< 0 . 0 5 .
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Lung戸sh. C a n n u l a t i o no fa o r t a and a v e i nwas a t t e m p t e d on s e v e r a ls u b a d u l tl u n g f i s hbutp a t e n t c a n n u l a ec o u l dn o tbe m a i n t a i n e di nanyo ft h e s e p r e p a r a t i o n s .A s i n g l ea d u l tmalel u n g f i s hweighing5 kgwasa v a i l a b l et ostudyv a s o p r e s s o ra c t i v i t yi nMay 1 9 9 8 .T h i sf i s h was a n e s t h e t i z e d with MS222 and c a n n u l a ei n s e r t e di nt h ed o r s a la o r t aandi nt h eh e p a t i c v e i n( a c c e s sd e v i c e ,g i f tfromBectonD i c k e n s o n )f o r measuremento fa r t e r i a lp r e s s u r eandf o r1吋ections, r e s p e c t i v e l y .Ont h ef o l l o w i n gdayt h ef i s hr e c e i v e dan i吋ection o f 1 mg a m p i c i l l i nv i at h eh e p a t i cv e i n c a n n u l a .Ondays2 ,3 ,7 ,8 ,and9f o l l o w i n gsurger予 bloodp r e s s u r echangesi nr e s p o n s et ot h r e eformso f ANGI I( [ A s n 1 ,V a l 5 ] ,[ A s p 1 ,V a l 5 ] ,[ A s p 1 ,I l e 5 ] ;Sigma) and t ot h ev e h i c l e( 0 . 6 % NaCl) were r e c o r d e d by c o n n e c t i n gt h ea o r t i cc a n n u l at oap r e s s u r et r a n s d u c e r (XTM-190-250SG,K u l i t eSemiconductorP r o d u c t s ,N J ) and pen r e c o r d e r . The f i s hr e c e i v e d at o t a lo f3 0 i吋ections v i at h eh e p a t i cv e i nc a n n u l ao v e rt h e5d a y s , withaminimumo f3 0minbetweene a c h1吋ection. The d o s e so fANGI Irangedfrom9t o1 2 0pmol/kgi n5 0, u l 0.6%s a l i n eandwerea d m i n i s t e r e drandomlテ R a t s . Male Sprague-Dawley r a t s weighing 2 8 6 . 9: t :1 . 9g( n= 7 ) were a n e s t h e t i z e dby an i p i吋ection o f50mg/kgp e n t o b a r b i t a lsodium( A b b o t t , NorthC h i c a g o ,I L )f o l l o w e dbyani . v .i吋ection o f5 mg/kgpentoliumt a r t r a t e(DainipponP h a r m a c e u t i c a l C o . ,O s a k a ,J a p a n ) .Thef e m o r a la r t e r ywasc a n n u l a t e d f o rmeasuremento fa r t e r i a lp r e s s u r e ,andt h ef e m o r a l v e i nwasc a n n u l a t e df o ri吋ections. Thea r t e r i a lc a t h ｭ e t e rwasc o n n e c t e dt oap r e s s u r et r a n s d u c e r( C P 0 1 ,
Kidneyt i s s u e ,c o n t a i n i n gt h ea d r e n a lhomolog( C a l l andJ a n s s e n s ,1 9 7 5 ) ,wast a k e nfromtwol u n g f i s h ,a maturemale( 5k g )andamaturef e m a l e( 7 . 5k g ) ,i n June1 9 9 6andminceda sd e s c r i b e dunderA n i m a l s . Themincedt i s s u ewasd i v i d e di n t o200-mgs a m p l e s , e a c ho fwhichwasi n c u b a t e di n2mlHepes七uffered L e i b o v i t zmedium(FlowL a b o r a t o r i e s ,S c o t l a n d )a t2 2 ｰ onar o c k i n gp l a t f o r m .A f t e r 1h t h emediumwas r e p l a c e dwithf r e s hmediumandi n c u b a t i o nc o n t i n u e d f o raf u r t h e rh o u r .T h i smediumwasd r a i n e d ,r e t a i n e d f o ra n a l y s i s andr e p l a c e d withmediumc o n t a i n i n g one o ft h ef o l l o w i n gc o n c e n t r a t i o n so fs y n t h e t i c [ A s n 1 ,V a l 5 ]ANGI I( S i g m a ,S tL o u i s , MO): 0 ,0 . 5 , 2 . 5 ,5 . 0 ,2 5 . 0 nmol/m l .I n c u b a t i o n was c o n t i n u e d f o raf u r t h e rh o u r .Eachdoseo fANGI Iwasr e p e a t e d on3ﾗ200-mgsampleso fmincedk i d n e y .Themedium from each i n c u b a t i o n ,b e f o r e and a f t e rt h ea d d i ｭ t i o no fANGI Iwasf r o z e na t-80ｰ Cu n t i la s s a y e df o r a l d o s t e r o n e .
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A l d o s t e r o n eRIA Two a l i q u o t so f3 0 0, u l from each o ft h e above mediumsamplesweree x t r a c t e di n5 0 0, u lo fmethanol and2ﾗ 1 5 0 , u la l i q u o t so feache x t r a c t i o nwered r i e d downi na s s a yt u b e s .Thea l d o s t e r o n ea s s a yhasbeen d e s c r i b e dp r e v i o u s l y( J o s se talリ 1994), usingahighly s p e c i f i ca n t i s e r u m( u c bB i o p r o d u c t s ,B e l g i u m ,< 0 . 0 0 1% c r o s s r e a c t i v i t ywitho t h e rs t e r o i d s ) .A l lsampleswere runw i t h i na s i n g l ea s s a y .I n t r a a s s a yv a r i a t i o nwas 6.1%（η ＝ 1 2 0 ) .Thea l d o s t e r o n ev a l u e sf o rt h emedium
209
Lun g f i s hA n g i o t e n s m samplespriort oadditionofANGI Iweresubtracted fromthosefollowing1hofincubationa f t e radditionof
revealedt h a tthepurifiedpeptidehadthesequenceof H-Asn-Arg-ValTyrVal-His-Pro-Phe-Thr-Leu-OH
ANGI It ogivepgaldosteronereleased/100m gadreｭ nal-kidneyt i s s u e .
( T a b l e1 ) . Themeasuredmassvalue ofthe puri白ed peptide (MH+= 1240) was almost i d e n t i c a lt o the t h e o r e t i c a lvalueof1 2 3 9 . 6 .Furthermore,thepurified peptidecomigratedwiththesyntheticlung白sh ANGI intwod i f f e r e n tHPLCsystems.Novasopressora c t i v ｭ i t ywasdetectedinthee e la tthepositionwhere[ Asp1] ANGIshouldbeelutedinthecation-exchangeHPLC. The mean baseline dorsal a o r t i c pressure of the A s n 1 ,V a l 5 ] lungfishwas10: ' :2m mHg.Bothlungfish[ and tetrapod [ A s p 1 ,V a l 5 ] ANG I I induced doseｭ
RESULTS 句
AnANGI l i k evasopressorsubstancewasproduced a f t e rincubationoflungfishplasmawithhomologous kidneye x t r a c t s .Thesubstancewaspurifiedbyc a t i o n ｭ exchangeHPLC,gel-permeationHPLC,andtwosteps ofreverse-phaseHPLC( F i g .1) .Thef i n a lyieldwas2 . 6 2 nmolasdeterminedbycomparingabsorbancea t220 .Aminoacidsequencing n mwitht h a tofe e lANGI
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60
. C h r o m a t o g r a m sf o rp u r i f i c a t i o no fl u n g f i s hANGI .( a )C a t i o n e x c h a n g eHPLC( S P 2 S W ,4 . 6ﾗ2 5 0mm,T o s o h )o ft h ef r a c t i o ne l u t e d F I G.1 w i t hd i e t h y l a m i n ef r o mDowex50W-X2r e s i n .Theo b l i q u el i n eshowsal i n e a rg r a d i e n tf r o ms o l v e n tA( 1 0 %CH 3 CNi n1 0m Mammonium a c e t a t e )t os o l v e n tB( 1 0 %CH3CNi n1M ammoniuma c e t a t e )i n1 2 0mina t1m l / m i n .F r a c t i o n sw e r ec o l l e c t e de v e r y2 . 5m i n .Onlyt h emarked f r a c t i o ne x h i b i t e dv a s o p r e s s o ra c t i v i t yi nt hee e l .Arrowsshowe l u t i o np o s i t i o n so ff o w l[ A s p 1 ,V a l 3 ,V a l 5 ,S e r 9 ]ANGI ,e e l[ A s n 1 ,V a l 3 ,V a l 5 ,G l y 9 ] .( b )G e lp e r m e a t i o nHPLC( S u p e r d e xP e p t i d eHR1 0 / 3 0 ,2 4m l ,P h a r m a c i aB i o t e c h )o ft h e ANGI ,a n dd o g f i s h[ A s n 1 ,P r o 3 ,I l e 5 ,G l n 9 ]ANGI f r a c t i o n1 5o fc a t i o n e x c h a n g eHPLC.S a m p l ewase l u t e dw i t h30%CH3CNi n0 . 1 %TFAf o r1 0 0mina t0 . 2 5m l / m i n .F r a c t i o n sw e r ec o l l e c t e d e v e r y2m i n .Or吐y t h emarkedf r a c t i o ne x h i b i t e dv a s o p r e s s o ra c t i v i t y .Ana r r o wshowst h ee l u t i o np o s i t i o no fe e lANGI .V O,voi dv o l u m e .( c ) 5 0mm,T o s o h )o ff r a c t i o n5o fg e l p e r m e a t i o nHPLC.Theo b l i q u el i n eshowsal i n e a rg r a d i e n tf r o m1 0 R e v e r s ep h a s eHPLC( O D S 1 2 0 T ,4 . 6ﾗ2 t o60%CH3CNi n0 . 1 %TFAf o r6 0mina t1m l / m i n .F r a c t i o n sw e r ec o l l e c t e df o re a c hp e a k .Thep e a kmarkedw i t ha na r r o we x h i b i t e d e v e r s ep h a s eHPLC( 2 1 9 T 5 4p h e n y l ,4. 6ﾗ2 5 0mm,V y d a c )o ft h ef r a c t i o nmarkedbyana r r o wi n( c ) .Theo b l i q u el i n e v a s o p r e s s o ractivit予（d) R a s o p r e s s o ra c t i v i t yand showsal i n e a rg r a d i e n tf r o m1 5t o35%CH3CNi n0 . 1%TFAf o r4 0mina t1m l /m i n .Thes i n g l es p i k eobtained 巴泣libited v wass u b m i t t e dt os e q u e n c ea n a l y s i s .
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TABLE1 AminoA c i dS e q u e n c e so fANGIi nR e p r e s e n t a t i v eS p e c i e s o fV e r t e b r a t e s
80
A s n A r g P r o T y r I l e H i s P r o P h e G l n L e u AspArgV a lT y rV a lH i sP r oPheAsn-Leu A s n A r g V a l T y r V a l H i s P r o P h e A s n L e u
AspA r g V a l T y r V a l H i s P r oPheAsnLeu A s p A r g V a l T y r V a l H i s P r o P h e H i s L e u A s p A r g V a l T y r V a l H i s P r o P h e S e r L e u A s p A r g V a l T y r V a l H i s P r o P h e H i s L e u A s p A r g V a l T y r I l e H i s P r o P h eH i s L e u
Ox
R a t
EO 田口里 δ 官。。一回
A s n A r g V a l T y r V a l H i s P r o P h e T h r L e u
70
60
50
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Asn-Arg-Val-Tyr-Val-HisーPro-Phe-Gly-Leu
szEE ） 22ωωω
F i s h D o g f i s h B o w f i n Chums a l m o n E e l L u n g f i s h T e t r apods B u l l f r o g T u r t l e Fowl
90
4 0 30
2 0 1 0
N o t e .F o rr e f e r e n c e ss e eK o b a y a s h iandT a k e i( 1 9 9 6 )andT a k e ie ta l . ( 1 9 9 8 ) .
A N GI l s .Onedoseof6 0pmol/kgof[Asn1,Val5]A N G I Iwasadministered,whichproducedabloodpressure r i s e in excess of 40 m mHg, overshooting the pen
。
3
。
10
30
100
300
1000
D o s eo fA N G I(pmollkg ，’・v.) F I G .3 . D o s e r e s p o n s er e l a t i o n s h i pf o rt h ev a s o p r e s s o re f f e c to f ANGI(n= 7 )a ndr a tANGI(n= 6 )i nr a t s .V e r t i c a lb a r s ft h em e a n . r e p r e s e n tｱSEo
lung白sh
recorderrange.Asa consequencet h i sdosewasnot repeatedf o re i t h e rthe[Asn1,V a l 5 ]orthe[Asp1,V a l 5 ]
baselinea r t e r i a lpressureofther a twas6 8 . 8: ' :0 . 7m m
Ihadno formsofANGILInc o n t r a s t ,[Asp1,I l e 5 ]A N GI
Hg ( n= 7 ) . Both lungfish and r a t A N G I induced
e f f e c tonbloodpressurea tdosesbelow6 0pmol/kg andproducedonlyaverys l i g h tr i s eina o r t i cpressure
dose-dependent increases in a r t e r i a l pressure with s出lilar potencywhenex紅凶ned i nd i f f e r e n tr a t s( F i g .3 ) .
0and120pmol/kgdoses( F i g .2 )and inresponset o6
Aldosterone was released from minced lungfish
theser i s e sweresustainedf o rapproximately1h .The
kidney-adrenalt i s s u einresponset o[Asn1,V a l 5 ]A N G
I Iinadose dependent >
manner( F i g .4 ) .
45 60
40
35
50
2 5 20
1 5 10
5
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30
1 0
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9
1 5 30 ANGI( p m o l / k g, i . v ) .
60
120
F I G .2 . D o s e r e s p o n s er e l a t i o n s h i pf o rt h ev a s o p r e s s o ra c t i v i t yo f I ,[ A s p 1,V a l 5 ]ANGI I ,a nd[ A s p 1 ,I l e 5 ]ANGI Ii n [ A s n 1 ,V a l 5 ]ANGI e o c e r a t o d u s( n= 1 ) .V e r t i c a lb a r sr e p r e s e n tｱSEo ft h e t h el u n g f i s h ,N of o u rr e p e a t so ft h esamed o s eandh o r m o n e . meano fthr官e t
C o p y r i g h tｩ1 9 9 9b yA c a d e m i cP r e s s A l lr i g h t so fr e p r o d u c t i oni na n yf o r mr e s e r v e d
。
。
0 . 5
2 . 5 n m o l / m l ) ANGI(
2 5
. D o s e r e s p o n s ec u r v ef o ra l d o s t e r o n er e s p o n s ebyl u n g f i s h F I G .4 nv i t r ow i t h[ A s n 1 ,V a l 5 ]ANGI If o r1h . a d r e n a l k i d n e yi n c u b a t e di fmean （π ＝ 1 2 ) . V e r t i c a lb a r srepresent 土 SE o
9
L u n g f i s hA n g i o t e n s m
211
DISCUSSION
r a t i oo f4: 1( K h o s l ae ta l . ,1 9 8 5 ) ,ap o s s i b l ee x p l a n a t i o n f o rwhichi st h a tt h e r ea r etwoANGp r e c u r s o rg e n e si n t h i ss p e c i e sa tl e a s t . L u n g f i s hANGIwasf u r t h e rc o n f i r m e da sana n g i o ｭ t e n s i nbyi t sa b i l i t yt oi n d u c ev a s o p r e s s o ra c t i o ni nt h e r a t .I nt h el u n g f i s h ,t h et e l e o s t / l u n g f i s hformo fANG I Ia l s o induced v a s o p r e s s o ra c t i o n and i na d o s e ｭ dependantmanner .Thebloodp r e s s u r er e s p o n s e st o l u n g f i s hANGI Ii nt h i sonef i s hweres i m i l a rt ot h o s e measuredbySawyere ta l .i n1 9 7 6i nr e s p o n s et ot h i s sameformo fANGI I ,e x c e p tf o rt h e6 0pmol/kgdose whichproducedamuchmored r a m a t i cr i s ei na o r t i c p r e s s u r ei nours t u d y .Evens o ,Sawyere ta l .( 1 9 7 6 ) remarkedont h ev a s c u l a rs e n s i t i v i t yo fN e o c e r a t o d u st o t h i sp e p t i d ea sb e i n gmuch g r e a t e rt h a nt h e yhad foundi nt h eA f r i c a nlung白sh, P r o t o p t e r u s .Wehave c o n f i r m e dt h i shighs e n s i t i v i t yf o rN e o c e r a t o d u sand have gone on t o show t h a tN e o c e r a t o d u si sa l m o s t e q u a l l ys e n s i t i v et ot h et e t r a p o dformo fANGI Ii n whicht h eo n l ys u b s t i t u t i o ni si nt h ef i r s tp o s i t i o n .The ANGI Ii nwhichbotht h ef i r s tandt h ef i f t hr e s i d u e sa r e a l t e r e d( i . e . ,[ A s p 1 ,I l e 5 ]ANGI I ) ,howeve乙 was i n e f f e c ｭ t i v ei nr a i s i n gbloodp r e s s u r eo v e rt h esamer a n g eo f d o s e swhichproducedana l m o s tt h r e e f o l di n c r e a s e with[ V a l 5 ]ANGI I .T h i ss u g g e s t st h a tt h ev e r ys m a l l d i f f e r e n c ebetweenv a l i n eandi s o l e u c i n ei nt h e( 5 ) ｭ p o s i t i o ni ss u f f i c i e n tt o produce a c o n f o r m a t i o n a l changei nt h eo c t a p e p t i d e ,whichr e s u l t si ni n c o m p a t ｭ i b i l i t ywitht h er e c e p t o r si n v o l v e dwitht h ev a s o p r e s ｭ s o ra c t i o no fANGI Ii nN e o c e r a t o d u s .However,i tmust bep o i n t e do u th e r et h a to n l yonel u n g f i s hwasc a n n u ｭ l a t e df o rt h e s ev a s o p r e s s o r measurements. S i m i l a r d i f f e r e n c e si np o t e n c ybetween[ V a l 5 ]and[ I l e 5 ]ANGI I haven o tbeenfoundi nanyo t h e rv e r t e b r a t e st e s t e d fromt e l e o s tf i s ht omammals(Watanabedαl., 1 9 7 7 ; T a k e iandHasegawa,1 9 9 0 ) .Thesames i m i l a r i t i e sand d i f f e r e n c e si nv a s o p r e s s o rs e n s i t i v i t yt ot h ed i f f e r e n t formso fANGI Iweren o te n c o u n t e r e df o rt h ea c t i o no f ANGI Iont h el u n g f i s ha d r e n a ls t e r o i d o g e n i cc e l l s . S y n t h e t i cANGI Iwitht h esames e q u e n c ea st h a tf o r t e l e o s t sa n d ,a swehaveshown,f o rl u n g f i s h ,h a sbeen foundp r e v i o u s l yt or a i s ea l d o s t e r o n el e v e l sandt o havenos i g n i f i c a n te f f e c tonc o r t i c o s t e r o n eo rc o r t i s o l l e v e l si nt h ebloodo fl u n g f i s h( J o s se ta l . ,1 9 9 4 ) .I nt h i s sames t u d yi twasfoundt h a ta l t e r a t i o no fa s p a r a g i n e t oa s p a r t i ca c i di nt h ef i r s tp o s i t i o nn e g a t e dt h i s s e l e c t i v es t e r o i d o g e n i cr e s p o n s e ,whereast h ea d d i t i o n o ft h ev a l i n et oi s o l e u c i n es u b s t i t u t i o ni nt h e (5 ）ー
Av a s o p r e s s o rm a t e r i a lwasproduceda f t e ri n c u b a ｭ t i o no ft h eplasmafromN .J o r s t e r iwithhomologous kidneye x t r a c t s ,whena s s a y e di nt h ee e l .Thep u r i f i e d v a s o p r e s s o rm a t e r i a lwassequenceda sANGI ,with a s p a r a g i n ei nt h e( 1 ) p o s i t i o n ,v a l i n ei nt h e( 5 ) ｭ p o s i t i o n ,andt h r e o n i n ei nt h e( 9 ) p o s i t i o n .A l lr e m a i n ｭ i n g amino a c i d s were homologous with a l lo t h e r sequencedANGI s ,witht h ee x c e p t i o no ft h ee l a s m o ｭ b r a n c h ,T .s c y l l i a ,whichhasap r a l i n ei np l a c eo fa v a l i n ei n( 3 ) p o s i t i o n( T a k e ie talリ 1993). Whilet h eT h r 9 i suniquet ot h el u n g f i s hs e q u e n c e ,t h eAsn1andV a l 5 a r es h a r e dwitht e l e o s tANGI s( T a b l e1 ) .Theo n l y o t h e rn o n t e l e o s tbonyf i s hf o rwhichANGs e q u e n c e d a t ai sa v a i l a b l ei st h eb o w f i n ,A m i a ,whichhas[ A s p 1 , V a l 5 ,A s n 9 ]ANGI( T a k e ie talリ 1998). T h i si si d e n t i c a lt o t h es e q u e n c eo fANGId e s c r i b e df o rb u l l f r o g .Given e o c e r a t o d u si sas a r c o p t e r y g i a nf i s handAmiaan t h a tN a c t i n o p t e r y g i a nf i s h ,t h es i m i l a r i t ybetweent h ebowfin andb u l l f r o gANGIi su n l i k e l yt or e p r e s e n tana n c i e n t o r i g i nf o rt h i sp a r t i c u l a rf o r m .Theweighto fe v i d e n c e ( t e l e o s t s ,e l a s m o b r a n c h ,andd i p n o a n )f a v o r sa s p a r a ｭ g i n eo v e ra s p a r t i ca c i da st h er e s i d u ei n( 1) p o s i t i o no f t h egnathostomea n c e s t r a lm o l e c u l e . Atp r e s e n t ,a n g i o t e n s i ni st h eo n l yp e p t i d ehormone c h a r a c t e r i z e df o rl u n g f i s hwhicha l i g n switht e l e o s t s r a t h e rt h a n with t e t r a p o d s .F o re x a m p l e ,l u n g f i s h p o s s e s sm e s o t o c i nandI o rphenymesotocin（恥1ichel e t alリ 1993; Hyodoe ta l . ,1 9 9 7 )a st h e i rn e u t r a lneurohyｭ p o p h y s i a lp e p t i d e ,w h i l et e l e o s t sp o s s e s si s o t o c i n ( A c h e r ,1 9 9 6 ) .F u r t h e r m o r e ,l u n g f i s hGnRH( J o s se talリ 1 9 9 4 ) ,ACTH( D o r e se ta l . ,1 9 9 0 ) ,e n k e p h a l i n(McDonｭ a l de talリ 1991), p r o l a c t i n(Nosoe talリ 1993), growth t alリ 1994), and common a l p h a hormone ( R u b i ne s u b u n i tf o rp i t u i t a r yg l y c o p r o t e i nhormones( A r a ie t a l . ,1 9 9 8 )havea l lbeenshownt os h a r emores i m i l a r i t y witht h et e t r a p o dt h a nwitht h et e l e o s tl i n e a g e s .Thus , t h i se x c e p t i o n a lm o l e c u l a ra l i g n m e n to fl u n g f i s ha n g i o ｭ t e n s i nwitht h a to ft e l e o s t ss e r v e st oemphasizet h e h i g h l yc o n s e r v e dn a t u r eo ft h ea c t i v eo c t a p e p t i d e , ANG1 1 ,w h i c h ,i nt u r n ,s t r o n g l ys u g g e s t st h a tt h etwo o t h e rformso fANGI Ifoundi nt e t r a p o d sandAmia have o c c u r r e d through m u t a t i o n si nt h e (1 ）ー and ( 5 ) p o s i t i o n sandn o tthroughgened u p l i c a t i o ne v e n t s . Havings a i dt h a t ,h o w e v e r ,i ti sknownt h a te e lh a s both[Asn1]ANGand[Asp1]ANGi nt h eplasmaa tt h e
Copy r i gh t 。 1 999 b yA c a d e m i cP r e s s A l l i r g h t s o fr e p r o d u c i t o nina n y f o r mr e s e r v e d
212
J o s se ta l
positionrestoredthes e l e c t i v i t ybutreducedthesensiｭ t i v i t y . These differences in action suggest t h a t "the
Neoceratodus RAS may provide a useful model for studyofA N GI Ireceptorsingeneral. Thea b i l i t yoflungfishA N GI Itostimulatealdosteｭ ronereleasefromlungfishadrenalsteroidogenicc e l l s
nv i t r orather hasbeenconfirmedint h i sstudy,usingi thani nv i v otechniques.Thefactthati tdoessoina dose-dependentmanner i s further evidence that the
RASplaysaroleinadrenalsteroidogenesisinlung白sh. Theselectivestimulationofaldosteronesecretion, to the exclusion of c o r t i s o l and corticosterone, by the
Isuggeststhataldosteronemay lungfishformofA N GI besynthesizedbyadistinctpopulationofadrenalc e l l s inlungfishasi ti sinmammals(Nishimura,1 9 8 7 ) .This possibilityi scurrentlyunderinvestigation.Inconcluｭ s i o n ,lungfishprovideaninterestingintermediatebeｭ tweenf i s handtetrapodsinthattheypossessateleost formofangiotensinwhich appears to a f f e c t adrenal steroidogenesisin a mammalianmanne乙 i.e., stimuｭ l a t e s aldosterone to the exclusion of corticosterone andI orc o r t i s o l .
ACKNOWLEDGMENTS
Wet h a n kt h ef o l l o w i n gf o rt h e i rencouragementanda s s i s t a n c e w i t ht h ec a n n u l a t i o nandr e c o r d i n go fbloodp r e s s u r ei nt h el u n g f i s h : B r i a nA t w e l l ,RobinM c A l l e n ,L iK e r s h a w ,LauraGammon,andJohn B l a i r W e s t .Thes t u d ywass u p p o r t e di np a r tbyg r a n t sfromt h e A u s t r a l i a nR e s e a r c hC o u n c i lt oJ . M . P . J .andt h eM i n i s t r yo fEducaｭ t i o n ,S c i e n c e ,andC u l t u r eo fJ a p a n( 0 8 6 4 0 8 4 3 )t oY . T .
REFERENCES
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Copyright 。 1999
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A c h e r ,R .( 1 9 9 6 ) .M o l e c u l a re v o l u t i o no ff i s hn e u r o h y p o p h y s i a l hormon e s :N e u t r a lands el e c t i v ee v o l u t i o n a r ymechanisms.Gen 0 2 ,1 5 7 1 7 2 . C o m p .E n d o c r i n o l .1 A r a i ,Y . ,Kubokawa,K . ,I s h i i ,S . ,andJ o s s ,J.M.P .( 1 9 9 8 ) .C l o n i n go f cDNAe n c o d i ngt h ecommona l p h as u b u n i tp r e c u r s o rm o l e c u l eo f p i t u i t a r ygl y c o p r o t e i nhormonesi nt h eA u s t r a l i a nl u n g f i s h ,Neo Endocri1叫 110, 1 0 91 1 7 . c e r a t o d u sf o r s t e r i .Gen. Comp. .N . ,andJ a n s s e n s ,P . A .( 1 9 7 5 ) .H i s t o c h e m i c a ls t u d i e so ft h e C a l l ,R a d r e n o c o r t i c a lhomologuei nt h ekidneyo ft h eA u s t r a l i a nl u n g ｭ f i s h ,N e o c e r a t o d u sf o r s t e r i .C e l lT i s s .R e s .1 5 6 ,5 3 3 5 3 8 . Do児島 R M . ,Adamczyk,D .L . ,andJ o s s ,J .M.P .( 1 9 9 0 ) .A n a l y s i so f ACTH-relatedandC L I P r e a l t e dp e p t i d e sp a r t i a l l yp u r i f i e dfrom t h ep i t u i t a r yo ft h eA u s t r a l i a nl u n g f i s h ,N e o c e r a t o d u sf o r s t e r i .Gen. 9 ,6ι73. C o m p .E n d o c r i n o l .7
Hyodo,S . ,I s h i i ,S . ,andJ o s s ,J .M.P .( 1 9 9 7 ) .A u s t r a l i a nl u n g f i s h n e u r o h y p o p h y s i a lg e n e sencodev a s o t o c i nand[ P h e 2 ] m e s o t o c i n r o c .N a t l . p r e c u r s o r shomologoust ot e t r a p o d t y p ep r e c u r s o r s .P c i .USA9 4 ,1 3 3 3 91 3 3 4 4 . A c a d .S J o s s ,J . M .P . ,K i n g ,J .A . ,andM i l l a r ,R .P .( 1 9 9 4 ) .I d e n t i f i c a t i o no ft h e m o l e c u l a rformso fands t e r o i dhormoner e s p o n s et og o n a d o t r o p i n ｭ e o c e r a t o d u sf o r s t e r i. r e l e a s i n ghormonei nt h eA u s t r a l i a nl u n g f i s h ,N G e n .C o m p .E n d o c r i n o l .9 6 ,3 9 2 4 0 0 . J o s s ,J .M.P . ,ArnoldR e e d ,D .E . ,andB a l m e n t ,R .J .( 1 9 9 4 ) .The s t e r o i d o g e n i cr e s p o n s et oa n g i o t e n s i nI Ii nt h eA u s t r a l i a nl u n g f i s h , N e o c e r a t o d u sf o r s t e r i .. JComp.P h y s i o l .B1 6 4 ,3 7 83 8 2. K o b a y a s h i ,H . ,andT a k e i ,Y .( 1 9 9 6 ) .Ther e n i n a n g i o t e n s i ns y s t e m .A c o m p a r a t i v ea p p r o a c h .Zoophysiolo幻t 3 5 ,7 7 9 2 K h o s l a ,M.C . ,N i s h i m u r a ,H . ,Hasegawa,Y . ,andBumpus,F .M. ( 1 9 8 5 ) .I d e n t i f i c a t i o n and s y n t h e s i so f[ 1a s p a r a g i n e ,5 v a l i n e , 9g l y c i n e]a n g i o t e n s i nIproducedfromplasmao fAmericane e l A n g u i l l ar o s t r a t a .Gen. C o m p .E n d o c r i n o l. 5 7 ,2 2 3 2 3 3 . .K . ,J o s s ,J . M. P . , and D a r e s ,R . M. ( 1 9 9 1 ) . The McDonald, L phylogenyo fMete n k e p h a l i nandL e u e n k e p h a l i n :S t u d i e sont h e e p i s o s t e u splaty仇incus andt h eA u s t r a l i a nl u n g f i s h , h o l o s t e a nf i s hL N e o c e r a t o d u sf o r s t e r i .G e n .C o m p .E n d o c r i n o l .8 4 ,2 2 82 3 6 . M e y e r ,A .( 1 9 9 5 ) .M o l e c u l a re v i d e n c eont h eo r i g i no ft h et e t r a p o d s 0 ,1 1 11 1 6 . andt h er e l a t i o n s h i p so ft h ec o e l a c a n t h .TREE1 .( 1 9 9 3 ) .L u n g f i s h M i c h e l ,G . ,C h a u v e t ,J . ,J o s s ,J .M.P . ,andA c h e r ,R n e u r o h y p o p h y s i a lhormones:Chemicali d e n t i f i c a t i o no fmesoto c i ni nt h en e u r o i n t e r m e d i a t ep i t u i t a r yo ft h eA u s t r a l i a nl u n g f i s h N e o c e r a t o d u sf o r s t e r i. G e n .Comp. E n d o c r i n o l .9 1 ,3 3 0 3 3 6 . N i s h i m u r a , H. ( 1 9 8 7 ) .R o l eo ft h er e n i na n g i o t e n s i n system i n o s m o r e g u l a t i o n .I n" V e r t e b r a t eEndocrino logy ”( P. K.T .Pangand M.P .S c h r e i b m a n ,E d s . ) ,V o l .2 ,p p .1 5 7 1 8 7 .AcademicP r e s s ,New Y o r k . . ,N i c o l l ,C .S . ,andKawauchi,H.( 1 9 9 3 ) .L u n g f i s hp r o l a c t i n N o s o ,T E泣1ibits c l o s et e t r a p o dr e l a t i o n s h i p s .B i o c h i m.B i o p h y s. A c t a.1 1 6 4 , 1 5 9 1 6 5 . .M.( 1 9 9 4 ) .C l o n i n go fagrowthhormone R u b i n ,D .A . ,andD o r e s ,R fromp r i m i t i v ebonyf i s handi t sp h y l o g e n e t i cr e l a t i o n s h i p s .Gen 5 ,7 1 8 3. C o m p .E n d o c r i n o l .9 . ,B l a i r W e s t ,J .R . ,S i m p s o n ,P . A . ,andS a w y e r ,M.K . S a w y e r ,W.H ( 1 9 7 6 ) .R e n a lr e s p o n s e so fA u s t r a l i a nl u n g f i s ht ov a s o t o c i n ,a n g i o ｭ . . JPhysiol.231,593602. t e n s i nI I ,andNaCli n f u s i o n .Amer T a k e i ,Y .( 1 9 8 8 ) . Changes i n blood volume a f t e ra l t e r a t i o no f n g u i l l aj a p o n i c a .C o m p . h y d r o m i n e r a lb a l a n c ei nc o n s c i o u se e l s ,A 9 3 2 9 7 . B i o c h e m .P h y s i o l .91A,2 a s o p r e s s o randd e p r e s s o re f f e c t s T a k e i ,Y . ,andHasegawa，主（ 1990). V o fn a t i v ea n g i o t e n s i n s and i n h i b i t i o no ft h e s ee f f e c t si nt h e C o m p .Endocriπol. 7 9 ,1 2 2 2 . J a p a n e s eq u a i l .Gen. .X . ,Nak句ima, K . ,andHazon, T a k e i ,Y . ,Hasegawa,Y . ,W a t a n a b e ,T N.( 1 9 9 3 ) .A novela n g i o t e n s i nIi s o l a t e dfromanelasmobranch f i s h .. JEndocrinol.139,281-285. T a k e i ,Y . ,I t a h a r a ,Y . ,B u t l e r ,D .G . ,\
