650

Brain Research, 107 (1976) 650-657 (~') Elsewer Scientific Pubhshmg Company, Amsterdam - Printed m The Netherlands

A morphine-like factor 'enkephalin' in

rat

brain: subceilular localization

RABI SIMANTOV, ADELE M. SNOWMAN ANDSOLOMON H. SNYDER Departments o f Pharmacology and Experimental Therapeutics, and Psychiatry and Behavioral Science~, Johns Hopkins University School of Medicine, Baltimore, Md. 21205 (U.S.A.)

(Accepted February 4th, 1976)

Pharmacologically relevant opiate receptor binding has been identified m vertebrate brain 13,16,17,21, intestine 3A~ and certain cell cultures s. The marked substrate specificity3,16A7,21, regional 5,9, cellular 14 and subcellular 15 distribution of opiate receptors suggests that they may be receptor sites for a normally occurring substance. Hughes et al. 6,7 identified a morphine-like factor by its ability to mimic the effects of morphine on smooth muscle in a fashion that can be antagonized by the opiate antagonist naloxone. A morphine-like substance 'enkephalin' with essentially the same chemical properties has been identified in mammalian brain extracts by its ability to compete for opiate receptor binding ~1,~z,22,23, while a chemically different substance which mimics morphine effects on smooth muscle has been reported in pituitary gland extracts 2. Enkephalin activity of mammalian brain, whether assayed by its effects on smooth muscle or opiate receptor binding, appears to be a peptide 6,7,11A2,22,23. Pig brain enkephalin activity is attributable to two peptides whose amino acid sequences have been shown by Hughes et al. 7 to be H-Tyr-Gly-Gly-Phe-Met-OH and H-TyrGly-Gly-Phe-Leu-OH respecUvely. Recently we have isolated enkephalin activity from bovine brain and shown it to be attributable to the same two peptides as in pig brain though with a greater proportion of leucine--enkephalin (Simantov and Snyder, in preparation). In binding studies it behaves like an opiate agonist, since its effects are diminished by sodium and enhanced by manganesellAL Its regional distribution in the brain is closely similar to that of opiate receptor binding itself 6,x~,12. In preliminary subcellular fractionation experiments, we found enkephalin activity to be most concentrated m crude mitochondrial fractions which contain both free mitochondria and pinched-off nerve terminals, synaptosomes 4,24. In the present study we have examined in detail the distribution of enkephalin activity and opiate receptor binding in subcellular fractions of rat brain. Male Sprague-Dawley rats (150-200 g) were killed by decapitation and their brains minus cerebella were homogenized in 20 vols of ice cold 0.32 M sucrose and centrifuged for 10 min at 1000 × g. All the following treatments were also performed at 0-4 °C. The pellet of the 1000 × g centrifugation, (Px), was washed m an equal volume of 0.32 M sucrose. A crude mitochondrial-synaptosomal pellet (P~) was

651 obtained by centrifuging the combined supernatant fractions for 20 min at 17,500 × g. For unlysed subfractionation the P~. pellet was suspended in an amount of 0.32 M sucrose equal to the original homogenizing medium and 10 ml of this suspension was layered on a 0.8 and 1.2 M two-step discontinuous sucrose gradient15. Subfractionation of osmotically shocked P2 suspension was performed by reconstitution in water 11. The water suspended P~ fraction was centrifuged for 20 min at 10,000 × g and 5 ml of the resulting supernatant fluid was layered on discontinuous sucrose gradients (0.4, 0.6, 0.8, 1.0 and 1.2 M sucrose). The gradients were centrifuged at 61,000 × g f o r 2 h . For extraction of enkephalin, each fraction from either the lysed or the nonlysed gradients was heated for 15 min in a boiling water bath. The suspension was then centrifuged at 100,000 × g for 1 h and the clear supernatant fluid lyophilized. The freeze-dried material was suspended in Tris.HCl buffer, pH 7.7 at 25 °C and assayed for enkephalin activity by the opiate binding assay11,12. Membranes from Sprague-Dawley rat brains minus cerebella were prepared and the stereospecific binding of [3H]naloxone was performed in 200 #1 of 0.01 M Tris.HC1 buffer (pH 7.7 at 25 °C) containing 4200 counts/min [3H]naloxone (5 #Ci/0.04 mg). Stereospecific opiate binding is defined as the difference in binding in the presence of 1/~M levallorphan from that in its absence. One unit of enkephalin is defined as that amount of enkephalin which yields 50 ~ receptor occupancy in the standard assay and determined according to Colquhoun 1, assuming classical binding interactions. Opiate receptor binding of the various fractions was performed in standard binding assays11,12,16. All the values are opiate specific binding expressed as the mean of triplicate determinations which varied less than 15 ~o, ± S.E.M. Uptake of [3H]norepinephrine (27.7 Ci/mmole) was performed in 4 ml of Krebs-Ringer buffer19. Monoamine oxidase was measured using [14C]tryptamine (55.2 mCi/mmole) as a substrate 25. Protein concentration was determined according to Lowry et al. lo. Among the primary subcellular fractions obtained by differential centrifugation, both enkephalin activity and opiate receptor binding are most enriched in the crude mitochondrial (P2) fraction (Table I). This fraction, which contains both free mitochondria and synaptosomes, also contains the highest [3H]norepinephrine uptake, which presumably involves synaptosomes, and monoamine oxidase activity, which is most concentrated in free mitochondria but also occurs in mitochondria within nerve terminals. When P2 pellets are subfractionated on discontinuous sucrose gradients designed to separate synaptosomes from mitochondria, both enkephalin activity and opiate receptor binding are most concentrated in fraction B at the interface of 0.81.2 M sucrose, a band which is enriched in synaptosomes. Enkephalin activity and opiate receptor binding in layer B are respectively 3 and 4 times greater than in layer C, which primarily contains free mitochondria. [3H]Norepinephrine uptake is also enriched about 3-fold more in the synaptosome-containing layer B than in layer C. By contrast monoamine oxidase activity is almost 3 times greater in layer C than layer B. When one takes into account the protein content of each fraction, the tel-

652 TABLE I S U B C E L L U L A R DISTRIBUTION OF E N K F P H A L I N ACTIVITY AND OPIATE RECEPTOR BINDING IN U N L Y S F D FRACTIONS OF R A T BRAIN

Enkephalln activity and stereospecific [3H]naloxone binding to various subfractions of rat brain homogenate were determined after differential centrlfugation or discontinuous sucrose gradient fractlonation as described in the text. Uptake of [3H]norepinephrlne and the activity of monoamlne oxidase were determined as markers for synaptosomes and mitochondrla respectively Values are the mean of 3 experiments each determined in triplicate, _-L-S.E M

Fraction

Enkephahn activity ( units/mg protein)

Opiate receptor ([moles .' ZH]nalox-

: '~Hj Norepinephrine uptake (fmoles/mg protein)

mg protein)

Monoamine Protetn oxtdase (mg/ml) activity (counts/rain~ mgprotein 10-3)

one bound/

Whole homogenate PI(1000 x g x 10min) Pa(17,500 x g- >~ 20rain) S~

1.58 0.44 3.64 1.05

0.14 0.06 0.20 0.15

49.3 ± 6.2 47.7 ± 5.2 2.1 ± 0 . 3 41.8 ± 3 9 7 4 . 8 ± 1 1 . 5 158.2 ± 7 2 21.8± 1 6 177 L 1.1

131 ± 224 ± 220± 22 9 ÷

Subfractlonation of P2 on sucrose gradients 0.32 M sucrose (A) 0.32-0.8 M (myelin, etc.) (B) 0.8-1.2 M(synaptosomes) (C) 1.2 M + pellet (mitochondria)

0.25±0.05 0.65 ± 0.05 2 90 ± 0 24

9.4±0.85 87.5±76 24.9 ± 3.1 57.2 ± 6.6 80.8 ± 6.6 172.0 ± 11.0

037±0.07 18 8 ± 1.4 95.7 ± 10.7

1.35:010 2.1 ± 0.20 2.6 ± 0.23

0.96 ± 0.14

20.8 ± 1.7

265 _t: 23 9

1.5 ± 0 14

± ± ± ±

54.4 ± 4.4

77 6.4 ± 0.30 2.0 1.6±015 16.5 3 . 9 0 ± 0 4 0 14 3.50 ± 0.20

ative percentages of total activity for enkephalin, opiate receptor binding, [aH]norepinephrine uptake and monoamine oxidase activity are distributed among the subcellular fractions in a fashion similar to their specific activity. The enrichment of norepinephrine uptake, which depends on intact nerve terminals, in layer B and of monoamine oxidase, which is primarily localized in free mitochondria, in layer C supports the notion that layers B and C predominantly contain synaptosomes and mitochondna respectively. This supposition is confirmed by electron microscopic examinations of these fractions prepared the same way as in previous studies in our laboratory 15 showing that fraction A contains mostly myelin and synaptosomal ghosts, fraction B is highly enriched in synaptosomes and fraction C contains primarily free mitochondria (M. J. Kuhar, personal communication). The localization of enkephalin activity to synaptosomal fractions is consistent with suggestions that enkephalin may be a neurotransmitter of neuronal systems for which the opiate receptor represents postsynaptic receptor siteslS, 20. To examine the subcellular localization of enkephalin in greater detail, we subjected crude mitochondrial (Pz) pellets to hypotonic lysis and centrifuged the lysate on discontinuous sucrose gradients which can resolve fractions enriched in synaptic vesicles, membrane fragments and free mitochondria ~4. Opiate receptor binding is most enriched in membrane containing fractions F (0.6-0.8 M sucrose) and G (0.8-1.0 M sucrose) with considerable activity also in fraction H (1.0-1.2 M sucrose) (Table II). These

653 TABLE II SUBCELLULARDISTRIBUTIONOF ENKEPHALINACTIVITYAND OPIATERECEPTOR BINDING OF LYSED P,~ PELLETS Enkephahn activity and stereospecific [gH]naloxone binding to various subfractions of rat brain homogenate were determined after lysis and centrifugation of the crude mltOChondrial-synaptosomal pellet, P~, on discontmuous sucrose gradients. Monoamme oxldase activity was determined as a marker of mitochondria. Values are the mean of 3 experiments each determined m triplicate, ± S.E.M. Fraction

Enkephalin activity (units/ mg protein)

Opiate receptor (fmoles [gH]naloxone bound/mg protein)

Monoamme Protein oxidase activity (mg/ml) (counts/rain~rag protein × 10 -a)

Whole homogenate P1 (1000 x g x 10 rain) P~(17,500 × g × 20rain)

1.66 4- 0.21 0.51 4- 0.07 3.83 4- 0.44

51.0 4- 7.2 2.5 ± 0.2 79.0 -I- 6.5

166 4- 14.9 26.5 4- 3.9 249 4- 16.0

6.25 4- 0.46 1.80 4- 0.20 3.65 4- 0.31

8.7 ± 1.1 78.7 4- 0.7

1.70 4- 0.20 1.50 4- 0.15

1564-15.0

1.104-0.20

195 4- 14.5 165 4- 14.3

1.25 ± 0.15 1.45 4- 0.10

233 4- 16.2

1.35 4- 0.13

467±41.0

1.304-0.17

Subfractionation of lysed P~ (10,000 × g supernatant fluid) Lysate (O) 0.77 4-0.09 0 (13) 0-0.4 Msucrose 0.75 :t: 0.06 9.0 4- 0.7 (synapt~c vesicles) (E) 0.4--~.6 M 0.94 i 0.09 14.9±0.8 (microsomes, etc.) (F) 0.6-0.8 M 1.46 4- 0.13 26.1 4- 1.4 (G) 0.8-1.0 M 1.15 4- 0.20 22.9 4- 1.5 (synaptosome ghost, membrane fragments) (H) 1.0-1.2 M 1.30 4- 0.11 21.8 4- 1.2 (damaged synaptosomes) (I) 1.2 M + pellet 0.784-0.10 9.64-0.8 (mltOChondria, shrunken synaptosomes)

three fractions each c o n t a i n m o r e t h a n twice the o p i a t e r e c e p t o r b i n d i n g o f f r a c t i o n I which possesses free m i t o c h o n d r i a with s o m e s h r u n k e n s y n a p t o s o m e s . F r a c t i o n s F , G a n d H each c o n t a i n 5 0 - - 8 0 ~ m o r e r e c e p t o r b i n d i n g t h a n f r a c t i o n E which is enriched in m i c r o s o m e s a n d o t h e r m e m b r a n e fragments. F r a c t i o n s D, which p o s sesses the s y n a p t i c vesicles, displays very little o p i a t e r e c e p t o r binding, while n o n e is detectable in the lysate. These findings confirm a previous e v a l u a t i o n o f the d i s t r i b u tion o f o p i a t e r e c e p t o r b i n d i n g in identically p r e p a r e d sucrose g r a d i e n t s using [3H]d i h y d r o m o r p h i n e , a different ligand, to label the o p i a t e r e c e p t o r 15. In earlier p r e l i m i n a r y studies, when crude m i t o c h o n d r i a l pellets were subjected to h y p o t o n i c lysis, s o m e e n k e p h a l i n activity was released into the s u p e r n a t a n t fluid, b u t a m a j o r p o r t i o n r e m a i n e d in the pellet 11. I n the p r e s e n t study, e n k e p h a l i n activity can be detected in the lysate b u t is also present in all the o t h e r layers o f the g r a d i e n t . W h e n one t a k e s into c o n s i d e r a t i o n the p r o t e i n c o n t e n t o f the v a r i o u s g r a d i e n t fractions, the high p r o t e i n c o n c e n t r a t i o n in fraction O (the lysate) indicates

654 TABLE III RELATIVEPARTICULATELOCALIZATIONOF ENKEPHALINACTIVITYIN SUCROSEGRADIENTFRACTIONS Enkephalln activity of unlysed (1) and lysed (11) subfract~onsof crude synaptosomal-mltochondrml P~ fraction was determined m the pellet and the supernatant after further centrlfugatlon at 100,000 × g for 60 mm. Data are the average of two experiments which varied less than 15 %. Fraction

Enkephalin activity

(untts/mg protein)

Pellet

Supernatant

Intact P2 (I) Sub]racttons, unlysed P2 0.32 M sucrose (A) 0.3243.8 M (B) 0.8-1.2 M (C) 1.2 M ÷ pellet

4.90

1.33

0 30 0.90 3.61 1 13

0.15 0 22 0.47 0.40

( H) Subfractions, lysed P2 lysate (D) 0-0.4 Msucrose (E) 0.44).6 M (F) 0.643.8 M (G) 0.8-1.0 M (H) 1.0-1.2 M (I) 1.2 M 4- pellet

0 34 0 35 0 27 0.41 0 35 0 22 0.20

4.36 3.97 3 70 4.20 3.30 2 16 1 05

that the greatest proportion of total enkephahn activity occurs m the lysate. The relative enrichment of enkephalin activity throughout the gradient resembles that of the opiate receptor. Thus the highest specific activity of enkephalin occurs m fractions F, G and H. Monoamine oxidase activity, by contrast, is greatest in fraction I which contains the highest density of free mltochondria. Electron microscopic examination of these gradient fractions 15 (M. J. Kuhar, personal commumcation) confirms that fractions F and G contain primarily membrane fragments and synaptosomal ghosts, while fraction D is most ennched in synaptic vesicles, fraction E contains microsomes and other membrane fragments, fraction H contains membranes and damaged synaptosomes, whde fraction I contains free mitochondria and some shrunken synaptosomes. To determine whether enkephahn actiwty in the gradient fractions from the P2 lysate is contained within intact particles or is only loosely bound to membrane surfaces, we centrifuged these fractions at 100,000 × g for 60 min and assayed both pellets and supernatants for enkephalin (Table III). Layers from gradient fractionation of the unlysed P2 pellet were centrifuged and assayed in the same way. The great majority of enkephalin activity in layers from fractionation of the unlysed P2 pellet remains particulate after centrifugation. Thus enkephalin activity in the unlysed P2 pellet is probably contained within subcellular particles. By contrast, when layers from gradients employing lysed P~ pellets are centrifuged, about 90 ~'o of the enkephalin activity in each layer is recovered in the supernatant fraction.

655

INSULIN ANGIOTENSIN H (6000)~, (1030)~

TYRAMINE (1~0)

60,,~ UNLYSED,O.8-1.2 M ( B ] • --* UNLYSED, I. 2 M (C) X--xLYSED,O6-O8(F]

40-

o,

Z0200

,b

~.o ~o FRACTJON NO

~o

Fig. 1. Blogel P2 chromatography of enkephalin. Three different subfractions from unlysed and lysed P2 gradients were tested for enkephalin activity by filtration through Blogel P2 column (200-400 mesh, 25 cm × 1.1 cm column). Three other peptides with known molecular wezght were also run through the same column (msuhn -- 6000, angiotensin II -- 1030, and tyramlne -- 170). Enkephalin actzvlty was assayed in the opiate receptor binding assay m triphcate. This indicates that the enkephalin activity of the lysed P2 pellet probably is only loosely bound to membrane surfaces. The relative enrichment of enkephalin activity in gradient fractions of the lysed P2 pellets resembles that of opiate receptor binding, suggesting that enkephalin may associate with opiate receptors on particle surfaces. In assessing enkephalin activity of subcellular fractions by its ability to compete for opiate receptor binding, one should ensure that the same material is being assessed in the various fractions. Brain enkephalin activity of presumed physiological relevance elutes from Biogel P~ columns in a single peak corresponding to a molecular weight of about 1000 (refs. 12, 20). We examined the chromatographic properties of enkephalin activity from 6 subcellular fractions including the intact P2 pellet, layers B and C from sucrose gradient fractionatlon of unlysed P2 fractions and layers O and F from gradient fractionation of the lysed P2 pellet. The chromatographic profiles for three of these are depicted in Fig. 1. In all 6 cases enkephalin activity is retained on the Biogel Pz column and elutes m a single peak corresponding to a molecular weight of about 1000. Another unique characteristic of brain enkephalin activity is its enhancement by manganese but not by calcium ions, and its depression by sodium but not potassium ions. For most subcellular fractions examined in the present study, the effects of sodium, potassium, manganese and calcium on enkephalin activities of all subcellular fractions was examined. As previously demonstrated for crude and semipurified enkephalin preparations, activity is enhanced by 1 m M manganese but not by a similar concentration of calcium, and is inhibited by 10 m M

656 s o d m m b u t n o t by the same c o n c e n t r a t i o n of potassium. Thus both Jn zts c h r o m a t o graphic properties a n d ion sensztwmes, the mater)al measured m these subcellular fractions appears identical to the b r a i n enkephalin actiwty described m earlier studies

6,7,11,12,20. It has been suggested that brain enkephahn might represent the neurotransmRter of neuronal pathways whose nerve terminals synapse upon opiate receptorsTM 20. In subcellular fracttonation studies, neurotransmltters are confined pNmartly to synaptosomal fractions. The localizatlon of enkephahn actlwty to synaptosomal fractions is consistent wlth a role as neurotransmltter or neuromodulator. When ]ysed crude mltochondrml pellets are centrifuged through sucrose gradients, enkephalin activity tends to be looselybound to membrane fragments which dlstribute throughout the gradlents m a fashion resembling opmte receptor binding. This suggests that enkephahn actlwty in these experiments may blnd loosely to the opiate receptor located on membranes of subcellular fractions. Supported by U S P H S G r a n t DA-00266, R S D A A w a r d MH-33128 to S H.S. a n d an E l e a n o r Roosevelt Fellowship to R.S.

1 COLQUHOUN,D., The relaUonshlp between classical and cooperative models for drug action. In H. P. RANG(Ed.), Drug Receptors, University Park Press, Baltimore, Md., 1973, pp. 149-182. 2 Cox, B. M., OPHEIM,K. E., TESCa~MACt-mR,H., AND GOLDSTEIN,A., A peptide-hke substance from pituitary that acts like morphine. 2. Purification and properties, Life Sci., 16 (1975) 17771782. 3 CRE~E, I., AND SNYDER, S. H., Receptor binding and pharmacological activity of opiates in the guinea pig intestine, J. PharmacoL exp. Ther, 194 (1975) 205-219. 4 GRAY, E. G., AND WHITTAKER, V. P, The isolation of nerve endings from brain: an electronmicroscopic study of fragments derived from homogemzatlon and centrifugat~on, J. Anat. (Lond.), 96 (1962) 79-86 5 HILLER, J. M., PEARSON, J., AND SIMON, E. J., Distribution of stereospecific binding of the potent narcotic analgesic etorphme in the human brain: predominance in the hmbic system, Res. Cornmun. chem. path PharmacoL, 6 (1973) 1052-1061. 6 HUGHES, J, Isolation of an endogenous compound from the brain with pharmacologzcal properties similar to morphine, Brain Research, 88 (1975) 295-308. 7 HUGHES,J, SMITH,T., KOSTERLITZ,H. W , FOlrHERGILL,L A., MORGAN,B. A., AND MORRIS, H. R., Identification of two related pentapeptldes from the brain with potent opiate agonlst actiwty, Nature (Lond.), 258 (1975) 577-579. 8 KLEE, W. A., AND NIRENaERG, M A , A neuroblastoma ghoma hybrid cell hne wlth morphine receptors, Proc. nat. Acad. Sci., ( Wash ) 71 (1974) 3474-3477. 9 KUHAR, M. J., PERT, C B, ANO SNYDER, S. H., Regional dlstributlon of opiate receptor binding in monkey and human brain, Nature (Lond.), 245 (1973) 443-451. 10 LOWRY, O. H., ROSEBROUGH, N. H., FARR, A. L., AND RANDALL, R. J., Protein measurement with the Folin phenol reagent, J. b~ol Chem., 193 (1951) 265-275 I 1 PASTERNAK,G. W., GOODMAN,R., AND SNYDER, S. H., An endogenous morphme-hke factor m mammahan brain, Life Sci., 16 (1975) 1765-1769. 12 PASTERNAK, G. W , SIMANTOV, R., AND SNYDER, S. H., Characterization of an endogenous morphine-hke factor (enkephalin) m mammahan brain, Molec. Pharmacol, in press. 13 PERT, C. B., APOSHIAN, D., AND SNYDER, S. H., Phylogenehc dlstrzbutlon of opmte receptor binding, Brain Research, 75 (1974) 356-361. 14 PERT, C. B., KUHAR, M. J, AND SNYDER, S. H , Autoradlographlc localization of the opiate receptor m rat brain, Life Sct., 16 (1975) 1849-1854. 15 PERT, C. B., SNOWMAN, A. M., AND SNYDER, S. H., Locahzatlon of opiate receptor binding m synaptic membranes of rat brain, Brain Research, 70 (1974) 184-I 88

657 16 PERT, C. B., AND SNYDER, S. H., Opiate receptor: demonstration in nervous tissue, Science, 174 (1973) 1011-1014. 17 SIMON, E. J., HILLER, J. M., AND EDELMAN, I., Stereospecific binding of the potent narcotic analgesic [aH]etorphine to rat brain homogenate, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 19471949. 18 SNYDER,S. H., The opiate receptor in normal and drug altered brain function, Nature (Lond.), 257 (1975) 185-189. 19 SNYDER,S. H., AND COYLE,J., Regional differences in 3H-norepinephrine and SH-dopamine uptake into rat brain homogenates, J. Pharmacol. exp. Ther., 165 (1969) 78-86. 20 SNYDER, S. H., SIMANTOV,R., AND PASTERNAK,G. W., The brain's own morphine, enkephalin, a peptlde neurotransmitter? Neurosci. Syrup., 1 (1975) in press. 21 TERENIUS,L., Characterization of receptor for narcotic analgesics in synaptic membrane fractions from rat brain, Acta pharmacol. (Kbh.), 33 (1973) 377-384. 22 TERENIUS,L., AND WAHLSTROM,A., Inhibitor(s) of narcotm receptor binding in brain extracts and c..¢rebrospinal fluid, Acta pharmacol. (Kbh.), 35, Suppl. I (1974) 55. 23 TERENIUS,L., AND WAHLSTROM,A., Morphine-like ligand for opiate receptors in human CSF, Life Sci., 16 (1975) 1759-1764 24 WHITTAKER,V. P., MICHAELSON,I. A., AND KIRKLAND, R. J. A., The separation of synaptic vesicles from nerve ending particles (synaptosomes), Biochem. J., 90 (1964) 293-303. 25 WURTMAN,R. J., AND AXELROO, J., A sensitive and specific assay for the estimation of monoamine oxldase, Biochem. PharmacoL, 12 (1963) 1439-1440.

A morphine-like factor 'enkephalin' in rat brain: subcellular localization.

650 Brain Research, 107 (1976) 650-657 (~') Elsewer Scientific Pubhshmg Company, Amsterdam - Printed m The Netherlands A morphine-like factor 'enkep...
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