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Potassium-induced release of enkephalins from rat striatal slices
European Journal of Ph~maco~gy, 48 (1978) 219--221 © Ehe~e~Nor~-Holland ~ o m e ~ c M Pros
219
Short communication POTASSIUM-INDUCED RELEASE OF ENKEPHALINS FROM RAT STRIATAL SLICES HILLMAN OSBORNE *, VOLKER H~LLT and ALBERT HERZ Depa~ment of Neuroph~maco~gy, Max-Planck-Ins~t for Psych&tH~ K ~ e p e l i n s t ~ e ~ 8000 M~nchen 40, Germany
ReceNed 16 January 1978, accepted 20 January 1978
H. OSBORNE, V. H~LLT and A. HERZ, Po~sMum-~duced ~ e
ofenkephalins ~om ~ t stHa~l sHce& European J. P h ~ m a c ~ . 48 (1978) 219--221. The rate of release of enkephalin from rat ~ a t ~ Mices increased 7--10 f ~ d in response to d e p ~ a ~ z ~ n by 50 mM p o t a ~ m ~ns in ~ o . The p o t ~ u m ~ v o k e d r ~ e ~ e of enkephM~s w ~ a b a s h e d in a cMc~m defi~ent m e , u r n . Uptake ~ u ~ ~ d ~ a t e d that the potassium-stimulated efflux w ~ not due to the ~ t ~ n of an act~e uptake mechan~m ~ r these pepfides. These f i n ~ n ~ pro~de suppo~ for the ~ew that enkephalins may ~ n ~ n as n e u r o ~ a n s m ~ ~ bra~.
Enkephalin ~ e
Ra~mmuno~y
Corpus ~ H ~ u m
1. I n ~ o d u c t i o n T h e r e is i n c r e a ~ n g s u p p o ~ f o r t h e n o t i o n t h a t e n k e p h ~ i n s m a y f u n c t i o n as n e u r o modulator/neurotransmitter agents in t h e mamm~ian cen~ nervous sy~em. Much of t h ~ s u p p o ~ has c o m e ~ o m ~ u d ~ s o f t h e r e ~ o n ~ a n d subceHular d ~ t ~ b u t i o n o f t h e p e p t i d e s in ~ r ~ n , w h ~ h s h o w e n k e p h ~ i n s to be u n e v e n l y d ~ i b u t e d in t h e b r ~ n o f s e v e r ~ s p e c k s ( S m ~ h et M., 1 9 7 6 ; Wesche e t ~ . , 1 9 7 7 ; Y a n g e t M., 1 9 7 7 ) a n d l o c ~ i z e d pred o m i n a n t l y in ~ a c t i o n s c o n t ~ n i n g t h e n e r v e endings ( P a s t e r n a k et M., 1 9 7 5 ; S i m a n t o v et M., 1 9 7 6 ; O s b o r n e et ~ . , 1 9 7 7 ) . R e c e n t l y , sever~ ~ud~s have shown that enkeph~ins exe~ a modulatory influence on the r~ease o f s e v e r ~ p u t a t i v e n e u r o ~ a n s m i t t e r s in v i ~ o ( T a u b e et ~ . , 1 9 7 6 ; S u b r a m a n i a n et ~ . , 1 9 7 7 ; Jessel a n d Iversen, 1 9 7 7 ) . I f e n k e p h ~ i n s are to be c o n ~ d e r e d as n e u r o t r a n s m ~ t e r candidates t h e y s h o u l d fulfil t h e i m p o r t a n t crite~ o n o f b ~ n g r e ~ a s e d ~ o m n e r v e t e r m i n u s in * Send repent ~ques~ to: H. Osborne at the above add~.
Neuro~ansm~r can~d~
r e s p o n s e t o d e p o l a r ~ a t i o n . In this r e p o ~ we p r e s e n t e v i d e n c e t h a t m e t h i o n i n e (met-)- a n d ~udne (~u-)-enkeph~in immunoreact~e mm terial are r e ~ a s e d ~ o m r a t c o r p u s ~ a t u m sHces in r e s p o n s e to p o t a ~ i u m d e p o l a r i z a t i o n in v i ~ o , a n d s h o w t h a t t h ~ e f f e c t is cMcium dependent.
2. M a t e r i a ~ a n d m e t h o d s M ~ e S p r a g u e - - D a w l e y rats ( 1 8 0 - - 2 2 0 g) w e r e killed b y d e c a p ~ a t i o n , t h e b r a n s r a p i d l y r e m o v e d ~ o m t h e skull a n d the striata d ~ s e c t e d o u t . Using a M c ~ w ~ n c h o p p e r , tissue slices w e r e p r e p a r e d b y ~ o n t ~ s e c t i o n i n g o f the s~tum at 2 6 0 p m i n t e r v ~ s , a n d t h e n b y resectioning a~er rotation of the turntable t h r o u g h 45 ° . T h e s5ces ~ o m 6 t o 8 rats w e r e i n c u b a t e d at 3 7 ° C in 50 m l K r e b s - - b ~ a r b o n ate m e d i u m c o n t ~ n i n g 11 m M ~ u c o s e a n d gased c o n t i n u o u ~ y w i t h a 5% c a r b o n d i o x i d e - 95% o x y g e n m i x t u r e ( c a r b o g e n ) . I n c u b a t i o n s w e r e c o n t i n u e d f o r 30 m i n d u ~ n g w h ~ h t i m e t h e m e d i u m was c h a n g e d t w i c e a n d t h e ~ices (500~600 m g w e t w e ~ h t ) t r a n s f e r r e d t o a
220
H. OSBORNE ET AL.
superfu~on chamber. The chamber, which was housed in a water bath, con~sted of a sy~nge with two movable plungers. The volume of the superfu~on chamber was adjured to 1.2 ml. Oxygenated medium was pumped upwards through the chamber at a flow rate of 1.0 ml/min, and co~ected at 4 min inte~ vals into cold acidified test tubes (0.2 ml 1 N
~eo ~ ~6 ~24
HC1/ 4aO:t HaC~5oeXcPeri N ,aln~orSUperfusate)ly mentm"ophilizedl . m 5int,he samplen SeutrahzeA d~hethllophilizeW de°nCl erC U nhncubates ~°w ~ i dam1 _~f
~12
ples were red~soNed in 50 mM NaH~PO~ soluo ~on, pH 7.4 with 1 N NaOH at 25°C, and subm~ted to an~y~s by a radioimmunoassay wh~h exhib~s a high degree of specifi~ty for met- and ~u~nkeph~in as previou~y d e s c ~ b e d (Wesche et ~., 1977). Expe~men~
~ ~ ~ 1~ ~ ~g. 1. R~e~e of m ~ n k e p h a l i n immuno~a~ivi~ ~om mt ~ a t ~ slic~ in norm~ (~) and c~c~m
showed that the Krebs--b~arbonate
medium
had no effect on the radioimmunoa~ays,
~
~ . . . . . .
defi~ent (©) m e d ~ m . The ho~zontM bar h d ~ the duration during w ~ c h the p o ~ i u m ~ v d w ~ devated to 50 mM. The resu~s are e x p ~ e d as percentage of the total t i ~ u e m ~ n k e p h a l i n content r d e ~ e d per min ( o r d h ~ e ) and are the means ± S.E. of 5 e x p e r i m e n t . A b ~ s a : 4 - m ~ c ~ c t i o n s .
3. Resul~
The mean rate of spontaneous efflux of met-enkeph~in immunoreact~e m a t e ~ ~om ~ a t ~ sfices superfused w~h Krebs-bicarbonate buffer was about 0.05% of the tot~ met~nkeph~in content of the ~ e s (1140 ± 158 pmo~/g, mean ± S.E.; n = 6) per min. As shown in fig. 1, elevation of the potas~um concen~a~on in the medium to 50 mM for 4 min resumed in a 10 fold increase in the efflux of met~nkeph~in immunoreactive m a t e ~ , the spontaneous rate of efflux b~ng re~ored 12 min a~er reapphca~on of standard superfu~on medium. 80--85% of the inifi~ met~nkeph~in content of the ~ices could be recovered at the end of superfu~on; approximately 25--30% of the decrease in peptide content can be accounted for in the samples, In another set of expe~men~, superfu~on of the ~ e s w~h a modified Krebs solu~on containing reduced Ca:÷ (0.1 mM) and elevated Mg:÷ (7 mM) comp~tely abo~shed the K ÷ evoked effect without markedly ~te~ng the spontaneous rate of efflux (fig. 1).
In sever~ experimen~ where samp~s were an~yzed for both leu- and met~nkeph~in immunoreactivities, the pattern of leuenkeph~in release pare~eled that obt~ned for met~nkeph~in. The mean rate of spontaneous efflux of about 0.15% of the leuenkeph~in content in ~ e s per min (220 ± 28 pmole/g, mean ± S.E.; n = 6) was increased 7 fold during a 4 min exposure to 50 mM potas~um. 4. D~cu~ion The resul~ presented here show ~hat leuand met~nkeph~in immunoreacfi~ty is released ~om rat ~ a t ~ s~ces in response to K÷ depola~zation. We sugge~ that the re~ase of enkeph~ins reflects the re~ase of these pep~des ~om nerve endings and not ~om non-neuron~ cells which are ~so constituents of the ~ices. Th~ proposal would be c o n ~ tent with previous data wh~h have shown enkeph~ins to be predominantly present in ~ructures wh~h cont~n the nerve endings,
ENKEPHALIN RELEASE Me ~ n ~ m m e s (for e x ~ p ~ see Osborne et ~ . , 1977). ~ ~ems u~k~y ~ a po~s~umfinduced ~ o n of an ~ p h ~ n reuptake m e c h ~ n ~ m c o ~ d h ~ e been the b ~ for Me e ~ c t s of K ÷ in these e x p e ~ m e n ~ . E x p e ~ m e n ~ , in which the uptake of the tritiated m ~ a b o ~ c ~ y stable m e t ~ n k e p h ~ ~ o ~ e (~:~ n ~ n ~ e by crude s t f i ~ s y n ~ t o s o m e p ~ p ~ a f i o n s was ~ d , f ~ l e d to i n d ~ a t e an active uptake m e ~ m for the pepfide ~ the concentration r ~ g e ~ - ~ M even after 60 min ~ c u b a f i o n ~ 37°C (un~ ~ data). ~ o u ~ the lack of ~ ~ t ~ e uptake m ~ h ~ m for an ~ p h ~ n ~o~e c ~ n o t be ~ ~ be t ~ e n as ev~ence for the absence of an a c t ~ e uptake m~h~m ~ r endogenous ~ p h ~ n , ~ ~ wo~hwhfle noting t h ~ uptake m ~ h ~ m s for pepfides are l ~ g ~ y u n k n o w n in mamm ~ n ~ s ~ m s {Hab~mann, 1977). The m ~ k e d d e p e n d e n c e of the K ÷ - ~ o ~ d release of ~ ~ on c ~ u m ions is ~ ogous to the cond~ions desc~bed ~or the evoked r ~ e a ~ of n ~ s m ~ in ~ t r o . ~ f l e t h ~ f i n i n g may therefore be t ~ e n as pm~ng support for the role of ~ ~ as ~ o ~ s m ~ s , the ~ ~ y that these pepfides m a y function as m o d ~ o ~ of ~ n a p f i c a c f i ~ t y c ~ n o t be n e ~ e c t e d .
~
o
~
m
~
We w~h to thank Dr. C. ~ ~ r h~ ad~se and as~stance h the preparation of tissue s~ces. ~ e
221 ~ c h n ~ a ~ t a n c e of M~. Ch~s~ S c h ~ r and Mi~ Gab~e~ ~ t ~~ y ~ o ~ d . Re~nc~ Hab~mann, E., 1977, ~ s m e m ~ and ~ a c ~ lular transport of ~ m ~ o ~ c ~ y active proteins and p o ~ p e p t ~ , ~ ~ . Arch. ~h~m~(.M. an~97~.L~uP~sen~511~977,O p ~ a n ~ g e ~ ~ t sub~ance P ~ e ~om rat ~ e m ~ M nu~eus, Nature 268,549. ~me, H.H., V. H~lt and A. Herz, 1977, Subc ~ u h r d~tfibufion of enkeph~ins and endogenous op~id acfi~ty ~ r~ br~n, ~ Sci. (in ~). ~ k , G ~ . , R. Goodman and S.H. Snyder, 1975, ~ endogenous morph~e~ke ~ctor (MLF) in mammM~n br~n, ~ Sci. 16, 1765. ~man~v, R., A ~ . Snowman and S.H. Snyder, 1976, A morph~e~ke ~ctor ' ~ n ' in rat br~n, ~ n Res. 107,650. SmRh, T.W., J. Hughes, H.W. Ko~efl~z and R.P. So~, 1976, E n ~ p h ~ , ~ a f i o n , d~tfibu~on and ~ncfion, ~: Op~t~ and Endo~nous Op~id Pepfid~, ed. H.W. Ko~efl~z ( E ~ e ~ / N o ~ h : Hofiand ~ o m e ~ c ~ P ~ , ~ m ) p . 57. Submma~an~~,S~'~om~hke~"M~zne~.Wfi~c~' an~Pr~g~.Dem ~" ~ng, 1977, Influence of ~ p h ~ n on K ÷ evoked efflux of putative n e u m ~ a n s m ~ in rat br~n, ~ ~ e ~ b . ~ c h . P h ~ m ~ . 299,163. ~ u b e , H.D., E. ~ m ~ , T. Endo and K. Starke, ~976' n ~ ~ En~ph~n~easei : ra~t~rNn, m°duN~uropeanJ ~ h~°r" ~ maeM. 38,377. W~che, D., V. H6~t and A. Herz, 1977, Ra~Mm~. ~m~~uno~yof e~h~~01,79. Naunyn~chm~deb. Yang, H.Y., J.S. Hong and E. Co~a, 1977, Re~on~ ~ f i b u ~ o n of leu- and m ~ n k e p h ~ in rat br~n, N e ~ o p h ~ m ~ o g y 16,303.
219
Short communication POTASSIUM-INDUCED RELEASE OF ENKEPHALINS FROM RAT STRIATAL SLICES HILLMAN OSBORNE *, VOLKER H~LLT and ALBERT HERZ Depa~ment of Neuroph~maco~gy, Max-Planck-Ins~t for Psych&tH~ K ~ e p e l i n s t ~ e ~ 8000 M~nchen 40, Germany
ReceNed 16 January 1978, accepted 20 January 1978
H. OSBORNE, V. H~LLT and A. HERZ, Po~sMum-~duced ~ e
ofenkephalins ~om ~ t stHa~l sHce& European J. P h ~ m a c ~ . 48 (1978) 219--221. The rate of release of enkephalin from rat ~ a t ~ Mices increased 7--10 f ~ d in response to d e p ~ a ~ z ~ n by 50 mM p o t a ~ m ~ns in ~ o . The p o t ~ u m ~ v o k e d r ~ e ~ e of enkephM~s w ~ a b a s h e d in a cMc~m defi~ent m e , u r n . Uptake ~ u ~ ~ d ~ a t e d that the potassium-stimulated efflux w ~ not due to the ~ t ~ n of an act~e uptake mechan~m ~ r these pepfides. These f i n ~ n ~ pro~de suppo~ for the ~ew that enkephalins may ~ n ~ n as n e u r o ~ a n s m ~ ~ bra~.
Enkephalin ~ e
Ra~mmuno~y
Corpus ~ H ~ u m
1. I n ~ o d u c t i o n T h e r e is i n c r e a ~ n g s u p p o ~ f o r t h e n o t i o n t h a t e n k e p h ~ i n s m a y f u n c t i o n as n e u r o modulator/neurotransmitter agents in t h e mamm~ian cen~ nervous sy~em. Much of t h ~ s u p p o ~ has c o m e ~ o m ~ u d ~ s o f t h e r e ~ o n ~ a n d subceHular d ~ t ~ b u t i o n o f t h e p e p t i d e s in ~ r ~ n , w h ~ h s h o w e n k e p h ~ i n s to be u n e v e n l y d ~ i b u t e d in t h e b r ~ n o f s e v e r ~ s p e c k s ( S m ~ h et M., 1 9 7 6 ; Wesche e t ~ . , 1 9 7 7 ; Y a n g e t M., 1 9 7 7 ) a n d l o c ~ i z e d pred o m i n a n t l y in ~ a c t i o n s c o n t ~ n i n g t h e n e r v e endings ( P a s t e r n a k et M., 1 9 7 5 ; S i m a n t o v et M., 1 9 7 6 ; O s b o r n e et ~ . , 1 9 7 7 ) . R e c e n t l y , sever~ ~ud~s have shown that enkeph~ins exe~ a modulatory influence on the r~ease o f s e v e r ~ p u t a t i v e n e u r o ~ a n s m i t t e r s in v i ~ o ( T a u b e et ~ . , 1 9 7 6 ; S u b r a m a n i a n et ~ . , 1 9 7 7 ; Jessel a n d Iversen, 1 9 7 7 ) . I f e n k e p h ~ i n s are to be c o n ~ d e r e d as n e u r o t r a n s m ~ t e r candidates t h e y s h o u l d fulfil t h e i m p o r t a n t crite~ o n o f b ~ n g r e ~ a s e d ~ o m n e r v e t e r m i n u s in * Send repent ~ques~ to: H. Osborne at the above add~.
Neuro~ansm~r can~d~
r e s p o n s e t o d e p o l a r ~ a t i o n . In this r e p o ~ we p r e s e n t e v i d e n c e t h a t m e t h i o n i n e (met-)- a n d ~udne (~u-)-enkeph~in immunoreact~e mm terial are r e ~ a s e d ~ o m r a t c o r p u s ~ a t u m sHces in r e s p o n s e to p o t a ~ i u m d e p o l a r i z a t i o n in v i ~ o , a n d s h o w t h a t t h ~ e f f e c t is cMcium dependent.
2. M a t e r i a ~ a n d m e t h o d s M ~ e S p r a g u e - - D a w l e y rats ( 1 8 0 - - 2 2 0 g) w e r e killed b y d e c a p ~ a t i o n , t h e b r a n s r a p i d l y r e m o v e d ~ o m t h e skull a n d the striata d ~ s e c t e d o u t . Using a M c ~ w ~ n c h o p p e r , tissue slices w e r e p r e p a r e d b y ~ o n t ~ s e c t i o n i n g o f the s~tum at 2 6 0 p m i n t e r v ~ s , a n d t h e n b y resectioning a~er rotation of the turntable t h r o u g h 45 ° . T h e s5ces ~ o m 6 t o 8 rats w e r e i n c u b a t e d at 3 7 ° C in 50 m l K r e b s - - b ~ a r b o n ate m e d i u m c o n t ~ n i n g 11 m M ~ u c o s e a n d gased c o n t i n u o u ~ y w i t h a 5% c a r b o n d i o x i d e - 95% o x y g e n m i x t u r e ( c a r b o g e n ) . I n c u b a t i o n s w e r e c o n t i n u e d f o r 30 m i n d u ~ n g w h ~ h t i m e t h e m e d i u m was c h a n g e d t w i c e a n d t h e ~ices (500~600 m g w e t w e ~ h t ) t r a n s f e r r e d t o a
220
H. OSBORNE ET AL.
superfu~on chamber. The chamber, which was housed in a water bath, con~sted of a sy~nge with two movable plungers. The volume of the superfu~on chamber was adjured to 1.2 ml. Oxygenated medium was pumped upwards through the chamber at a flow rate of 1.0 ml/min, and co~ected at 4 min inte~ vals into cold acidified test tubes (0.2 ml 1 N
~eo ~ ~6 ~24
HC1/ 4aO:t HaC~5oeXcPeri N ,aln~orSUperfusate)ly mentm"ophilizedl . m 5int,he samplen SeutrahzeA d~hethllophilizeW de°nCl erC U nhncubates ~°w ~ i dam1 _~f
~12
ples were red~soNed in 50 mM NaH~PO~ soluo ~on, pH 7.4 with 1 N NaOH at 25°C, and subm~ted to an~y~s by a radioimmunoassay wh~h exhib~s a high degree of specifi~ty for met- and ~u~nkeph~in as previou~y d e s c ~ b e d (Wesche et ~., 1977). Expe~men~
~ ~ ~ 1~ ~ ~g. 1. R~e~e of m ~ n k e p h a l i n immuno~a~ivi~ ~om mt ~ a t ~ slic~ in norm~ (~) and c~c~m
showed that the Krebs--b~arbonate
medium
had no effect on the radioimmunoa~ays,
~
~ . . . . . .
defi~ent (©) m e d ~ m . The ho~zontM bar h d ~ the duration during w ~ c h the p o ~ i u m ~ v d w ~ devated to 50 mM. The resu~s are e x p ~ e d as percentage of the total t i ~ u e m ~ n k e p h a l i n content r d e ~ e d per min ( o r d h ~ e ) and are the means ± S.E. of 5 e x p e r i m e n t . A b ~ s a : 4 - m ~ c ~ c t i o n s .
3. Resul~
The mean rate of spontaneous efflux of met-enkeph~in immunoreact~e m a t e ~ ~om ~ a t ~ sfices superfused w~h Krebs-bicarbonate buffer was about 0.05% of the tot~ met~nkeph~in content of the ~ e s (1140 ± 158 pmo~/g, mean ± S.E.; n = 6) per min. As shown in fig. 1, elevation of the potas~um concen~a~on in the medium to 50 mM for 4 min resumed in a 10 fold increase in the efflux of met~nkeph~in immunoreactive m a t e ~ , the spontaneous rate of efflux b~ng re~ored 12 min a~er reapphca~on of standard superfu~on medium. 80--85% of the inifi~ met~nkeph~in content of the ~ices could be recovered at the end of superfu~on; approximately 25--30% of the decrease in peptide content can be accounted for in the samples, In another set of expe~men~, superfu~on of the ~ e s w~h a modified Krebs solu~on containing reduced Ca:÷ (0.1 mM) and elevated Mg:÷ (7 mM) comp~tely abo~shed the K ÷ evoked effect without markedly ~te~ng the spontaneous rate of efflux (fig. 1).
In sever~ experimen~ where samp~s were an~yzed for both leu- and met~nkeph~in immunoreactivities, the pattern of leuenkeph~in release pare~eled that obt~ned for met~nkeph~in. The mean rate of spontaneous efflux of about 0.15% of the leuenkeph~in content in ~ e s per min (220 ± 28 pmole/g, mean ± S.E.; n = 6) was increased 7 fold during a 4 min exposure to 50 mM potas~um. 4. D~cu~ion The resul~ presented here show ~hat leuand met~nkeph~in immunoreacfi~ty is released ~om rat ~ a t ~ s~ces in response to K÷ depola~zation. We sugge~ that the re~ase of enkeph~ins reflects the re~ase of these pep~des ~om nerve endings and not ~om non-neuron~ cells which are ~so constituents of the ~ices. Th~ proposal would be c o n ~ tent with previous data wh~h have shown enkeph~ins to be predominantly present in ~ructures wh~h cont~n the nerve endings,
ENKEPHALIN RELEASE Me ~ n ~ m m e s (for e x ~ p ~ see Osborne et ~ . , 1977). ~ ~ems u~k~y ~ a po~s~umfinduced ~ o n of an ~ p h ~ n reuptake m e c h ~ n ~ m c o ~ d h ~ e been the b ~ for Me e ~ c t s of K ÷ in these e x p e ~ m e n ~ . E x p e ~ m e n ~ , in which the uptake of the tritiated m ~ a b o ~ c ~ y stable m e t ~ n k e p h ~ ~ o ~ e (~:~ n ~ n ~ e by crude s t f i ~ s y n ~ t o s o m e p ~ p ~ a f i o n s was ~ d , f ~ l e d to i n d ~ a t e an active uptake m e ~ m for the pepfide ~ the concentration r ~ g e ~ - ~ M even after 60 min ~ c u b a f i o n ~ 37°C (un~ ~ data). ~ o u ~ the lack of ~ ~ t ~ e uptake m ~ h ~ m for an ~ p h ~ n ~o~e c ~ n o t be ~ ~ be t ~ e n as ev~ence for the absence of an a c t ~ e uptake m~h~m ~ r endogenous ~ p h ~ n , ~ ~ wo~hwhfle noting t h ~ uptake m ~ h ~ m s for pepfides are l ~ g ~ y u n k n o w n in mamm ~ n ~ s ~ m s {Hab~mann, 1977). The m ~ k e d d e p e n d e n c e of the K ÷ - ~ o ~ d release of ~ ~ on c ~ u m ions is ~ ogous to the cond~ions desc~bed ~or the evoked r ~ e a ~ of n ~ s m ~ in ~ t r o . ~ f l e t h ~ f i n i n g may therefore be t ~ e n as pm~ng support for the role of ~ ~ as ~ o ~ s m ~ s , the ~ ~ y that these pepfides m a y function as m o d ~ o ~ of ~ n a p f i c a c f i ~ t y c ~ n o t be n e ~ e c t e d .
~
o
~
m
~
We w~h to thank Dr. C. ~ ~ r h~ ad~se and as~stance h the preparation of tissue s~ces. ~ e
221 ~ c h n ~ a ~ t a n c e of M~. Ch~s~ S c h ~ r and Mi~ Gab~e~ ~ t ~~ y ~ o ~ d . Re~nc~ Hab~mann, E., 1977, ~ s m e m ~ and ~ a c ~ lular transport of ~ m ~ o ~ c ~ y active proteins and p o ~ p e p t ~ , ~ ~ . Arch. ~h~m~(.M. an~97~.L~uP~sen~511~977,O p ~ a n ~ g e ~ ~ t sub~ance P ~ e ~om rat ~ e m ~ M nu~eus, Nature 268,549. ~me, H.H., V. H~lt and A. Herz, 1977, Subc ~ u h r d~tfibufion of enkeph~ins and endogenous op~id acfi~ty ~ r~ br~n, ~ Sci. (in ~). ~ k , G ~ . , R. Goodman and S.H. Snyder, 1975, ~ endogenous morph~e~ke ~ctor (MLF) in mammM~n br~n, ~ Sci. 16, 1765. ~man~v, R., A ~ . Snowman and S.H. Snyder, 1976, A morph~e~ke ~ctor ' ~ n ' in rat br~n, ~ n Res. 107,650. SmRh, T.W., J. Hughes, H.W. Ko~efl~z and R.P. So~, 1976, E n ~ p h ~ , ~ a f i o n , d~tfibu~on and ~ncfion, ~: Op~t~ and Endo~nous Op~id Pepfid~, ed. H.W. Ko~efl~z ( E ~ e ~ / N o ~ h : Hofiand ~ o m e ~ c ~ P ~ , ~ m ) p . 57. Submma~an~~,S~'~om~hke~"M~zne~.Wfi~c~' an~Pr~g~.Dem ~" ~ng, 1977, Influence of ~ p h ~ n on K ÷ evoked efflux of putative n e u m ~ a n s m ~ in rat br~n, ~ ~ e ~ b . ~ c h . P h ~ m ~ . 299,163. ~ u b e , H.D., E. ~ m ~ , T. Endo and K. Starke, ~976' n ~ ~ En~ph~n~easei : ra~t~rNn, m°duN~uropeanJ ~ h~°r" ~ maeM. 38,377. W~che, D., V. H6~t and A. Herz, 1977, Ra~Mm~. ~m~~uno~yof e~h~~01,79. Naunyn~chm~deb. Yang, H.Y., J.S. Hong and E. Co~a, 1977, Re~on~ ~ f i b u ~ o n of leu- and m ~ n k e p h ~ in rat br~n, N e ~ o p h ~ m ~ o g y 16,303.