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(Stroke. 1996;27:941-950.)
© 1996 American Heart Association, Inc.

Articles

Relationships Between ATP Depletion, Membrane Potential, and the Release of Neurotransmitters in Rat Nerve Terminals

An In Vitro Study Under Conditions That Mimic Anoxia, Hypoglycemia, and Ischemia

Maria S. Santos, PhD; António J. Moreno, PhD Arsélio P. Carvalho, PhD

From Centro de Neurociências de Coimbra, Departamento de Zoologia, Universidade de Coimbra (Portugal).

Correspondence to M.S. Santos, Centro de Biologia Celular, Departamento de Zoologia, Universidade de Coimbra, 3049 Coimbra Codex, Portugal.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Background and Purpose It is known that the extracellular accumulation of glutamate during anoxia/ischemia is responsible for initiating neuronal injury. However, little information is available on the release of monoamines and whether the mechanism of its release resembles that of glutamate, which may itself influence the release of monoamines by activating presynaptic receptors. This study was designed to characterize the release of both amino acids and monoamines under chemical conditions that mimic anoxia, hypoglycemia, and ischemia.

Methods The contents of synaptosomes in adenine nucleotides (ATP, ADP, and AMP), amino acids (aspartate, glutamate, taurine, and -aminobutyric acid), and monoamines (dopamine, noradrenaline, and 5-hydroxytryptamine) were measured by high-performance liquid chromatography, after the synaptosomes were subjected to anoxia (KCN+oligomycin), hypoglycemia (2 mmol/L 2-deoxyglucose in glucose-free medium), and ischemia (anoxia plus hypoglycemia).

Results The anoxia- and ischemia-induced release of noradrenaline, dopamine, 5-hydroxytryptamine, and glutamate correlated well with ATP depletion. The correlation observed between glutamate levels and the release of dopamine and 5-hydroxytryptamine in ischemic conditions suggests a functional linkage between the two transmitter systems. However, the antagonists of presynaptic glutamate receptors failed to alter the amount of monoamines released. The inhibition of Na⁺,K⁺-ATPase by ouabain had an effect similar to that produced by ischemia.

Conclusions The decrease in Na⁺ and K⁺ gradients resulting from the energy depletion of the synaptosomes under ischemic conditions or resulting from the inhibition of Na⁺,K⁺-ATPase by ouabain promotes the reversal of the neurotransmitter transporters. The decrease in uptake of neurotransmitters may also contribute to the rise in the extracellular concentration of different transmitters observed during brain ischemia.

Key Words: anoxia • cerebral ischemia • glutamates • hypoglycemia • neuronal damage • neurotransmitters • rats

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
There is considerable evidence that extracellular accumulation of the excitatory amino acids aspartate and glutamate contributes to the neuronal damage observed in many central nervous system diseases, including hypoglycemia and ischemia.¹ Studies in vivo^{2 3 4 5 6 7} and in vitro^{8 9 10 11} show that ischemia and/or anoxia releases glutamate and GABA. In vivo dialysis experiments have also shown that severe insulin-induced hypoglycemia causes the release of aspartate, glutamate, and GABA.^{12 13}

The neurotoxicity of excitatory amino acids, mainly glutamate, is caused by an excessive activation of postsynaptic glutamate receptors, which increases the intracellular free Ca²⁺ concentration ([Ca²⁺]_i), causing a cascade of metabolic events leading to cell death.^{1 2 3 4 5 6 7 8 9 10 11 12 13 14} However, other neurotransmitters have also been implicated in the ischemia-induced process of ischemic neuronal injury. It has been well documented that during ischemia there is also a massive release of DA, NA, and 5-HT, which may modulate the final responses of nerve cells to the ischemic neuronal injury.^{5 15 16 17 18 19}

In synaptosomes, evidence has been found that anoxia/ischemia causes the release of various neurotransmitters by a Ca²⁺-dependent^{4 20 21} and a Ca²⁺-independent mechanism.^{9 22} Many studies have shown that the Ca²⁺-independent component of the ischemia-induced release of neurotransmitters may be due to the reversal of Na⁺-dependent neurotransmitter carriers,^{23 24 25} as a result of the alteration in intracellular ions occurring during ATP depletion.²⁶ In addition, the inhibition of energy-dependent processes leads to an impaired operation of the synaptic vesicle transporters and plasma membrane reuptake mechanisms, resulting in an increase in cytoplasmic and extracellular levels of neurotransmitters.²⁷

Although changes in the level of extracellular amino acids after hypoglycemia and ischemia have been reported, little information obtained in a single study on the same preparation is available on the relationships between energy metabolism, membrane depolarization, and the correlation between extracellular changes in both amino acids and monoamines (see Reference 28²⁸ ). In particular, there is no information regarding the release of monoamines from synaptosomes under ischemic conditions and whether the mechanism of this release resembles that of amino acids, which may themselves influence the release of monoamines by activating presynaptic receptors.²⁹ Therefore, we measured the ATP levels, membrane potential, Ca²⁺ influx, and release of the amino acids aspartate, glutamate, taurine, and GABA and the release of monoamines DA, NA, and 5-HT under conditions that mimic anoxia, hypoglycemia, and ischemia (anoxia plus hypoglycemia) in rat brain synaptosomes as the in vitro model of the nerve terminal. Additionally, we tested whether the release of monoamines (DA, NA, and 5-HT) was a direct effect of ischemia or was mediated by the action of the glutamate released through its specific presynaptic receptors.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Preparation of Synaptosomes
Crude synaptosomes were prepared from brain of male Wistar rats (weight, 180 to 220 g) according to the method of Hajós,³⁰ with some modifications. After animal decapitation, the whole cerebral cortices were rapidly removed and homogenized in 10 vol of 0.32 mol/L sucrose buffered at pH 7.4 with Tris. The homogenate was centrifuged at 1000g for 10 minutes, and the synaptosomes were isolated from the supernatant by centrifugation at 12 000g for 20 minutes. The white and fluffy synaptosome layer was then resuspended, respun, and resuspended in the sucrose medium at a protein concentration of 15 to 20 mg/mL, as determined by the biuret method. Experiments were carried out within 2 hours of preparation.

Incubation Conditions
Synaptosomes were incubated at a concentration of 3 to 4 mg/mL for 10 minutes at 30°C in a standard medium containing the following (mmol/L): NaCl 118, KCl 3, MgCl₂ 1.2, NaH₂PO₄ 1, CaCl₂ 1.2, glucose 10, HEPES adjusted to pH 7.4 with Tris, and 0.1% of bovine serum albumin. When catecholamine release was studied, pargyline (100 µmol/L) was added in incubation medium to prevent catecholamine oxidation and metabolism. The incubation was terminated by centrifugation (120 seconds in an Eppendorf microcentrifuge), and the supernatant was assayed by HPLC for amino acids and monoamines released. Levels of the total amino acids and monoamines were measured in preparations lysed with sonication before centrifugation. Adenine nucleotides were extracted from synaptosomal incubations by the addition of cold perchloric acid, and the measurements were performed, after neutralization, as described.³¹ The values for ATP, ADP, and AMP were determined by HPLC, as described below.

Chemical anoxia, hypoglycemia, and ischemia were induced in synaptosomes by addition of the following: (1) 2 mmol/L KCN+5 µg/mL oligomycin in standard medium, (2) 2 mmol/L 2-DG in glucose-free medium, and (3) 2 mmol/L 2-DG+2 mmol/L KCN+5 µg/mL oligomycin in glucose-free medium. KCN inhibits the respiratory chain, oligomycin inhibits the mitochondrial ATP synthesis, and 2-DG inhibits competitively glycolysis and glycogenolysis. The composition of choline medium was identical to the Na⁺ medium, except that the NaCl was replaced by choline chloride. Test compounds were introduced into the incubation medium before the addition of protein. In experiments in which glucose was omitted, NaCl was raised to 123 mmol/L.

HPLC Determination of Adenylates, Amino Acids, and Catecholamines
Adenine nucleotides (ATP, ADP, and AMP) were separated by reverse-phase HPLC as described by Stocchi et al.³² The chromatographic apparatus was a Beckman System Gold, consisting of a binary pump (model 126) and a variable UV detector (model 166), controlled by a computer. The detection wavelength was 254 nm, and the column was a Lichrospher 100 RP-18 (5 µm) from Merck. An isocratic elution with 100 mmol/L phosphate buffer (KH₂PO₄) (pH 6.5) and 1.0% methanol was performed with a flow rate of 1 mL/min. The time required for each analysis was 6 minutes.

Amino acids were analyzed in a Gilson-ASTED system according to the manufacturer's manual. The amino acid derivatives resulting from the precolumn derivatization with orthophthaldialdehyde/2-mercaptoethanol were separated on a Spherisorb ODS column (particle size, 5 µm; 150 mm long, 4.6 mm ID), at a flow rate of 2.5 mL/min, with the use of the following ternary solvent system: buffer A (250 mmol/L sodium phosphate, 15%; 200 mmol/L propionic acid, 20%; acetonitrile, 7%; DMSO, 3%; pH 6.2); buffer B (acetonitrile, 40%; methanol, 33%; DMSO, 7.1%); and buffer C (250 mmol/L sodium phosphate, 25%; 250 mmol/L propionic acid, 20%; acetonitrile, 71%; DMSO, 3.1%; pH 5.5). The effluent was monitored by a fluorescent detector (Gilson, model 121; excitation and emission wavelengths at 340 and 410 nm, respectively). The integration of the amino acid peak area and further calculations were performed by the Gilson system software, and quantification was allowed by running standard amino acids solutions in the same conditions. The time required for each analysis was 45 minutes.

Concentrations of NA, DA, 5-HT, and metabolites (dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid) in the supernatants were determined according to the method described by Warnhoff.³³ The HPLC system consisted of a pump (Gilson, model 305) combined with an electrochemical detector (Gilson, model 141) with a glassy carbon electrode maintained at a potential of 0.65 V with a sensitivity of 2 nA/V. Separation was achieved by the use of a 250x4.6-mm reversed-phase analytical column (Spherisorb ODS, 5 µm) and a mobile phase consisting of 0.1 mol/L citric acid, 0.5 mmol/L sodium octyl sulfate, 0.15 mmol/L EDTA, 1 mmol/L dibutylamine, and 10% methanol (vol/vol). The flow rate was 1 mL/min, and the time required for each analysis was 30 minutes. Calculations were performed by the Integrator Spectra-Physics system (model SP 4600), and quantification was allowed by comparing the heights of the peaks in the samples with the heights of the peaks in the standard solutions, injected before each experiment.

Measurement of Membrane Potential
Membrane potential was estimated from the accumulation of TPP⁺ as described previously,³⁴ with some modifications. Synaptosomes (4 mg/mL) were preincubated for 10 minutes at 30°C, in the presence of test substances, before membrane potential studies. The membrane potential studies were performed by transferring aliquots of synaptosomal suspensions (0.6 mg protein) into 850 µL of medium containing 4 µmol/L TPP⁺ (final concentration). The values given for the membrane potential of synaptosomes were determined after correcting for the TPP⁺ taken up in the absence of a K⁺ gradient, which approximately gives the contribution of the mitochondria and that of TPP⁺ binding for the total TPP⁺ accumulation.

Evaluation of Synaptosomal Integrity
Synaptosomal integrity was determined by detecting the activity of LDH in the supernatant of synaptosomes exposed for 10 minutes to the different described conditions. The activity was measured by means of a spectrophotometric assay³⁵ and was expressed as percentage of the total LDH present in synaptosomes.

Measurement of ⁴⁵Ca²⁺ Uptake
Calcium uptake was determined by a filtration technique as previously described by Coutinho et al.³⁶ Essentially, synaptosomes were incubated in the indicated media with 1 µCi/mL of ⁴⁵Ca²⁺, and the reaction was initiated by the addition of synaptosomes. After 10 minutes of incubation, 120-µL aliquots (0.3 to 0.5 mg protein), were removed and filtered through Whatman GF/B filters. The filters were rinsed twice with 5 mL of cold buffer with the following composition (mmol/L): choline chloride 125, KCl 3, MgCl₂ 1.2, Na₂HPO₄ 1, EGTA 0.1, HEPES-Tris 10 (pH 7.4), and 0.1% bovine serum albumin. The ⁴⁵Ca²⁺ retained by the filters was determined by liquid scintillation counting in a Packard Tri-Carb 460-CD.

Statistical Analysis
Statistical analysis of data was performed with the use of the two-tailed Student's t test. Values are presented as mean±SE; differences with a value of P<.05 were considered significant.

Materials
ATP, ADP, AMP, and standard amino acids for HPLC analysis were obtained from Sigma Chemical Co. CNQX was obtained from NOVO, and MK-801 was obtained from Merck Sharp and Dohme Research Laboratory. ⁴⁵Ca²⁺ was obtained from Amersham Laboratories. HPLC-grade methanol was from Riedel-de-Häen AG. All the other chemicals were of the highest grade of purity commercially available.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of Conditions That Mimic Anoxia, Hypoglycemia, and Ischemia
Synaptosomal Membrane Integrity
To investigate the possibility that the experimental conditions used caused damage to the plasma membrane, we measured the activity of LDH in the supernatant of the synaptosomes under the different experimental conditions. Table 1 shows that when synaptosomes were incubated alone for 10 minutes at 30°C, LDH released was 7.7±1.7% of the total intrasynaptosomal LDH content. At 10 minutes of anoxia, hypoglycemia, and ischemia, the release values were 6.9±1.5%, 8.3±1.4%, and 8.3±2.7%, respectively. The lack of a significant release of LDH compared with control suggests that the damage of synaptosomes was not responsible for the observed increase in the release of amino acids and monoamines reported below.

View this table: [in this window] [in a new window]   Table 1. Effect of Anoxic, Hypoglycemic, and Ischemic Conditions on Integrity of Synaptosomes

Synaptosomal Adenine Nucleotide Contents
The effects of in vitro anoxia-, hypoglycemia-, or ischemia-like conditions on the adenine nucleotide levels in synaptosomes were evaluated (Fig 1). Freshly isolated synaptosomes contained 3.30±0.32 nmol ATP per milligram protein, 0.54±0.04 nmol ADP per milligram protein, and 2.70±0.38 nmol AMP per milligram protein. Thus, the ratio of ATP to ADP initially was 6.15±1.05. Exposure of synaptosomes for 10 minutes to the different experimental conditions resulted in a significant decrease in the intrasynaptosomal ATP level, and this decrease in ATP was reflected in an increase in ADP and AMP (Fig 1).

View larger version (33K): [in this window] [in a new window]   Figure 1. Levels of ATP, ADP, and AMP in synaptosomes incubated in standard medium (controls); standard medium+KCN+oligomycin (anoxia); glucose-free, 2-DG-containing medium (hypoglycemia); and glucose-free, 2-DG-containing medium+KCN+oligomycin (ischemia) for 10 minutes at 30°C. Each result is the mean±SE (bars) of six different experiments. Values statistically different from the respective control are indicated (*P<.005; **P<.05).

Preincubation with KCN+oligomycin in glucose-free medium (ischemic condition) caused decreases of ATP from 2.68±0.26 in the control to 0.72±0.11 nmol/mg protein, whereas the ADP and AMP increased from 0.44±0.04 to 0.90±0.08 and from 2.92±0.13 to 3.96±0.46 nmol/mg protein, respectively. The ratio of ATP to ADP in synaptosomes declined from 6.06±0.20 to 0.87±0.08. In hypoglycemic conditions (glucose-free medium+2-DG), the decrease in synaptosomal ATP and the increases in ADP and AMP were similar to those observed in anoxia, and ischemia (glucose-free medium+2-DG+KCN+oligomycin) caused the ATP levels to fall to 0.11±0.02 nmol/mg protein (4% of the control value) and the ratio of ATP to ADP to fall to 0.18±0.01, which suggests a near complete depletion of the ATP content (Fig 1).

Synaptosomal Membrane Potentials
In Fig 2, we report the effects of anoxia, hypoglycemia, and ischemia on the synaptosomal membrane potential monitored with a TPP⁺-sensitive electrode. The membrane potential of freshly isolated synaptosomes was -53.95±1.78 mV, and this value decreased to -39.43±2.43 mV during the incubation of synaptosomes for 10 minutes. The membrane potential was decreased by anoxic and hypoglycemic conditions (-28.35±2.46 and -31.70±0.84 mV, respectively), and ischemia reduced the plasma membrane to a even lower value of -16.30±1.09 mV (Fig 2).

View larger version (58K): [in this window] [in a new window]   Figure 2. Effects of anoxia, hypoglycemia, and ischemia on synaptosomal membrane potential. Membrane potential was determined by following the uptake of TPP+ in media containing 4 µmol/L TPP+ as described in "Materials and Methods." Each result is the mean (bars) of four different experiments. Values statistically different from control are indicated (*P<.01; **P<.05).

Release of Monoamines and Amino Acid
The changes in the levels of monoamines released under conditions of anoxia, hypoglycemia, or ischemia are shown in Fig 3. The intrasynaptosomal levels of monoamines were 19.99±1.13 pmol NA per milligram protein, 28.33±1.96 pmol DA per milligram protein, and 16.27±1.61 pmol 5-HT per milligram protein. In ischemic conditions the release of DA or 5-HT increased by approximately sixfold compared with the respective values in control conditions (from 4.12±0.64 to 23.04 pmol/mg protein for DA and from 1.76±0.18 to 10.89±0.31 pmol/mg protein for 5-HT). The increase in the release of monoamines was less marked in anoxic than in ischemic conditions (Fig 3). Hypoglycemia was without significant effects on the release of NA, DA, and 5-HT.

View larger version (33K): [in this window] [in a new window]   Figure 3. Release of NA, DA, and 5-HT from synaptosomes incubated in standard medium (controls); standard medium+KCN+oligomycin (anoxia); glucose-free, 2-DG-containing medium (hypoglycemia); and glucose-free, 2-DG-containing medium+KCN+oligomycin (ischemia) for 10 minutes at 30°C. Each result is the mean±SE (bars) of six different experiments for control and ischemic conditions and four different experiments for anoxic and hypoglycemic conditions. Significant differences from control are indicated (*P<.005; **P<.05).

As reported previously by several investigators,^{4 20 21 37} we found that anoxia-, hypoglycemia-, or ischemia-like conditions caused release of amino acid neurotransmitters. In Fig 4 we summarize our results, showing that our synaptosomal preparation responds to the ischemic insult as expected. Synaptosomes contained 68.41±3.89 nmol aspartate per milligram protein, 64.85±3.01 nmol glutamate per milligram protein, 32.8±1.80 nmol taurine per milligram protein, and 23.82±2.78 nmol GABA per milligram protein. The basal release (10 minutes of incubation at 30°C) of aspartate, glutamate, taurine, and GABA was approximately 3.0, 6.9, 12.3, and 0.4 nmol/mg protein, respectively, and anoxia or ischemia significantly increased the release of aspartate, glutamate, and GABA (Fig 4). No significant changes in taurine release occurred in either experimental condition. Ischemia evoked the largest increase in the release of glutamate (28.98±1.08 nmol/mg protein).

View larger version (33K): [in this window] [in a new window]   Figure 4. Release of aspartate (ASP), glutamate (GLU), taurine (TAU), and GABA from synaptosomes incubated in standard medium (controls); standard medium+KCN+oligomycin (anoxia); glucose-free, 2-DG-containing medium (hypoglycemia); and glucose-free, 2-DG-containing medium+KCN+oligomycin (ischemia) for 10 minutes at 30°C. Each result is the mean±SE (bars) of six to eight different experiments. Significant differences from control are indicated (*P<.005; **P<.05).

Thus, exposure of synaptosomes to ischemia for 10 minutes increased the glutamate release fourfold compared with the basal value, and the release represents approximately 50% of the total intrasynaptosomal glutamate concentration. The increase in the amount of aspartate release was less marked than that of glutamate, and under ischemic conditions its release was almost threefold that of the control value. Under ischemic conditions, a massive release of GABA was also observed (approximately 12-fold the basal release; 20% of the total synaptosomal GABA). The basal release of taurine and nontransmitter amino acids (result not shown) was not significantly influenced by anoxic, hypoglycemic, or ischemic conditions (Fig 4).

⁴⁵Ca²⁺ Uptake
In Fig 5 we report the ⁴⁵Ca²⁺ uptake by synaptosomes in conditions similar to those previously used in our release experiments. Under control conditions and after 10 minutes of incubation, the amount of ⁴⁵Ca²⁺ accumulated was approximately 30 nmol/mg protein. Anoxia, hypoglycemia, and ischemia reduced the uptake to 15.76±3.52, 22.47±2.04, and 12.87±1.96 nmol/mg protein, respectively.

View larger version (49K): [in this window] [in a new window]   Figure 5. Effects of anoxia, hypoglycemia, and ischemia on 45Ca2+ uptake into synaptosomes. Synaptosomes were incubated at 30°C in different media supplemented with 45CaCl2 (1 µCi/mL). At 10 minutes the reaction was stopped by rapid filtration. Each result is the mean±SE (bars) of four different experiments. Values statistically different from control are indicated (*P<.05; **P<.005).

Relationships Between Ratio of ATP to ADP, Membrane Potential, Release of Glutamate and Monoamines, and ⁴⁵Ca²⁺ Uptake
Our results show that there is a linear correlation between the ratio of ATP to ADP in synaptosomes and membrane depolarization (Fig 6A); between the ratio of ATP to ADP and the stimulation of the release of glutamate, DA, and 5-HT (Fig 6B); and between the ratio of ATP to ADP and the inhibition of ⁴⁵Ca²⁺ uptake (Fig 6C). This is despite the fact that the various values were obtained for hypoglycemic, anoxic, or ischemic conditions. Changes in aspartate and GABA release were not well correlated with changes in the ratio of ATP to ADP.

View larger version (21K): [in this window] [in a new window]   Figure 6. Relationships between ratio of ATP to ADP and membrane potential (A), glutamate (GLUT) and monoamines released (B), and 45Ca2+ uptake (C) under hypoglycemic, anoxic, and ischemic conditions. Values are from Figs 1 through 5.

Relationship Between Release of Glutamate and Release of Monoamines
Fig 7 shows that the levels of glutamate and the amounts of DA and 5-HT released under hypoglycemic, anoxic, or ischemic conditions were highly correlated, suggesting that the release of monoamines could be mediated by the activation of presynaptic glutamate receptors. To examine whether the ischemia-induced release of these monoamines was a consequence of the stimulation of NMDA and AMPA/kainate receptors by glutamate, we investigated the effect of glutamate antagonists on the release of monoamines. We observed that the levels of NA, DA, and 5-HT released in ischemia were not reduced by the addition of 10 µmol/L CNQX and 3 µmol/L MK-801 (Table 2). The above results indicate that the activation of presynaptic inotropic glutamate receptors was not responsible for the anoxia- or ischemia-induced release of monoamines.

View larger version (27K): [in this window] [in a new window]   Figure 7. Relationship between the hypoglycemia-, anoxia-, or ischemia-induced release of glutamate and levels of NA, DA, and 5-HT released. Values are from Figs 3 and 4.

View this table: [in this window] [in a new window]   Table 2. Effect of CNQX/MK-801 on Ischemia-Induced Release of NA, DA, and 5-HT in Synaptosomes

Effect of Ca²⁺ or Na⁺ on Ischemia-Induced Release of Glutamate and Monoamines
The dependence of the ischemia-induced release of glutamate and monoamines on Ca²⁺ was tested by incubating synaptosomes in a medium without added Ca²⁺ and containing 0.1 mmol/L EGTA and in a medium containing the normal Ca²⁺ concentration (1.2 mmol/L). Fig 8 shows that the presence of Ca²⁺ did not affect the ischemia-induced release of glutamate, NA, DA, and 5-HT from synaptosomes. In the absence of Ca²⁺, ischemia increased the release of NA, DA, and 5-HT from 8.78±0.55, 3.48±0.71, and 1.8±0.51 pmol/mg protein to 16.17±2.98, 24.91±2.82, and 17.17±2.11 pmol/mg protein, respectively. The basal release of the monoamines was not influenced by Ca²⁺, but the basal release of glutamate was significantly increased by the absence of Ca²⁺ (from 4.86±0.47 to 16.02±1.78 nmol/mg protein); Ca²⁺ did not influence the maximal release of glutamate caused by ischemia (Fig 8A).

View larger version (35K): [in this window] [in a new window]   Figure 8. Role of Ca2+ in the ischemia-induced release of glutamate (A) and NA, DA, and 5-HT (B) in synaptosomes. The ischemic condition (ISCH) was induced in a standard glucose-free, 2-DG-containing medium+KCN+oligomycin or in a similar medium devoid of added Ca2+ and containing 0.1 mmol/L EGTA. Each result is the mean±SE (bars) of four different experiments for glutamate and five different experiments for monoamines. Values statistically different from respective controls (CONT) obtained in the presence of Ca2+ are indicated (*P<.05; **P<.01).

Data of Fig 9 indicate that the absence of extracellular Na⁺ induced the release of amino acids and monoamines even in the absence of ischemia, and the release of neurotransmitters in choline chloride medium (glutamate, 31.15±3.33 nmol/mg; NA, 12.98±2.48 pmol/mg; DA, 27.97±3.21 pmol/mg; and 5-HT, 8.99±0.56 pmol/mg) was similar to that induced by ischemia in Na⁺ medium (glutamate, 27.79±1.29 nmol/mg; NA, 12.46±2.45 pmol/mg; DA, 23.04±1.74 pmol/mg; and 5-HT, 10.89±0.31 pmol/mg). Thus, when synaptosomes were incubated in choline medium, it was not possible to study the effect of ischemia since under control conditions there was an increase in glutamate, NA, DA, and 5-HT in the medium comparable to that induced by ischemia in Na⁺ medium (Fig 9), and ischemia did not produce further release in the absence of Na⁺ (Fig 9).

View larger version (38K): [in this window] [in a new window]   Figure 9. Role of Na+ in the ischemia-induced release of glutamate (A) and NA, DA, and 5-HT (B) in synaptosomes. The ischemic condition (ISCH), glucose-free, 2-DG-containing medium+KCN+oligomycin, was induced in a standard Na+ medium or in a medium containing choline chloride instead of NaCl. Each result is the mean±SE (bars) of five different experiments for glutamate and four different experiments for monoamines. Values statistically different from respective controls (CONT) obtained in the presence of Ca2+ are indicated (*P<.05; **P<.01).

Effect of Ouabain on Ischemia-Induced Release of Glutamate and Monoamines
When 1 mmol/L ouabain was included in the incubation medium, the amount of glutamate and monoamines released did not differ significantly from the values obtained after ischemic conditions. Thus, as shown in Fig 10, the amounts of glutamate, NA, DA, and 5-HT released by ouabain during 10 minutes of incubation (24.75±1.43 nmol/mg, 15.53±3.47 pmol/mg, 23.65±2.77 pmol/mg, and 11.49±2.57 pmol/mg, respectively) were similar to those induced by ischemia (29.07±0.48 nmol/mg for glutamate, 15.97±2.73 pmol/mg for NA, 27.03±3.24 pmol/mg for DA, and 13.13±1.94 pmol/mg protein for 5-HT). Fig 10 shows also that the amount of neurotransmitters released by the combined effects of ouabain and ischemia was not significantly different from that released by ouabain or ischemia alone. Thus, when Na⁺,K⁺-ATPase is inhibited, the maximal release has already occurred, and ischemia does not produce further release of either amino acids or monoamines (Fig 10).

View larger version (42K): [in this window] [in a new window]   Figure 10. Effect of ouabain on ischemia-induced release of glutamate (A) and NA, DA, and 5-HT (B). The ischemic condition (ISCH), glucose-free, 2-DG-containing medium+KCN+oligomycin, was induced in the absence or in the presence of 1 mmol/L ouabain. Each result is the mean±SE (bars) of four different experiments. Values statistically different from respective controls (CONT) obtained in the absence of ouabain are indicated (*P<.01).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In these experiments we studied the effect of conditions that mimic anoxia, hypoglycemia, and ischemia on the release of endogenous amino acids and monoamines from rat brain synaptosomes. Our results show that under the different experimental conditions used, the release of NA, DA, and 5-HT was correlated with the decrease in the ratio of ATP to ADP and membrane potential, and that the release of monoamines correlated with the release of glutamate. The observation that the glutamate antagonists MK-801 and CNQX failed to decrease the release of NA, DA, and 5-HT induced by ischemia suggests that the activation of presynaptic ionotropic excitatory amino acid receptors does not account for the ischemia-induced release of monoamines and that the correlation observed may reflect that the release of the two groups of neurotransmitters has a similar cause.

As reported previously,^{4 20 37} we found that ischemia caused an increase in the extrasynaptosomal accumulation of glutamate, aspartate, and GABA and that the release of glutamate may be caused by a Ca²⁺-dependent and a Ca²⁺-independent mechanism. Thus, our synaptosomal preparation responds to the anoxic or ischemic insult as expected. However, the endogenous contents in amino acids of these synaptosomes are higher than those reported in purified synaptosomes.³⁷ We found threefold higher values for aspartate and GABA concentrations and a 1.5-fold higher value for glutamate concentration than that reported previously. These differences are probably due to different isolation methods used for preparing the synaptosomes.

Of particular interest in our study is the finding that anoxia or ischemia also causes massive release of NA, DA, and 5-HT from synaptosomes. We found that the effect of hypoglycemia on the release of monoamines, as well as that of amino acids, is much less marked than that of anoxia or ischemia (Figs 3 and 4). The evidence that in synaptosomes glycolysis is not an important mechanism for ATP production and consequently for the function of Na⁺,K⁺-ATPase may explain why hypoglycemia alone, for 10 minutes, did not significantly affect the basal release of neurotransmitters.³⁷ It is well known that in brain, mitochondrial oxidative phosphorylation provides 95% of total ATP synthesis and is the predominant source of energy.^{26 37}

We observed that conditions which cause the release of glutamate also cause the release of monoamines. The correlation between the release of glutamate and the release of monoamines in synaptosomes during hypoglycemia, anoxia, or ischemia (Fig 7) would suggest a priori that glutamate may produce its effect by activating presynaptic glutamate receptors, resulting in the release of NA, DA, and 5-HT, since evidence exists that under some conditions glutamate may induce the release of DA and NA from isolated nerve terminals.^{17 38 39 40 41 42 43} However, when we investigated the effect of blocking the glutamate receptors during ischemia on the release of NA, DA, and 5-HT, we found that MK-801 and CNQX, which are antagonists of NMDA and non-NMDA receptors, were unable to decrease the amount of NA, DA, and 5-HT released by ischemia. Thus, the ionotropic NMDA or AMPA receptors are not involved in the regulation of monoamine release mechanisms, under our experimental conditions. We should point out that the levels of extracellular glutamate attained in our experimental conditions are much lower than those reported to be effective in releasing dopamine.⁴⁴ We are now testing the possible involvement of the metabotropic glutamate receptors, which have been shown to be present in presynaptic nerve terminals,^{45 46} in the modulation of ischemia-induced release of monoamines.

It is of interest that extracellular Ca²⁺ is not required for the effect of ischemia on the release of NA, DA, or 5-HT (Fig 8). This finding suggests that the monoamines are not released by an exocytotic mechanism. Several mechanisms may be responsible for the Ca²⁺-independent increase in extrasynaptosomal neurotransmitters during ischemia. Under conditions of ATP depletion, as in the case of anoxic or ischemic conditions, it is expected that the monoamine transporters at the level of synaptic vesicle membrane would not be operating, and therefore the monoamines would accumulate in the cytoplasm.²⁵ This in turn makes the monoamines available to be transported across the plasma membrane by the electrogenic transport systems working in the outward direction.^{25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47} The depolarization of the plasma membrane and changes in the Na⁺ gradient observed during ischemia would promote the carrier-mediated release of NA, DA, and 5-HT from the cytoplasm. Additionally, it is well known that exocytosis is inhibited after few minutes of ischemia and after the complete depletion of ATP.^{9 48}

Although there were no differences between control and ischemic conditions regarding the total amount of glutamate release, the Ca²⁺-dependent release of glutamate was significantly increased in ischemic conditions. When the total release was subtracted from the basal release, a significant effect of Ca²⁺ was observed (from 12.72 nmol/mg protein in the absence of Ca²⁺ to 26.02 nmol/mg protein in the presence of Ca²⁺), suggesting that, under our experimental conditions, Ca²⁺ may be involved in the ischemia-induced release mechanism of glutamate. This implies that the Ca²⁺-independent release of glutamate after a 10-minute period of ischemia should be proceeded by an initial Ca²⁺-dependent exocytotic release from synaptosomes, as has been shown by numerous groups (see Reference 28²⁸ ).

It is generally believed that the levels of K⁺ that are reached during hypoxia and ischemia⁴⁹ are sufficient to cause membrane depolarization and reversal of electrogenic uptake systems (data not shown and References 50 and 51^{50 51} ). If synaptic plasma membrane is depolarized and the Na⁺ gradient decreased, the direction of neurotransmitters is reversed from inward to outward.^{23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47} Additionally, the increase in extracellular K⁺ would also inhibit the presynaptic carrier responsible for the reuptake of the released neurotransmitters from synaptic cleft.⁵¹

The possibility that glutamate, NA, DA, and 5-HT release during ischemia is due to a change in the Na⁺ and K⁺ gradients was evaluated by using ouabain, a glycoside that inhibits Na⁺,K⁺-ATPase but preserves energy metabolism.⁵² The inhibition of Na⁺,K⁺-ATPase, the enzyme responsible for the active transport of Na⁺ and K⁺ across the cell membrane, causes the dissipation of the ionic gradients, inducing a nonvesicular release of neurotransmitters^{34 47 52 53} by reversal of the Na⁺-dependent plasma membrane carrier. Ouabain did not have a significant effect on the release under ischemic conditions, in which Na⁺,K⁺-ATPase was already inhibited as a result of ATP depletion (Figs 1 and 10). However, ouabain by itself increased the release of glutamate and monoamines to values very similar to those observed in ischemic conditions. This similarity between the effects of ouabain and ischemia on the release and the observation that ischemia does not increase the ouabain-induced release suggest that the release of both glutamate and monoamines is mediated by changes in synaptosomal ionic homeostasis resulting from the direct (presence of ouabain) or indirect (ischemic conditions) inhibition of Na⁺,K⁺-ATPase. Recent work by other investigators is consistent with our results. It was demonstrated that a decrease in the activity of the enzyme was neurotoxic in vivo⁵⁴ and that deprivation of glucose and/or oxygen and metabolic inhibitors resulted in a decrease in ATP content and a reduction of Na⁺,K⁺-ATPase activity.⁵⁵ Decreased Na⁺,K⁺-ATPase activity and reduced binding of ouabain have been also detected after ischemia in vivo.^{56 57} The effect of ouabain and ischemia on amino acid release in our studies is similar to that seen by Madl and Burgesser⁵⁸ in 1994 in rat hippocampal slices.

The observation that anoxia or ischemia decreased the influx of ⁴⁵Ca²⁺ in synaptosomes (Fig 5) is further evidence that the release of neurotransmitters mainly occurs through a Ca²⁺-independent mechanism. However, since the uptake of ⁴⁵Ca²⁺ was measured at 10 minutes of ischemia, we cannot exclude the possibility of an early rapid uptake followed by a decrease, which may be responsible for the initial Ca²⁺ dependency of the release reported by other authors.^{20 21} The observation that the ratio of ATP to ADP decreased as the Ca²⁺ uptake decreased suggests that the level of ATP probably determines the Ca²⁺ buffering capacity of the synaptosomes, which would decrease as the ATP level is reduced, preventing the massive increase in intracellular calcium. Hypoxia and hypoglycemia have also been shown to decrease presynaptic calcium currents in hippocampal neurons.^{59 60}

The increase in extracellular monoamines and glutamate levels due to ischemia can also be the result of the imbalance between the release of neurotransmitters from synaptosomes and its reuptake mechanisms by ischemia. It is generally assumed that the high-affinity Na⁺-dependent transporters inactivate synaptically released neurotransmitters, maintaining its concentration below those that are neurotoxic.^{27 61 62} Our finding that in ischemic conditions the extracellular accumulation of glutamate, NA, DA, or 5-HT is very similar to that observed in control synaptosomes incubated in choline medium (Fig 9) indicates that the extracellular accumulation of neurotransmitters is probably due to the failure of transport systems to reaccumulate the released neurotransmitters. In control conditions and in the absence of Na⁺, the observed release of glutamate and monoamines can be ascribed to the spontaneous release from synaptosomes resulting from the favorable outward Na⁺ concentration, which cannot be reaccumulated because the carriers were inhibited in the absence of Na⁺.^{25 47} The effects of the inhibition of the DA carrier by nomifensine, a specific inhibitor of the reuptake mechanism, on control and ischemic conditions are similar to those observed in choline medium (data not shown). It is interesting that in ischemic conditions the extrasynaptosomal glutamate and dopamine levels in Na⁺ medium are significantly lower than those observed in choline medium (Fig 9). This difference may be due to the fact that at 10 minutes of ischemia, some of the released glutamate and DA can still be taken up into synaptosomes. Prolonged ischemia and/or hypoxia inactivates the Na⁺-dependent uptake of several neurotransmitters.^{8 63 64 65 66}

In summary, we showed that the release of monoamines NA, DA, and 5-HT increased significantly during anoxia and ischemia in synaptosomes, in parallel with the release of glutamate, but this amino acid did not modulate the release of monoamines. The release correlated well with the decrease in membrane potential and with the ratio of ATP to ADP. The ischemia-induced release of monoamines was completely Ca²⁺ independent, but a component of glutamate release was Ca²⁺ dependent. In parallel studies, we showed that inhibition of Na⁺,K⁺-ATPase by ouabain had an effect similar to that produced by ischemia on the release of the different neurotransmitters.

	Selected Abbreviations and Acronyms

 

AMPA = -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid CNQX = 6-cyano-7-nitroquinoxaline-2,3-dioxine DA = dopamine 2-DG = 2-deoxyglucose DMSO = dimethyl sulfoxide GABA = -aminobutyric acid HPLC = high-performance liquid chromatography 5-HT = 5-hydroxytryptamine LDH = lactate dehydrogenase MK-801 = (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine NA = noradrenaline NMDA = N-methyl-D-aspartate TPP+ = tetraphenylphosphonium

	Acknowledgments

 
This study was supported by JNICT (Portuguese Research Council, PBICT/SAU/1801/93 and STRDA/C/SAU/318/92) and Human Capital Mobility Program (No. ERB 4050PL932039). The authors are very grateful to Drs T. Morgadinho and C. Palmeira for their help with the HPLC techniques.

Received May 25, 1995; revision received January 5, 1996; accepted January 16, 1996.

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