Journal of Neurochemistry, 1977. Vol. 29. pp. 335-343. Pergarnon Press. Printed in Great Britain.
O N THE SITE OF ORIGIN OF TRANSMITTER AMINO ACIDS RELEASED BY DEPOLARIZATION OF NERVE TERMINALS I N VITRO J. S. DE BELLEROCHE' and H. F. BRADFORD Biochemistry Department, Imperial College, London S.W.7. U.K. (Received 17 November 1976. Revised 18 February 1977. Accepted 25 February 1977)
Abstract-The site of origin of transmitter amino acids released by depolarizing agents from nerve endings was studied. The model used was the incubated and depolarized synaptosome preparation from which the component soluble, synaptic vesicle, membrane and mitochondria1 sub-fractions were obtained. Synaptosomal amino acids were radioactively labelled from ~-[U-'~C]glucosein viuo by intraventricular injection and in uitro during subsequent incubation. The specific radioactivities of amino acids released in response to K' (56 mM) or veratrine (75 p ~ were ) found to closely resemble those of the soluble cytoplasmic fraction, in most cases differing significantly from those of the other fractions. The specific radioactivity of the GABA and aspartate released by K + stimulation and the GABA and glutamate released by veratrine were significantly different from that of the vesicles in each case. The specific radioactivities of glutamate released by both agents, and also GABA with K' stimulation, were approximately double that of the amino acid released in control conditions. Depletion of the soluble cytoplasmic pools of glutamate, GABA and aspartate occurred following stimulation, corresponding to the induced-release of these compounds. Turnover of the amino acids in the other subfractions was too low to account for their participation in the release process in addition to the soluble cytoplasmic pool. A cytoplasmic origin of release of neurotransmitter amino acids from nerve endings is proposed.
IN SPITE of clear evidence for quanta1 release of trans- amino acids released by depolarizing agents was used mitter at certain CNS synapses (KATZ & MILEDI, as an index of their site of origin. 1963; KUNO,1964; WEAKLY,1969) direct evidence implicating synaptic vesicles in this process has not been METHODS forthcoming and the purpose of the present investigaIntrauentricular injection of D-[U-'4C]ghcoSe tion was t o localise the subcellular origin of released Eight female Sprague-Dawley rats (22W250 g) were used transmitter. A synaptosome preparation from cerebral per experiment. These were anaesthetized with diethyl cortex was the in vitro system chosen for the study. ether-air and the junction of the coronal and sagittal It is now well established that a selective release of sutures of the skull was exposed to use as a reference point. endogenous and preloaded transmitter amino acids A molded Perspex guide was used for injection into the is induced by treatment of synaptosomes with such lateral ventricles. The injection was made at a point 1 mm depolarizing stimuli as electrical pulses, potassium posterior to the coronal suture and 2mm lateral to the and veratrine. Thus, glycine is released from spinal sagittal suture and on one side only. A microlitre syringe cord synaptosomes and glutamate, GABA and aspar- and needle with a nylon stop were used to inject to a tate from cortical synaptosomes by calcium-depen- depth of 3.5-4mm. Injection of 12.5pCi of aqueous ~-[U-'~C]glucose(283 mCi/mmol) containing 3% ethanol 1970; DE BELLEROCHE dent mechanisms (BRADFORD, & BRADFORD, 1972; OSBORNE et a!., 1973; REDBURN was made in a volume of 50 pl. Rats recovered from anaes& COTMAN,1974). This in vitro stimulus-coupled thesia 3 4 min after injection and were killed 30 min later. The brains were removed and the cerebral cortices placed release is blocked by agents shown t o prevent transin 0.32 M-sucrose. Although injection was into one hemimitter release in vivo. Thus tetanus toxin blocks gly- sphere only, it was shown that both ventricles became filled et al., 1973), tetro- with injection fluid by the use of aqueous methylene blue. cine and GABA release (OSBORNE dotoxin prevents noradrenaline release (BLAUSTEIN et al., 1972) and botulinum toxin prevents acetylcholine Preparation and incubation of synaptosomes & MARCHBANKS, 1976). These release (WONNACOTT Synaptosomes were prepared by the method of GRAY and other properties together form a strong case for & WHITTAKER (1962) as modified by BRADFORDet al. considering synaptosomes a valid in vitro model for (1973). Synaptosomes (40 mg) were suspended in Krebs studying neurotransmitter release mechanisms. In the bicarbonate medium (7.5 ml) of composition: (mM); NaC1, experiments reported here different degrees of radio- 124; KCI, 5: Na2HP04, 1.2; MgSO,, 1.3; CaCI,, 0.75; active labelling of transmitter amino acids in vesicles, NaHCO,, 26; pH 7.5 containing 10.3mM-glucose cytoplasm and other synaptosome compartments (0.934pCi/pmol) and gassed with 95% 0,/5% CO,. Incuwere established, and the specific radioactivities of bation was at 37°C for periods of up to 20 min as indicated 335
J. S . DE BELLEROCHE and H. F. BRADFORD
in the legends. Potassium stimulation was applied after an initial 10 min incubation, 1.0 M-KCI,made up in Krebsbicarbonate medium, being added to give a final concentration of K + of 56 mM. An equivalent volume of Krebsbicarbonate medium was added to the controls. Veratrine stimulation was carried out by adding 75 pl 7.5 mM-Veratrine (Sigma, London) after an initial 10 min incubation. This preparation contains a mixture of cavadilline, sebadine. cevadine and veratridine. Veratrine was made up in 30% ethanol and an equivalent volume (75~1)of 30% ethanol was added to the controls. Incubation of synaptosomes was terminated by centrifugation of the synaptosome suspension st to00 g for 5 min. The supernatant was added to 2 ~ - P C A to give a final concentration of 0.4 N-PCA.
added to the PCA extracts. Precipitated protein was sedimented by centrifugation at lOOOg for 10min at 0°C and the extracts were used for amino acid analysis. Preliminary purification of amino acids was carried out by cation exchange chromatography using Zeocarb 225 (BRADFORD & THOMAS, 1969). Fractionation and quantitation of amino acids and their radioactivity was carried out by means of a modified automated amino acid analyser, the details of which are described elsewhere (DE BELLEROCHE et al., 1976). The samples were applied to a column of Zeocarb 225 (8-10 pm beads), eluted with a programmed gradient and the eluate stream split, part (33%) being analysed
Preparation of synaptic uesicles
The sedimented synaptosomes were hypo-osmotically ruptured with 5 rnM-Tris-EDTA, pH 6.5 (5 ml/g starting material; 5 m1/10 mg synaptosome protein) as described by MARCHBANKS (1968). The method used for the preparation of synaptic vesicles was a modified form of that used by WHITTAKER and SHERIDAN (1965). The osmotically ruptured synaptosome fraction was pipetted onto a discontinuous sucrose gradient (1.6/0.6/0.4/0.2M-sucrose), using the additional 0.2 M sucrose layer to separate the soluble cytoplasmic components in the Tris-EDTA region of the gradient from the vesicle fraction in the 0.4 M-sucrose region (MARCHBANKS, 1968). The gradient was centrifuged at 75,000 g for 2 h. At the end of centrifugation, the different regions of the gradient were removed with a Pasteur pipette according to the scheme indicated in Figure l a and extracted into 0.4 M-PCA (final concentration). The question of whether there was any contamination of the vesicle fraction by diffusion of free amino acids was checked by placing an amino acid mixture onto similar sucrose gradients in the presence or absence of tissue and centrifuging as above. In the absence of tissue, a small degree of contamination of all fractions occurred to a similar extent due to diffusion from the soluble fraction (Fig. Synaptic I Light I lc). This was less than 0.5% for the vesicle fraction. When membrane I memb- Vesicle Soluble I I fraction /mitocho- I rane an animo acid mixture (20 nCi ~-[U-'~C]asparate ndria I I (100 mCi/mmol), 30 nCi ~-[U-'~C]glutamate (125 mCi/ I I I mmol) and 10 nCi L-[U-'~C]GABA (224 mCi/mmol); enough to double the free amino acid concentration) was added to a lysed synaptosome extract in 5 mM-Tris-EDTA, FIG.1. Distribution of [U-'4C]amino acids on a disconp.H. 6.5 and stood at W C prior to centrifugation on tinuous sucrose gradient used to prepare synaptic vesicles. the sucrose gradient, a very similar pattern was obtained. Figure l(a) shows the scheme of sucrose concentrations In three experiments the percentage contamination of the used for preparation of synaptic vesicles. Synaptosomes vesicle and light membrane fractions from the soluble frac- were incubated in Krebs-bicarbonate medium containing tion was found to be 0.55% (range: 0.45-0.67%) and 0.39% 10.3m~-~-[U-'~C]glucose (0.934 mCi/mmol) at 37°C for (range: 0.2&0.57%), respectively. These experiments 10 min. The synaptosomes were hyper-osmotically shocked showed that the amino acid present in the 0 . 4 ~layer in 5mM-Tris-EDTA, pH 6.5 and placed on the sucrose (vesicle fraction) after separation of synaptosome lysates gradient indicated above and this was centrifuged at was a significant portion, clearly distinguishable above the 75,000 g for 2 h. The distribution of [U-14C]aspartate and level of free amino acids present due to contamination. glutamate is shown in Fig. l(b), as percentage of total Morphological characterization of the synaptic vesicle counts in the 4 fractions defined by Fig. l(a). The values fraction prepared by this method has shown that it consists are means with the S.E.MS indicated by bars (n = 5). In almost entirely of 50 nm profiles with occasional extended Fig. l(c) the distribution of [U-'4C]aspartate and gluta& BRADFORD,mate is shown when similar sucrose gradients are loaded membrane fragments (DE BELLEROCHE 1973b). with an amino acid mixture containing 30 nCi [U-14C]aspartate (100 pCi/pmol), 30 nCi [U-'4C]glutamate (100 pCi/ Extruction and analysis of amino acids pmol), 130 nmol GABA and 25 nmol alanine in 5 mM TrisAn internal radioactively labelled amino acid standard EDTA, pH 6.5 and centrifuged and separated as above. (6-12 nCi [U-'4C]valine) and an internal standard for The values are means (n = 3) with the S.E.MS indicated by bars. fluorometric estimation (37.5-100 nmol norleucine) were
Origin of released transmitter
Crude mitochondria1 fraction
Synaptic membranes/ mitochondria
0 Aspartate IGlutamate LY GABA
FIG.2. Specific radioactivities of amino acids in fractions of cerebral cortex following intraventricular injection of [U-' 4C]glucose. Sprague-Dawley rats were injected intraventricularly with 12.5 pCi (50 pl) ~-[U-'~C]glucose(283 mCi/mmol). After 30 min the animals were killed and the cerebral cortices were removed and homogenised (whole homogenate). The homogenate was centrifuged at lo00 g for 10 min, the resulting pellet (nuclear pellet) was sampled and the supernatant was centrifuged at 17,000g for 20 min. The resulting supernatant (microsomal fraction) and pellet (crude mitochondrial fraction) were sampled and the latter was used to prepare synaptosomes. Synaptosomes were incubated in Krebsbicarbonate medium containing 10 mwglucose at 37°C for 20 min. The synaptosomes were then hypoosmotically shocked and the synaptosome subfractions were obtained as defined in Fig. 1. The protein content and specific radioactivities of amino acids in each fraction are shown as histograms. The values are means, the bars indicating the S.E.MS and the number of experiments is shown above the bars.
fluorometrically following reaction with buffered u-phalaldialdehyde/mercaptoethanol and the remainder (67%) was passed through the continuous flow cell of a liquid scintillation counter. Protein was analysed by the method of LOWRYet al. (1951) using bovine serum albumin as standard.
of glutamate and GABA being about 50% greater than those of aspartate. Radioisotopic labelling of synaptosomal amino acids by ~ [ U - ' ~ C ] g h c o s ein vitro
During incubation (up to 20 min) in D-[u-'4c]glUcose (0.934gCi/pmol), cortical synaptosomal suspenRadioisotopic labelling of synaptosomal amino acids by sions prepared from ~-[U-'~C]injectedbrains syntheintraventricular injection of ~-[U-'~C]glucose sised further radioactively labelled aspartate, glutaSpecific radioactivities of amino acids in the cere- mate, GABA and alanine (Fig. 3). Radioactively bral cortex homogenate and other subcellular frac- labelled aspartate, glutamate and GABA were found tions showed close similarities in pattern to pre- in all synaptosome sub-fractions. Alanine was only et present in the soluble fraction. The specific radioactiviously published data (CREMER,1964; GAITONDE a!., 1965; VAN DEN BERG, 1973). The synaptosomal vities of aspartate and glutamate in the soluble fracamino acids radioactively labelled by intraventricular tion, and of aspartate and GABA in the vesicle fracinjection of ~-[U-'~C]glucose(50 p1 per rat contain- tion showed a regular increase with time. In contrast, ing 12.5 pCi, 283 mCi/mmol) were glutamate, GABA vesicular glutamate and mitochondrial/synaptic memand aspartate (Fig. 2). All three radioactively labelled brane aspartate, glutamate and GABA showed a amino acids were found in the soluble fraction and more non-linear relationship with a sharp increase the synaptic membrane/mitochondrial fraction. occurring between 10 and 20 min. During this period Labelled glutamate and GABA were found in the of incubation the only significant change in pool size synaptic vesicle fraction. The specific radioactivities was an increase in glutamate in the vesicles and of of each amino acid were similar for all fractions, those aspartate in soluble and vesicle fractions (Fig. 4). RESULTS
J. S . DE BELLEROCHE and H. F. BRADFORD
FIG. 3. Specific radioactivities of amino acids in synaptosome subfractions and their increase with time. SpragueDawley rats were injected intraventricularly with 12.5 pCi (50 pl) ~-[U-'~C]glucose (283 mCi/mmol). The animals were killed after 30 min and the cerebral cortices were used to prepare synaptosomes which were then either sampled or incubated in Krebs-bicarbonate medium containing 10.3rn~-~-[U-'~C]glucose (0.934pCi/pmol) at 37°C for periods of 10 or 20 min. The synaptosomes were ruptured hypo-osmoticallyand the synaptosome subfractions prepared. The specific radioactivities of amino acids (pCi per nmol) in the fractions are means with the bars indicating the S.E.MS. The number of experiments was 4-6.
The effect of'depolarizing agents on amino acid release
Application of potassium (56 mM) or veratrine (75 p ~ increased ) the release of endogenous glutamate, GABA and aspartate from synaptosomes, both agents producing a 24-fold elevation in the levels found in the incubation medium (Fig. 5). The effects of the depolarizing agents on the specific radioactivities of labelled amino acids recovered in the incubation medium and in the synaptosomal subfractions were analysed and the specific radioactivities of amino acid released from the subfractions was estimated (Figs. 6 and 7). Stimulation with 56mM-K+ produced significant increases in the specific radioactivities of glutamate and GABA released compared with that released under control conditions, whilst veratrine increased only that of glutamate significantly. This indicated that stimulated release of glutamate, and possibly of GABA, was from a different compartment to that of the background release from the control. The specific radioactivities of the amino acid released to the medium by K t stimulation were equivalent to those of the soluble cytoplasmic amino acids (Fig. 6). The specific radioactivities of vesicular GABA and vesicular aspartate and those of the membrane fractions were significantly different from the
levels for the released amino acids. The specific radioactivity of vesicular amino acids was either higher (aspartate) or lower (GABA) than that of the soluble fraction, whereas the membrane fraction specific radioactivity was always greater than that of the soluble fraction. For glutamate, the specific radioactivities of the soluble and vesicle fractions were not significantly different. After exposure t o veratrine the specific radioactivities of glutamate and of GABA recovered in vesicles were significantly lower than the values for those amino acids recovered in the medium (Fig. 7). The soluble cytoplasmic pool showed specific activities closest to those of the released amino acid, being slightly larger than those of the mitochondria1 fraction. The specific radioactivity of aspartate released by stimulation was similar to that of the vesicle and membrane fraction. Veratrine did not significantly affect the specific activity of synaptic membrane/mitochondrial fractions indicating that part of the effect of K + could well be due to its known activation of pyruvate kinase (MCILWAIN& BACHELARD, 1971). The effect of depolarizing agents on size and turnover of amino acid pools
Both veratrine and K + stimulation caused substan-
Origin of released transmitter Asp
ments described in Fig. l(c) and the Methods Section, at least demonstrate that the amino acid pools attributed to vesicles are not present simply due to diffusion from other fractions of the preparative gradient. Further, since the vesicle fraction shows no propensity to take up labelled amino acids added to the lysate (Fig. l(c) and DE BELLEROCHE & BRADFORD, 1973b), it is likely to represent a pool that was present before rupture of the synpatosome. It remains possible, though unlikely, that a proportion of the amino acids were lost from the vesicles during preparation. The similarity of the specific activities of the soluble cytoplasmic amino acids to those released by stimujynaptic vesicles lation, together with the corresponding depletion of this pool, point to a cytoplasmic rather than a vesicular origin for the released amino acid. However, before the involvement of vesicles in the release process is discounted other evidence needs to be brought to bear. For instance, it was possible that the amino acids had passed into the vesicles from the cytoplasm before being discharged to the incubation fluid, and m that this cycle of vesicle filling and emptying was d I I I I I I I I I ~ accelerated by depolarization. However, when the .ighi membranesA data of Fig. 3 is converted into rates of turnover of T the vesicle pool it is seen that both the control rates and the rates during depolarization are too low by T Synaptic several orders of magnitude to account for the amounts of amino acid released to the medium (Table 1). Acceptable equation of the vesicle pool turnover with the extent of transmitter release in this way is strongly dependent upon there being a high recovery (yield) of the vesicles and their content by the method €-a h A of preparation employed. The yield of synaptosomal acetylcholine in vesicles prepared by this technique 10 20 10 20 I0 20 10 20 was found to be 70% (WHITTAKER, 1969), indicating that both vesicles and their content might be reTime, min covered in high proportion in the experiments FIG. 4. The effect of incubation on amino acid pools in reported here. A small percentage (3.7%) of the total synaptosome subfractions. Experimental conditions as in Fig. 3. Amino acids in the synaptosome subfractions were synaptosomal protein appeared in the vesicle fraction. analysed and related to the protein content of the subfrac- This yield would have to be very large before a single tion. The values are means, the bars indicating the S.E.M.S. emptying of the vesicle amino acid pool (1.94 x lo3%, or multiple emptying which is limited by the rate of The number of experiments was 4-6. filling 2.3 x lo5%), could be large enough to equal the quantity of transmitter amino acid released. tial decreases in the soluble cytoplasmic pool sizes Moreover, if the transmitter passed from cytoplasm of aspartate, glutamate and GABA. This effect with to vesicles before discharge, the specific activity of K f stimulation could account for more than 80% of cytoplasmic and vesicular amino acids should be the extra amino acid recovered in the incubation fluid identical, unless compartmentation exists within the (Fig. 8). Veratrine stimulation caused a greater de- cytoplasm. The data of Figs. 6 and 7 show that the crease in the soluble cytoplasmic pools of amino acid specific radioactivities of these two pools are in all (Fig. 9). Both agents caused small but significant cases significantly different. After treatment with veralosses of amino acid from the vesicle and membrane trine, the mitochondrial/membrane fraction contained fractions. some amino acids at specific radioactivities close to those of the released amino acid (e.g. GABA and aspartate, Fig. 7). The remote possibility that this DISCUSSION fraction is the source of released transmitter seems In a study of the distribution of soluble compounds to be contraindicated by the absence of a similar corfollowing osmotic lysis and centrifugation procedures, relation where potassium was the depolarizing agent the problem of redistribution during these procedures employed (Fig. 6). In this case the specific radioactivicannot be eliminated. However, the control experi- ties in the mitochondrial/membrane fraction are very
J. S. DE BELLEROCHE and H. F. BRADFORD
K t ond verotrine
m K + (56rn~)
ats were inFIG.5. Release of transmitter amino acid by- potassium and veratrine. Sprague-Dawle! . jetted intraventricularly with 12.5 pCi (SO PI) ~-[U-'~C]glucose(283 mCi/mmol). The animals were killed after 30min and the cerebral cortices were used to prepare synaptosomes which were then incubated in Krebs-bicarbonate medium containing 10.3mM-D-[U-'4C]ghCOSe (0.934pCi/pnol) at 37°C for 20 min (control). Stimulation was carried out by adding K + (56 mM) or veratrine (75 pM) after 10 min initial incubation and continuing for a further 10 min incubation. Final concentrations are shown in brackets. The values of amino acid release t o the incubation (nmol per 100 mg synaptosome protein) are means, the bars indicating the S.E.MSand the number of experiments is shown above the bars. The asterisk indicates that stimulated release of amino acid was significantly greater than the controls P i0.01. high due probably to the stimulation of pyruvate
kinase by K'.
0 Control K+ stimulcted Asporta te Soluble fraction
Mitochondrla / membranes n = 4-5
5 ~ 0
The general conclusion from the data presented above is that transmitter amino acids are released directly from synaptosomal cytoplasm during treatment with depolarizing agents, and that the vesicles do not appear to be involved as intermediate carriers in the process. It is assumed that each transmitter amino acid is being released from one sub-population of synaptosomes derived from neurones employing that
FIG.6. The effect of potassium stimulation on the specific radioactivities of amino acids released from the synaptosome subfractions. Experimental conditions as for Fig. 5. Specific radioactivities (nCi/pnol) of amino acids released to the incubation medium (release) were measured in control and K + stimulated conditions. Amino acid pool sizes ( p o l ) and radioactivity (nCi) in the synaptosome subfractions were measured after incubation under control or stimulated conditions and the specific radioactivities of the amino acid released by stimulation were determined (synaptosomal source) from the difference in pool size and radioactivity between control and stimulated paired samples. The values are means with the S.E.M.Sshown by bars. The black asterisk indicates that the specific radioactivity of amino acid released by K + stirnupation was significantly greater than the control released amino acid P i0.01. The unfilled asterisk indicates that the value is significantly different from that of the amino acid released to the medium by stimulation, P i0.01.
Origin of released transmitter
I Release I Synaptosomal source I I Glutomate I I
The effect of veratrine on synaptosomol ainiro acid pools S V
Soluble fraction Vesicles
M Mitochadria/membranes n = 5
0 Control IVeratrine stimulated Soluble fraction Vesicles
Mitochondria / membranes
FIG. 7. The effect of veratrine stimulation on the specific radioactivities of amino acids released from the synaptosome subfractions. Experimental conditions as for Fig. 6, using veratrine stimulation. Effect of K t on synaptosomol amino acid pools
S Soluble fraction v Vesicles L Light membranes M Mitochondria/membranes
,m [sip Alonine
FIG. 9. The effect of veratrine stimulation on amino acid release and loss from the synaptosome subfractions. Experimental conditions as for Fig. 6 using veratrine (75 PM) stimulation instead of K ' . The release of amino acid induced by veratrine stimulation is shown as histograms (R) with the decrease in pool size of amino acid in the synaptosome subfractions (S, V, M) shown by the side. The values are means (nmo1/100mg synaptosomal protein) with the bars indicating the S.E.M.S and the number of experiments was 5 .
transmitter at its terminals. The limits of the application of this conclusion to the cortical synapse in vitro are set (a) by the validity of synaptosomes as a model, and (b) by the extent to which the synaptosoma1 sub fractions prepared and studied here match the size and composition of the in situ structures from which they are derived. The validity of the synaptosome as an isolated working presynpase has been argued positively and in detail elsewhere (DEBELLEROCHE & BRADFORD, 19730; BRADFORD, 1975). Since amino acid and other transmitter release from synaptosomes is blocked by agents which prevent their release in viuo, it seems likely to be a good model (see introduction). As the yields of vesicles from synaptosomes appear to be in the expected range, and calculation shows that an unacceptably high proportion Aspartate GABA of the synaptosome would have to consist of vesicles FIG.8. The effect of potassium stimulation on amino acid for this pool to be commensurate with the amount release and loss from the synaptosome subfraction. Ex- released, this fraction does appear to be representative perimental conditions as for Fig. 6. The release of amino of its in situ counterpart. It remains possible that acid induced by K + (56mM) stimulation is shown as a amino acids leak from vesicles to a major extent durhistogram (R) with the decrease in pool size of amino acid ing preparation and are recovered in the soluble cytoin the synaptosome subfractions (S, V, L and M) by the plasmic fraction as suggested previously by MANGAN side. The decrease in pool size was obtained by subtracting (1966). However, this does not appear the value of the stimulated amino acid pool from the con- & WHITTAKER to be the case for acetylcholine, and the ability of trol value. The values are means (nmo1/100 mg synaptosoma1 protein) with the bars indicating the S.E.M.S and the vesicles (subsequently isolated) to show substantial increases in pool size during synaptosome incubation number of experiments shown by the bars. I
and H. F. BRADFORD J. S. DE BELLEROCHE
TAHLE 1. RATESOF
Synaptosome subfraction Glutamate VesicI es Soluble Membranes
A M I N O ACID TURNOVER IN SYNAPTOSOME FRACTIONS
Calculated rates of turnover of amino acids in fractions obtained from 100 mg synaptosomal protein Control K' Stimulated
673.2 x lo3 18.9 20.5
2.8 22.4 8.5
1.9 66.3 8.5
3.5 44.2 3.7
Aspartate Vesicles Soluble Membranes
2.9 77.9 17.8
2.1 21.3 4.9
K + stimulated release of amino acid from 100mg synaptosomal protein in 10 min
154.3 x lo3
Values were calculated for the final 10min of incubation during which K t stimulation was carried out, and are denved from the measurements of each rate of increase of specific radioactivity of amino acid given in Figs 3, 4 and 8, and represent 4 9 experiments Units (pmo1/100mg protein of incubated synaptosomes/lOmin)
(Fig. 4 aspartate and glutamate) provides evidence against this view. Since differences in specific radioactivities are among the key observations in this paper on which sites of origin of transmitter are based, the extent to which these differences are clear-cut needs comment. If cytoplasm and vesicles constitute clearly different compartments which are not directly functionally related, then large differences in the specific radioactivities of their amino acids might be expected. Our data did not give this picture, although the differences were both substantial and significant. Taken together with the additional data on vesicle pool turnover, considerable confidence may be given to assigning the site of transmitter release to the cytoplasmic compartment. One striking reason why the expected large, clear-cut differences were not seen may relate to recent demonstrations that incubated synaptosomes actively endocytose, and this process is greatly accelerated by depolarization as judged by uptake of horseradish peroxidase (COOKE r t ul., 1975; FRIED & BLAUSTEIN, 1976). Endocytosis must involve uptake of incubation medium and therefore also of compounds such as released amino acids which this would contain. Thus, part of the amino acid extracted from isolated vesicles may have come from the extracellular fluids by this process, having been recently released from cytoplasm. This would be consistent with the recent finding that stimulation of the Torpedo electric organ causes uptake of labelled dextran into a vesicle fraction which contains the highest acetylcholine specific activity (ZIMMERMANN, 1976), the latter being the recently released transmitter.
Acknowlrdgemrnts-This project was financed as part of an M.R.C. Programme Grant.
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(LAJTHAA. ed.) Vol. 2, pp. 327-364. Plenum Press, New York. V. P. & SHERIDAN N. M. (1965) J. Neurochem. WHITTAKER 12, 36S372. WONNACOTT S. J. & MARCHBANKS R. (1976) Biochem. J . 156, 701-712. ZIMMERMANN H . (1976) 1st Meet. Eur. SOC. Neurochem. Abstr. 6C.