1 Medical Clinic of Hamamatsu Base, Japan Air Self Defense Force, Hamamatsu, 2 Self Defense Force, Gifu Hospital, Kagamihara, 3 Department of Anesthesiology, Self Defense Force, Hanshin Hospital, Kawanishi, 4 Department of Anesthesia, Shiki Citizen Hospital, Shiki and 5 Department of Anesthesiology, National Defense Medical College, Tokorozawa, Japan
* Corresponding author: Medical Clinic of Hamamatsu Base, Nishiyama-cho-1, Hamamatsu city, Shizuoka, 432-8551 Japan. E-mail: yuadachi{at}poppy.ocn.ne.jp
Accepted for publication May 15, 2005.
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Abstract |
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Methods. Microdialysis probes were implanted into male SpragueDawley rats and perfused with artificial cerebrospinal fluid (CSF). The dialysate was injected directly into an HPLC every 20 min. Each group of rats (n=57) was administered saline, apomorphine 100 µg kg1, pargyline 7.5 or 75 mg kg1, reserpine 2 mg kg1 or -methyl-p-tyrosine (AMPT) 250 mg kg1. Another set of rats was perfused with artificial CSF containing tetrodotoxin (TTX) 1 µM or calcium-free CSF containing 10 mM EGTA. Rats were anaesthetized with halothane 0.5 or 1.5% 1 h after pharmacological treatments.
Results. In rats pretreated with apomorphine, despite a decrease in DA concentration, halothane induced a increase in DA metabolites. Pargyline (high dose) and reserpine completely and AMPT partially antagonized the increase in DA metabolites induced by halothane anaesthesia. TTX perfusion reduced the increase in DA, whereas calcium-free CSF perfusion did not.
Conclusions. Our data suggest that halothane accelerates DA metabolism at presynaptic sites by releasing DA from reserpine-sensitive storage vesicles to the cytoplasm in a calcium-independent manner. The metabolic oxidative stress of inhalation anaesthesia requires future investigation.
Keywords: anaesthetics volatile, halothane ; measurement techniques, microdialysis ; model, rat brain ; pharmacology, dopamine
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Introduction |
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It is widely believed that anaesthesia modulates synaptic communication within the CNS.610 Dopamine is a major CNS neurotransmitter,11 and numerous experiments have been carried out to examine the effects of anaesthetic drugs on dopaminergic neural activity,4 5 8 9 12 mainly by examining the release of DA from nerve terminals. In addition, changes in the extracellular concentrations of DA metabolites are also thought to result from the modification of dopaminergic activity.13
Fink-Jensen et al.5 speculated that halothane anaesthesia might increase DA release from axon terminals and might simultaneously accelerate DA reuptake via a proportional increase in the activity of DA transporters, thereby maintaining a steady extracellular concentration of DA and increasing the production of DA metabolites. Although an increase in dopaminergic neuronal activity causes an increase in DA release and enhances the subsequent metabolism of DA, the increase in the concentrations of DA metabolites that occurs during anaesthesia may be independent of DA release.2 3 We hypothesized that halothane might act by enhancing the metabolism of presynaptically stored or synthesized DA within axon terminals, rather than by affecting the metabolism of DA taken up from the extracellular space.
In the present study, we used in vivo microdialysis to examine the mechanism(s) by which the concentrations of DA metabolites are increased during halothane anaesthesia.2 3 We investigated the relationships between halothane anaesthesia and the effects of apomorphine (a DA D2 receptor agonist),14 pargyline (an inhibitor of monoamine oxygenase, MAO),15 reserpine (an inhibitor of catecholamine transport into vesicles),16 -methyl-p-tyrosine (AMPT; a tyrosine hydroxylase inhibitor),17 tetrodotoxin (TTX) and calcium-free artificial cerebrospinal fluid (CSF).
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Methods |
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Microdialysis
Rats were anaesthetized with sevoflurane and ventilated through an orotracheal tube. Surgery was performed by topical application of lidocaine 1%. Using a stereotaxic apparatus, a unilateral guide cannula was implanted just above the striatum (AP +0.6 mm, ML +3.0 mm, DV 3.8 mm) following the atlas of Paxinos and Watson.18 The rats were allowed to recover for at least 2 days before the experiment began. After each experiment, the rats were killed by inhalation of isoflurane and i.v. injection of thiopental. The brain was removed, and the placement of the microdialysis probe was identified histologically.
Microdialysis probes were obtained from Eicom (Kyoto, Japan) (o.d. 0.22 mm, membrane length 3 mm, polycarbonate tubing, cutoff molecular weight 50 000). At about 7:00 a.m. on the day of the experiment, the rat was briefly anaesthetized with sevoflurane. The probe was inserted carefully into the striatum through a guide cannula and fixed to the cannula with a screw. This procedure was performed within 5 min of brief anaesthesia, after which the rat was immediately placed in a clear open Plexiglas box (15 litres capacity, 27 cm diameter x 26 cm high) for recovery. The rats regained consciousness within approximately 3 min. After recovery, the probe was continuously perfused with artificial CSF (Na+ 145.4 mEq litre1, K+ 2.8 mEq litre1, Ca2+ 2.3 mEq litre1, Mg2+ 2.2 mEq litre1, 23.1 mEq litre1, Cl 128.5 mEq litre1, P 1.1 mmol litre1, glucose 61 mg dl1) at a flow rate of 2 µl min1 using a microinfusion pump (ESP-64, EICOM, Kyoto, Japan) to determine the baseline concentrations of DA and its metabolites. Samples were collected every 20 min and injected directly into an online analytical system with an auto-injector (EAS-20; Eicom), as described elsewhere.2 3 The concentrations of DA, 3-MT, DOPAC and HVA in each dialysate fraction (40 µl per 20 min) were determined by HPLC with an electrochemical detector (ECD-300; Eicom). These compounds were separated by reverse-phase ion-pair chromatography with a 5-µm C-18 column (MA5-ODS, 150 x 2.1 mm; Eicom) using an isocratic mobile phase (0.1 M sodium acetate, 0.1 M citric acid, 1.4 mM sodium 1-octanesulfonate, 5 µM EDTA-Na2 and methanol 1314%, pH 3.9), delivered at a flow rate of 230 µl min1 by a high-pressure pump (EP-300; Eicom). A guard column (MA, 5 x 4 mm; Eicom) prevented deterioration and plugging of the analytical column. The compounds were quantified by electrochemical detection using a glassy carbon working electrode set at 650 mV against a AgAgCl reference electrode. The detection limit for each of the compounds was roughly 0.5 pg per sample.
DA and its metabolites reached stable baseline concentrations within about 4.5 h after implantation of the microdialysis probe. Therefore, at least six dialysate samples (each 40 µl collected in 20 min) were collected before starting the experiment. The mean value obtained from the last three samples was used as the baseline concentration. The time at which manipulation started is hereafter called fraction number 1 (Fr. 1).
Experimental procedure
Each group consisted of five to seven rats. The rats were given saline (2 ml kg1), the same volume of apomorphine 100 µg kg1, pargyline 7.5 or 75 mg kg1, reserpine 2 mg kg1 and AMPT 250 mg kg1 intraperitoneally with or without 1-h halothane anaesthesia (0.5 or 1.5%). Apomorphine was administered at the time of induction of anaesthesia and administration of other drugs was followed by anaesthesia after 80 min for pargyline, 24 h for reserpine and 4 h for AMPT, in order to acquire the maximum pharmacological effects. Two other groups were perfused with a different dialysate starting 1 h before anaesthesia, with TTX 10 µM or with calcium-free artificial CSF containing 10 mM EGTA (Ca2+-free CSF).
Each rat was anaesthetized in a semi-closed Plexiglas box, into which halothane 3% was initially introduced at a rate of 3 litre min1 for about 5 min until a steady state was achieved. Subsequently, halothane 0.5 or 1.5% was applied at a rate of 2 litre min1, using air (23% oxygen) as a carrier. The rectal temperature of the rat was monitored and maintained at 37°C with an electrical heating pad, except in the control and 0.5% halothane groups, because the animals were consistently awake during inhalation. The concentrations of volatile gas and oxygen in the box were monitored using an infrared anaesthetic gas analyser (Capnomac Ultima; Datex, Helsinki, Finland). Immediately after the 1-h anaesthesia, the gas in the box was exchanged with room air by forced ventilation.
Drugs
Halothane was obtained from Takeda Chemical Industries (Osaka, Japan). Apomorphine, reserpine, AMPT and TTX were purchased from Sigma (St Louis, MO, USA). Pargyline was purchased from ICN (ICN Biomedicals, Costa Mesa, CA, USA).
Statistics
Data are presented as mean (SEM). Data were analysed by two-way analysis of variance with drugs as between-subjects variable and time as within-subject variable. The values of fractions between 3 and 8 were calculated as the effect of anaesthesia. For significant (P<0.05) drug or time interactions, a subsequent NewmanKeuls post hoc multiple comparison test was performed (NCSS 2000; Number Cruncher Statistical Systems, Kaysville, UT, USA).
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Results |
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Discussion |
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It has been proposed that the marked increase in the concentrations of DA metabolites during halothane anaesthesia is attributable to an increase in DA turnover, including the release and reuptake of DA.4 5 In our previous studies,2 3 halothane increased the concentrations of extracellular DA metabolites and markedly enhanced the DA release that was induced by the systemic administration of nomifensine.2 3 However, the increase in DA concentration after local administration of nomifensine into the striatum was not increased by halothane anaesthesia.3 The local administration of nomifensine might have been sufficient to inhibit DA transport,20 which suggested that halothane probably did not accelerate DA turnover from the intra- to extracellular space at the axon terminal.5 Furthermore, halothane anaesthesia increased the extracellular concentrations of DA metabolites in rats that were given nomifensine both systemically and locally.3 We hypothesized that halothane might act by enhancing the metabolism of presynaptically stored or synthesized DA released from the axon terminal, rather than by altering the metabolism of DA that is returned to the terminal from the synaptic cleft.
In the first experiment, we confirmed that the extracellular concentrations of DA and DA metabolites were modulated independently by halothane anaesthesia. Apomorphine is an agonist of presynaptic DA D2 receptors. Pretreatment with apomorphine reduces the release of DA and decreases the extracellular concentrations of DA and DA metabolites by potentiating the negative feedback regulation of DA.14 In the present study, halothane did not affect the apomorphine-induced reduction in extracellular DA concentrations. The increase in the concentrations of DA metabolites was independent of extracellular DA and was not regulated by the DA D2 receptor. Therefore, the origin of the DA metabolites that increase in concentration in the presence of halothane is likely to be unrelated to released or transported DA.
Pargyline, an inhibitor of MAO, increases the extracellular concentration of DA by inhibiting the oxidation of DA to DOPAC and by inhibiting the oxidation of 3-MT to HVA.15 21 In our study, pretreatment with pargyline decreased the concentrations of DOPAC and HVA and increased the concentrations of DA and 3-MT (Figs 3 and 4). The 3-MT is synthesized from DA by catechol-o-methyltransferase (COMT).15 22 COMT is found only at the postsynaptic site,23 and changes in the concentration of 3-MT have been reported to be a good indicator of dopaminergic neural activity.13 In the present study, the increase in the concentrations of both DA and 3-MT due to the inhibition of MAO was enhanced during halothane anaesthesia. Halothane anaesthesia itself might increase dopaminergic activity and accelerate DA release and turnover; however, the extracellular concentration of DA was maintained at a constant level during anaesthesia by the oxidation of DA in the absence of pargyline.
At nerve endings, reserpine depletes vesicular stores of catecholamines and inhibits the release of neurotransmitters.16 The administration of 2 mg kg1 reserpine 24 h before halothane anaesthesia was sufficient to deplete DA in the present study. After reserpine administration, the extracellular concentration of DA was too low to be detected before and during anaesthesia, and the halothane- induced increase in the concentrations of DA metabolites was completely abolished (Fig. 5). Therefore, the increase in the concentrations of DA metabolites during anaesthesia may be attributable to the release of DA from vesicles.
AMPT inhibits the synthesis of 3-(3,4-dihydroxyphenyl)-DL-alanine from tyrosine by the enzyme tyrosine hydroxylase and depletes catecholamines such as DA at the axon terminal.17 Pretreatment with AMPT reduced the concentrations of DA and DA metabolites in the present study (Table 1). Parker and Cubeddu24 reported that the amphetamine-induced release of extracellular DA was reduced by inhibiting DA synthesis with AMPT and was increased by inhibition of MAO. Amphetamine-induced DA release depends mainly on a reserpine-resistant (non-vesicular) DA pool.16 In contrast to the effect of reserpine, AMPT failed to completely inhibit the increase in the concentrations of DA metabolites during halothane anaesthesia in the present study (Fig. 5).
The results of the present study indicate that halothane may stimulate the release of DA from a reserpine-sensitive vesicular store into the cytoplasm at the axon terminal. DA released in this way may be metabolized immediately in a compensatory manner by MAO within the cytoplasm.21 This hypothesis is supported by the results of Keita and colleagues,8 who investigated the effect of halothane on DA release in slice preparations and synaptosomes and demonstrated that halothane increased the spontaneous release of DA in vitro. In the present study, microdialysis was used to measure the extracellular concentrations of DA and DA metabolites, which included substances that were released or that diffused from axon terminals. Therefore, we could not distinguish between changes in the absolute amount of intracellular cytoplasmic DA and the total extracellular concentration of DA.21 However, the pharmacological manipulations that we used revealed the effects of halothane on DA release and metabolism.
Finally, we investigated the effects of TTX-containing or calcium-free perfusate on DA metabolism during halothane anaesthesia. TTX reduced the halothane-induced increase in the concentrations of DA metabolites (Fig. 6), whereas the calcium-free perfusate had no such effect (Fig. 7). As the increase in the concentrations of metabolites was sensitive to TTX, the effect of halothane on DA metabolism must be related to neuronal activity. Calcium-dependent release of vesicular DA into the extracellular space is reduced in calcium-free perfusates.2527 Our results indicate that the halothane-induced DA release from vesicles of the axon terminal might depend on neuronal activity and sodium channels,28 but is independent of calcium entry. The increase in the concentration of 3-MT during anaesthesia was reduced in the calcium-free perfusate. The calcium-sensitive exocytotic transport of DA into the extracellular space and the reuptake of DA into the postsynaptic site might be inhibited in the absence of extracellular calcium ions, and the halothane-induced acceleration of the metabolism of DA to 3-MT was diminished.25
Our results are similar to the findings of Woodward and colleagues,29 who suggested that the slow phase of potassium-stimulated DA release from rat striatal synaptosomes in vitro did not require calcium, but might be mediated via reversal of the sodium-linked DA transport system. Our experiments did not reveal any difference in the change of the phase of DA release, but it is possible that halothane induces the release of vesicular DA via a similar mechanism. It has been amply documented that during depolarization-evoked neurotransmitter release, a calcium-dependent fraction of neurotransmitter is released into the extracellular space via exocytosis, while a sodium-dependent fraction is released from the cytoplasm via a reversal in the activity of neurotransmitter transporters.26 30 Halothane decreases the nerve impulse-dependent release of exocytotic DA.8 31 but may increase the release of extravesicular DA into the cytoplasm and may accelerate the oxidation of DA by MAO.
In conclusion, we demonstrated that halothane enhanced DA metabolism at the axon terminal. DA was released from a reserpine-sensitive vesicular pool in a calcium-independent manner, and MAO simultaneously metabolized DA that was released into the cytoplasm at presynaptic sites.
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Acknowledgments |
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References |
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