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Pain Medicine  |   January 2012
Lidocaine Metabolites Inhibit Glycine Transporter 1: A Novel Mechanism for the Analgesic Action of Systemic Lidocaine?
Author Affiliations & Notes
  • Robert Werdehausen, M.D.
    *
  • David Kremer, M.D.
  • Timo Brandenburger, M.D.
    *
  • Lukas Schlösser, M.D.
  • Janusz Jadasz, M.Sc.
    §
  • Patrick Küry, Ph.D.
    §
  • Inge Bauer, Ph.D.
    #
  • Carmen Aragón, Ph.D.
  • Volker Eulenburg, Ph.D.
    **
  • Henning Hermanns, M.D.
  • *Resident in Anesthesiology, Staff Anesthesiologist, #Associate Professor, Department of Anesthesiology, Medical Faculty, Heinrich Heine University of Düsseldorf, Düsseldorf, Germany. Resident in Neurology, §Research Scientist, Department of Neurology, Medical Faculty, Heinrich Heine University of Düsseldorf. Professor, Department of Molecular Biology and Centre of Molecular Biology “Severo Ochoa,” Autonomous University of Madrid, Madrid, Spain. **Research Associate, Institute of Biochemistry, University of Erlangen-Nürnberg, Nürnberg, Germany.
Article Information
Pain Medicine / Pain Medicine / Pharmacology
Pain Medicine   |   January 2012
Lidocaine Metabolites Inhibit Glycine Transporter 1: A Novel Mechanism for the Analgesic Action of Systemic Lidocaine?
Anesthesiology 1 2012, Vol.116, 147-158. doi:10.1097/ALN.0b013e31823cf233
Anesthesiology 1 2012, Vol.116, 147-158. doi:10.1097/ALN.0b013e31823cf233
What We Already Know about This Topic
  • Glycine, a major inhibitory neurotransmitter, is rapidly removed after release by glycine transporters, including type 1, expressed in astrocytes

  • Whether lidocaine, which reduces neuropathic pain, acts in part by inhibiting glycine transporter 1 has not been studied

What This Article Tells Us That Is New
  • In cultured astrocytes, lidocaine failed to alter glycine transporter 1 function, but its metabolites inhibited directly or competitively glycine transporter 1 at clinically relevant concentrations, suggesting a possible target for the analgesic action of lidocaine in neuropathic pain

THE amino acid glycine is, besides γ-aminobutyric acid, the major inhibitory neurotransmitter in the central nervous system. In addition, glycine is involved in excitatory neurotransmission as obligatory coagonist of glutamate at N-methyl-D-aspartate (NMDA) receptors. The synaptic glycine concentration is tightly regulated by the Na+/Cl−-dependent glycine transporters (GlyT). GlyT type 1 (GlyT1) is predominantly expressed in astrocytes and a subset of presumably glutamatergic neurons, whereas GlyT type 2 (GlyT2) is exclusively expressed in glycinergic neurons.1 At glycinergic synapses, GlyT1 removes glycine from the synaptic cleft, while GlyT2 mediates the reuptake of glycine into nerve terminals.2,3 
The role of inhibitory, especially glycinergic, neurotransmission in pathologic pain is well documented.4,5 Spinal synaptic disinhibition is one of the proposed mechanisms of pathologically increased pain sensitivity, and restoring synaptic inhibition may serve to restore physiologic conditions.6,7 Likewise, inhibition of glycine transporters has evolved as a potential new treatment of neuropathic pain.8  11 In particular, increasing the extracellular level of glycine via  blockade of GlyT1 has been proposed very recently as a potential therapeutic approach for chronic pain with cognitive impairment.12 
The local anesthetic lidocaine has been shown to exert effects in vivo  that cannot be explained by its action on voltage-gated sodium channels. These effects comprise, among others, antiinflammatory13,14 and antinociceptive15 properties. The significant antineuropathic effect of systemic lidocaine is also well established,16,17 although its mechanism remains elusive. Early evidence from in vitro  investigations suggested a glycine-like action of lidocaine in the central nervous system.18,19 Furthermore, results from a recent study by our own group supported the hypothesis that at least part of the analgesic activity of systemic lidocaine might originate from a glycinergic action by lidocaine or one of its metabolites.20 
Lidocaine is mainly metabolized hepatically by cytochrome P450 isoforms CYP1A2 and 3A4 in a sequential process of oxidative N-dealkylation, although only a small amount (10%) is excreted unchanged renally.21 Its major metabolites are monoethylglycinexylidide (MEGX), glycinexylidide (GX), and N-ethylglycine (EG), all of which have a glycine-like moiety. MEGX shows about 80% potency of the parent drug at voltage-dependent sodium channels, while GX and EG are nearly ineffective. Based on structural similarities to the alternative GlyT1 substrate sarcosine,22 lidocaine metabolites might also affect GlyT1 function. This hypothesis would explain most of the antinociceptive effects of systemic lidocaine application in neuropathic pain.
Therefore, the aim of the present study was to investigate the effect of lidocaine and its major metabolites on glycine transport. At first, different central nervous system cell types from primary cell preparations of the rat brain cortex were screened for GlyT expression. After confirming high expression levels of GlyT1 in cultured primary astrocytes, these were used to evaluate the effects of lidocaine and its metabolites on glycine uptake. Since GlyT1 makes use of the sodium gradient as an energy source for the intracellular accumulation of glycine, its function can be accessed by electrophysiological methods.23 Following this approach we analyzed the effect of lidocaine and its metabolites on GlyT1 function in more detail by two-electrode voltage clamp analysis in GlyT1-expressing Xenopus laevis  oocytes.
Materials and Methods
Reagents
Lidocaine hydrochloride and the metabolites MEGX and GX were provided by AstraZeneca, Research and Development (Södertälje, Sweden). ALX5407, ALX1393, sarcosine, glycine, L-alanine, EG, laminin, poly-D-lysine and tetrazolium salt (XTT) were purchased in their highest available purity from Sigma-Aldrich (St. Louis, MO). Polyclonal antibodies against GlyT1 and GlyT2 originating from rabbit were described previously.24,25 Monoclonal mouse anti-glial fibrillary acidic protein and mouse antineuron-specific nuclear protein antibodies were purchased from Millipore (Billerica, MA). A goat antirabbit IgG antibody conjugated to horseradish peroxidase was used as secondary antibody in western blot analysis (Dianova, Hamburg, Germany). Secondary antibodies goat antirabbit and donkey antimouse (Alexa Fluor 488 and 592) for fluorescence microscopy were purchased from Invitrogen (Carlsbad, CA). Phosphate buffered saline without calcium and magnesium was obtained from Invitrogen. Trypsin/EDTA was purchased from Biochrom AG (Berlin, Germany).
Astrocyte, Microglial, and Oligodendrocyte Cell Culture
All animal experiments for this study were performed in accordance with the regulations of the local Animal Use and Care Committee (North Rhine-Westphalia State Agency for Nature, Environment and Consumer Protection, Recklinghausen, Germany). To screen for GlyT expression in primary cultures of the central nervous system, cells were prepared from cerebral cortices of Wistar rats.26 In brief, rats were anesthetized using isoflurane (Abbott, Abbott Park, IL), decapitated, and the brains quickly removed. To avoid fibroblast or endothelial contamination, cortical tissue was carefully isolated from blood vessels and meninges, rinsed with Dulbecco's modified Eagle medium (DMEM; Invitrogen), dissociated by trypsinization, and suspended in DMEM supplemented with 10% fetal calf serum (Invitrogen), penicillin (80 units/ml), and streptomycin (0.2 mg/ml). Dissociated cells were plated in 75 cm2culture flasks (Corning Incorporated Life Sciences, Lowell, MA). After 5 days, cultures were washed with DMEM to remove cellular debris and maintained until subconfluency. Cellular debris, microglia, oligodendrocytes, and their early precursor cells were then removed by shaking flasks overnight at 250 rpm at 37°C. The remaining cell population harvested from shaking flasks consisted of more than 98% primary rat astrocytes, as determined by immunocytochemical analysis using antibodies against glial fibrillary acidic protein. For the following glycine uptake experiments, astrocytes were detached by incubation with Accutase (Invitrogen) for 3 min at 37°C, counted, and replated on 6-well cell culture plates coated with poly-D-lysine (0.1 mg/ml). For preparation of microglia and oligodendrocytes, supernatants from shaking steps were seeded on uncoated plastic Petri dishes and microglia were allowed to sediment and attach for 15 min. Oligodendrocyte enriched supernatant was then collected and used for further quantitative real time polymerase chain reaction analysis. Microglia were immediately lysed.
Cortical Neuron Preparation
Pregnant Wistar rats were sacrificed by anaesthetization with isoflurane 15 days after conception, and embryos were removed with sterile technique. Embryonic cortices were removed, gently dissected into small pieces, and collected in DMEM. Following centrifugation (2,000 rpm/30 s), DMEM was discarded, and 10 ml of 0.05% trypsin/EDTA (Invitrogen) were added. The tissue was then incubated for 8 min at 37°C and 10% CO2. The reaction was stopped by adding 10 ml of DMEM medium containing 10% fetal calf serum and centrifuged again as described above. Cell pellets were carefully resuspended in 1 ml DMEM and filtered through a sterile nylon mesh (pore diameter: 30 μm). The filtered cell suspension was transferred to a new tube into a total volume of 50 ml DMEM and spun down at 1,500 rpm for 5 min. Afterward, neurons were resuspended in astrocyte-conditioned medium prepared from cortical rat astrocyte cultures after a conditioning period of between 3 and 4 days. Neurons were then plated on dishes coated with 0.1 mg/ml poly-D-lysine and 3 μg/ml laminin.
Quantitative Real Time Polymerase Chain Reaction
RNA from cultured cells was purified using the RNeasy kit (Qiagen, Hilden, Germany) following the supplier's protocols. Isolated RNA was reversely transcribed using the high capacity complimentary DNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA). Quantitative determination of gene expression levels was performed on an ABI 7900 sequence detection system using TaqMan® universal PCR master mix (Applied Biosystems). TaqMan® probes (Applied Biosystems) for GlyT1 (Assay ID: Rn01416529_m1) and GlyT2 (Assay ID: Rn01475607_m1) genes were selected. Conditions for quantitative real time polymerase chain reaction were 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 60 s. GAPDH (Assay ID: Rn01775763_g1) was used as a reference gene and relative gene expression levels were determined according to the comparative CT method.27 Each sample was measured in quadruplicate.
Immunofluorescence Microscopy of Glycine Transporter Expression
To confirm GlyT expression with immunofluorescence microscopy in primary rat astrocytes from the cortical brain, cells were detached by incubation with Accutase (Invitrogen) for 3 min at 37°C, counted, and replated on sterile 8-well Lab-Tek chamber glass slides (Nalge Nunc Int., Rochester, NY). After 24 h, cells were washed with phosphate buffered saline and fixed in 4% paraformaldehyde for 15 min at room temperature. After additional washing with phosphate buffered saline, samples were blocked and permeabilized with blocking buffer containing 1% bovine serum albumin, 3% of normal serum of the second antibody host species, and 0.05% saponine in phosphate buffered saline.
For immunostaining of frozen brain and spinal cord cryosections, 10 μm-thick sections were fixed in 4% paraformaldehyde for 10 min at room temperature and washed with phosphate buffered saline. Subsequently, cryosections were blocked and permeabilized for 1 h with blocking buffer containing 10% normal goat serum and 0.2% saponine in phosphate buffered saline.
Primary antibodies were diluted (1:500) in phosphate buffered saline (containing 1% bovine serum albumin for cell cultures, or 2% normal goat serum and 0.2% saponine for cryosections) and were applied by incubation overnight at 4°C. Secondary antibodies were diluted (1:1,000) in the same buffer and applied for 1 h (between 3 and 4 h for cryosections) at room temperature. Subsequently, glass slides were washed with phosphate buffered saline and mounted to coverslips with mounting medium (for cell cultures: Vectashield; Vector Laboratories Inc., Burlingame, CA; for cryosections: ProLong Gold antifade reagent with DAPI; Invitrogen), evaluated with a Leica DM LB fluorescence light microscope, and documented with a digital camera (Leica Microsystems, Wetzlar, Germany).
Western Blot Analysis of Glycine Transporter Expression
To analyze GlyT1 and GlyT2 protein expression, cells were lysed for 20 min in lysis buffer (150 MM NaCl, 50 MM Tris-HCl pH 7.5, 1% NP-40, 1 μM pepstatin, 0.1 μM phenylmethylsulphonylfluoride, 0.15 μM aprotinin, and 1 μM leupeptin). Lysates were centrifuged at 10,000 g at 4°C for 15 min, and the supernatants were harvested. The protein content was measured using the bicinchoninic acid assay (Pierce, Rockford, IL). Sodium dodecyl sulfate polyacrylamide gel electrophoresis separated equal amounts of protein (20 μg per lane), which were transferred to a polyvinylidene difluoride membrane (Amersham Pharmacia, Piscataway, NJ). In a transblot SD cell blotting was performed at 1 mA/cm2for 1h (Bio-Rad, Munich, Germany). The membrane was blocked for 2 h with 0.05% Tween 20 in phosphate buffered saline containing 4% bovine serum albumin and incubated with the primary antibodies overnight at 4°C. The secondary antibody was applied for 1 h at room temperature after washing with 0.05% Tween 20 in phosphate buffered saline. The membrane was washed in phosphate buffered saline with 0.05% Tween 20, and bound antibodies were visualized using the enhanced chemiluminescence system (Amersham Buchler, Braunschweig, Germany).
Glycine Uptake Analysis
Primary astrocytes were pretreated for 30 min at 37°C with glycine uptake buffer (120 MM NaCl, 2 MM KCL, 1 MM CaCl2, 1 MM MgCl2, 10 MM HEPES, pH 7.5) alone as negative control. Additional samples contained an excessive concentration of cold glycine (10 MM) for saturation, ALX5407 and sarcosine as positive controls for GlyT1 inhibition,28 ALX1393 as positive control for GlyT2 inhibition,29 or indicated concentrations of lidocaine and the three major metabolites of lidocaine. All components were diluted in glycine uptake buffer.
After completed incubation, cells were gently washed twice by adding 500 μl phosphate buffered saline into wells. After the final wash, the cells were incubated in 400 μl of glycine uptake buffer. In addition, glycine uptake buffer contained 2.5 MM L-alanine to reduce unspecific (not GlyT-mediated) glycine uptake. For inhibition experiments, compounds were dissolved and diluted in glycine uptake buffer to prepare a 5x stock solution. Cells were pretreated at room temperature for 30 min by adding 100 μl of this stock solution, followed by 10 μl glycine working solution, which yielded a final concentration of 2.5 μM [14C]-glycine mixed with 22.5 μM of unlabeled glycine. Plates were incubated at room temperature for 30 min. Subsequently cells were washed gently with phosphate buffered saline to remove excessive glycine. Finally, cells were lysed by addition of high salt buffer (0.5 M sodium chloride and 2% sodium dodecyl sulfate) and incorporated [14C]-glycine was quantified by liquid scintillation counting. Therefore, cell lysates were transferred to analyzing tubes containing 4 ml of liquid scintillation cocktail (Ultima gold; PerkinElmer, Waltham, MA). Subsequently, radioactivity was measured using a liquid scintillation counter (Tricarb 2100 TR; Packard, Berkshire, United Kingdom).
Detection of Mitochondrial Metabolic Activity
For in vitro  determination of mitochondrial metabolic activity as a marker of cell viability, we used the tetrazolium hydroxide (XTT) assay. XTT, a yellow tetrazolium salt, is cleaved to a soluble orange formazan dye, which can be measured by absorbance. To measure cell viability using XTT, samples were prepared with 100 μl of cell suspension in 96-well cell culture plates, and cells were allowed to adhere overnight at a density of 100,000 cells/well. Subsequent to incubation with lidocaine or one of its metabolites, 50 μl of XTT assay solution (XTT 1 mg/ml and phenazine methosulfate 50 μM, diluted in cell culture medium without phenol red) were added to each well. Mixing the samples gently for 1 min was followed by incubation for 120 min at 37°C. After additional mixing for 3 min the absorbance was measured spectrophotometrically at a wavelength of 450 nm. Resulting values (n = 4 for each condition) for absorbance of untreated controls were subsequently normalized and compared with all other treatments as a measure of metabolic activity.
Electrophysiology in Xenopus laevis  Oocytes
Xenopus laevis  toads were obtained from Nasco International (Fort Atkinson, WI). Isolation, defolliculation, and storage of Xenopus laevis  oocytes were performed as described previously.30,31 Subsequently, oocytes were injected with 52 nl aliquots of rat GlyT1 complimentary RNA (0.5 μg/μl) and stored in sterile oocyte solution (in MM: 96 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.4), supplemented with gentamycin (50 μg/ml; Sigma-Aldrich). GlyT-mediated currents were recorded in the two-electrode whole cell voltage clamp configuration as previously described.30 Oocytes were injected with rat GlyT1 complimentary RNA 2–4 days earlier; noninjected oocytes were used as controls. The electrodes contained 3 M KCl and had resistances of 0.3–1 MΩ. The superfusion solution consisted of 90 MM NaCl, 1 MM KCl, 2 MM MgCl2, and 5 MM HEPES–NaOH, pH 7.4. All recordings were performed with the membrane potential held at −50 mV. Only recordings from oocytes with no significant change in control responses throughout the experiment were included for analysis. Substrates were applied for 30 s followed by a wash-out of 30 s. A fast and reproducible solution exchange was achieved by using a custom-built oocyte chamber with a volume of 40 μl combined with a fast solution exchange (150 μl/s). Data were recorded with Cellworks software at 300 Hz and low pass filtered at 20 Hz using the Turbo TEC-05 amplifier (NPI Electronics, Göttingen, Germany). Solutions exchange was software controlled (Cellworks, NPI Electronics). All measurements were performed at room temperature (20–22°C). Accounting for the variability in GlyT1-expression in the oocyte system, currents were normalized to the current induced by glycine. All data presented as mean ± SD from six independent experiments. EC50values were calculated from a nonlinear fit of the Hill equation to the data using Graph Pad Prism Software version 5.0 (GraphPad Software Inc., La Jolla, CA).
Statistics
Each experiment was performed three times unless stated otherwise. Results are expressed as mean ± SD and compared with ANOVA followed by post hoc  Bonferroni correction (two-tailed testing) using Graph Pad Prism Software. P  < 0.05 was considered significant.
Results
GlyT1 Is Differentially Expressed in Primary Central Nervous System Cell Cultures and Tissue
To screen for a primary cultured cell type that allows the analysis of pharmacological effects of lidocaine and its metabolites on GlyT1 function, we analyzed the expression of GlyT based on quantitative real time polymerase chain reaction in different cultured cortical central nervous system cell types. GlyT1 messenger RNA was strongest expressed in astrocytes, while expression levels were considerably lower in neurons and oligodendrocytes. GlyT1 messenger RNA was barely detectable in microglial cells (fig. 1A). GlyT2-expression was not detectable in either investigated cortical cell type (data not shown).
Fig. 1. (A  ) Comparison of glycine transporter (GlyT) messenger RNA expression in different cell types of the central nervous system. In order to screen the different cell types obtained from rat cortical brain preparation for GlyT messenger RNA expression, quantitative real time polymerase chain reaction analysis was employed. Data are presented as mean ± SD (n = 4). (B–E  ) GlyT1 and GlyT2 protein expression levels in primary astrocytes cell culture. Western blot analysis of GlyT protein expression revealed high GlyT1 protein (70 kDa) levels (B  ) in primary astrocytes, whereas GlyT2 protein (87 kDa) (C  ) was not detected. Homogenized tissue from brain and spinal cord was used as a positive control in both analyses, including brainstem regions, explaining the positive result for GlyT2 expression. Similarly, immunofluorescence microscopy in astrocytes indicated expression of GlyT1 (D  ), but not of GlyT2 (E  ). GlyT1 = glycine transporter 1; GlyT2 = glycine transporter 2.
Fig. 1. (A 
	) Comparison of glycine transporter (GlyT) messenger RNA expression in different cell types of the central nervous system. In order to screen the different cell types obtained from rat cortical brain preparation for GlyT messenger RNA expression, quantitative real time polymerase chain reaction analysis was employed. Data are presented as mean ± SD (n = 4). (B–E 
	) GlyT1 and GlyT2 protein expression levels in primary astrocytes cell culture. Western blot analysis of GlyT protein expression revealed high GlyT1 protein (70 kDa) levels (B 
	) in primary astrocytes, whereas GlyT2 protein (87 kDa) (C 
	) was not detected. Homogenized tissue from brain and spinal cord was used as a positive control in both analyses, including brainstem regions, explaining the positive result for GlyT2 expression. Similarly, immunofluorescence microscopy in astrocytes indicated expression of GlyT1 (D 
	), but not of GlyT2 (E 
	). GlyT1 = glycine transporter 1; GlyT2 = glycine transporter 2.
Fig. 1. (A  ) Comparison of glycine transporter (GlyT) messenger RNA expression in different cell types of the central nervous system. In order to screen the different cell types obtained from rat cortical brain preparation for GlyT messenger RNA expression, quantitative real time polymerase chain reaction analysis was employed. Data are presented as mean ± SD (n = 4). (B–E  ) GlyT1 and GlyT2 protein expression levels in primary astrocytes cell culture. Western blot analysis of GlyT protein expression revealed high GlyT1 protein (70 kDa) levels (B  ) in primary astrocytes, whereas GlyT2 protein (87 kDa) (C  ) was not detected. Homogenized tissue from brain and spinal cord was used as a positive control in both analyses, including brainstem regions, explaining the positive result for GlyT2 expression. Similarly, immunofluorescence microscopy in astrocytes indicated expression of GlyT1 (D  ), but not of GlyT2 (E  ). GlyT1 = glycine transporter 1; GlyT2 = glycine transporter 2.
×
Consistent with these findings, western blot analysis showed robust protein levels of GlyT1 in cultivated astrocytes (fig. 1B), whereas GlyT2 protein was absent in astrocytes (fig. 1C). In addition, GlyT1 expression was found in immunofluorescence microscopy of rat astrocytes (fig. 1D), while GlyT2 was not detectable (fig. 1E).
To exclude that the GlyT1 expression in cultured cortical astrocytes resulted from a tissue culture artifact, we also investigated the expression of GlyT1 in cortical brain coronal sections of the parietal lobe by immunofluorescence microscopy. Here, GlyT1 immunoreactivity was observed in both astrocytes and neurons, as indicated by costaining with glial fibrillary acidic protein and neuron-specific nuclear protein, respectively (fig. 2A and B). Similar results regarding astrocytes were found in spinal cord cryosections, especially in white matter regions, although neuronal expression of GlyT1 seemed to be less abundant (see Supplemental Digital Content 1, , which is a figure displaying GlyT1 immunoreactivity in spinal cord cryosections).
Fig. 2. Immunofluorescence microscopy of glycine transporter 1 (GlyT1) expression in rat brain cortex coronal cryosections. (A  ) GlyT1-related signals were found in the most superficial areas of the cortex at the parietal lobe, predominantly in cells that stained positive for glial fibrillary acidic protein, a specific marker for astroglial cells. (B  ) Less superficial, most GlyT1-related fluorescence was found in cells that costained positive for neuron-specific nuclear protein. Note that digital overlays (Merge) demonstrate the colocalization of astrocytes and neurons with GlyT1. Nuclei were stained using DAPI. Similar results from spinal cord cryosections can be found in Supplemental Digital Content 1, . GlyT1 = glycine transporter 1; NeuN = neuron-specific nuclear protein.
Fig. 2. Immunofluorescence microscopy of glycine transporter 1 (GlyT1) expression in rat brain cortex coronal cryosections. (A 
	) GlyT1-related signals were found in the most superficial areas of the cortex at the parietal lobe, predominantly in cells that stained positive for glial fibrillary acidic protein, a specific marker for astroglial cells. (B 
	) Less superficial, most GlyT1-related fluorescence was found in cells that costained positive for neuron-specific nuclear protein. Note that digital overlays (Merge) demonstrate the colocalization of astrocytes and neurons with GlyT1. Nuclei were stained using DAPI. Similar results from spinal cord cryosections can be found in Supplemental Digital Content 1, . GlyT1 = glycine transporter 1; NeuN = neuron-specific nuclear protein.
Fig. 2. Immunofluorescence microscopy of glycine transporter 1 (GlyT1) expression in rat brain cortex coronal cryosections. (A  ) GlyT1-related signals were found in the most superficial areas of the cortex at the parietal lobe, predominantly in cells that stained positive for glial fibrillary acidic protein, a specific marker for astroglial cells. (B  ) Less superficial, most GlyT1-related fluorescence was found in cells that costained positive for neuron-specific nuclear protein. Note that digital overlays (Merge) demonstrate the colocalization of astrocytes and neurons with GlyT1. Nuclei were stained using DAPI. Similar results from spinal cord cryosections can be found in Supplemental Digital Content 1, . GlyT1 = glycine transporter 1; NeuN = neuron-specific nuclear protein.
×
Lidocaine Metabolites Reduce Glycine Uptake in Primary Astrocytes
[14C]-labeled glycine incorporation assays using cultured cortical astrocytes revealed a high affinity uptake system in these cells. Consistent with an active transporter mediating these effects, [14C]-labeled glycine incorporation was strikingly reduced by cooling to 4°C (physical inhibition; negative control), or preincubation with a high concentration of glycine. Moreover, glycine uptake activity was considerably reduced by incubation with known inhibitors like ALX5407 or the alternative GlyT1 substrate sarcosine, thus indicating that GlyT1 mediates the majority of the uptake activity. No significant reduction of glycine uptake was found after treatment with the GlyT2 inhibitor ALX1393 (fig. 3A).
Fig. 3. Glycine uptake by astrocytes. (A  ) Detectable radioactivity from intracellular [14C]-glycine after 30 min uptake at an extracellular glycine concentration of 25 μM in untreated controls was used a measure for glycine uptake. A further control cooled to 4°C during uptake incubation to physically inhibit uptake indicated very low unspecific background. Pretreatment with a very high glycine concentration saturated the system, leading to inhibited uptake of radio-labeled glycine during uptake incubation. The inhibitors ALX5407 and sarcosine of glycine transporter (GlyT) 1 were found to exert a strong inhibition of glycine uptake in the investigated model, indicating a high relevance of GlyT1-mediated transport in this system. In contrast, with an inhibitor of GlyT2 and ALX1393, no detectable inhibition of glycine uptake was observed. (B  ) Pretreatment with mounting concentrations of lidocaine did not lead to a decrease in glycine uptake compared with untreated controls, except the highest and already slightly cytotoxic concentration (1 MM). All investigated metabolites of lidocaine, monoethylglycinexylidide (MEGX), glycinexylidide (GX) and N-ethylglycine (EG) were found to exert a significant inhibition compared with untreated controls. Data are presented as mean ± SD (n = 3). An asterisk indicates a significant difference compared with controls (P  < 0.05).
Fig. 3. Glycine uptake by astrocytes. (A 
	) Detectable radioactivity from intracellular [14C]-glycine after 30 min uptake at an extracellular glycine concentration of 25 μM in untreated controls was used a measure for glycine uptake. A further control cooled to 4°C during uptake incubation to physically inhibit uptake indicated very low unspecific background. Pretreatment with a very high glycine concentration saturated the system, leading to inhibited uptake of radio-labeled glycine during uptake incubation. The inhibitors ALX5407 and sarcosine of glycine transporter (GlyT) 1 were found to exert a strong inhibition of glycine uptake in the investigated model, indicating a high relevance of GlyT1-mediated transport in this system. In contrast, with an inhibitor of GlyT2 and ALX1393, no detectable inhibition of glycine uptake was observed. (B 
	) Pretreatment with mounting concentrations of lidocaine did not lead to a decrease in glycine uptake compared with untreated controls, except the highest and already slightly cytotoxic concentration (1 MM). All investigated metabolites of lidocaine, monoethylglycinexylidide (MEGX), glycinexylidide (GX) and N-ethylglycine (EG) were found to exert a significant inhibition compared with untreated controls. Data are presented as mean ± SD (n = 3). An asterisk indicates a significant difference compared with controls (P 
	< 0.05).
Fig. 3. Glycine uptake by astrocytes. (A  ) Detectable radioactivity from intracellular [14C]-glycine after 30 min uptake at an extracellular glycine concentration of 25 μM in untreated controls was used a measure for glycine uptake. A further control cooled to 4°C during uptake incubation to physically inhibit uptake indicated very low unspecific background. Pretreatment with a very high glycine concentration saturated the system, leading to inhibited uptake of radio-labeled glycine during uptake incubation. The inhibitors ALX5407 and sarcosine of glycine transporter (GlyT) 1 were found to exert a strong inhibition of glycine uptake in the investigated model, indicating a high relevance of GlyT1-mediated transport in this system. In contrast, with an inhibitor of GlyT2 and ALX1393, no detectable inhibition of glycine uptake was observed. (B  ) Pretreatment with mounting concentrations of lidocaine did not lead to a decrease in glycine uptake compared with untreated controls, except the highest and already slightly cytotoxic concentration (1 MM). All investigated metabolites of lidocaine, monoethylglycinexylidide (MEGX), glycinexylidide (GX) and N-ethylglycine (EG) were found to exert a significant inhibition compared with untreated controls. Data are presented as mean ± SD (n = 3). An asterisk indicates a significant difference compared with controls (P  < 0.05).
×
Treatment with lidocaine using clinically relevant (1–10 μM) and higher concentrations (100 μM) did not influence glycine uptake. Only a very high and therefore cytotoxic concentration (1 MM) led to a moderate reduction of glycine uptake (fig. 3B). In contrast, all three investigated metabolites of lidocaine led to a significant dose-dependent decrease of glycine uptake (fig. 3B). Evaluation of mitochondrial metabolism with XTT assay and cell morphology by light microscopy indicated no reduction of metabolism and cell viability by all investigated substances and concentrations (data not shown), except with lidocaine at the highest applied concentration (1 MM), which led to a reduction of mitochondrial activity by 24 ± 2% (P  < 0.05) compared with untreated controls. Therefore, cytotoxic effects can be ruled out as the mechanism of glycine uptake reduction in our experiments.
Glycine Uptake Inhibition by Lidocaine in Combination with Its Metabolites Depends on Extracellular Glycine Concentration
To investigate the relationship between extracellular glycine concentration and uptake inhibition by lidocaine in combination with its three major metabolites, increasing concentrations of glycine were used (fig. 4). For these experiments, a clinically relevant concentration of lidocaine (4 μM) was combined with a fixed concentration ratio of its metabolites (MEGX 2.5 μM, GX 0.3 μM, EG 30 μM) to mimic the clinical situation, especially after continuous application of lidocaine.32  34 At the lowest glycine concentration (10 μM), glycine uptake was very low and therefore no inhibition could be detected either with ALX5407 as the positive control or with the combination of lidocaine and the three major metabolites. With increasing glycine concentrations (25–100 μM), glycine uptake increased considerably (fig. 4A), whereas the degree of inhibition by ALX5407 decreased from 69 to 40% (P  < 0.05; fig. 4B). Similarly, the degree of glycine uptake inhibition by lidocaine combined with metabolites decreased significantly from 43 to 27% (P  < 0.05; fig. 4B).
Fig. 4. Glycine concentration-dependent uptake and inhibition. (A  ) Glycine uptake in untreated controls (red squares  ) and effects of glycine transporter (GlyT) inhibition by ALX5407 as a specific GlyT1-inhibitor (blue circles  ) and a clinically relevant combination of lidocaine and its major metabolites (green triangles  ) were investigated by using mounting concentrations of extracellular glycine (12.5–100 μM). Lidocaine (4 μM) was combined with monoethylglycinexylidide (MEGX; 2.5 μM), glycinexylidide (GX; 0.3 μM), and N-ethylglycine (EG; 30 μM) to mimic clinically relevant unbound plasma concentrations after continuous systemic or epidural application of lidocaine. (B  ) The degree of inhibition by ALX5407 (blue bars  ) as well as by lidocaine in combination with its metabolites (green bars  ) significantly decreases with increasing extracellular glycine concentrations, indicating a competitive mechanism of glycine transport inhibition. Data are presented as mean ± SD (n = 3). An asterisk indicates a significant difference between groups (P  < 0.05).
Fig. 4. Glycine concentration-dependent uptake and inhibition. (A 
	) Glycine uptake in untreated controls (red squares 
	) and effects of glycine transporter (GlyT) inhibition by ALX5407 as a specific GlyT1-inhibitor (blue circles 
	) and a clinically relevant combination of lidocaine and its major metabolites (green triangles 
	) were investigated by using mounting concentrations of extracellular glycine (12.5–100 μM). Lidocaine (4 μM) was combined with monoethylglycinexylidide (MEGX; 2.5 μM), glycinexylidide (GX; 0.3 μM), and N-ethylglycine (EG; 30 μM) to mimic clinically relevant unbound plasma concentrations after continuous systemic or epidural application of lidocaine. (B 
	) The degree of inhibition by ALX5407 (blue bars 
	) as well as by lidocaine in combination with its metabolites (green bars 
	) significantly decreases with increasing extracellular glycine concentrations, indicating a competitive mechanism of glycine transport inhibition. Data are presented as mean ± SD (n = 3). An asterisk indicates a significant difference between groups (P 
	< 0.05).
Fig. 4. Glycine concentration-dependent uptake and inhibition. (A  ) Glycine uptake in untreated controls (red squares  ) and effects of glycine transporter (GlyT) inhibition by ALX5407 as a specific GlyT1-inhibitor (blue circles  ) and a clinically relevant combination of lidocaine and its major metabolites (green triangles  ) were investigated by using mounting concentrations of extracellular glycine (12.5–100 μM). Lidocaine (4 μM) was combined with monoethylglycinexylidide (MEGX; 2.5 μM), glycinexylidide (GX; 0.3 μM), and N-ethylglycine (EG; 30 μM) to mimic clinically relevant unbound plasma concentrations after continuous systemic or epidural application of lidocaine. (B  ) The degree of inhibition by ALX5407 (blue bars  ) as well as by lidocaine in combination with its metabolites (green bars  ) significantly decreases with increasing extracellular glycine concentrations, indicating a competitive mechanism of glycine transport inhibition. Data are presented as mean ± SD (n = 3). An asterisk indicates a significant difference between groups (P  < 0.05).
×
The Lidocaine Metabolite N-ethylglycine Is a Substrate of GlyT1
To study the effects of lidocaine metabolites on GlyT1 more directly and thus excluding possible indirect mechanisms, we investigated the effects of the respective substances on GlyT1 in Xenopus laevis  oocytes. Here the application of glycine to oocytes expressing GlyT1 resulted in a typical concentration-dependent current response (fig. 5A). Consistent with these currents being exclusively mediated by GlyT1, they were efficiently inhibited by preincubation with 5 μM ALX5407. Moreover, application of sarcosine resulted in inward directed currents similar to those seen after glycine incubation. In noninjected oocytes, no currents were observed with any of the substances applied here.
Fig. 5. Oocyte electrophysiology of glycine transporter 1. (A  ) Representative traces of induced currents in Xenopus laevis  oocytes expressing glycine transporter 1 are depicted. Glycine, N-ethylglycine, and sarcosine were applied for 30 s followed by a washout of 30 s. ALX5407 (1 μM) pretreatment for 30 s before stimulation with substrates led to an almost complete suppression of inducible currents. (B  ) Current registrations with seven different concentrations (glycine 1–1,000 μM; EG 10–1,000 μM) from six different oocytes were normalized to maximal inducible currents (glycine 1,000 μM) and used to calculate concentration-response curves for glycine and EG. Data are presented as mean ± SD (n = 6). Gly = glycine; EG = N-ethylglycine.
Fig. 5. Oocyte electrophysiology of glycine transporter 1. (A 
	) Representative traces of induced currents in Xenopus laevis 
	oocytes expressing glycine transporter 1 are depicted. Glycine, N-ethylglycine, and sarcosine were applied for 30 s followed by a washout of 30 s. ALX5407 (1 μM) pretreatment for 30 s before stimulation with substrates led to an almost complete suppression of inducible currents. (B 
	) Current registrations with seven different concentrations (glycine 1–1,000 μM; EG 10–1,000 μM) from six different oocytes were normalized to maximal inducible currents (glycine 1,000 μM) and used to calculate concentration-response curves for glycine and EG. Data are presented as mean ± SD (n = 6). Gly = glycine; EG = N-ethylglycine.
Fig. 5. Oocyte electrophysiology of glycine transporter 1. (A  ) Representative traces of induced currents in Xenopus laevis  oocytes expressing glycine transporter 1 are depicted. Glycine, N-ethylglycine, and sarcosine were applied for 30 s followed by a washout of 30 s. ALX5407 (1 μM) pretreatment for 30 s before stimulation with substrates led to an almost complete suppression of inducible currents. (B  ) Current registrations with seven different concentrations (glycine 1–1,000 μM; EG 10–1,000 μM) from six different oocytes were normalized to maximal inducible currents (glycine 1,000 μM) and used to calculate concentration-response curves for glycine and EG. Data are presented as mean ± SD (n = 6). Gly = glycine; EG = N-ethylglycine.
×
Lidocaine, MEGX, and GX (each 33 μM and 100 μM) did not lead to a detectable response (data not shown), whereas EG elicited a glycine-like response in a concentration-dependent manner, suggesting that this substance is an alternative GlyT1 substrate (fig. 5A). Similar to glycine-induced currents, the preincubation of the oocytes with ALX5407 resulted in a complete inhibition of EG and sarcosine elicited currents (fig. 5A).
Analysis of concentration-response relationship of EG on GlyT1 resulted in an EC50of 55 μM (CI 95%: 49, 62 μM) and a shift of the sigmoid concentration-response curve to the right compared with glycine (EC5027 μM; CI 95%: 23, 31 μM), which was evaluated in the same experimental setup (fig. 5B). EG-induced currents were compared with those of sarcosine, which is known as a naturally occurring inhibitor of GlyT1. At equimolar concentrations, similar GlyT1-mediated currents were observed (fig. 6A), indicating that both substances act as substrates of GlyT1 and thereby might inhibit glycine uptake. Finally, simultaneous incubation with EG and glycine increased glycine-induced currents in an under-additive and concentration-dependent manner, as would be expected when combining two regular substrates of a transporting system (fig. 6B).
Fig. 6. (A  ) Glycine transporter 1 (GlyT1)-mediated currents recorded in Xenopus laevis  oocytes in response to application of N-ethylglycine (EG) and sarcosine for 30 s were compared. (B  ) To investigate the additive effect of EG and glycine at GlyT1, EG was applied to Xenopus laevis  oocytes expressing GlyT1 in mounting concentrations alone or in combination with a fixed concentration of glycine (33.3 μM). Simultaneous application of EG and glycine further increased inducible GlyT1-mediated currents compared with glycine application alone. This under-additive effect further underlines that both substrates compete for transport capacity at GlyT1, as expectable for regular substrates. An asterisk indicates a significant difference compared with glycine-induced currents (P  < 0.05). Values are given as a fraction of glycine-induced currents (33.3 μM). Data are presented as mean ± SD (n = 6).
Fig. 6. (A 
	) Glycine transporter 1 (GlyT1)-mediated currents recorded in Xenopus laevis 
	oocytes in response to application of N-ethylglycine (EG) and sarcosine for 30 s were compared. (B 
	) To investigate the additive effect of EG and glycine at GlyT1, EG was applied to Xenopus laevis 
	oocytes expressing GlyT1 in mounting concentrations alone or in combination with a fixed concentration of glycine (33.3 μM). Simultaneous application of EG and glycine further increased inducible GlyT1-mediated currents compared with glycine application alone. This under-additive effect further underlines that both substrates compete for transport capacity at GlyT1, as expectable for regular substrates. An asterisk indicates a significant difference compared with glycine-induced currents (P 
	< 0.05). Values are given as a fraction of glycine-induced currents (33.3 μM). Data are presented as mean ± SD (n = 6).
Fig. 6. (A  ) Glycine transporter 1 (GlyT1)-mediated currents recorded in Xenopus laevis  oocytes in response to application of N-ethylglycine (EG) and sarcosine for 30 s were compared. (B  ) To investigate the additive effect of EG and glycine at GlyT1, EG was applied to Xenopus laevis  oocytes expressing GlyT1 in mounting concentrations alone or in combination with a fixed concentration of glycine (33.3 μM). Simultaneous application of EG and glycine further increased inducible GlyT1-mediated currents compared with glycine application alone. This under-additive effect further underlines that both substrates compete for transport capacity at GlyT1, as expectable for regular substrates. An asterisk indicates a significant difference compared with glycine-induced currents (P  < 0.05). Values are given as a fraction of glycine-induced currents (33.3 μM). Data are presented as mean ± SD (n = 6).
×
The Lidocaine Metabolite MEGX Has a Direct Inhibitory Effect on GlyT1
In order to identify direct GlyT1-inhibiting effects, glycine-induced currents were recorded before and after incubation with each metabolite of lidocaine for 30 min. Glycine-induced currents (33 μM) were reduced to 63 ± 11% by incubation with MEGX (33 μM) for 30 min (P  < 0.05), indicating a direct inhibitory effect of MEGX on GlyT1 (fig. 7A). After thorough washout of MEGX (60 s), glycine-induced currents remained at 58 ± 9% of previous glycine response (P  < 0.05). In contrast, incubation with GX (33 μM) did not alter glycine-induced currents (fig. 7B).
Fig. 7. Effect of lidocaine metabolites on glycine transporter 1 (GlyT1)-mediated currents. To indentify direct GlyT1-inhibiting effects of the lidocaine metabolites monoethylglycinexylidide (MEGX) and glycinexylidide (GX) in Xenopus laevis  oocytes expressing GlyT1, glycine-induced currents were recorded immediately during simultaneous application (first column), after pretreatment of the same oocytes with MEGX and GX respectively for 30 min (second column), and after thorough washout (60 s) of lidocaine metabolites (third column). Whereas MEGX (33.3 μM) led to a significant and prolonged reduction (A  ), GX (33.3 μM) did not alter glycine-induced currents (B  ). An asterisk indicates a significant difference compared with initial glycine-induced currents (P  < 0.05). Values are given as a fraction of initial glycine-induced currents (33.3 μM). Data are presented as mean ± SD (n = 6).
Fig. 7. Effect of lidocaine metabolites on glycine transporter 1 (GlyT1)-mediated currents. To indentify direct GlyT1-inhibiting effects of the lidocaine metabolites monoethylglycinexylidide (MEGX) and glycinexylidide (GX) in Xenopus laevis 
	oocytes expressing GlyT1, glycine-induced currents were recorded immediately during simultaneous application (first column), after pretreatment of the same oocytes with MEGX and GX respectively for 30 min (second column), and after thorough washout (60 s) of lidocaine metabolites (third column). Whereas MEGX (33.3 μM) led to a significant and prolonged reduction (A 
	), GX (33.3 μM) did not alter glycine-induced currents (B 
	). An asterisk indicates a significant difference compared with initial glycine-induced currents (P 
	< 0.05). Values are given as a fraction of initial glycine-induced currents (33.3 μM). Data are presented as mean ± SD (n = 6).
Fig. 7. Effect of lidocaine metabolites on glycine transporter 1 (GlyT1)-mediated currents. To indentify direct GlyT1-inhibiting effects of the lidocaine metabolites monoethylglycinexylidide (MEGX) and glycinexylidide (GX) in Xenopus laevis  oocytes expressing GlyT1, glycine-induced currents were recorded immediately during simultaneous application (first column), after pretreatment of the same oocytes with MEGX and GX respectively for 30 min (second column), and after thorough washout (60 s) of lidocaine metabolites (third column). Whereas MEGX (33.3 μM) led to a significant and prolonged reduction (A  ), GX (33.3 μM) did not alter glycine-induced currents (B  ). An asterisk indicates a significant difference compared with initial glycine-induced currents (P  < 0.05). Values are given as a fraction of initial glycine-induced currents (33.3 μM). Data are presented as mean ± SD (n = 6).
×
Discussion
Aim of the study was to investigate the effect of lidocaine and its major metabolites on glycine transport in vitro  to elucidate a possible mechanism by which lidocaine could exert its antinociceptive effects in vivo  . Therefore, we first confirmed the predominant expression of functional GlyT1 in rat cortical astrocytes, whereas GlyT2 was barely detectable in this cell type. Furthermore, GlyT1 was found to be expressed not only in astrocytes, but also in neuronal cells of the cerebral cortex.35,36 In contrast, expression of GlyT2 was not detected in neuronal cells from the rat cortex, which is in line with previous investigations.24,25 
Known inhibitors of GlyT1 diminished glycine uptake in primary astrocytes almost completely, whereas GlyT2 inhibition had no effect on glycine uptake. These results indicate that the glycine uptake in astrocytes is predominantly mediated by GlyT1. The local anesthetic lidocaine did not exert significant effects on glycine uptake in vitro  at clinically relevant concentrations. Only at toxic concentrations of lidocaine was glycine uptake slightly diminished. In contrast, all investigated metabolites of lidocaine dose-dependently reduced the GlyT1-mediated uptake by up to 60% at concentrations known to occur after systemic application of lidocaine.34,37 In line with the results obtained with glycine uptake assay, data from electrophysiological experiments suggest that lidocaine itself has no effect on GlyT1, although its metabolite MEGX reduces GlyT1 function as demonstrated by inhibition of glycine-induced currents. Furthermore, EG, which is structurally similar to the known GlyT1-inhibitor sarcosine, was shown to be a hitherto unknown substrate for GlyT1.
Numerous effects of lidocaine have been described that cannot be explained by its principal mechanism of action, i.e  ., the blockade of voltage-gated sodium channels. In vitro  investigations suggest lidocaine-mediated effects among others on Gαi protein function,38 NMDA receptors,39 thromboxane A2 signaling, 40 and glycinergic neurotransmission.19 Clinically, intravenous lidocaine exerts a variety of effects, some of which are of benefit in perioperative settings.41,42 Thus, lidocaine and other local anesthetics have an increasingly broad spectrum of indications beyond analgesia and antiarrhythmic effects, most of which still remain insufficiently investigated.43 
Furthermore, systemic lidocaine has been shown to act antinociceptive in numerous investigations in animals and humans.17,44 The precise mechanism underlying this effect on nociception is still not well understood. In a previous investigation we could demonstrate that at least part of the analgesic effect is mediated by glycinergic mechanisms and, consequently, possibly by NMDA receptor signaling.20 
During continuous systemic or epidural application of lidocaine, unbound plasma levels of lidocaine and MEGX have been reported to remain stable at values of up to 1–6 μg/ml (4.3–25.6 μM) lidocaine45 and 0.5–0.6 μg/ml (2.4–2.9 μM) MEGX, whereas GX and EG have been reported to accumulate at least for the first 48 h33 to unbound plasma concentrations of 0.06–0.1 μg/ml (0.3–0.4 μM) and 2.5–3 μg/ml (25–30 μM) respectively.34,37,46 Therefore, especially with continuous application of lidocaine, unbound plasma concentrations of respective metabolites reach levels that lead to significant glycine transport inhibition in vitro  .
GlyT regulate the glycine concentration in the synaptic cleft at glycinergic inhibitory and glutamatergic excitatory synapses.1 At glycinergic synapses, glycine is removed from the extracellular space by complementary activity of GlyT1 mainly expressed by astrocytes and neuronal GlyT2.23 At glutamatergic synapses, GlyT1-mediated uptake maintains the glycine concentration below the saturation level of the glycine-binding site of the NMDA receptor.
Inhibition of the glycine transport is thought to have beneficial effects in patients with schizophrenia by indirectly increasing the glycine concentration at glutamatergic synapses and thereby restoring NMDA receptor activation.47 Therefore, GlyT1 seems to play a crucial role in regulating NMDA receptor function.48 Accordingly, GlyT1 is present in the vicinity of NMDA receptors, which are known to be critically involved in nociception, especially in the context of pathologic pain.49  52 
Interestingly, glycine transporter inhibitors have been demonstrated to reduce pain in different in vivo  models of neuropathic pain. GlyT1 has been suggested to lower extracellular glycine concentration at glycinergic synapses.23,53 This would be in line with the notion that inhibition of GlyT1 function, leading to accumulation of glycine at the site of inhibitory glycine receptors, can ameliorate pathologic pain. In fact, a study in mice demonstrated that intrathecal strychnine–induced dynamic allodynia was reduced by intrathecal sarcosine,54 supporting the ability of GlyT1 inhibition to enhance glycinergic inhibition. Furthermore, in neuropathic rats after spared nerve injury, GlyT1 inhibition by oral or intrathecal sarcosine has been shown to be effective in reducing pathologic mechanical sensitivity. The authors of this study concluded that inhibition of GlyT1 at multiple central sites induces acute analgesia, as well as acute and long-term reduction in neuropathic pain behavior.11 In addition to that, Tanabe et al  . found that an increase of endogenous glycine concentration following GlyT1 inhibition not only leads to inhibition of pain transmission at the spinal level but also to protection against impairment of hippocampal long-term potentiation. Therefore, the authors concluded that GlyT1 inhibition might also be capable of ameliorating the cognitive disturbances often observed in patients with chronic pain.9 However, although numerous further studies point to the importance of GlyT1 inhibition as a potential treatment option for chronic pain therapy,810,55 several other studies also underline the relevance of GlyT2 inhibition.8,54,56 
Although in our study, the lidocaine metabolite MEGX reduced GlyT1 function and EG was found to inhibit glycine uptake as a novel substrate of GlyT1, the effects on GlyT2 remain unknown. According to our results, a possible mechanism of lidocaine-mediated antinociceptive effects is the increase of extracellular glycine concentration, resulting in enhanced activity at inhibitory glycinergic synapses by inhibition of GlyT1-mediated glycine reuptake. However, it is also conceivable that an increase of glycine concentration may also enhance the activation of excitatory glutamatergic synapses by local transporter inhibition or neurotransmitter spillover.57 In summary, the direct consequences of a general or selective inhibition of glycine transporters with respect to resulting glycine concentrations and activity of inhibitory and excitatory synapses still remain unclear and need to be further elucidated.
Direct comparison of the chemical structures of GlyT1 inhibitors, like sarcosine, with the molecular structures of lidocaine and its metabolites shows an intriguing similarity. As might have been expected as a result of this structural similarity, we could show that the lidocaine metabolite EG acts as a substrate of GlyT1, whereas MEGX directly interferes with GlyT1 function. Correspondingly, increasing extracellular glycine concentrations led to a reduced glycine transport inhibition by a combination of lidocaine metabolites. The fact that the lidocaine metabolite GX alone induced a significant decrease in glycine uptake in astrocytes, although it did not have an effect on glycine-induced currents, may be attributed to indirect effects on glycine uptake, although this remains speculative.
Hitherto, various explanations for the antinociceptive activity of systemic lidocaine, including both central and peripheral mechanisms, have been discussed.15 Although there is evidence suggesting that systemic lidocaine may have beneficial effects on pathologic pain by suppression of ectopic firing,58,59 the exact mechanism still remains unclear.60 Until now, very few investigations have been conducted to clarify whether lidocaine might exert some of its antinociceptive activity through glycinergic pathways. In contrast to our hypothesis that systemic lidocaine or its metabolites act indirectly through enhancing glycinergic activity by GlyT1 inhibition, another study has proposed that lidocaine and also procaine directly activate glycine and distinct γ-aminobutyric acid receptors mediating the observed antinociceptive effect.61 Whether or not these two proposed glycinergic mechanisms possibly act in concert with each other to produce an antinociceptive effect remains to be clarified.
Of note, various other effects of systemic lidocaine application have been described for the central nervous system.62 Neuropsychiatric adverse effects included dysphoria, depressive mood with or without paranoid ideation, agitation with hallucinations, dizziness, light headiness or drowsiness, perioral or limb paresthesia, visual disturbances, confusion, disorientation, and cognitive dysfunction. Since these undesired effects were most likely induced by lidocaine itself or its sodium channel-blocking metabolites MEGX and GX, when aiming for glycinergic enhancement, application of metabolites like EG, which has no relevant effect on sodium channels, could be similarly effective, but associated with a lower probability of inducing undesired effects.
In conclusion, the major lidocaine metabolites, but not lidocaine itself, inhibit the GlyT1-mediated uptake of glycine. While MEGX was found to inhibit glycine-induced response of GlyT1, EG was found to interfere by acting as a previously unknown substrate for GlyT1. This effect can be observed at concentrations of EG that regularly occur systemically during epidural anesthesia or therapeutic systemic application. Furthermore, a combination of lidocaine and its metabolites, mimicking the clinical situation in which the parent drug and the metabolites are present simultaneously, even increases the observed effects. Since inhibition of GlyT1 has been shown to significantly reduce hyperalgesia in a variety of pathologic pain states in vivo  , glycine transporter inhibition by lidocaine metabolites might provide a novel molecular mechanism for the well-established antinociceptive effect of systemic lidocaine. However, whether the pharmacological effects observed in our in vitro  investigation account for the antinociceptive effect of systemic lidocaine in vivo  needs to be further elucidated.
The authors thank Nicole Eichhorst (Technician, Department of Gastroenterology, Hepatology and Infectiology, Medical Faculty, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany) for technical guidance in scintillation counting, and Enrique Nuñez, B.Sc. (Technician, Department of Molecular Biology and Centre of Molecular Biology “Severo Ochoa,” Autonomous University of Madrid, Madrid, Spain), for the GlyT1 and GlyT2 polyclonal antibodies affinity purification.
References
Gomeza J, Armsen W, Betz H, Eulenburg V: Lessons from the knocked-out glycine transporters. Handb Exp Pharmacol 2006; 457–83
Aragón C, López-Corcuera B: Glycine transporters: Crucial roles of pharmacological interest revealed by gene deletion. Trends Pharmacol Sci 2005; 26:283–6
Eulenburg V, Gomeza J: Neurotransmitter transporters expressed in glial cells as regulators of synapse function. Brain Res Rev 2010; 63:103–12
Zeilhofer HU: Synaptic modulation in pain pathways. Rev Physiol Biochem Pharmacol 2005; 154:73–100
Zeilhofer HU: The glycinergic control of spinal pain processing. Cell Mol Life Sci 2005; 62:2027–35
Zeilhofer HU: Loss of glycinergic and GABAergic inhibition in chronic pain–contributions of inflammation and microglia. Int Immunopharmacol 2008; 8:182–7
Zeilhofer HU, Zeilhofer UB: Spinal dis-inhibition in inflammatory pain. Neurosci Lett 2008; 437:170–4
Hermanns H, Muth-Selbach U, Williams R, Krug S, Lipfert P, Werdehausen R, Braun S, Bauer I: Differential effects of spinally applied glycine transporter inhibitors on nociception in a rat model of neuropathic pain. Neurosci Lett 2008; 445:214–9
Tanabe M, Takasu K, Yamaguchi S, Kodama D, Ono H: Glycine transporter inhibitors as a potential therapeutic strategy for chronic pain with memory impairment. ANESTHESIOLOGY 2008; 108:929–37
Dohi T, Morita K, Kitayama T, Motoyama N, Morioka N: Glycine transporter inhibitors as a novel drug discovery strategy for neuropathic pain. Pharmacol Ther 2009; 123:54–79
Centeno MV, Mutso A, Millecamps M, Apkarian AV: Prefrontal cortex and spinal cord mediated anti-neuropathy and analgesia induced by sarcosine, a glycine-T1 transporter inhibitor. Pain 2009; 145:176–83
Kodama D, Ono H, Tanabe M: Increased hippocampal glycine uptake and cognitive dysfunction after peripheral nerve injury. Pain 2011; 152:809–17
Hollmann MW, Durieux ME: Local anesthetics and the inflammatory response: A new therapeutic indication? ANESTHESIOLOGY 2000; 93:858–75
Hollmann MW, Durieux ME: Prolonged actions of short-acting drugs: Local anesthetics and chronic pain. Reg Anesth Pain Med 2000; 25:337–9
Mao J, Chen LL: Systemic lidocaine for neuropathic pain relief. Pain 2000; 87:7–17
Tremont-Lukats IW, Challapalli V, McNicol ED, Lau J, Carr DB: Systemic administration of local anesthetics to relieve neuropathic pain: A systematic review and meta-analysis. Anesth Analg 2005; 101:1738–49
Challapalli V, Tremont-Lukats IW, McNicol ED, Lau J, Carr DB: Systemic administration of local anesthetic agents to relieve neuropathic pain. Cochrane Database Syst Rev 2005; CD003345
Biella G, Lacerenza M, Marchettini P, Sotgiu ML: Diverse modulation by systemic lidocaine of iontophoretic NMDA and quisqualic acid induced excitations on rat dorsal horn neurons. Neurosci Lett 1993; 157:207–10
Biella G, Sotgiu ML: Central effects of systemic lidocaine mediated by glycine spinal receptors: An iontophoretic study in the rat spinal cord. Brain Res 1993; 603:201–6
Muth-Selbach U, Hermanns H, Stegmann JU, Kollosche K, Freynhagen R, Bauer I, Lipfert P: Antinociceptive effects of systemic lidocaine: Involvement of the spinal glycinergic system. Eur J Pharmacol 2009; 613:68–73
Wang JS, Backman JT, Taavitsainen P, Neuvonen PJ, Kivistö KT: Involvement of CYP1A2 and CYP3A4 in lidocaine N-deethylation and 3-hydroxylation in humans. Drug Metab Dispos 2000; 28:959–65
Harsing LG Jr., Juranyi Z, Gacsalyi I, Tapolcsanyi P, Czompa A, Matyus P: Glycine transporter type-1 and its inhibitors. Curr Med Chem 2006; 13:1017–44
Roux MJ, Supplisson S: Neuronal and glial glycine transporters have different stoichiometries. Neuron 2000; 25:373–83
Zafra F, Aragón C, Olivares L, Danbolt NC, Giménez C, Storm-Mathisen J: Glycine transporters are differentially expressed among CNS cells. J Neurosci 1995; 15:3952–69
Zafra F, Gomeza J, Olivares L, Aragón C, Giménez C: Regional distribution and developmental variation of the glycine transporters GLYT1 and GLYT2 in the rat CNS. Eur J Neurosci 1995; 7:1342–52
McCarthy KD, de Vellis J: Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 1980; 85:890–902
Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001; 29:e45
Atkinson BN, Bell SC, De Vivo M, Kowalski LR, Lechner SM, Ognyanov VI, Tham CS, Tsai C, Jia J, Ashton D, Klitenick MA: ALX 5407: A potent, selective inhibitor of the hGlyT1 glycine transporter. Mol Pharmacol 2001; 60:1414–20
Xu TX, Gong N, Xu TL: Inhibitors of GlyT1 and GlyT2 differentially modulate inhibitory transmission. Neuroreport 2005; 16:1227–31
Laube B, Hirai H, Sturgess M, Betz H, Kuhse J: Molecular determinants of agonist discrimination by NMDA receptor subunits: Analysis of the glutamate binding site on the NR2B subunit. Neuron 1997; 18:493–503
Nicke A, Bäumert HG, Rettinger J, Eichele A, Lambrecht G, Mutschler E, Schmalzing G: P2X1 and P2X3 receptors form stable trimers: A novel structural motif of ligand-gated ion channels. EMBO J 1998; 17:3016–28
Fukuda T, Kakiuchi Y, Miyabe M, Okubo N, Yaguchi Y, Kohda Y, Toyooka H: Plasma lidocaine, monoethylglycinexylidide, and glycinexylidide concentrations after epidural administration in geriatric patients. Reg Anesth Pain Med 2000; 25:268–73
Dickey EJ, McKenzie HC 3rd, Brown KA, de Solis CN: Serum concentrations of lidocaine and its metabolites after prolonged infusion in healthy horses. Equine Vet J 2008; 40:348–52
Nakayama S, Miyabe M, Kakiuchi Y, Inomata S, Osaka Y, Fukuda T, Kohda Y, Toyooka H: Propofol does not inhibit lidocaine metabolism during epidural anesthesia. Anesth Analg 2004; 99:1131–5
Cubelos B, Giménez C, Zafra F: Localization of the GLYT1 glycine transporter at glutamatergic synapses in the rat brain. Cereb Cortex 2005; 15:448–59
Raiteri L, Raiteri M: Functional ‘glial’ GLYT1 glycine transporters expressed in neurons. J Neurochem 2010; 114:647–53
Roberts RT, Alexander NM, Kelner MJ: Definitive liquid-chromatographic demonstration that N-ethylglycine is the metabolite of lidocaine that interferes in the Kodak sarcosine oxidase-coupled method for creatinine. Clin Chem 1988; 34:2569–72
Benkwitz C, Garrison JC, Linden J, Durieux ME, Hollmann MW: Lidocaine enhances Galphai protein function. ANESTHESIOLOGY 2003; 99:1093–101
Hahnenkamp K, Durieux ME, Hahnenkamp A, Schauerte SK, Hoenemann CW, Vegh V, Theilmeier G, Hollmann MW: Local anaesthetics inhibit signalling of human NMDA receptors recombinantly expressed in Xenopus laevis  oocytes: Role of protein kinase C. Br J Anaesth 2006; 96:77–87
Hahnenkamp K, Theilmeier G, Van Aken HK, Hoenemann CW: The effects of local anesthetics on perioperative coagulation, inflammation, and microcirculation. Anesth Analg 2002; 94:1441–7
Herroeder S, Pecher S, Schönherr ME, Kaulitz G, Hahnenkamp K, Friess H, Böttiger BW, Bauer H, Dijkgraaf OG, Durieux ME, Hollmann MW: Systemic lidocaine shortens length of hospital stay after colorectal surgery: A double-blinded, randomized, placebo-controlled trial. Ann Surg 2007; 246:192–200
McCarthy GC, Megalla SA, Habib AS: Impact of intravenous lidocaine infusion on postoperative analgesia and recovery from surgery: A systematic review of randomized controlled trials. Drugs 2010; 70:1149–63
Borgeat A, Aguirre J: Update on local anesthetics. Curr Opin Anaesthesiol 2010; 23:466–71
Abram SE, Yaksh TL: Systemic lidocaine blocks nerve injury-induced hyperalgesia and nociceptor-driven spinal sensitization in the rat. ANESTHESIOLOGY 1994; 80:383–91
Ikeda Y, Oda Y, Nakamura T, Takahashi R, Miyake W, Hase I, Asada A: Pharmacokinetics of lidocaine, bupivacaine, and levobupivacaine in plasma and brain in awake rats. ANESTHESIOLOGY 2010; 112:1396–403
Kihara S, Miyabe M, Kakiuchi Y, Takahashi S, Fukuda T, Kohda Y, Toyooka H: Plasma concentrations of lidocaine and its principal metabolites during continuous epidural infusion of lidocaine with or without epinephrine. Reg Anesth Pain Med 1999; 24:529–33
Shim SS, Hammonds MD, Kee BS: Potentiation of the NMDA receptor in the treatment of schizophrenia: Focused on the glycine site. Eur Arch Psychiatry Clin Neurosci 2008; 258:16–27
Scimemi A: The interrelated lives of NMDA receptors and glycine transporters. J Physiol 2009; 587:3061–2
Omote K, Kawamata T, Kawamata M, Namiki A: Formalin-induced release of excitatory amino acids in the skin of the rat hindpaw. Brain Res 1998; 787:161–4
Carlton SM, Coggeshall RE: Inflammation-induced changes in peripheral glutamate receptor populations. Brain Res 1999; 820:63–70
Wu LJ, Zhuo M: Targeting the NMDA receptor subunit NR2B for the treatment of neuropathic pain. Neurotherapeutics 2009; 6:693–702
Qu XX, Cai J, Li MJ, Chi YN, Liao FF, Liu FY, Wan Y, Han JS, Xing GG: Role of the spinal cord NR2B-containing NMDA receptors in the development of neuropathic pain. Exp Neurol 2009; 215:298–307
Jursky F, Nelson N: Developmental expression of the glycine transporters GLYT1 and GLYT2 in mouse brain. J Neurochem 1996; 67:336–44
Nishikawa Y, Sasaki A, Kuraishi Y: Blockade of glycine transporter (GlyT) 2, but not GlyT1, ameliorates dynamic and static mechanical allodynia in mice with herpetic or postherpetic pain. J Pharmacol Sci 2010; 112:352–60
Morita K, Motoyama N, Kitayama T, Morioka N, Kifune K, Dohi T: Spinal antiallodynia action of glycine transporter inhibitors in neuropathic pain models in mice. J Pharmacol Exp Ther 2008; 326:633–45
Haranishi Y, Hara K, Terada T, Nakamura S, Sata T: The antinociceptive effect of intrathecal administration of glycine transporter-2 inhibitor ALX1393 in a rat acute pain model. Anesth Analg 2010; 110:615–21
Ahmadi S, Muth-Selbach U, Lauterbach A, Lipfert P, Neuhuber WL, Zeilhofer HU: Facilitation of spinal NMDA receptor currents by spillover of synaptically released glycine. Science 2003; 300:2094–7
Amir R, Argoff CE, Bennett GJ, Cummins TR, Durieux ME, Gerner P, Gold MS, Porreca F, Strichartz GR: The role of sodium channels in chronic inflammatory and neuropathic pain. J Pain 2006; 7:S1–29
Xiao WH, Bennett GJ: C-fiber spontaneous discharge evoked by chronic inflammation is suppressed by a long-term infusion of lidocaine yielding nanogram per milliliter plasma levels. Pain 2008; 137:218–28
Jänig W: What is the mechanism underlying treatment of pain by systemic application of lidocaine? Pain 2008; 137:5–6
Hara K, Sata T: The effects of the local anesthetics lidocaine and procaine on glycine and gamma-aminobutyric acid receptors expressed in Xenopus  oocytes. Anesth Analg 2007; 104:1434–9
Gil-Gouveia R, Goadsby PJ: Neuropsychiatric side-effects of lidocaine: Examples from the treatment of headache and a review. Cephalalgia 2009; 29:496–508
Fig. 1. (A  ) Comparison of glycine transporter (GlyT) messenger RNA expression in different cell types of the central nervous system. In order to screen the different cell types obtained from rat cortical brain preparation for GlyT messenger RNA expression, quantitative real time polymerase chain reaction analysis was employed. Data are presented as mean ± SD (n = 4). (B–E  ) GlyT1 and GlyT2 protein expression levels in primary astrocytes cell culture. Western blot analysis of GlyT protein expression revealed high GlyT1 protein (70 kDa) levels (B  ) in primary astrocytes, whereas GlyT2 protein (87 kDa) (C  ) was not detected. Homogenized tissue from brain and spinal cord was used as a positive control in both analyses, including brainstem regions, explaining the positive result for GlyT2 expression. Similarly, immunofluorescence microscopy in astrocytes indicated expression of GlyT1 (D  ), but not of GlyT2 (E  ). GlyT1 = glycine transporter 1; GlyT2 = glycine transporter 2.
Fig. 1. (A 
	) Comparison of glycine transporter (GlyT) messenger RNA expression in different cell types of the central nervous system. In order to screen the different cell types obtained from rat cortical brain preparation for GlyT messenger RNA expression, quantitative real time polymerase chain reaction analysis was employed. Data are presented as mean ± SD (n = 4). (B–E 
	) GlyT1 and GlyT2 protein expression levels in primary astrocytes cell culture. Western blot analysis of GlyT protein expression revealed high GlyT1 protein (70 kDa) levels (B 
	) in primary astrocytes, whereas GlyT2 protein (87 kDa) (C 
	) was not detected. Homogenized tissue from brain and spinal cord was used as a positive control in both analyses, including brainstem regions, explaining the positive result for GlyT2 expression. Similarly, immunofluorescence microscopy in astrocytes indicated expression of GlyT1 (D 
	), but not of GlyT2 (E 
	). GlyT1 = glycine transporter 1; GlyT2 = glycine transporter 2.
Fig. 1. (A  ) Comparison of glycine transporter (GlyT) messenger RNA expression in different cell types of the central nervous system. In order to screen the different cell types obtained from rat cortical brain preparation for GlyT messenger RNA expression, quantitative real time polymerase chain reaction analysis was employed. Data are presented as mean ± SD (n = 4). (B–E  ) GlyT1 and GlyT2 protein expression levels in primary astrocytes cell culture. Western blot analysis of GlyT protein expression revealed high GlyT1 protein (70 kDa) levels (B  ) in primary astrocytes, whereas GlyT2 protein (87 kDa) (C  ) was not detected. Homogenized tissue from brain and spinal cord was used as a positive control in both analyses, including brainstem regions, explaining the positive result for GlyT2 expression. Similarly, immunofluorescence microscopy in astrocytes indicated expression of GlyT1 (D  ), but not of GlyT2 (E  ). GlyT1 = glycine transporter 1; GlyT2 = glycine transporter 2.
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Fig. 2. Immunofluorescence microscopy of glycine transporter 1 (GlyT1) expression in rat brain cortex coronal cryosections. (A  ) GlyT1-related signals were found in the most superficial areas of the cortex at the parietal lobe, predominantly in cells that stained positive for glial fibrillary acidic protein, a specific marker for astroglial cells. (B  ) Less superficial, most GlyT1-related fluorescence was found in cells that costained positive for neuron-specific nuclear protein. Note that digital overlays (Merge) demonstrate the colocalization of astrocytes and neurons with GlyT1. Nuclei were stained using DAPI. Similar results from spinal cord cryosections can be found in Supplemental Digital Content 1, . GlyT1 = glycine transporter 1; NeuN = neuron-specific nuclear protein.
Fig. 2. Immunofluorescence microscopy of glycine transporter 1 (GlyT1) expression in rat brain cortex coronal cryosections. (A 
	) GlyT1-related signals were found in the most superficial areas of the cortex at the parietal lobe, predominantly in cells that stained positive for glial fibrillary acidic protein, a specific marker for astroglial cells. (B 
	) Less superficial, most GlyT1-related fluorescence was found in cells that costained positive for neuron-specific nuclear protein. Note that digital overlays (Merge) demonstrate the colocalization of astrocytes and neurons with GlyT1. Nuclei were stained using DAPI. Similar results from spinal cord cryosections can be found in Supplemental Digital Content 1, . GlyT1 = glycine transporter 1; NeuN = neuron-specific nuclear protein.
Fig. 2. Immunofluorescence microscopy of glycine transporter 1 (GlyT1) expression in rat brain cortex coronal cryosections. (A  ) GlyT1-related signals were found in the most superficial areas of the cortex at the parietal lobe, predominantly in cells that stained positive for glial fibrillary acidic protein, a specific marker for astroglial cells. (B  ) Less superficial, most GlyT1-related fluorescence was found in cells that costained positive for neuron-specific nuclear protein. Note that digital overlays (Merge) demonstrate the colocalization of astrocytes and neurons with GlyT1. Nuclei were stained using DAPI. Similar results from spinal cord cryosections can be found in Supplemental Digital Content 1, . GlyT1 = glycine transporter 1; NeuN = neuron-specific nuclear protein.
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Fig. 3. Glycine uptake by astrocytes. (A  ) Detectable radioactivity from intracellular [14C]-glycine after 30 min uptake at an extracellular glycine concentration of 25 μM in untreated controls was used a measure for glycine uptake. A further control cooled to 4°C during uptake incubation to physically inhibit uptake indicated very low unspecific background. Pretreatment with a very high glycine concentration saturated the system, leading to inhibited uptake of radio-labeled glycine during uptake incubation. The inhibitors ALX5407 and sarcosine of glycine transporter (GlyT) 1 were found to exert a strong inhibition of glycine uptake in the investigated model, indicating a high relevance of GlyT1-mediated transport in this system. In contrast, with an inhibitor of GlyT2 and ALX1393, no detectable inhibition of glycine uptake was observed. (B  ) Pretreatment with mounting concentrations of lidocaine did not lead to a decrease in glycine uptake compared with untreated controls, except the highest and already slightly cytotoxic concentration (1 MM). All investigated metabolites of lidocaine, monoethylglycinexylidide (MEGX), glycinexylidide (GX) and N-ethylglycine (EG) were found to exert a significant inhibition compared with untreated controls. Data are presented as mean ± SD (n = 3). An asterisk indicates a significant difference compared with controls (P  < 0.05).
Fig. 3. Glycine uptake by astrocytes. (A 
	) Detectable radioactivity from intracellular [14C]-glycine after 30 min uptake at an extracellular glycine concentration of 25 μM in untreated controls was used a measure for glycine uptake. A further control cooled to 4°C during uptake incubation to physically inhibit uptake indicated very low unspecific background. Pretreatment with a very high glycine concentration saturated the system, leading to inhibited uptake of radio-labeled glycine during uptake incubation. The inhibitors ALX5407 and sarcosine of glycine transporter (GlyT) 1 were found to exert a strong inhibition of glycine uptake in the investigated model, indicating a high relevance of GlyT1-mediated transport in this system. In contrast, with an inhibitor of GlyT2 and ALX1393, no detectable inhibition of glycine uptake was observed. (B 
	) Pretreatment with mounting concentrations of lidocaine did not lead to a decrease in glycine uptake compared with untreated controls, except the highest and already slightly cytotoxic concentration (1 MM). All investigated metabolites of lidocaine, monoethylglycinexylidide (MEGX), glycinexylidide (GX) and N-ethylglycine (EG) were found to exert a significant inhibition compared with untreated controls. Data are presented as mean ± SD (n = 3). An asterisk indicates a significant difference compared with controls (P 
	< 0.05).
Fig. 3. Glycine uptake by astrocytes. (A  ) Detectable radioactivity from intracellular [14C]-glycine after 30 min uptake at an extracellular glycine concentration of 25 μM in untreated controls was used a measure for glycine uptake. A further control cooled to 4°C during uptake incubation to physically inhibit uptake indicated very low unspecific background. Pretreatment with a very high glycine concentration saturated the system, leading to inhibited uptake of radio-labeled glycine during uptake incubation. The inhibitors ALX5407 and sarcosine of glycine transporter (GlyT) 1 were found to exert a strong inhibition of glycine uptake in the investigated model, indicating a high relevance of GlyT1-mediated transport in this system. In contrast, with an inhibitor of GlyT2 and ALX1393, no detectable inhibition of glycine uptake was observed. (B  ) Pretreatment with mounting concentrations of lidocaine did not lead to a decrease in glycine uptake compared with untreated controls, except the highest and already slightly cytotoxic concentration (1 MM). All investigated metabolites of lidocaine, monoethylglycinexylidide (MEGX), glycinexylidide (GX) and N-ethylglycine (EG) were found to exert a significant inhibition compared with untreated controls. Data are presented as mean ± SD (n = 3). An asterisk indicates a significant difference compared with controls (P  < 0.05).
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Fig. 4. Glycine concentration-dependent uptake and inhibition. (A  ) Glycine uptake in untreated controls (red squares  ) and effects of glycine transporter (GlyT) inhibition by ALX5407 as a specific GlyT1-inhibitor (blue circles  ) and a clinically relevant combination of lidocaine and its major metabolites (green triangles  ) were investigated by using mounting concentrations of extracellular glycine (12.5–100 μM). Lidocaine (4 μM) was combined with monoethylglycinexylidide (MEGX; 2.5 μM), glycinexylidide (GX; 0.3 μM), and N-ethylglycine (EG; 30 μM) to mimic clinically relevant unbound plasma concentrations after continuous systemic or epidural application of lidocaine. (B  ) The degree of inhibition by ALX5407 (blue bars  ) as well as by lidocaine in combination with its metabolites (green bars  ) significantly decreases with increasing extracellular glycine concentrations, indicating a competitive mechanism of glycine transport inhibition. Data are presented as mean ± SD (n = 3). An asterisk indicates a significant difference between groups (P  < 0.05).
Fig. 4. Glycine concentration-dependent uptake and inhibition. (A 
	) Glycine uptake in untreated controls (red squares 
	) and effects of glycine transporter (GlyT) inhibition by ALX5407 as a specific GlyT1-inhibitor (blue circles 
	) and a clinically relevant combination of lidocaine and its major metabolites (green triangles 
	) were investigated by using mounting concentrations of extracellular glycine (12.5–100 μM). Lidocaine (4 μM) was combined with monoethylglycinexylidide (MEGX; 2.5 μM), glycinexylidide (GX; 0.3 μM), and N-ethylglycine (EG; 30 μM) to mimic clinically relevant unbound plasma concentrations after continuous systemic or epidural application of lidocaine. (B 
	) The degree of inhibition by ALX5407 (blue bars 
	) as well as by lidocaine in combination with its metabolites (green bars 
	) significantly decreases with increasing extracellular glycine concentrations, indicating a competitive mechanism of glycine transport inhibition. Data are presented as mean ± SD (n = 3). An asterisk indicates a significant difference between groups (P 
	< 0.05).
Fig. 4. Glycine concentration-dependent uptake and inhibition. (A  ) Glycine uptake in untreated controls (red squares  ) and effects of glycine transporter (GlyT) inhibition by ALX5407 as a specific GlyT1-inhibitor (blue circles  ) and a clinically relevant combination of lidocaine and its major metabolites (green triangles  ) were investigated by using mounting concentrations of extracellular glycine (12.5–100 μM). Lidocaine (4 μM) was combined with monoethylglycinexylidide (MEGX; 2.5 μM), glycinexylidide (GX; 0.3 μM), and N-ethylglycine (EG; 30 μM) to mimic clinically relevant unbound plasma concentrations after continuous systemic or epidural application of lidocaine. (B  ) The degree of inhibition by ALX5407 (blue bars  ) as well as by lidocaine in combination with its metabolites (green bars  ) significantly decreases with increasing extracellular glycine concentrations, indicating a competitive mechanism of glycine transport inhibition. Data are presented as mean ± SD (n = 3). An asterisk indicates a significant difference between groups (P  < 0.05).
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Fig. 5. Oocyte electrophysiology of glycine transporter 1. (A  ) Representative traces of induced currents in Xenopus laevis  oocytes expressing glycine transporter 1 are depicted. Glycine, N-ethylglycine, and sarcosine were applied for 30 s followed by a washout of 30 s. ALX5407 (1 μM) pretreatment for 30 s before stimulation with substrates led to an almost complete suppression of inducible currents. (B  ) Current registrations with seven different concentrations (glycine 1–1,000 μM; EG 10–1,000 μM) from six different oocytes were normalized to maximal inducible currents (glycine 1,000 μM) and used to calculate concentration-response curves for glycine and EG. Data are presented as mean ± SD (n = 6). Gly = glycine; EG = N-ethylglycine.
Fig. 5. Oocyte electrophysiology of glycine transporter 1. (A 
	) Representative traces of induced currents in Xenopus laevis 
	oocytes expressing glycine transporter 1 are depicted. Glycine, N-ethylglycine, and sarcosine were applied for 30 s followed by a washout of 30 s. ALX5407 (1 μM) pretreatment for 30 s before stimulation with substrates led to an almost complete suppression of inducible currents. (B 
	) Current registrations with seven different concentrations (glycine 1–1,000 μM; EG 10–1,000 μM) from six different oocytes were normalized to maximal inducible currents (glycine 1,000 μM) and used to calculate concentration-response curves for glycine and EG. Data are presented as mean ± SD (n = 6). Gly = glycine; EG = N-ethylglycine.
Fig. 5. Oocyte electrophysiology of glycine transporter 1. (A  ) Representative traces of induced currents in Xenopus laevis  oocytes expressing glycine transporter 1 are depicted. Glycine, N-ethylglycine, and sarcosine were applied for 30 s followed by a washout of 30 s. ALX5407 (1 μM) pretreatment for 30 s before stimulation with substrates led to an almost complete suppression of inducible currents. (B  ) Current registrations with seven different concentrations (glycine 1–1,000 μM; EG 10–1,000 μM) from six different oocytes were normalized to maximal inducible currents (glycine 1,000 μM) and used to calculate concentration-response curves for glycine and EG. Data are presented as mean ± SD (n = 6). Gly = glycine; EG = N-ethylglycine.
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Fig. 6. (A  ) Glycine transporter 1 (GlyT1)-mediated currents recorded in Xenopus laevis  oocytes in response to application of N-ethylglycine (EG) and sarcosine for 30 s were compared. (B  ) To investigate the additive effect of EG and glycine at GlyT1, EG was applied to Xenopus laevis  oocytes expressing GlyT1 in mounting concentrations alone or in combination with a fixed concentration of glycine (33.3 μM). Simultaneous application of EG and glycine further increased inducible GlyT1-mediated currents compared with glycine application alone. This under-additive effect further underlines that both substrates compete for transport capacity at GlyT1, as expectable for regular substrates. An asterisk indicates a significant difference compared with glycine-induced currents (P  < 0.05). Values are given as a fraction of glycine-induced currents (33.3 μM). Data are presented as mean ± SD (n = 6).
Fig. 6. (A 
	) Glycine transporter 1 (GlyT1)-mediated currents recorded in Xenopus laevis 
	oocytes in response to application of N-ethylglycine (EG) and sarcosine for 30 s were compared. (B 
	) To investigate the additive effect of EG and glycine at GlyT1, EG was applied to Xenopus laevis 
	oocytes expressing GlyT1 in mounting concentrations alone or in combination with a fixed concentration of glycine (33.3 μM). Simultaneous application of EG and glycine further increased inducible GlyT1-mediated currents compared with glycine application alone. This under-additive effect further underlines that both substrates compete for transport capacity at GlyT1, as expectable for regular substrates. An asterisk indicates a significant difference compared with glycine-induced currents (P 
	< 0.05). Values are given as a fraction of glycine-induced currents (33.3 μM). Data are presented as mean ± SD (n = 6).
Fig. 6. (A  ) Glycine transporter 1 (GlyT1)-mediated currents recorded in Xenopus laevis  oocytes in response to application of N-ethylglycine (EG) and sarcosine for 30 s were compared. (B  ) To investigate the additive effect of EG and glycine at GlyT1, EG was applied to Xenopus laevis  oocytes expressing GlyT1 in mounting concentrations alone or in combination with a fixed concentration of glycine (33.3 μM). Simultaneous application of EG and glycine further increased inducible GlyT1-mediated currents compared with glycine application alone. This under-additive effect further underlines that both substrates compete for transport capacity at GlyT1, as expectable for regular substrates. An asterisk indicates a significant difference compared with glycine-induced currents (P  < 0.05). Values are given as a fraction of glycine-induced currents (33.3 μM). Data are presented as mean ± SD (n = 6).
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Fig. 7. Effect of lidocaine metabolites on glycine transporter 1 (GlyT1)-mediated currents. To indentify direct GlyT1-inhibiting effects of the lidocaine metabolites monoethylglycinexylidide (MEGX) and glycinexylidide (GX) in Xenopus laevis  oocytes expressing GlyT1, glycine-induced currents were recorded immediately during simultaneous application (first column), after pretreatment of the same oocytes with MEGX and GX respectively for 30 min (second column), and after thorough washout (60 s) of lidocaine metabolites (third column). Whereas MEGX (33.3 μM) led to a significant and prolonged reduction (A  ), GX (33.3 μM) did not alter glycine-induced currents (B  ). An asterisk indicates a significant difference compared with initial glycine-induced currents (P  < 0.05). Values are given as a fraction of initial glycine-induced currents (33.3 μM). Data are presented as mean ± SD (n = 6).
Fig. 7. Effect of lidocaine metabolites on glycine transporter 1 (GlyT1)-mediated currents. To indentify direct GlyT1-inhibiting effects of the lidocaine metabolites monoethylglycinexylidide (MEGX) and glycinexylidide (GX) in Xenopus laevis 
	oocytes expressing GlyT1, glycine-induced currents were recorded immediately during simultaneous application (first column), after pretreatment of the same oocytes with MEGX and GX respectively for 30 min (second column), and after thorough washout (60 s) of lidocaine metabolites (third column). Whereas MEGX (33.3 μM) led to a significant and prolonged reduction (A 
	), GX (33.3 μM) did not alter glycine-induced currents (B 
	). An asterisk indicates a significant difference compared with initial glycine-induced currents (P 
	< 0.05). Values are given as a fraction of initial glycine-induced currents (33.3 μM). Data are presented as mean ± SD (n = 6).
Fig. 7. Effect of lidocaine metabolites on glycine transporter 1 (GlyT1)-mediated currents. To indentify direct GlyT1-inhibiting effects of the lidocaine metabolites monoethylglycinexylidide (MEGX) and glycinexylidide (GX) in Xenopus laevis  oocytes expressing GlyT1, glycine-induced currents were recorded immediately during simultaneous application (first column), after pretreatment of the same oocytes with MEGX and GX respectively for 30 min (second column), and after thorough washout (60 s) of lidocaine metabolites (third column). Whereas MEGX (33.3 μM) led to a significant and prolonged reduction (A  ), GX (33.3 μM) did not alter glycine-induced currents (B  ). An asterisk indicates a significant difference compared with initial glycine-induced currents (P  < 0.05). Values are given as a fraction of initial glycine-induced currents (33.3 μM). Data are presented as mean ± SD (n = 6).
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