Differential effect of the benzophenanthridine alkaloids sanguinarine and chelerythrine on glycine transporters

Neurochemistry International 58 (2011) 641–647

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Differential effect of the benzophenanthridine alkaloids sanguinarine and chelerythrine on glycine transporters Frantisek Jursky *, Martina Baliova Laboratory of Neurobiology, Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 842 51 Bratislava, Slovakia

A R T I C L E I N F O

A B S T R A C T

Article history: Received 6 September 2010 Received in revised form 29 January 2011 Accepted 1 February 2011 Available online 17 February 2011

Glycine transporter inhibitors modulate the transmission of pain signals. Since it is well known that extracts from medicinal plants such as Chelidonium majus exhibit analgesic properties, we investigated the effects of alkaloids typically present in this plant on glycine transporters. We found that chelerythrine and sanguinarine selectively inhibit the glycine transporter GlyT1 with comparable potency in the low micromolar range while berberine shows no inhibition at all. At this concentration both alkaloids only minimally affected transport of the closely related glycine transporter GlyT2, suggesting that the effect is not mediated by the inhibitory activity of these alkaloids on the Na+/K+ ATPase. GlyT1 inhibition was time-dependent, noncompetitive and increased with glycine concentration. While chelerythrine inhibition was reversible, the effect of sanguinarine was resistant to wash out. These results suggest that benzophenanthridine alkaloids interact with glycine transporters and at low micromolar range selectively target glycine transporter GlyT1. ß 2011 Elsevier Ltd. All rights reserved.

Keywords: Neurotransmitter Transporter Glycine Protein kinase C PKC GlyT1 GlyT2 Chelidonium majus Inhibitor Pain Chelerythrine Sanguinarine Benzophenanthridine Alkaloid Ouabain

1. Introduction Neurotransmitter transporters are a family of membrane proteins, which regulate the availability of several neurotransmitters including glycine (Nelson, 1998; Masson et al., 1999; Aragon and Lopez-Corcuera, 2003; Eulenburg et al., 2005; Gether et al., 2006). Glycine is predominantly involved in the inhibitory neurotransmission of the mammalian hindbrain (Betz, 1992). However it also modulates neurotransmission in the forebrain, where its major effect is believed to be caused by acting on the coagonist site of the NMDA receptor (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988). Both glycine and D-serine can potentiate the NMDA receptor, but the individual contribution of these two agonists is still not clearly defined, mainly because they share a common binding site on the NMDA receptor (Mothet et al., 2000; Panatier et al., 2006; Wolosker, 2007; Shimazaki et al., 2010). The recently developed specific glycine transporter inhibitors are

* Corresponding author. Tel.: +421 2 5930 7437; fax: +421 2 5930 7416. E-mail address: [email protected] (F. Jursky). 0197-0186/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2011.02.001

able to balance the hypofunction of the NMDA receptor. This hypofunction is presumably associated with schizophrenia and inhibitors of GlyT1 represent promising drugs for the treatment of this serious psychiatric disease (Sur and Kinney, 2007; Javitt, 2009). Accumulating evidence indicates that glycinergic and GABAergic transmission has a very specific role in the development of neuropathic pain. In some pathological states, the inflammatory prostaglandin PGE2 activates the EP2 subtype of the prostaglandin E receptor, which leads to protein kinase A dependent inhibition a3 containing glycine receptors in the superficial dorsal horn in the vicinity of nociceptive afferents terminals. The disinhibition of a3 containing glycine receptors increases the firing of superficial dorsal horn neurons and elevates the transmission of nociceptive signals to higher brain pain centers (Ahmadi et al., 2002; Harvey et al., 2004; Zeilhofer and Zeilhofer, 2008; Dohi et al., 2009). According to another hypothesis, neuropathic pain could originate from changes in the chloride gradient, resulting in insufficient glycinergic and GABAergic inhibitory input (Coull et al., 2003, 2005; Prescott et al., 2006). Additionally, glycine can also potentiate the NMDA receptor and influence the periaqueductal

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gray opioid-based antinociception (Wink et al., 1998; Maione et al., 2000; Martins et al., 2008). Glycine transporter inhibitors ameliorate the symptoms of neuropathic pain most likely via an increase in the local glycine concentration (Dohi et al., 2009; Morita et al., 2008; Tanabe et al., 2008). Even though the exact mechanism still needs to be elucidated, pain relief represents a second important line of potential pharmaceutical use for glycine transporter inhibitors. Several pharmacophores acting on glycine transporters were described recently (for review see Bridges et al., 2008; Gilfillan et al., 2009). Since each substance likely has its own specific mode of action and potential pharmacological side effects, there is a constant need for new pharmacophores, which could be adapted to make improved drugs. Alkaloids are a group of biological substances, which exhibit a wide spectrum of anti-inflammatory, antimicrobial, antitumor, analgesic and spasmolytic effects (Wink et al., 1998; Verpoorte, 1998; Simanek et al., 2003). The alkaloids investigated here, chelerythrine and sanguinarine, have many cellular targets, which is probably caused by their quaternary nitrogen, polycyclic and planar structure interacting with the nucleophilic and anionic moieties of amino acids in biomacromolecules (Schmeller et al., 1997). The inhibitory effect of chelerythrine and sanguinarine on several protein kinases and Na+/K+ ATPase activity is well known (Herbert et al., 1990; Wang et al., 1997). The potency of these effects can vary significantly, however. Chelerythrine blocks the ATP-induced cation fluxes mediated by the P2X7 receptor (Shemon et al., 2004), while sanguinarine binds to the angiotensine AT1 receptor (Caballero-George et al., 2003) and inhibits phosphatase PP2C (Aburai et al., 2010), aminopeptidase N and dipeptidyl peptidase IV (Sedo et al., 2002). Two types of interactions between benzophenanthridine alkaloids and DNA have been described. Sanguinarine intercalates into DNA in a similar way as ethidium bromide (Maiti et al., 1982). Sanguinarine and chelerythrine are both metabolized by hepatic microsomes to species, which form covalently bound DNA, adducts under certain conditions (Stiborova et al., 2002). Benzophenanthridine alkaloids affect apoptosis and despite their structural similarities, chelerythrine and sanguinarine target different binding sites on the pro-survival Bcl-XL protein (Zhang et al., 2006). Concerning the effects on the glycine neurotransmitter system, Chelidonium herba, having a high content of benzophenanthridine alkaloids, inhibits glycine-activated currents and potentiates glutamate-activated ion currents in rat periaqueductal gray neurons (PAG) (Shin et al., 2003). Additionally, interaction with several other receptors has been described previously (Wink et al., 1998). In this work we report that the benzophenanthridine alkaloids chelerythrine and saguinarine inhibit glycine transporters and in low micromolar concentration preferentially target the glycine transporter GlyT1. 2. Materials and methods 2.1. Materials Dulbecco’s modified Eagles medium (DMEM), fetal calf serum, streptomycin, penicillin, L-glutamine, Trypsine-EDTA solution, sanguinarine in 98.1% purity, M.P. (279–282 8C) and chelerythrine in 95% purity, M.P. (200–204 8C), berberine, ouabain octahydrate (MP. 182–205 8C), luminol, PCA, hydrogen peroxide, 2mercaptoethanol, PEI 80 kDa were purchased from Sigma Chemicals (St. Louis, MO, USA). Affinity purified primary antibodies (epitope: 554–625) against mouse GlyT1C terminus were used as previously described (Baliova and Jursky, 2010). Secondary horseradish peroxidase conjugated antibodies were purchased from Millipore (Temecula, CA, USA). [3H]Glycine (2.08 TBq/mmol) was supplied by ICN (Irvine, CA, USA). Tris(Hydroxymethyl)aminomethane (Tris) free base, HEPES free acid, sodium chloride (NaCl), lithium chloride (LiCl), all molecular biology grade were from Merck Chemicals, Slovakia (Bratislava). Stock solutions of alkaloids used

for subsequent dilutions were prepared as follows: 3 mg of berberine (MW 371.81) was dissolved in 1 ml of methanol to a final concentration 8 mM; 1 mg of chelerythrine (MW 383.8) was dissolved in 0.3 ml of redistilled water to make 8.68 mM; 5 mg of sanguinarine (MW 367.78) was dissolved in 1.550 ml of methanol to make 8.77 mM. Stock solutions 200 diluted in redestilled water had the following absorbances at 328 nm. Berberine 0.532, chelerythrine 0.552, sanguinarine 0.441. 2.2. Glycine transporter genes and cell lines Rat GlyT2a (Liu et al., 1993), human GlyT1c (gift from Prof. Vandenberg, Department of Pharmacology, Institute for Biomedical Research, University of Sydney, Australia) and human GlyT2a (gift from Prof. Robert Harvey, Department of Pharmacology, School of Pharmacy, London, UK) were transferred to a pEDFPN1D plasmid containing a G418 resistance gene. This plasmid was obtained from pEGFPN1 (Clontec, Palo Alto, CA, USA) in the following way: the GFP gene was excised with ApaI/XbaI, and then the DNA ends were filled with the Klenow fragment and ligated. Glycine transporter genes were inserted downstream of the CMV promoter. HEK293T-hGlyT1c and HEK293T-rGlyT2a stable cell lines were prepared via the integration of ApaLI linearized pEDFPN1D plasmids bearing glycine transporter genes into the HEK293T cell line (ATCC CRL-1573 293) using PEI transfection (Boussif et al., 1995). Single colonies were isolated after selection with G418 at a concentration of 0.5 mg/ml. Cell lines were further propagated in complete DMEM 0.25 mg/ml G418. For uptake purposes HEK293T-hGlyT1c cells and HEK293T-hGlyT2a cells were seeded on 24-well plates. Following transfer of the cells to atmospheric CO2 concentration, the original medium (0.5 ml) was replaced with the same volume of DMEM medium buffered with 20 mM HEPES-NaOH pH 7.4. 2.3. Dose–response of hGlyT1 and hGlyT2 glycine uptake to chelerythrine and sanguinarine Before the assay, HEK293T-hGlyT1c and HEK293T-hGlyT2a expressing cells seeded on 24-well plates were washed once with 0.5 ml of uptake buffer (25 mM Tris–HCl, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, 5 mM alanine, pH 7.4) (Alanine was added to inhibit the endogenous general amino-acid transport system in HEK293T cells). Uptake buffer (0.2 ml) containing various amounts of chelerythrine or sanguinarine (Fig. 2A and B) was then added. Cells were preincubated with alkaloids for 3 min at 23 8C, then the solution was aspirated and 0.2 ml of uptake buffer containing 10 mM [3H] glycine with an alkaloid concentration equal to that in the preincubation solution was added to assay glycine uptake for 1 min (GlyT1) or 5 min (GlyT2) at 23 8C. Uptake was stopped by two 0.5 ml washes of uptake buffer containing lithium instead sodium (25 mM Tris–HCl, 150 mM LiCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, 5 mM alanine). Cells were dissolved in Triton buffer (0.1 M Tris–HCl pH 8.0, 0.5% TritonX100), transferred into scintillation liquid and the samples were measured with a scintillation counter. 2.4. Time dependence of chelerythrine and sanguinarine inhibition Cells HEK293T-hGlyT1c seeded on 24-well plates were washed once with 0.5 ml of uptake buffer. Uptake buffer (0.2 ml) containing 10 mM chelerythrine, 10 mM sanguinarine or 1 mM ouabain was then added. Cells were preincubated with alkaloids for distinct time intervals (see Fig. 2C) at 23 8C, then the solution was aspirated and 0.2 ml of uptake buffer containing 10 mM [3H] glycine with 10 mM alkaloid or 1 mM ouabain was added to assay the glycine uptake for 1 min. In control samples the uptake assay was preformed in the same way, except that alkaloids were omitted during the pre-incubation and uptake assay. Uptake was stopped by two 0.5 ml washes of lithium containing uptake buffer. Cells were dissolved in Triton buffer, transferred into scintillation liquid and samples were measured with a scintillation counter. 2.5. Reversibility of chelerythrine and sanguinarine inhibition HEK293T-hGlyT1c cells seeded on 24-well plates were washed once with 0.5 ml of uptake buffer. Uptake buffer (0.2 ml) containing 10 mM chelerythrine or 5 mM sanguinarine was then added and preincubated for 3 min. Following aspiration of the liquid, the cells were washed 2 with 0.5 ml of uptake buffer and covered with 0.7 ml of uptake buffer containing 10 mM glucose. Cells were then placed on a gentle shaker. At various time intervals, the solution was aspirated and cells were additionally washed once with 0.5 ml uptake buffer. Glycine uptake was then assayed by adding 0.2 ml of uptake solution containing 10 mM [3H] labeled glycine for 1 min at 23 8C. Uptake was stopped by two 0.5 ml washes of lithium-containing uptake buffer. Cells were dissolved in Triton buffer, transferred into scintillation liquid and samples were measured on scintillation counter. 2.6. Determination of inhibition type HEK293T-hGlyT1c cells plated on 24-well plates were washed with 0.5 ml of uptake buffer and preincubated 3 min in the same buffer with and without 2.5 mM

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Fig. 1. (A) Chemical structures of chelerythrine, sanguinarine and berberine. (B) Effect of ouabain (OB), berberine (BE), chelerythrine (CH), sanguinarine (SA) and sarcosine (SR) on glycine uptake of human GlyT1c expressed in HEK293T cells (Cells were preincubated for 3 min with 10 mM BE and CH, 5 mM SA, 1 mM OB, 150 mM SC then uptake was measured for 1 min at 23 8C in the presence of substances equal to that in the preincubation solution. In control sample substances were omitted. Control represents 1.290 + 0.045 nM of glycine/mg/min.) Data show row means  SEM from two separate experiments performed in triplicates.

sanguinarine and 5 mM chelerythrine. Uptake of glycine was then assayed for 1 min at 23 8C in the presence or absence of 2.5 mM sanguinarine and 5 mM chelerythrine, at various glycine concentrations. Saturating curves were transferred to a linear Eadie-Hofstee plot. Surmountability of inhibition was tested by assaying glycine uptake for 1 min in the presence or absence of 5 mM chelerythrine, 2.5 mM sanguinarine, or 150 mM sarcosine at two 0.01 mM and 0.5 mM glycine concentrations. 2.7. Influence of chelerythrine and sanguinarine on the surface expression of GlyT1 HEK293T-hGlyT1c cells were washed 2 with HEPES-buffered saline solution and incubated 3 min in the presence or absence of 10 mM chelerythrine and sanguinarine. Cells were transferred on ice, washed 3 with ice-cold PBS and

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changes in surface localized hGlyT1c were assayed using surface biotinylation as described previously (Baliova et al., 2004). 2.8. Recovery of GlyT1 transport by thiol compounds HEK293T-hGlyT1c cells seeded on a 24-well plate were washed once with 0.5 ml uptake buffer. Uptake buffer (0.2 ml) with and without 2.5 mM sanguinarine was then added. Following a 3 min preincubation at 23 8C the solution was aspirated. Cells were overlaid with 0.2 ml of 10 mM [3H] glycine containing uptake buffer with and without 2.5 mM sanguinarine and glycine uptake was assayed for 1 min at 23 8C. In parallel samples uptake assay was preformed with the same solutions mixed additionally with 2-mercaptoethanol (final concentration 14 mM) 10 min before the assay. In all samples uptake was stopped by two 0.5 ml washes of

Fig. 2. Dose–response curves of hGlyT1c and hGlyT2a to chelerythrine and sanguinarine (A) and (B) and the effect of pre-incubation time with alkaloids on hGlyT1c glycine uptake (C) in HEK293T cells. The dose–response curves were obtained following 3 min preincubation with or without the indicated concentration of alkaloids. (D) Effect of 2mercaptoethanol (2ME) on inhibition of hGlyT1c by 2.5 mM sanguinarine (SA). See Section 2 for details (in A control represents 1.134  0.031 nM of glycine/mg/min for GlyT1 and 0.569  0.036 nM of glycine/mg/5 min for GlyT2; in B control represents 1.281  0.013 nM of glycine/mg/min for GlyT1 and 0.506  0.022 nM of glycine/mg/5 min for GlyT2; in C control represents 1.238  12 nM of glycine/mg/min; in D control represents 1.306  0.026 nM of glycine/mg/min). The quantification data show row means  SEM from three separate experiments performed in triplicates.

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lithium-containing uptake buffer, cells were dissolved in Triton buffer, transferred into scintilation liquid and measured. 2.9. Data analysis In all assays the basal transport rate, measured as the uptake of tritiated glycine in the presence of 20 mM cold glycine, was subtracted from the data measured for hGLYT1c and hGLYT2a. Data were measured in triplicates and analysed using GraphPad Prism 4.00 for Windows (GraphPad Software, San Diego, CA, USA). The relative intensity of immunostaining was quantified with UN-SCAN-IT (Silk Scientific Inc. Utah, USA) and plotted with GraphPad Prism.

3. Results During our work we found a profound difference in the inhibitory effect of the PKC inhibitor chelerythrine on the two closely related glycine transporters GlyT1 and GlyT2. A similar effect was produced by the chelerythrine structural analog sanguinarine (Fig. 1B), which indicated that the effect of the alkaloids is not associated with their PKC inhibition and they might interact with the transporter directly. Fig. 1B shows the effect of chelerythrine, sanguinarine, berberine and the Na+/K+ ATPase inhibitor ouabain on GlyT1 mediated glycine uptake in HEK293T cells. While both chelerythrine and sanguinarine significantly inhibited the accumulation of glycine by transporter GlyT1, berberine and ouabain had no effect under these conditions. Fig. 2A and B shows that in the low micromolar range the inhibitory effect is specific for GlyT1 and no inhibition is observed for the closely related human glycine transporter GlyT2a. To show some inhibibitory activity on GlyT2a much higher concentration of alkaloids (about one order of magnitude) was required (Fig. 2A and B). Both chelerythrine and sanguinarine inhibited GlyT1 in a timedependent manner (Fig. 2C). Onset of inhibition was rapid in the beginning and decline of activity was maximal within about 3 min of preincubation. To obtain better reproducibility, we chose a 3 min preincubation interval with alkaloids in most of the experiments before the uptake assay. Because the commercial alkaloids used contained up to 5% impurities we tested their molecular identity in the inhibition process by including thiol compounds that are known to interact with benzophenantridine alkaloids (Vespalec et al., 2003). As shown in Fig. 2D, the presence of 2-mercaptoethanol significantly interfered with the inhibitory potency of sanguinarine. Fig. 2D also shows that 2-mercaptoethanol itself did not exhibit any effect on hGlyT1c mediated uptake. In the next experiment we investigated the possibility that GlyT1 inhibition reflects surface redistribution of transporters induced by alkaloids. Isolation of the plasma membrane fraction of GlyT1 using surface biotinylation in control cells and in cells treated with alkaloids (Fig. 3) did not show any significant differences, which could be correlated with their inhibitory activity. This indicated that surface redistribution is not likely responsible for the observed inhibitory effect of benzophenanthridine alkaloids. To determine the type of inhibition we performed a substratesaturating assay in the presence or absence of sanguinarine and chelerythrine. Transformation of the saturating curves to linear Eadie Hofstee plots (Fig. 4A and B) indicated that both alkaloids exhibit noncompetitive inhibition. To further verify the mode of inhibition we performed a so-called surmountability test (Mezler et al., 2008). The method is based on the idea that an increasing amount of glycine in the uptake mix should competitively displace a competitive inhibitor such as sarcosine, but not noncompetitive inhibitor acting on a site different from the substrate (glycine) binding site. As shown in Fig. 4C, co-application of inhibitors with low 10 mM glycine and 50-times more concentrated 0.5 mM glycine resulted, in the case of the typically competitive inhibitor

Fig. 3. Effect of chelerythrine (CH) and sanguinarine (SA) on the surface distribution of GlyT1. Two sets of cells were incubated with 10 mM alkaloids for 3 min at 23 8C. Cells in the first set were washed and uptake was measured for 1 min at 23 8C (control represents 1.401  0.035 nM of glycine/mg/min). Another set of cells was transferred on ice and washed 3 with ice cold PBS. Cells were biotinylated on the surface and the biotinylated membrane protein fraction was isolated on streptavidin agarose according to Baliova et al. (2004). Proteins were resolved in PAGE, transferred on immobilon and GlyT1 was detected and quantified using antiGlyT1C554-625 antibodies. The quantification data show row means  SEM from two separate experiments performed in triplicates.

sarcosine, in about 50% decrease in inhibition. In contrast, inhibition of glycine uptake by chelerythine and saguinarine under the same conditions increased by about 50%, indicating a noncompetitive mode of inhibition. Reversibility of inhibition was tested by measuring different washout times following a 3-min incubation with alkaloids (Fig. 4D). While chelerythrine inhibition was reversible and we achieved 50% recovery of GlyT1 activity in 1 h (the conditions described in Section 2) the reversibility of sanguinarine inhibition under the same conditions was minimal. Because the alkaloids contain up to 5% of the impurities, we have tested their molecular identity in the process of inhibition by including the thiol compounds that are known to interact with the benzophenantridine alkaloids. The presence of 2-mercaptoethanol significantly interfered with the inhibitory activity of sanguinarine (Fig. 2D). 4. Discussion In this work we show that the benzophenanthridine alkaloids chelerythrine and saguinarine inhibit glycine transporters and, at low micromolar concentration selectively target the glycine transporter GlyT1. Benzophenanthridine alkaloids have multiple cellular targets resulting in several effects on living cells. For this

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Fig. 4. Effect of alkaloids on hGlyT1c glycine uptake kinetics and surmountability of inhibition. (A) and (B) Lines represent glycine saturating curves and Eadie-Hofstee plot (inset) for HEK293T-hGlyT1c uptake in the absence or presence of alkaloids (2.5 mM sanguinarine, SA; 5 mM chelerythrine, CH). (C) Surmountability of inhibition was tested by assaying glycine uptake in the presence or absence of 2.5 mM sanguinarine, 5 mM chelerythrine, or 150 mM sarcosine (SC) at two 0.01 mM and 0.5 mM glycine concentrations. (D) Reversibility of alkaloid inhibition. Chelerythrine and sanguinarine were preincubated with cells for 3 min at 10 mM and 5 mM concentrations. Then alkaloids were washed out for 0, 10, 30, 60 min as described in Section 2 and assayed 1 min for glycine uptake at 23 8C (in C control represents 1.104  0.045 nM of glycine/mg/ min; in D control represents 1.348  0.055 nM of glycine/mg/min). The quantification data show row means  SEM from two separate experiments performed in triplicates.

reason we performed several experiments, which exclude the possibility that the observed inhibition is caused by changes in some of the transport-associated processes. Sanguinarine inhibits Na+/K+ ATPase (Pitts and Meyerson, 1981) and it could modify the transmembrane sodium gradient, which is the major driving force of glycine uptake via sodium-dependent transporters. Even though interaction of the Na+/K+ ATPase with ouabain or sanguinarine results in immediate reduction in the electrogenic current produced by the ATPase, the initial change in intracellular sodium concentration will be small within a few minutes following application of inhibitors (Helms et al., 2006). In our experiments we observed a 20% inhibition of GlyT1 following the first 30 s of uptake (15 s uptake plus 15 s 2 wash, when 20 mM chlerythrine was applied without preincubation, results not shown). Even though the extracellular concentration of sodium is buffered by the large amount of sodium in the uptake buffer on the surface of the cells, the possibility exists that inhibition of the Na+/K+ ATPase by the alkaloids could affect the sodium in the unstirred layer of the cell surface. In low micromolar concentrations, however, both chelerythrine and sangunarine had only a minimal effect on the uptake of the closely related transporter GlyT2, which also uses the driving force of the sodium gradient to transport its substrate. Additionally, the presence of 1 mM ouabain during the 3 min preincubation period and subsequent 1 min uptake did not inhibit GlyT1 uptake (Fig. 1B). This indicates that the major inhibitory effect on GlyT1 uptake by chelerythrine and sanguinarine is not caused by inhibition of the Na+/K+ ATPase. Chelerythrine is a potent inhibitor of several protein kinases (Herbert et al., 1990). Of these, PKC is especially involved in the surface redistribution of neurotransmitter transporters (Sato et al., 1995; Holton et al., 2005; Morioka et al., 2008). While the differences in PKC inhibition for chelerythrine (Ki 0.66 mM) and

saguinarine (Ki 217 mM) are more than two orders of magnitude (Herbert et al., 1990; Wang et al., 1997), in our experiments both alkaloids had an almost identical inhibitory effect on GlyT1c, when used at the comparable concentration. Quantification of surface localized transporters in both control and chelerythrine exposed HEK293 cells expressing hGlyT1c did not show any significant change in transporter surface expression on the time scales used for the uptake assay. In addition, the down regulation on glycine transporters from the cellular surface and suppression of transport activity of membrane glycine transporters are not caused by the inhibition, but by the activation of PKC (Sato et al., 1995; Morioka et al., 2008). These results suggest that inhibition of GlyT1 is not caused by chelerythrine mediated inhibition of PKC and associated GlyT1 redistribution. Alkaloids have similar physical properties and it is therefore difficult to purify them to homogeneity. The commercially purchased substances used contained 95% chelerythrine and 98% sanguinarine. Since GlyT1 inhibition occurred in the micromolar range, the possibility exists that small amounts of impurities could account for the observed inhibition of GlyT1. The source of chelerythrine used in this work was Chelidonium majus, while sanguinarine originated from Macleaya cordata. Because of their different sources it is unlikely that the molecular identity and amount of the minor accompanying impurities in the commercial preparations will be the same. Both alkaloids when used at comparable concentration showed similar inhibition potency and 2-mercaptoethanol also prevented the alkaloid mediated inhibition of GlyT1. Interaction of benzophenatridine alkaloids with thiol containing substances is well described (Vespalec et al., 2003) and suggests that inhibition was not caused by minor contaminants. The effects of alkaloids on microtubules (Wolff and Knipling, 1993; Lopus and Panda, 2006) or membrane permeability (Nichols

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et al., 1978) which could potentially affect the uptake have previously been described. These effects are unlikely to be responsible for the inhibition of GlyT1, because no effect on the closely related GlyT2 transporter was observed in low micromolar concentrations. Increasing the amount of glycine in the presence of a constant amount of the GlyT1 inhibitor sarcosine decreased the inhibition of glycine accumulation. This effect is typically observed for competitive inhibitors (Mezler et al., 2008). Increasing the glycine concentration in the presence of a constant amount of chelerythrine or sanguinarine did not cause such an effect, suggesting that inhibition is not competitive. On the contrary, increased glycine enhanced the inhibitory potency of both compounds. This indicates that glycine might induce a conformation change in GlyT1 or in some other way facilitate binding of alkaloids. Taking together the above results and the Eadie Hofstee plot we suggest that these alkaloids act by noncompetitive inhibition. Chelerythrine and saguinarine have multiple targets and it is difficult to predict which of the physiological effects previously described would potentially account for the inhibition of glycine transporter GlyT1. In natural medicine, aqueous extracts from Chelidonium herba have been used for analgesic effects. The PAG region of the brain is known to be heavily involved with nociception. In the work of Shin et al. (2003), the authors studied the modulation of Chelidonium herba on glycine-activated and glutamate-activated ion currents in rat periaqueductal gray neurons. The Chelidonium extract inhibited the glycine-activated ion current and increased the glutamate-activated ion current. Recent experiments indicate that inhibition of glycine transporter GlyT1 significantly enhances the glutamate-activated ion current through the NMDA receptor, which uses glycine as a coagonist (Martina et al., 2004; Whitehead et al., 2004). The NMDA receptor is involved in pain transmission, which is supported by the fact that glutamate analgesic action is blocked by NMDA blocker MK801, when microinjected into the PAG area. Thus inhibition of GlyT1 by the sanguinarine and chelerythrine present in Chelidonium preparations could increase glycine in the vicinity of NMDA receptor and results in stimulation of glutamate induced currents as observed in Shin et al. (2003). On the other hand, it has also been reported that antinociceptive effects of GLYT1 blockers occur through potentiation of glycinergic inhibitory neurotransmission following GLYT1 blockade (Dohi et al., 2009; Tanabe et al., 2008). Thus, it is possible that the analgesic effects of Chelidonium alkaloids could be, in part, due to such a mechanism. There are additional reports indicating that in higher concentrations, water extracts from C. majus have stronger analgesic effects than aspirin (Yilmaz et al., 2007). Interestingly, our results show that the inhibitory effect of alkaloids significantly increases with concentration of glycine and time. Glycine in synapses can reach concentrations of tenths of millimoles. Thus in vivo, even lower doses of these substances might lead to small but sufficient GlyT1 inhibition in synapses, which operate under high local glycine concentrations. Acknowledgements This work was supported by Slovak Academy of Sciences grants VEGA 2/0052/10 and 2/0045/10. References Aburai, N., Yoshida, M., Ohnishi, M., Kimura, K., 2010. Sanguinarine as a potent and specific inhibitor of protein phosphatase 2C in vitro and induces apoptosis via phosphorylation of p38 in HL60 cells. Biosci. Biotechnol. Biochem. 74, 548–552.

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