Xenon reduces glutamate-, AMPA-, and kainate-induced membrane currents in cortical neurones

A. Dinse1, K. J. Föhr1,*, M. Georgieff1, C. Beyer2, A. Bulling3 and H. U. Weigt1

1 Clinic for Anesthesiology, Ulm, Germany. 2 Department of Anatomy, Tübingen, Germany. 3 flyion GmbH, Tübingen, Germany

* Corresponding author. E-mail: karl.foehr{at}medizin.uni-ulm.de

Accepted for publication December 13, 2004.


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. The anaesthetic, analgesic, and neuroprotective effects of xenon (Xe) are believed to be mediated by a block of the NMDA (N-methyl-D-aspartate) receptor channel. Interestingly, the clinical profile of the noble gas differs markedly from that of specific NMDA receptor antagonists. The aim of this study was, therefore, to investigate whether Xe might be less specific, also inhibiting the two other subtypes of glutamate receptor channels, such as the {alpha}-amino-3-hydroxy-5-methyl-4-isoxazolole propionate (AMPA) and kainate receptors.

Methods. The study was performed on voltage-clamped cortical neurones from embryonic mice and SH-SY5Y cells expressing GluR6 kainate receptors. Drugs were applied by a multi-barreled fast perfusion system.

Results. Xe, dissolved at approximately 3.45 mM in aqueous solution, diminished the peak and even more the plateau of AMPA and glutamate induced currents. At the control EC50 value for AMPA (29 µM) these reductions were by about 40 and 56% and at 3 mM glutamate the reductions were by 45 and 66%, respectively. Currents activated at the control EC50 value for kainate (57 µM) were inhibited by 42%. Likewise, Xe showed an inhibitory effect on kainate-induced membrane currents of SH-SY5Y cells transfected with the GluR6 subunit of the kainate receptor. Xe reduced kainate-induced currents by between 35 and 60%, depending on the kainate concentration.

Conclusions. Xe blocks not only NMDA receptors, but also AMPA and kainate receptors in cortical neurones as well as GluR6-type receptors expressed in SH-SY5Y cells. Thus, Xe seems to be rather non-specific as a channel blocker and this may contribute to the analgesic and anaesthetic potency of Xe.

Keywords: anaesthetics volatile, xenon ; measurement techniques, electrophysiology ; model, brain cortical neurones ; model, neuroblastoma cells ; pharmacology, glutamate ligand gated ion channel ; receptors, amino acid, AMPA ; receptors, amino acid, kainate ; receptors ; amino acid, NMDA


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The pharmacological properties of xenon (Xe) as an effective and safe anaesthetic with neuroprotective properties may at least in part be explained by its effect on excitatory synapses where it has been shown to inhibit mainly the currents conducted by the NMDA (N-methyl-D-aspartate) receptor.1 2 As the clinical profile of Xe is markedly different from that of other NMDA antagonists, it is most likely that the noble gas has additional targets. A blocking effect of Xe on two-pore K+ channels as well as on nicotinic acetylcholine and on serotonin receptors has been described, whereas an effect on non-NMDA receptors has so far been denied, although recently also confirmed.3 As to inhibitory synapses, the effects of Xe on glycine and GABA receptors have been examined, and also here, the results were controversial.4 5

The three subtypes of glutamate receptor channels have been named after their preferred ligands (i.e. NMDA {alpha}-amino-3-hydroxy-5-methyl-4-isoxazolole propionate (AMPA), and kainate). All three receptors have been suspected to play crucial roles in various diseases.68 AMPA and kainate receptors (also referred to as ‘non-NMDA receptors’) are suspected to be involved in spinal pain pathways,9 10 while NMDA receptors receive increasing attention in the context of anaesthetic mechanisms.11 Non-NMDA receptors modulate the release of a variety of neurotransmitters.1214 In addition, depolarization of the post-synaptic membrane via AMPA or kainate receptors facilitates relief of the Mg2+ block of the NMDA receptor.6 Therefore, glutamate receptor antagonists for any subtype have considerable potential as therapeutic agents.

NMDA receptor antagonists have been more intensively studied with respect to clinical application, such as neuroprotection and pain, than AMPA or kainate receptor antagonists. There is evidence from animal studies that AMPA and kainate receptor antagonists may also have neuroprotective effects in ischaemic stroke and epilepsy.1520 Moreover, the proposal of chronic pain being relieved by antagonists of kainate and perhaps also of AMPA receptors receives growing support.9 10 15

An antagonistic effect of Xe on AMPA receptors was not apparent in a study using an indirect approach at native neurones, but was postulated after currents conducted by AMPA receptor channels in heterologous expression systems had been directly investigated.21 22 An antagonistic effect was detectable in these systems, however, only when receptor desensitization was overcome by either using kainate as a non-selective and non-desensitizing agonist or by applying glutamate in the presence of cyclothiazide. Fast desensitizing currents evoked by the natural agonist glutamate in the absence of cyclothiazide were suppressed only poorly and in a subunit-specific manner. From these results, the authors22 concluded that, in vivo, Xe effects are not mediated via AMPA receptors. A direct investigation of the effects of Xe on kainate-controlled channels is not available.

As the structures of the three glutamate receptors are highly homologous, especially in domains that could be relevant for an interaction with anaesthetics, these channels may all be relevant molecular targets for Xe.23 The aim of this study was, therefore, to examine the action of Xe on non-NMDA receptors using AMPA, kainate, and glutamate as agonists.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Preparation and culture of cortical neurones
Adult BALB/c mice were fed a standard pellet diet and kept in a 12-h dark/light cycle, as approved by the institutional animal care and use committee. The mice were allowed to mate during a 12-h period and the day after mating was defined as day 0 of pregnancy. On embryonic day 15, pregnant mice were anaesthetized with chloral hydrate 25% and killed by decapitation. After removal of the striatal and hippocampal tissue, the cerebral cortex of the fetal brains was dissected and dissociated both enzymatically (trypsin 0.1%) and mechanically. Cortical neurones were plated to a density of 1.8x105 cells per cm2 on poly-DL-ornithine-coated (0.5 mg ml–1) plastic culture dishes (10 cm2). The cultures were maintained in minimum essential medium (Invitrogen, Karlsruhe, Germany) supplemented with 5% fetal calf serum, and grown for at least 2 weeks. The medium was exchanged every third day. Neurones were used for experimentation after 10–14 days culture.

Transient transfection of SH-SY5Y cells with GluR6 receptors
SH-SY5Y cells (ATCC#CRL 2266) were maintained at 37°C in a humidified atmosphere at air 95%, CO2 5% in a mixture of DMEM and HAM F12 (1:1) supplemented with 50 u penicillin ml–1, 50 µg ml–1 streptomycin (Gibco, Eggenstein, Germany), 2 mM L-glutamine (Boehringer, Mannheim, Germany), and 15% (v/v) fetal calf serum (Gibco). The cells were grown on uncoated culture dishes (Sarstedt, Nümbrecht, Germany) to 40% confluency and transfected using the LipofectamineTM2000 transfection reagent (Invitrogen) with the cDNA encoding the VCR version of the rat GluR6 subunit (NIH: GeneBank Nucleotide Sequence, accession number Z11548).24 The plasmid encoding the GluR6 cDNA in pBSSK(+) was kindly provided by S. Heinemann (La Jolla, CA) and subcloned into the eukaryotic expression vector pcDNA3.1 (Invitrogen, Karlsruhe, Germany). For identification of GluR6-expressing cells, the cDNA of green fluorescent protein was co-transfected. Transfected cells were treated with ConA (0.4 mg ml–1, 10 min room temperature (RT)) in order to prevent receptor desensitization25 and used for experimentation within 24–48 h.

Standard experimental procedures
The cells were transferred to plastic dishes that were perfused continuously at about 4.5 ml min–1 with an ‘extracellular’ medium containing 140 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1.5 mM CaCl2, 6 mM glucose, 12 mM HEPES, and 100 µM strychnine, pH adjusted to 7.3. Membrane currents were determined at room temperature (23–25°C) using the whole-cell recording technique with the membrane potential constantly clamped at –80 mV. The equipment consisted of an EPC-9 amplifier and TIDA software (HEKA, Lambrecht, Germany). The recording pipettes were drawn from borosilicate glass with a tip resistance of 3–6 M{Omega} when filled with an ‘intracellular’ medium containing 140 mM KCl, 2 mM ATP x 2 Na, 2 mM MgCl2, 2 mM EGTA, 10 mM HEPES, pH adjusted to 7.2. To improve sealing, the pipettes had been briefly dipped into dimethyldichlorosilane 2%, dissolved in methylene chloride.

Chemicals
Xe was purchased from Messer (Griesheim, Germany). AMPA, (S-{alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), kainate, and SYM2206 (-4-[4-aminophenyl]-1,2 dihydro-1-methyl-2-propylcarbamoyl-6,7-methylene-dioxyphthalazine) were from Tocris (Köln, Germany). ConA (type IV) and all other chemicals were obtained from Sigma (Deisenhofen, Germany).

Preparation and application of test solutions
Xe solutions were prepared by equilibrating 15 ml Xe gas with 40 ml of the particular test solution. The monitored average concentrations of Xe were 91.4 (10.0) µl ml–1 during the experiments using AMPA, and 88.3 (5.2) µl ml–1 during the experiments using kainate. A Xe concentration of 90 µl ml–1 corresponds to 3.54 mM at standard conditions in our laboratory (~500 m above sea level, room temperature 22–25°C), a partial pressure of 0.84 atm or an anaesthetic potency of about 1.1 MAC for humans. Xe concentrations were measured from the fluid actually perfusing the cells by static headspace gas chromatography mass-spectrometry (headspace GCMS).

Agents were applied to the cells by the L/M-SPS-8 superfusion system (List, Darmstadt, Germany). To restrict the presence of the agent to a small volume within the dish, a combination of two perfusion systems was installed, that is (i) a global bath perfusion with the inflow set at 4.5 ml min–1 and an outflow that removed any excess fluid and (ii) a local inlet for the generation of a continuous stream of test solution. For a rapid change between various test solutions the local inlet consisted of the tip of an eight-barreled pipette that was positioned at a distance of 50–100 µm upstream of the measuring field. All test solutions were administered at 1 ml min–1 using infusion pumps (Braun, Melsungen, Germany). When AMPA-induced currents were recorded, an additional inlet, positioned 50–100 µm upstream from the measurement field was used for the pre-application of Xe. The time of solution exchange in the measuring field was estimated from the changes in liquid junction potential to be about 1 ms.

Statistical analysis
Results are presented as means (SD). Paired t-test was used for comparing the mean results from the same sample and the unpaired t-test was used for comparing different samples. A difference between results was considered significant when P<0.05. Sigmoidal concentration–response curves were fitted by non-linear regression using the Levenberg–Marquardt method. To compare EC50 values obtained with different agents, 95% confidence intervals were calculated.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Xe effects on AMPA-induced membrane currents in cortical neurones
Application of AMPA to cortical neurones activated inward currents. A typical current response was characterized by a fast initial peak, which rapidly declined to a level that was almost stable at the end of a 5-s drug application pulse (plateau current) (Fig. 1, inset). Both peak and plateau current increased with increasing AMPA concentration. EC50 values for the AMPA-induced peak and plateau currents were 29 and 4.6 µM, respectively. The currents were abolished upon washout periods of less than 1 s (Fig. 1, inset). Repeated applications of the same AMPA concentration resulted in identical responses.



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Fig 1 Inhibition of AMPA-induced peak currents in cortical neurones by pre-applied Xe. Cells were voltage-clamped at –80 mV. The concentration of 5-s AMPA pulses was varied between 1 and 400 µM (abscissa). Peak currents were normalized to the mean peak obtained with 400 µM AMPA. Currents obtained in the presence of Xe were significantly smaller than control. Data points represent means (SD) of 4–13 cells. The inset shows representative traces of currents induced by 50 µM AMPA in the absence (left and right, controls) and presence of the dissolved gas (middle, Xe pre-applied for 10 s). Applications of AMPA and Xe (at about 90 µl ml–1 solution) are denoted by horizontal bars.

 
Simultaneous application of AMPA plus Xe resulted in reduced currents. Both peak and plateau were affected, but to different degrees. At 25 µM AMPA, which is close to the EC50 value, the reduction was 24.2 (6.6) (n=12) and 60.4 (8.5)% (n=12), respectively. Pre-application of Xe for 10 s significantly increased the reduction of the peak (by 40.9 (10.7)%; n=13; P<0.05), but not that of the plateau (56.1 [10.3]%, n=13; P=0.2758). Longer pre-incubation times (60 s) did not further enhance the reduction of peak currents (39.2 [8.4]%; n=6; P<0.05). In all cases, the inhibitory effect of Xe was fully reversible upon washout (Fig. 1, inset).

Because of the stronger effect, we decided to pre-apply Xe for 10 s in all of the following experiments on AMPA receptors. For AMPA concentrations varying between 1 and 400 µM and pre-applied Xe the reduction of the peaks ranged between 59.9 (6.4) and 29.1 (9.7)%, and that of the plateau ranged between 61.8 (6.5) and 48.6 (15.2)%, respectively (n=12 each, Fig. 1). Thus, the relative reduction of AMPA-induced peak currents by Xe was stronger at lower AMPA concentrations.

In the presence of Xe, the EC50 for AMPA-induced peak currents was increased from 29.9 to 48.7 µM. This shift was, however, statistically not significant as the 95% confidential intervals of EC50 values overlapped. From this result we concluded that the mechanism by which Xe blocks AMPA receptors is mainly non-competitive.

To test for a possible cross-excitation of NMDA receptors by AMPA, we added 1 mM Mg2+ to the extracellular fluid, a concentration that completely blocks NMDA receptors at the negative holding potential of –80 mV. We also applied AMPA (50 µM) in combination with the selective NMDA receptor antagonists MK-801 (100 µM) and ketamine (100 µM). These blockers had little or no inhibitory effect on AMPA-induced membrane currents in cortical neurones (n=9 cells; data not shown). Therefore, the observed effects of Xe on AMPA-induced currents were most likely mediated by AMPA receptors and not by NMDA receptors.

Speed of Xe reversibility
After termination of the combined application of AMPA and Xe, a brief additional inward current consistently appeared before the signal returned to baseline (‘hump’ in Fig. 2A). A possible explanation for this might be that the action of Xe was reversed quicker than that of AMPA. To test this hypothesis we co-applied AMPA and Xe for 5 s and terminated the application of AMPA while Xe was maintained for an additional 5 s. The ‘hump-like’ inward current did not appear in this case (Fig. 2B). If the application of Xe was terminated while AMPA was still present, the current amplitude rapidly rose to the control plateau current (Fig. 2C).



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Fig 2 Fast reversibility of the Xe effect on the AMPA-evoked plateau currents in cortical neurones (‘hump’ current). (A) Simultaneous termination of the application of AMPA and Xe induces a fast transient inward current (‘hump’, indicated by arrow, also seen in inset of Fig. 1). (B) The ‘hump’ is absent, when the application of Xe outlasts the AMPA application. (C) The level of the plateau current turns to the control value when the application of Xe is terminated before that of AMPA. Applications of AMPA and Xe are denoted by bars.

 
Xe effects on kainate-induced membrane currents in cortical neurones
Application of kainate to cortical neurones evoked inward currents that showed no sign of desensitization. Within the tested kainate concentration range of 12.5–400 µM, current amplitude increased with increasing kainate concentration.

The kainate-induced currents were also reduced when Xe was co-applied. The reduction was 54.8 (5.7)% at 12.5 µM kainite and by 29.2 (5.3)% at 400 µM kainate (Fig. 3, containing a representative recording as inset). Again, the reduction was fully reversible upon washout. The computed EC50 for kainate-induced membrane currents was 57.5 µM in the absence and 61.9 µM in the presence of Xe.



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Fig 3 Inhibition of kainate-induced membrane currents in cortical neurones by Xe. The concentration of 5-s kainate pulses was varied between 12.5 and 400 µM. Current responses are normalized to the mean current induced by 400 µM kainate. Data points represent means (SD) of 8–14 cells. Currents obtained in the presence of Xe were significantly smaller than their respective controls. Representative traces of currents induced by 50 µM kainate show the sequence of control, test with Xe and another control (inset). Applications of kainate and Xe are denoted by bars. Cells were voltage-clamped at –80 mV.

 
Xe effects on glutamate-induced currents in cortical neurones
Under conditions where NMDA-receptors are completely blocked (1 mM Mg2+ and 100 µM ketamine), a supramaximal concentration of glutamate (3 mM) induced fast desensitizing currents with a rise time (10–90%) of 7.1 (1.3) ms, a desensitization time constant of 15.1 (2.7) ms and a plateau to peak ratio of 0.17. Xe inhibited these responses with the initial peak reduced less (by 44.6 [7%]) than the final plateau (by 65.9 [5.5]%; n=5, Fig. 4).



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Fig 4 Inhibition of glutamate-induced currents in cortical neurones by Xe. (A) Pulses (5 s) of glutamate (3 mM) were applied to neurones in the absence of (controls left and right) and with pre-applied (middle) Xe. (B) Superimposed rising signal component from the left and middle trace at expanded time scale. Rise times (10–90%) for control or in the presence of Xe were 6.2 and 6.4 ms and desensitization time constants were 12.7 and 12.1 ms, respectively. Applications of glutamate and Xe (at about 90 µl ml–1 solution) are denoted by horizontal bars. Neurones were voltage-clamped at –80 mV.

 
Kainate-induced currents in GluR6-transfected SH-SY5Y cells
It is well known that kainate also activates AMPA receptors in neuronal cells.26 In order to test for a contribution of kainate-activated AMPA receptors to the above-reported currents, we also performed experiments using cells containing the GluR6 subtype of kainate receptors which, in contrast to GluR5, shows no sensitivity to AMPA.8 Application of kainate (0.31–20 µM) to SH-SY5Y-cells transfected with GluR6 (and treated with 0.4 mg ml–1 ConA for 10 min) induced membrane currents that showed no desensitization.

These currents were similarly reduced by Xe (Fig. 5). The reduction was 59.6 (3.7)% when the currents were induced by 0.31 µM kainate and by 34.5 (4.1)% with 20 µM kainate. As the EC50 values in the absence (0.97 µM) and presence of Xe (at 1.31 µM kainate) were not significantly different, we postulate a non-competitive action of Xe. Furthermore, terminating the simultaneous application of kainate and Xe resulted in ‘hump’-currents as described above for AMPA-induced currents (see also Fig. 5, inset). Using identical protocols as described for the analysis of AMPA hump-currents, qualitatively similar results were obtained.



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Fig 5 Reduction of kainate-induced membrane currents in GluR6-transfected SH-SY5Y cells by Xe. The concentration of kainate pulses was varied between 312.5 nM and 20 µM. Current responses are normalized to the mean amplitude induced by 20 µM kainate. Currents obtained in the presence of Xe were significantly smaller than their respective controls. Data points represent means (SD) of 5–14 cells. The inset shows representative original current traces. Applications of kainate and Xe (at about 90 µl ml–1 solution) are denoted by bars. Cells were voltage-clamped at –80 mV.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our results suggest that Xe inhibits neuronal AMPA and kainate receptors of neuronal cells. Currents conducted by non-NMDA receptors were reduced equally effectively by the noble gas, no matter whether they had been induced by subtype-selective agonist AMPA, the non-selective agonist kainate, or the naturally occurring agonist glutamate. The data indicate that the inhibitory effect of Xe on non-NMDA glutamate receptors does neither depend on the chemical nature of the agonist used nor whether the agonists evoke a desensitizing or a non-desensitizing response.

Mechanism of block
Because all glutamate receptors are inhibited to a similar extent by Xe one might speculate whether NMDA, AMPA, and kainate receptors are influenced by similar interaction sites.

A point mutation in the GluR1 (L503Y) and GluR4 (L507Y) genes has been described which strongly reduces Xe sensitivity.22 It seems rather unlikely, that corresponding sites in the other glutamate receptor subtypes are responsible for the described inhibitory effects of Xe because in the GluR6 gene this site predicts a tyrosin. This is not 7ompatible with our findings at this receptor. On the other hand, the sensitivity to Xe seems related to receptor desensitization or receptor sensitivity towards cyclothiazide,22 the most powerful tool to suppress desensitization of AMPA receptors.27 28 However, the interaction site of Xe remains so far unknown.

Our data on the ‘hump’ current (Fig. 2), which suggest that the dissociation of Xe might be faster than channel closure, may be taken as a strong indication that the block occurs only when the channel is in the open state because such ‘humps’ are a characteristic feature of fast open-channel blockers.29 Another argument for an open-channel block might be that the plateau currents were more reduced than the peak. Such a difference was not observed with NMDA receptors.30 31 It remains unclear, whether this variance results from different kinetics of desensitization of AMPA and NMDA receptors, or is a consequence of the speed of our solution exchange, or whether this reflects a special characteristic of AMPA receptors also observed with other antagonists.32

Xe sensitivity of AMPA receptors does not depend on the agonist
Whereas NMDA specifically activates NMDA receptors, AMPA and kainate show cross-reactivity.15 The affinity of the AMPA receptor for AMPA is at least two orders of magnitude higher than for kainate.22 Application of AMPA to native neurones mainly activates fast-desensitizing AMPA currents whereas kainate activates a non-desensitizing AMPA current. The current signals, as a result of both agonists, were reduced to a similar degree by Xe, indicating that native AMPA receptors are Xe sensitive. Furthermore, Xe sensitivity was also confirmed for fast application of the naturally occurring agonist glutamate when applied at a supramaximal concentration (3 mM). Both the fast and persistent components of glutamate-induced current were inhibited. The latter finding is in contrast to the observation of Plested and co-workers who revealed Xe sensitivity for AMPA receptors only when receptor desensitization was overcome either by applying the partial agonist kainate or by inclusion of cyclothiazide but not by ultra-fast application of glutamate.22 From this finding the authors concluded that the noble gas has little or no effect on AMPA receptors. As a possible explanation of this discrepancy they suggested that the pharmacological sensitivity of a receptor might critically depend on the agonist or the expression system.

Whether these different findings arise from the different cell systems (native neurones vs expression system) or measuring configurations (whole cell vs outside-out) is unclear. If AMPA receptors behave identically in expression systems and native neurones we would not have expected Xe to block AMPA receptors, as heteromeric expressed GluR receptors of the flip variant, which dominate in cortical neurones,33 are completely insensitive to Xe.22

Role of AMPA receptors in synaptic transmission
Stimulation of AMPA receptors causes a fast inflow of cations into the post-synaptic cell, which results in membrane depolarization.34 The rapid activation and brief open time of the AMPA receptor facilitates removal of the block of NMDA receptors exerted by Mg2+. This allows the more slowly activating NMDA receptor also to conduct synaptic currents.6 Therefore, AMPA and NMDA receptors are not only co-localized but also act in synergy. Interactions between certain glutamate antagonists at NMDA and AMPA receptors have also been demonstrated.16 35 Thus, during anaesthesia, a block of AMPA receptors by Xe may additionally enhance the reduction of NMDA-induced currents by Xe. Evidence has emerged that non-NMDA receptors may interact with other neurotransmitter systems by modulating pre-synaptic release of the corresponding neurotransmitters. This has been shown for GABA as well as for acetylcholine.12 13 Thus, Xe might in addition affect receptors conducting inhibitory membrane currents, but this is speculative.

Clinical implications
The fact that Xe blocks not only the NMDA receptor but also AMPA and kainate receptors may explain several attractive pharmacological properties of this noble gas in the context of anaesthesia, such as the observation that profound analgesia occurs without psychomimetic or other undesirable side effects, except for some minimal effects on the cardiovascular system.36 37 However, further sub-studies of other inhaled anaesthetics like isoflurane and sevoflurane will be required to fully ascertain the differences to Xe regarding effects on AMPA and kainate receptors.

Evidence has long been available for a favourable physiological interaction between NMDA and AMPA receptors. It is well-established that antagonism of AMPA receptors alone produces a substantial anaesthetic effect with minimal haemodynamic effects.38 Combined application of NMDA and AMPA receptor antagonists has the advantage that the combination is supra-additive. This was suggested by the resulting reduction on halothane MAC in rats.39 Dizocilpine (MK-801) and ketamine antagonize the NMDA receptor in a non-competitive fashion. These drugs produce psychomimetic effects that have limited their clinical application.39 In contrast, psychomimetic side effects were not observed with AMPA receptor antagonists.

Uniquely amongst anaesthetics with known NMDA receptor antagonist action, Xe exhibits neuroprotective properties without co-existing neurotoxicity.40 A potent neuroprotection by Xe even at subanaesthetic concentrations could be demonstrated in in vivo and in vitro models.40 Studies have shown that NMDA, AMPA, and kainate as well as glutamate have excitotoxic properties in vitro, as well as in vivo.41 42 There is convincing evidence that NMDA receptor antagonism is an important neuroprotective mechanism.41 42 The AMPA/kainate receptors are other possible sites of glutamate-mediated neurodegeneration. Indeed, application of 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinolyline (NBQX), a potent AMPA antagonist, is effective in reducing glutamate-induced brain swelling and neuronal damage in rats.18 43 Furthermore, a delayed neuroprotection was found with GYKI-5246632 when administered 15 min or 3 h following experimental brain injury indicating a potentially longer time-window for therapy with AMPA/kainate receptor antagonists than with NMDA antagonists.44 It is of interest that in an in vitro study a supra-additive neuroprotectant effect was observed by a combination of AMPA receptor antagonism with NBQX and NMDA receptor antagonism with dizocilpine.16 A recent study indicated that even in subanaesthetic concentration, Xe attenuates cardiopulmonary bypass-associated neurologic and neurocognitive dysfunction more efficaciously when compared with MK801, a selective NMDA receptor antagonist.45

In conclusion, the clinical features of Xe as an anaesthetic or analgesic over pure NMDA antagonists might stem from its variant pharmacological properties, that is the additional antagonism of AMPA and kainate receptors.


    Acknowledgments
 
We thank Dr R. Rüdel (Department of General Physiology) and Dr A. Sobolevsky (Columbia University, New York) for helpful discussions and Mrs Margot Autenrieth-Kronenthaler for excellent technical assistance. This work was supported by the German Research Association (DFG; We1837/2-1).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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