Xenon reduces activation of transient receptor potential vanilloid type 1 (TRPV1) in rat dorsal root ganglion cells and in human TRPV1-expressing HEK293 cells

Life Sciences 88 (2011) 141–149

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Xenon reduces activation of transient receptor potential vanilloid type 1 (TRPV1) in rat dorsal root ganglion cells and in human TRPV1-expressing HEK293 cells John P.M. White a, Guy Calcott a, Agnes Jenes a,b, Mahmuda Hossein a, Cleoper C. Paule a, Peter Santha c, John B. Davis d, Daqing Ma a, Andrew S.C. Rice a, Istvan Nagy a,⁎ a

Anaesthetics, Pain Medicine and Intensive Care Section, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Chelsea and Westminster Hospital, 369, Fulham Road, London, SW10 9NH, United Kingdom Department of Physiology, University of Debrecen, Medical and Health Science Centre, Debrecen, H-4012, Hungary c Department of Physiology, Faculty of Medicine, University of Szeged, Dom Ter 10, Szeged, H-6720, Hungary d GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, United Kingdom b

a r t i c l e

i n f o

Article history: Received 5 July 2010 Accepted 27 October 2010 Available online 4 November 2010 Keywords: Analgesia Post-operative pain Xenon TRPV1

a b s t r a c t Aims: Xenon provides effective analgesia in several pain states at sub-anaesthetic doses. Our aim was to examine whether xenon may mediate its analgesic effect, in part, through reducing the activity of transient receptor potential vanilloid type 1 (TRPV1), a receptor known to be involved in certain inflammatory pain conditions. Main methods: We studied the effect of xenon on capsaicin-evoked cobalt uptake in rat cultured primary sensory neurons and in human TRPV1 (hTRPV1)-expressing human embryonic kidney 293 (HEK293) cells. We also examined xenon's effect on the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the rat spinal dorsal horn evoked by hind-paw injection of capsaicin. Key findings: Xenon (75%) reduced the number of primary sensory neurons responding to the TRPV1 agonist, capsaicin (100 nM–1 μM) by ~ 25% to ~ 50%. Xenon reduced the number of heterologously-expressed hTRPV1 activated by 300 nM capsaicin by ~ 50%. Xenon (80%) reduced by ~ 40% the number of phosporylated ERK1/2expressing neurons in rat spinal dorsal horn resulting from hind-paw capsaicin injection. Significance: Xenon substantially reduces the activity of TRPV1 in response to noxious stimulation by the specific TRPV1 agonist, capsaicin, suggesting a possible role for xenon as an adjunct analgesic where hTRPV1 is an active contributor to the excitation of primary afferents which initiates the pain sensation. © 2010 Elsevier Inc. All rights reserved.

Introduction The transient receptor potential vanilloid type 1 (TRPV1) receptor is a non-selective cationic channel, which is expressed by the great majority of nociceptive primary sensory neurons (Caterina et al. 1997; Michael and Priestley, 1999; Guo et al. 1999; Nagy et al. 2008). Acute activation of TRPV1 in primary sensory neurons by capsaicin, or some other TRPV1 agonist, results in a short-lasting burning pain sensation. Moreover, sustained TRPV1 activation in these neurons, resulting from inflammation of peripheral tissues, leads to the development of heat hyperalgesia and visceral hyper-reflexia (Caterina et al. 2000; Davis et al. 2000; Charrua et al., 2007).

⁎ Corresponding author. Anaesthetics, Pain Medicine and Intensive Care Section, Department of Surgery and Cancer, Imperial College London, Faculty of Medicine, Chelsea and Westminster Hospital, Room G3.45, 369 Fulham Road, London SW10 9NH, United Kingdom. Tel.: + 44 20 8746 8897; fax: + 44 20 8237 5109. E-mail address: [email protected] (I. Nagy). 0024-3205/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2010.11.002

Xenon is an inert gas which binds to a remarkable range of proteins, including ion channels, such as the ionotropic N-methyl-Daspartate (NMDA) receptor for glutamate, as well as certain nonNMDA glutamate receptors (Franks et al. 1998; Gruss et al. 2004; Dinse et al. 2005; Dickinson et al., 2007; Salmi et al. 2008). In addition to its anaesthetic effect, xenon, in sub-anaesthetic doses, provides analgesia (Yagi et al. 1995; Petersen-Felix et al. 1998). The xenoninduced analgesia, at least in part, is produced in the spinal cord; xenon reduces noxious stimulation-evoked spiking activity and long term potentiation in spinal cord neurons (Utsumi et al. 1997; Watanabe et al. 2004; Benrath et al. 2007). It appears likely that xenon-induced reduction of spinal nociceptive processing is not mediated solely through inhibition of NMDA receptor activity. Thus, it is recognised that all components of spinal cord ventral root potentials (VRPs) evoked by stimulation of primary afferents are reduced by xenon (Watanabe et al. 2004). Individual components of these VRPs are dependent on one, or other, of NMDA receptors, non-NMDA receptors, or tachykinin receptors (Nagy et al. 1994; Thompson et al. 1994; Nagy and Woolf 1996). Hence, an inhibitory effect of xenon on VRPs may result from xenon's action on these receptors in the spinal

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cord. In addition, or in the alternative, such an effect may result from xenon's action at one, or more, receptor types on primary nociceptive neurons resulting in reduced nociceptive input into the spinal dorsal horn. Xenon reduces pain-related behaviour evoked by formalin injection, which induces an inflammatory reaction in tissues (Fukuda et al. 2002; Ma et al. 2004). Recently, it has been shown that painrelated behaviour resulting from formalin injection is reduced by TRPV1 antagonists (Garcia-Martinez et al. 2006; Tang et al. 2007). These data suggest that, if xenon indeed reduces the nociceptive input into the spinal dorsal horn, that reduction may be mediated by an inhibitory effect of xenon on TRPV1. Our in vitro experiments examined the effect of xenon on TRPV1-mediated responses evoked by capsaicin in cultured rat primary sensory neurons and in hTRPV1expressing human embryonic kidney 293 (HEK293) cells. Our in vivo experiments studied the effect of xenon on spinal nociceptive processing (as evidenced by phosphorylation of ERK1/2) resulting from activation of TRPV1 expressed by primary nociceptive afferents caused by hind-paw injection of capsaicin in rat. Materials and methods Animals All procedures were performed in accordance with the UK Animals (Scientific Procedures) Act, 1986 under the project licence No70/6104 that was approved by the Ethics Review Process Committee at Imperial College London under the direction of Central Biological Services. All animals were treated in conformity with the requirements of Directive 86/609/EEC, as amended, and in conformity with the requirements of the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals and the Guide for the Care and Use of Laboratory Animals (1996), prepared by the National Academy of Sciences' Institute for Laboratory Animal Research. In total, 31 female Sprague Dawley rats (each ~ 100 g in weight) were used in this study. In addition, 4 TRPV1 knockout and 4 wild type mice were employed. The animals were kept at 12 h light–dark cycles at controlled temperature and humidity, with food and water ad libitum. Preparation of cultured primary sensory neurons Rats and mice were terminally anaesthetised by Enflourane (Abbott, Maidenhead, UK) and dorsal root ganglia (DRG) from the first cervical to the first sacral segments were dissected out and maintained in culture medium (Ham's nutrient F12 (Invitrogen, UK), supplemented with: 1 ml L-glutamine (Invitrogen, UK); 50 IU/ml penicillin (Invitrogen, UK); 50 μg/ml streptomycin (Invitrogen, UK); and 4% Ultroser G (Pall BioPharmaceuticals, France)). Ganglia were incubated in collagenase (2000 U/ml, Sigma) for 3 h at 37 °C in an atmosphere of 95% air and 5% CO2. Following washes in the supplemented medium, the ganglia were triturated through a firepolished Pasteur pipette, before being re-suspended and plated on poly-DL-ornithine-coated glass coverslips. The cells were then kept at 37 °C in an atmosphere of 95% air and 5% CO2 for 2 days in the presence of nerve growth factor (NGF 2.5 S, 50 ng/ml, Promega, UK). Each animal provided one culture. We then used the cobalt uptake technique, infra, to assess the effect of xenon on TRPV1 activity. Preparation of HEK293 cells transiently transfected with human TRPV1 HEK293 cells were maintained and propagated using standard procedures. Briefly, cells were grown as monolayers in minimum essential medium (MEM, Invitrogen, UK) containing 10% foetal bovine serum (Invitrogen, UK), 2 mM L-glutamine (Invitrogen, UK), penicillin (5,000 IU/ml, Invitrogen, UK), streptomycin (5 mg/ml, Invitrogen,

UK) and MEM non-essential amino acids (Invitrogen, UK) in an atmosphere of 95% air and 5% CO2 at 37 °C. Cells were harvested every 3–4 days, using trypsin-EDTA. Only cells with a passage number lower than 22 were used in this study. Sub-confluent HEK cells were transiently transfected with human TRPV1 (hTRPV1) using Plus Reagent and Lipofectamine (Invitrogen, UK). The hTRPV1 was cloned in a pcDNA3.1/V5-His-TOPO vector (Invitrogen, UK). Transfected cells were plated onto 13 mm coverslips coated with poly-DL-ornithine (Sigma, UK) in 25,000 cells/cm2 density and cultured in the growth media for 1 to 4 days. We then used the cobalt uptake technique to assess the effect of xenon on TRPV1 activity. Electrophysiology To confirm that HEK293 cells do not express TRPV1, or any other ion channel which responds to the specific TRPV1 agonist, capsaicin, we subjected HEK293 cells transfected with hTRPV1 and non-transfected HEK293 cells to capsaicin at different concentrations (3 nM to 10 μM) and measured the ionic flux across the cell membrane by conventional whole-cell voltage-clamp recordings. Further, to confirm that our technique for transfecting hTRPV1was reliably expressing hTRPV1 in HEK293 cells, we also made whole-cell voltage clamp recordings of the responses to capsaicin application of random samples of HEK293 cells after transfection to confirm that these cells exhibited a current characteristic for capsaicin activation of TRPV1 expressed by HEK 293 cells. The recordings were performed at 37 °C. The bath and pipette solutions consisted of (mM): NaCl, 130; KCl, 5; CaCl2, 2; MgCl2, 2; glucose, 10; HEPES, 10; (pH 7.4) and NaCl, 5; KCl, 130; MgCl2, 1.26; HEPES, 10 (pH 7.4), respectively. The holding potential was −60 mV. Electrodes (between 4 and 6 MΩ) were pulled from borosilicate glass capillaries using a DMZ-Universal Puller (Harvard Apparatus Ltd). Recordings were done with an Axopatch 200B amplifier and Digidata 1200 digitizer (Molecular Devices, USA). Solutions including capsaicin were perfused at a rate of 2–3 ml/min through a small plastic tube, placed within 100 μm of the cell which the recording was done from. Data were collected with the pClamp 8 software package (Axon Instruments, USA) with a sampling rate of 1 kHz and 5 kHz filtering. Capsaicin (Tocris, UK) was dissolved first in DMSO to obtain 1 mM stock solution and was further diluted with the extracellular solution. The final DMSO dilution was 1:2000. Labelling by cobalt uptake on activation of TRPV1 by capsaicin in the presence, and absence, of xenon The cobalt uptake experiments were performed as described previously (Sathianathan et al. 2003; Singh Tahim et al. 2005). Briefly, cultured primary sensory neurons, or HEK293 cells attached to their cover-slips, were washed in buffer comprising, in mM: NaCl, 57.5; KCl, 5; MgCl2, 2; HEPES, 10; glucose, 12; sucrose, 139; at pH7.4 (hereinafter described as “cobalt-free uptake-buffer”) twice for 2 min. Then, cells were pre-incubated in that buffer saturated with a mixture of 75% nitrogen and 25% oxygen (control) or 75% xenon and 25% oxygen. The first experiment with primary sensory neurons was performed at room temperature. The pre-incubation of cells in this experiment lasted for 15 min and was performed using cobalt-free uptake-buffer that had been previously exposed by bubbling to the appropriate gas mixture for 15 min. The pre-incubation was done in four-well plates which were covered by parafilm in order to prevent the escape of the gases. That buffer was next replaced with cobalt-containing uptakebuffer (+5 mM CoCl2) which had been exposed by bubbling to the appropriate gas mixture for 15 min and either contained, or lacked, capsaicin (100 nM or 1 μM; stock dissolved in DMSO). Incubation of the cells in the cobalt-containing uptake-buffer lasted for 5 min in four well plates covered by parafilm.

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In an attempt to improve xenon delivery to the cells, the preincubation of primary sensory neurons was performed in a subsequent experiment in an air-tight chamber filled with one, or other, of the gas mixtures. The cobalt-free uptake-buffer was saturated by exposing it to the gases in the chamber for 45 min before the preincubation. The flow-rate of the gas mixtures through the chamber was adjusted to 2.1 l/min both during the buffer saturation and preincubation, while the chamber itself had a volume of 3 l. The preincubation time was 15, 30, or 60 min. Cobalt-containing uptakebuffer (which had also been saturated by exposing it to the gases for 45 min in the chamber and contained, or lacked, capsaicin (500 nM)) was applied to the neurons for 5 min. The temperature within the chamber throughout the experiment was maintained at 37 °C. Having regard to the results obtained from the above experiments on primary sensory neurons, the pre-incubation of the transfected HEK293 cells was performed for 45 min in the chamber with cobaltfree uptake-buffer (which buffer had been previously exposed to the appropriate gas mixture in the chamber for 45 min). Incubation of the cells in the cobalt-containing uptake-buffer (which had also been exposed in the chamber to the appropriate gasses for 45 min either with, or without, capsaicin (10 nM–1 μM)) lasted for 5 min. The temperature of the chamber was maintained at 37 °C. Following the removal of the cobalt-containing uptake-buffer both primary sensory neurons and HEK293 cells were washed briefly in cobalt-free uptake-buffer. Cells were then transferred into 0.4% mercaptoethanol in cobalt-free uptake-buffer for 2 min, then into 70% ethanol for 30 min, and mounted on glass slides using glycerol. Analysis of the cobalt labelling was carried out as described previously. Briefly, the optical intensities of more than 100 systematically randomly chosen cells were measured on each coverslip by a Leica light microscope attached to a CCD camera and a PC running the QWin software package (Leica) following subtracting the background in order to compensate for any misalignment of the optical axis of the microscope. Background subtraction results in the transformation of images into negatives, in which cobalt labelled cells appear as bright cells, while non-labelled cells appear as dark ones. The distributions of the optical intensities of cells in negative images taken from each coverslip were then fitted by normal distributions. The cut-off intensity distinguishing between responding and non-responding cells was established as the 95% confidence interval of the normal distribution fitting the intensities of the labelled cells.

Capsaicin injection-induced expression of phosphorylated extracellular signal regulated kinase 1/2 Sixteen female Sprague Dawley rats were randomly assigned to four groups. Xenon (80%) and oxygen (20%) was administered as a mixture at atmospheric pressure to the animals from two of these groups in a sealed Perspex chamber. The flow-rate of the gas mixture through the chamber was 2.1 l/min, while the chamber itself had a volume of 3 l. After exposure to the xenon/oxygen mixture for 15 min, the animals in one of these groups received a subcutaneous injection of capsaicin (3 mg/ml dissolved in 10% Tween 80; 25 μl) into the right hind-paw. The other xenon-treated group of animals were injected with vehicle (10% Tween 80; 25 μl). The remaining two groups of animals were anaesthetised with sodium pentobarbital (60 mg/kg, i.p.), after which one group was injected with capsaicin, while the other group was injected with vehicle. Following 5 minute survival under anaesthesia, all animals were perfused transcardially with saline followed by 4% paraformaldehyde. The lumbar spinal cord was removed and placed into the same fixative for an additional 4 h. Tissues were then put into 30% sucrose until they sank. The ventral horn of the L4–L5 segments was labelled by a shallow longitudinal cut on the left side. Then, 20 μm transverse sections were cut on a crysostat and thaw-mounted on Superfrost Plus microscope slides

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(VWR, West Chester, PA). Sections were air dried over-night and stored at −20 °C until required. Extracellular signal-regulated kinase 1/2 (ERK1/2), when activated by phosphorylation (pERK1/2) in spinal dorsal horn neurons, is a recognised marker of spinal nociceptive processing (Ji et al. 1999). Capsaicin injection into the hind-paw of laboratory animals results in increased pERK1/2 expression in the spinal dorsal horn in a somatotopically relevant manner (Ji et al. 1999). The effect of xenon on subcutaneous capsaicin injection-evoked activation of spinal cord neurons was assessed using immunohistochemistry by studying the number of cells expressing pERK1/2 in the dorsal horn of the spinal cords of the experimental animals. Spinal cord sections were washed in 0.01 M phosphate-buffered saline (PBS) and incubated in PBS containing 0.3% Triton X (PBST) for 10 min followed by incubation of the sections in 10% normal donkey serum (NDS, Jackson Labs, USA) in PBST for 20 min. Following brief washes in PBST containing 1% NDS, sections were incubated in a polyclonal primary antibody raised in rabbit against the residues surrounding T202/Y204 of pERK1/2 (1:700; Neuromics, USA) overnight. The immunoreaction was visualized by incubation with donkey anti-rabbit IgG conjugated to fluorescein isothiocyanate (1:500; Jackson Labs, USA) for 2 h. Sections were covered by glass cover-slips using Vectashield (Vector, UK). All the washes and incubations were done at room temperature in PBST. Spinal cord sections were analysed in a Leica epifluorescent microscope. Immunopositive neurons with clearly visible perikarya were manually counted on both the ipsi- and contra-lateral dorsal horn in every second of 10 adjacent sections.

Statistical analysis The relative number of cobalt-labelled cells in each set of experiments in each culture was normalized to the highest relative number found in that set of experiments in order to make comparisons between primary sensory neurons and HEK293 cells easier. Normalized data obtained in the same conditions were then averaged. The average number of pERK1/2-expressing neurons per section in each spinal cord was established. Then, the average number of pERK1/ 2-expressing cells of animals subjected to the same treatment was calculated. Differences between relevant groups were assessed by one way or multiple analyses of variance as appropriate. The statistical significance of the differences was assessed by the Dunnett's test. Differences were regarded as significant at p b 0.05. Data are expressed as mean ± SEM. “n” shows the number of cultures or animals which received the same treatment.

Results Reliability of hTRPV1 transfection of HEK293 cells To confirm that our transfection procedure resulted in the expression of functional hTRPV1, we made whole-cell voltage clamp recordings on non-transfected HEK293 cells and on hTRPV1-transfected HEK293 cells, when these respective cell-types were stimulated by capsaicin. As expected, non-transfected HEK293 cells exhibited no response when challenged by capsaicin (not shown). However, in HEK293 cells transfected with hTRPV1, capsaicin elicited concentrationdependent capsaicin-evoked inward currents (not shown). Whole-cell voltage clamp recordings of the responses to capsaicin application of random samples of HEK293 cells after transfection confirmed that these cells exhibited a current characteristic for capsaicin activation of TRPV1 expressed by HEK 293 cells in about 60% of cells examined.

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Capsaicin-evoked cobalt entry into neurons and HEK293 cells occurs only via TRPV1 To confirm that capsaicin-evoked cobalt entry in neurons and HEK293 cells occurs only via TRPV1, we compared with, and without, stimulation by capsaicin (500 nM) in cobalt uptake buffer: (a) the relative number of cobalt-labelled cells in primary sensory neuron cultures prepared from wild-type (WT) mice and TRPV1−/− (KO) mice, respectively; and (b) the relative number of cobalt-labelled cells in non-transfected HEK293 cells and in hTRPV1-transfected HEK293 cells, respectively. Incubating primary sensory neurons in capsaicin-free cobalt uptake buffer produced similar degrees of labelling in both WT and KO mice (4.1 ± 1.1% (n = 3) and 3.6 ± 0.8% (n = 3), respectively, (Fig. 1). However, addition of capsaicin (500 nM) to the cobalt uptake buffer significantly increased the proportion of cobalt-labelled cells in cultures prepared from WT mice (14.6 ± 1.2%, n = 3; Fig. 1). This proportion of labelled neurons corresponds with the proportion of cells expressing TRPV1 in mouse DRG and the EC50 of capsaicin on these cells. In contrast to the cultures prepared from the WT mice, no significant additional labelling resulted from the addition of capsaicin to the cobalt uptake buffer in cultures prepared from KO mice (1.7 ± 0.6%, n = 3; Fig. 1). Only a negligible number of non-transfected, and hTRPV1transfected HEK293, cells showed any sign of cobalt accumulation when no capsaicin was added to the cobalt uptake buffer (Fig. 2). In agreement with the results obtained from the whole-cell recordings, 42.4 ± 3.1% (n = 6) of the hTRPV1-transfected HEK293 cells accumulated cobalt, while only an insignificant number of non-transfected cells showed signs of cobalt-labelling (Fig. 2). These results confirm that capsaicin results in cobalt entry into both primary sensory neurons and hTRPV1-transfected HEK293 cells only through TRPV1. Consequently, any change observed in the capsaicin-induced cobalt accumulation following exposure of the cells

Fig. 2. Capsaicin-induced cobalt uptake in untransfected and hTRPV1-transfected HEK293 cells. In control experiments (cont) cells were incubated in capsaicin-free cobalt uptake buffer. Note that capsaicin (caps) induces cobalt accumulation only in hTRPV1 transfected cells. Note also that the images were taken after subtracting the background. Therefore, the images are negative images. Bar indicates 15 μm.

to any gases is referable to an effect of the gases on the activation of TRPV1. Hence, in our subsequent experiments in which, on stimulation with capsaicin, cobalt accumulation in primary sensory neurons and in HEK293 cells transfected with hTRPV1 was altered after

Fig. 1. Capsaicin-induced cobalt uptake in cultured dorsal root ganglion neurons obtained from wild type (WT) and TRPV1−/− (KO) mice. (A) In control experiments (cont) cells were incubated in capsaicin-free cobalt uptake buffer. Note that capsaicin (caps) induces a response only in cells obtained from wild type mice. Arrows indicate labelled neurons. Note also that the images were taken after subtracting the background. Therefore, the images are negative images. Bar indicates 30 m. (B) Bar chart shows relative number of cobalt labelled cells taken from wild type and TRPV1−/− mice. n = 3 in each group.

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exposure to xenon, this change in cobalt accumulation was properly referred to an effect of xenon on the activation of TRPV1.

Effect of xenon on capsaicin-evoked activation of TRPV1 in primary sensory neurons Cultured primary sensory neurons placed in capsaicin-free cobaltcontaining uptake-buffer in the presence of a nitrogen/oxygen gas mixture showed only a negligible proportion of labelled neurons. However, addition of 1 μM capsaicin to the cobalt-containing buffer resulted in labelling in 37.5 ± 0.9% of the total population of cells (n = 3; Fig. 3). The proportion of neurons labelled by 100 nM capsaicin-evoked cobalt influx was significantly less than that produced by 1 μM capsaicin. While exposed to a xenon/oxygen mixture, cultured neurons placed in the cobalt-containing uptake-buffer without capsaicin showed only a negligible proportion of labelled neurons (Fig. 3C). There was no significant difference between the number of cells so labelled and the number of cells previously labelled by the cobaltcontaining uptake-buffer alone in the presence of nitrogen/oxygen. However, when the nitrogen/oxygen mixture was replaced with a xenon/oxygen mixture, capsaicin application to neurons in cobaltcontaining uptake-buffer resulted in a decrease in the proportion of labelled neurons (Fig. 3C). This diminution in the number of neurons labelled on exposure to a xenon/oxygen mixture, when compared with a nitrogen/oxygen mixture, occurred at both concentrations of capsaicin employed. However, the reduction was significant only at 100 nM capsaicin. The xenon-evoked reduction in labelling at 100 nM capsaicin was about 30% (Fig. 3C).

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When primary sensory neurons were pre-incubated, and then maintained, with xenon, in the air-tight chamber at 37 °C, xenon reduced the proportion of primary sensory neurons labelled by capsaicin-induced cobalt-entry by ~15% (with 15 min pre-incubation; n = 2), ~50% (with 30 min pre-incubation; n = 2) and 54.7 ± 8.7% (with one hour pre-incubation; n = 3; data are not shown). These findings indicated that increasing the exposure time to xenon in an air-tight chamber can increase the efficacy of the xenon-induced inhibitory effect on TRPV1. Effect of xenon on capsaicin-evoked activation of hTRPV1 expressed in HEK293 cells Having regard to the data obtained on cultured primary sensory neurons, the experiments studying the effect of xenon on hTRPV1transfected HEK293 cells stimulated with capsaicin were performed in an air tight chamber with 45 min pre-incubation. To confirm that the 45 minute pre-incubation does not per se result in cobalt-labelling, we first compared the capsaicin-induced responses of the hTRPV1expressing HEK293 cells exposed to the usual twice 2 min wash in cobalt-free uptake-buffer with the same responses after a 2 minute wash in the same buffer followed by a 45 minute pre-incubation in that buffer when saturated with nitrogen/oxygen. In both conditions, capsaicin (10 nM-1 μM) evoked a concentration-dependent increase in the number of labelled cells (Fig. 4A, B) but there was no significant difference between the respective responses (Fig. 4C). The relative number of cobalt-labelled cells at any capsaicin concentration was significantly higher than that produced by the cobalt-containing uptake-buffer alone (Fig. 4C). We found that the 300 nM capsaicin application following only the usual twice 2 minute wash produced

Fig. 3. Effect of xenon on capsaicin-evoked TRPV1 responses of rat cultured primary sensory neurons. (A) Controls: a negligible number of neurons accumulated cobalt. (B) Incubation with Co2+ and capsaicin for 5 min: a substantial proportion of cells accumulated cobalt (dark cells). Bar: 20 μm. Both (A) and (B) were taken without background subtraction. All analysis however, were done after background subtraction. (C) Averaged normalised relative number of cultured primary sensory neurons showing cobalt accumulation in 3–5 cultures. White columns: cultures incubated with 75% nitrogen and 25% oxygen. Black columns: cultures incubated with 75% xenon and 25% oxygen. Note that the proportion of capsaicin-responding neurons is reduced when the cells are pre-incubated in the buffer saturated with 75% xenon and 25% oxygen. This reduction was significant only at 100 nM capsaicin. * Indicates significant difference (p b 0.05).

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Fig. 4. Effect of xenon on capsaicin-evoked responses of hTRPV1 expressed by HEK293 cells after transient transfection. (A) Controls: a negligible number of cells accumulated cobalt. (B) Incubation with Co2+ and capsaicin for 5 min, a substantial proportion of cells accumulated cobalt (dark cells). Bar: 20 μm. (C) Averaged normalised relative number of hTRPV1expressing HEK293 cells showing cobalt accumulation in 3 cultures at each concentration. Empty bars: incubation with Co2+ and capsaicin for 5 min. Gray bars: incubation for 45 min in 75% nitrogen–25% oxygen. Black bars: incubation for 45 min with 75% xenon–25% oxygen. Note that incubation of cells in the presence of xenon (black bars) resulted in reduction of the number of capsaicin-responding neurons. This reduction was significant only at 100 nM and 300 nM. * Indicates significant difference (p b 0.05).

labelling in about half of the cells on average (51.9 ± 17.9%, n = 3; Fig. 4C). The relative number of labelled cells at 1 μM capsaicin in both conditions was lower than at 300 nM, which was probably due to fact that Co2+ entry has saturated at these concentrations (Fig. 4C). In the presence of xenon, capsaicin also produced a concentrationdependent increase in the number of cobalt-labelled cells between 10 nM and 300 nM (Fig. 4C). Comparison of the relative number of cells labelled by capsaicin-induced cobalt accumulation in the presence of nitrogen/oxygen and xenon/oxygen, respectively, revealed that xenon reduced the capsaicin-evoked responses at concentrations ranging from 10 nM to 300 nM capsaicin. However, a significant difference in the reduction in labelling was found only at concentrations of 100 nM and 300 nM capsaicin. On average, the xenon-induced reduction in the proportion of cobalt-labelled cells was approximately 50% over the range from 10 nM to 300 nM capsaicin. The maximum reduction induced by xenon was found at 100 nM capsaicin, where the labelling was reduced by about 55%.

tralateral dorsal horn of the spinal cord, regardless of whether the animals had been treated with sodium pentobarbital or xenon (Fig. 5C). Injection of capsaicin into the paws produced negligible pERK1/2 expression in the contralateral side of the spinal dorsal horn also (Fig. 5C). In contrast, hind-paw injection of capsaicin resulted in a substantial number of pERK1/2-immunopositive cells in the ipsilateral spinal dorsal horn both in the sodium pentobarbital-, and xenontreated, rats (Fig. 5C). In the sodium pentobarbital-anaesthetised rats, capsaicin injection resulted in pERK1/2 immunolabelling in 12.4 ± 0.4 neurons/section (n = 3), which was significantly different from that found in the ipsilateral side of the vehicle-injected animals (Fig. 5C). Xenon anaesthesia significantly reduced the capsaicin injectionevoked pERK1/2 expression in the ipsilateral spinal dorsal horn (7.6 ± 1.1 neurons/section; n = 3; Fig. 5C). The reduction in the number of pERK1/2 expression neurons was about 40%. Collectively, these results suggest that xenon, at the dose which we employed, reduced capsaicinevoked activation of rat spinal dorsal horn neurons.

Capsaicin-evoked phosphorylation of ERK1/2 in spinal dorsal horn neurons

Discussion

The immunostaining was readily recognisable in the dorsal horn of the spinal cord (Fig. 5A and B). In agreement with previous findings from Ji et al. (1999) capsaicin injection evoked pERK1/2 expression in perikarya and processes which appeared to belong to neurons. The great majority of the immunolabelled perikarya were distributed in the superficial layers of the dorsal horn. Immunostained neurons could only very rarely be seen in the deep dorsal horn or around the central canal. Injection of the vehicle into the hind-paw of rats resulted in negligible pERK1/2 expression in either the ipsilateral or the con-

We examined the effect of xenon on capsaicin-induced activation of TRPV1 in rat cultured primary sensory neurons and in hTRPV1expressing HEK 293 cells using the agonist-activated cobalt uptake technique. This technique was introduced by Pruss et al. (1991), and has proved successful in determining the expression and extent of activation of non-selective ligand-gated cation channels, such as TRPV1 (Pruss et al. 1991; Sathianathan et al. 2003; Singh Tahim et al. 2005; Nagy et al. 1993; Lorusso et al. 1998). When the extent of TRPV1 activity is assessed by the cobalt uptake technique, the proportion of labelled cells, which depends on the concentration of TRPV1 agonists

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Fig. 5. The effect of xenon on the activation of spinal cord neurons evoked by subcutaneous capsaicin injection was assessed by studying the expression of phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) in the dorsal horn. Adult rats were anaesthetised either by pentobarbital (Pento) or 80% xenon (Xe) and capsaicin was injected into the right hind paw. pERK1/2 expression was revealed by immunoflourescent staining. (A) A typical image of the ipsilateral spinal dorsal horn from a pentobarbital-anaesthetised rat. Note that there are several pERK1/2-immunopositive neurons in lamina I and outer lamina II. (B) A typical image of the ipsilateral spinal dorsal horn from a xenon-anaesthetised rat. Note that the number of pERK1/2-immunoposive neurons is less than in the spinal cord of the pentobarbital-anaesthetised animals. Scale on (B) is 100 μm. (C) The number of pERK1/2-immunolabelled neurons was counted in every second section of 10 adjacent sections prepared from the L4–L5 spinal cords. Then, the number of immunopositive cells per section was calculated. Note that xenon application resulted in a significant decrease in the average number of pERK1/2-expressing neurons (n = 3 in each data point).

(Sathianathan et al. 2003; Singh Tahim et al. 2005) is established. In DRG cultures this requires differentiation between neurons and other cells (satellite cells and fibroblasts), which can reliably be done by examining the shape and size of the cells (Sathianathan et al., 2003; Singh Tahim et al. 2005). Although the cobalt uptake technique effectively identifies the extent of ion channel activation, including that of TRPV1, in a group of cells, the analysis of the labelling cannot provide the resolution necessary to assess channel activity at single cell level, which requires the use of more sensitive methods, such as whole-cell voltage-clamp recordings or calcium imaging. We found in our rat cultured DRG neurons that xenon (75%), depending on the pre-incubation time and the capsaicin concentration, reduced by ~ 25% to ~50% the proportion of primary sensory neurons responding to the archetypical TRPV1 agonist, capsaicin (100 nM–1 μM). DRG neurons from mice lacking TRPV1 are nonresponsive to capsaicin (Caterina et al. 2000; Davis et al. 2000). Since capsaicin is a specific agonist for TRPV1, it follows that the reduction in cobalt labelling resulted from an inhibitory action by xenon on TRPV1 activation by capsaicin rather than on an effect of xenon on a different ion channel expressed by the DRG neurons, e.g., TRPA1. We further confirmed that this effect of xenon on TRPV1 is not speciesdependent by studying the effect of xenon on the activation by capsaicin of hTRPV1-transfected HEK293 cells. HEK293 cells do not express any receptor type known to be activated by capsaicin, which is supported by the fact that we have found that in these cells, before hTRPV1 transfection, capsaicin does not evoke any ionic flux. However, after such transfection, a distinctive capsaicin-evoked ionic flux is detected. Therefore, the ingress of cobalt found after application of capsaicin to hTRPV1-transfected HEK293 cells in our experiment should be attributed to the activation of hTRPV1 ion channels expressed by these HEK293 cells after transfection. Hence, the reduction in cobalt-labelling by capsaicin of these transfected cells in the presence of xenon shows an inhibitory effect of xenon on the activation of hTRPV1. Our data suggest that xenon inhibited the

activity of heterologously-expressed hTRPV1 by ~ 50% when hTRPV1 activation was evoked by 300 nM capsaicin. We should also mention that the effect of a given dosage of xenon differs between human and rodent subjects. In humans (Cullen et al. 1969), the MAC of xenon is 71 vol.%, whereas, in rats, the MAC of xenon is 86 vol.% (David et al. 2003). Thus, in the present experiments, we used xenon at concentrations which, while of anaesthetic potency in humans, are only of sedative potency in rats, i.e., sufficient to induce an analgesic effect in rat without inducing surgical anaesthesia. We also employed immunohistochemistry to examine the extent to which xenon affected pERK1/2 expression in spinal dorsal horn neurons (a marker of spinal nociceptive processing) resulting from peripheral capsaicin-induced activation of TRPV1 expressed by primary nociceptive afferents (Ji et al. 1999). The existence of capsaicinactivated neuronal ERK1/2 in the spinal dorsal horn was demonstrated by the pattern of pERK1/2 labelling observed in the absence of xenon. This was entirely consistent with established patterns of such activation (Ji et al. 1999) and was confirmed by the observations of a neuroanatomist. Here, we demonstrated an inhibitory effect of xenon on the activation of spinal dorsal horn neurons evoked by peripheral capsaicin injection and consequential TRPV1 activation in primary nociceptive afferents. Xenon (80%) reduced the number of pERK1/2expressing spinal dorsal horn neurons in rat by ~40%. In this context, however, we do not attribute the diminution in spinal nociceptive signalling which resulted from exposure to xenon to an effect of xenon on TRPV1 alone because, as previously observed, xenon is well known to also bind to the ionotropic N-methyl-D-aspartate (NMDA) receptor for glutamate, as well as certain non-NMDA glutamate receptors (Franks et al. 1998; Gruss et al. 2004; Dickinson et al. 2007; Salmi et al. 2008). Indeed, not only do central terminals of primary afferents expressing TRPV1 employ glutamate transmission, but it is also well known that there is enhanced phosphorylation of the NMDA receptor 1 subunit (NR1) in dorsal horn and spinothalamic tract

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neurons after intradermal injection of capsaicin in rats and these receptors are, therefore, also obvious targets for inhibition by xenon (Zou et al. 2000). Unlike volatile anaesthetics, xenon and nitrous oxide, at subanaesthetic concentrations, can reduce heat-induced pain (Yagi et al. 1995, 1996; Petersen-Felix et al. 1998). In rats, xenon suppresses both the pain-related behaviour, and the spinal expression of the nociceptive marker, cFos, induced by formalin injection (Fukuda et al., 2002; Ma et al., 2004). This finding of a significant inhibitory effect of xenon on the activity of hTRPV1 may well have important clinical implications for the control of severe inflammatory-type pain to which TRPV1 is a recognised contributor (Caterina et al. 2000; Davis et al. 2000; Charrua et al. 2007; Garcia-Martinez et al. 2006; Tang et al. 2007). The findings in our experiments that xenon is capable of exercising an inhibitory effect on hTRPV1 is, moreover, consistent with its known inhibitory effect on other ligand-gated ion channels, including glutamatergic (Franks et al. 1998; Dinse et al. 2005), serotonergic (Suzuki et al. 2002) and nicotinic acetylcholine channels (Suzuki et al. 2003). Among the glutamatergic channels, all types seem to be inhibited by xenon (Franks et al. 1998; Dinse et al. 2005). Under gas pressures ranging from 8 to 20 bar, xenon is able to bind to discrete sites in hydrophobic cavities, ligand-binding and substratebinding pockets and into the pore of channel-like structures. Xenon, through weak van der Waals forces, can bind to pre-existing atomicsized cavities in the interior of certain globular protein molecules (Schoenborn and Nobbs, 1966; Tilton et al. 1986; Tilton et al. 1988; Schiltz et al. 1995; Prange et al. 1998; Labella et al. 1999). Xenon demonstrates an exceptional promiscuity, even binding to tissueplasminogen activator, which is a serine protease (David et al., 2010). Urate oxidase is a prototype of a variety of intracellular globular proteins, and annexin V has structural and functional characteristics that allow it to be considered as a prototype for the NMDA receptor. Colloc'h et al. (2007) have shown that in both urate oxidase and annexin V, xenon has its primary binding site at a flexible gas cavity with no visible water, leading them to propose that xenon would bind to the same site within its main physiological target, the pore of the NMDA receptor. Based on the already demonstrated promiscuity of xenon and the homology of various TRP channels, we propose that other TRP channels which play role in the development of pain, such as TRPA1 may also be inhibited by xenon. These questions remain to be addressed. Nonetheless, it is considered that the substantial inhibitory effect on hTRPV1 which we have demonstrated suggests that xenon may have a possible role as an adjunct analgesic in at least several inflammatory pain conditions where hTRPV1 is known to be an active contributor to the excitation of primary afferents which initiates the pain sensation. Conclusion Xenon substantially reduces the activity of hTRPV1 in response to noxious stimulation by the specific TRPV1 agonist, capsaicin, suggesting a possible role for xenon as an adjunct analgesic where hTRPV1 is an active contributor to the excitation of primary afferents which initiates the pain sensation. Conflict of interest statement The authors declare that there are no conflicts of interest.

Acknowledgments This work has been supported by the Joint Research Committee of the Chelsea and Westminster NHS HealthCare Trust, London, United Kingdom. The funding source has had no input into either the design or execution of the experimental work, the interpretation of data, or otherwise. Agnes Jenes has been supported by a BJA/RCoA Project

Grant. Cleoper C. Paule has been supported by a BBSRC-GSK CASE award. Peter Santha has been supported by a Marie Curie IntraEuropean Fellowship (MCIEF 500960). The results published here have been presented in summary form at the 12th World Congress on Pain, Glasgow, UK, August, 2008.

References Benrath J, Kempf C, Georgieff M, Sandkuhler J. Xenon blocks the induction of synaptic long-term potentiation in pain pathways in the rat spinal cord in vivo. Anesthesia & Analgesia 2007;104(1):106–11. Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen-Zeitz KR, et al. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 2000;288(5464):306–13. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 1997;6653:816–24. Charrua A, Cruz CD, Cruz F, Avellino A. Transient receptor potential vanilloid subfamily 1 is essential for the generation of noxious bladder input and bladder overactivity in cystitis. Journal of Urology 2007;177(4):1537–41. Colloc'h N, Sopkova-de Oliveira Santos J, Retailleau P, Vivarès D, Bonneté F, Langlois d'Estainto B, et al. Protein crystallography under xenon and nitrous oxide pressure: comparison with in vivo pharmacology studies and implications for the mechanism of inhaled anesthetic action. Biophysical Journal 2007;92(1):217–24. Cullen SC, Eger II EI, Cullen BF, Gregory P. Observations of the anesthetic effect of the combination of xenon and halothane. Anesthesiology 1969;31(4):305–9. David HN, Haelewyn B, Risso J-J, Colloc'h N, Abraini JH. Xenon is an inhibitor of tissueplasminogen activator: adverse and beneficial effects in a rat model of thromboembolic stroke. J Cerebral Blood Flow Metabolism 2010;30(4):718–28. David HN, Leveille F, Chazalviel L, MacKenzie ET, Buisson A, Lemaire M, et al. Reduction of ischemic brain damage by nitrous oxide and xenon. Journal of Cerebral Blood Flow and Metabolism 2003;23(10):1168–73. Davis JB, Gray J, Gunthorpe MJ, Hatcher JP, Davey PT, Overend P, et al. Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature 2000;405(6783):183–7. Dickinson R, Peterson BK, Banks P, Simillis C, Martin JC, Valenzuela CA, et al. Competitive inhibition at the glycine site of the N-methyl-D-aspartate receptor by the anesthetics xenon and isoflurane: evidence from molecular modeling and electrophysiology. Anesthesiology 2007;107(5):756–67. Dinse A, Frohr KJ, Georgieff M, Beyer C, Bulling A, Weigt HU. Xenon reduces glutamate-, AMPA-, and kainate-induced membrane currents in cortical neurons. British Journal of Anaesthesia 2005;94(4):479–85. Franks NP, Dickinson R, de Souza SL, Hall AC, Lieb WR. How does xenon produce anaesthesia? Nature 1998;396(6709):324. Fukuda T, Nishimoto C, Hisano S, Miyabe M, Toyooka H. The analgesic effect of xenon on the formalin test in rats: a comparison with nitrous oxide. Anesthesia & Analgesia 2002;95(5):1300–4. Garcia-Martinez C, Fernandez-Carvajal A, Valenzuela B, Gomis A, Van Den Nest W, Ferroni S, et al. Design and characterization of a noncompetitive antagonist of the transient receptor potential vanilloid subunit 1 channel with in vivo analgesic and anti-inflammatory activity. Journal of Pain 2006;7(10):735–46. Gruss M, Bushell TJ, Bright DP, Lieb WR, Mathie A, Franks NP. Two-pore domain K+ channels are a novel target for the anesthetic gases xenon, nitrous oxide, and cyclopropane. Molecular Pharmacology 2004;65(2):443–52. Guo A, Vulchanova L, Wang J, Li X, Elde R. Immunocytochemical localisation of the vanilloid receptor (VR1): relationship to neuropeptides, the P2X3 purinoceptor and IB4 binding sites. European Journal of Neuroscience 1999;11(3):946–58. Ji R-R, Baba H, Brenner GJ, Woolf CJ. Nociceptive-specific activation of ERK in spinal neurons contributes to pain hypersensitivity. Nature Neuroscience 1999;2(12): 1114–9. LaBella FS, Stein D, Queen G. The site of general anesthesia and cytochrome P450 monoxygenases: occupation of the enzyme heme pocket by xenon and nitrous oxide. European Journal of Pharmacology 1999;381(1):R1-3. Lorusso GF, De Stasio G, Gilbert B, Perret D, Perfetti P, Margaritondo G, et al. High sensitivity quantitative analysis of cobalt uptake in rat cerebellar granule cells with and without excitatory amino acids. Neuroscience Letters 1998;248(1): 9-12. Ma D, Sanders RD, Halder S, Rajakumaraswamy N, Franks NP, Maze M. Xenon exerts age-independent antinociception in Fischer rats. Anesthesiology 2004;100(5): 1313–8. Michael GJ, Priestley JV. Differential expression of the mRNA for the vanilloid receptor subtype 1 in cells of the adult rat dorsal root and nodose ganglia and its downregulation by axotomy. Journal of Neuroscience 1999;19(5):1844–54. Nagy I, Miller BA, Woolf CJ. NK1 and NK2 receptors contribute to C-fibre evoked slow potentials in the spinal cord. Neuroreport 1994;5(16):2105–8. Nagy I, Pabla R, Matesz C, Dray A, Woolf CJ, Urban L. Cobalt uptake enables identification of capsaicin- and bradykinin- sensitive subpopulations of rat dorsal root ganglion cells in vitro. Neuroscience 1993;56(1):241–6. Nagy I, Paule C, White J, Urban L. Functional molecular biology of the transient receptor potential vanilloid type 1 (TRPV1) ion channel. In: Kofalvi A, editor. Cannabinoids and the Brain. New York: Springer; 2008. p. 101–30. Nagy I, Woolf CJ. Lignocaine selectively reduces C fibre-evoked neuronal activity in rat spinal cord in vitro by decreasing N-methyl-D-aspartate and neurokinin receptormediated post-synaptic depolarizations; implications for the development of novel centrally acting analgesics. Pain 1996;64(1):59–70.

J.P.M. White et al. / Life Sciences 88 (2011) 141–149 Petersen-Felix S, Luginbuhl M, Schnider TW, Curatolo M, Arendt-Nielsen L, Zbinden AM. Comparison of the analgesic potency of xenon and nitrous oxide in humans evaluated by experimental pain. British Journal of Anaesthesia 1998;81(5):742–7. Prange T, Schiltz M, Pernot L, Colloc'h N, Longhi S, Bourguet W, et al. Exploring hydrophobic sites in proteins with xenon or krypton. Proteins 1998;30(1):61–73. Pruss RM, Akeson RL, Racke MM, Wilburn JL. Agonist-activated cobalt uptake identifies divalent cation-permeable kainate receptors on neurons and glial cells. Neuron 1991;7(3):509–18. Salmi E, Laitio RM, Aalto S, Maksimow AT, Langsjo JW, Kaisti KK, et al. Xenon does not affect y-aminobutyric acid type A receptor binding in humans. Anesthesia & Analgesia 2008;106(1):129–34. Sathianathan V, Avelino A, Charrua A, Santha P, Matesz K, Cruz F, et al. Insulin induces cobalt uptake in a subpopulation of rat cultured primary sensory neurons. European Journal of Neuroscience 2003;18(9):2477–86. Schiltz M, Fourme R, Broutin I, Prange T. The catalytic site of serine proteinases as a specific binding cavity for xenon. Structure 1995;3(3):309–16. Schoenborn BP, Nobbs CL. The binding of xenon to sperm whale deoxymyoglobin. Molecular Pharmacology 1966;2(5):495–8. Singh Tahim A, Santha P, Nagy I. Inflammatory mediators convert anandamide into a potent activator of the vanilloid type 1 transient receptor potential receptor in nociceptive primary sensory neurones. Neuroscience 2005;136(2):539–48. Suzuki T, Koyama H, Sugimoto M, Uchida I, Mashimo T. The diverse actions of volatile and gaseous anesthetics on human-cloned 5-hydroxytryptamine 3 receptors expressed in Xenopus oocytes. Anesthesiology 2002;96(3):699–704. Suzuki T, Ueta K, Sugimoto M, Uchido I, Mashimo T. Nitrous oxide and xenon inhibit the human (a7)5 nicotinic acetylcholine receptor expressed in Xenopus oocytes. Anesthesia & Analgesia 2003;96(2):443–8.

149

Tang L, Chen Y, Chen Z, Blumberg PM, Kozikowski AP, Wang ZJ. Antinociceptive pharmacology of N-(4-chlorobenzyl)-N'-(4-hydroxy-3-iodo-5-methoxybenzyl) thiourea, a high-affinity competitive antagonist of the transient receptor potential vanilloid 1 receptor. Journal of Pharmacology and Experimental Therapeutics 2007;321(2):791–8. Thompson SW, Dray A, Urban L. Injury-induced plasticity of spinal reflex activity: NK1 neurokinin receptor activation and enhanced A- and C-fiber mediated responses in the rat spinal cord in vitro. Journal of Neuroscience 1994;14(16):3672–87. Tilton Jr RF, Singh UC, Weiner SJ, Connolly ML, Kuntz Jr ID, Kollmann PA, et al. Computational studies of the interaction of myoglobin and xenon. Journal of Molecular Biology 1986;192(2):443–56. Tilton Jr RF, Singh UC, Kuntz Jr ID, Kollman PA. Protein-ligand dynamics. A 96 picosecond simulation of a myoglobin-xenon complex. Journal of Molecular Biology 1988;199(1):195–211. Utsumi J, Adachi T, Miyazaki Y, Kurata J, Shibata M, Murakawa M, et al. The effect of xenon on spinal dorsal horn neurons: a comparison with nitrous oxide. Anesthesia & Analgesia 1997;84(6):1372–6. Watanabe I, Takenoshita M, Sawada T, Uchida I, Mashimo T. Xenon suppresses nociceptive reflex in newborn rat spinal cord in vitro; comparison with nitrous oxide. European Journal of Pharmacology 2004;496(1–3):71–6. Yagi M, Mashimo T, Kawaguchi T, Yoshiya I. Analgesic and hypnotic effects of subanaesthetic concentrations of xenon in human volunteers: comparison with nitrous oxide. British Journal Anaesthesia 1995;74(6):670–3. Zou X, Lin Q, Willis WD. Enhanced phosphorylation of NMDA receptor 1 subunits in spinal cord dorsal horn and spinothalamic tract neurons after intradermal injection of capsaicin in rats. Journal of Neuroscience 2000;20(18):6989–97.