Juur.nol of Neurochrt?~istry,1976. Vol. 26. pp. 561-511. Pergamon Press. Prmted in Great Britain

A STUDY OF THE METABOLISM AND RELEASE OF DOPAMINE AND AMINO ACIDS FROM NERVE ENDINGS ISOLATED FROM SHEEP CORPUS STRIATUM J. S. DE BELLEROCHE, H . F. BRADFORD and D. G. JONES' Department of Biochemistry, Imperial College of Science and Technology, Prince Consort Road, London, S.W.7 (Received 1 April 1975. Revised 13 June 1915. Accepted 30 July 1975)

Abstract-Synaptosomes prepared from sheep corpus striatum showed a linear rate of respiration over a 90 min period of incubation in Krebs-bicarbonate medium containing glucose (10 mM) and the rate of respiration was stimulated by electrical pulses. Dopamine was released from synaptosome beds to the medium by either electrical pulses or 56mM-K+ (lomin), increasing 108% and 76% respectively above control levels of release. The presence of d- or ]-amphetamine ( 0 . 1 2 m ~ )in the inctlbation medium (40 min) increased the accumulation of dopamine in the medium by 310 and 275% respectively and 56mM-K+ also caused a significant increase in the release of glutamate, GABA and aspartate. Radioactively labelled dopamine was synthesized by the synaptosomes from L-[ '4C]tyrosine, L-DOPA or DL-DOPA, and electrical pulses caused a 35% increase in the rate of dopamine production from [U-'"C] tyrosine. No increased release of ['4C]dopamine in response to depolarizing stimuli was found to occur when synaptosome beds were transferred from medium containing radioactive precursors to fresh medium for further incubation (20 min). In the presence of 1- and d-amphetamine, accumulation of ''C-labelled doparnine in the incubation media was increased 129% and 380% respectively, the latter was partially depressed by absence of calcium from the medium. Three radioactively labelled metabolites formed by synaptosomes during incubation in DL-[~-'~C]DOPA were detected; the major ones were dihydroxyphenylacetic acid and homovanillic acid and the third was unidentified. When the synaptosome beds were transferred to medium containing no radioactive precursors, it was found that labelled dihydroxyphenylacetic acid was 7 times more abundant than labelled dopamine in the incubation medium (20 min) and one-third as abundant in the synaptosomes. The dihydroxyphenylacetic acid n Ci/dopamine n Ci ratio was greatly affected by K + stimulation, decreasing 52% and 34% in the incubation medium and synaptosomes respectively. A pathway of dihydroxyphenylacetic acid degradation was shown to occur through decarboxylation. These results are discussed in terms of the compartmentation of dopamine and its metabolism. It is proposed that one pool of dopamine is released by depolarizing agents and during the period of incubation it is replaced by synthesis from the endogenous tyrosine (193 nmo1/100 mg protein) and not by the labelled dopamine in the synaptosome. The synaptosomal pool of dopamine which is radioactively labelled after pulse labelling with DL-[2''C]DOPA appears to be prone to oxidation to DOPAC and hornovanillic acid which are preferentially released from thc synaptosomes. pulses and can be affected by other agents including amphetamine both in uiuo from corpus striatum (MCLEIWAN,1965; PORTIG & VOGS, 1969; RIDDELL & SzERB, 1971; BEESONet d., 1971) and in uitro from Striatal Slices (BALDESSARIhI & KOPIN, 1966; FARNEBO et a/., 1971; NG et a/., 1972; BEESONet al., 1969; AZARRO& RUTLEDGE,1973) or from striatal synaptosomes preloaded with radioactively labelled dopaet al., 1972). mine (FERRIS The aim of the present study was t o examine the disease (HORNYKIEWICZ, 1966) strongly suggest that dopamine is an inhibitory transmitter in this region. metabolic comparmentation and release of dopamine Biochemical evidence for a transmitter role for dopa- synthesized endogenously in isolated striatal nerve mine stems from measurement of its release. This has endings a n d compare the actions of releasing agents been induced by depolarizing agents such as electrical such as electrical pulses, K + and amphetamine. The synaptosomal preparation seemed well suited for this purpose, since it retains many of the complex meta'Department of Anatomy, University of Western Austra- bolic processes of the nerve ending in situ. When incubated in physiological salines, preparations from the lia, Nedlands, Western Australia cerebral cortex, for example, show calcium-dependent Abbreoiations used: DOPAC, 3,4dihydroxyphenylacetic release of putative CNS transmitters including ACh acid; HVA, homovanillic acid. 561

DOPAMINE is highly concentrated in the mammalian corpus striatum, where histochemical evidence shows that it is associated with nerve terminals of the region, especially those of the nigrostriatal tract (reviewed by FUXE & ANDEN,1966), and electrophysiological evidence indicates that the firing of a significant proportion of the neurons is inhibited by its application (CONNOR, 1970). This evidence together with its severe depletion in dyskinesia such as occurs in Parkinson's

562

J . S . DE BELLEROCHE, H. F. BRADFORD and D. G. JONES

out by adding 1.0 M-KCI(0.25 ml) made up in Krebs-bicar(DE BELLEROCHE & BRADFORD,1972a), glutamate, bonate medium to the 5 ml saline in which the SYnaPtoGABA, aspartate (DE BELLEROCHE & BRADFORD, Some bed was being incubated, thus raising the Potassium 19726) and noradrenaline (BLAUSTER'J rt al., 1972). level by 50 mM. Respiration of synaptosomes and their response to electrical stimulation was measured by Warburg METHODS manometry as described by BRADFORD (1970). Incubation of synaptosome beds with radioactiueb labelled Preparation of synuptosomes from sheep corpus striaturn. Brains were removed from the isolated head of the sheep tyrosine and DOPA and extraction for radioactiuify analysis. within a few minutes of death by stunning and serverage Synaptosome beds were incubated for 40 min in Krebs-biof the spinal cord at the neck. The corpus striatum, consist- carbonate medium (5 ml) containing either 2.5 pCi L-[U''C]tyrosine (10 mCi/mmol) or 1.0 pCi DL-3,4-dihydroxying of caudate nucleus, globus pallidus and putamen was rapidly dissected out. A transverse section was made at phenyl-[2-14Cjalanine (51 mCi/mmol). The Quick Transfer the level of the optic chiasma. The central masses of the Holder with synaptosome bed was then lifted out, the surcaudate heads were dissected out from the cerebral cortex face liquid allowed to drain off and the system transferred to fresh incubation medium. At the end of incubation, the in the anterior portion. The remaining caudate/putamen/ globus pallidus were dissected out from the posterior por- bed was placed in either 0.4 M-perchtoric acid (3 ml) contion with the lateral ventricles and corpus callosum as in- taining 0.1% sodium rnetabisulphite or in 0.2 M-acetic ternal and external limits. The hypothalamus, ventrally and acid/0.2 M-HC1 (3 ml) containing 0.1% sodium metabisulthalamus, posteriorly were also removed. The dissected phite. Non-isotopic noradrenaline and dopamine carrier were also included as indicated. The samples were stored portions were immediately placed .in ice-cold 0.32 M-SUCat -22°C. Analysis of the medium containing radioactively rose and transported back to the laboratory in this solulabelled DOPA following incubation in DL-DOPA is tion. A lo?; suspension in 0.32 M-SUCTOSe was homogenized and used for the preparation of synaptosomes (BRADFORD, shown in Table 2. All other measurements of dopamine 1969). The 10% homogenate was centrifuged at l000g for and 3,4-dihydroxyphenylaceticacid (DOPAC) release were 10min. the resulting supernatant at 19,620g for 2 0 m h made in the second period of incubation in fresh medium and a suspension from the resulting crude mitochondria1 during which stimulation and drug addition was made. Measurement of l4CO2 euolued from 3,4-dihydroxyphenyl pellet was layered on a 1.2/08 M discontinuous sucrose gradient and centrifuged at 75,000 g for 1 h. The synaptbsome [2-'4C]aianine. Synaptosomes (approx 6 mg/ml) were suslayer at the 1.2/0,8M-sucrose interface was diluted to 045 M pended in 'Krebs-phosphate medium containing bicarand centrifuged at 55,000 g for 20 min. The pelleted synap- bonate, having the following composition (mM); NaCI, 124; tosomes were suspended in Krebs-bicarbonate medium KCI, 5 ; KHzPO4, 1.2; MgSO,, 1.3; CaCI,, 0.75; Na,and used to prepare beds of synaptosomes by sedimen- HPO,, 19.3; NaHCO,, 1.0;L-ascorbic acid, 0.5 and glucose, Portions of the suspension 10 (pH 7.4 and gassed with 02). tation on to rectangles of nylon gauze by the methods & BRADFORD, 1972a). (2 ml) were incubated in Warburg flasks for 60 min at 37°C. described elsewhere (DE BELLEROCHE Electron microscopy. Suspensions of synaptosomes in The centre wells of the Warburg flasks contained moisKrebsbicarbonate medium were fixed at 4°C with veronal tened filter paper wicks to which 0.1 ml hyamine solution acetate buffered 17" OsO, for 1 h. The pellets of fixed (hyamine diluted 1 :1 with water) was added. To release synaptosomes were dehydrated with alcohol and embed- carbon dioxide dissolved in the medium, 0.1 ml 1.0 M-HCI ded in Araldite. A Phillips 300 electron microscope was was placed in the side arms of the flasks. At the end of used for examination of the specimens. Sections were incubation the HCI was added to the medium and incubastained on the grids with a 1% ethanol solution of uranyl tion continued for a further 10min. After incubation the acetate followed by lead citrate. wicks were removed and placed in vials together with InCUbUtiCJII of synaptosomes and extraction for Juoro0.5mI water used to rinse the inside of the centre well. metric analysis. Synaptosome beds (approx 10 mg protein) Bray's scintillation fluid was added to the vials and the & were anchored in Quick Transfer Holders (MCILWAIN samples were counted in a Packard Tri-Carb Scintillation RODNIGHT, 1962) and were incubated at 37°C in Krebs-bicounter. carbonate medium (5 ml) of composition (mM); NaCI, 124; Fluorometric analysis of dopamine. Extracts of synaptoKCI, 5 ; KHZPO,, 1.2; MgSO,, 1.3; CaCI2, 0.75; NaHCO,, some beds and their incubation media were first purified pH 7.5, containing 10 mM-glUCOse and 0.5 mM-L-ascorbic by adsorption on alumina using the method of ANTON & COz. At acid. The medium was gassed with 95% oz/5% SAYRE(1962) as described by WEIL-MALHERBE (1971). The the end of the incubation (40min), the synaptosome beds resulting catecholamine extract was used for fluorometric were placed in 0.42 M-perchloric acid (9.5 ml) and mixed determination of dopamine using the hydroxyindole method by vortex agitation. The medium bathing the synaptosome of LAVERTY & TAYLOR(l968), a separate tissue blank beds was placed in 0.8 M-perchloric acid (5 ml). The precipi- and internal standard being determined for each sample. tated protein was sedimented by centrifugation at 9009 The recovery of radioactively labelled dopamine added to for 15 rnin at 0°C and used for protein determination by the tissue sample in PCA was 67-76% (28 determinations) the method of LOWRYet ul. (1951). The supernatant fluid after purification on activated alumina. Allowance has not was used either (i) for amino acid analysis by automated been made for recovery in the data presented on Fig. 2, & THOMAS,1969) or hut has been used in the estimates of specific activities ion exchange techniques (BRADFORD (ii) for fluorometric determination of dopamine. The ex- in the text. tracts were stored at -22°C until analysed. Electrical Analysis of radiouctiuely labelled dopamine. Dopamine stimulation was by square-wave pulses of 10 V, 0-4 ms du- was separated from other radioactively labelled comration and alternating in polarity, applied at 100/s without pounds chromatographically, using a Zeocarb 225 ion exdelay between pulses to the two electrodes of the Quick change resin column (350 x 3mm, 8% cross-linked resin, Transfer Holder which were positioned on either side of kept at 38°C). The sample was applied to the column and the synaptosome bed. Potassium stimulation was carried eluted using a buffered system of increasing pH containing

Dopamine and amino acids in striatal synaptosomes

563

lithium salts. The serially eluted fractions were collected, blended with Bray's scintillation fluid and analysed using a Packard Tri-Carb Scintillation counter. Recovery of dopamine radioactivity by this procedure was Y5-104% (25 determinations).Later adaptation of the method consisted of incorporating an 'on line' scintillation counter, U.V. absorptiometer and fluorometric detector to analyse the et al., 1975). This method column effluent~(D)e BELLEROCHE separates the catecholamines, dopamine, noradrenaline, adrcnaline,their methoxy derivatives, 3-methoxy-4-hydroxytyramine, metanephrine, normetanephrine and the acid metabolites, homovanillic acid (HVA) and dihydroxyphenylacetic acid. Primary amines were detected fluorometrically and the remaining compounds localized by their U.V. absorption (280nm). Marerials. Analar grade chemicals were used throughout. Radioisotopes were obtained from the Radiochemical Centre, Amersham.

Metabolic peuformance and amino acid content gf isolated .yaptosarnes. When synaptosomes were incubated in Krebs-bicarbonate medium containing lOmM-glucose, there was a linear rate of oxygen uptake during at least 90min of incubation. The mean rate t_ S.D. was 61 k 4(6 values) pmol 0,/100 mg protein/h. Application of electrical pulses during the second 30 min of incubation increased this respiratory rate to 74 _+ 7 (6) pmol 0,/100 mg protein/h ( P < 0.05). Both basal and stimulated values are in the range obtained for synaptosomes isolated from other species and brain regions (BRADFORD,1974). The amino acid content of synaptosomes and the proportions relcased to the medium during 40 min incubation are shown in Table 1. Amino acid release in response to potassium stimulation. Raising the medium K + concentration from 6 mM to 56 mM for the last 10 min of incubation led RESULTS to significant increases only in the release of GABA, Morphology of synaptosomes isolated )om sheep glutamate and aspartate to the medium. Thc net inr corpus striatum. As depicted in Fig. 1, the synaptoso- creases were similar in absolute quantity (within 15%) ma1 preparations derived from basal ganglia consisted to those obtained from cerebral cortex synaptosomes principally of readily recognized synaptosomes. This incubated under similar conditions (DE BELLEROCHE 1972n). The other amino acids studied, applied to both 30 and 60min incubated material. & BRADFORD, Contamination by isolated mitochondria and myelin glutamine, serine, glycine and alanine showed no fragments was limited, while microsomes were seen change in their release pattern due to 5 6 m ~ - K + in some instances (Fig. la). Small membranous pro- (Table 1). Synaptosomal dopamine levels and the effect of elecfiles were also present, although there was no way trical pulses avui potassium stimulation on the release of determining with certainty their origin. It is more than likely, however, that at least some of them were of dopamine to the medium. Stimulation for IOmin grazing sections through synaptosomes. The synapto- with either electrical pulses or 56mM medium K + somes in general resembled incubated synaptosomes produced significant (P < 0.01 for both cases) indescribed elsewhere, e.g. cerebral cortex (JONES & creases in release of dqpamine to the medium of 108% BRADFORD, 1971) and spinal cord/medulla (OSBORNE and 76% above control values respectively (Fig. 2). et al., 1973). It is noticeable however, that in addition m e presence of d or 1-amphetamine (0.12 mM) during to the routinely described synaptic vesicles, mitochon- the incubation period (40 min) led to large increases dria and vacuoles, some of these basal ganglia synap- ( P < 0.001 for both agents) in dopamine released to tosomes also contained large, irregularly-shaped the medium of 3fi and 275% above control respectvacuoles and multivesicular bodies (Figs. l a and b). ively. These large increases due to amphetamine did Dense cored vesicles which are normally associated not seem to influence the amounts of dopamine rewith the presence of catecholamines in nerve ter- leased by depolarizing treatments. This was not minals could not be absolutely identified, as 0 ~ 0 4 proved statistically as the K C effect was small compared to that due to amphetamine. In addition to rather than KMnO, was used for fixation. TABLE1.

AMINO ACID LEVELS IN SYhAPTOSOME BEDS AND RELEASED TO THE MEDIUM INCUBGTIOY: THE EFFECT OF RAISED K' Amino acid Synaptosome bed Control

Aspartate Glutamine Serine Ghitamate Glycine Alanine GABA

Number of experiments

19067 f 145.6 614.5 + 62.0 429

383.0

36620

205 8 700.3 f 26.2 409.3 3 7 4 1174-9 f 71.5

+ 5

nmol per 100 mg protcin Released 10 the medium

K' stimulation

Control

*

K + ~timuIatinii

+

1675.8 2 608 579.4 i 76.5 340.5 i 22-4 32251 I 45.1 5549 i 19.4 310.3 k 11.9 1123.0 +. 38.1

3038 23.0 412-9 k 25.0 335.6 k 33.3 815 2 i 37.5 483.5 t 16.3 31 7.0 k 47.0 1724 i 13.7

464.9 56.3' 442.4 26-5 351.3 f 6.9 1180.3 i ?4.?* 553.4 27 4 348.7 i 35-4 262-9 t 24 1'

5

6

4

+

Increase in rclcasc with stirnulacion 164.1 29 5 15.7 365-1

69.9 31.7

90.9

Synaptosome beds were incubated at 37°C: in Krebs bicarbonate medium containing 10 mM glucose for 40 min. Potassium stimulation was carried out by adding KC1 to give a final concentration of 56 mM K' after 30 min incubation, the incubation being continued for a further 10 min. Values are means S.E.M. * Denotes significantly greater than the control with P i0,001.

J. S. DE BELLEROCHE, H. F. BRADFORD and D. G. JONES

564

Endogenous dopamine Release

12mM

Pomph

4 s

012mM 0 0 5 m

domph

d0mfphM

T

hydroxylase was avoided, and a value of 130 nmo1/100 mg protein/h was obtained. In DL-[I4qDOPA ( 3 . 9 ~ the ~ ) minimum rate of synthesis was 25.6 nmo1/100 mg proteinh. Electrical stimulation caused a 35% increase (P < 0.05) in the total labelled dopamine recovered with [14C]tyrosine as precursor. The rates of dopamine synthesis from tyrosine were similar to maximal rates of dopamine synthesis (VmJ from tyrosine in striatal slices reported previously (BEESONet ul., 1971) and to rates in the caudate nucleus in viuo (JAVOY & GLOWINSKI, 1971; COSTA& NEFF,1966), which are in the range 17-18.6 nmol/g/h. The calculated synaptosomal rates from ~-['~C]tyrosine are in fact likely to be considerably larger than 19.8 nmo1/100 mg protein/h since the figure does not allow for metabolite formation from dopamine or for Rodioctivel y lo belied dopam ine

FIG. 2. Synaptosome beds were incubated at 37°C for 40 min in Krebs-bicarbonate medium containing 10 mglucose and 0-5 mM-L-ascorbate; d- or I-amphetamine were present in the medium as indicated. Potassium stimulation (K stim) was carried out by adding KCl made up in the above saline to give a final concentration of 56mM (from 6 mM) at 30 min. Electrical stimulation (elec. stim) was by application of electrical pulses for the last 10 min of incubation. The histograms represent values of dopamine in nmo1/100 mg protein in the synaptosome beds (lower) and released to the incubation medium (upper). The values are means, the bars represent the S.E.M.and the number of experiments are indicated.

this effect of amphetamine (0.12mM) the d isomer was also found to decrease the total levels of dopamine (0.02 < P < 0.05). Incubation in the presence of the monoamine oxidase inhibitor, nialamide (10 p ~ ) did , not significantly affect the distribution of dopamine between tissue and medium and it was not used routinely in the experiments reported here. Amphetamine at a concentration of 0.12 mM however was employed routinely (i) because it facilitated measurement of dopamine in the medium and (ii) in order to gain further information about its mode of action. Synthesis of' radioactively labelled dopamine from Ltyrosine, L-DOPA and DL-DOPA. During 40 min incubation in 4-50 pm-t-Ci4C]tyrosine, L-['~C]DOPA or DL-DOPAthere was synthesis of radioactive dopamine (Fig. 3). From the levels of labelled dopamine present in the synaptosomes after incubation together with that released from the synaptosomes during incubation, a minimum rate of dopamine synthesis could be calculated. This, however, did not take into account the formation of dopamine metabolites during incubation. Thus, with Ci4C]tyrosine ( 5 0 ~ as ~ ) a precursor, dopamine synthesis was 19.8 nmo1/100 mg proteinh. In L-['~C]DOPA (20 m), the rate of synthesis was considerably greater as the rate limiting control step catalysed by tyrosine

J

y .:si

S y noptosomes

FIG. 3. Synaptosome beds were incubated at 37°C for 40 min in Krebs-bicarbonate medium containing 10 mMglucose, 0.5 mM-L-ascorbate and the radioactively labelled precursor as indicated on the figure. The beds were then transferred to fresh medium with no isotope, and incubated for a further 20min at 37°C. Potassium stimulation was by adding KCl to give a final concentration of 56 mM after 10min and elcctrical stimulation was by application of electrical pulses for the last 10min of incubation. A t the end of incubation the synaptosome beds were removed from the medium and extracted separately into 0 4 M-perchloric acid (3 ml) containing Olp/, sodium metabisulphite, 0 1 mwdopamine, 0.1 mM-noradrenaline. The incubation medium was added to 2 M-perchloric acid (1.2ml) containing 0.1% sodium mctabisulphite and non-isotopic carriers. The histograms represent values of dopamine in nCi/100 mg protein in the synaptosome bed (lower) and released to the medium (upper). The values are means, with the S.E.M.indicated by bars and the number of experiments indicated beside. * Denotes that the total radioactively labelled dopamine (released + synaptosomal) formed from L-tyrosine is significantly increased with electrical stimulation relative to the control with P < 005.

FIG.1. (a) Basal ganglia synaptosomes incubsted for 60 min in Krebs-bicarbonate. Synaptosomes (s) are distributed throughout the field, with occasional mitochondria (mit) and microsomes (m) as contaminants. Adherent postsynaptic membranes (pm) are associated with some synaptosomes. Non-specific membranous profiles (mp) are also present. AII illustrated material prepared using OsO, fixation, uranyl and lead staining; x 46,000. l(b) Example of a synaptosome present in this fraction. Its contents include vacuoles (v) and multivesicular bodies (mvb) in addition to the usual synaptic vesicles (sv); x 46,000. l(c) Basal ganglia, 30 min incubation. This synaptosome has an incomplete limiting membrane (arrow), although it is otherwise intact; x 94,500. NC--564

Dopamine and amino acids in striatal synaptosomes

dilution of the external radioactive tyrosine pool due to the presence of an endogenous tyrosine pool. This tyrosine pool was found to be 19.5 _+ 0.8 nmo1/1@) mg protein (mean value _+ S.E.M. for 3 determinations) which would give a concentration in the synaptosome of approx 2 0 p ~and this could significantly dilute the specific activity of the externally added tyrosine (50 w). The minimum contribution of labelled dopamine to the synaptosomal dopamine pool after 40min incubation in DL-[' 4C]DOPA represents 10.5 nmo1/100 mg protein (calculated from the specific activity of the precursor and the values in Table 2) which is 503% of the total synaptosomal dopamine at this point (Table 2). Synaptosomal pool sizes of dopamine were very similar in the presence (Table 2) or absence (Fig. 2) of ~ . ~ ~ M - D L - D OThe P A .pool sizes of released dopamine were not measured in the experiments where radioactivity was determined because of the need to add excess non-isotopic carrier. However, greater release of unlabelled dopamine does appear to occur in the presence of DL-DOPA (3.9 p ~ ) .This is evident from the high levels of dopamine (nci) released in the first 40min of incubation (Table 2), which, since the specific activity of the dopamine cannot exceed the precursors must imply a high level of unlabelled dopamine release. Release of radioactively labelled dopamine. The two series of experiments show that a larger proportion (13% in 20min) of the total labelled as distinct from the unlabelled (6% in 40 min) dopamine pool was recovered in the medium following incubation with DL-['~C]DOPA suggesting the presence of dopamine compartmentation. Further evidence for this compartmentation came from the finding that electrical pulses or raised K + did not increase the release of labelled dopamine from the beds when any of the labelled precursors were used (Fig. 3), although these agents did produce a significant increase in the total dopamine released (Fig. 2). For reasons of cost and

565

availability with high specfic activity, DL-[ 14c]DOPA was used in the subsequent studies rather than ['4C]tyrosine or [14C]DOPA. The possible conversion of DL-DOPA by the DOPA decarboxylase of non-neural tissue was assumed to be minimal as a reasonably pure synaptosome preparation was used. However, the metabolism of DOPA conversion by synaptosomes derived from 5-hydroxytryptamine neurons cannot be discounted. The release of radioactively labelled dopamine formed $-om DL-[2-'4C]DOPA in the presence of dand l-amphetamine. There were large significant increases ( P < 0.01) in the release of labelled dopamine to the incubation medium in the presence of d- or I-amphetaniine (Fig. 4). The d-isomer (0.12 mM) was considerably more potent in this respect, causing a 380% increase, and the I-isomer (0.12m~)caused a 129% increase in release. This reflects the relative potencies of these isomers as shown in a number of situations e.g. in oiuo induction of stereotyped behaviour (SCHEEL-KRUGER, 1972), inducing release of dopm i n e into the cerebroventricular system of anaesthetized cat (CHIUEH & MOORE,1974) and inducing dopamine release from superfused caudate nucleus (BEESONet al., 1971) and in vitro in their action in inhibiting dopamine uptake by synaptosomes (THORNBURG & MOORE,1973). In the present experiments d-amphetamine also produced a 36% increase in total isotopic dopamine compared to the control, whilst the 1-isomer was without significant effect. Time course studies showed labelled dopamine was being continuously released from synaptosomes in the presence of d-amphetamine (Fig. 5), the rate gradually diminishing over a 60min period, presumably due either to turnover of labelled dopamine or to a reduction in pool size over this period. If the medium K f was raised to 56+ mM during the last 10 min of incubation in amphetamine there was a significant depression in the release of labelled dopamine measured at 20min (Table 3). This contrasts with the increase in

TABLE2. THE FORMATION

OF DOPAMINE AND ACID METABOLITES BY SYNAPTOSOMES DURING INCUBATION IN RADIOACTIVELY LABELLED DL-DOPA

nCi/lM) mg protein Spaprosome bedl dopamine Incubation mrdivm dopamine dihydroxyphenyl acetic acid homovanillic acid unidentified acid metabolite

535.2 ? 24.3 (4)

52648

nmol/lM)mg protem

Spccific radioactivity nCihmol

2089 f 2.1 (4)

25.6

k 61.4(9)

k 607(6) 297.28 ? 45-5 (6) 317,08

139.82 k l0.0(5)

Synaptosome beds were incubated in Krebs-bicarbonate medium containing 10 mM glucose, 0.5 m M L-ascorbate and 3.9 ,UM ~~-3,4-dihydroxyphenyl-[2-'~C]-alanine (51 nCi/nmol) for 40min at 37°C. At the end of incubation the synaptosome bed was removed from the medium and extracted separately into 0.4NPCA (3 ml) containing 0.1% sodium metabisulphite. The incubation medium was added to 2N PCA to give a final concentration of 04N PCA containing 0.1% sodium metabisulphite and 0.1 mM dopamine. The values are means S.E.M.S with the number of experiments in brackets. These figures refer to pre-incubation conditions in the presence of the labelled precursor whereas all subsequent figures and Tables refer to values obtained following the final incubation.

+

566

J. S. DE BELLEROCHE, H. F BRADFORDand D. G . JONES

roxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) as shown from both in viva and in oitro studies (RUTLEDGE & JONASON, 1967; BREESEet a/., 1969; SPANO& NEFF,1972). As shown in the present study by their incorporation of radioactivity from labelled DOPA, these two compounds are also formed I amph in synaptosomes. The major metabolite formed from labelled DOPA which was recovered both in synaptosomes and their incubation media chromatographed with the R , of dihydroxyphenylacetic acid. This was distinct from homovanillic acid and was not a product of decomposition during extraction and fractionation procedure. DOPAC was predominantly recovered from the incubation medium (20min) where it was 7 times more concentrated (in terms of nCi) than the radioactively labelled dopamine in this fraG tion. In the synaptosome bed, however, radioactive DOPAC was a minor component being present to one-third of the extent of labelled dopamine. The pattern of distribution is shown by the chromatograms Svnaotasomes of extracts of these metabolites from tissue and incubation medium (Fig. 6). FIG. 4. Synaptosome beds were incubated at 37°C for Potassium stimulation caused a significant decrease 40 min in Krebs-bicarbonate medium containing 10 m ~ - (52%) in the ratio of DOPAC (nCi):dopamine (nCi) glucose, 0 5 mM-L-ascorbate and 3.9 ~~-3,4-dihydroxy- present in the medium and in synaptosome beds phenyl-[2-14C]alanine (51 mCi/mmol). The beds were then (34%)(Table 5). This was seen primarily as a decrease transferred to fresh medium without DL-DOPAand incuin ['4C]DOPAC, since the dopamine levels were unbated for a further 20 min at 37°C. The incubation medium changed by K + stimulation (Fig. 3). The reduction contained d- or I-amphetamine (012 mM) as indicated. The method of stimulation, extraction and expression of results of [14C]DOPAC presumably occurs by an increase was as indicated in the legend to Fig. 3. * Denotes that in its breakdown, since no parallel increase in the the total radioactively labelled dopamine (release + synap- dopamine pool occurred which would indicate a retosomes) is significantly increased by the presence of d-am- duced DOPAC formation. No radioactive derivative phetamine (control) compared to in its absence with with the DOPAC carbon skeleton was recovered P < 0.05. the extracts to substantiate this. However, isotopically Radioactively labelled dopamine

from DL DOPA

Doparnine release in d amphetamine

total dopamine release which occurred under these

conditions (Fig. 2) and again indicates a differential effect on labelled and unlabelled dopamine pools. However, after longer periods of incubation in amphetamine (3MO min) the amount of labelled dopamine released by K + stimulation approached that released in the controls (Table 3). The dopamine pool released by K', thus, had a different rate of turnover from that released by amphetamine. The influence of calcium. Since there was no difference in the release of labelled dopamine in calciumfree medium (Table 4), the process responsible for this release has no calcium dependence. This contrasts with the dopamine releasing effect of amphetamine which was significantly reduced ( P < 0.02) in the calcium-free medium. In contrast to the control condition in calcium-free medium, potassium now showed a dopamine releasing effect. There were no significant changes in the levels of isotopic dopamine present in synaptosomes incubated in calcium-free medium under the 4 conditions studied in Table 4. Metabolites formed fiom radioactively labelled DOPA. Dopamine is catabolized in the brain principally by oxidation to the acid metabolites 3,4-dihyd-

C

+ a 400-

006mM

10

20

30

Time,

40

50

60

min

FIG. 5. Synaptosome beds were incubated at 37°C for 40 min in Krebs-bicarbonate medium containing 10 mglucose, 0.5 mM-L-ascorbate and 3.9 phi-dihydroxyphenyl-[2-14C]alanine (51 mCi,'mmol). The beds were then transferred to fresh medium containing d-amphetamine as indicated and no DL-DOPA.The incubation was carried out for times between 20 and 60 min. At the end of incubation, medium (5 ml) was extracted into 2 hi-perchloric acid (1.2 ml) containing 0.1% sodium metabisulphite, 0.1 mdopamine and 0.1 mwnoradrenaline. The S.L.M. are indicated as bars with the number of experiments beside.

Dopamine and amino acids in striatal synaptosomes

TABLE3.

DOPAMINE RELEASE FROM SYNAPTOSOME BEDS INCUBATED IN THE PRESENCE OF d-AMPHETAMINE ~

Incubation time (min)

Dopamine nCi/100 mg protein Control K + stimulation

0 1 2 mM d-amphetamine

20 30 40 60

338.5 i 29.2 (4) 400.5 f 34.7 (3) 421.6 & 3 6 5 (9) 5766 f 500 151

222.6 t 24.9 (5)' 400.5 f 101.8 (3) 436.9 f 47.3 (9) 596.5 f 80.2 (41

Synaptosome beds were incubated for 40 min at 37°C in Krebs-bicarbonate medium containing 3.9 PM DL-DOPA2-14C (51 mCi!mmol) and 10 mM glucose. The beds were then transferred to fresh Krebs-bicarbonate medium containing 1 0 m M glucose and d-amphetamine (0.12m M ) and incubation was continued for the times indicated (20-60min). Potassium stimulation was by adding K+ to give a final concentration of 56 mw 10 min before the end of incubation. Values are means f S.E.M.S for the number of experiments in brackets. * Value significantly smaller than control, 0.01 < P < 0.02.

561

special significance in the biochemical properties of striatal synaptosomes is their ability to synthesize and degrade dopamine, noradrenaline and their metabolites, showing the presence of organized multienzyme systems for these purposes. The work reported here shows that the now wellestablished metabolic response to depolarizing stimuli shown by synaptosomes from other brain regions extends to synaptosomes from the corpus striatum. In addition to the preferential release of putative amino acid transmitters that occursI we have shown here that this preparation also releases dopamine. Stimulus-induced release of dopamine. Both electrical pulses and 56 m - K t induced the release of dopamine. In the presence of amphetamine (d or I), there was a large increase in dopamine release from synaptosome beds and a decrease in total dopamine recovered with d-amphetamine (0.12mM). The latter is consistent with data from striatum in uiw (JAVOY et al., 1968) and from striatal slices (BENSON et al., 1969), where amphetamine caused decreased synthesis. These effects may be due directly or indirectly to the inhibitory effect produced by amphetamine on tyrosine hydroxylase in striatal synaptosomes (PATRICK & BARCHAS,1974). Synthesis of labelled dopamine. Both isotopic L-tyrosine and DL-DOPAprovided rapid labelling of synap tosomal dopamine, the minimum rates estimated being comparable to in viuo rates. The more rapid synthesis of dopamine from DOPA rather than tyrosine was expected since the use of DOPA avoids the involvement of the rate limiting enzyme tyrosine hydroxylase. Analysis of the radioactively labelled

labelled carbon dioxide was found to be evolved during incubation of synaptosomes with 3,4-dihydroxyphenyl-[2-'4C]alanine (Table 6) but not with 3,4-dihydro~yphenyl-[3-'~C]alanine. This labelled carbon dioxide is likely to arise from decarboxylation of DOPAC and represents a significant pathway of DOPAC degradation. In control incubations in the absence of calcium, the levels of labelled DOPAC fell substantially as shown by the ratio (Table 5) which fell to half its original value. Since the labelled dopamine showed no change, this indicates an increased rate of DOPAC breakdown in calcium-free medium. The effect of potassium stimulation on labelled DOPAC levels was much reduced. When d- and 1-amphetamine were present both labelled dopamine and labelled DOPAC TABLE4. THE EFFECT OF ABSENCE OF CALCIUM ON SYNAPTOincreased their levels in the medium. Since the ratio SOMAL LEVELS OF DOPAMINE A N D ITS RELEASE TO THE INCUBATION MEDIUM increases (13- and 14-fold respectively) far more than the dopamine counts (which increase 5- and 2.5-fold Dopamine nCiilOOmg protein Calcium free respectively), this shows a greatly accelerated breakCalcium medium containing down of DOPAC. Although there are no data availcontaining 0 5 mu EGTA medium able to decide the question, it is possible that the constancy of the counts in the dopamine pool could Release to the medium 829 k 10.1 (8) 6 8 0 f 6 0 (? be achieved by rapid resynthesis of dopamine. Unlike KNo+ additions stimulation 91.4 k 167(9) 102.8 I2-0(8)t dopamine itself, the metabolites DOPAC and HVA 0119 mM d-amphetamine 338.5 i 29.2(4) 258.6 k 204(9)! 19 mM d-amphetamine,K' formed from labelled dopamine are largely recovered 01stimulation 2226 *24-9(5)* 213.0 t 152(9r in the medium. This is particularly well seen following Synaptosome beds were incubated for 20min at 37°C incubation in the medium used for the radioactive Krebs-bicarbonate medium containing 10 m M glucose, preincubation period in DL-DOPA(40min). A third in (see legend after 40 min pre-incubation in DL-DOPA-~-'~C acid metabolite was also recovered in this medium to Table 3). K t stimulation was by adding KC1 to give (see Table 2). The production of [I4C]DOPAC and a final concentration of 56 mM, 10 min before the end of its release to the medium in the presence of d-amphet- incubation. Values are means +_ S.E.M.S for the number amine increased with time (Fig. 7), and even at 60 min of experiments in brackets. *Denotes a significantly decreased release due to K+ showed no plateau-effect. (both in the presence of d-amphetaminc), @Of < P < 0.02. DISCUSSION

Synaptosomes isolated from sheep corpus striatum show the same intact morphological appearance and equivalent enrichment to those isolated from other brain regions. They also display high rates of respiration and a wide range of metabolic activities. Of

t Denotes that release due to K + stimulation (calcium free medium) is significantly increased when compared to incubation with no additions (calcium-free medium) with P < 0.01. 1Denotes that release due to 0.119 mM d-amphetamine in calcium-free medium is significantly decreased when compared to release due to 0 . 1 1 9 m M d-amphetamine in calcium containing medium, 001 < P < 0 . 0 2 .

J. S. DE BELLEROCHE, H. F. BRADFORD and D. G. JONES

568

precursor, suggesting that the events accompanying depolarization by electrical pulses can modulate the activity of tyrosine hydroxylase. Elevation of the pool sizes of other putative transmitters including ACh and glutamate has been shown t o occur following stimulation of cortical synaptosomes (DE BELLEROCHE &

DOPAC

BRADFORD,1972a,b). Release of labelled dopamine. During incubation of synaptosome beds following radioactive labelling of the dopamine pool, radioactively labelled dopamine was found to accumulate in the medium. The proportion of labelled dopamine recovered here was considerably @fold) larger than the unlabelled dopamine TABLE6. T m CUBATED IN

EVOLUTION OF 14C02 BY SYNAPTOSOMES INTHE PRESENCE OF 3,4-DMYDROXYf'HENYL

[2-14c]ALANINE ''COi (d p.m.1

FIG. 6. Radioactive analysis of extracts of synaptosomes and their incubation media. Three representative patterns of Zeocarb 225 cation exchange chromatographic separ(DOPA) ation of ~~-3,4dihydroxyphenyl-[2-'~CJalanine and the two major products formed by synaptosomes, dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC). Elution is from acid to basic buffer (right to left) and the peaks are monitored by serial analysis by liquid scintillation counting. (a) Incubation medium from 20 min incubation of synaptosome bed. (b) Incubation medium from 20min incubation of synaptosome bed in 0.12 mM-d-amphetamine. (c) Synaptosome extract from 10 min incubation. Conditions of incubations are described in the legend to Fig. 4,for the initial preincubation period in the presence of radioactively labelled DL-DOPA. products of synaptosomes showed that dopamine and DOPAC were the major metabolites, labelling of other catecholamines being insignificant. Electrical pulses caused a 35% increase in the total labelled dopamine content when L-tyrosine was used as precursor, an effect which was not seen with L-DOPA as TABLE5. THERATIO OF

Control No additions No Ca", 0.5 mM EGTA

d-amphetamine 0-119 m~ d-amphetamine 0 1 1 9 m ~ No . Ca" 0.5 mM EGTA I-amphetamine 0.119m~ Apomorphine hydrochloride 01 mM

NO

Synaptosomes + 3.4-dih ydrox yphenyl -[2-"CJ-alanine No tissue + 3.4-dihydroxyphenyl -[2-i*C]-alanine Synaptosomes + 3.4-dihydroxyphenyl -[3-'4q-alanine No tissue + 3,4dihydroxyphenyl -[3-"q-alanine

0341 f 0043 (8) 0154 f 0.009(4)

0225' f OW7 (7) 0.148 f 0009 (4)

-

-

0145 f 0.023 (4)

0161 f (>022(4)

-

-

0,274 f 0049 (2)

0.187 f 0028 ( 2 )

HCI added at the end of incubation

1424 i 54 (8)'

1781 f 1.5616)'

173 f 37 (3)

261 i 38 (41

505 k 115 (4)

217 f 6(4)

123 & 8(4)

181 f 8(3)

Synaptosomes were incubated in Warburg flasks for 60min at 37°C in Krebs-phosphate medium containing lOmM glucose, 1 mM NaHCO,, 0 5 mM Lascorbate and 3.9 PM 3,4dihydroxyphenyl acetic acid containing either 3,4-dihydro~yphenyl-C3-'~C]-aIanineor 3,4-dihydroxyphenyl-[2-14C]-alanine (51 nCi/nmol). Krebs-phosphate medium was also incubated without tissue. Hyamine soaked wicks used to trap evolved COz were removed at the end of incubation (No HCI) or after addition of 0.1 ml 1 . 0 HCl ~ to the flask (HCI added) and a further 10min incubation. Values are means f S.E.M.Sfor the number of experiments in brackets. * Denotes significantly greater than 'no tissue' control P < 0001.

RADIOACTIVE LABELLING IN DOPAC Ratio: nCi DOPAC/nCi Dopamine

Synaptosomes K + (54mm) stimulation

na

% decrease with K +

TO THAT IN DOPAMINE

Released to the medium K (56 mM) Control stimulation

":change with K'

7.25 k 1.63(12) 3.02 i 0.29(7).

3.48. 0-47(8) 2 l l t f 031 181

- 52 - 30

056 i 012(3)

044 i 0-18(3)

+ 50

NS

070&0l6(101

0~62i0-08(10)

NS

1.87 i 0.05 (2)

1.52 k 0 1 8 ( 2 )

NS

32

3 37 i 0.45 (4)

2.71 f 0.01 (2)

- 20

34 NS

Synaptosome beds were incubated for 20 min at 37°C in Krebs-bicarbonate medium containing 10 mM glucose, after 40min pre-incubation in DL-DOPA [2-'*C]. See Legend to Table 4. Values are means f S.E.M.Sfor number of experiments in brackets. Values significantly smaller than controls with *P < 002, t P < 0.1.

Dopamine and amino acids in striatal synaptosomes

Time,

min

FIG.7. Radioactively labelled DOPAC present in synaptosome beds and released to the medium during different times of incubation in d-amphetamine. Synaptosome beds were incubated at 37°C for 40min in Krebs-bicarbonate medium containing 10 mwglucose, 0 5 mM-L-ascorbate and 3.9 p~-dihydroxyphenyl-[2-’~C]alanine (51 mCi/ mmol). The synaptosome beds were then transferred to fresh medium containing 0.12 mM-d-ampheMmine and no isotope. Jncubation was carried on for 20-60 min periods of incubation. Potassium stimulation was by adding KCI to raise the medium K’ concentration from 6 to 5 6 m ~ , 10 min before the end of incubation. DOPAC (nCi) present in the synaptosome beds is indicated by t---.for confor K + stimulated samples. trol values and A-A DOPAC released to the medium is indicated by e-p-0 for control samples and A--.-A for K+ stimulated samples. Values are means from 4 9 experiments and S.E.M.are less than 10%.

recovered in separate experiments under similar conditions. This shows that the labelled dopamine was preferentially released to the medium. This ready release of newly-synthesized transmitter is now a common observation and has been shown for NA (KOPIN et al., 1968) and ACh (COLLIER,1969; POTTER, 1970). It provides convincing evidence for compartmentation of the synthesis and storage of these transmitters. It is therefore rather striking that depolarizing agents which were clearly affecting metabolism in the normal manner caused no detectable increase in the release of the substantial pool of labelled dopamine. Compartmentation of dopamine. Although labelled dopamine appeared to be readily released during incubation and handling of control samples, the application of depolarizing stimuli induced release from an unlabelled pool. The separate dopamine pool released by these agents is either a very rapidly labelling pool with short turnover time or one that has not become labelled by the time of release. The former explanation, which required the presence of adequate non-isotopic precursor (e.g. tyrosine) is most

569

consistent with the observations, firstly, with respect to the influence of electrical pulses in producing detectable elevation of rates of synthesis during their application. Secondly, the turnover rate of the small functional (i.e. releasable) dopamine pool in the cat caudate nucleus in vivo as determined by JAVOY & GLOWINSKI (1971) was rapid and found to be 69 nmol/g/h which represented 23% of the total dopamine. Since the in zivo average value for tyrosine hydroxylase activity (29 nmol/g/h) was comparable to the value in the present experiments, one could expect it to take 4min or less for the ‘functional pool’ in synaptosomes to be formed from the unlabelled tyrosine. In the present study, during the 20min incubation following an acute labelling period of Mmin, the activity of the rapidly turning pool would be expected to result in the synthesis of only unlabelled dopamine. The residual pool of labelled precursor (DOPA) present in the synaptosomes (Fig. 6) therefore appears to be separate from the metabolic pathway involved in the stimulated release. Similar patterns of compartmentation have been shown in more intact systems such as sympathetic ganglion, where the acetylcholine pool (‘surplus ACh’) labelled from superfusion with [3H]choline is not released by prolonged nerve stimulation, in medium containing unlabelled choline (approx 15 p ~ ) ,although unlabelled ACh is released. The labelled ACh does however contribute to the spontaneous ACh & KATZ, 1971). output in eserinized solution (COLLIER Similarly, hemidiaphragm of rat when charged with labelled ACh and then superfused in medium containing unlabelled choline releases non-radioactive ACh more rapidly than labelled ACh on stimulation (POTTER, 1970). The same conclusions about the release of the most recently synthesized ACh upon stimulation have also derived from experiments on superfused rabbit cerebral cortex (CHAKRINet al., 1972) and electric organ of Torpedo (DUNANT et al., 1972). It is apparent from the above mentioned pulse labelling experiments that synthesis and release of non-isotopic dopamine and ACh does occur, even though a plentiful supply of labelled transmitter and labelled precursor are present intracellularly. The ACh (COLLIER & KATZ, 1971) or dopamine (see above) released by nerve impulses in the former case or depolarizing stimuli in the latter is apparently replaced by synthesis at the site of storage and not by movement from the labelled pool. Thus, this ‘newly synthesized’ pool is released before equilibration with the preformed stores and may represent a pool formed either in tissue other than dopaminergic terminals or formed in a distinct intrasynaptosomal compartment. In the experiments of COLLIER & KATZ(1971), the amount of ACh released by stimulation was not altered by ACh esterase inhibitors of varying effectiveness, in whose presence the ‘surplus’ ACh was detected, which shows that this ACh was not available for breakdown by intracellular esterases. A parallel

570

J. S. DE BELLEROCHE, H. F. BRADFORD and D. G. JONES

may be drawn with respect to dopamine, Since the showed that more than 77% of the total was redopamine release by stimulus is not sensitive to Or covered in the medium (20 min incubation). During available for breddown whereas the ‘surplus’ labelled depolarization total counts found in DOPAC were pool is rapidly oxidized to the acid metabolites. In diminished by 44% (calculated from data on Tables cat caudate nucleus continuously superfused With 3 and 4) whilst there was no comparable rise in the [3H]tyrosine, a pulse of K + ( 3 0 m ~ )leads to in- counts recovered in dopamine. However, the evolucreased release of C3H]dopamine, but the effect is aP- tion of labelled carbon dioxide from 3,4-dihydroxyparently partly obscured if an acute labelling tech- phenyl-[2-I4C]alanine during incubation with synapet al., 1971). These authors were tosomes showed that a pathway of DOPAC degradanique is used (BEESON not able to demonstrate a consistent increase in re- tion by decarboxylation existed. The likely product lease of labelled dopamine following electrical stimu- of DOPAC decarboxylation is dihydroxybenzoic acid lation of substantia nigra. This further illustrates the which has been demonstrated in human urine followdifficulty in labelling the small pool that rapidly turns ing intravenous infusion of DOPAC (ALTON & GOODALL,1969). Diversion to this pathway particuover. Eflect of amphetamine. The presence of d- and I-am- larly during depolarization, therefore, appears to be a phetamine produced large increases in the accumu- new alternative catabolic route in brain. Synaptosomal amino acid pools and release. The patlation of dopamine in the medium, up to 35% of the total dopamine being released during 40 rnin incuba- terns of release of glutamate, GABA and aspartate tion (Fig. 2). The difference in potencies of the two in response to 56mM-K+ were similar to those obisomers was most obvious in their effects on radioac- tained from cerebral cortex synaptosomes. The pool tively labelled dopamine, where the d-isomer was ap- sues of amino acids in corpus striatum synaptosomes prox 3-4 times more potent. These differences in po- compared to those derived from cerebral cortex (DE tency parallel those obtained for dopamine accumu- BELLEROCHE & BRADFORD,1972a, b) did however lation in vim as mentioned above and in particular show some differences: GABA, aspartate and glutaparallel the potencies in inhibition of dopamine up- mine were considerably more concentrated than in take by amphetamine in striatal synaptosomes (FER- the cortex being increased by 160, 104 and 89% reRIS et a[., 1972; HARRIS & BALDESSARINI, 1973). From spectively. Glycine and glutamate were increased 54 this one could speculate that amphetamine was oper- and 49% respectively relative to cortex and serine and ating largely as an inhibitor of dopamine uptake alanine showed no difference. rather than as a releasing agent which is the more usual explanation of its action. Certainly the dopa- Acknowledgements-We would like to thank the M.R.C. mine uptake system of synaptosomes would be suffi- for financial support in the form of a Programme grant. ciently potent to remove dopamine from the medium in the absence of an uptake inhibitor since a V,, REFERENCES of 300nmol/g/h ( K , = 0.1 ,UM) has been obtained for a synaptosome preparation from rat corpus striatum ALTON H. & GOODALLMcC. (1969) Biochem. Pharmac. 18, 1373-1379. (HOLTZ& COYLE, 1974). ANTON A. H. & SAYRED. F. (1962) J. Pharmac. exp. Ther. Specific activities of dopamine pool. Under the pres138, 36&375. ent experimental conditions. the specific activity of AZARRO A. J. & RUTLEDGEC. 0. (1973) Biochem. Pharmac. dopamine in the synaptosome bed (40 min incubation 22, 2801-2813. in DL-[14C]DOPA, followed by 20 min further incu- BALDESSARINI R. J. & KOPINI. J. (1966) Science, N.1: 152, bation without DL-DOPA) only reached 35% of the 1630- 1631. A., FELTZP. & GLOWINSKI J. precursor value. However, the dopamine released in BEESONM. J., CHERAMY (1969) Proc. natn. A c d . Sci. U.S.A. 62, 741-748. the absence of depolarizing stimuli or in the presence A., FELTZP. & GLDWINSKIJ. of amphetamine was almost equivalent to that of the B m i x M. J., CHERAMY (1971) Brain Res. 32, 407424. precursor. This can be seen from the release of isoM. ,P.,JOHNSON E. M., JR. & NEEDLEMAN P. topic dopamine in the presence of amphetamine BLAUSTEIN (1972) Proc. natn. A c d . Sci. U.S.A. 69, 2237-2240. (40 min) in Fig. 5, where all the dopamine of this pool BRADFORD H. F. (1969) J . NeuroEhem. 16, 675-684. has been synthesized from the radioactive precursor BRADFORD H. F. (1970) Brain Res. 19, 239-247. and amounts to 8.3 nmoljl00 mg protein as calculated BRADFORD H. F. (1974) Biochem. Soc. Ems. 2, 680-682. from the specific activity of the precursor. This com- BRADFORD H. F. & THOMAS A. J. (1969) J . Neurochem. pares with the value of 7.6nmolj100mg protein for 16, 1495-1504. C., NIELSENM. & SCHEEL-KRU~~ER J. (1974) the non-isotopic dopamine released under these con- BRAESTRUP J. Neurochem. 23, 569-579. ditions and measured fluorometrically. These results show the presence within the bed of a dopamine pool BREESEG. R., CHASET. N. & KOPINI. J. (1969) J . Pharmac. exp. Ther. 165, 9-1 3. of specific activity comparable to that of the precurR. M., MITCHELL J. F. & sor, in addition to a pool which labels to a much CHAKRINL. W., MARCHBANKS WHITTAKER V. P. (1972) J. Neurochem. 19, 2727-2736. smaller extent. CHIUEHC. & MOOREK. E. (1974) Res. Commun. Chem. Dnpamine metabolites and depolarization. The distriPath Pharmac. 7, 189-199. bution of DOPAC, the major metabolite of dopamine COLLIER B. (1969) J. Physiol., L.ond. 205, 341 352. 9

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