Ac ta Physiol Scand 1979, 107: 205-212
Metabolic properties of nerve endings isolated from rat brain S.-E. J A N S S O N , M. H. H A R K O N E N a n d H. HELVEt Department of Clinical Chemistry, University of Helsinki, Meilahti Hospital, Helsinki, Finland
JANSSON, S.-E., HARKONEN, M. H. & HELVE, H.: Metabolic properties of nerve endings isolated from rat brain. Acta Physiol Scand 1979, 107: 205-212. Received 23 Jan. 1979. ISSN 0001-6772. Department of Clinical Chemistry, University of Helsinki, Finland. Energy metabolism was studied in nerve endings isolated from 3-week-old rat brain. Concentrations of glycogen, glucose, ATP, phosphocreatine and lactate were lower in synaptosomes than in the intact brain. The consumption of these endogenous substrates, the ability to generate high-energy phosphate, and the production of ammonia were determined in aerobic and anaerobic conditions. Unlike nerve tissue in general, synaptosomes preferentially utilized endogenous ATP and phosphocreatine stores which, on incubation in the absence of exogenous substrates, were emptied long before glycogen stores were exhausted. The optimal medium for respiratory studies was found to have electrolyte concentrations equal to the extracellular fluid. The synaptosomes had an endogenous respiration rate of 6.3 nmol 0, mg prot. min, measured with an oxygen electrode, and it probably reflects consumption of their glycogen stores. Glucose usually had no effect on the respiration rate of synaptosomes, but sometimes increased it slightly. However, after incubation in the presence of arsenate synaptosomes showed an increase in respiration when glucose was added. ADP, when added with glucose, also stimulated respiration. Pyruvate and succinate always increased respiration, succinate usually having the stronger effect. The present results show that isolated nerve endings are metabolically intact, which justifies their use in research on neurotransmission. In addition, opposite to the present consensus, synaptic transmission does not seem primarily to depend on the availability of glucose but rather on local stores of high-energy phosphate compounds. Key words: Synaptosomes, respiration, energy metabolism, synaptic function
Present knowledge of t h e metabolism of nerve endings is limited and based mainly on t h e results of studies on t h e oxygen consumption of isolated nerve endings (synaptosomes). Such studies h a v e show n that synaptosomes respire using glucose, pyruvate (Bradford 1969), glutamate and succinate (Verity 1972) as substrates, and t h a t ATP a n d phosphocreatine can b e synthesized in the presence of glucose (Bradford 1969). Little attention h a s b e e n paid to the substrates that provide energy for synaptosomes, despite the observations that synaptic transmission is the aspect of nerve function most vulnerable to shortage of glucose (Larrabee & Klingman 1962, Harkonen et al. 1969). T h e present study concerned t h e oxygen consumption, high-energy phosphate production a n d rates of energy usage by synaptosomes in various conditions. T h e results indicate that synaptosomes are metabolically intact a n d that local energy metabolism plays a n important role in synaptic transmission.
MATERIAL AND METHODS Preparation of synaptosomes. Three-week-old SpragueDawley rats of both sexes were decapitated and the brain, except for the cerebellum, was dissected out and homogenized in 0.32 mol/l ice-cold, oxygenated sucrose in a glass homogenizer with a Plexiglass pestle (clearance 250 pm) at 850 rpm for 2 min. In some experiments, the cerebral cortices were dissected out and used instead of whole-brain homogenates. The nuclear and crude mitochondria1 fractions, and the subfractions containing myelin, synaptosomes and mitochondria were isolated as described by Gray & Whittaker (1962). After one wash the synaptosomes were resuspended in 0.32 mol/l sucrose. The protein of the synaptosome suspension was determined according to Lowry et al. (1951). The purity of the fractions were checked by electron microscopy. Incubation techniques. When oxygen consumption was determined, a sample of the synaptosome suspension in sucrose was diluted with 1 ml of incubation medium and incubations were carried out in a thermostated Plexiglass vessel at 30°C. In other expts., the diluted synaptosome preparations were incubated at 37°C in stoppered 50-ml polypropylene centrifuge tubes (gentle shaking), into which gases (95% 0 , + 5 % CO, or N,) were bubbled through venipucture needles. Samples were withdrawn Actu P h y ~ i o lScoiid 107
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Table 1. Effect of substrates or inhibitors on endogenous respiration of synuptosornes Endogenous respiration was 6.3k1.0 nmol O,/mg prot./min (mean? S.E. of 8 expts.). All expts. were carried out at 30°C. The increase (+) or decrease (-) in oxygen uptake induced by an agent was calculated in comparison to the control respiration in the particular experiments. Values are means ? S.E. of 4-14 expts. indicated as the number of different batches of synaptosomes/the total number of tests Effect of oxygen uptake nmol O2 mg protein-' min-'
Change
Agent added Glucose, 10 mmol/l Pyruvate, 10 mmol/la Succinate, 10 mmol/l ADP, 1 mmol/lb ADP, 5 mmol/l FCCP, mmol/lc Oligomycin, 1 pg/mld
+0.33+0.30 +5.90&0.45 6.24 ?0.48 +1.46?0.16 +5.08?1.35 +2.54 k0.28 -0.10
+ 5
a
+
(%)
+89+6 +83+4 +24+3 +52+2 +34?6 - 7
No. of expts. 8/ 14 3/10 4/12 2/7 4/4 2/7 4/4
During the first linear minute. The effect of ADP was studied in the presence of 10 mmolll glucose. FCCP was added after glucose or glucose plus 1 or 5 mmol/l ADP. The effect of oligomycin was studied in the presence of 10 mmol/l glucose.
with a long needle without interruption of incubation and fixed immediately with an equal volume of 0.6 mol/l icecold perchloric acid containing 2 mmol/l EDTA. The mixture was centrifuged in the cold at 3 500Xg for 5 min, and a portion of the supernatant was neutralized (pH 6.5) with 2.5 mol/l KHCO,. Determination of oxygen consumption. The oxygen consumption of synaptosome suspensions was determined with a Clark-type oxygen electrode (Clark et al. 1953). Determination of metabolites. Glycogen, ATP, ADP, AMP and lactate in the neutralized perchloric acid extract were measured by fluorometric enzymatic methods with a Farrand A-3 fluorometer (Lowry et al. 1964, Passonneau et al. 1967). As sucrose is partially hydrolysed in perchloric acid, glucose was determined in a Ba(OH),ZnSO, extract of synaptosomes (Harkonen et al. 1969). Ammonia production was determined by a fluorometric enzymatic method (Folbergrova et al. 1969). Semi-quantitative evaluation of the vesicle content of incubated synaptosomes. For electron microscope studies, a sample of the synaptosome suspension was diluted with ice-cold 2.5 % glutaraldehyde solution in 0.1 mol/l phosphate buffer, pH 7.4. After fixation, the synaptosomes were centrifuged and the pellet processed according to a conventional EM routine. Photographic negatives of representative areas were taken at 10500xmagnification and after framing and coding, projected onto a white, lined background. The entire coded negative was independently inspected by two of the authors and every recognizable synaptosome was evaluated for its vesicle content by expressing the area filled with vesicles as a percentage of the whole area of the synaptosomes (except for the intrasynaptosomal mitochondria). Thus 100% denotes synaptosomes packed with vesicles whereas 0% denotes particles identified as synaptosome ghosts or as synaptosomes without intact vesicles. Every negative contained about 20 synaptosomes and each author evaluated about 100 synaptosomes in various states for each of the different incubation conditions. T h e validity of this subjective Acrii
Plrv.wd S c ~ n d107
method of quantification was confirmed by planimetry and actual counting of vesicles. Reagents. The water used had been deionized, and passed through charcoal and Millipore filters. Substrates and pyridine nucleotides were obtained from the Sigma Chemical Company (St. Louis, Mo., USA) or from Boehringer and Sohne (Mannheim, West Germany); heart lactate dehydrogenase was purchased from Worthington (Freehold, N.J., USA). Other chemicals used were commercially available products of analytical grade.
RESULTS Purity of the synnptosomal fraction
The purity of every synaptosomal fraction prepared was checked by electron microscopy. The nerve endings were mainly well-preserved but the fractions also contained some unidentified membrane profiles, either empty nerve endings of fragments of glial origin. Mitochondria1 contamination was less than 10% of all particles and consisted almost solely of small, apparently synaptosomal mitochondria. Oxygen consumption Respiration was studied routinely in a medium consisting of NaCl 136 (values in mmol per liter), KCI 5.6, CaCI, 2.2, NaH,PO, 1.2, MgC12 1.3 and imidazole buffer, pH 7.4, 30. The effects of Ca2+,Mg2+,K+ and PO,3- ions on synaptosomal consumption of oxygen in the presence of 10 mmolll glucose were tested by increasing the concentration of each ion from zero to 10-20
Energy metabolism in synaptosomes
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Table 2. The concentrations of various substrates of energy metabolism in isolated nerve endings and in the cerebral cortex of the rat (rnmollkg prot.) Values referring to synaptosomes and cerebral cortex are means of at least two determinations or means k S.E. of 4 determinations Cerebral cortex ~
Metabolite
Synaptosomes
Glycogen Glucose ATP P-creatine Lactate
4.20f0.51 0.30 2.81f0.34 2.29f0.43 5.22k 1.41 2.94f0.36 4.93k0.95 0.031
ADP AMP
CAMP' a
~
Immediately frozen"
Delayed frozen*
28.8f0.66 4.05 ?0.68 17.8k0.06 11.4k 1.98 24.4k0.83
18.0k0.22 1.47k 0.36 5.86k0.82 2.19k0.65 34.0k2.64
0.020
Head frozen in liquid nitrogen immediately after decapitation. Skull opened and brain removed before freezing in liquid nitrogen. Determined according to Raij & Harkonen 1976.
mmol/l. At 0 . 5 4 mmol/l Ca2+, respiration was slightly enhanced (%lo%), but at higher concentrations (8 mmol/l) clearly depressed. Mg2+also slightly enhanced respiration at concentrations of 0.5-5.0 mmol/l. Neither ion had any stimulatory effect on respiration in the presence of 10 mmol/l pyruvate or succinate. No change in oxygen uptake occurred in glucose, pyruvate or succinate medium when the PO?- (0.5-10 mmol/l) or K+ (1-20 mmol/l) concentration was increased stepwise. The negative results with high K+ concentrations could in part be due to the fact that the synaptosomes might have been depolarized during storage in the cold sucrose solution. The short incubation time in the electrolyte medium was not sufficient to repolarize the synaptosomal membrane. In tests with various buffers (imidazole-HC1, Tris-HC1, glycylglycine, bicarbonate), none seemed to be superior to the others. The isolated synaptosomes contained large amounts of endogenous substrates and respired at an appreciable rate without addition of substrates ((6.3k1.0 nmol OJmg prot./min, mean fS.E. of 8 synaptosomal preparations, range 4.02-12.4 nmol OJmg prot./min). Oxygen consumption was linear during the first 3-5 min (the values given in Table 1 were measured within this time), but then often tended to decrease. Thus, when synaptosomes were incubated in oxygenated substrate-free medium at 3WC, respiration fell to about 50% in 30 min and to 20 % in 60 min. Endogenous respiration was not significantly lowered when synaptosomes were stored at 0°C for as long as 6 h, although
sometimes the response to added substrates seemed to be weaker than that of freshly prepared synaptosomes . Addition of 10 mmol/l glucose to the incubation medium did not have a constant effect (Table 1). Mostly respiration was unaffected, but in some experiments a small increase was seen. Even with synaptosomes which had been aged by incubation at 30°C for 30-60 min in the absence of substrates, addition of glucose did not raise oxygen consumption significantly. However, when synaptosomes were first incubated in 40 mmol/l arsenate for 1 h, which by itself did not stimulate respiration, addition of glucose induced a 2-fold increase in oxygen consumption. Addition of 5 mmol/l ADP to a glucose medium produced a 70-100% increase in respiration (Table 1). The response was not linear and the highest values were measured during the first few minutes. Pyruvate and succinate at a concentration of 10 mmol/l stimulated oxygen uptake, raising it to twice the level without any added substrate (Table 1). Respiration with pyruvate was not linear and after a few minutes only 40 % of the effect remained. FCCP (carbonyl cyanide p-trifluoromethoxy phenylhydrazone), and oligomycin were used to study the mechanisms controlling the respiration of synaptosomes. In a glucose medium, 1 mmol/l of FCCP had a slight stimulatory effect (34% mean increase) on the rate of oxygen consumption (Table 1). The effect of oligomycin was also small but in 4 expts. the addition of 1 pg/ml oligomycin after AGIO f'livsiol Scand 107
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z
W A
T E 0
”
0 5
15
30 60 INCUBATION TIME, MINUTES
120
Fig. I . Utilization of energy metabolites in isolated nerve endings. Synaptosomes were incubated at 37°C in oxygenated, substrate-free electrolyte solution buffered with imidazole-HCI, pH 7.4. At the times indicated samples were withdrawn from the suspension. Results of a typical experiment. Gly=glycogen, Lac=lactate and PCr=phosphocreatine.
glucose inhibited the respiration by 7 %. Likewise when oligomycin was added after 40 mmol/l arsenate, 18% inhibition was obtained. Metabolite levels
Table 2 shows the concentration of energy metabolites measured in perchloric acid extracts of freshly prepared synaptosomes. The metabolite concentrations were considerably lower in the synaptosomes than in cerebral cortex which had been frozen immediately. The decrease was very rapid during the initial 15-20 s following decapitation while the brain was being dissected out and thus before it was
,
I
0
15 30 0 15 INCUBATION TIME MINUTES
30
Fig. 3. Generation of ATP and phosphocreatine in synaptosomes in an oxygenated electrolyte medium at 37°C. The medium was supplemented with 10 mmol/l glucose or with 10 mmol/l glucose+5 mmol/l ADP. Results of a typical experiment.
chilled. This became evident when the metabolites were measured in extracts of cerebral cortices obtained either after freezing the head of the rat in liquid nitrogen immediately after decapitation or after dissection in the same way as for preparation of synaptosomes followed by freezing (Table 2 ) . ATP and phosphocreatine concentrations were only a little lower in the synaptosomes than in the “delayed frozen” cerebral cortex and corresponded well to levels previously reported (Barberis & McIlwain 1976, Hawthorne & Pickard 1977, Nelson-Krause & Howard 1978). Thus, during the actual isolation procedure, the decrease was relatively slow. This was apparently due to the fact that the synaptosomes had adequate supplies of oxygen and nutrients and metabolism was slowed down by chilling. The differences in glucose and lactate levels between the synaptosomes and the “delayed frozen” cerebral cortex is probably due to diffusion of these compounds into the surrounding medium. Changes in rnetabolites during incubation in oxygen
0
10 20 INCUBATION TIME W T E S
30
Fig. 2. Effect of anoxia on the utilization of energy metabolites in isolated nerve endings. A suspension of synaptosomes was incubated under nitrogen in the absence of added substrates at 3 7 T , and samples were taken .it the times indicated. Results of a typical experiment. Acto P l i v ~ i i Jcotid ~l 107
The concentrations of glycogen, ATP, phosphocreatine and lactate were followed during incubation in oxygen for 2-3 h in a substrate-free medium in 3 different synaptosomal preparations (Fig 1). When synaptosomes are isolated in a sucrose gradient, measurement of glucose is difficult, because perchloric acid produces glucose via hydrolysis of sucrose. For measurements in freshly prepared synaptosomes, we therefore used the Ba(OH),ZnSO, precipitation method, and found glucose to be approximately 0.3 mmol/kg prot. (Table 2 ) . As the concentration was so low and extracts are
Energy metabolism in synaptosomes
209
the P : 0 ratio is taken as 3, this rate of use of -P is equivalent to an oxygen consumption of 3.3 nmol/ mg prot./min. This is somewhat lower than the value measured with an oxygen electrode. Generation of ATP and phosphocreatine
I
0
I
I
I
15 30 0 15 INCUBATKIN TIME, MINUTES
I
30
Fig. 4 . Metabolism of high-energy phosphate compounds in synaptosomes in an oxygenated electrolyte medium at 37°C. The medium was supplemented with 10 mmol/l glucose (A) or with 10 mmol/l glucose+5 mmol/l ADP (B)
(the ADP content of the blank incubation solution was subtracted from the ADP values). Results of a typical experiment.
difficult to prepare, glucose was not measured during incubation studies. During the first 5 rnin glycogen dropped to 65 % of the initial level, and after 15 rnin only about half of the original concentration was left (Fig. 1). The rate of consumption then slowed down, but at 3 h practically all glycogen had been used up. Lactate increased during the first 5 rnin and then decreased at a constant rate, and at 3 h only trace amounts were detected. ATP and phosphocreatine fell at comparable rates; after 5 rnin the values were only about half the initial level, and after 1 h less than 10% was left. Changes in metabolites during incubation in nitrogen
When synaptosomes ( 3 separate preparations) were incubated in a nitrogen-saturated medium in the absence of added energy substrates, glycogen fell to half the initial concentration in 10 min, then decreased more slowly (Fig. 2). After an initial lag of 1 rnin the lactate concentration rose and the increase was roughly proportional to the decrease in glycogen. ATP and phosphocreatine fell rapidly during the first min reaching 30 % and 15% of the respective initial levels. After 10-15 min, there was less than 10% of these substrates left. An approximate metabolic rate can be calculated for the synaptosomes from the rate of use of highenergy phosphate (-P) seen as changes in ATP, phosphocreatine, glucose and glycogen (see e.g. Harkonen et al. 1969). During the first rnin of anoxia -P was used at a rate of 10 mmol/kg prot./min. If 14-79588 1
Synaptosomes were incubated with constant oxygenation in a medium containing 10 mmol/l glucose alone or 10 mmol/l glucose and 5 mmol/l ADP in order to study their ability to synthesize highenergy phosphate. Neither alone nor with ADP could glucose maintain the initial level of these high-energy phosphate compounds (Fig. 3). However, in the presence of glucose the decrease was smaller than during endogenous respiration, e.g. ATP decreased by 1.3 mmol compared with 2.2 mmol, and phosphocreatine by 0.3 mmol compared with 1.7 mmol per kg protein during the first 15 rnin (values are means of 2 separate expts.). Furthermore, after this initial fall, the level of both compounds remained the same (ATP 1.0 and phosphocreatine 0.8 mmol/kg prot.) for 1 h while during endogenous respiration, high-energy phosphates were practically exhausted. The presence of 10 mmol/l glucose could not prevent the concentrations of ADP and AMP from decreasing during incubation, especially during the first 15 rnin (Fig. 4A). In Fig. 4 B (incubation medium supplemented with 10 mmol glucose + 5 mmol/l ADP) the linear decrease of ADP, and the linear increase of AMP, as a function of time, suggest the presence of enzymatic ADPase activity. Generation of high-energy phosphate was also studied in a Ca-free medium containing 20 mmol/l
t
4
INCUBATION TIME, MINUTES
Fig. 5 . Ammonia production in isolated nerve endings in
an electrolyte solution without added energy substrates in the presence or absence of oxygen at 37°C. Results of a
typical experiment. Acto Phr~sinlScand I07
S . - E . Jutisson et al.
210
80 1
Empty-.,.
were badly damaged (empty of vesicles). Whether performed in the absence or presence of oxygen, incubation had the same morphological effect.
Half-flied
DISCUSSION
I
J
I\---;----*/’- -.-.-.a
_./-
-.-
40 _._.-.-.-’-
---__---_ .
20
?I
30 WUBATION TIME, MINUTES
1 0I 60
Fig. 6 . The effect of incubation at 37°C in oxygenated electrolyte solution without added energy substrates on
the preservation of synaptosomes. The “filled” synaptosomes contain the normal quantity of vesicles (occupying 60-100% of the synaptosome area), those which are “half-filled” have lost about half their vesicles, and the “empty” synaptosomes contain few, if any, vesicles (occupying at most 20% of the synaptsome area). The points represent the result of two independent investigators.
During the first 15 min, 50% of the ATP and phosphocreatine were lost, but after this initial fall, there was no further loss of either compound. Amrnoniu production and vesicle preservation in incubated synaptosomes
For synaptosomes incubated in an oxygenated substrate-free medium ammonia production was, after an initial lag, about 1.5 mmol/kg prot./min during the first 30 min and then levelled off (Fig. 5 ) . Under nitrogen, ammonia production was much slower, averaging 0.4 mmol/kg prot./min (Fig. 5 ) . Synaptosomes incubated at 37°C without substrates showed clear-cut morphological alterations as compared with unincubated synaptosomes stored at 4°C on ice. The number of vesicles decreased drastically, and often enlarged vesicles as well as swollen intrasynaptosomal mitochondria could be seen. These changes were apparent after as little as 30 min, but after incubation for 1 h most synaptosomes contained hardly any intact vesicles and were filled with debris. These changes were evaluated semi-quantitatively (see Material and methods). At zero time well preserved synaptosomes and synaptosomes with a very low vesicular content were about equal in number (Fig. 6). However, after l h at 37°C only about 10% of the synaptosomes were in good condition and nearly 70%
Pattern of utilizution of energy reserves Despite the low concentration of endogenous substrates, the metabolic changes that occurred when the tissue was incubated in an oxygen or nitrogen atmosphere show that the synaptosomes are viable and utilize their energy reserves according to their requirements. In both conditions glycogen, ATP and phosphocreatine, the major energy-yielding compounds in nervous tissue (besides glucose, which in vivo originates from blood), were consumed in a pattern quite different from that seen in immature nerve cells (Hemminki & Harkonen 1974) or in the sympathetic ganglion (Harkonen et al. 1969). In synaptosomes, ATP and phosphocreatine were used rapidly in both the presence and the absence of oxygen, while in the other tissues they lasted much longer, especially in oxygen. The faster initial decline of glycogen in synaptosomes as compared to ganglion cells or in immature nerve cells could be due to the fact that they are practically devoid of endogenous glucose, which is the substrate utilized, initially at least, in the ganglion. In most of our synaptosome preparations, glycogen was detectable at substantial concentrations long after ATP and phosphocreatine were used up. It is obvious from the small decrease in glycogen after 15 min in oxygen that this is not the main substrate supporting the substantial respiration. However, a considerable amount of ammonia was produced between 15 and 30 min after the start of incubation and would explain the oxygen consumption, because most of the ammonia formed in nervous tissue during incubation in vitro originates from amino acids that are oxidatively deaminated (Weil-Malherbe 1962). Oxygen consumption of isolated nerve endings
It has been shown previously that synaptosomes have an adequate endogenous respiration rate, which can be stimulated by glucose or pyruvate (Bradford 1969, Verity 1972, Diamond & Fishman 1973, Bradford et al. 1978). With our technique, the endogenous respiration rate was higher than with manometric techniques (Bradford 1969) but, even in
Energj niettrbolism in .~yticiptosorne~ 21 1
aged synaptosomes, we were only occasionally able to show a slight stimulation of oxygen consumption by glucose. The insensitiveness of our synaptosome preparation to glucose is probably due to the short initial observation time during which they were still respiring on endogenous glycogen stores. The failure to increase respiration in aged synaptosomes, which had consumed most of their glycogen stores, may have been due to tissue catabolism and damage to vital enzymatic structures during the aging process. This explanation is supported by the observation that during the first 30 min of incubation, ammonia production was considerable. The insensitiveness of our synaptosome preparations to uncouplers apparently reflects the fact that in the complex metabolic system which the synaptosome represents, respiration is controlled at a step which is not affected by uncouplers. The stimulating effect of ADP on glucose respiration leads us to assume that ADP, despite its positive charge, does to some extent penetrate the synaptosomal membrane. Taking the ratio of the respiration rate in the presence of ADP to that in its absence as an indication of the respiratory control in the system (Chance & Williams 1956), a ratio of 1.7 is obtained with 5 mmol/l ADP which would suggest fair respiratory control. When intrasynaptosomal mitochondria have been isolated in Ficollsucrose media they have also shown good metabolic activity and respiratory control (Lai & Clark 1976).
Generation o f high-rnrrgy phosplzute3
Bradford (1969) reported that, with glucose as a substrate, synaptosomes synthesized both ATP and phosphocreatine. Closer scrutiny of his data, however, shows that actually he measured the levels of A T P and phosphocreatine in synaptosomes after incubation for 1 h in various media in the presence of metabolic poisons. In our generation experiments we followed the concentration of both compounds during the course of incubation. The rapid initial decrease in ATP concentration could be due to the membrane Na+-K+-ATPase ( Abdel-Latif & Abood 1964) activated by temperature and Na+ and K + in the incubation medium. The simultaneous decrease in phosphocreatine could thus be due to creatine kinase activity tending to synthesize A T P from ADP and phosphocreatine. After the initial fall
there was hardly any change in A T P o r phosphocreatine concentration during the next 2-3 h. Our interpretation of these results is that NA+-K+ATPase activity, creatine kinase activity, and oxidative phosphorylation supported by glucose (or glycogen), came to equilibrium, and A T P and phosphocreatine reached a steady-state-concentration in the synaptosomes. The AMP deaminase activity localized in the synaptosomes (Jansson & Harkonen, to be published) would explain the decreasing sum of adenylates during incubation.
Energy nzetabolisni und synaptic firnction
The function of nerve tissue, especially synaptic transmission (Larrabee & Klingman 1962, Harkonen et al. 1969), is very sensitive to glucose deprivation. It has been shown that failure of transmission occurs long before A T P and phosphocreatine are exhausted in the ganglion, and this seems to coincide with depletion of hexoses (Harkonen et al. 1969). Evidence also indicates that the locus of the failure is at the synaptic level and several explanations have been offered without direct experimental support (see Larrabee & Klingman 1962, Nicolescu et al. 1966, Harkonen et al. 1969). The present results show that 2 h after the start of incubation (when transmission through the ganglion is known to fail, Harkonen et al. 1969), the synaptosomes were depleted of A T P and phosphocreatine, although glycogen was still present. Therefore, blockage of impulse propagation at the synaptic level appears to be due to local depletion of A T P in the nerve endings, followed by failure of the ion pump. The other possible explanation would be the catabolism of tissue elements in the absence of glucose; this hypothesis gains support from the high production of ammonia and disappearance of vesicles containing the transmitter. It might well be that failure of chemical transmission is due to the two phenomena together. The earlier hypothesis of depletion of acetyl-CoA, suggested because failure of transmission coincides with depletion of hexoses, seems no longer tenable (Harkonen et al. 1969).
This work was supported by grants from the Medical Research Council in the Academy of Finland, the Sigrid Juselius Foundation. Helsinki, and the Finska Lakaresallskapet . Actn Pliv.%iolScotid 107
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Metabolism of glucose and oxygen in mammalian sympathetic ganglia at and in action. In: Neurochemistry (ed. K, A, c, Elliott, I , H. page and J . H. Quastel), pp. 150-176. Thomas, Springfield. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. 1951. Protein measurement with the Fohn phenol reagent. J Biol Chem 193: 265-275. LOWRY, 0. H., PASSONNEAU, J. V., HASSELBERGER, F. X. & SCHULZ, D. W. 1964. Effects of ischemia on known substrates and co-factors of the glycolytic pathway in brain. J Biol Chem 239: 18-30. NELSON-KRAUSE, D. C. & HOWARD, B. D. 1978. Energy utilization in the induced release of yaminobutyric acid from synaptosomes. Brain Res 147: 91-105. NICOLESCU, P., DOLIVO, M., ROUILLER, C. & FOROGLOU-KERAMEUS, C. 1966. The effect of deprivation of glucose on the ultrastructure and function of the superior cervical ganglion of the rat in vitro. J Cell B i o l 2 9 267-285. PASSONNEAU, J. V., GATFIELD, P. D., SCHULZ, D. W. & LOWRY, 0. H. 1967. An enzymic method for measurement of glycogen. Analyt Biochem 19: 315326. RAIJ, K. & HARKONEN, M. 1976. Determination of cyclic adenosine 3‘3’-monophosphate in urine. Scand J Clin Lab Invest 36: 161-168. WEIL-MALHERBE, H. 1962. Ammonia metabolism in the brain. In: Neurochemistry (ed. K. A. C. Elliott, I. H. Page and J. H. Quastel), pp. 321-330. Thomas, Springfield. VERITY, M. A. 1972. Cation modulation of synaptosoma1 respiration. J Neurochem 19: 1305-1317.
Metabolic properties of nerve endings isolated from rat brain S.-E. J A N S S O N , M. H. H A R K O N E N a n d H. HELVEt Department of Clinical Chemistry, University of Helsinki, Meilahti Hospital, Helsinki, Finland
JANSSON, S.-E., HARKONEN, M. H. & HELVE, H.: Metabolic properties of nerve endings isolated from rat brain. Acta Physiol Scand 1979, 107: 205-212. Received 23 Jan. 1979. ISSN 0001-6772. Department of Clinical Chemistry, University of Helsinki, Finland. Energy metabolism was studied in nerve endings isolated from 3-week-old rat brain. Concentrations of glycogen, glucose, ATP, phosphocreatine and lactate were lower in synaptosomes than in the intact brain. The consumption of these endogenous substrates, the ability to generate high-energy phosphate, and the production of ammonia were determined in aerobic and anaerobic conditions. Unlike nerve tissue in general, synaptosomes preferentially utilized endogenous ATP and phosphocreatine stores which, on incubation in the absence of exogenous substrates, were emptied long before glycogen stores were exhausted. The optimal medium for respiratory studies was found to have electrolyte concentrations equal to the extracellular fluid. The synaptosomes had an endogenous respiration rate of 6.3 nmol 0, mg prot. min, measured with an oxygen electrode, and it probably reflects consumption of their glycogen stores. Glucose usually had no effect on the respiration rate of synaptosomes, but sometimes increased it slightly. However, after incubation in the presence of arsenate synaptosomes showed an increase in respiration when glucose was added. ADP, when added with glucose, also stimulated respiration. Pyruvate and succinate always increased respiration, succinate usually having the stronger effect. The present results show that isolated nerve endings are metabolically intact, which justifies their use in research on neurotransmission. In addition, opposite to the present consensus, synaptic transmission does not seem primarily to depend on the availability of glucose but rather on local stores of high-energy phosphate compounds. Key words: Synaptosomes, respiration, energy metabolism, synaptic function
Present knowledge of t h e metabolism of nerve endings is limited and based mainly on t h e results of studies on t h e oxygen consumption of isolated nerve endings (synaptosomes). Such studies h a v e show n that synaptosomes respire using glucose, pyruvate (Bradford 1969), glutamate and succinate (Verity 1972) as substrates, and t h a t ATP a n d phosphocreatine can b e synthesized in the presence of glucose (Bradford 1969). Little attention h a s b e e n paid to the substrates that provide energy for synaptosomes, despite the observations that synaptic transmission is the aspect of nerve function most vulnerable to shortage of glucose (Larrabee & Klingman 1962, Harkonen et al. 1969). T h e present study concerned t h e oxygen consumption, high-energy phosphate production a n d rates of energy usage by synaptosomes in various conditions. T h e results indicate that synaptosomes are metabolically intact a n d that local energy metabolism plays a n important role in synaptic transmission.
MATERIAL AND METHODS Preparation of synaptosomes. Three-week-old SpragueDawley rats of both sexes were decapitated and the brain, except for the cerebellum, was dissected out and homogenized in 0.32 mol/l ice-cold, oxygenated sucrose in a glass homogenizer with a Plexiglass pestle (clearance 250 pm) at 850 rpm for 2 min. In some experiments, the cerebral cortices were dissected out and used instead of whole-brain homogenates. The nuclear and crude mitochondria1 fractions, and the subfractions containing myelin, synaptosomes and mitochondria were isolated as described by Gray & Whittaker (1962). After one wash the synaptosomes were resuspended in 0.32 mol/l sucrose. The protein of the synaptosome suspension was determined according to Lowry et al. (1951). The purity of the fractions were checked by electron microscopy. Incubation techniques. When oxygen consumption was determined, a sample of the synaptosome suspension in sucrose was diluted with 1 ml of incubation medium and incubations were carried out in a thermostated Plexiglass vessel at 30°C. In other expts., the diluted synaptosome preparations were incubated at 37°C in stoppered 50-ml polypropylene centrifuge tubes (gentle shaking), into which gases (95% 0 , + 5 % CO, or N,) were bubbled through venipucture needles. Samples were withdrawn Actu P h y ~ i o lScoiid 107
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Table 1. Effect of substrates or inhibitors on endogenous respiration of synuptosornes Endogenous respiration was 6.3k1.0 nmol O,/mg prot./min (mean? S.E. of 8 expts.). All expts. were carried out at 30°C. The increase (+) or decrease (-) in oxygen uptake induced by an agent was calculated in comparison to the control respiration in the particular experiments. Values are means ? S.E. of 4-14 expts. indicated as the number of different batches of synaptosomes/the total number of tests Effect of oxygen uptake nmol O2 mg protein-' min-'
Change
Agent added Glucose, 10 mmol/l Pyruvate, 10 mmol/la Succinate, 10 mmol/l ADP, 1 mmol/lb ADP, 5 mmol/l FCCP, mmol/lc Oligomycin, 1 pg/mld
+0.33+0.30 +5.90&0.45 6.24 ?0.48 +1.46?0.16 +5.08?1.35 +2.54 k0.28 -0.10
+ 5
a
+
(%)
+89+6 +83+4 +24+3 +52+2 +34?6 - 7
No. of expts. 8/ 14 3/10 4/12 2/7 4/4 2/7 4/4
During the first linear minute. The effect of ADP was studied in the presence of 10 mmolll glucose. FCCP was added after glucose or glucose plus 1 or 5 mmol/l ADP. The effect of oligomycin was studied in the presence of 10 mmol/l glucose.
with a long needle without interruption of incubation and fixed immediately with an equal volume of 0.6 mol/l icecold perchloric acid containing 2 mmol/l EDTA. The mixture was centrifuged in the cold at 3 500Xg for 5 min, and a portion of the supernatant was neutralized (pH 6.5) with 2.5 mol/l KHCO,. Determination of oxygen consumption. The oxygen consumption of synaptosome suspensions was determined with a Clark-type oxygen electrode (Clark et al. 1953). Determination of metabolites. Glycogen, ATP, ADP, AMP and lactate in the neutralized perchloric acid extract were measured by fluorometric enzymatic methods with a Farrand A-3 fluorometer (Lowry et al. 1964, Passonneau et al. 1967). As sucrose is partially hydrolysed in perchloric acid, glucose was determined in a Ba(OH),ZnSO, extract of synaptosomes (Harkonen et al. 1969). Ammonia production was determined by a fluorometric enzymatic method (Folbergrova et al. 1969). Semi-quantitative evaluation of the vesicle content of incubated synaptosomes. For electron microscope studies, a sample of the synaptosome suspension was diluted with ice-cold 2.5 % glutaraldehyde solution in 0.1 mol/l phosphate buffer, pH 7.4. After fixation, the synaptosomes were centrifuged and the pellet processed according to a conventional EM routine. Photographic negatives of representative areas were taken at 10500xmagnification and after framing and coding, projected onto a white, lined background. The entire coded negative was independently inspected by two of the authors and every recognizable synaptosome was evaluated for its vesicle content by expressing the area filled with vesicles as a percentage of the whole area of the synaptosomes (except for the intrasynaptosomal mitochondria). Thus 100% denotes synaptosomes packed with vesicles whereas 0% denotes particles identified as synaptosome ghosts or as synaptosomes without intact vesicles. Every negative contained about 20 synaptosomes and each author evaluated about 100 synaptosomes in various states for each of the different incubation conditions. T h e validity of this subjective Acrii
Plrv.wd S c ~ n d107
method of quantification was confirmed by planimetry and actual counting of vesicles. Reagents. The water used had been deionized, and passed through charcoal and Millipore filters. Substrates and pyridine nucleotides were obtained from the Sigma Chemical Company (St. Louis, Mo., USA) or from Boehringer and Sohne (Mannheim, West Germany); heart lactate dehydrogenase was purchased from Worthington (Freehold, N.J., USA). Other chemicals used were commercially available products of analytical grade.
RESULTS Purity of the synnptosomal fraction
The purity of every synaptosomal fraction prepared was checked by electron microscopy. The nerve endings were mainly well-preserved but the fractions also contained some unidentified membrane profiles, either empty nerve endings of fragments of glial origin. Mitochondria1 contamination was less than 10% of all particles and consisted almost solely of small, apparently synaptosomal mitochondria. Oxygen consumption Respiration was studied routinely in a medium consisting of NaCl 136 (values in mmol per liter), KCI 5.6, CaCI, 2.2, NaH,PO, 1.2, MgC12 1.3 and imidazole buffer, pH 7.4, 30. The effects of Ca2+,Mg2+,K+ and PO,3- ions on synaptosomal consumption of oxygen in the presence of 10 mmolll glucose were tested by increasing the concentration of each ion from zero to 10-20
Energy metabolism in synaptosomes
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Table 2. The concentrations of various substrates of energy metabolism in isolated nerve endings and in the cerebral cortex of the rat (rnmollkg prot.) Values referring to synaptosomes and cerebral cortex are means of at least two determinations or means k S.E. of 4 determinations Cerebral cortex ~
Metabolite
Synaptosomes
Glycogen Glucose ATP P-creatine Lactate
4.20f0.51 0.30 2.81f0.34 2.29f0.43 5.22k 1.41 2.94f0.36 4.93k0.95 0.031
ADP AMP
CAMP' a
~
Immediately frozen"
Delayed frozen*
28.8f0.66 4.05 ?0.68 17.8k0.06 11.4k 1.98 24.4k0.83
18.0k0.22 1.47k 0.36 5.86k0.82 2.19k0.65 34.0k2.64
0.020
Head frozen in liquid nitrogen immediately after decapitation. Skull opened and brain removed before freezing in liquid nitrogen. Determined according to Raij & Harkonen 1976.
mmol/l. At 0 . 5 4 mmol/l Ca2+, respiration was slightly enhanced (%lo%), but at higher concentrations (8 mmol/l) clearly depressed. Mg2+also slightly enhanced respiration at concentrations of 0.5-5.0 mmol/l. Neither ion had any stimulatory effect on respiration in the presence of 10 mmol/l pyruvate or succinate. No change in oxygen uptake occurred in glucose, pyruvate or succinate medium when the PO?- (0.5-10 mmol/l) or K+ (1-20 mmol/l) concentration was increased stepwise. The negative results with high K+ concentrations could in part be due to the fact that the synaptosomes might have been depolarized during storage in the cold sucrose solution. The short incubation time in the electrolyte medium was not sufficient to repolarize the synaptosomal membrane. In tests with various buffers (imidazole-HC1, Tris-HC1, glycylglycine, bicarbonate), none seemed to be superior to the others. The isolated synaptosomes contained large amounts of endogenous substrates and respired at an appreciable rate without addition of substrates ((6.3k1.0 nmol OJmg prot./min, mean fS.E. of 8 synaptosomal preparations, range 4.02-12.4 nmol OJmg prot./min). Oxygen consumption was linear during the first 3-5 min (the values given in Table 1 were measured within this time), but then often tended to decrease. Thus, when synaptosomes were incubated in oxygenated substrate-free medium at 3WC, respiration fell to about 50% in 30 min and to 20 % in 60 min. Endogenous respiration was not significantly lowered when synaptosomes were stored at 0°C for as long as 6 h, although
sometimes the response to added substrates seemed to be weaker than that of freshly prepared synaptosomes . Addition of 10 mmol/l glucose to the incubation medium did not have a constant effect (Table 1). Mostly respiration was unaffected, but in some experiments a small increase was seen. Even with synaptosomes which had been aged by incubation at 30°C for 30-60 min in the absence of substrates, addition of glucose did not raise oxygen consumption significantly. However, when synaptosomes were first incubated in 40 mmol/l arsenate for 1 h, which by itself did not stimulate respiration, addition of glucose induced a 2-fold increase in oxygen consumption. Addition of 5 mmol/l ADP to a glucose medium produced a 70-100% increase in respiration (Table 1). The response was not linear and the highest values were measured during the first few minutes. Pyruvate and succinate at a concentration of 10 mmol/l stimulated oxygen uptake, raising it to twice the level without any added substrate (Table 1). Respiration with pyruvate was not linear and after a few minutes only 40 % of the effect remained. FCCP (carbonyl cyanide p-trifluoromethoxy phenylhydrazone), and oligomycin were used to study the mechanisms controlling the respiration of synaptosomes. In a glucose medium, 1 mmol/l of FCCP had a slight stimulatory effect (34% mean increase) on the rate of oxygen consumption (Table 1). The effect of oligomycin was also small but in 4 expts. the addition of 1 pg/ml oligomycin after AGIO f'livsiol Scand 107
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z
W A
T E 0
”
0 5
15
30 60 INCUBATION TIME, MINUTES
120
Fig. I . Utilization of energy metabolites in isolated nerve endings. Synaptosomes were incubated at 37°C in oxygenated, substrate-free electrolyte solution buffered with imidazole-HCI, pH 7.4. At the times indicated samples were withdrawn from the suspension. Results of a typical experiment. Gly=glycogen, Lac=lactate and PCr=phosphocreatine.
glucose inhibited the respiration by 7 %. Likewise when oligomycin was added after 40 mmol/l arsenate, 18% inhibition was obtained. Metabolite levels
Table 2 shows the concentration of energy metabolites measured in perchloric acid extracts of freshly prepared synaptosomes. The metabolite concentrations were considerably lower in the synaptosomes than in cerebral cortex which had been frozen immediately. The decrease was very rapid during the initial 15-20 s following decapitation while the brain was being dissected out and thus before it was
,
I
0
15 30 0 15 INCUBATION TIME MINUTES
30
Fig. 3. Generation of ATP and phosphocreatine in synaptosomes in an oxygenated electrolyte medium at 37°C. The medium was supplemented with 10 mmol/l glucose or with 10 mmol/l glucose+5 mmol/l ADP. Results of a typical experiment.
chilled. This became evident when the metabolites were measured in extracts of cerebral cortices obtained either after freezing the head of the rat in liquid nitrogen immediately after decapitation or after dissection in the same way as for preparation of synaptosomes followed by freezing (Table 2 ) . ATP and phosphocreatine concentrations were only a little lower in the synaptosomes than in the “delayed frozen” cerebral cortex and corresponded well to levels previously reported (Barberis & McIlwain 1976, Hawthorne & Pickard 1977, Nelson-Krause & Howard 1978). Thus, during the actual isolation procedure, the decrease was relatively slow. This was apparently due to the fact that the synaptosomes had adequate supplies of oxygen and nutrients and metabolism was slowed down by chilling. The differences in glucose and lactate levels between the synaptosomes and the “delayed frozen” cerebral cortex is probably due to diffusion of these compounds into the surrounding medium. Changes in rnetabolites during incubation in oxygen
0
10 20 INCUBATION TIME W T E S
30
Fig. 2. Effect of anoxia on the utilization of energy metabolites in isolated nerve endings. A suspension of synaptosomes was incubated under nitrogen in the absence of added substrates at 3 7 T , and samples were taken .it the times indicated. Results of a typical experiment. Acto P l i v ~ i i Jcotid ~l 107
The concentrations of glycogen, ATP, phosphocreatine and lactate were followed during incubation in oxygen for 2-3 h in a substrate-free medium in 3 different synaptosomal preparations (Fig 1). When synaptosomes are isolated in a sucrose gradient, measurement of glucose is difficult, because perchloric acid produces glucose via hydrolysis of sucrose. For measurements in freshly prepared synaptosomes, we therefore used the Ba(OH),ZnSO, precipitation method, and found glucose to be approximately 0.3 mmol/kg prot. (Table 2 ) . As the concentration was so low and extracts are
Energy metabolism in synaptosomes
209
the P : 0 ratio is taken as 3, this rate of use of -P is equivalent to an oxygen consumption of 3.3 nmol/ mg prot./min. This is somewhat lower than the value measured with an oxygen electrode. Generation of ATP and phosphocreatine
I
0
I
I
I
15 30 0 15 INCUBATKIN TIME, MINUTES
I
30
Fig. 4 . Metabolism of high-energy phosphate compounds in synaptosomes in an oxygenated electrolyte medium at 37°C. The medium was supplemented with 10 mmol/l glucose (A) or with 10 mmol/l glucose+5 mmol/l ADP (B)
(the ADP content of the blank incubation solution was subtracted from the ADP values). Results of a typical experiment.
difficult to prepare, glucose was not measured during incubation studies. During the first 5 rnin glycogen dropped to 65 % of the initial level, and after 15 rnin only about half of the original concentration was left (Fig. 1). The rate of consumption then slowed down, but at 3 h practically all glycogen had been used up. Lactate increased during the first 5 rnin and then decreased at a constant rate, and at 3 h only trace amounts were detected. ATP and phosphocreatine fell at comparable rates; after 5 rnin the values were only about half the initial level, and after 1 h less than 10% was left. Changes in metabolites during incubation in nitrogen
When synaptosomes ( 3 separate preparations) were incubated in a nitrogen-saturated medium in the absence of added energy substrates, glycogen fell to half the initial concentration in 10 min, then decreased more slowly (Fig. 2). After an initial lag of 1 rnin the lactate concentration rose and the increase was roughly proportional to the decrease in glycogen. ATP and phosphocreatine fell rapidly during the first min reaching 30 % and 15% of the respective initial levels. After 10-15 min, there was less than 10% of these substrates left. An approximate metabolic rate can be calculated for the synaptosomes from the rate of use of highenergy phosphate (-P) seen as changes in ATP, phosphocreatine, glucose and glycogen (see e.g. Harkonen et al. 1969). During the first rnin of anoxia -P was used at a rate of 10 mmol/kg prot./min. If 14-79588 1
Synaptosomes were incubated with constant oxygenation in a medium containing 10 mmol/l glucose alone or 10 mmol/l glucose and 5 mmol/l ADP in order to study their ability to synthesize highenergy phosphate. Neither alone nor with ADP could glucose maintain the initial level of these high-energy phosphate compounds (Fig. 3). However, in the presence of glucose the decrease was smaller than during endogenous respiration, e.g. ATP decreased by 1.3 mmol compared with 2.2 mmol, and phosphocreatine by 0.3 mmol compared with 1.7 mmol per kg protein during the first 15 rnin (values are means of 2 separate expts.). Furthermore, after this initial fall, the level of both compounds remained the same (ATP 1.0 and phosphocreatine 0.8 mmol/kg prot.) for 1 h while during endogenous respiration, high-energy phosphates were practically exhausted. The presence of 10 mmol/l glucose could not prevent the concentrations of ADP and AMP from decreasing during incubation, especially during the first 15 rnin (Fig. 4A). In Fig. 4 B (incubation medium supplemented with 10 mmol glucose + 5 mmol/l ADP) the linear decrease of ADP, and the linear increase of AMP, as a function of time, suggest the presence of enzymatic ADPase activity. Generation of high-energy phosphate was also studied in a Ca-free medium containing 20 mmol/l
t
4
INCUBATION TIME, MINUTES
Fig. 5 . Ammonia production in isolated nerve endings in
an electrolyte solution without added energy substrates in the presence or absence of oxygen at 37°C. Results of a
typical experiment. Acto Phr~sinlScand I07
S . - E . Jutisson et al.
210
80 1
Empty-.,.
were badly damaged (empty of vesicles). Whether performed in the absence or presence of oxygen, incubation had the same morphological effect.
Half-flied
DISCUSSION
I
J
I\---;----*/’- -.-.-.a
_./-
-.-
40 _._.-.-.-’-
---__---_ .
20
?I
30 WUBATION TIME, MINUTES
1 0I 60
Fig. 6 . The effect of incubation at 37°C in oxygenated electrolyte solution without added energy substrates on
the preservation of synaptosomes. The “filled” synaptosomes contain the normal quantity of vesicles (occupying 60-100% of the synaptosome area), those which are “half-filled” have lost about half their vesicles, and the “empty” synaptosomes contain few, if any, vesicles (occupying at most 20% of the synaptsome area). The points represent the result of two independent investigators.
During the first 15 min, 50% of the ATP and phosphocreatine were lost, but after this initial fall, there was no further loss of either compound. Amrnoniu production and vesicle preservation in incubated synaptosomes
For synaptosomes incubated in an oxygenated substrate-free medium ammonia production was, after an initial lag, about 1.5 mmol/kg prot./min during the first 30 min and then levelled off (Fig. 5 ) . Under nitrogen, ammonia production was much slower, averaging 0.4 mmol/kg prot./min (Fig. 5 ) . Synaptosomes incubated at 37°C without substrates showed clear-cut morphological alterations as compared with unincubated synaptosomes stored at 4°C on ice. The number of vesicles decreased drastically, and often enlarged vesicles as well as swollen intrasynaptosomal mitochondria could be seen. These changes were apparent after as little as 30 min, but after incubation for 1 h most synaptosomes contained hardly any intact vesicles and were filled with debris. These changes were evaluated semi-quantitatively (see Material and methods). At zero time well preserved synaptosomes and synaptosomes with a very low vesicular content were about equal in number (Fig. 6). However, after l h at 37°C only about 10% of the synaptosomes were in good condition and nearly 70%
Pattern of utilizution of energy reserves Despite the low concentration of endogenous substrates, the metabolic changes that occurred when the tissue was incubated in an oxygen or nitrogen atmosphere show that the synaptosomes are viable and utilize their energy reserves according to their requirements. In both conditions glycogen, ATP and phosphocreatine, the major energy-yielding compounds in nervous tissue (besides glucose, which in vivo originates from blood), were consumed in a pattern quite different from that seen in immature nerve cells (Hemminki & Harkonen 1974) or in the sympathetic ganglion (Harkonen et al. 1969). In synaptosomes, ATP and phosphocreatine were used rapidly in both the presence and the absence of oxygen, while in the other tissues they lasted much longer, especially in oxygen. The faster initial decline of glycogen in synaptosomes as compared to ganglion cells or in immature nerve cells could be due to the fact that they are practically devoid of endogenous glucose, which is the substrate utilized, initially at least, in the ganglion. In most of our synaptosome preparations, glycogen was detectable at substantial concentrations long after ATP and phosphocreatine were used up. It is obvious from the small decrease in glycogen after 15 min in oxygen that this is not the main substrate supporting the substantial respiration. However, a considerable amount of ammonia was produced between 15 and 30 min after the start of incubation and would explain the oxygen consumption, because most of the ammonia formed in nervous tissue during incubation in vitro originates from amino acids that are oxidatively deaminated (Weil-Malherbe 1962). Oxygen consumption of isolated nerve endings
It has been shown previously that synaptosomes have an adequate endogenous respiration rate, which can be stimulated by glucose or pyruvate (Bradford 1969, Verity 1972, Diamond & Fishman 1973, Bradford et al. 1978). With our technique, the endogenous respiration rate was higher than with manometric techniques (Bradford 1969) but, even in
Energj niettrbolism in .~yticiptosorne~ 21 1
aged synaptosomes, we were only occasionally able to show a slight stimulation of oxygen consumption by glucose. The insensitiveness of our synaptosome preparation to glucose is probably due to the short initial observation time during which they were still respiring on endogenous glycogen stores. The failure to increase respiration in aged synaptosomes, which had consumed most of their glycogen stores, may have been due to tissue catabolism and damage to vital enzymatic structures during the aging process. This explanation is supported by the observation that during the first 30 min of incubation, ammonia production was considerable. The insensitiveness of our synaptosome preparations to uncouplers apparently reflects the fact that in the complex metabolic system which the synaptosome represents, respiration is controlled at a step which is not affected by uncouplers. The stimulating effect of ADP on glucose respiration leads us to assume that ADP, despite its positive charge, does to some extent penetrate the synaptosomal membrane. Taking the ratio of the respiration rate in the presence of ADP to that in its absence as an indication of the respiratory control in the system (Chance & Williams 1956), a ratio of 1.7 is obtained with 5 mmol/l ADP which would suggest fair respiratory control. When intrasynaptosomal mitochondria have been isolated in Ficollsucrose media they have also shown good metabolic activity and respiratory control (Lai & Clark 1976).
Generation o f high-rnrrgy phosplzute3
Bradford (1969) reported that, with glucose as a substrate, synaptosomes synthesized both ATP and phosphocreatine. Closer scrutiny of his data, however, shows that actually he measured the levels of A T P and phosphocreatine in synaptosomes after incubation for 1 h in various media in the presence of metabolic poisons. In our generation experiments we followed the concentration of both compounds during the course of incubation. The rapid initial decrease in ATP concentration could be due to the membrane Na+-K+-ATPase ( Abdel-Latif & Abood 1964) activated by temperature and Na+ and K + in the incubation medium. The simultaneous decrease in phosphocreatine could thus be due to creatine kinase activity tending to synthesize A T P from ADP and phosphocreatine. After the initial fall
there was hardly any change in A T P o r phosphocreatine concentration during the next 2-3 h. Our interpretation of these results is that NA+-K+ATPase activity, creatine kinase activity, and oxidative phosphorylation supported by glucose (or glycogen), came to equilibrium, and A T P and phosphocreatine reached a steady-state-concentration in the synaptosomes. The AMP deaminase activity localized in the synaptosomes (Jansson & Harkonen, to be published) would explain the decreasing sum of adenylates during incubation.
Energy nzetabolisni und synaptic firnction
The function of nerve tissue, especially synaptic transmission (Larrabee & Klingman 1962, Harkonen et al. 1969), is very sensitive to glucose deprivation. It has been shown that failure of transmission occurs long before A T P and phosphocreatine are exhausted in the ganglion, and this seems to coincide with depletion of hexoses (Harkonen et al. 1969). Evidence also indicates that the locus of the failure is at the synaptic level and several explanations have been offered without direct experimental support (see Larrabee & Klingman 1962, Nicolescu et al. 1966, Harkonen et al. 1969). The present results show that 2 h after the start of incubation (when transmission through the ganglion is known to fail, Harkonen et al. 1969), the synaptosomes were depleted of A T P and phosphocreatine, although glycogen was still present. Therefore, blockage of impulse propagation at the synaptic level appears to be due to local depletion of A T P in the nerve endings, followed by failure of the ion pump. The other possible explanation would be the catabolism of tissue elements in the absence of glucose; this hypothesis gains support from the high production of ammonia and disappearance of vesicles containing the transmitter. It might well be that failure of chemical transmission is due to the two phenomena together. The earlier hypothesis of depletion of acetyl-CoA, suggested because failure of transmission coincides with depletion of hexoses, seems no longer tenable (Harkonen et al. 1969).
This work was supported by grants from the Medical Research Council in the Academy of Finland, the Sigrid Juselius Foundation. Helsinki, and the Finska Lakaresallskapet . Actn Pliv.%iolScotid 107
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.IULI. A-.
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