Neuroscience Letters, 122 (1991) 83-86
83
Elsevier Scientific Publishers Ireland Ltd. NSL 07469
Neurocatin induces changes in release and level of serotonin in synaptosomal fraction from rat brain Lucia Fernandez-Novoa and Anna Pastuszko Department of Biochemistry and Biophysics, Medical School, University of Pennsylvania, Philadelphia, PA 19104 (U.S.A.)
(Received 2 May 1990; Revisedversion received 10 August 1990; Accepted 3 October 1990) Key words: Neurocatin; Serotonin; 5-Hydroxyindoleaceticacid; Rat brain; Synaptosome
A newly isolated factor from mammalian brain, neurocatin, is shown to increase both the level and release of serotonin in suspensions of synaptosomes isolated from rat brain. Incubation of synaptosomes for 10 min with approximately 20 nM neurocatin resulted in release of about 50% of the total pool of serotonin. Quantification of serotonin and its major metabolite, using an electrochemicaldetector, indicated that the presence of neurocatin also caused an increase in the absolute level of serotonin and decrease in its catabolism to 5-hydroxyindoleaceticacid (5-HIAA). The effect is dependent on the time of incubation and concentration of neurocatin. At a concentration of 20 nM, neurocatin increased about 60% the level of serotonin and decreased about 50% the level of 5-HIAA. Depolarization conditions - - 50 gM veratridine and 50 mM K ÷ medium - increased release of serotonin by 50% and 30% respectivelywithout affectingthe level of serotonin or its catabolism to 5-HIAA.
In earlier reports we presented evidence that bovine brain contains a factor which, when added to rat brain synaptosomes, causes decreased formation of 3,4-dihydroxyphenylacetic acid, increased formation of norepinephrine and its N-methyl derivatives and increased release of catecholamines [6]. This factor (neurocatin) has now been purified to a single chromatographic peak by high resolution H P L C . It is a small peptide with a molecular weight in the range of 2,000 to 2,500 Da [10]. In the present paper, it is reported that addition of neurocatin to synaptosomes from rat brain stimulated release of serotonin and increased the level of this monoamine. It also decreased ~production of the catabolic product of serotonin, 5-hydroxyindoleacetic acid (5-HIAA). Male Sprague-Dawley rats, approximately 200g, were used throughout the study. Crude synaptosomal preparations (P2 fraction) were prepared from homogenates of combined cerebral hemispheres and brain stem in buffered 0.25 M sucrose solution by centrifugation of the initial low speed supernatant (3 min at 1300 g) for 10 min at 17,000 g. The final synaptosomal pellets were suspended at approximately 6 mg protein/ml in modified Krebs-Henseleit buffer (140 m M NaC1, 5 m M KCI, 1.3 m M MgSO4, 10 m M TrisHEPES, 1 m M Na2HPO4; p H 7.4) containing 10 m M glucose and 1 m M CaCI2 and incubated aerobically for Correspondence: A. Pastuszko, Department of Biochemistry and Biophysics, Medical School, University of Pennsylvania, Philadelphia, PA 19104, U.S.A.
0304-3940/91/$ 03.50 © 1991 ElsevierScientific Publishers Ireland Ltd.
1, 2.5, 5 and 10 min in a Dubnoff metabolic shaker bath at 37°C. The effect of neurocatin was tested by adding approximately 0 (control), 5, 10, 20 and 40 nM neurocatin to the incubation medium. At the end of the incubation periods, the reactions were quenched by adding cold trichloroacetic acid (TCA) to a final concentration o f 1% by weight. The samples were then centrifuged to remove the precipitated material. The clear supernatants were analyzed by high pressure liquid chromatography [3, 4]. In some experiments, synaptosomes were incubated as described above but also in the presence of 3/iCi/ml of [3H]serotonin (5hydroxy G-[3H]tryptamine creatinine sulphate), Amersham, specific activity 12.5 Ci/mmol. In addition to measuring the changes in the level of serotonin and 5H I A A by an electrochemical detector, the eluate from the chromatographs was collected in 0.15 ml fractions and the radioactivity in each fraction was measured. The neurocatin used during this study was obtained after elution from a semipreparative column (last step before final purification). Neurocatin in this fraction was 25-40% of the total protein measured by the absorption at 220 nm. The concentration o f the final product can only be estimated as the exact structure and molecular weight are not yet known. The concentrations of neurocatin are based upon an estimated molecular weight of 2,000 Da. Uptake of [aH]serotonin (0.4 /tM) was initiated by adding radioactive neurotransmitter to the synaptosomal suspension (approx. 1 mg protein) in Krebs-Hense-
84 leit buffer, pH 7.4 with 10 m M glucose and 1 m M CaC12, and with or without 20 nM neurocatin. At the end of incubation periods of 1-10 min, 100 pl samples were placed in 400 pl tubes containing 50 pl silicone oil (specific gravity 1.03) and rapidly centrifuged for 2 min (Backman Model B Microfuge). The radioactiviy in the supernatant and pellet fractions was determined. Release of serotonin was measured by incubating suspensions of synaptosomes at 6 mg protein/ml for 10 min at 37°C in a Krebs medium containing 10 m M glucose and 1 m M CaC12 with or without 5, 10, 20, or 40 nM neurocatin (all final concentrations). In some experiments, the incubation medium also contained 1 p M clorgyline, an inhibitor of monoamine oxidase, 50/~M veratridine or 50 m M [K +]. At the end of the incubation the samples were treated in one of two ways: (1) Aliquots (250/tl) were withdrawn, acidified with cold T C A (1.5 % final concentration) and centrifuged to remove the precipitated material. The acid-soluble fraction was used for determination of the total content of serotonin and 5H I A A by HPLC. (2) Aliquots (100/~1) were withdrawn and rapidly centrifuged (Beckman Model B Microfuge) through a layer of silicone oil into 100 pl of 1.5% TCA in 3 % NaCI. The top layer, which contained the extrasynaptosomal medium, was pipetted off, acidified with trichloroacetic acid to a final concentration of 1.5% and used to determine the content of serotonin and 5-HIAA by H P L C with electrochemical detection. The release experiments were designed in such a way (without reuptake inhibitors) that values may be affected by changes in reuptake as well as in synthesis of neurotransmitter.
Protein concentration was determined by the method of Lowry et al. [5] with bovine serum albumin as the standard. All data are presented as means _+ S.E.M. The results are compared using the paired t-test. When the data are presented as percent of control, the statistical analysis was carried out on the raw data. As may be seen in Fig. IA,B, 10 min incubation of synaptosomes in the presence of 20 nM neurocatin had marked effects on the level of serotonin and 5-HIAA. Neurocatin caused an increase of about 60 % in the level of serotonin and an about 50% decrease in the level of 5-HIAA. The c h r o m a t o g r a m presented in Fig. 1 is typical of those obtained from 11 experiments. The effect of neurocatin was examined also when the incubation medium contained [3H]serotonin. After 10 min incubation, the presence of 20 nM neurocatin in the incubation medium resulted in the radioactivity in the serotonin fraction being substantially greater than that in the control (169% of control) and decreased the radioactivity in the 5-HIAA fraction by about 37%. The changes in the levels of serotonin and 5-HIAA were dependent on the concentration of neurocatin (see Table I, total) and on the interval of incubation of synaptosomes with this peptide (Fig. 2A,B). The presence of 20 nM neurocatin resulted in an increase in serotonin which became statistically significant (P < 0.05) by 2.5 min and was significant at all longer times of incubation (Fig. 2A). The results are plotted in the inset of Fig. 2A as the increase in level of serotonin in the presence of 20 nM neurocatin relative to control. The changes in the level of 5-HIAA are shown in Fig. 2B with the differ-
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Fig. 1. The effect of neurocatin on the levels of serotonin and 5-HIAA in synaptosomes from rat brain. The synaptosomes were incubated for 10 min at 37°C with 0 (A) and 20 nM neurocatin (B). At the end of the incubation period, aliquots were withdrawn, acidified with TCA and centrifuged to remove the precipitated material. The acid extract was assayed by HPLC with electrochemical detector. The flow rate of 0.75 ml/min gave a typical retention time for 5-HIAA and serotonin (5-HT) of 8.1 and 13.6 min, respectively.The chromatogram in the figure is representativeof those from 11 independent experiments.
85 TABLE I EFFECT OF NEUROCATIN, DEPOLARIZATION AND CLORGYLINE ON THE LEVEL AND RELEASE OF SEROTONIN AND 5-HIAA IN RAT BRAIN SYNAPTOSOMES The synaptosomes (approximately 6 mg/ml) were incubated for 10 min in the presence of different concentrations of neurocatin or in the presence of I /tM clorgyline, 50 mM K÷-medium or 50/LM veratridine. The total concentration of serotonin and 5-HIAA was measured after quenching the sample directly with TCA, whereas the values for the supernatant are from experiments in which the synaptosomes were removed by centrifugation through a layer of silicon oil (see methods). The data are presented as the means ± S.E.M. for 5 experiments except for conditions marked with an asterisk, where 3 experiments are presented. Conditions
5-HT (pmol/mg protein)
Control Neurocatin (5 nM) Neurocatin (10 nM) Neurocatin (20 nM) Neurocatin (40 nM) Clorgyline (1/~M)* Veratridine (50/~M)* High K ÷ (50 mM)*
5-HIAA (pmol/mg protein)
Total
Supernatant
Total
Supernatant
22.7 + 4.1 22.9 ___2.0 28.4_ 1.7 36.9 _+2.1 44.1 _ 1.8 40.2 _ 0.9 17.5 ±_5.2 20.4+2.2
1.0 ±_0.6 1.7 + 0.5 2.7 + 0.9 18.4 + 3.1 28.7 5:4.2 5.5 5-0.6 8.4 ± 1.9 6.3±2.0
33. I _ 6.2 31.0 _ 3.1 27.8 _ 1.7 20.1 5- 0.9 9.3 + 1.6 14.1 5:2.2 34.7 ± 3.8 32.1 +2.0
23.0__+7.1 20.0 _ 4.1 19.0 + 4.7 12.4 _ 5.0 7.0 + 2.0 10.7 + 0.6 21.0 5- 4.5 19.0+3.1
ence (decrease) between the level of 5-HIAA in the presence and absence of 20 nM neurocatin plotted in the inset. The decrease in 5-HIAA was statistically significant by 5 min incubation time (P < 0.02). Similar effects of neurocatin were observed in synaptosomes specifically isolated from the brainstem, a region particularly rich in serotonergic neurons. After 20 min incubation, synaptosomes from brainstem contained 92 pmol serotonin/mg protein (control) while those incubated in the presence of 20 nM neurocatin contained 145 pmol serotonin/mg protein. The level of 5-HIAA was 171 pmol/mg protein in the controls and 40"
A
•
CONTROL
this decreased to 58 pmol/mg protein in the presence of neurocatin. Incubation of synaptosomes with neurocatin stimulated release of serotonin into the suspending medium, in a concentration dependent manner (Table I). Twenty nM neurocatin caused release of about 50 % of the total content of serotonin, whereas 40 nM released about 65 % of the total serotonin. The effect of neurocatin on the total level and on the release of serotonin can be compared with that induced by 50/~M veratridine or by 50 mM K+-medium. Depolarization of synaptosomal membranes with veratridine or high K ÷ caused release of about 50% and 31% of total serotonin, respectively. In contrast to the addition of neurocatin neither of these 40-
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83
Elsevier Scientific Publishers Ireland Ltd. NSL 07469
Neurocatin induces changes in release and level of serotonin in synaptosomal fraction from rat brain Lucia Fernandez-Novoa and Anna Pastuszko Department of Biochemistry and Biophysics, Medical School, University of Pennsylvania, Philadelphia, PA 19104 (U.S.A.)
(Received 2 May 1990; Revisedversion received 10 August 1990; Accepted 3 October 1990) Key words: Neurocatin; Serotonin; 5-Hydroxyindoleaceticacid; Rat brain; Synaptosome
A newly isolated factor from mammalian brain, neurocatin, is shown to increase both the level and release of serotonin in suspensions of synaptosomes isolated from rat brain. Incubation of synaptosomes for 10 min with approximately 20 nM neurocatin resulted in release of about 50% of the total pool of serotonin. Quantification of serotonin and its major metabolite, using an electrochemicaldetector, indicated that the presence of neurocatin also caused an increase in the absolute level of serotonin and decrease in its catabolism to 5-hydroxyindoleaceticacid (5-HIAA). The effect is dependent on the time of incubation and concentration of neurocatin. At a concentration of 20 nM, neurocatin increased about 60% the level of serotonin and decreased about 50% the level of 5-HIAA. Depolarization conditions - - 50 gM veratridine and 50 mM K ÷ medium - increased release of serotonin by 50% and 30% respectivelywithout affectingthe level of serotonin or its catabolism to 5-HIAA.
In earlier reports we presented evidence that bovine brain contains a factor which, when added to rat brain synaptosomes, causes decreased formation of 3,4-dihydroxyphenylacetic acid, increased formation of norepinephrine and its N-methyl derivatives and increased release of catecholamines [6]. This factor (neurocatin) has now been purified to a single chromatographic peak by high resolution H P L C . It is a small peptide with a molecular weight in the range of 2,000 to 2,500 Da [10]. In the present paper, it is reported that addition of neurocatin to synaptosomes from rat brain stimulated release of serotonin and increased the level of this monoamine. It also decreased ~production of the catabolic product of serotonin, 5-hydroxyindoleacetic acid (5-HIAA). Male Sprague-Dawley rats, approximately 200g, were used throughout the study. Crude synaptosomal preparations (P2 fraction) were prepared from homogenates of combined cerebral hemispheres and brain stem in buffered 0.25 M sucrose solution by centrifugation of the initial low speed supernatant (3 min at 1300 g) for 10 min at 17,000 g. The final synaptosomal pellets were suspended at approximately 6 mg protein/ml in modified Krebs-Henseleit buffer (140 m M NaC1, 5 m M KCI, 1.3 m M MgSO4, 10 m M TrisHEPES, 1 m M Na2HPO4; p H 7.4) containing 10 m M glucose and 1 m M CaCI2 and incubated aerobically for Correspondence: A. Pastuszko, Department of Biochemistry and Biophysics, Medical School, University of Pennsylvania, Philadelphia, PA 19104, U.S.A.
0304-3940/91/$ 03.50 © 1991 ElsevierScientific Publishers Ireland Ltd.
1, 2.5, 5 and 10 min in a Dubnoff metabolic shaker bath at 37°C. The effect of neurocatin was tested by adding approximately 0 (control), 5, 10, 20 and 40 nM neurocatin to the incubation medium. At the end of the incubation periods, the reactions were quenched by adding cold trichloroacetic acid (TCA) to a final concentration o f 1% by weight. The samples were then centrifuged to remove the precipitated material. The clear supernatants were analyzed by high pressure liquid chromatography [3, 4]. In some experiments, synaptosomes were incubated as described above but also in the presence of 3/iCi/ml of [3H]serotonin (5hydroxy G-[3H]tryptamine creatinine sulphate), Amersham, specific activity 12.5 Ci/mmol. In addition to measuring the changes in the level of serotonin and 5H I A A by an electrochemical detector, the eluate from the chromatographs was collected in 0.15 ml fractions and the radioactivity in each fraction was measured. The neurocatin used during this study was obtained after elution from a semipreparative column (last step before final purification). Neurocatin in this fraction was 25-40% of the total protein measured by the absorption at 220 nm. The concentration o f the final product can only be estimated as the exact structure and molecular weight are not yet known. The concentrations of neurocatin are based upon an estimated molecular weight of 2,000 Da. Uptake of [aH]serotonin (0.4 /tM) was initiated by adding radioactive neurotransmitter to the synaptosomal suspension (approx. 1 mg protein) in Krebs-Hense-
84 leit buffer, pH 7.4 with 10 m M glucose and 1 m M CaC12, and with or without 20 nM neurocatin. At the end of incubation periods of 1-10 min, 100 pl samples were placed in 400 pl tubes containing 50 pl silicone oil (specific gravity 1.03) and rapidly centrifuged for 2 min (Backman Model B Microfuge). The radioactiviy in the supernatant and pellet fractions was determined. Release of serotonin was measured by incubating suspensions of synaptosomes at 6 mg protein/ml for 10 min at 37°C in a Krebs medium containing 10 m M glucose and 1 m M CaC12 with or without 5, 10, 20, or 40 nM neurocatin (all final concentrations). In some experiments, the incubation medium also contained 1 p M clorgyline, an inhibitor of monoamine oxidase, 50/~M veratridine or 50 m M [K +]. At the end of the incubation the samples were treated in one of two ways: (1) Aliquots (250/tl) were withdrawn, acidified with cold T C A (1.5 % final concentration) and centrifuged to remove the precipitated material. The acid-soluble fraction was used for determination of the total content of serotonin and 5H I A A by HPLC. (2) Aliquots (100/~1) were withdrawn and rapidly centrifuged (Beckman Model B Microfuge) through a layer of silicone oil into 100 pl of 1.5% TCA in 3 % NaCI. The top layer, which contained the extrasynaptosomal medium, was pipetted off, acidified with trichloroacetic acid to a final concentration of 1.5% and used to determine the content of serotonin and 5-HIAA by H P L C with electrochemical detection. The release experiments were designed in such a way (without reuptake inhibitors) that values may be affected by changes in reuptake as well as in synthesis of neurotransmitter.
Protein concentration was determined by the method of Lowry et al. [5] with bovine serum albumin as the standard. All data are presented as means _+ S.E.M. The results are compared using the paired t-test. When the data are presented as percent of control, the statistical analysis was carried out on the raw data. As may be seen in Fig. IA,B, 10 min incubation of synaptosomes in the presence of 20 nM neurocatin had marked effects on the level of serotonin and 5-HIAA. Neurocatin caused an increase of about 60 % in the level of serotonin and an about 50% decrease in the level of 5-HIAA. The c h r o m a t o g r a m presented in Fig. 1 is typical of those obtained from 11 experiments. The effect of neurocatin was examined also when the incubation medium contained [3H]serotonin. After 10 min incubation, the presence of 20 nM neurocatin in the incubation medium resulted in the radioactivity in the serotonin fraction being substantially greater than that in the control (169% of control) and decreased the radioactivity in the 5-HIAA fraction by about 37%. The changes in the levels of serotonin and 5-HIAA were dependent on the concentration of neurocatin (see Table I, total) and on the interval of incubation of synaptosomes with this peptide (Fig. 2A,B). The presence of 20 nM neurocatin resulted in an increase in serotonin which became statistically significant (P < 0.05) by 2.5 min and was significant at all longer times of incubation (Fig. 2A). The results are plotted in the inset of Fig. 2A as the increase in level of serotonin in the presence of 20 nM neurocatin relative to control. The changes in the level of 5-HIAA are shown in Fig. 2B with the differ-
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Fig. 1. The effect of neurocatin on the levels of serotonin and 5-HIAA in synaptosomes from rat brain. The synaptosomes were incubated for 10 min at 37°C with 0 (A) and 20 nM neurocatin (B). At the end of the incubation period, aliquots were withdrawn, acidified with TCA and centrifuged to remove the precipitated material. The acid extract was assayed by HPLC with electrochemical detector. The flow rate of 0.75 ml/min gave a typical retention time for 5-HIAA and serotonin (5-HT) of 8.1 and 13.6 min, respectively.The chromatogram in the figure is representativeof those from 11 independent experiments.
85 TABLE I EFFECT OF NEUROCATIN, DEPOLARIZATION AND CLORGYLINE ON THE LEVEL AND RELEASE OF SEROTONIN AND 5-HIAA IN RAT BRAIN SYNAPTOSOMES The synaptosomes (approximately 6 mg/ml) were incubated for 10 min in the presence of different concentrations of neurocatin or in the presence of I /tM clorgyline, 50 mM K÷-medium or 50/LM veratridine. The total concentration of serotonin and 5-HIAA was measured after quenching the sample directly with TCA, whereas the values for the supernatant are from experiments in which the synaptosomes were removed by centrifugation through a layer of silicon oil (see methods). The data are presented as the means ± S.E.M. for 5 experiments except for conditions marked with an asterisk, where 3 experiments are presented. Conditions
5-HT (pmol/mg protein)
Control Neurocatin (5 nM) Neurocatin (10 nM) Neurocatin (20 nM) Neurocatin (40 nM) Clorgyline (1/~M)* Veratridine (50/~M)* High K ÷ (50 mM)*
5-HIAA (pmol/mg protein)
Total
Supernatant
Total
Supernatant
22.7 + 4.1 22.9 ___2.0 28.4_ 1.7 36.9 _+2.1 44.1 _ 1.8 40.2 _ 0.9 17.5 ±_5.2 20.4+2.2
1.0 ±_0.6 1.7 + 0.5 2.7 + 0.9 18.4 + 3.1 28.7 5:4.2 5.5 5-0.6 8.4 ± 1.9 6.3±2.0
33. I _ 6.2 31.0 _ 3.1 27.8 _ 1.7 20.1 5- 0.9 9.3 + 1.6 14.1 5:2.2 34.7 ± 3.8 32.1 +2.0
23.0__+7.1 20.0 _ 4.1 19.0 + 4.7 12.4 _ 5.0 7.0 + 2.0 10.7 + 0.6 21.0 5- 4.5 19.0+3.1
ence (decrease) between the level of 5-HIAA in the presence and absence of 20 nM neurocatin plotted in the inset. The decrease in 5-HIAA was statistically significant by 5 min incubation time (P < 0.02). Similar effects of neurocatin were observed in synaptosomes specifically isolated from the brainstem, a region particularly rich in serotonergic neurons. After 20 min incubation, synaptosomes from brainstem contained 92 pmol serotonin/mg protein (control) while those incubated in the presence of 20 nM neurocatin contained 145 pmol serotonin/mg protein. The level of 5-HIAA was 171 pmol/mg protein in the controls and 40"
A
•
CONTROL
this decreased to 58 pmol/mg protein in the presence of neurocatin. Incubation of synaptosomes with neurocatin stimulated release of serotonin into the suspending medium, in a concentration dependent manner (Table I). Twenty nM neurocatin caused release of about 50 % of the total content of serotonin, whereas 40 nM released about 65 % of the total serotonin. The effect of neurocatin on the total level and on the release of serotonin can be compared with that induced by 50/~M veratridine or by 50 mM K+-medium. Depolarization of synaptosomal membranes with veratridine or high K ÷ caused release of about 50% and 31% of total serotonin, respectively. In contrast to the addition of neurocatin neither of these 40-
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