189

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIVERSITY OF MASSACHUSETTS on 04/04/15 For personal use only.

ARTICLE Pyridoxal-5=-phosphate (MC-1), a vitamin B6 derivative, inhibits expressed P2X receptors Olivier Thériault, Hugo Poulin, George R. Thomas, Albert D. Friesen, Waleed A. Al-Shaqha, and Mohamed Chahine

Abstract: P2X receptors are cation-permeable ligand-gated ion channels that open in response to the binding of ATP. These receptors are present in many excitable cells, including neurons, striated muscle cells, epithelial cells, and leukocytes. They mediate fast excitatory neurotransmission in the central and peripheral nervous systems and are thought to be involved in neuropathic pain, inflammation, and cell damage following ischemia–reperfusion injuries. P2X receptors are thus a target for the development of new therapeutics to treat chronic pain and inflammation. In this study, we characterized the inhibition caused by pyridoxal-5=-phosphate, a natural metabolite of vitamin B6 (MC-1), of P2X2, P2X4, P2X7, and P2X2/3 receptors stably expressed in HEK293 cells using the patch-clamp technique in the whole-cell configuration. We also tested a new approach using VC6.1, a modified cameleon calcium-sensitive fluorescent protein, to characterize the inhibition of P2X2 and P2X2/3. MC-1 blocked these two P2X receptors, with an IC50 of 7 and 13 ␮mol/L, respectively. P2X2 exhibited the highest affinity for VC6.1, and the chimeric receptor P2X2/3, the lowest. The patch-clamp and imaging approaches gave similar results and indicated that VC6.1 may be useful for high throughput drug screening. Pyridoxal-5=-phosphate is an efficient P2X blocker and can be classified as a P2X antagonist. Key words: P2X receptors, purinergic receptors, patch-clamp, ATP, HEK293 cells, pyridoxal-5=-phosphate, calcium. Résumé : Les récepteurs P2X sont des canaux ioniques opérés par un ligand, perméables aux cations, qui s'ouvrent en réponse a` la liaison de l'ATP. Ces récepteurs sont présents chez plusieurs cellules excitables, notamment les neurones, les cellules de muscle strié, les cellules épithéliales et les leucocytes. Ils servent d'intermédiaires a` la transmission excitatrice rapide dans les systèmes nerveux central et périphérique et semblent impliqués dans la douleur neuropathique, l'inflammation et le dommage cellulaire qui suit une ischémie–reperfusion. Les récepteurs P2X constituent ainsi des cibles de développement de nouvelles thérapies visant le traitement de la douleur chronique et l'inflammation. Dans cette étude, nous avons caractérisé l'inhibition causée par le pyridoxal-5=-phosphate, un métabolite naturel de la vitamine B6 (MC-1), des récepteurs P2X2, P2X4, P2X7 et P2X2/3 exprimés de façon stable dans les cellules HEK293, grâce a` la technique de patch-clamp sur cellules entières. Nous avons aussi testé une nouvelle approche a` l'aide de VC6.1, une protéine fluorescente Caméléon modifiée sensible au calcium, afin de caractériser l'inhibition de P2X2 et P2X2/3. Le MC-1 bloquait ces deux récepteur P2X, avec des CI50 de 7 ␮mol/L et 13 ␮mol/L, respectivement. Le P2X2 présentait l'affinité la plus forte pour VC6.1, et le récepteur chimère P2X2/3, la plus faible. Les approches en patch-clamp et par imagerie produisaient des résultats similaires et indiquaient que VC6.1 pourrait être utile lors d'un criblage a` haut débit. Le pyridoxal-5=-phosphate est une bloqueur efficace de P2X et peut être classé parmi les antagonistes de P2X. [Traduit par la Rédaction] Mots-clés : récepteurs P2X, récepteurs purinergiques, patch-clamp, ATP, HEK293, pyridoxal-5’-phosphate, calcium.

Introduction P2 receptors are a family of receptors that respond to the binding of extracellular ATP, ADP, UTP, and UDP. The family is composed of 2 subfamilies that differ in their molecular structures and signaling pathways (Abbracchio and Burnstock 1994). Ionotropic P2X receptors are expressed mainly in the heart and vascular smooth muscle, as well as the central (CNS) and peripheral (PNS) nervous systems. There are 7 subtypes of P2X receptors (P2X1 through P2X7) (North 2002). P2Y receptors are expressed in vascular smooth muscle, endothelium, and the immune system (platelets and lymph nodes) (Abbracchio et al. 2006). P2Y receptors belong to the G protein coupled receptor (GPCR) family. The stim-

ulation of P2Y receptors leads to the activation of intracellular signal transduction pathways. P2X receptors are involved in different neurotransmission pathways in both the CNS and PNS in biological and pathological conditions (Burnstock 2007). They are also involved in neuroeffector transmission in some immune responses, bone formation, and sensory epithelia. P2X3 appears to be involved in the perception of a full bladder. P2X3 knockout mice lose their voiding reflex and have a distended bladder (Cockayne et al. 2000). P2X3 and P2X2/3 antagonists attenuate thermal hyperalgesia and mechanical allodynia following chronic nerve constriction injuries (Jarvis et al. 2002). Microglia are activated following peripheral nerve injuries and upregulate the expression of P2X4. This is thought to

Received 21 October 2013. Accepted 4 December 2013. O. Thériault, H. Poulin, and M. Chahine. Le Centre de recherche de l'institut universitaire en santé mentale de Québec, and Department of Medicine, Université Laval, 2601 chemin de la Canardière, Quebec City, QC G1J 2G3, Canada. G.R. Thomas. CanAm BioResearch Inc., 6–1200 Waverley Street, Winnipeg, MB R3T 0P4, Canada. A.D. Friesen. Medicure Inc., 4-1200 Waverley Street, Winnipeg, MB R3T 0P4, Canada. W.A. Al-Shaqha. College of Medicine, Al-Imam Muhammad Ibn Saud Islamic University, Riyadh, Kingdom of Saudi Arabia. Corresponding author: Mohamed Chahine (e-mail: [email protected]). Can. J. Physiol. Pharmacol. 92: 189–196 (2014) dx.doi.org/10.1139/cjpp-2013-0404

Published at www.nrcresearchpress.com/cjpp on 6 December 2013.

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIVERSITY OF MASSACHUSETTS on 04/04/15 For personal use only.

190

lead to pain hypersensitivity via microglia–neuronal signaling. The disruption of the P2X7 gene in knockout mice causes a decrease in allodynia following nerve injuries. These results indicate that P2X receptors could be targeted for the development of therapies to treat hyperalgesia and allodynia. The physiology of P2X receptors differs depending on the pathology. For example, P2X1 levels are higher in the case of an unstable bladder (O'Reilly et al. 2001), as well as congestive heart failure (Hou et al. 1999), while P2X3 and P2X5 are lower in human detrusor in adults with urge incontinence (Moore et al. 2001). These isoforms are thus attractive targets for novel treatments for people afflicted with these diseases (Burnstock 2006). When P2X receptors are expressed in a heterologous expression system they can assemble as either homotrimers or heterotrimers. Heterotrimeric receptors are interesting in that they present characteristics that are not only intermediate between the 2 isoforms but also display new biophysical and pharmacological characteristics (North 2002). P2X1 and P2X3 exhibit rapid desensitization (within a few dozen milliseconds), P2X2, P2X4, and P2X5 exhibit slow desensitization, and P2X7 exhibits very slow desensitization and stronger activation by Bz-ATP. In general, P2X receptors are blocked by suramin and PPADS (pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid; tetrasodium salt hydrate) (North and Surprenant 2000). In this study, we tested MC-1 (pyridoxal-5=-phosphate monohydrate), a P2X receptor antagonist, on HEK293 cells stably expressing P2X receptors, and compared the results with those obtained with PPADS, a non-selective P2X receptor antagonist. We also compared the results obtained with P2X2 and P2X2/3 using the patch-clamp technique with those obtained with a novel approach using a calcium sensitive fluorescent protein (VC6.1). Our results revealed that VC6.1 can be used for high-throughput drug screening.

Materials and methods Cell cultures and cell lines All HEK293 cell lines used in this study were grown in Dulbecco's minimal essential medium (DMEM/F12) (Gibco BRL Life Technologies, Burlington, Ontario, Canada), supplemented with fetal bovine serum (10%), penicillin (100 U/mL), and streptomycin (10 mg/mL) (Gibco). The cells were incubated at 37 °C in a 5% CO2 humidified atmosphere. VC6.1 was a gift from Philip G. Haydon (University of Pennsylvania, Philadelphia, USA). VC6.1/P2X2 and VC6.1/P2X2/3 double cell lines were generated from P2X2 or P2X2/3 cell lines by stable transfection with VC6.1. Five micrograms of pIREShyg3/VC6.1 vector were transfected using the calcium phosphate method. The following day, the cells were split and serially diluted. The next day, 150 ␮g/mL of hygromycin was added to the medium to select transfected cells. After 3 weeks, individual clones were randomly picked and expanded in the same medium; except that the concentration of hygromycin was lowered to 75 ␮g/mL. The clones were analyzed using a fluorescence microplate reader and cells that generated a FRET signal in the presence of 100 ␮mol/L of ATP were selected for further study. Patch-clamp experiments and P2X current recordings Macroscopic inward currents generated by applying ATP to HEK293 cells were recorded using the whole-cell configuration of the patch-clamp technique. The measurements were carried out at room temperature (≈22 °C). Fire-polished, low-resistance patch electrodes (2–5 M⍀) were pulled from Corning 8161 glass (Dow Corning, Midland, Michigan, USA) using a model P-97 Flaming/ Brown micropipette puller (Sutter Instrument Company, Novato, California, USA). Patch-clamp experiments were performed using an Axopatch 200B amplifier with a CV 203BU headstage (Molecular Devices, Sunnyvale, Calif.). Voltage command pulses were gen-

Can. J. Physiol. Pharmacol. Vol. 92, 2014

Fig. 1. Chemical structure of pyridoxal-5=-phosphate, a natural metabolite of vitamin B6 (MC-1).

erated using a personal computer equipped with an AD converter (Digidata 1322A; Molecular Devices) and pCLAMP software version 8.0 (Molecular Devices). When appropriate, linear leakage current artifacts were removed using online leak subtraction. P2X currents were filtered at 5 kHz and were digitized at 20 kHz. The digitized currents were stored on a computer for later offline analysis. Typically, the cells were allowed to stabilize for 10 min after the whole-cell configuration was established before recordings. The cells were allowed to recover for 3 (P2X7 and P2X2/3) to 5 min (P2X2 and P2X4) between each application of ATP. The recovery times were determined based on the kinetics of recovery (slow for P2X2 and P2X4; faster for P2X7 and P2X2/3). The dose–response curves were constructed by normalizing the current induced by selected ATP concentrations to the concentration that was reported in the literature to induce a maximum to near maximum effect (Khakh et al. 2001). The concentration of ATP used for normalization was 150 ␮mol/L ATP for P2X2, 100 ␮mol/L for P2X2/3 and P2X4, and 2 mmol/L ATP for P2X7. Data were then fitted to a Hill equation to calculate the pEC50. The Hill curve might therefore not be from 0 to 1, as the concentration used might not induce the maximal value. Dose–response curves to MC-1 and PPADS were constructed from several concentrations of drugs. We applied a test pulse of ATP 10 min after the G⍀ seal was obtained. Then the cells were superfused with the selected concentration of drug for 5–10 min. The current evoked by the agonist in the presence of the drug were recorded. The dose–response curves were plotted against the normalized current from the test pulse. The agonist used for the dose–response curve was 100 ␮mol/L ATP for P2X2, P2X2/3, and P2X4. For P2X7, we used 2 mmol/L ATP for the dose–response curve to MC-1 and 100 ␮mol/L Bz-ATP for the dose–response curve to PPADS. FRET-based measurements of P2X channel function Stably transfected VC6.1/P2X cells were trypsinized, washed, resuspended in the assay solution (130 mmol/L NaCl, 2 mmol/L KCl, 10 mmol/L CaCl2, 1 mmol/L MgCl2, 10 mmol/L glucose, and 10 mmol/L HEPES, at pH 7.4) and seeded at 100 000 cells per well in 96 well plates (Corning) just prior the assay. The assays were conducted using a fluorescence imaging plate reader (FusionTM; Packard), where the cells were excited at 425 nm, and the emissions were recorded at 485 (FCFP) and 535 nm (FVenus) for 0.5 s. For ATP dose responses, a first reading (Fi) was carried out without ATP and then the cells were incubated for 5 min with various concentrations of ATP before the second reading (Ff). For drug-block experiments, a first reading (Fi) was carried without ATP and then the cells were incubated for 30 min with increasing amounts of MC-1 or PPADS before adding ATP (100 ␮mol/L) and performed the second reading (Ff). The change in the fluorescence resonance energy transfer (FRET) ratio (Rf/Ri) was calculated using the following equation: Rf/Ri = (FVenus/FCFP)f/(FVenus/FCFP)i, where (F)i and (F)f represent the Published by NRC Research Press

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIVERSITY OF MASSACHUSETTS on 04/04/15 For personal use only.

Thériault et al.

191

Fig. 2. MC-1 (pyridoxal-5=-phosphate) is P2X2 receptor antagonist. (A) Representative current traces of maximal inward currents evoked by 100 ␮mol/L ATP in cells expressing P2X2 (left) and by 100 ␮mol/L ATP after a 10 min incubation with 10 ␮mol/L MC-1 (right). ATP was applied for 2 s. Currents were generated using a conventional whole-cell configuration (see the Materials and methods). Cells were held at a holding potential of –60 mV. (B) Dose–response curve of ATP on P2X2 receptors. The pEC50 was 4.53 ± 0.02 mol/L. Each data point corresponds to the mean ± SEM of 4–10 experiments. Currents were normalized to 150 ␮mol/L of ATP, which is near the maximal response. (C) Dose–response curve for MC-1 on P2X2. A first current was evoked with 100 ␮mol/L ATP 10 min after the seal was obtained, and cells were then incubated in the presence of MC-1 for 10 min and were tested by applying 100 ␮mol/L ATP. The graph shows the normalized current of the evoked response in the presence of the MC-1 over the response in the absence of drugs (IDrug/IATP). Each data point corresponds to the mean ± SEM of 4–7 experiments. (D) Dose–response curve for PPADS on P2X2. A first current was evoked with 100 ␮mol/L ATP 10 min after the seal was obtained and cells were then incubated in the presence of PPADS for 10 min and were tested by applying 100 ␮mol/L ATP. The graph shows the normalized current of the evoked response in the presence of the PPADS over the response in the absence of drugs (IDrug/IATP). Each data point corresponds to the mean ± SEM of 4–5 experiments. In B, the response to ATP was normalized to the response evoked by 150 ␮mol/L ATP. In C and D, the responses were normalized to the maximal response evoked by 100 ␮mol/L ATP. Values for pIC50 or pEC50, Hill coefficient (n), and R2 are shown.

fluorescence emissions at the specified wavelength before and after adding the ATP, respectively. The ATP and the drug solutions were applied manually into the wells using multichannel pipets. Solutions and reagents For the conventional whole-cell configuration, the extracellular solution was composed of the following (in mmol/L): 145 NaCl, 2 KCl, 2 CaCl2, 1 MgCl2, and 10 HEPES, and was titrated to pH 7.4 using 1 mol/L CsOH. The intracellular pipette solution was composed of the following (in mmol/L): 35 NaCl, 105 CsF, 10 EGTA, and 10 HEPES, and was titrated to pH 7.4 using 1 mol/L CsOH. The perforated patch method was used with P2X4 and P2X7, since these receptors exhibit a high current rundown. For these experiments, the cells were perforated by adding 80–120 ␮g/mL of amphotericin-B to the patch pipette solution (Fountain and North 2006). A fresh 25 mg/mL stock solution of amphotericin-B in DMSO was prepared each week and was stored at 4 °C. The intracellular pipette solution was composed of the following (in mmol/L): 145 NaCl, 10 HEPES, and 10 EGTA, and was titrated to pH 7.3 using 1 mol/L NaOH. An intracellular solution containing amphotericin-B was prepared every 2 h from stock solution (Horn and Korn 1992). The cells were typically perforated 10 min after the formation of the G⍀ seal. Typically the series resistance exhibits little or no variation. We did not observe any increase in series resistance, in fact, sometimes we saw a slight decrease at the end of our experiments.

ATP was applied using a perfusion system (ValveLink 8.2 controller for solution delivery, Automate Scientific, Berkeley, Calif.), with a few improvements for fast agonist and drug delivery. Three needles pointing at the cell were fixed together to avoid crosscontamination and allow fast perfusion to ensure rapid activation of the agonist-induced current. ATP (adenosine 5=-triphosphate, magnesium salt), Bz-ATP (2=(3=)O-(4-benzoylbenzoyl), adenosine 5=-triphosphate triethylammonium salt), PPADS (pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt hydrate), and all of the other chemicals were from Sigma (Saint Louis, Missouri, USA) and were handled as recommended by the company. MC-1 (pyridoxal-5=-phosphate monohydrate) (Fig. 1) was provided by Medicure Inc. (Winnipeg, Manitoba, Canada) as a yellow powder. Stock solutions in extracellular solution were made weekly and were kept in the dark at 4 °C. Data analysis Data were analyzed using a combination of pCLAMP software, Microsoft Excel, SigmaPlot 8.0 (SPSS Inc., Chicago, Illinois, USA), and GraphPad Prism version 6.0 (San Diego, Calif.). Dose–response data were analyzed using a logistic Hill equation in GraphPad Prism. Maximum and minimum were fixed at 1 and 0 respectively for antagonist dose–response and minimum fixed at 0 for agonist dose–response. To calculate the pEC50 and pIC50, the following equaa , where “n” is the Hill coefficient, “x0” tion was used: y ⫽ 1 ⫹ 10共x0⫺x兲n Published by NRC Research Press

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIVERSITY OF MASSACHUSETTS on 04/04/15 For personal use only.

192

Can. J. Physiol. Pharmacol. Vol. 92, 2014

Fig. 3. MC-1 (pyridoxal-5=-phosphate) is a P2X2/3 receptor antagonist. (A) Representative current traces of maximal inward currents evoked by 100 ␮mol/L ATP in cells expressing P2X2/3 (left) and by 100 ␮mol/L ATP following a 5 min incubation with 250 ␮mol/L MC-1 (right). ATP was applied for 2 s. Currents were generated using a conventional whole-cell configuration. Cells were held at a holding potential of –60 mV. (B) Dose–response curve for ATP on P2X2/3. The pEC50 was 4.70 ± 0.07 mol/L. Each data point corresponds to the mean ± SEM of 3–7 experiments. Currents were normalized to 100 ␮mol/L of ATP, which is near the maximal response. (C) Dose–response curve for MC-1 on P2X2/3. A first current was evoked with 100 ␮mol/L ATP 10 min after the seal was obtained and cells were then incubated in the presence of MC-1 for 5 min and were tested by applying 100 ␮mol/L ATP. The graph shows the normalized current of the evoked response in the presence of the MC-1 over the response in the absence of drugs (IDrug/IATP). Each data point corresponds to the mean ± SEM of 4–6 experiments. (D) Dose–response curve for PPADS on P2X2/3. A first current was evoked with 100 ␮mol/L ATP 10 min after the seal was obtained and cells were then incubated in the presence of PPADS for 5 min and were tested by applying 100 ␮mol/L ATP. The graph shows the normalized current of the evoked response in the presence of the PPADS over the response in the absence of drugs (IDrug/IATP). Each data point corresponds to the mean ± SEM of 3–5 experiments. In B, C, and D, the responses were normalized to the maximal response evoked by 100 ␮mol/L ATP. Values for pIC50 or pEC50, Hill coefficient (n), and R2 are shown.

is the log(IC50), “x” is the log concentration, and “a” is the theoretical maximum. Statistical differences for IC50 and Hill coefficient were assessed using GraphPad Prism. Values were considered different for a p-value < 0.05. Fluorescence experiments with the cameleon calcium-sensitive fluorescent protein data were fitted a , where “y0” with a 4 parameter Hill equation: y ⫽ y0 ⫹ 1 ⫹ 10共x0⫺x兲n is the minimum of the curve, “a” is max.(y) – min.(y), “n” is the Hill coefficient, and “c” is the pIC50. Values are the mean ± SEM.

Results MC-1 blocks P2X2 receptors Figure 2A (left panel) shows an example of the current evoked by 100 ␮mol/L ATP on P2X2-expressing cells. Figure 2B shows the dose–response of ATP-evoked P2X2 current activation. A 14% current was evoked with 10 ␮mol/L ATP, while a near maximal current was evoked with 150 ␮mol/L ATP. Fitting the data points to a Hill function revealed that the pEC50 is 4.53 ± 0.02 mol/L (EC50 = 30 ␮mol/L). The pEC50 values are shown in the figures. The effect of MC-1 was first assessed on P2X2 currents evoked by 100 ␮mol/L ATP. MC-1 potently blocked P2X2 receptors, with a pIC50 of 5.14 ± 0.03 mol/L (IC50 = 7.3 ␮mol/L) (Fig. 2C. The effect of PPADS was then assessed on P2X2 currents evoked by 100 ␮mol/L ATP using similar conditions. PPADS had a pIC50 of 6.22 ± 0.06 mol/L (IC50 = 0.6 ␮mol/L) (Fig. 2D). Similar results have been reported by Valera

et al. (1994). While MC-1 was shown to be a P2X2 receptor antagonist, it was less potent than PPADS. MC-1 blocks P2X2/3 receptors To determine whether MC-1 blocks P2X2/3 receptors, we studied the effect of MC-1 on ATP-evoked P2X2/3 currents using the conventional whole-cell configuration of the patch-clamp technique, as described in the Materials and methods. Figure 3A (left panel) shows an example of a P2X2/3 current evoked by 100 ␮mol/L ATP. A dose–response curve was generated by applying different ATP concentrations, with 100 ␮mol/L evoking the maximum current. The pEC50 was 4.70 ± 0.07 mol/L (EC50 = 20 ␮mol/L) (Fig. 3A, right panel, and Fig. 3B). The effect of MC-1 on P2X2/3 currents evoked by 100 ␮mol/L ATP was assessed. MC-1 blocked P2X2 receptors, with a pIC50 4.06 ± 0.07 mol/L (IC50 = 86 ␮mol/L) (Fig. 3C). For comparison purposes, the effect of PPADS on P2X2/3 currents evoked by 100 ␮mol/L ATP was also assessed using similar conditions (Fig. 3D). Comparable results have been reported previously. For a review, see North and Surprenant (2000). While MC-1 blocked P2X2 receptors, its IC50 was higher than that of PPADS, indicating that MC-1 is less potent than PPADS. MC-1 blocks P2X4 receptors To determine the effect of MC-1 on P2X4 receptors, ATP-evoked P2X4 currents were recorded using the perforated patch technique (see the Materials and methods). An example of a current trace Published by NRC Research Press

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIVERSITY OF MASSACHUSETTS on 04/04/15 For personal use only.

Thériault et al.

193

Fig. 4. MC-1 (pyridoxal-5=-phosphate) is a P2X4 receptor antagonist. (A) Representative current traces of maximal inward currents evoked by 100 ␮mol/L ATP in cells expressing P2X4 (left) and by 100 ␮mol/L ATP after a 5 min incubation with 50 ␮mol/L MC-1 (right). ATP was applied for 2 s. Currents were generated using the amphotericin-B perforated patch configuration (see the Materials and methods). Cells were held at a holding potential of –60 mV. (B) Dose–response curve of ATP on P2X4. The pEC50 was 5.37 ± 0.05 M. Each data point corresponds to the mean ± SEM of 3–5 experiments. (C) Dose–response curve of MC-1 on P2X2/3. A first current was evoked with 100 ␮mol/L ATP 10 min after the seal was obtained and cells were then incubated in the presence of MC-1 for 5 min and were tested by applying 100 ␮mol/L ATP. The graph shows the normalized current of the evoked response in the presence of the MC-1 over the response in the absence of drugs (IDrug/IATP). Each data point corresponds to the mean ± SEM of 3–5 experiments. (D) Dose–response curve for PPADS on P2X4. A first current was evoked with 100 ␮mol/L ATP 10 min after the seal was obtained and cells were then incubated in the presence of PPADS for 5 min and were tested by applying 100 ␮mol/L ATP. The graph shows the normalized current of the evoked response in the presence of the PPADS over the response in the absence of drugs (IDrug/IATP). Each data point corresponds to the mean ± SEM of 3–4 experiments. In panels B, C, and D, the responses were normalized to the maximal response evoked by 100 ␮mol/L ATP. Values for pIC50 or pEC50, Hill coefficient (n), and R2 are shown.

is shown in Fig. 4A (left panel). ATP evoked a dose-dependent increase in current, with a pEC50 of 5.37 ± 0.05 mol/L (EC50 = 4.4 ␮mol/L) (Fig. 4B). MC-1 caused a dose-dependent block of P2X4, with a pIC50 of 4.41 ± 0.04 mol/L (IC50 = of 44 ␮mol/L) (Fig. 4A, right panel, and Fig. 4C). For comparison purposes, the effect of PPADS was tested on P2X4 receptors. The pIC50 for PPADS was 4.78 ± 0.04 mol/L (IC50 = 16 ␮mol/L) (Fig. 4D), which is similar to the 9.6 ␮mol/L reported by Jones et al. (2000). MC-1 blocks P2X7 receptors The effect of MC-1 on P2X7 receptors was studied using the perforated-patch technique. An example of an ATP-evoked P2X7 current trace is shown in Fig. 5A (left panel). ATP-evoked currents were concentration-dependent, with a pEC50 of 3.13 ± 0.8 mol/L (EC50 = 750 ␮mol/L) (Fig. 5B). MC-1 caused a dose-dependent block of the P2X7 receptor, with a pIC50 = 5.01 ± 0.05 mol/L (IC50 = 10 ␮mol/L) (Fig. 5A, right panel, and Fig. 5C). The pIC50 of PPADS on 100 ␮mol/L Bz-ATP-evoked P2X7 currents was 4.97 ± 0.08 (IC50 = 11 ␮mol/L) (Fig. 5D), which was comparable with the IC50 value obtained with MC-1. FRET-based measurements of P2X channel function Since P2X channels conduct calcium ions (Egan and Khakh 2004), we investigated the possibility that an increase in intracellular calcium levels is a reflection of the opening of P2X channels. We measured changes in intracellular calcium levels using VC6.1, a calcium-sensitive fluorescent protein. VC6.1 is a modified cameleon-2 protein that produces a strong FRET signal when it binds to cal-

cium. The change in the fluorescence resonance energy transfer (FRET) ratio (Rf/Ri) was calculated using the following formula: Rf/Ri = (FVenus/FCFP)f/(FVenus/FCFP)i, where (F)i and (F)f are the fluorescence emissions at the specified wavelength before and after the addition of ATP, respectively. HEK293 cells endogenously express P2X and other ion channels and transporters. We first verified whether an increase in the concentration of ATP affected the FRET signal of cells stably transfected with VC6.1 alone (Fig. 6a), and did not detect any change in fluorescence. To evaluate the sensitivity of the method, we measured the changes in the FRET signals of stable cell lines expressing VC6.1 and P2X2 or VC6.1 and P2X2/3 (VC6.1/P2X2 and VC6.1/ P2X2/3) associated with increasing concentrations of ATP. We then calculated the dose–responses of P2X2 and P2X2/3 to ATP from these results. The pEC50 was 4.80 ± 0.03 mol/L for P2X2 (EC50 = 16 ␮mol/L) and 4.92 ± 0.03 mol/L for P2X2/3 (EC50 = 12 ␮mol/L) (Figs. 6A and 7A, respectively). We used the same cell lines (VC6.1/ P2X2 and VC6.1/P2X2/3) to calculate the inhibition efficiencies of MC-1 and PPADS. For P2X2, the pIC50 was 5.13 ± 0.02 mol/L (IC50 = 7.4 ␮mol/L) for MC-1 (Fig. 6B) and 5.09 ± 0.02 mol/L (IC50 = 8.1 ␮mol/L) for PPADS (Fig. 6C). For P2X2/3, the pIC50 was 4.93 ± 0.03 (IC50 = 13 ␮mol/L) for MC-1 (Fig. 7B) and 5.72 ± 0.02 (IC50 = 1.9 ␮mol/L) for PPADS (Fig. 7C). These results were similar to those obtained from the patch-clamp experiments.

Discussion and conclusions We studied the P2X2, P2X2/3, P2X4, and P2X7 receptors. While MC-1 blocked all 4 P2X receptors, it exhibited a higher affinity for Published by NRC Research Press

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIVERSITY OF MASSACHUSETTS on 04/04/15 For personal use only.

194

Can. J. Physiol. Pharmacol. Vol. 92, 2014

Fig. 5. MC-1 (pyridoxal-5=-phosphate) is a P2X7 receptor antagonist. (A) Representative current traces of the maximal inward currents evoked by 2 mmol/L ATP in cells expressing P2X4 (left) and by 100 ␮mol/L ATP after a 5 min incubation with 50 ␮mol/L MC-1 (right). ATP was applied for 2 s. Currents were generated using the amphotericin-B perforated patch configuration (see the Materials and methods). Cells were held at a holding potential of –60 mV. (B) Dose–response curve for ATP on P2X7 receptors. The pEC50 was 3.13 ± 0.08 mol/L. Each data point corresponds to the mean ± SEM of 3–4 experiments. (C) Dose–response curve of MC-1 on P2X7. A first current was evoked with 100 ␮mol/L ATP 10 min after the seal was obtained and cells were then incubated in the presence of MC-1 for 5 min and were tested by applying 2 mmol/L ATP. The graph shows the normalized current of the evoked response in the presence of MC-1 over the response in the absence of drugs (IDrug/IATP). Each data point corresponds to the mean ± SEM of 4–8 experiments. (D) Dose–response curve for PPADS on P2X7. A first current was evoked with 100 ␮mol/L ATP 10 min after the seal was obtained and cells were then incubated in the presence of PPADS for 5 min and were tested by applying 100 ␮mol/L Bz-ATP. The graph shows the normalized current of the evoked response in the presence of the PPADS over the response in the absence of drugs (IDrug/IATP). Each data point corresponds to the mean ± SEM of 3–4 experiments. In panels B and C, the responses were normalized to the maximal response evoked by 2 mmol/L ATP. In panel D, the responses were normalized to 100 ␮mol/L Bz-ATP. Values for pIC50 or pEC50, Hill coefficient (n), and R2 are shown.

P2X2 and P2X7. The effect of PPADS was comparable with that reported in the literature. For a review, see North and Surprenant (2000). The IC50 values of P2X blocked with MC-1 were equal to (P2X7) or higher (P2X2, P2X2/3, and P2X4) than with PPADS. Clearly, the Hill coefficients for the dose–response curves of MC-1 and PPADS are different (p < 0.05) for P2X2, P2X2/3, and P2X7, which suggests that the blocking mechanism for these 2 drugs on these receptors may differ. The Hill coefficients for P2X2 and P2X7 (MC-1) and for P2X2/3, P2X4, and P2X7 (PPADS) are significantly different from 1; a plausible explanation may lie in the fact that these 2 drugs act allosterically on these receptors. Further studies are required to elucidate the specific allosteric sites. Two studies on the effect of MC-1 on P2 receptors have been performed on native P2X receptors. The first, by Trezise et al. (1994) on the rat vagus nerve and vas deferens, revealed that MC-1 may be a potent blocker of P2X receptors. MC-1 has the ability to block ␣-␤-meATP-evoked currents, suggesting that it may block P2X1 and P2X3. P2X1 and P2X3 are also expressed in the heart and do not seem to be involved in excitation–contraction coupling. The second study, by Wang et al. (1999), revealed that MC-1 can block ATP-induced calcium intake in rat cardiomyocytes. This is an important finding given that calcium intake is thought to induce cell damage to the ischemic heart. These 2 studies also indicated that MC-1 is a general P2X antagonist and could be used in treatments that target P2X receptors. A clinical study tested the hypothesis that MC-1 reduces cardiovascular morbidity and mortality in patients undergoing a coro-

nary artery bypass graft by blocking ATP-induced calcium entry in the myocardium (Tardif et al. 2007). The primary end points of the study were a decreased incidence of cardiovascular death, nonfatal myocardial infarction, and non-fatal cerebral infarction up to day 30 post-operation. The study revealed that MC-1 did not significantly reduce the primary end points. However MC-1 did significantly reduce the concentration of creatinine kinasemyocardial band (CK-MB) in the blood. CK-MB is an isoenzyme that is almost exclusively expressed by the myocardium and is used to diagnose myocardial infarct and estimate cell damage. Tardif et al. (2007) suggested that a larger cohort of patients was required to evaluate the cardioprotective effects of MC-1. Based on the results of this study, MC-1 may reduce the influx of calcium into cells and thus contribute to reducing cell damage. Antagonists derived from pyridoxal-5=-phosphate with a better selectivity, such as PPADS and PPNDS, have been identified (Lambrecht et al. 2000). These 2 compounds are valuable tools for in-vitro experiments and are more effective and selective than MC-1. However they have not been tested in humans. MC-1, on the other hand, has successfully undergone many safety tests (Hirai et al. 1998; Lonn et al. 2006). Since MC-1 is a P2X antagonist, it should be studied under pathological conditions with a view to treating patients. It should also be tested for its potential to treat chronic inflammatory diseases and neuropathic pain by blocking P2X7. Blocking P2X4 and P2X7 has been shown to reduce neuropathic pain and allodynia caused by nerve injuries, respectively (Tsuda et al. 2003; Honore et al. 2006). Since MC-1 blocks both of Published by NRC Research Press

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIVERSITY OF MASSACHUSETTS on 04/04/15 For personal use only.

Thériault et al.

195

Fig. 6. P2X2 fluorescence microplate assays. (A) VC6.1/P2X2 (filled circle) or VC6.1 (open triangle) cells were incubated with various concentrations of ATP, and changes in the FRET signal were monitored (FVenus/FCFP). (B and C) VC6.1/P2X2 cells were incubated with increasing concentrations of MC-1 (pyridoxal-5=-phosphate) (B) or PPADS (C). A first reading was taken (FVenus/FCFP)i and is referred to as Ri. The channels were activated by adding 100 ␮mol/L ATP. A second reading was taken (FVenus/FCFP)f and is referred to as Rf. The results are expressed as the Rf/Ri ratio, which represents the change in the FRET signal as a function of drug concentration. Error bars represent the standard deviation of experiments performed in triplicate. The ratios were directly translated into dose–response curves using a Hill 4-parameter fitting routine.

Fig. 7. P2X2/3 fluorescence microplate assays. (A) VC6.1/P2X2/3 cells were incubated with various concentrations of ATP, and changes in the FRET signal were monitored (FVenus/FCFP). (B and C) VC6.1/P2X2 cells were incubated with increasing concentrations of MC-1 (pyridoxal-5=-phosphate) (B) or PPADS (C). A first reading was taken (FVenus/FCFP)i and is referred to as Ri. The channels were activated by 100 ␮mol/L ATP. A second reading was taken (FVenus/FCFP)f and is referred to as Rf. The results are expressed as the Rf/Ri ratio, which represents the change in the FRET signal as a function of drug concentration. Error bars represent the standard deviation of experiments performed in triplicate. The ratios were directly translated into dose–response curves using a Hill 4-parameter fitting routine.

these receptors and is safe for use in humans, we believe that valuable information could be obtained from testing it on patients with neuropathic pain and allodynia. Various calcium-reactive fluorescent proteins have been engineered over the past few years. Since P2X channels are permeable to calcium ions, we tested the hypothesis that this type of protein might be a useful and powerful tool for high-throughput drug screening of P2X channels. VC6.1 was engineered by Evanko and Haydon and produces a strong FRET signal when it binds to calcium (Evanko and Haydon 2005). We showed that VC6.1 can be used as an indicator of the function of P2X2 and P2X2/3 channels. Since the FRET results were similar to those obtained using the patch-clamp technique, and

since our method does not require a modified P2X protein, we believe that it could be useful for high-throughput drug screening. However, we observed a significant difference in the block of MC-1 on P2X2/3. This difference observed in both techniques may rely on the differences in the stimulation protocol and recording. The recording of the fluorescence in the FRET method rely on the accumulation of calcium over 5 min compare with the patchclamp technique that is based on the maximum amplitude in the presence of the agonist. Furthermore, P2X2/3 is known to desensitize in the presence of the agonist that might influence the results after few minutes. Richler reported that genetically engineered P2X receptors can also be detected by calcium imaging, a powerful in-vivo tool (Richler et al. 2008). We also showed that the affinity Published by NRC Research Press

196

of MC-1 for P2X receptors was similar to that of PPADS, a nonselective P2X receptor antagonist. In conclusion, MC-1 blocked several P2X receptors and can thus be classified as a P2X receptor antagonist.

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIVERSITY OF MASSACHUSETTS on 04/04/15 For personal use only.

Acknowledgements We thank Laimonis Gailis for his critical reading of the manuscript. During these studies M. Chahine was an Edwards Senior Investigator (Joseph C. Edwards Foundation).

References Abbracchio, M.P., and Burnstock, G. 1994. Purinoceptors: are there families of P2X and P2Y purinoceptors? Pharmacol. Ther. 64(3): 445–475. doi:10.1016/ 0163-7258(94)00048-4. PMID:7724657. Abbracchio, M.P., Burnstock, G., Boeynaems, J.M., Barnard, E.A., Boyer, J.L., Kennedy, C., et al. 2006. International Union of Pharmacology LVIII: update on the P2Y G protein-coupled nucleotide receptors: from molecular mechanisms and pathophysiology to therapy. Pharmacol. Rev. 58(3): 281–341. doi: 10.1124/pr.58.3.3. PMID:16968944. Burnstock, G. 2006. Pathophysiology and therapeutic potential of purinergic signaling. Pharmacol. Rev. 58(1): 58–86. doi:10.1124/pr.58.1.5. PMID:16507883. Burnstock, G. 2007. Physiology and pathophysiology of purinergic neurotransmission. Physiol. Rev. 87(2): 659–797. doi:10.1152/physrev.00043.2006. PMID: 17429044. Cockayne, D.A., Hamilton, S.G., Zhu, Q.M., Dunn, P.M., Zhong, Y., Novakovic, S., et al. 2000. Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice. Nature, 407(6807): 1011–1015. doi:10.1038/35039519. PMID:11069181. Egan, T.M., and Khakh, B.S. 2004. Contribution of calcium ions to P2X channel responses. J. Neurosci. 24(13): 3413–3420. doi:10.1523/JNEUROSCI.5429-03. 2004. PMID:15056721. Evanko, D.S., and Haydon, P.G. 2005. Elimination of environmental sensitivity in a cameleon FRET-based calcium sensor via replacement of the acceptor with Venus. Cell Calcium, 37(4): 341–348. doi:10.1016/j.ceca.2004.04.008. PMID: 15755495. Fountain, S.J., and North, R.A. 2006. A C-terminal lysine that controls human P2X4 receptor desensitization. J. Biol. Chem. 281(22): 15044–15049. doi:10. 1074/jbc.M600442200. PMID:16533808. Hirai, K., Seki, T., and Takuma, Y. 1998. Cerebrospinal fluid somatostatin in West syndrome: changes in response to combined treatment with high-dose pyridoxal phosphate and low-dose corticotropin. Neuropeptides, 32(6): 581–586. doi:10.1016/S0143-4179(98)90089-0. PMID:9920458. Honore, P., Donnelly-Roberts, D., Namovic, M.T., Hsieh, G., Zhu, C.Z., Mikusa, J.P., et al. 2006. A-740003 [N-(1-{[(cyanoimino)(5-quinolinylamino) methyl]amino}-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide], a novel and selective P2X7 receptor antagonist, dose-dependently reduces neuropathic pain in the rat. J. Pharmacol. Exp. Ther. 319(3): 1376–1385. doi:10. 1124/jpet.106.111559. PMID:16982702. Horn, R., and Korn, S.J. 1992. Prevention of rundown in electrophysiological recording. Methods Enzymol. 207: 149–155. doi:10.1016/0076-6879(92)07010-L. PMID:1382181. Hou, M., Malmsjo, M., Moller, S., Pantev, E., Bergdahl, A., Zhao, X.H., et al. 1999. Increase in cardiac P2X1-and P2Y2-receptor mRNA levels in congestive heart

Can. J. Physiol. Pharmacol. Vol. 92, 2014

failure. Life Sci. 65(11): 1195–1206. doi:10.1016/S0024-3205(99)00353-7. PMID: 10503935. Jarvis, M.F., Burgard, E.C., McGaraughty, S., Honore, P., Lynch, K., Brennan, T.J., et al. 2002. A-317491, a novel potent and selective non-nucleotide antagonist of P2X3 and P2X2/3 receptors, reduces chronic inflammatory and neuropathic pain in the rat. Proc. Natl. Acad. Sci. U.S.A. 99(26): 17179–17184. doi:10. 1073/pnas.252537299. PMID:12482951. Jones, C.A., Chessell, I.P., Simon, J., Barnard, E.A., Miller, K.J., Michel, A.D., et al. 2000. Functional characterization of the P2X(4) receptor orthologues. Br. J. Pharmacol. 129(2): 388–394. doi:10.1038/sj.bjp.0703059. PMID:10694247. Khakh, B.S., Burnstock, G., Kennedy, C., King, B.F., North, R.A., Séguéla, P., et al. 2001. International union of pharmacology. XXIV. Current status of the nomenclature and properties of P2X receptors and their subunits. Pharmacol. Rev. 53(1): 107–118. PMID:11171941. Lambrecht, G., Rettinger, J., Baumert, H.G., Czeche, S., Damer, S., Ganso, M., et al. 2000. The novel pyridoxal-5=-phosphate derivative PPNDS potently antagonizes activation of P2X(1) receptors. Eur. J. Pharmacol. 387(3): R19–R21. doi:10.1016/S0014-2999(99)00834-1. PMID:10650184. Lonn, E., Yusuf, S., Arnold, M.J., Sheridan, P., Pogue, J., Micks, M., et al. 2006. Homocysteine lowering with folic acid and B vitamins in vascular disease. N. Engl. J. Med. 354(15): 1567–1577. doi:10.1056/NEJMoa060900. PMID:16531613. Moore, K.H., Ray, F.R., and Barden, J.A. 2001. Loss of purinergic P2X(3) and P2X(5) receptor innervation in human detrusor from adults with urge incontinence. J. Neurosci. 21(18): RC166. PMID:11549755. North, R.A. 2002. Molecular physiology of P2X receptors. Physiol. Rev. 82(4): 1013–1067. PMID:12270951. North, R.A., and Surprenant, A. 2000. Pharmacology of cloned P2X receptors. Annu. Rev. Pharmacol. Toxicol. 40: 563–580. doi:10.1146/annurev.pharmtox. 40.1.563. PMID:10836147. O'Reilly, B.A., Kosaka, A.H., Chang, T.K., Ford, A.P., Popert, R., Rymer, J.M., et al. 2001. A quantitative analysis of purinoceptor expression in human fetal and adult bladders. J. Urol. 165(5): 1730–1734. doi:10.1016/S0022-5347(05)66403-8. PMID:11342965. Richler, E., Chaumont, S., Shigetomi, E., Sagasti, A., and Khakh, B.S. 2008. Tracking transmitter-gated P2X cation channel activation in vitro and in vivo. Nat. Methods, 5(1): 87–93. doi:10.1038/nmeth1144. PMID:18084300. Tardif, J.C., Carrier, M., Kandzari, D.E., Emery, R., Cote, R., Heinonen, T., et al. 2007. Effects of pyridoxal-5=-phosphate (MC-1) in patients undergoing highrisk coronary artery bypass surgery: results of the MEND-CABG randomized study. J. Thorac. Cardiovasc. Surg. 133(6): 1604–1611. doi:10.1016/j.jtcvs.2007. 01.049. PMID:17532963. Trezise, D.J., Bell, N.J., Khakh, B.S., Michel, A.D., and Humphrey, P.A. 1994. P2 purinoceptor antagonist properties of pyridoxal-5-phosphate. Eur. J. Pharmacol. 259(3): 295–300. doi:10.1016/0014-2999(94)90656-4. PMID:7982456. Tsuda, M., Shigemoto-Mogami, Y., Koizumi, S., Mizokoshi, A., Kohsaka, S., Salter, M.W., et al. 2003. P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. Nature, 424(6950): 778–783. doi:10.1038/ nature01786. PMID:12917686. Valera, S., Hussy, N., Evans, R.J., Adami, N., North, R.A., Surprenant, A., et al. 1994. A new class of ligand-gated ion channel defined by P2x receptor for extracellular ATP. Nature, 371(6497): 516–519. doi:10.1038/371516a0. PMID: 7523951. Wang, X., Dakshinamurti, K., Musat, S., and Dhalla, N.S. 1999. Pyridoxal 5=-phosphate is an ATP-receptor antagonist in freshly isolated rat cardiomyocytes. J. Mol. Cell. Cardiol. 31(5): 1063–1072. doi:10.1006/jmcc.1999.0936. PMID: 10336844.

Published by NRC Research Press

Pyridoxal-5'-phosphate (MC-1), a vitamin B6 derivative, inhibits expressed P2X receptors.

P2X receptors are cation-permeable ligand-gated ion channels that open in response to the binding of ATP. These receptors are present in many excitabl...
682KB Sizes 0 Downloads 3 Views