Neurochemical Research (1)233-249 (1976)
R E L E A S E OF S E R O T O N I N F R O M N E R V E ENDINGS BY L-5-HY D R O X Y T R Y P T O P H A N , a-METHYL-m-TYRAMINE, AND ELE V A T E D POTASSIUM IONS W. J. MCBRIDE AND M. H. APR~SON Section of Basic Neural Sciences The Institute of Psychiatric Research and Departments of Psychiatry and Biochemistry Indiana University Medical Center Indianapolis, Indiana 46202
Accepted February 10, 1976
The release of [3H]5-hydroxytryptamine ([3H]5-HT) by L-5-hydroxytryptophan (L-5-HTP), c~-methyl-m-tyramine (c~-MMTA), and elevated levels of K + was studied using crude synaptosomal preparations (Pz) isolated from the telencephalon of the rat and pigeon. Studies were conducted in vitro in the presence of either 2x 10-5 M tranylcypromine, which inhibited the MAO activity of both the extrasynaptosomal mitochondria and the mitochondria contained within the nerve endings (intrasynaptosomal mitochondria), or 2x 10-5 M nialamide, which inhibited the M A O activity of the extrasynaptosomal mitochondria under the experimental conditions used. In the Pz fraction isolated from the rat, either 55 mM K +, 0.10 mM L-5-HTP, or 0.03 mM a - M M T A significantly increased the release of [~H]5-HT above control levels, regardless of which MAO inhibitor was present in the medium. In the presence of tranylcypromine, this increased release by 55 mM K + or 0.10 mM L-5-HTP was partially suppressed if Ca z+ was omitted from the medium. In the presence of nialamide, the release by 55 mM K + was completely prevented if Ca -~+ was omitted; the release by L-5-HTP was only partially affected. The release of [3H]5-HT by ~ - M M T A did not appear to be markedly affected by removal of Ca 2+, regardless of which MAO inhibitor was present. Very similar data were obtained in the presence of nialamide using the P2 fraction isolated from the telencephalon of the pigeon, with the exception that 0.10 mM L-5-HTP caused an increase in the release of [3H]5-HIAA (which was not calcium-dependent) instead of [3H]5-HT. The data are discussed in
233 O I976 Plermm Publishing Corporation, 227 West I7th Street, New York, N.Y. 1001t. No part of this publication may be reproduced, stored in a retrieval system, or transmitted ill any form or by any means, electronic, mechanical photocopying, microfilming, recording, or otherwise, without wlitten permission of the publisher.
234
McBRIDE AND APRISON
terms of an intrasynaptosomal storage pool of 5-HT, which is released extraneuronally by a calcium-dependent process.
INTRODUCTION In studies involving the search for neurochemical correlates of behaviors exhibited by animals, Aprison and coworkers (1,2) have shown that intramuscularly administered D,L-5-hydroxytryptophan (which raises the tissue levels of serotonin) or ot-methyl-m-tyrosine '(which decreases the tissue levels of serotonin) produced the same behavioral depression (reduced responding rates) in pigeons or rats working on an approach schedule of reinforcement. In order to explain why the same behavioral effect was observed when the levels of total serotonin (5-HT) changed in opposite directions, Hingtgen and Aprison (3) and Aprison and Hingtgen (1,2,4) postulated that, in both cases, the physiologically effective pool of 5-HT (that available for release into the synaptic cleft plus that already released into the cleft) increased, and it was this 5-HT acting on certain " k e y " synapses that caused the observed depression in the animal's behavior. Recent studies by McBride and Aprison (5) and McBride et al. (6,7) provided evidence to support this hypothesis. The latter studies demonstrated that, in vitro, either a-methyl-m-tyramine (a major metabolite of o~-methyl-m-tyrosine) or L-5-hydroxytryptophan (L5-HTP) could cause a release of radioactive 5-HT from isolated preparations of nerve endings (P~). This study was undertaken to provide information on the mechanism of release of 5-HT from nerve endings by 5-HTP, c~-methyl-m-tyramine (o~-MMTA), and membrane-depolarizing conditions, as caused by elevated levels of potassium ions.
EXPERIMENTAL PROCEDURE Male and female pigeons (white Carneaux, 8-9 mo old, wt approx. 0.5 kg) and female Wistar rats, wt approx. 200 g, were used in these studies. The telencephalon was used for the preparation of the crude synaptosomal fraction (P2) according to a procedure based on that of Gray and Whittaker (8) as previously described (6,9). Incubation Conditions. The Pz fraction was resuspended in standard bathing medium to give a protein concentration of approximately 2-3 mg/ml. This medium is similar to that previously described by Mcllwain et al. (10) and consisted of (in mM): 120 NaC1, 4.75 KC1, 1.2 K H 2 P Q , 1.2 MgSO4, 0.75 CaCI2, 25 NaHCO3, and 10 o-glucose in equilibrium with 95% 02-5% CO2. An MAO inhibitor, either 2• 10-5 M tranylcypromine (Smith, Kline & French Labs., Phila., Pa.) or 2• -5 M nialamide (Chas. Pfizer & Co., New York), was present in the medium during all the incubation and washing procedures unless otherwise noted. The presence of an MAO inhibitor is necessary when using the P2
RELEASE OF SEROTONIN FROM NERVE ENDINGS
235
fraction to study effiux of [3H]5-HT, since the free mitochondria present in this fraction would readily convert the released [3H]5-HT to [3H]5-HIAA. The suspension was transferred in 1-rnl portions to separate test tubes (13• ram). Samples were then placed in a water bath at 37~ for 5 rain prior to the addition of 0.20 /~Ci [~H]5-hydroxytryptamine (sp act 6.8 Ci/mmol; Amersham-Searle, Arlington Heights, Ill.) to give a final concentration of 2.5• -8 M in 1.20 ml of incubation medium. Suspensions were then incubated for 10 rain at 37~ under a continuous stream of 95% 025% COz. Detailed procedures for termination of the incubation and subsequent washing steps for preparation of the synaptosomal fraction for efflux studies were previously published (5). The method for studying the effiux of radioactive 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) has also been described in detail elsewhere (6). For the efflux studies, samples were taken at " 0 " and 10 rain; the amount of [~H]5-HT and [3H]5-HIAA appearing in the medium at the " 0 " time point was then subtracted from values obtained at 10 rain for samples obtained from the particulate and medium. Separation of the incubated P2 fraction (particulate fraction) from the incubation medium was achieved by rapid, highspeed centrifugation (6). The procedure of Kariya and Aprison (11) was used to separate and isolate radioactive 5-HT and 5-HIAA; the adopted procedure, as used in the present experiments, has also been described elsewhere in detail (5). In those cases where the particle-bound radioactivity was to be determined, the pellet was resuspended, after incubation, in 0.400 ml 3% Triton X-100 in 0.20 M phosphate buffer (pH 6.1), and was then taken through the ion-exchange column chromatography procedure. Synaptosomal Distribution of [3H]5-HT in the Incubated and Lysed P2 Fraction. In these experiments, the telencephalons from 4 rats were used to prepare the crude synaptosomal fraction (P2). The P~ pellets were resuspended and incubated as described above, except that the concentration of [aH]5-HT was 5• 10-8 M. After incubation, the pellets were washed by a procedure that included resuspension and centrifugation in 0.32 M sucrose to remove extrasynaptosomal ions that might interfere with the separation of the subsynaptosomal fractions. This procedure was described previously (6). At the step where the pellets were washed with 0.32 M sucrose, the suspensions were pooled to give 2 samples (each sample containing the incubated Pz fractions isolated from 2 telencephalons). The resulting washed pellets were resuspended in ice-cold H~O (adjusted to pH 7.07.4 with dilute NaOH) and osmotically shocked (12). This suspension was then layered over a discontinuous gradient consisting of 6 ml 0.20 M, 6 ml 0.40 M, 6 ml 0.81 M, and 8 ml 1.26 M sucrose. The pH of the sucrose solutions had been previously adjusted to 7.07.4. Separation of the subsynaptosomal components was achieved by centrifuging the SW 27 rotor (Beckman Instruments) for 105 min at 75,000g, as previously described (13). The upper water layer contained soluble material; synaptic vesicles were found at the interface of the 0.20 M and 0.40 M sucrose layers (13-15). The addition of the 0.20 M layer in the gradient was necessary to prevent contamination of the synaptic vesicle fraction by the soluble fraction. The fraction at the interface of the 0.40 M and 0.81 M sucrose layers appears to contain predominantly myelin and synaptic plasma membranes (9,15,16). Synaptic plasma membranes (SPM) and unlysed or partially lysed nerve endings are found at the interface of the 0.81 M and 1.26 M sucrose layers (9,12,15,16). Mitochondria sediment through the 1.26 M sucrose layer (9,12,13). Morphological examination of subcellular fractions from incubated P2 fractions was done with a Hitachi HS-7 electron microscope. Samples were prepared by standard procedures (9), which included fixation in gluteraldehyde, postfixation with O s Q , dehydration, staining with uranyl acetate, and embedding in Epon. Silver to gold sections were prepared, mounted, and counterstained with lead citrate. All radioactivity measurements were made with a scintillation counter (Packard Tricarb,
236
McBRIDE AND A P R I S O N
Model 3375, Packard Instrument Co., Inc., Downers Grove, Ill.) equipped with automatic external standardization to monitor counting efficiency. Protein determinations were done with the procedure of Lowry et al. (17). Statistical analysis was done with the standard Student t test. There were small differences in the control values from day to day. Therefore, statistical analyses were done on data obtained in the same expedment for test samples and control samples.
RESULTS
Effects of Nialamide and Tranylcypromine on Uptake and Metabolism of [3H]5-HT The uptake and metabolism of [3H]5-HT into the P2 fractions of rat and pigeon were studied in the presence and absence of two inhibitors of monoamine oxidase (MAO) activity (Table I). The concentration of inhibitors chosen was 2• -5 M, since this concentration of nialamide had previously been reported t6 prevent the metabolism of [~H]5-HT in tissue slices (19). However, with our experimental conditions, nialamide had no apparent effect on the accumulation of [~H]5-HIAA from [3H]5HT in the P2 fraction isolated from the telencephalon of the rat or pigeon. On the other hand, 2x10 -5 M nialamide inhibited the M A O activity of the extrasynaptosomal mitochondria (mitochondria present in the P2 fraction that are not enclosed within the pinched-off nerve endings) approximately 90%. Tranylcypromine appeared to be a very effective inhibitor of [~H]5-HIAA formation (95%) in the P2 fraction and by the extrasynaptosomal mitochondria. Neither nialamide nor tranylcypromine appeared to inhibit the uptake or increase the release of [3H]5-HT, since the amount of [3H]5-HT accumulated in the P2 fraction in the presence of either drug was not less than the amount accumulated in the absence of drug.
Subcellular Distribution of [3H]5-HT and [aH]5-HIAA in the Lysed Pz Preliminary experiments were carried out to determine whether the [~H]5-HT accumulated within the P2 fraction could be found bound to synaptic vesicles (Table II). These experiments were done using the P2 fraction isolated from the telencephalon of the rat. When the P2 fraction was incubated with [3H]5-HT in the presence of nialamide and subsequently lysed and fractionated by centrifugation through discontinuous sucrose gradients, the distribution of [3H]5-HT was approximately 50% soluble and 50% particulate; the distribution of [~H]5-HIAA in contrast,
RELEASE OF SEROTONIN FROM NERVE ENDINGS
237
TABLE I EFFECTS OF NIALAMIDE AND TRANYLCYPROMINE ON UPTAKE AND METABOLISM OF [3H]5-HYDROXYTRYPTAMINE USING CRUDE SYNAPTOSOMAL PREPARATION (])2) AND ON M A O ACTIVITY OF EXTRASYNAPTOSOMAE MITOCHONDRIA ISOLATED FROM R A T AND PIGEON TELENCEPHALONS a
5-HT Inhibitor Rat None 2 x 10 -5 2 x 10-5 Pigeon None 2 • 10-5 2 x 10 -5
5-HIAA
d p m / m g protein in P2 ~
M nialamide M tranylcypromine
21,860 -+ 4,180 27,760 -+ 4,450 33,760 -+ 7,540
18,600 -+ 780 19,730 -+ 380 840 -+ 87 ~
M nialamide M tranylcypromine
23,200 -+ 2,400 14,230 -+ 890 27,360 --- 2,280 14,600 -+ 700 48,260 -+ 2,930 b 990 -+ 310 c
M A O activity of extrasynaptosomal mitochondria (pmole 5 - H I A A formed/30 min/mg protein) b
35.0 + 0.7 (4) 3.8 -+ 0.5 ~ 0.03 -+ 0.03 c 36.2 -+ 1.8 (4) 4.4 -+ 0.8 c 1.8 -+ 0.7 c
a T h e P2 fraction was isolated and incubated as previously described (5-7). Samples were preincubated for 5 min at 37~ in the p r e s e n c e of the inhibitor before addition of [3H]5h y d r o x y t r y p t a m i n e to give a final concentration of 2.5 • 10-8 M. Samples were then incubated for 10 rain at 37~ T h e P~ s u s p e n s i o n s were then centrifuged and the radioactive 5 - H T and 5 - H I A A were determined in the particulate fraction (Pz), which contained myelinated a x o n s , m e m b r a n e s , synaptosornes (some of which contain mitochondria, hereafter designated as i n t r a s y n a p t o s o m a l mitochondria), and e x t r a s y n a p t o s o mal mitochondria. T h e e x t r a s y n a p t o s o m a l mitochondria were isolated in a separate e x p e r i m e n t by centrifuging the P2 fraction t h r o u g h a d i s c o n t i n u o u s gradient of 0.80 M and 1.30 M s u c r o s e according to the procedure o f Gray and W h i t t a k e r (8). The e x t r a s y n a p t o s o m a l mitochondria sedimented t h r o u g h the 1.30 M s u c r o s e layer and formed a pellet at the b o t t o m o f the centrifuge tube. M A O activity was determined using this r e s u s p e n d e d pellet with the p r o c e d u r e of M c C a m a n et al. (18), using [*H]5-HT as the substrate at a concentration of 2.5 x 10 -s M. b Data r e p r e s e n t the m e a n s -+so o f 3 determinations unless otherwise noted. c Significance of differences between control and drug-treated preparations is as follows: P < 0.005.
was 86% soluble and only 14% particulate. With respect to the particulate-bound [3H]5-HT, significant radioactivity was found in the synaptic vesicle and myelin membrane fractions, but only relatively low levels were found in the synaptic plasma membrane (SPM) and mitochondrial fractions. The specific radioactivity (dpm/mg protein) of [3H]5-HT in the synaptic vesicle fraction was 3 times greater than the specific radioactivity of [3H]5-HT in the soluble fraction, which had the next highest value. On the other hand, the specific radioactivity of [3H]5-HIAA in the
238
McBRIDE
AND
APRISON
r ,.d O
b., O
b-,
uz b.,
,..]
z~ >., 9
R E L E A S E OF S E R O T O N I N F R O M N E R V E ENDINGS BY L-5-HY D R O X Y T R Y P T O P H A N , a-METHYL-m-TYRAMINE, AND ELE V A T E D POTASSIUM IONS W. J. MCBRIDE AND M. H. APR~SON Section of Basic Neural Sciences The Institute of Psychiatric Research and Departments of Psychiatry and Biochemistry Indiana University Medical Center Indianapolis, Indiana 46202
Accepted February 10, 1976
The release of [3H]5-hydroxytryptamine ([3H]5-HT) by L-5-hydroxytryptophan (L-5-HTP), c~-methyl-m-tyramine (c~-MMTA), and elevated levels of K + was studied using crude synaptosomal preparations (Pz) isolated from the telencephalon of the rat and pigeon. Studies were conducted in vitro in the presence of either 2x 10-5 M tranylcypromine, which inhibited the MAO activity of both the extrasynaptosomal mitochondria and the mitochondria contained within the nerve endings (intrasynaptosomal mitochondria), or 2x 10-5 M nialamide, which inhibited the M A O activity of the extrasynaptosomal mitochondria under the experimental conditions used. In the Pz fraction isolated from the rat, either 55 mM K +, 0.10 mM L-5-HTP, or 0.03 mM a - M M T A significantly increased the release of [~H]5-HT above control levels, regardless of which MAO inhibitor was present in the medium. In the presence of tranylcypromine, this increased release by 55 mM K + or 0.10 mM L-5-HTP was partially suppressed if Ca z+ was omitted from the medium. In the presence of nialamide, the release by 55 mM K + was completely prevented if Ca -~+ was omitted; the release by L-5-HTP was only partially affected. The release of [3H]5-HT by ~ - M M T A did not appear to be markedly affected by removal of Ca 2+, regardless of which MAO inhibitor was present. Very similar data were obtained in the presence of nialamide using the P2 fraction isolated from the telencephalon of the pigeon, with the exception that 0.10 mM L-5-HTP caused an increase in the release of [3H]5-HIAA (which was not calcium-dependent) instead of [3H]5-HT. The data are discussed in
233 O I976 Plermm Publishing Corporation, 227 West I7th Street, New York, N.Y. 1001t. No part of this publication may be reproduced, stored in a retrieval system, or transmitted ill any form or by any means, electronic, mechanical photocopying, microfilming, recording, or otherwise, without wlitten permission of the publisher.
234
McBRIDE AND APRISON
terms of an intrasynaptosomal storage pool of 5-HT, which is released extraneuronally by a calcium-dependent process.
INTRODUCTION In studies involving the search for neurochemical correlates of behaviors exhibited by animals, Aprison and coworkers (1,2) have shown that intramuscularly administered D,L-5-hydroxytryptophan (which raises the tissue levels of serotonin) or ot-methyl-m-tyrosine '(which decreases the tissue levels of serotonin) produced the same behavioral depression (reduced responding rates) in pigeons or rats working on an approach schedule of reinforcement. In order to explain why the same behavioral effect was observed when the levels of total serotonin (5-HT) changed in opposite directions, Hingtgen and Aprison (3) and Aprison and Hingtgen (1,2,4) postulated that, in both cases, the physiologically effective pool of 5-HT (that available for release into the synaptic cleft plus that already released into the cleft) increased, and it was this 5-HT acting on certain " k e y " synapses that caused the observed depression in the animal's behavior. Recent studies by McBride and Aprison (5) and McBride et al. (6,7) provided evidence to support this hypothesis. The latter studies demonstrated that, in vitro, either a-methyl-m-tyramine (a major metabolite of o~-methyl-m-tyrosine) or L-5-hydroxytryptophan (L5-HTP) could cause a release of radioactive 5-HT from isolated preparations of nerve endings (P~). This study was undertaken to provide information on the mechanism of release of 5-HT from nerve endings by 5-HTP, c~-methyl-m-tyramine (o~-MMTA), and membrane-depolarizing conditions, as caused by elevated levels of potassium ions.
EXPERIMENTAL PROCEDURE Male and female pigeons (white Carneaux, 8-9 mo old, wt approx. 0.5 kg) and female Wistar rats, wt approx. 200 g, were used in these studies. The telencephalon was used for the preparation of the crude synaptosomal fraction (P2) according to a procedure based on that of Gray and Whittaker (8) as previously described (6,9). Incubation Conditions. The Pz fraction was resuspended in standard bathing medium to give a protein concentration of approximately 2-3 mg/ml. This medium is similar to that previously described by Mcllwain et al. (10) and consisted of (in mM): 120 NaC1, 4.75 KC1, 1.2 K H 2 P Q , 1.2 MgSO4, 0.75 CaCI2, 25 NaHCO3, and 10 o-glucose in equilibrium with 95% 02-5% CO2. An MAO inhibitor, either 2• 10-5 M tranylcypromine (Smith, Kline & French Labs., Phila., Pa.) or 2• -5 M nialamide (Chas. Pfizer & Co., New York), was present in the medium during all the incubation and washing procedures unless otherwise noted. The presence of an MAO inhibitor is necessary when using the P2
RELEASE OF SEROTONIN FROM NERVE ENDINGS
235
fraction to study effiux of [3H]5-HT, since the free mitochondria present in this fraction would readily convert the released [3H]5-HT to [3H]5-HIAA. The suspension was transferred in 1-rnl portions to separate test tubes (13• ram). Samples were then placed in a water bath at 37~ for 5 rain prior to the addition of 0.20 /~Ci [~H]5-hydroxytryptamine (sp act 6.8 Ci/mmol; Amersham-Searle, Arlington Heights, Ill.) to give a final concentration of 2.5• -8 M in 1.20 ml of incubation medium. Suspensions were then incubated for 10 rain at 37~ under a continuous stream of 95% 025% COz. Detailed procedures for termination of the incubation and subsequent washing steps for preparation of the synaptosomal fraction for efflux studies were previously published (5). The method for studying the effiux of radioactive 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) has also been described in detail elsewhere (6). For the efflux studies, samples were taken at " 0 " and 10 rain; the amount of [~H]5-HT and [3H]5-HIAA appearing in the medium at the " 0 " time point was then subtracted from values obtained at 10 rain for samples obtained from the particulate and medium. Separation of the incubated P2 fraction (particulate fraction) from the incubation medium was achieved by rapid, highspeed centrifugation (6). The procedure of Kariya and Aprison (11) was used to separate and isolate radioactive 5-HT and 5-HIAA; the adopted procedure, as used in the present experiments, has also been described elsewhere in detail (5). In those cases where the particle-bound radioactivity was to be determined, the pellet was resuspended, after incubation, in 0.400 ml 3% Triton X-100 in 0.20 M phosphate buffer (pH 6.1), and was then taken through the ion-exchange column chromatography procedure. Synaptosomal Distribution of [3H]5-HT in the Incubated and Lysed P2 Fraction. In these experiments, the telencephalons from 4 rats were used to prepare the crude synaptosomal fraction (P2). The P~ pellets were resuspended and incubated as described above, except that the concentration of [aH]5-HT was 5• 10-8 M. After incubation, the pellets were washed by a procedure that included resuspension and centrifugation in 0.32 M sucrose to remove extrasynaptosomal ions that might interfere with the separation of the subsynaptosomal fractions. This procedure was described previously (6). At the step where the pellets were washed with 0.32 M sucrose, the suspensions were pooled to give 2 samples (each sample containing the incubated Pz fractions isolated from 2 telencephalons). The resulting washed pellets were resuspended in ice-cold H~O (adjusted to pH 7.07.4 with dilute NaOH) and osmotically shocked (12). This suspension was then layered over a discontinuous gradient consisting of 6 ml 0.20 M, 6 ml 0.40 M, 6 ml 0.81 M, and 8 ml 1.26 M sucrose. The pH of the sucrose solutions had been previously adjusted to 7.07.4. Separation of the subsynaptosomal components was achieved by centrifuging the SW 27 rotor (Beckman Instruments) for 105 min at 75,000g, as previously described (13). The upper water layer contained soluble material; synaptic vesicles were found at the interface of the 0.20 M and 0.40 M sucrose layers (13-15). The addition of the 0.20 M layer in the gradient was necessary to prevent contamination of the synaptic vesicle fraction by the soluble fraction. The fraction at the interface of the 0.40 M and 0.81 M sucrose layers appears to contain predominantly myelin and synaptic plasma membranes (9,15,16). Synaptic plasma membranes (SPM) and unlysed or partially lysed nerve endings are found at the interface of the 0.81 M and 1.26 M sucrose layers (9,12,15,16). Mitochondria sediment through the 1.26 M sucrose layer (9,12,13). Morphological examination of subcellular fractions from incubated P2 fractions was done with a Hitachi HS-7 electron microscope. Samples were prepared by standard procedures (9), which included fixation in gluteraldehyde, postfixation with O s Q , dehydration, staining with uranyl acetate, and embedding in Epon. Silver to gold sections were prepared, mounted, and counterstained with lead citrate. All radioactivity measurements were made with a scintillation counter (Packard Tricarb,
236
McBRIDE AND A P R I S O N
Model 3375, Packard Instrument Co., Inc., Downers Grove, Ill.) equipped with automatic external standardization to monitor counting efficiency. Protein determinations were done with the procedure of Lowry et al. (17). Statistical analysis was done with the standard Student t test. There were small differences in the control values from day to day. Therefore, statistical analyses were done on data obtained in the same expedment for test samples and control samples.
RESULTS
Effects of Nialamide and Tranylcypromine on Uptake and Metabolism of [3H]5-HT The uptake and metabolism of [3H]5-HT into the P2 fractions of rat and pigeon were studied in the presence and absence of two inhibitors of monoamine oxidase (MAO) activity (Table I). The concentration of inhibitors chosen was 2• -5 M, since this concentration of nialamide had previously been reported t6 prevent the metabolism of [~H]5-HT in tissue slices (19). However, with our experimental conditions, nialamide had no apparent effect on the accumulation of [~H]5-HIAA from [3H]5HT in the P2 fraction isolated from the telencephalon of the rat or pigeon. On the other hand, 2x10 -5 M nialamide inhibited the M A O activity of the extrasynaptosomal mitochondria (mitochondria present in the P2 fraction that are not enclosed within the pinched-off nerve endings) approximately 90%. Tranylcypromine appeared to be a very effective inhibitor of [~H]5-HIAA formation (95%) in the P2 fraction and by the extrasynaptosomal mitochondria. Neither nialamide nor tranylcypromine appeared to inhibit the uptake or increase the release of [3H]5-HT, since the amount of [3H]5-HT accumulated in the P2 fraction in the presence of either drug was not less than the amount accumulated in the absence of drug.
Subcellular Distribution of [3H]5-HT and [aH]5-HIAA in the Lysed Pz Preliminary experiments were carried out to determine whether the [~H]5-HT accumulated within the P2 fraction could be found bound to synaptic vesicles (Table II). These experiments were done using the P2 fraction isolated from the telencephalon of the rat. When the P2 fraction was incubated with [3H]5-HT in the presence of nialamide and subsequently lysed and fractionated by centrifugation through discontinuous sucrose gradients, the distribution of [3H]5-HT was approximately 50% soluble and 50% particulate; the distribution of [~H]5-HIAA in contrast,
RELEASE OF SEROTONIN FROM NERVE ENDINGS
237
TABLE I EFFECTS OF NIALAMIDE AND TRANYLCYPROMINE ON UPTAKE AND METABOLISM OF [3H]5-HYDROXYTRYPTAMINE USING CRUDE SYNAPTOSOMAL PREPARATION (])2) AND ON M A O ACTIVITY OF EXTRASYNAPTOSOMAE MITOCHONDRIA ISOLATED FROM R A T AND PIGEON TELENCEPHALONS a
5-HT Inhibitor Rat None 2 x 10 -5 2 x 10-5 Pigeon None 2 • 10-5 2 x 10 -5
5-HIAA
d p m / m g protein in P2 ~
M nialamide M tranylcypromine
21,860 -+ 4,180 27,760 -+ 4,450 33,760 -+ 7,540
18,600 -+ 780 19,730 -+ 380 840 -+ 87 ~
M nialamide M tranylcypromine
23,200 -+ 2,400 14,230 -+ 890 27,360 --- 2,280 14,600 -+ 700 48,260 -+ 2,930 b 990 -+ 310 c
M A O activity of extrasynaptosomal mitochondria (pmole 5 - H I A A formed/30 min/mg protein) b
35.0 + 0.7 (4) 3.8 -+ 0.5 ~ 0.03 -+ 0.03 c 36.2 -+ 1.8 (4) 4.4 -+ 0.8 c 1.8 -+ 0.7 c
a T h e P2 fraction was isolated and incubated as previously described (5-7). Samples were preincubated for 5 min at 37~ in the p r e s e n c e of the inhibitor before addition of [3H]5h y d r o x y t r y p t a m i n e to give a final concentration of 2.5 • 10-8 M. Samples were then incubated for 10 rain at 37~ T h e P~ s u s p e n s i o n s were then centrifuged and the radioactive 5 - H T and 5 - H I A A were determined in the particulate fraction (Pz), which contained myelinated a x o n s , m e m b r a n e s , synaptosornes (some of which contain mitochondria, hereafter designated as i n t r a s y n a p t o s o m a l mitochondria), and e x t r a s y n a p t o s o mal mitochondria. T h e e x t r a s y n a p t o s o m a l mitochondria were isolated in a separate e x p e r i m e n t by centrifuging the P2 fraction t h r o u g h a d i s c o n t i n u o u s gradient of 0.80 M and 1.30 M s u c r o s e according to the procedure o f Gray and W h i t t a k e r (8). The e x t r a s y n a p t o s o m a l mitochondria sedimented t h r o u g h the 1.30 M s u c r o s e layer and formed a pellet at the b o t t o m o f the centrifuge tube. M A O activity was determined using this r e s u s p e n d e d pellet with the p r o c e d u r e of M c C a m a n et al. (18), using [*H]5-HT as the substrate at a concentration of 2.5 x 10 -s M. b Data r e p r e s e n t the m e a n s -+so o f 3 determinations unless otherwise noted. c Significance of differences between control and drug-treated preparations is as follows: P < 0.005.
was 86% soluble and only 14% particulate. With respect to the particulate-bound [3H]5-HT, significant radioactivity was found in the synaptic vesicle and myelin membrane fractions, but only relatively low levels were found in the synaptic plasma membrane (SPM) and mitochondrial fractions. The specific radioactivity (dpm/mg protein) of [3H]5-HT in the synaptic vesicle fraction was 3 times greater than the specific radioactivity of [3H]5-HT in the soluble fraction, which had the next highest value. On the other hand, the specific radioactivity of [3H]5-HIAA in the
238
McBRIDE
AND
APRISON
r ,.d O
b., O
b-,
uz b.,
,..]
z~ >., 9
