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Anesth Analg 1999;88:1401
© 1999 International Anesthesia Research Society

GENERAL ARTICLES

Clonidine Inhibits and Phorbol Acetate Activates Glutamate Release from Rat Spinal Synaptoneurosomes

Tetsutaro Shinomura, MD*, Shin-ichi Nakao, MD{dagger}, Takehiko Adachi, MD*, and Koh Shingu, MD{dagger}

*Department of Anesthesia, Kyoto University Hospital, Kyoto; and {dagger}Department of Anesthesiology, Kansai Medical University and Hospital, Osaka, Japan

Address correspondence and reprint requests to T. Shinomura, Department of Anesthesia, Kyoto University Hospital, Sakyo-ku, Kyoto 606-8507, Japan. Address e-mail to shino{at}kuhp.kyoto-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Glutamate is a major neural transmitter of noxious stimulation in the spinal cord. We measured glutamate release from rat spinal synaptoneurosomes by using an enzyme-linked fluorimetric assay. Glutamate was released from spinal cord synaptoneurosomes in response to the addition of 30 mM potassium chloride, 1 mM 4-aminopyridine, or 1 µM ionomycin in the presence of external calcium. There was less release of glutamate in the absence, versus the presence, of external calcium. Clonidine significantly reduced the level of glutamate released from the spinal cord synaptoneurosomes. Tetradecanoyl phorbol acetate, an activator of protein kinase C, enhanced glutamate release. Forskolin, a protein kinase A activator, had no effect on the glutamate efflux. Our data indicate that glutamate released in the spinal cord is dependent on protein kinase C but is independent of the protein kinase A pathway. They also suggest that the inhibition of glutamate release may be the underlying mechanism of antinociception by clonidine at the spinal cord level.

Implications: We demonstrated that synaptoneurosomes from rat spinal cord could release glutamate in response to depolarization. We showed that an activator of protein kinase C increased glutamate released from spinal cord synaptoneurosomes but that clonidine decreased it. Glutamate release may be one of the mechanisms of antinociception at the spinal cord level.


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Glutamate, an excitatory amino acid, is involved not only in excitatory synaptic transmission, but also in synaptic plasticity during memory acquisition and in pathological states, such as hypoxic neuronal death (1), drug addiction (2), and nociception (3,4). Glutamate plays an essential role in relaying noxious stimuli in the spinal cord. The N-methyl-D-aspartate (NMDA) receptor, one of the glutamate receptors, is thought to be involved in spinal hyperexcitability after peripheral nerve injury, in which a light touch can cause severe pain (5,6). The NMDA receptor mediates tonic nociceptive transmission in the dorsal horn of the spinal cord. In contrast, the {alpha}-amino-3-hydroxyisoxazolepropionic acid receptor, which is another type of ionotropic glutamate receptor, mediates the acute phase of nociception (7). Spinal metabotropic glutamate receptors are required for the generation of inflammation-evoked hyperexcitability of spinal cord neurons (8).

{alpha}2-adrenergic agonists, administered intrathecally or epidurally, have potent antinociceptive effects (9). They alter the release of neurotransmitters in the spinal dorsal horn (10,11) and hyperpolarize dorsal horn wide dynamic-range neurons (12). On the one hand, there have been few reports on the effect of {alpha}2-adrenergic agonists on glutamate release in the spinal cord. On the other hand, the intracellular signal transduction system of noxious stimulation has been studied intensively, and protein kinase C (PKC) has been reported to play an important role in the regulation of nociception (13). However, it has not yet been determined whether PKC increases glutamate release from the spinal cord.

In the present study, the effects of clonidine (an {alpha}2-adrenergic agonist), tetradecanoyl phorbol acetate (TPA; an activator of PKC), and forskolin (an activator of protein kinase A [PKA]) on the calcium-dependent glutamate release from spinal synaptoneurosomes were studied using a fluorimetric system.


   Methods
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 Abstract
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 Methods
Results
 Discussion
 References
 
The protocol for this study followed the guidelines for animal experiments of Kyoto University. Synaptoneurosomes were prepared essentially as previously described (14). Adult male Wistar rats weighing 250–350 g were stunned and decapitated. The spinal cord from the cervical to the lumbar region was removed and gently homogenized in a glass-glass homogenizer in 7 vol (wt/vol) of a physiological HEPES buffer consisting of 125 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.2 mM Na2HPO4, 10 mM glucose, and 20 mM HEPES/NaOH, pH 7.4. The homogenate was passed through a prefilter consisting of three layers of nylon mesh (pore size 100 µm), then filtered through a 10-µm filter. The filtered solution was then centrifuged at 1000g for 15 min. The pellet was resuspended in the physiological HEPES buffer to give a protein concentration of 0.67 mg/mL. The protein concentration was measured by using the Bradford method (15). Synaptoneurosomes were stored as a resuspension at 0°C until use, and all experiments were performed within 5 h of preparation.

The release of endogenous glutamate was determined fluorimetrically as described previously (16,17). First, we examined the effects of KCl (30 mM), 4-aminopyridine (4-AP; 1 mM), and ionomycin (1 µM), all of which induce intracellular calcium increase. Resuspended synaptoneurosomes, 2 mL, were stirred at 37°C in a thermostatted fluorimeter cuvette. Five minutes after the beginning of incubation, 0.1 mM CaCl2 was added, and at 35 min, the assay was initiated by addition of 1 mM NADP+, 1.2 mM CaCl2, and 104 U of glutamate dehydrogenase. Immediately after these additions, recording of fluorescence was started (0 s). At 200 s, KCl, 4-AP, ionomycin, or vehicle (double-distilled water or 0.01% ethanol in water) was added. Second, we investigated the effects of tetradecanoyl phorbol acetate, forskolin, and clonidine on depolarization-induced glutamate release. In this experiment (100 s), tetradecanoyl phorbol acetate (TPA; 100 nM), forskolin (1 and 10 µM), clonidine (1, 10, 100 µM), or vehicle (double-distilled water) was added. At 200 s, 4-AP was added to induce depolarization. Fluorescence was monitored in a spectrofluorimeter at 340 nm (excitation) and 460 nm (emission). In assays without external calcium, 0.5 mM EGTA was added instead of calcium. Data were recorded at 0.5-s intervals up to 500 s and were transferred to a computer for storage and analysis. Calcium-dependent release of glutamate was calculated as the difference in the amount of glutamate released between the presence and absence of external calcium for 300 s after the addition of depolarizers.

Glutamate dehydrogenase was purchased from Toyobo Chemicals, Osaka, Japan. Ionomycin was obtained from Boehringer Mannheim GmbH, Mannheim, Germany. All other reagents were from Nacalai Chemicals, Kyoto, Japan or Sigma Chemicals, St. Louis, MO.

The mean value of at least five determinations of each experiment was used as a representative value of each group. Statistical comparisons of each group were performed by one-way analysis of variance, followed by Bonferroni's modification of the t-test. Differences at P < 0.05 were considered statistically significant.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
KCl- and 4-AP–evoked glutamate release has two components: one calcium-dependent and the other calcium-independent. Figure 1 shows the change of glutamate concentration induced by KCl depolarization in the presence or absence of calcium. At 200 s, synaptoneurosomes were depolarized by adding KCl to a final concentration of 30 mM. The increase of glutamate from 0 to 200 s shows no significant difference between the presence and absence of calcium, and this increase may include basal release of glutamate from synaptoneurosomes. KCl induced a drastic increase in the glutamate release in the presence of external calcium but had almost no effect when 0.5 mM EGTA was added instead of calcium, i.e., in the absence of external calcium. The difference between the presence and absence of calcium at 500 s was considered as the calcium-dependent glutamate release.



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  		Figure 1. KCl stimulates glutamate release from spinal cord synaptoneurosomes. Synaptoneurosomes (0.67 protein mg/mL) were incubated in the presence of 1.3 mM CaCl2 (a) or 0.5 mM EGTA (b). Synaptoneurosomes were depolarized by 30 mM KCl at 200 s. Glutamate release was assayed fluorimetrically. Each trace is the mean of five independent experiments.

 
Figure 2 shows the amount of glutamate released calcium-dependently 300 s after the addition of either KCl, 4-AP, or ionomycin. The difference in glutamate release between parallel experiments performed in the presence and absence of external calcium was obtained at specific time points. We used 30 mM KCl and 1 mM 4-AP to induce depolarization. When double-distilled water (the vehicle of KCl and 4-AP) or 0.01% ethanol in water (the vehicle of ionomycin; it has no effect on glutamate release) was added at 200 s, almost no increase in glutamate efflux was observed. The increase in glutamate levels after the addition of 30 mM KCl, 1 mM 4-AP, or 1 µM ionomycin was significantly different from that in the vehicle. The difference among the increase in release caused by 30 mM KCl, 1 mM 4-AP, and ionomycin was not statistically significant.



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  		Figure 2. Calcium-dependent release of glutamate after the addition of 30 mM KCl, 1 mM 4-aminopyridine (4-AP), 1 µM ionomycin, or vehicle. Data are mean values ± SEM (bars) from experiments 300 s after the addition of each drug. *P < 0.001 versus vehicle.

 
The involvement of PKA and PKC in glutamate release was examined in spinal cord synaptoneurosomal preparations. Synaptoneurosomes were depolarized by 1 mM 4-AP. Table 1 shows that TPA, a potent PKC activator, potentiated the glutamate efflux. Neither 1 µM forskolin nor 10 µM forskolin (data not shown) increased the amount of glutamate released.


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  		Table 1. Effects of Tetradecanoyl Phorbol Acetate (TPA) and Forskolin on Glutamate Released from the Spinal Cord Synaptoneurosomes
 
Clonidine (1, 10, and 100 µM), an {alpha}2-adrenergic agonist, reduced the calcium-dependent glutamate release from the spinal cord synaptoneurosomes dose-dependently (Fig. 3).



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  		Figure 3. Dose response of clonidine on the amount of glutamate released from spinal cord synaptoneurosomes in 300 s after the addition of 1 mM 4-aminopyridine. Vehicle and 1, 10, and 100 µM clonidine were added 100 s before the addition of 4-aminopyridine. Data are mean values ± SEM (bars) from more than five independent experiments. *P < 0.05 versus vehicle. **P < 0.001 versus vehicle.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
We showed that depolarization-induced glutamate release from spinal synaptoneurosomes was augmented in response to the addition of TPA. Clonidine, an {alpha}2-adrenergic agonist used clinically, decreased the amount of glutamate released from spinal synaptoneurosomes.

Synaptosomes and synaptoneurosomes are models used widely to study neurotransmitter release. Using these preparations, we can study the role of glutamate in the spinal cord. Synaptosomes are composed of the presynaptic sac, whereas synaptoneurosomes are entities in which a presynaptic sac is attached to a resealed postsynaptic sac (14). We previously reported that depolarization-induced glutamate release from cerebrocortical synaptoneurosomes is reduced by a hemeoxygenase inhibitor (18).

The amount of purified synaptosomes obtained from spinal cord was much smaller than that obtained from brain; furthermore, the glutamate release per 1 mg of protein from spinal cord synaptosomes was approximately one-tenth that from brain synaptosomes (unpublished observation). As far as we know, all previous reports used crude spinal cord synaptosomes, instead of purified synaptosomes, to measure glutamate release (19,20). Thus, we used purified spinal cord synaptoneurosomes instead of crude spinal cord synaptosomes, because much more purified synaptoneurosomes (as protein) were easily obtained from one spinal cord.

We measured glutamate efflux using a fluorimetric method, which has been used to quantitate the amount of glutamate released from cerebrocortical synaptosomes (17,21). However, some reports have measured glutamate release from the spinal synaptosomes by using means other than the fluorimetric method. One group assessed glutamate release from spinal cord synaptosomes using a combination of sampling at certain intervals and high-performance liquid chromatography analysis of glutamate (20). Another group loaded spinal cord synaptosomes with [3H]glycine and [3H]D-aspartic acid as markers of glycine and glutamate/aspartate release, respectively (19). In contrast, the method that we used reflects the amount of intrinsic glutamate released from spinal cord synaptoneurosomes in real time.

Tibbs et al. (21) reported that 30 mM KCl and 1 mM 4-AP increase intrasynaptosomal calcium concentrations to a similar extent and induce glutamate release in cerebrocortical synaptosomes. Our experiments also demonstrated that the amount of glutamate released by 30 mM KCl and 1 mM 4-AP showed no significant difference. KCl initiates depolarization by changing the membrane potential, which is normally maintained by an electrochemical gradient. 4-AP induces depolarization by blocking potassium channels. Both of these drugs induce depolarization, thereby causing calcium influx and glutamate release. The origin of calcium-dependent glutamate release is the synaptic vesicles, and the origin of calcium-independent release is the cytosol (22). Synaptoneurosomes were depolarized by 1 mM 4-AP to investigate the effect of TPA, forskolin, and clonidine because 4-AP is reported to cause more "physiological" depolarization than KCl (22).

Kamisaki et al. (20) showed that clonidine reduced glutamate release from a spinal cord synaptosome preparation. However, they used a crude fraction of spinal cord that was not purified synaptosome in a strict sense and that contained mitochondria, etc. They also showed that clonidine reduced glutamate release from a spinal cord synaptosome preparation (20). The effective clonidine concentrations reported (1–10 µM) were nearly equivalent to the clinically effective range in cerebrospinal fluid (23). Our results are compatible with the report in synaptosomes (20), although our preparation included both pre- and postsynaptic components.

Cyclic AMP enhances the depolarizing responses of a proportion of dorsal horn neurons to ionotropic glutamate receptor agonists (24). The role of PKA in nociception at the spinal cord has been suggested, but the potentiation of glutamate efflux from synapses has not yet been proven. The fact that forskolin failed to affect glutamate release rules out the involvement of PKA in the potentiation of the glutamate efflux.

TPA increases the amount of glutamate released from cerebrocortical synaptosomes (16). PKC plays an important role in the regulation of nociception at the spinal cord level (4). Our study is the first to show that TPA can increase the amount of glutamate released from spinal cord synaptoneurosomes. Combined with recent data from knockout mice that PKC{gamma} is essential in establishing the state of chronic pain (13), we speculate that the potentiation of glutamate efflux by PKC plays an important role in nociception in the spinal cord.

In conclusion, we showed that glutamate was released from spinal cord synaptoneurosomes in response to calcium influx using a real-time fluorimetric system. The effects of PKC and {alpha}2-adrenergic agonists on the regulation of glutamate release may explain their involvement in nociceptive mechanisms at the spinal cord level.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication February 22, 1999.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 1999 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press