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- Non-technical summaryAbstract
Nitrous oxide (N2O) has long been used as a pain reliever, but little is known of its targets in the body. We show that N2O indirectly inhibits T-type calcium channels through free radical reactions. These reactions depend on a histidine residue on the channel that binds metal ions. If we prevent this histidine from binding metals, N2O cannot inhibit T-currents. Mice that are treated with EUK-134 to remove free radicals show little pain relief after N2O administration. This report provides new information on how N2O interacts with ion channels, helping our understanding of how this popular pain reliever works.
Nitrous oxide (N2O, laughing gas) has been used as an anaesthetic and analgesic for almost two centuries, but its cellular targets remain unclear. Here, we present a molecular mechanism of nitrous oxide's selective inhibition of CaV3.2 low-voltage-activated (T-type) calcium channels in pain pathways. Using site-directed mutagenesis and metal chelators such as diethylenetriamine pentaacetic acid and deferoxamine, we reveal that a unique histidine at position 191 of CaV3.2 participates in a critical metal binding site, which may in turn interact with N2O to produce reactive oxygen species (ROS). These free radicals are then likely to oxidize H191 of CaV3.2 in a localized metal-catalysed oxidation reaction. Evidence of hydrogen peroxide and free radical intermediates is given in that N2O inhibition of CaV3.2 channels is attenuated when H2O2 is neutralized by catalase. We also use the adrenochrome test as an indicator of ROS in vitro in the presence of N2O and iron. Ensuing in vivo studies indicate that mice lacking CaV3.2 channels display decreased analgesia to N2O in response to formalin-induced inflammatory pain. Furthermore, a superoxide dismutase and catalase mimetic, EUK-134, diminished pain responses to formalin in wild-type mice, but EUK-134 and N2O analgesia were not additive. This suggests that reduced ROS levels led to decreased inflammation, but without the presence of ROS, N2O was not able to provide additional analgesia. These findings reveal a novel mechanism of interaction between N2O and ion channels, furthering our understanding of this widely used analgesic in pain processing.
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- Non-technical summaryAbstract
N2O has been used as an anaesthetic and excellent analgesic for close to two centuries in clinical practice. It is also a common drug of abuse. In spite of this, the molecular mechanisms underlying its clinical effects are poorly understood, partly because of difficulties in working with this agent in vitro. Ion channels as targets for N2O have only recently been taken into consideration. The first papers describing specific interactions between N2O and N-methyl-d-aspartate (NMDA) receptors in the CNS (Jevtovic-Todorovic et al. 1998; Mennerick et al. 1998) have begun to explore this mechanism. These effects may be the basis of N2O anaesthesia since reduction of excitatory drive in CNS neurons is one of the key theories of anaesthesia. The first study of the effects of N2O on ion channels in the pain pathway showed that N2O inhibition of T-type channels (T-channels) is specific for the CaV3.2 isoform (Todorovic et al. 2001b). This isoform is abundantly expressed in the major pain processing regions of the peripheral nervous system (PNS) and central nervous system (CNS): the peripheral sensory neurons and superficial layers of the dorsal horn in the spinal cord (Talley et al. 1999). In addition, CaV3.2 channels were directly implicated in pain using both knock-out and knock-down strategies (Bourinet et al. 2005; Choi et al. 2007), strongly suggesting that at least part of N2O's potent analgesia could be mediated by blockade of CaV3.2 T-channels. However, a mechanistic link between these two phenomena has not been established.
Much of the research about the interaction of N2O and metals has been in bacterial systems, such as the clusters of copper and iron bound by histidine residues in N2O reductase and the membrane protein NosR (Brown et al. 2000; Wunsch & Zumft, 2005). While another study found evidence of free radical intermediates during N2O metabolism in human intestinal contents (Bosterling et al. 1980), no one has been able to establish an interaction between N2O and metal ions in a mammalian system.
Reactive oxygen species (ROS) have been implicated in a number of pathological conditions, including chronic and inflammatory pain (Chung, 2004). Intraplantar and intrathecal application of free radical scavengers have been shown to reduce oxidative stress, inflammation and pain (Salvemini et al. 1999; Nishiyama & Ogawa, 2005). Synthetic superoxide dismutase (SOD) and catalase mimetics, such as EUK-134, have been shown to prevent oxidative stress and have neuroprotective properties in ischaemic brain injury (Baker et al. 1998; Rong et al. 1999). By minimizing the ROS level, these agents are believed to prevent the induction of stress-related gene responses, as well as reducing molecular damage, such as lipid peroxidation and protein oxidation. Affected proteins may also include potassium channels and NMDA receptors, which are known to have altered responses after exposure to ROS (Aizenman et al. 1990; Aizenman, 1995; Ichinari et al. 1996).
Until recently, very little was known about the role of T-channels in sensory processing, specifically in nociception. However, recent data indicate that these channels may play an important role in boosting peripheral and central sensory transmission (reviewed in Todorovic & Jevtovic-Todorovic, 2006; Nelson et al. 2007b). Thus, sensory neurons provide an excellent in vitro model for studies of T-channels in nociception. Here, we use recombinant and native CaV3.2 channels expressed in sensory neurons to study the molecular mechanisms of selective inhibition of these channels by N2O, a commonly used anaesthetic and analgesic.
Ethical approval was obtained for all experimental protocols from the University of Virginia Animal Care and Use Committee, Charlottesville, VA, USA. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals adopted by the US National Institutes of Health. Every effort was made to minimize animal suffering and the number of animals used. For removal of tissues, animals were deeply anaesthetized with inhaled isoflurane and rapidly killed with a guillotine.
For recordings of recombinant T-currents, we used both transiently transfected and stably expressed CaV3.1 or CaV3.2 channels in human embryonic kidney (HEK-293) cells as reported previously (Joksovic et al. 2006). HEK-293 cells (CRL-1573; American Type Culture Collection, Manassas, VA, USA) were grown in DMEM/F-12 media (Invitrogen) supplemented with 10% fetal bovine serum, penicillin G (100 mg ml−1) and streptomycin (0.1 mg ml−1). Transiently transfected cell lines were made using cDNA encoding human CaV3.1 or CaV3.2, and a plasmid encoding the CD8 antigen. Cells were then incubated with polystyrene microbeads coated with CD8 antibody (Dynabeads M-450 CD8, Dynal GmbH, Hamburg, Germany). After ∼48 h, cells with microbeads bound were selected for electrophysiology.
Three- to eight-week-old Sprague–Dawley rats were briefly anaesthetized with isoflurane and decapitated. Dorsal root ganglia were removed and incubated in a mixture of collagenase H (2 mg ml−1, Roche) and Dispase I (0.17 mg ml−1, Roche) for 1 h at 37°C. Ganglia were dissociated using Pasteur pipettes and plated on glass coverslips. Neurons were used for electrophysiology 2–8 h later.
Nitrous oxide preparation
Nitrous oxide solutions were prepared by bubbling 80% N2O and 20% O2 (GT&S Welco, Richmond, VA, USA) through a bubbling stone into external recording solutions. N2O concentration in the gas mixture was constantly monitored by an infrared gas analyser (Datex-Engtstrom, Helsinki, Finland). During experiments, test solutions were maintained in tightly sealed, all-glass syringes, and replaced every 30 min. However, due to extremely low solubility of N2O in water, it is likely that actual concentrations of N2O in our in vitro experiments were much lower than 80%.
Other drug preparations
Stock solutions of all drugs were made within 48 h of use. Agents were dissolved in appropriate solvents as follows: formalin (Fisher Scientific), 4% paraformaldehyde in saline; deferoxamine (Calbiochem), 10 μm in external solution; DTPA (Sigma), 100–300 μm in external solution; H2O2 (Fisher Scientific), 0.1% by volume in deionized water for adrenochrome test or external solution for electrophysiology; catalase (Sigma), 500 U ml−1 in external solution; adrenaline (epinephrine; Sigma) in deionized water; EUK-134 (Cayman Chemical), 2 mg ml−1 in saline.
All recordings were made using standard whole-cell techniques. Electrodes were fabricated from borosilicate microcapillary tubes (Drummond Scientific, Broomall, PA, USA) and fire-polished to final resistances of 2–3 MΩ. Voltage commands and digitization of membrane voltages and currents were done with Clampex 9.2 of the pClamp software package (Axon Instruments), running on an IBM-compatible computer. Membrane voltages and currents were recorded with an Axopatch 200B patch-clamp amplifier (Axon Instruments). Data were analysed using Clampfit 9.2 (Axon Instruments) and Origin 7.0 (Microcal Software). Currents were filtered at 2 kHz. Series resistance (Rs) and membrane capacitance (Cm) values were taken directly from readings on the amplifier following electronic subtraction of the capacitative transients. We compensated for 50–80% of Rs. Cells were held (Vh) at −90 mV and depolarized to a test potential (Vt) of −30 mV every 10–20 s to evoke inward Ba2+ currents, unless otherwise noted. In most experiments, a P/N protocol was used for online leak subtraction.
Multiple independently controlled glass syringes served as reservoirs for a gravity-driven local perfusion system, where manually controlled valves allowed for switching between syringes. Solution exchange was accomplished by constant suction through a glass capillary tube at the opposite end of the recording dish. All drugs were prepared as stock solutions and freshly diluted to appropriate concentrations at the time of experiments. The external recording solution used to isolate Ba2+ currents contained (in mm): BaCl2, 2–10; TEA-Cl, 152; Hepes, 10; adjusted to pH 7.4 with TEA-OH. In a limited number of experiments we used 2 mm Ca2+ in our external recording solution. We did not notice any difference in the effects of N2O, metal chelators or H2O2 regardless of which external solution was used. The internal solution contained (in mm): caesium methane sulfonate, 110; phosphocreatine, 14; Hepes, 40; Mg-ATP, 2; Na-GTP, 0.1; adjusted to pH 7.2 with KOH.
Oxidation of adrenaline to adrenochrome
Detection of adrenochrome production was performed using a spectrophotometer (ThermoSpectronic, Rochester, NY, USA). Optical absorbance at 485 nm was measured every 5 min over a 30 min period. Reagents were placed in a 2 ml sealed glass cuvette and immediately inserted into the spectrophotometer for data collection. All measurements were corrected against blanks containing 20 mm epiHCl (Sigma) in 0.1 m HCl and 50 mm Hepes/KOH buffer (pH 10). To the blanks, we added the required amounts of FeCl3, H2O2 or 80% N2O. 80% N2O was bubbled into Hepes/KOH buffer for 30 min prior to experiments.
Data analysis and statistical procedures
Peak currents and exponential fits to currents were calculated using Clampfit, while curve fitting was performed using Origin 7 software. For general statistical evaluations, we used one- and two-way ANOVAs with values <0.05 judged to be statistically significant, and paired or unpaired Student's t tests as appropriate. Average data are presented as mean ± standard errors of the mean (s.e.m.).
Adult mice of either sex were used for all behavioural studies. CaV3.2 knock-out mice (CaV3.2−/−) and their age-matched wild-type littermates (CaV3.2+/+) were studied at 16 to 60 weeks of age. The initial breeding pair for our colony of CaV3.2−/− mice was obtained through the courtesy of Dr Kevin Campbell (University of Iowa, Iowa City, IA, USA). These mice were repeatedly backcrossed for at least six generations against wild-type (C57BL/65J) mice. Animals were genotyped using tail biopsy. Mutant mice were normal in appearance, weight and overt behaviour. They demonstrated a brisk righting reflex and in a previous study showed no differences in motor abilities under control conditions (Choi et al. 2007).
Behavioural tests for inflammatory pain after formalin injection were performed in a clear Plexiglass chamber prefilled with 20% N2O, 20% O2 and 60% N2 at a flow rate of 6 l min−1. The mouse was first placed in the prefilled chamber to accommodate for 30 min. The mouse was then rapidly removed from the chamber, injected with 20 μl of 4% formalin or saline into the plantar side of the right paw and returned to the test chamber. The time in seconds spent licking and biting the paw was measured for an hour and recorded for every 5 min interval. Afterwards, the mouse's temperature was measured to ensure normothermia. Control mice were tested under the same protocol, with normal room air flow instead of the N2O mixture.
To test the effects of EUK-134 (EUK; Cayman Chemical), 5 mg of EUK was dissolved in 2.5 ml of saline by vortexing for 2.5 h. A mouse was injected intraperitoneally (i.p.) with fresh EUK solution (25 mg kg−1). Thirty minutes after the EUK injection, the mouse was placed inside the chamber to equilibrate and familiarize itself with the environment. One hour after the EUK injection, the mouse was injected with formalin, and the rest of the experiment was performed as described above. Control mice received equal volumes of saline in an i.p. injection.
The effects of EUK on inflammation were quantified after i.p. injection of EUK. After 1 h following i.p. saline or EUK injections, control paw volume was measured by insertion into a beaker of water on a weight scale (Fereidoni et al. 2000). During this time, the mouse is mechanically restrained and fastened to an immobile structure. Afterwards, formalin was injected, and paw volume measured again after 1 h. The difference of the two volumes was then calculated.
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- Non-technical summaryAbstract
Nitrous oxide is frequently used in clinical settings as a potent analgesic, but little is known of its molecular mechanism of action. Here, we present evidence that N2O-based inhibition of T-channels in vitro and in vivo is mediated by free radical signalling.
Recent publications describe an essential role of histidine 191 for three other CaV3.2-selective agents: nickel (Kang et al. 2006), ascorbate (Nelson et al. 2007a) and zinc (Nelson et al. 2007b). Accordingly, we began with H191 in our search for the means of N2O inhibition of CaV3.2 channels. Mutation of this histidine residue to glutamine (H191Q) in CaV3.2 abolished N2O inhibition, while the reverse mutation (Q172H) in N2O-insensitive CaV3.1 produced a gain of N2O susceptibility.
The H191-dependent increase in CaV3.2 currents with DFX suggests removal of a tonically bound iron ion, whose presence inhibits the channel. This increased current was significantly reduced in the H191Q mutant, which further emphasizes that this histidine plays a key role in the metal binding site. Since the potentiating actions of DFX were fast and reversible (Fig. 3), it is unlikely that the observed increase in T-current was due to a side effect of DFX as a potent hypoxia-inducing factor. This also makes recruitment or incorporation of new T-channels an improbable mechanism of action of DFX in our experiments, in contrast to the chronic DFX application seen in Carabelli et al. (2007). To confirm that the observed changes were due to the chelating ability of DFX, we tested another metal chelator, DTPA. This non-specific lower-affinity chelator also increased CaV3.2 currents, but most importantly, DTPA also greatly diminished N2O inhibition of CaV3.2 channels, indicating a need for a bound metal for N2O effectiveness. In the Fenton reaction, iron and H2O2 combine to produce the hydroxyl radical ·OH. This highly unstable complex then reacts with a small number of amino acid residues in the immediate vicinity of the metal binding site (Stadtman & Oliver, 1991) in MCO. Targets for MCO may include histidine, cysteine, lysine, arginine, proline and tyrosine residues due to their participation in metal binding sites (Stadtman & Berlett, 1991; Stadtman & Levine, 2003). However, histidine is one of the most vulnerable amino acids to oxidation reactions (Uchida, 2003). Accordingly, we found that N2O had significantly smaller inhibitory effect in the H191Q mutant than the H191C mutant of CaV3.2. This could be due to the requirement for at least one additional nearby free cysteine residue to be cross-linked during an oxidation reaction involving cysteine residues.
Application of H2O2 produced T-current inhibition similar, but not additive to that of N2O, supporting our hypothesis that H2O2 is an intermediate in the reactions between N2O and CaV3.2 channels. Indeed, decomposition of H2O2 by catalase interrupts this process. Furthermore, quantification of the oxidation of adrenaline to adrenochrome allowed us to more directly assess the role of N2O in producing ROS. Again, the action of N2O paralleled that of H2O2.
MCO of the same histidine residue that we examine here (H191) in T-channels was recently implicated as the mechanism of ascorbate selective inhibition of CaV3.2 current (Nelson et al. 2007a). Ascorbate has well-known pro-oxidant properties; it is able to generate ROS by reducing transition metals via a one-electron transfer mechanism (Stadtman, 1991). Here, we propose a similar, but novel role for N2O in a molecular mechanism of inhibition of CaV3.2, as presented in our model showing a two-electron oxidation of iron and consequent reduction of N2O to N2. Fig. 7A shows a CaV3.2 channel in its normal state, passing inward calcium current, and a metal ion (in this case Fe2+) bound to H191. Upon encountering N2O (Fig. 7B), one of two sets of reactions can occur: a one-electron transfer that requires an N2O− intermediate and Fenton chemistry (eqn (1)), or a two-electron transfer with an Fe4+O intermediate (eqn (2)). Both pathways could result in the appearance of hydroxyl radicals and oxidation of Fe2+ to Fe4+ as follows:
• OH is so unstable that it reacts locally, oxidizing H191 to 2-oxo-histidine (Fig. 7B). We hypothesize that this results in decreased channel open probability, similar to that reported in NMDA receptors after histidine modification (Donnelly & Pallotta, 1995). Indeed, Bartels and colleagues (2009) have recently used single channel recordings to demonstrate that nitrous oxide inhibits recombinant CaV3.2 currents by decreasing single channel open time duration and probability of channel opening. To complete the cycle, both histidine and iron may be reduced to their original state by elements in the endogenous electron donor system (e.g. NADH or NADPH), and CaV3.2 resumes its normal passage of calcium current (Fig. 7C). Further extensive chemical studies are needed to precisely delineate proposed pathways of N2O interaction with this critical histidine residue of CaV3.2. In addition to these proposed pathways, we were able to demonstrate that similar mechanisms may exist in vivo and at least partly mediate N2O analgesia in inflammatory pain.
Figure 7. Proposed mechanism of MCO of CaV3.2 channels in the presence of N2O and iron A, at rest Fe2+ (or possibly another redox-sensitive transition metal such as Cu2+) is bound to H191 and several additional, unidentified residues that comprise the high-affinity metal binding site on the external surface of Cav3.2. B, N2O interacts with bound iron to produce H2O2, an unstable intermediate ROS such as •OH, and iron is oxidized to Fe4+. Consequently, the C-2 position of the imidazole ring of H191 is modified by •OH, most probably resulting in 2-oxo-H191. This modification causes conformational changes of CaV3.2 and ultimately reduced T-currents via a presumed allosteric mechanism. C, Fe4+ is reduced back to Fe2+ by the electron donor system, allowing the cycle to begin again.
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Though N2O is often thought of as an inert gas, interactions between N2O, metals and free radicals have been documented. For example, transition metals and N2O may form a metal oxide and N2 (Banks et al. 1968; Raiche & Belbruno, 1987; Delabie et al. 2001). Similar reactions between N2O and alkali metals have also been studied (Tishchenko et al. 2004). Koppenol (1991) found the generation of hydroxyl radicals from N2O to be energetically unfavourable, agreeing with our observation that N2O alone is unable to significantly oxidize adrenaline. However, Koppenol's calculations do not take into account the presence of redox-reactive transition metal ions, which could lower activation energies. Also, even if conditions remain unfavourable, even a small amount of highly reactive ROS could have significant effects in vivo. Indeed, Bosterling et al. (1980) found evidence of free radical intermediates during N2O metabolism by intestinal bacteria. ROS have been shown to be intermediates in the metabolic reduction of other nitrogen compounds (Mason & Holtzman, 1975), while irradiation of aqueous solutions of N2O produced ROS that proved toxic to Escherichia coli (Brustad & Wold, 1976). Interactions between N2O and metals are also critical clinically. Serious side-effects of exposure to N2O such as megaloblastic erythropoiesis (pernicious anaemia) and degeneration of the spinal cord are caused by oxidation of cobalamin in vitamin B12 (Hadzic et al. 1995). This oxidation renders vitamin B12 useless as a co-enzyme to methionine synthase, disrupting several pathways (Lumb et al. 1980, 1981). In fact, previous studies have shown that free radical scavengers such as dimethylthiourea and SOD are able to significantly reduce N2O-mediated inactivation of methionine synthase (Koblin & Tomerson, 1990; Nair et al. 1995).
Each of these studies supports our hypothesis that N2O indirectly inhibits T-currents by oxidizing the channel-bound metal and producing ROS, which then participate in MCO of the CaV3.2 T-channel. Our chelator experiments provide evidence of the dependence of N2O on channel-bound redox-reactive metal ions such as iron or possibly copper (Nelson et al. 2007b), while application of catalase demonstrated the necessity of H2O2 and hydroxyl radicals. Furthermore, our occlusion experiment with H2O2 and N2O strongly suggests that there is a common pathway of their inhibition of T-currents. The oxidation of adrenaline to adrenochrome allowed us to model the generation of ROS by N2O and iron in a cell-free system. Lastly, we show that intraperitoneal application of an SOD/catalase mimetic, EUK-134, is able to reduce pain and inflammation in the formalin test, and consequently abolish analgesic effects of N2O in this model. Importantly, the potent analgesic effect of sub-anaesthetic concentrations of N2O in the formalin test was almost completely absent in CaV3.2−/− mice. Thus, we present novel evidence that N2O inhibits T-channels in the pain pathway via ROS in a cell-free solution, individual neurons and the whole animal.
In addition, NMDA receptors are prominent contributors in pain transmission in the dorsal horn of the spinal cord, an important pain processing region. Importantly, the synergistic action of T-channels, NMDA receptors and neurokinin receptors in lamina I of the dorsal horn is required for sensitization to inflammatory stimuli in the formalin test (Ikeda et al. 2006). Since CaV3.2 is expressed in both peripheral sensory neurons and the dorsal horn of the spinal cord (Talley et al. 1999), it is likely that N2O inhibition of T-channels via ROS works together with inhibition of NMDA receptors to reduce inflammatory pain.
Though N2O has been in use for centuries, little is known of its mechanism of action. The data presented here provide much needed information to understanding the actions of N2O. This information also has wider applications: MCO of proteins in the presence of trace metals such as iron or copper is becoming widely recognized as a key step in many pathological conditions. These include inflammatory diseases such as rheumatoid arthritis, atherosclerosis and ischaemia reperfusion damage (Stadtman & Oliver, 1991; Wang et al. 2004). Furthermore, N2O is implicated in neurocognitive decline after general anaesthesia due to neurotoxicity (Jevtovic-Todorovic et al. 1998, 2001, 2003). Future studies are necessary to investigate these possibilities. Further research will also be needed to determine whether other ion channels (e.g. NR1/NR2A NMDA receptors) that are known to contain high-affinity metal binding sites (Herin & Aizenman, 2004) show inhibition by N2O (Jevtovic-Todorovic et al. 1998; Mennerick et al. 1998; Georgiev et al. 2008) and undergo modification by free radicals (Aizenman et al. 1990; Gao et al. 2007) are subject to similar regulation.