Prevents Disruption

8/14/2019 Prevents Disruption

1/4

E L S E V I E R Behavioural Brain Research 83 (1997) 205-208

VIOURALBRAIN

RESEARCH

R e s e a r c h r e p o r t

N o. 302, a newly synthesized [pGlu4,Cyt 6 ]A V P (4 -9 ) analogue, preventsthe disruption of avoidance behav ior

K e n j i H i r a te a ,, , M a s u h a r u H i r a n o a, Yo s h ih a r u N a k a j i m a b , A k i o H i y a m a a , O s a m u M a e d a a,W a t a ru A s a k u r a ~

a Researc h Laboratories, Nippo n Chemiphar Co. Ltd., M isato, Saitama 341, Japanb Pharm aceuticals R esea rch Laboratories, Fu jirebio Inc., Hachioji, T ok yo 192, Japan

Received 8 July 1995; revised 5 February 1996; accepted 6 Feb ruary 1996

A b s t r a c t

We investigated the effects of [pG Iu4,C yt6]A VP (4-9) fragments and its analogues on cycloheximide (CHX)-induced learningimpairment in rats using the step-through-type passive avoidance test in rats. CH X (2 .8 mg/kg, s.c.) significantly shortened thestep-through latency in the retention trial, pG lu-A sn-C ys( Cy s)-P ro-A rg-G ly-N H2 ([pGlu4,Cyt6]AVP(4--9); 10 ng/kg, s.c.), amajor metabolite of arg inin e vasopressin, improved the CHX-induced learning impairment. Asn -C ys -P ro -A rg -O H(I-Cys 6]AVP(5-8); 1 ng/kg) corrected avoidance learning in the CHX-treated group, whereas neither Cys(C ys)- Pro -A rg- OH no rpG lu-A sn-C ys(C ys)-P ro-O H had any effect (1, 10 and 100 ng/kg, s .c.) , pG lu-A sn-Se r-Pro -Arg -Gly -NH 2 (No. 302), a newlysynthesized [pG lu4,C yt6]A VP (4-9) analogue, significantly prolonged the latency shortened b y CH X at do ses of 0.1, 1 and10 ng/kg (s.c.). As n- Se r-P ro -A rg -O H also improved the learning disruption induced by CH X, although the effective dose was100 time s higher than that of No. 302. The half-life of No. 302 in rat bloo d was ab out 5.5, 22 and 25 times longer than that ol-pGlu4,Cyt 6] AVP (4-9 ), [Cys6] AVP (5-8) and Asn -Ser- Pro -Ar g-O H, respectively. These results suggest that [Cys6] AVP (5-8)is the m inimal effective amino acid sequence in [pG Iu4,C yt6]A VP (4-9), an d show that No. 302 is a potent, pharmacologicallyactive peptide with high stability in the blood.

Keywords: No. 302; Arginine vasopressin analog; Avoidance behavior; Cycloheximide; Learning im pairme nt

1 . I n t r o d u c t i o n

Since the effect of arginine v asopress in (A VP (1-9 )) o nlea rn ing and memory in expe r imen ta l an ima l s wasrepor t ed by De Wied e t a l . [1 ] , s eve ra l i nves t iga to r sh a v e s h o w n t h a t AV P ( 1 - 9 ) m o d u l a t e s l e a r n i n g a n dmemory. For example , in the pass ive avoidance tes t ,AV P( 1- 9) d i rec t ly affects the re tent ion an d re t r ievalp rocesses o f l ong- t e rm m em ory in roden t s [2 ] .AV P(1- 9 ) , l y s ine vasopress in and desmo press in impro veinte l lec tual funct ions , a t tent iveness , and spontanei ty indemen t i a pa t i en t s and in hea l thy vo lun tee r s [3 ] .

AV P ( 1 - 9 ) i s m e t a b o l i z e d t o [ p G I u 4 , C y t 6 ] AV P ( 4 - 9 )a n d [ C y t 6 ] A V P ( 5 - 9 ) b y a m i n o p e p t i d a se a n d o th e r

* Corre spon ding autho r. T el.: (81) (489) 52-4311; Fax: (81) (489)52-0743.

enzymes in the bra in [4] . These metabol i tes fac i l i ta tel ea rn ing b ehav io r in avo idance and r ad ia l maze t a sks inra t s , and they a re abou t 1000- fo ld more po ten t t hanAV P ( 1 - 9 ) [ 5 - 7 ] . F u r th e rm o r e , [ C y t 6 ] AV P ( 5 " 8 )also improves learning behavior, suggest ing that[Cy t6 ]AVP(5-8 ) i s t he min ima l sequence in the e ff ec to f [ p G I u 4 , C y t 6 ] AV P ( 4 - 9 ) a n d [ C y t 6 ] A V P ( 5 - 9 ) [ 6 ] .However, i t i s not c lear whether or not[Cy t6 ]AVP(5-8 ) i s t he min ima l and ac t ive amino ac idsequence responsible for the effect of [pGlu 4,Cy t 6 ] AV P(4-9 ) .

We inves t iga t ed the e ff ec t o f [pGlu4 ,Cy t6 ]AVP(4-9 )f r agmen t s on the d i s rup t ion o f pass ive avo idance beha -vior induced by cycloheximide, a prote in synthes is inhib-i tor, in ra ts . Fur the rm ore , we invest igated the effect ofn e w l y s y n t h e s i z e d [ p G l u 4 , C y t 6 ] AV P ( 4 - 9 ) a n a l o g u e s ,and determined the biological s tabi l i ty (Fig . 1) .

0166-4328/97/$17.00 C opyr ight 1997 Elsevier Science B.V. A ll rights reservedPH S 0 1 6 6 - 4 3 2 8 ( 9 6 ) 0 0 1 1 8 - 0

8/14/2019 Prevents Disruption

2/4

206 tE Hirate et al./Behavioural Brain Research 83 (1997 ) 205 -20 8

A rg l n l n evasoprlmslnt

: AVP(1 -9 ) Cys -Tyr-Phe -GIn -Asn-Cys -P ro -Arg -Gly -NH21 2 3 4 5 6 7 8 D

cy s[pGIu4,Cyt6IAVP(4"9) pOlu.Asn.Cys-Pro.Arg.Gly.NH 2

4 5 6 7 8 9

|Cy lS ]AVP(5 -8 ) Asn -Cys -P ro -Arg -OH

No. 302 pGlu -Asn-Se r-P ro -Arg -Gly -NH2

Fig. 1. Amino acid sequences of AV P(1-9) , [pGlu4,C yt6]AV P(4-9)and related peptides.

2 . M a t e r i a ls a n d m e t h o d s

added to b lood ( f ina l conce n t ra t ion 0 .5 mM ) , an d incu-bated a t 37C. Sam ples (0 .25 ml) were col lected imm edi-ately after, and at 5, 10, 30, 60 and 120 min after startingthe incubat ion. Ice-cold t r ichloroacet ic acid solut ion(6.25%, 1 ml ) was ad ded to each sam ple and the mix tu rewas centr i fuged (2200 g , 4C) for 20 min. The superna-

t an t was f i l t e red th rough a membrane f i l t e r (Ge lmanSciences Jap an Ltd. , Toky o; Type 13, pore s ize 0.45 lam).The pep t ide concen t ra t ion in the superna tan t wasassayed by H PL C wi th a U V de tec to r, and the ha lf -l if eof the tes t pept ide in bloo d was calcula ted.

2.I . Animals

Male , 8 -week-o ld HL A-W is ta r r a t s were housed ingroups o f 5 in an a i r- cond i t ioned room ( t empera tu re ,23+ 2C ; hum idi ty, 55+ 10%; l ights on, 07.00-19.00 h) ,wi th f ree access to food and water.

2.2. P assive avoidanc e test

The appara tus fo r the s t ep - th rough- type pass iveavoid ance tes t con sis ted of one ac ryl ic-res in l ight( 8 x 3 9 .5 x 2 0 c m ) a n d o n e d a r k c o m p a r t m e n t( 14 19 .5 x 20 cm) . Each com par tm en t was f loored wi ths ta inless s teel gr ids . The two compartments were sepa.ra ted by a black wal l wi th a hole (6 6 era).

O n the f i rs t day o f t e s ting , ra t s were hab i tua ted to theappara tus . On the second day, an acqu i s i t ion t r i a l wasper fo rmed . Animals were ind iv idua l ly p laced in the l igh t

compar tmen t . One second a f t e r r a t s en te red the da rkcom par tme n t , an e l ec t ri c shock (100 V, 0 .5 mA ) wasde l ive red th rough the g r id f loo r. Imm edia te ly a f te r r a t ss t epped back in to the l igh t compa r tmen t , t hey werere tu rned to the i r home cages. For ty -e igh t hours l a te r,the an imal s were aga in p laced in the l ight com par tm en t(re tent ion t r ia l) . The t ime unt i l they entered th e darkcom par tm en t was mea sured as r e sponse l a t ency (max i -mum 600 s) .

Cyc lohex im ide (CHX; S igma Chemica l s , S t. Lou i s ,M Ot was d i s so lved in sa line , and subcu taneou s ly adm in-i s te red (2 .8 mg /kg) imm edia te ly a f t e r the acqu i s i t iontr ia l . The tes t pept ides were dissolved in sa l ine , andsubcu taneous ly admin i s t e red 1 h be fo re the acqu i s i t iontrial.

2.3. Statist ical analysis

The resu lt s a re r ep resen ted as the m ean + SEM , a ndana lyzed wi th two- ta i l ed Wi lcoxon ' s r ank sum test .

2.4. Stabili ty in blood

Un der e the r anes thesia , b lood w as co l l ec ted from theabdom ina l ao r t a o f ra t s. Tes t so lu t ion (12 .5 m M ) w as

3 . R e s u l t s

In the acqu i s i tion t r ia l , r a ts en te red the da rk com par t -men t abou t 2 s a f t e r they were p laced in the l igh tcompar tmen t . In the r e t en t ion t r i a l , t he r a t s tha t weregiven sal ine imm ediate ly af ter e lect r ic shock, requiredabou t 400 s to en te r the da rk co mp ar tmen t . The re sponsela t ency in r a t s t r ea ted wi th C HX (2.8 mg/kg) , a ne a rminimum le thal dose , was less than 100 s , and thedifference was s ignif icant between the sa l ine- and CH X-t rea ted g roups .

3.1. Investigation of active fra gm ent

As summ ar ized in F ig . 2 , [pGlu4 ,Cy t 6 ]A VP (4-9) a nd[ p G l u 4 , C y t 6 ] AV P ( 5 - 9 ) i m p r o v e d t h e C H X - i n d u c e davo idance l ea rn ing impa i rm en t a t a dose o f 10 ng /kg(s. c. ). [Cy t 6 ] AV P(6-9 ) tha t d id no t have pG lu-A sn a tthe N- te rmina l a l so improved the l ea rn ing d i s rup t ioninduce d by C HX at a d ose of 100 ng/ kg (s.c. ). Ne i ther[ C y t 6 ] AV P ( 6 - 8 ) n o r [ p G l u 4 , C y t r " ] AV P ( 4 - 7 ) a f f e c t e dthe r e sponse l a t ency in CHX- t rea ted ra t s .[Cys6 ]AV P(5-8) , i n w hich cys tine was r ep laced wi thcys te ine , improved the CH X- induced im pa i rmen t a t adose of l ng/ kg (s .c . ) (Fig . 3). The half -l ives of[ p G l u 4 , C y t 6 ] A V P ( 4 - 9 ) a n d [ C y s r" ] AV P ( 5 - 8) in t h eblood were 40 and 10 min, respect ively.

3.2. Investigation o f new ly synthesized peptides

p G l u - A s n - S e r - P r o - A r g - G l y - N H 2 ( N o . 3 02 ), a[ pGlu 4 ,Cyt6 " ]AV P (4 -9 ) ana logue , improved the l ea rn -ing d i s rup t ion induced by CH X a t doses o f 0.1, 1 and10 ng /kg , (s.c.) (Fig. 4). T he half-life o f No. 302 in bloodw a s 2 23 m i n . A s n - S e r - P r o - A rg - O H ( 10 n g /k g , s .c .) , a na n a l o g u e o f [ C y s 6 ] AV P ( 5 - 8 ) a l s o i m p r o v e d th e C H X -induced l ea rn ing impa i rm en t in the avo idance t e st . Thehalf -l i fe of th is pep t ide in th e bloo d was 9 min.

8/14/2019 Prevents Disruption

3/4

8/14/2019 Prevents Disruption

4/4

20 8 K~ Hirate et al./Behavioural Brain Research 83 (1997) 205-208

was abo u t 5 .5 - and 22-fo ld longer than tha t o f[ p O l u 4 , C y t 6 1 AV P ( 4 - 9 ) a n d [ C y s 6 ] AV P ( 5 - 8 ) , r e sp e c -t ively. These f indings show that No. 302 is a poten tpharm acolo gical ly act ive pept ide wi th high s tabi l i ty. Inadd i t ion , e i the r pyrog lu tamic ac id a t the N- te rmina l o rg lyc inamide a t the C- the rmal , o r bo th , in No . 302 con-t r ibute to i t s pha rma colog ical s ignificance and biologicals ta b il it y, s in c e A s n - S e r - P r o - A rg - O H w a s le ss p h a r m a -co log ica l ly po ten t a nd s t ab le in the b lood than No . 302 .

The effect of No . 302 in th is s tud y is appare nt ly to bedue to i t s d i rec t ac t ion on l ea rn ing and m em ory func t ionin the b ra in r a the r than by chang ing the sens i t iv i ty toCH X and /o r e l ec t ri c shock , since we showed tha tNo . 302 improv ed the CO2- induced l ea rn ing impa i rme n tin the pass ive avoidanc e tes t wi thout affect ing shocksens it ivi ty [8 ] . A l though the mechan i sm(s ) o f the e ffec to f No . 302 on the CH X- induce d l ea rn ing impa i rm en tremains un know n, No . 302 does no t seem to d i rec t lys t imula te AV P(1-9 ) r ecep to r, because i t ha rd ly d i splaces

3H-labeled AVP(1-9) specif ic b inding in the brain [81.In conclusion , the resul ts of th is s tudy show that[Cys61AVP(5-8) i s the min imal amino ac id sequenceof [pG Iu4 ,C yt6 ]A VP (4-9 ) in the im prov ing e ffec t o fa v o i d a n c e l e a r n i n g i m p a i r m e n t i n d u c e d b y C H X .No. 302 is a new highly s table pept ide that improves theavo idance l ea rn ing im pa i rme n t m ore e ffi cac ious ly than[pGlu4 ,Cyt 61AVP(4-9) .

References

[1] De W ied, D. and Bohus, B., Long -term and short- term effects onre tent ion of a con di t ioned avoidance response in ra ts by t rea t -ment w i th long ac t ing pi t ress in and ~t -MSH,Nature, 212 (1966)1484-1486.

[2] Gash, D.M., Herman, J.P. and Thomas G.J. , Vasopressin and

anim al behavior. In: D.M. G ash a nd G.J. Boer (Eds.),VasopressinPrinciples and Properties, Plenum Press, New York, 1987,pp. 517-547.

[3] Jolles, J. , Vasopressin and human behavior. In: D.M. Gash andG.J. Boer (Eds.),Vasopressin Principles and Properties,PlenumPress, Ne w York, 1987, pp. 548-578.

[4] Burbach, J.P.H. and Liu, B., Measurement of vasopressin-con-ver t ing aminopept idase ac t iv i ty and vasopress in metabot i tes ,Methods Enzymol.,168 (1989) 385-397.

[5] Burbach, J.P.H., Kovacs, G.L., De Wied, D., Van Nispen, J.W.and Greven, H.M., A m ajor metabol i te of arginine vasopress inin the bra in i s a h ighly potent neuropept ide ,Science,221 (1983)1310-1312.

[6] De W ied, D., Gaffori, O., Burba ch, J.P.H., K ovacs, G.L. and V an

Ree. J.M., Structure activity relationship studies with C-term inalf ragment of vasopress in and oxytocin on avoidance behaviors ofrats, J. Pharmacol. Exp. Ther.,241 (1987) 268-274.

[7] St rupp, B.J . , Improve ment of m emory by a vasopress in ,Behav.Neurosci.,103 (1989) 743-754.

[8] Hirate, K., Nakajima, Y., Maeda, O., Masui, S. and Hirano, M,Effect of newly synthesized analogue of [pGlu4,Cyt 6] AV P(4-9) on pass ive avoidance behavior,Jpn. J. Pharma-col., Suppl. 1, 64 (1994) 354P.