Differential modulation of striatonigral dynorphin and enkephalin by dopamine receptor subtypes

Bram Research 51)7 (1990) 57-64 Elsevier

57

BRES 151211

Differential modulation of striatonigral dynorphin and enkephalin by dopamine receptor subtypes H -K Jlang 1, J F McGlnty 2 and J S H o n g ~ ~Neuropharmacology Section, Laboratory of Molecular and Integrative Neurosctenee, National Institute o] Em tronmental Health Sciences National Institutes of Health, Research Triangle Park, NC 27709 (U S A ) and eDepartment o] Anatomy and (ell Btologv, East Carohna University, School o[ Medicine, Greenville NC 27858 (U ~ A ) (Accepted 13 June 1989)

Key words Enkephahn Dynorphin, Dopamlne receptor subtype, Strlatum, Substantla nlgra, Modulation

Prevmus studies indicate that dopammerglc transmission inhlblts the biosynthesis ot enkephahn and stimulates that ol dvnorphm m the striatomgral pathway of intact rat The purpose of this study was to determine which dopamlne (DA) receptor subtype(s) me&ate the modulatory actions of DA We measured stnatal and mgral levels of enkephahn and dynorphln m (1) intact rats repeatedly inlected with Dj (SKF-38393 5 mg/kg, i p ) or D 2 (LY-171555, 1 mg/kg, i p ) agonlsts, alone or in combination, (2) 6-hvdroxydopamlne (6-OHDA)-lesmned rats repeatedly injected with the same D~ or D 2 agonlsts, and (3) intact rats repeatedly mlected with D~ (SCH-23390 0 05 mg/kg s c ) or D. (sulplride, 100 mg/kg, s c ) antagomsts gwen alone or in comblnatmn with the mixed D1/D2 agomst apomorphme (5 mg/kg i p ) Repeated Inlectmns ot the D~ agonlst to intact rats (twice dady for 7 days) produced a small but not statistically significant increase m striatal levels of dynorphin, slmdar treatment with the D 2 agomst did not affect dynorphm levels at all Combined treatments with D~ and D 2 agomsts &d not potentmte the effect of the D 1 agonlst 6-OHDA lesmns of the mgrostriatal DA pathway alone decreased the level ol dynorphm m both the striatum and substantta mgra However, repeated D l agomst, but not D 2 inlections not only reversed the decrease m dynorphtn levels, but caused a significant increase above control levels In intact rats repeated reJections of the D~ or D 2 antagonist alone failed to alter the levels of dynorphln, but the D~ antagonist, not the D 2 antagonist, attenuated the apomorphine-indueed increase m strlatal dvnorphm Regulation of striatal [MetS]enkephahn (ME) was found to be different from that of dvnorphm Whde repeated D~ agonlst mlectlons In intact rats had no effect on stnatal ME D 2 agonlst treatment caused a modest increase m ME The effect ol the D 2 agonlst in intact rats ~as blocked by co-administration with the D~ agomst In addition 6-OHDA lesions alone increased striatal levels ol ME and this increase was blocked by the D~ agomst but not the D2 agonlst Finally the D~ antagonist increased the smatal level of ME in intact rats ~hereas the D2 antagonist had no effect These data indicate that Dt receptors me&ate excitatory control over the biosynthesis ol smatonigral dvnorphln and also exert a tonic inhibitor} influence on the enkephahn system Furthermore stimulation of the Dz subtype imposes an excitatory stimulus on enkephahn biosynthesis but has no effect on dvnorphm

INTRODUCTION

demonstrated produced

Previous work from our laboratory shown that nigrostriatal dopamlne

and others has

(DA)

exerts a tome

that

enhancement

by repeated

rejections

of

DA

transmission

of a p o m o r p h i n e

or

D - a m p h e t a m i n e i n c r e a s e s t h e a m o u n t ot d y n o r p h l n a n d the mRNA

e n c o d i n g its p r e c u r s o r

prodynorphIn m the

inhibitory Influence on the biosynthesis of enkephahn m r a t s t r l a t u m ~6 2~-26 s6 B l o c k a d e o f D A t r a n s m i s s i o n b y

s t n a t o m g r a l r e g i o n s 2° ~4 ~ ~7 4~ ~Ihese f i n d i n g s i l l u s t r a t e

e~ther r e p e a t e d r e j e c t i o n s o f t h e D A r e c e p t o r a n t a g o n i s t

the

h a i o p e r l d o i , o r by l e s i o n l n g t h e n I g r o s t r i a t a l D A p a t h w a y

i n t e r a c t i o n m t h e b a s a l g a n g l i a h a s s e r v e d as a u s e t u l

with 6-hydroxydopamlne (6-OHDA),

model system for understanding the cellular and molec-

i n c r e a s e s t h e levels

o f b o t h e n k e p h a h n a n d t h e m R N A e n c o d i n g ItS p r e c u r sor, p r o e n k e p h a l i n ~ 4. 42 47 so s~ ss s9 T h e s e r e s u l t s suggest t h a t b l o c k a d e of D A t r a n s m i s s i o n r e l i e v e s t h e t o m c m h i b l t t o n by D A o n e n k e p h a h n b i o s y n t h e s i s 24 26 T h i s mhtbltory influence, however,

is a l r e a d y o p e r a t i n g at

the &fferentlal regulation of enkephahn and dynorphm m basal

ganglia

ular mechamsms

by

DA

This

DA-oplold

peptide

u n d e r l y i n g t r a n s s y n a p t i c r e g u l a t i o n of

neuropeptldes by various neurotransm~tters m the central nervous system s m T h e r e a r e t w o k n o w n D A r e c e p t o r s u b t y p e s , D~ a n d D~

w h i c h are c o u p l e d to & f f e r e n t s e c o n d m e s s e n g e r

s t i m u l a t i o n o f D~- a n d D 2 - r e c e p t o r s fails t o a l t e r t h e level

s y s t e m s a n d m e d i a t e d d i e r e n t D A - l n d u c e d effects s 9 ~ ~0 ~ 4~ s4 E l u c i d a t i o n of t h e r e c e p t o r s u b t y p e s w h i c h

of enkephahn

In c o n t r a s t t o e n k e p h a -

mediate

laboratory

d y n o r p h m s h o u l d p r o v i d e n e e d e d m t o r m a t m n for f u r t h e r

m a x i m a l levels u n d e r n o r m a l c o n d m o n s ,

lin,

recent

m the stnatum ~ reports

Correspondence

from

our

since further

and

others

dopammergic

J S Hong NIEHS P O Box 12233, Research Triangle Park N( 27709 U S A

modulation

ot e n k e p h a h n

and

58 understandmg

o f t h e signal t r a n s d u c t , o n p a t h w a y s re-

volved m gene expression ot neuropepttdes release

M o c c h e t t l et al ~ r e p o r t e d

mediate the halopendol-mduced However

and their

that D:receptors

increase m smatal ME

~t is n o t k n o w n w h i c h s u b t y p e r e g u l a t e s t h e

e x p r e s s i o n of d y n o r p h m

In this s t u d y we u s e d s e l e c t i v e

D~ a n d D e a g o m s t s a n d a n t a g o m s t s to e x a m i n e t h e r o l e s of these two receptor

subtypes m the modulation

ot

d y n o r p h m a n d e n k e p h a h n in t h e b a s a l g a n g h a To f u r t h e r characterize

the receptor

specificity o t t h e d o p a m i n e

effect, we c o m p a r e d t h e effects o f d o p a m m e r g l c D~ a n d D e agonlsts depleted

m

intact

of stnatal

rats

and

dopamme

m

rats

permanently

by 6-hydroxydopamlne

(6-OHDA)

MATERIALS AND METHODS

Ammals Male Fischer-344 rats (Charles R~ver, Raleigh, NC) between 12 and 14 weeks of age were used They were housed 4 to a cage m a colony room maintained on a 12/12 h hght/dark cycle at 21 + 2 °C and 50 _ 10% humidity

Radtotmmunomam (RIA ) Frozen ussucs v,ere homogemzcd m 20 ~ols ol 2 N at.et~c acid The homogenates were divided into 2 ahquot~ on~ for monoammc determination, and one for RIA of dvnorphm ,\(i-81 d',norphm A(1-17) and [MetS]enkephahn (ME) For RIA, the homogenate~ were heated in a boding water bath for S mm then cenmfuged at 25000 g for 20 mln and an ahquot of the supernatant was l'¢ophdized The residues were reconstituted m RIA buller (0 (12 M phosphate bufter pH 74, containing 11 1"~ N NaCl tllc: bovmt serum albumin I1(11% thlmerosal and (1 IQ Inton "K-100) and the suspension was centrifuged again The le,,els ot ME dynorphm A(1-8) and dynorphln A(1-17) m the supernatant were determined by RIA, as prewously described zs Briefly, ahquots ot tissue extracts were incubated with specific antiserum and ~zsI-[abeled tracers at 4 °C overnight Free and bound CzSI-labeled tracers were separated bv the addmon of a charcoal slurry and subsequent centrlfugauon The antiserum against dynorphm A ( I - 8 ) does not crossreact with [Leu~]enkephahn (LE) or fl-endorphln but has a small crossreactlv,ty with dynorphin A( I - 13) (0 02e/c ) and dvnorpbm A( 1-17) (0 01%) The sensitw,ty of the assa) is approxlmatel2~ 5 fmol Fhe ant,serum against dynorphm A(1-17) does not crossreact with ME LE, fl-endorphln or DYN A ( I - 8 ) but has a t0(;- cross-rcactwlt'~ with DYN A(1-13) The sensmwty ot this assay is approximately 5 fmol The ant,serum against ME does not cross-react w~th dynorphm A ( I - 8 ) or dynorphm A(l-17) The sensmvltv ot this assay Is approx 10 fmol

Measurement oJ dopamme and its metabohtes Treatment of ammals Repealed mjecttons of D~ and De agontsts m mtact rats Rats recmved dady mtrapentoneal mlectmns (twice dally, 1 ml/kg body wmght) with e~ther SKF-38393 (2,3,4,5-tetrahydro-l-phenyl(1H)3-benzazepme-7,8-dml hydrochlonde, Research Bmchemlcals Inc , Nat,ck, MA, 5 mg/kg) or LY-171555 [trans-(-)-4aR-4aR-4,4a, 5,6,7,8,8a,9-octahydro-5-propyl-(1H)] or [2H-pyrozolo(3,4-g)] qmnohne monohydrochlonde, Ell Ldly Research Laboratories, Indlanapohs, IN, 1 mg/kg) or both drugs dissolved m water for 7 days Control rats were rejected wath water according to the same schedule Ammals were decapitated 16 h after the last mjectmn

Levels of DA and ,ts metabohtes ,n the stnatum of 6-OHDA lesioned rats were determined by an HPLC method described by Wdson et al 62

Statistics Analys~s of variance °~ wa~ used to test for overall staUstlcal s,gmficance If a s,gmflcant effect was observed by analysis of variance, post hoc comparisons between group means were made using F~sher's Least S,gnlflcant Difference test 3x A slgmficance of P < (1 05 was reqmred for rejection of the null hypothesis

Repeated mlecttons of D~ and De agontsts tn 6-hydroxydopamtne (6-OHDA) lesioned rats 6-OHDA (10-/~g-free base m 0 5 /~1 RESULTS

amficml cerebrospmal fluid (CSF), containing 0 1% ascorbate) was rejected undaterally into the substantla mgra (coordinates based on mteraural line anterior 3 5 mm, lateral +2 5 mm, vemca1-2 2 mm according to the atlas of Paxmos and Watson, 1986) The contralateral s~te was rejected w~th amflclal CSF, and served as control Two weeks after the 6-OHDA mlectmn, rats received dally mlectmns of either SKF-38393, LY-171555 or water (vehicle) according to the procedures described above Ammals were decapitated 16 h after the last injection

Levels o f D Y N A ( 1 - 8 ) , D Y N A ( 1 - 1 7 ) , and M E m the strtatum and substantta ntgra after repeated mlectlons o f selecttve D 1- and D2-receptor agontsts

Repealed mlecttons oJ D~ and De antagomsts, alone or m combination with apomorphme tn intact rats Rats were dwided into

A(I-81,

6 groups and recmved dady subcutaneous InJections (twice dady for 7 days) of one of the following compounds (1) H20 (1 ml/kg), (2) apomorphme (S~gma Chemical C o , St Louis, MO, 5 0 mg/kg, dissolved in H~O), (3) R(+)-SCH-23390 (R(+)-8-chloro-2,3 4, 5-tetrahydro-3-methyl-5-phenyl-lH-3-benzazepme-7-ol-HCI, Research Blochem~cals Inc , Natick, MA, 50/~g/kg, dissolved m HzO ), (4) R(+)-sulpmde (S,gma Chemical Co , St Louis, MO, 100 mg/kg, d~ssolved m a few drops of glacml acid, neutrahzed w~tb NaOH to pH 6 0, and then dduted w~th HzO), (5) apomorphme + SCH23390, and (6) apomorphlne + sulpmde Ammals were decapitated 16 h after the last mject~on

S e v e n d a l l y r e j e c t i o n s o f a D1 a g o m s t ( S K F - 3 8 3 9 3 , 5 m g / k g , i p , t w i c e dally) f a d e d to a l t e r t h e l e v e l s o f D Y N DYN

A(1-17),

substantla ntgra relatwe

and ME

m the stnatum

to c o n t r o l

values

and

Although

t h e r e was a s m a l l i n c r e a s e m t h e l e v e l s o t D Y N A ( 1 - 8 ) and DYN A(1-17) m the strtatum, this increase was not statistically s t g m f i c a n t ( T a b l e I) the D 2 agomst (LY-171555,

Repeated

mlecnons ol

1 mg/kg, l p,

t w i c e dally)

also d i d n o t c h a n g e t h e l e v e l s o f D Y N A ( 1 - 8 ) a n d D Y N A(1-17) increased

m the smatomgral the

level o f M E

regions, m

but significantly

the smatum

The

De

a g o m s t - m d u c e d i n c r e a s e m t h e s t n a t a l level o f M E was

Brain dissection

p r e v e n t e d w h e n t h e D 1 a g o m s t w a s c o - a d m i n i s t e r e d w~th

After decap~tatmn, brains were removed and the strmta were dissected according to the method of Glowlnskl and Iversen t7 The substant,a mgra was hand-dissected according to the method of Palkovtts 44 T, ssues were stored at -70 °C untd bmchem~cal assays were performed

t h e D 2 a g o m s t ( T a b l e I)

Stnatomgral

levels of DYN

A(1-8) and DYN A(1-17) after the combined treatment w e r e n o t s~gnlflcantly d~fferent f r o m t h o s e of t h e D1 t r e a t e d g r o u p ( T a b l e 1)

59 TABLE I

Levels ofD YNA (1-8), D YN A (1-171 and ME m the stnatum and suhstantta mgra after repeated mlecttons o] D : and De-receptor agontst~ Rats recewed dadv mlecnons of D I (SKF-38~193 5 mg/kg, t p , twice daffy) and D= (LY-171555, 1 mg/kg l p twice daffy) agonlsts alone or m combination for 7 days Rats were decapitated 16 h after the last injection Numbers represent mean _+ S E M (pmol/g ~et wt in strlatum fmol/mgra in substanUa mgra) n = 8

Pepttde

Bram area

DYNA(1-8)

Drug treatment

stnatum substanna mgra strmtum substantm mgra strmtum substanua mgra

DYNA(I-17) [Met~]enkephahn

Control

~KF-38 ~93

LY-171555

$KF-18~9? + LY-I71555

17 5_+ 1 3 993 + 100 11 8+_l/I 86 + 6 5 1854 _+ 89 2546 + 187

23 5 ± 3 3 1094 + 146 132±07 89 ± 5 8 1867 ± 164 2336 _+ 204

15 0_+ l 9 916 _+ 89 102_04 86 _+ 9 7 231(I ± 1~7' 2140 + 149

24 0 ± ~, 1 119 138_+(15 97 ± 10 1922 ± 57 b 2417 _+ 15~ 1085 +

~P < 0 05 vs control group b p ,~ 0 05 VSLY-171555 group

Levels oJ D Y N A ( 1 - 8 ) , D Y N A ( 1 - 1 7 ) and M E m the strmtum and substantla ntgra after repeated mlecttons o f selecttve D¢ and D 2 receptor agontsts m undaterally 6 - O H D A - l e s t o n e d rats S i m i l a r d r u g t r e a t m e n t s w e r e g i v e n to rats w i t h p r i o r undateral injection of 6-OHDA

into the substantla nigra

T w o w e e k s a f t e r t h e n l g r a l i n j e c t i o n of 6 - O H D A ,

r e v e r s e d b y t h e D I a g o n l s t (Fig

1) s p e c i t l c a l l y , r e p e a t e d

i n j e c t i o n s of S K F - 3 8 3 9 3 c a u s e d a l a r g e i n c r e a s e In t h e s t r I a t a l l e v e l s o f D Y N A ( 1 - 8 ) ( 1 5 0 % a b o v e c o n t r o l level) and DYN

A(1-17)

6-OHDA-lesloned returned

(100%

a b o v e c o n t r o l l e v e l ) in t h e

side (Fig

1)

the 6-OHDA-lnduced

The

same

decrease

treatment

in b o t h d y n -

there

o r p h i n s p e c i e s t o c o n t r o l v a l u e s in t h e s u b s t a n t i a n l g r a

w a s a p p r o x i m a t e l y 9 0 % d e p l e t i o n of s t r l a t a l levels o f D A

(Fig 2) In c o n t r a s t , r e p e a t e d i n j e c t i o n s o f LY-171555 to

(control

l e s i o n e d r a t s f a i l e d to c h a n g e t h e levels o f d y n o r p h l n s in

33 _+ 1 2 p m o l / m g w e t w t , 6 - O H D A

2 8 + 0 3

p m o l / m g w e t w t , n = 14) a n d its acid m e t a b o h t e s , H V A ,

the

and DOPAC

relative to the vehicle-treated

in t h e i n j e c t e d site r e l a t i v e t o t h e c o n t r a -

6-OHDA-lesioned

striatum

and

substantla

6-OHDA

nlgra

lesioned group

l a t e r a l c o n t r o l , t h e r e w a s n o c h a n g e in t h e s t r l a t a l level

(Figs

of 5-HIAA

d y n o r p h l n levels in t h e c o n t r a l a t e r a l c o n t r o l site r e l a t i v e

T h i s d e p l e t i o n of s t r i a t a i D A w a s a c c o m p a -

n i e d b y a 3 0 - 5 0 % d e c r e a s e in t h e levels of D Y N A ( 1 - 8 )

s t r l a t a l l e v e l s of d y n o r p h i n

2)

T h e d e c r e a s e in t h e

induced by 6-OHDA

was

N e i t h e r t h e D I o r D= a g o n i s t c h a n g e d

to n o n - l e s i o n e d , v e h i c l e c o n t r o l s (Figs

a n d D Y N A ( 1 - 1 7 ) In t h e s t r l a t u m (Fig 1) a n d s u b s t a n t l a m g r a r e l a t i v e to c o n t r o l (Fig

1 a n d 2)

In c o n t r a s t to t h e c h a n g e s In d y n o r p h t n s ,

~t

['--7 Control

#

[~

!e

6-OHDA Lesloned

6-OHDA

t r e a t m e n t c a u s e d a n i n c r e a s e in t h e s t r i a t a l level o f M E a n d this i n c r e a s e was p r e v e n t e d by t h e D~ a g o n l s t n o t b y t h e D 2 a g o n l s t (Fig

80

1 a n d 2)

30

[~

1400

but

3)

Control

70

6 - OHDA Lesioned

~7o --.

25 o

60 o.

-

E 20

50

40

~

a.

~

60~ 0

12OO

i

,%

-6 I 0 0 0 E

E ~5 -

--

50

I

800

E 40~

i

o

~" 6 0 0 Z

20 _

"6

o

I0

0

r_

Vehicle

SKF

38393

L¥ 171555

Vehicle ~(F-~S3ra3 LY-I"r1555

0

F;g 1 Effects of repeated rejections of D] and D 2 agonlsts on the stnatal levels of DYN A ( I - 8 ) and DYN A(I-17) m unilaterally 6-OHDA les~oned rats "Iwo weeks after mgral ;nlecuon o[ 6-OHDA rats were injected with SKF-38393 (5 mg/kg l p ) LY-171555 (1 mg/kg, i p ) or vehicle (H.O 1 mg/kg) twice daily lor 7 consecutwe days and were killed 16 h after the last rejection Each value ls the mean _+ S E M of 12-14 rats *P < (105, **P < 0 01 ***P < 0 001 compared w~th contralateral controls

30 ~

400

2O

:~ 2 0 0

10

0

r--

L w,a

Vehlcte

SKF-38393

t-Y-171555

z

0

Fig 2 Effects of repeated lnlecUons of D~ and D 2 agomsts on the mgral levels of DYN A ( I - 8 ) and DYN A(I-17) m unilaterally 6-OHDA lesioned rats Substanna mgra ~as hand-dissected from the same rats as described m Fig 1 Each value is the mean _+ S E M ol 12-14 rats *P < 005 **P ~ 001 compared with contralateral controls

60 1ABLI: III

Levels o] D YN A (1-8) and D YN A(1-17) m the substantta m~ra alter repeated m)ectton~ or apomorphme, alone or m t ombmatlon wtth D ~ and D e antagontst~

F"-] Control 6-OHDA Lesioned

3ooo

Drug t re a t me nt s were the same as in Table I1

ca

Drug treatment

T

~. 2ooo

Pepttde concentratton (fmol/mgra) D~N4(1-8)

DYNA(I-17)

2339 3912 2703 4067 2271 2626

65 121 64 139 74 59

uJ Control Apomorphme APO/SCH-23390

I

"~ 1000

APO/sulpmde SCH-23390 R(+)-sulpmde

+ 296 _ 293 b + 333 ~ + 113 ~' _+ 300 + 230

9 3 9 6 3 4

_+ 5 8 + 14 9 t' + 8 4~ +_ 7 8 I~ -+ 6 1 _+ 4 8

r-

0

Veh*cle

SKF-31B39:3

LY- 171555

'P <0 05 vs control---group~bP< 0 001 vs control group, ~P < 0 05 apomorphme

Fig 3 Effects of r e p e a t e d mject~ons of D~ and D e agomsts on the

stnatal levels of [MetS]enkephahn m unilaterally 6-OHDA lesioned rats Tissues were dissected from the same rats as described m F~g 1 Each value is the mean + S E M of 12-14 rats *P < 0001 compared with contralateral control

Effects o f D ~ and D 2 antagomsts on apomorphme-mduced increases m the levels of D Y N A(1-8) and D YN A (1-17) m the stnatum and sub~tantta mgra Seven daily reJections of the D 1 antagonist (SCH23390, 0 05 mg/kg, s c , twice daffy) or the D 2 antagonist (sulpmde, 100 mg/kg, s c , twice dally) failed to alter the levels of D Y N A ( 1 - 8 ) and D Y N A ( 1 - 1 7 ) m the strmtomgral regions (Tables II and III) Repeated mlectlons with a D A agomst, apomorphme (5 mg/kg, s c , twice daily for 7 days) caused significant increases m the levels of dynorphms These increases m dynorphm levels were greatly attenuated by the concurrent administration with SCH-23390, but not sulpmde m the strmtum and substantm mgra (Tables II and III)

T A B L E II

Levels of D Y N A (1-8), D Y N A (1-17) and M E m the stnatum after repeated m]ecttons o f apomorphme, alone or m combmatton wtth D 1 and D e antagomsts

Apomorphme (5 mg/kg), SCH-23390 (D~ antagomst, 0 05 mg/kg), sulpmde (100 mg/kg) were administered 1 p , twice daily for 7 days and rats were decapitated 16 h after last mjecnon Values are mean + SEMn=8 Drug treatment

Contro l Apomorphme APO/SCH-23390 APO/sulplrtde SCH-23390 R(+)-sulpmde

Pepttde concentratton (pmol/g wt ) DYNA(1-8)

DYNA(1-17)

[MetS]enkephahn

24 42 31 42 25 25

7 9 8 8 7 7

1928+ 129 2073 + 191 2258 + 258 1861 _+ 215 2885 + 312" 1910 + 186

4+ 1_ 8+ 7+ 7+ 8+

1 3 2 2 2 1

9 6b 2" ~ 6b 3 9

8 6 0 9 9 5

+ + + + + +

0 0 0 0 0 0

2 & 6¢ 6~ 5 3

"P < 0 05 vs control group, bp < 0 001 VS control group, ~P < 0 05 vs a p o m o r p h m e group

Stnatal levels of M E after repeated mjecttons of D 1 and De antagontsts, alone or m combmatton wtth apomorphme Seven daily mlect~ons of SCH-23390 resulted m a slgmficant increase m the stnatal level of ME (Table II) Th~s increase m M E was almost completely blocked by the co-administration of a p o m o r p h m e , which by itself showed no effect on the strmtal M E level In contrast, sulpmde treatment did not alter the stnatal level of ME alone or in combination with a p o m o r p h m e (Table II) DISCUSSION

We have previously reported that mgrostrlatal D A exerts phasic stlmulatory control over dynorphm biosynthesis and tonic inhibitory control over enkephahn biosynthesis m the mgrostnatal regions (22-26,33-35) The results of this study indicate that D 1 receptors mediate both the stimulatory effect on dynorphm and the mhibltory effect on enkephahn In addmon, we &scovered that D2-receptor stimulation increases strmtal enkephahn levels, an effect which was not apparent m our earher stu&es which used less selective agomsts such as apomorphme C o m p a r e d with apomorphme, the D 1 selectwe agomst showed lower efficacy m enhancmg the level of dynorphm m the strmtomgral regions of intact rats Repeated rejections of SKF-38393 caused a modest mcrease m stnatal D Y N A ( 1 - 8 ) and D Y N A(1-17), but these changes were not statistically sigmflcant A higher dose of SKF-38393 (15 mg/kg) resulted m a similar degree of mcrease (data not shown), suggesting the low intrinsic actwity of this D~ agomst To determine whether simultaneous actwatlon of D~ and D2 subtypes was necessary to achieve the maximal effect of a p o m o r p h m e and other d o p a m m e related agents, as has been demonstrated m certain behaworal and electrophyslologlcal stu&es TM 27 ~6 46 49 ~'~ 60 o l D l a n d D 2 a g o m s t s w e r e g i v e n t o g e t h e r

61 Results showed that the D e agonlst did not slgnlhcantly enhance the D~-lnduced trend toward an increase m the strlatal level of dynorphm Thus, one of the reasons why the DI agomst is not able to produce the same degree of increase m dynorphin levels as a p o m o r p h m e is because of the difference in the intrinsic activity of each drug toward D 1-receptors Although the D 1 agomst exerted a non-significant effect on the level of dynorphm in intact rats, it dramatically increased the level of dynorphin in the stnatum of 6-OHDA-lesloned rats A type of D~receptor supersensitivity after long-term blockade of D A transmission by the 6 - O H D A lesion may be responsible for this pronounced effect Although it is generalb beheved that a nlgral 6 - O H D A lesion produces an increase primarily in D. receptor supersensmvlty ~2 s7 other studies report that this lesion also produces behavioral supersensitivity to D~ agonlsts 2 is The exact receptor mechanisms that potentiate the effect of the D t agonlst in 6 - O H D A - l e s i o n e d rats remain to be studied Similar to the effect of apomorphlne (L1 et al , 1988), the SKF-38393-tnduced increase in dynorphln content in the 6 - O H D A injected side was accompanied by an increase in the abundance of m R N A encoding prodynorphln (100e7c above the contralateral side, Jlang et a l , preliminary observations) The increase of DYN A ( 1 - 8 ) and its immediate precursor D Y N A ( l - 1 7 ) in both the strlatum and substantla nlgra were proportional These data indicate that the increase in D Y N A ( 1 - 8 ) content is due to an increase in the biosynthesis ot dynorphln rather than due to an increase in the rate ot conversion of precursor to end product One observation worth noting from this experiment was that 6 - O H D A caused a significant decrease of stnatal and nlgral levels ot dynorphln Previous studies from our laboratory 34 and others 4s reported that long-term blockade of d o p a m m e transmission by repeated injections of haloperldol tails to decrease the strlatal level of dynorphln, suggesting that there is no tonic excitatory influence ol D A on the dynorphm system There are several possible explanations tor the difference between the haloperldol and 6 - O H D A results We found that a large degree ot D A depletion by 6 - O H D A (greater than 90%) is required in order to observe a decrease in strlatal dynorphln Thus, almost total elimination of D A transmission is required to see alterations ot dynorphln by 6 - O H D A Blockade oI D A transmission by daily Injections of halopendol may not be efficacious enough to cause a significant inhibition of dynorphm biosynthesis A n o t h e r reason could be related to the differences in the nature ot the blockade Halopendol-lnduced blockade is accompanied by an increase in D A and other substances co-stored in the nlgrostrlatal nerve terminals, whereas

6 - O H D A completely destroys the neurons Finally, the possibility that non-specific effects ot 6 - O H D A on the substantla nlgra may cause damage to the dynorphlncontaining neurons which results in the decrease In dynorphln levels should also be considered Although we cannot rule out the aforementioned possibility, the large increase in dynorphm levels of the 6-OHDA-lesloned side after the D~ agomst injections suggests that dynorphln-contalnlng neurons remained tunctlonal after 6O H D A injection The notion that D~-receptors mediate the stlmulatory effect ot dopamlne on strlatonlgral dynorphln was further supported by the trading that the D~ antagonist SCH23390 significantly antagonized the apomorphlne-lnduced increase in the strlatal level of dvnorphln, whereas LY-171555 (D2 antagonist) was devoid of this activity This conclusion is consistent with the report that a m e t h a m p h e t a m m e - m d u c e d increase in strlatal dynorphln is antagonized by SCH-23390, but not b~ sulplrlde 2~' The possible signal transductlon pathways coupling the activation of Di-receptors to the expression ol dynorphln in this brain region are not presently known However, it IS interesting to note the concentrated distribution of dynorphln~s ~s, Dl_receptors v ~ 14 > ~4 and DA-sensltlVe aden~l cyclase l~ in the strlosome (patch) compartment ot the strlatum This Interesting distribution pattern suggests a possible role of D 1 receptor-linked aden~l cyclase in regulating the metabolism 01 dynorphln Moreover, it is interesting to compare the D A receptor subtypes which regulate strlatonlgral dynorphln and substance P The pattern ot dopamlnergxc regulation ot substance P and dynorphln is very similar, both are under the excitatory influence of D A ~' > el 22 However, it has been clearly documented that substance P is regulated primarily by D 2 receptors~,a~ s2 Elucidation o| the signal transductlon pathways mediating the expression ol dvnorphln and substance P would increase our understanding ol the molecular mechanisms underlying the dopamlnerglc control of these two peptldes Regulation ol enkephalln by the two dopamlnc receptor subtypes has been studied by two groups Mocchettl et al w showed an increase ot strlatal levels o! enkephahn and m R N A encoding proenkephalln after repeated injections of the D~ antagonist SCH-23390 in the strlatum In contrast the D e antagonist sulplrlde caused a small decrease in the level of enkephahn and proenkephahn m R N A ~" Based on these results, ~t was suggested that D~-receptors mediate a tomc inhibitory influence on the metabolism ot enkephahn whereas D,-receptors pht~ an opposite role On the other hand, Morris et al 41 reported that SCH-23390 decreases and haloperldol increases the level ot enkephahn and m R N A encoding proenkephalln in the strlatum These authors concluded that the D~

~2 subtype plays a tomc e x o t a t o r y role, whereas D 2 plays a

levels m the normal state

It ~s m t er est m g to note that

tomc inhibitory role in the btosynthesls ot enkephalln aj

Dl-receptors

etlect oppostte

The different conclusions drawn by these two groups is

D~-receptors, as r e p e a t e d lnjectmns ot the D e agomst

possibly due to the doses of SCH-23390 m j e c te d

The

L Y - 1 7 1 5 5 5 caused a modest but slgmtmant increase m

htgh dose of SCH-23390 (2 4 mg/kg, s c ) used by M o r n s

strlatal e n k e p h a h n m tnta~t rats whtch was blocked by the

et al might c o m p r o m i s e the selectw~ty ot this c o m p o u n d

D~ agonlst

Furthermore

Dl-receptors play a dominant role over De-receptors m

the dose of haloperldol (2 4 mg/kg

s c )

medmte

an

to that

ot

In thts context, tt must be pointed out that

used by M o r n s et al ts not selective for D e receptors 41

regulating the expression of e n k e p h a h n

The effect of SCH-23390 on the level of enkephalln from the present study is conststent with the results reported

by the halopertdol (mtxed D 1 and De antagomst)-lnduced increase in e n k e p h a l m biosynthesis e~-e647ss and the

by M o cch et t l et al 3~, which ln&cated that the Dj-

blockade of the SCH-23390-mduced increase m enkeph-

r e c e p t o r subtype mediates the tonic lnh~bttory influence

ahn level by a p o m o r p h m e (mixed D 1 and D e agontst) (Table lI)

on the btosynthests of e n k e p h a h n

Thts concept was

further s u p p o rt ed by our study using selective D l and D e agomsts

Stmllar to the effects of a p o m o r p h m e 3~, re-

Fhts ts reflected

In summary, thts study d e m o n s t r a t e d that D A receptor subtypes exert differential regulatton on the biosyn-

p e a t e d m j e c t m n s of SKF-38393 alone failed to alter

thesis of d y n o r p h m and e n k e p h a h n m the s m a t o m g r a i

strtatal levels of e n k e p h a h n , but p r e v e n t e d the increase

regtons

of strtatal e n k e p h a h n m d u c e d by 6 - O H D A lesions m the

dynorphln

substantta nlgra

enkephahn

In contrast, the 6 - O H D A - m d u c e d in-

D~-receptors m ed i at e an excttatory effect on but exert a tome mhtbttory

mftuence on

In contrast, De-receptors m e & a t e an e x o t -

crease m e n k e p h a l m was not affected by the D 2 agomst

atory m o d u l a t m n on e n k e p h a l m but have no effect on

LY-171555

dynorphln

Taken together, these data show evidence

strongly favoring the conclusmn that the D j mechamsm exerts tomc mhlbttory influence over the blosynthests ot e n k e p h a l m and this mhlbttton is operating at maximal

Acknowledgements The authors would like to thank Mrs Loretta Moore and Dr Jennifer Shook for the excellent e&tonal assistance and helpful dlscussmns m preparing this manuscript

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