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Keywords:

  • ethanol;
  • HEK293 cells;
  • P2X3 receptor;
  • P2Y1 receptor;
  • trichloroethanol

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Culture and transfection procedures
  5. Electrophysiology
  6. [Ca2+]i measurement
  7. Reverse transcription and polymerase chain reaction
  8. Immunocytochemistry
  9. Drugs
  10. Statistics
  11. Results
  12. Presence of P2X3 receptors
  13. Electrophysiological effects of P2 receptor agonists and interaction with the respective antagonists
  14. Effects of P2 receptor agonists on the intracellular Ca2+ concentration and interaction with the respective antagonists
  15. Effects of ethanol and TCE on α,β-meATP-induced inward currents
  16. Effects of ethanol and TCE on ATP-, ADP-β-S- and UTP-induced rises of intracellular Ca2+
  17. Discussion
  18. Acknowledgements
  19. References

Membrane currents and changes in the intracellular Ca2+ concentration ([Ca2+]i) were measured in HEK293 cells transfected with the human P2X3 receptor (HEK293-hP2X3). RT-PCR and immunocytochemistry indicated the additional presence of endogenous P2Y1 and to some extent P2Y4 receptors. P2 receptor agonists induced inward currents in HEK293-hP2X3 cells with the rank order of potency α,β-meATP ≈ ATP > ADP-β-S > UTP. A comparable rise in [Ca2+]i was observed after the slow superfusion of ATP, ADP-β-S and UTP; α,β-meATP was ineffective. These data, in conjunction with results obtained by using the P2 receptor antagonists TNP-ATP, PPADS and MRS2179 indicate that the current response to α,β-meATP is due to P2X3 receptor activation, while the ATP-induced rise in [Ca2+]i is evoked by P2Y1 and P2Y4 receptor activation. TCE depressed the α,β-meATP current in a manner compatible with a non-competitive antagonism. The ATP-induced increase of [Ca2+]i was much less sensitive to the inhibitory effect of TCE than the current response to α,β-meATP. The present study indicates that in HEK293-hP2X3 cells, TCE, but not ethanol, potently inhibits ligand-gated P2X3 receptors and, in addition, moderately interferes with G protein-coupled P2Y1 and P2Y4 receptors. Such an effect may be relevant for the interruption of pain transmission in dorsal root ganglion neurons following ingestion of chloral hydrate or trichloroethylene.

Abbreviations used
ADP-β-S

adenosine 5′-O-(2-thiodiphosphate)

AMPA

α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid

α,β-meATP

α,β-methylene adenosine 5′-triphosphate

[Ca2+]i

intracellullar Ca2+ concentration

CPA

cyclopiazonic acid

GDP-β-S

guanosine 5′-O-(3-thiodiphosphate)

HEK293-hP2X3

human embryonic kidney cells transfected with human P2X3 receptors

MRS2179

2′-deoxy-N6-methyladenosine-3′,5′-diphosphate

PPADS

pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid

RT–PCR

reverse transcriptase–polymerase chain reaction

TCE

2,2,2-trichloroethanol

TNP-ATP

2′,3′-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate

A matter of particular interest in the past few years was the investigation of ethanol actions at ligand-activated neurotransmitter receptor-channels. It is generally accepted that ethanol inhibits ionotropic glutamate receptors of the NMDA and AMPA types, although some disagreement exists whether this effect occurs with similar or with different potencies (Faingold et al. 1998; Korpi et al. 1998; Wirkner et al. 1999). Facilitatory effects of ethanol have been reported for GABAA, serotonin 5-HT3, and glycine receptors (Faingold et al. 1998; Korpi et al. 1998).

Reports about direct effects of alcohols on G protein-coupled receptors are less frequent. Ethanol decreased the binding of the high-affinity form of the mouse cerebral cortical β-adrenoceptor for isoproterenol (Valverius et al. 1987) and negatively modulated opioid δ receptors in N18TG2 cells (Gomes et al. 2000). Alcohols were also described to inhibit the specific binding of agonists and antagonists to serotonin 5-HT1A receptors (Harikumar and Chattopadhyay 2000).

2,2,2-trichloroethanol (TCE) plays an important role in the field of industrial health and environmental toxicology as one of the main metabolites of the widely used solvent trichloroethylene (Maull et al. 1997), and also in medicine as a metabolite of the sedative-hypnotic drug chloral hydrate (Pershad et al. 1999). TCE is believed to be the active metabolite responsible for the pharmacological effects of these substances. In fact, TCE has previously been shown to potentiate currents mediated by GABAA receptors in mouse hippocampal neurons (Peoples and Weight 1994) and by glycine as well as 5HT3 receptors expressed in Xenopus oocytes (Pistis et al. 1997). Moreover, TCE was reported to inhibit both NMDA (Scheibler et al. 1999) and AMPA receptor (Fischer et al. 2000)-induced increases of the intracellular Ca2+ concentration ([Ca2+]i) in mesencephalic and cortical neurons of rats, respectively.

Adenosine 5′-triphosphate (ATP) has been shown to act as an extracellular signalling molecule in the central and peripheral nervous system, on the one hand as a neurotransmitter by its own right and on the other as a co-transmitter of, for example, noradrenaline (Starke et al. 1991; Poelchen et al. 2001) and acetylcholine (Cunha et al. 1994). In the last few years, two populations of ATP receptors have been cloned (Ralevic and Burnstock 1998; Illes et al. 2000). P2X receptors are ligand-gated cationic channels and mediate fast excitatory neurotransmission. P2Y receptors are coupled to G proteins and mediate slow synaptic signals or the release of Ca2+ from intracellular stores.

The inhibitory effect of ethanol on responses caused by ATP on P2X receptors was demonstrated originally in isolated dorsal root ganglion (DRG) neurons of bullfrogs (Li et al. 1993). The mammalian counterparts of bullfrog sensory neurons are known to possess the P2X3 receptor-type (Lewis et al. 1995) which is localized almost exclusively on sensory neurons and is postulated to play a major role in pain transmission (Lewis et al. 1995; Chizh and Illes 2001). TCE inhibited the ATP-induced inward current in cultured rat DRG neurons (A. Günther, R. Reinhardt and P. Illes, unpublished).

The aim of the present study was to find out whether TCE and ethanol alter P2X3 receptor-function in HEK293 cells transfected with the human P2X3 receptor (HEK293-hP2X3) by means of whole-cell patch-clamp recording and single-cell fura-2 microfluorimetry. In addition, endogenous human P2Y receptor subtypes were characterized in this cell line and their sensitivities to the two alcohols were compared with that of the P2X3 receptor. Some of the results have been published previously in a preliminary form (Köles et al. 2000).

Culture and transfection procedures

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Culture and transfection procedures
  5. Electrophysiology
  6. [Ca2+]i measurement
  7. Reverse transcription and polymerase chain reaction
  8. Immunocytochemistry
  9. Drugs
  10. Statistics
  11. Results
  12. Presence of P2X3 receptors
  13. Electrophysiological effects of P2 receptor agonists and interaction with the respective antagonists
  14. Effects of P2 receptor agonists on the intracellular Ca2+ concentration and interaction with the respective antagonists
  15. Effects of ethanol and TCE on α,β-meATP-induced inward currents
  16. Effects of ethanol and TCE on ATP-, ADP-β-S- and UTP-induced rises of intracellular Ca2+
  17. Discussion
  18. Acknowledgements
  19. References

HEK293 cells (European Collection of Cell Cultures, Porton Down, UK) were grown in 50-mL flasks (Sarstedt, Nürnberg, Germany) at 37°C and 5% CO2 in humidified air. The culture medium was Dulbecco's modified Eagle's medium (DMEM) also containing 25 mm HEPES, 110 μg/mL sodium pyruvate, 1 mg/mL d-glucose, 4 μg/mL pyridoxine (Life Technologies, Karlsruhe, Germany), 2 mm l-glutamine, 1% non-essential amino acids (NEAA) (all Sigma, Deisenhofen, Germany) and 10% fetal bovine serum (Life Technologies). Cells were split weekly and when about 60% confluent, transfected in serum-free DMEM of the above composition for 2 h with pcDNA3 (Invitrogen, Groningen, the Netherlands) carrying a human P2X3 cDNA (obtained from Dr J. N. Wood, University College, London, UK). Then, cells were kept in DMEM of the above composition supplemented with 10% fetal bovine serum and 50 μg/mL geneticin (Life Technologies) at 37°C and 10% CO2 in humidified air. Cells were maintained in 250 mL flasks (Sarstedt) and were used for 25–30 splits. They were plated on either 35-mm plastic dishes (Sarstedt) for electrophysiological recordings or on quarz glass coverslips coated with poly-l-lysine (Sigma) for Ca2+ measurements. Experiments were carried out 1–2 days (patch-clamp recordings) or 2–5 days (Ca2+ measurements) after replating.

Electrophysiology

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Culture and transfection procedures
  5. Electrophysiology
  6. [Ca2+]i measurement
  7. Reverse transcription and polymerase chain reaction
  8. Immunocytochemistry
  9. Drugs
  10. Statistics
  11. Results
  12. Presence of P2X3 receptors
  13. Electrophysiological effects of P2 receptor agonists and interaction with the respective antagonists
  14. Effects of P2 receptor agonists on the intracellular Ca2+ concentration and interaction with the respective antagonists
  15. Effects of ethanol and TCE on α,β-meATP-induced inward currents
  16. Effects of ethanol and TCE on ATP-, ADP-β-S- and UTP-induced rises of intracellular Ca2+
  17. Discussion
  18. Acknowledgements
  19. References

Recordings were made by using the conventional whole-cell patch-clamp method. All experiments were performed at room temperature (20–22°C). The pipette solution contained (in mM): CsCl, 140; MgCl2, 1; CaCl2, 2; HEPES, 10; EGTA, 11; MgATP, 1.5; GTP, 0.3; pH adjusted to 7.4 with CsOH. In some experiments, GTP (0.3 mm) was replaced by an equimolar concentration of GDP-β-S, while in other experiments, 5.5 mm BAPTA and 5.5 mm CsCl were used to substitute 11 mm EGTA. The pipette resistances were 3–5 MΩ. The liquid junction potential (VLJ) between the bath- and pipette solutions at 22°C was calculated according to Barry (1994) and was found to be 10.1 mV. Holding potential values given in this study were corrected for VLJ. Recordings were made at a holding potential of − 80 mV. Currents were filtered at 5 kHz with the inbuilt filter of the Axopatch 200B (Axon Instruments, Foster City, CA, USA). Data were then sampled at 10 kHz and stored on-line with a personal computer using the AxoScope software (Axon Instruments). Drugs were dissolved in an external solution of the following composition (in mM): NaCl, 135; KCl, 4.5; CaCl2, 2; MgCl2, 2; HEPES, 10; glucose, 10; pH adjusted to 7.4 with NaOH, and were applied by pressure using a DAD12 superfusion system (Adams and List, Westbury, NY, USA). The bath was continuously perfused with the normal external solution by one pressure-independent valve of this system, and the solution was removed from the bath with a vacuum pump.

Data acquisition and analysis were performed computer-controlled by using the pClamp 6.0 software (Axon Instruments). Concentration-response curves were fitted using the following three parametric logistic function (Origin; MicrocalTM Software Inc.): I = Imax/[1 + (EC50/agonist)n]. Where I is the steady state current produced by the agonist; Imax the maximal current at infinite agonist concentrations; n the Hill coefficient, and EC50 the concentration of agonist producing 50% of Imax.

[Ca2+]i measurement

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Culture and transfection procedures
  5. Electrophysiology
  6. [Ca2+]i measurement
  7. Reverse transcription and polymerase chain reaction
  8. Immunocytochemistry
  9. Drugs
  10. Statistics
  11. Results
  12. Presence of P2X3 receptors
  13. Electrophysiological effects of P2 receptor agonists and interaction with the respective antagonists
  14. Effects of P2 receptor agonists on the intracellular Ca2+ concentration and interaction with the respective antagonists
  15. Effects of ethanol and TCE on α,β-meATP-induced inward currents
  16. Effects of ethanol and TCE on ATP-, ADP-β-S- and UTP-induced rises of intracellular Ca2+
  17. Discussion
  18. Acknowledgements
  19. References

HEK293-hP2X3 cells were loaded with the Ca2+-sensitive fluorescent dye fura-2 acetoxymethyl ester (5 μm; Sigma) at 37°C for 30 min in culture medium. To remove extracellular traces of the dye, the cells were then washed in fura-2 free physiological saline containing (in mM): NaCl, 135; KCl, 5; MgCl2, 1; CaCl2, 2; HEPES, 10; glucose, 10; pH adjusted to 7.2 with NaOH. Subsequently, the coverslips were mounted into a perfusion chamber (250 μL), placed on the stage of an inverted microscope with epifluorescence optics (Diaphot 200; Nikon, Kanagawa, Japan). Throughout the experiments, cells were continuously superfused at 0.8 mL/min by means of a roller pump with drug-free or drug-containing solutions, respectively. In an additional series of experiments, the drug-containing solutions were applied directly to single cells by pressure, using a DAD12 application system (see Electrophysiology). The tissue chamber was continuously superfused with physiological saline by means of the roller pump (see above).

Fluorescence ratio measurements were made on single, morphologically identified cells with a dual wavelength spectrometer (alternating excitation at 340/380 nm). Fura 2-fluorescence was measured over the cell body by a microscope photometer attached to a photomultiplier detection system (Ratiomaster System; PTI, Lawrenceville, NJ, USA).

Data acquisition, presentation and analysis were performed computer-controlled using commercially available software (PTI, FeliX, Vers.1.1) Calibration of [Ca2+]i was performed with 10 μm ionomycin and 25 mm EGTA according to Grynkiewicz et al. (1985).

Reverse transcription and polymerase chain reaction

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Culture and transfection procedures
  5. Electrophysiology
  6. [Ca2+]i measurement
  7. Reverse transcription and polymerase chain reaction
  8. Immunocytochemistry
  9. Drugs
  10. Statistics
  11. Results
  12. Presence of P2X3 receptors
  13. Electrophysiological effects of P2 receptor agonists and interaction with the respective antagonists
  14. Effects of P2 receptor agonists on the intracellular Ca2+ concentration and interaction with the respective antagonists
  15. Effects of ethanol and TCE on α,β-meATP-induced inward currents
  16. Effects of ethanol and TCE on ATP-, ADP-β-S- and UTP-induced rises of intracellular Ca2+
  17. Discussion
  18. Acknowledgements
  19. References

Total RNA from HEK293-P2X3 cells (about 1.1 million cells) was extracted by using a Total Quick RNA mini kit (BIOZOL, Eching, Germany) from 4 days after plating in culture dishes. Genomic DNA was removed by twice-repeated digestion with RNase-free DNase I (50 units; Roche, Basel, Switzerland) for 2 h at 37°C. First-strand cDNA was synthesised by reverse transcription of 1 μg of total RNA with SuperscriptTM-II reverse transcriptase (Life Technologies) according to standard protocols. cDNA fragments of the various P2Y receptors were amplified using a set of sense and antisense primers specific for the respective P2Y receptor subtypes: P2Y1, P2Y2, P2Y4, P2Y6 (Jin et al. 1998), P2Y12 (Hollopeter et al. 2001) and P2Y13 (Communi et al. 2001). PCRs were run on a PTC-200 Thermocycler (MJ Research, Watertown, MA, USA) in a final volume of 25 μL containing 1 μL of the first strand cDNA, 1 unit Ampli-Taq DNA Polymerase (Applied Biosystems, Lincoln, CA, USA), and (anti)sense primers (200 nm, each). Negative controls lacking first-strand cDNA were run in parallel. Positive controls were performed with first-strand cDNA obtained from brain tissue. Amplification products were detected by ethidium bromide-staining subsequent to agarose gel electrophoresis (1.5%). The identity of the PCR products was verified by Southern blot analysis.

Immunocytochemistry

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Culture and transfection procedures
  5. Electrophysiology
  6. [Ca2+]i measurement
  7. Reverse transcription and polymerase chain reaction
  8. Immunocytochemistry
  9. Drugs
  10. Statistics
  11. Results
  12. Presence of P2X3 receptors
  13. Electrophysiological effects of P2 receptor agonists and interaction with the respective antagonists
  14. Effects of P2 receptor agonists on the intracellular Ca2+ concentration and interaction with the respective antagonists
  15. Effects of ethanol and TCE on α,β-meATP-induced inward currents
  16. Effects of ethanol and TCE on ATP-, ADP-β-S- and UTP-induced rises of intracellular Ca2+
  17. Discussion
  18. Acknowledgements
  19. References

After fixation in ice-cold methanol, washing with Tris buffered saline (TBS, 0.05 m; pH 7.6) and blocking with 5% fetal calf serum (FCS), the cultures were incubated with the rabbit anti-P2X3 receptor antibody (1 : 1000; Dr E. J. Kidd, GlaxoWellcome, Cambridge, UK) or with the rabbit anti-P2Y receptor antibodies (anti-P2Y1: 1 : 1500, Dr D. Moore, SmithKline Beecham Pharmaceuticals, Harlow, Essex, UK and Dr B. K. Kishore, Department of Internal Medicine, University of Cincinnati School of Medicine, Cincinnati, OH, USA; anti-P2Y2: 1 : 1000; Dr M. A. Knepper, Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, Bethesda, MA, USA; anti-P2Y4: 1 : 1000, Alomone Laboratories, Jerusalem, Israel) in TBS containing 0.1% Triton X-100 and 5% FCS for 12 h at 4°C. The preparations were washed three times for 5 min each in TBS and were then incubated with the secondary antibody Cy3-conjugated goat anti-rabbit IgG (1 : 800; Jackson ImmunoResearch, West Grove, PA, USA) in TBS containing 0.1% Triton X-100 and 5% FCS for 2 h at room temperature. After intensive washing, the cultures were dehydrated in a series of graded ethanol, processed through n-butylacetate and covered with entellan (Merck, Darmstadt, Germany). Control experiments were carried out without the primary antibody or by pre-adsorption of the antibody with the immunizing peptides. Microphotographs were made by using a fluorescence microscope (Axioskop; Zeiss, Oberkochen, Germany) equipped with a fluorescence filter set (BP 450–490, FT 510; LP 520) and with the Zeiss Axio Vision System 2.0 (Zeiss, Lawrenceville, NJ, USA).

Drugs

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Culture and transfection procedures
  5. Electrophysiology
  6. [Ca2+]i measurement
  7. Reverse transcription and polymerase chain reaction
  8. Immunocytochemistry
  9. Drugs
  10. Statistics
  11. Results
  12. Presence of P2X3 receptors
  13. Electrophysiological effects of P2 receptor agonists and interaction with the respective antagonists
  14. Effects of P2 receptor agonists on the intracellular Ca2+ concentration and interaction with the respective antagonists
  15. Effects of ethanol and TCE on α,β-meATP-induced inward currents
  16. Effects of ethanol and TCE on ATP-, ADP-β-S- and UTP-induced rises of intracellular Ca2+
  17. Discussion
  18. Acknowledgements
  19. References

The following drugs and chemicals were used: α,β-methyleneATP lithium salt (Biotrend, Köln, Germany); adenosine 5′-triphosphate disodium salt (ATP), adenosine 5′-triphosphate magnesium salt (MgATP), adenosine 5′-O-(2-thiodiphosphate) trilithium salt (ADP-β-S), cyclopiazonic acid, 2′-deoxy-N6-methyladenosine-3′,5′-diphosphate diammonium salt (MRS2179), ethanol, guanosine 5′-triphosphate lithium salt (GTP), guanosine 5′-O-(3-thiodiphosphate) trilithium salt (GDP-β-S), pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid tetrasodium salt (PPADS), 2,2,2-trichloroethanol (TCE), uridine 5′-triphosphate trisodium salt (UTP) (Sigma); 2′,3′-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate trisodium salt (TNP-ATP) (Molecular Probes, Leiden, the Netherlands).

Presence of P2X3 receptors

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Culture and transfection procedures
  5. Electrophysiology
  6. [Ca2+]i measurement
  7. Reverse transcription and polymerase chain reaction
  8. Immunocytochemistry
  9. Drugs
  10. Statistics
  11. Results
  12. Presence of P2X3 receptors
  13. Electrophysiological effects of P2 receptor agonists and interaction with the respective antagonists
  14. Effects of P2 receptor agonists on the intracellular Ca2+ concentration and interaction with the respective antagonists
  15. Effects of ethanol and TCE on α,β-meATP-induced inward currents
  16. Effects of ethanol and TCE on ATP-, ADP-β-S- and UTP-induced rises of intracellular Ca2+
  17. Discussion
  18. Acknowledgements
  19. References

In HEK293-hP2X3 cells cultured under identical conditions as cells subjected to electrophysiological recording or Ca2+ measurement, the expression of mRNA encoding the receptor subtypes P2Y1, P2Y2, P2Y4, P2Y6, P2Y12, and P2Y13 was investigated using RT-PCR. By the occurrence of a 528, 431, and 575 bp amplification product, mRNAs coding for P2Y1, P2Y4, and P2Y13 were detected, respectively (Fig. 1a). In RNA samples not subjected to cDNA synthesis, no amplification products were generated excluding contamination of the samples with genomic DNA. P2Y2, P2Y6 and P2Y12 receptor mRNA was not detectable. Immunocytochemical labelling demonstrated the expression of P2X3 (Fig. 1bi) and P2Y1 (Fig. 1bii) receptors but not of P2Y2 receptors at the plasma membrane of cultured HEK293-hP2X3 cells. In a low number of cells especially within cell aggregates, P2Y4 receptor-immunoreactivity could be detected (not shown).

image

Figure 1. Presence of P2 receptor-types in HEK293 cells transfected with the human P2X3 receptor (HEK293-hP2X3). (a) P2Y receptor mRNA expression in HEK293-hP2X3 cells. Subsequent to total RNA extraction and RT-PCR amplification with primers specific for distinct P2Y receptor subtypes, cDNA products were analysed by agarose gel electrophoresis (1.5%). A representative gel with ethidium bromide-stained cDNA fragments of the P2Y1 (528 bp), the P2Y4 (431 bp) and the P2Y13 (575 bp) receptor is given. P2Y2, P2Y6 and P2Y12 transcripts were not detectable (data not shown). (b) Microphotographs of HEK293-hP2X3 cells after immunocytochemical labelling of P2X3 (i) and P2Y1 receptors (ii) with the respective rabbit anti-P2 receptor antibodies (scale bars, 80 μm each). A P2Y4 receptor-immunoreactivity was detected in a very low number of cells only (not shown).

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Electrophysiological effects of P2 receptor agonists and interaction with the respective antagonists

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Culture and transfection procedures
  5. Electrophysiology
  6. [Ca2+]i measurement
  7. Reverse transcription and polymerase chain reaction
  8. Immunocytochemistry
  9. Drugs
  10. Statistics
  11. Results
  12. Presence of P2X3 receptors
  13. Electrophysiological effects of P2 receptor agonists and interaction with the respective antagonists
  14. Effects of P2 receptor agonists on the intracellular Ca2+ concentration and interaction with the respective antagonists
  15. Effects of ethanol and TCE on α,β-meATP-induced inward currents
  16. Effects of ethanol and TCE on ATP-, ADP-β-S- and UTP-induced rises of intracellular Ca2+
  17. Discussion
  18. Acknowledgements
  19. References

Pressure application of ATP or α,β-meATP for 1 s to HEK293-hP2X3 cells at a holding potential of − 80 mV evoked fast rising and rapidly desensitizing inward currents (Fig. 2a). Cells not transfected with P2X3 receptors failed to respond to ATP or α,β-meATP at any concentration used. Increasing concentrations of both agonists (0.1–100 μm each) were applied a single time to each HEK293-hP2X3 cell in order to establish concentration–response relationships (Fig. 2c). In order to find out why the maximum effect of α,β-meATP was considerably higher than that of ATP itself, experiments were made with micropipettes filled, either with the standard solution containing GTP, or, with a solution containing instead of GTP its enzymatically stable analogue GDP-β-S (300 μm) which is known to interfere with G protein-dependent reactions (Sternweis and Pang 1990) (Fig. 2b). ATP (30 μm) was pressure-ejected three times with 5-min intervals between applications, both under standard conditions (T1, − 487.2 ± 152.2 pA; T2, − 311.6 ± 104.7 pA; T3, − 310.6 ± 106.8 pA; n = 7 each) and under conditions when GDP-β-S (300 μm) replaced GTP (T1, − 978.8 ± 258.9 pA; T2, − 1009.4 ± 258.1 pA; T3, − 1231.3 ± 283.7 pA; n = 9 each). Hence, the response to ATP decreased from T1 to T2/T3 when a GTP-containing pipette solution was used, while it did not change in the presence of internally applied GDP-β-S. Moreover, the replacement of GTP by GDP-β-S almost doubled the ATP-induced current at T1 (note that the scale bars in Fig. 2bi and bii are different). Additional experiments showed that when a GDP-β-S-containing pipette was used and ATP (30 μm) was applied three times with 5-min intervals (T1, − 1422.5 ± 192.2 pA; T2 and T3 values are not shown) followed by three applications of α,β-meATP (10 μm) according to a similar time-schedule (T1, − 1517.5 ± 323.9 pA; n = 6 each), there was no difference between the agonist-induced currents, indicating that only ATP but not α,β-meATP effects were increased by this procedure. Furthermore, the replacement of the standard EGTA-buffered pipette solution with a BAPTA-buffered solution also resulted in stable and comparable inward currents to ATP (30 μm; T1, − 1405.0 ± 169.9 pA) and α,β-meATP (10 μm; T1, − 1584.0 ± 334.9 pA; n = 10 each).

image

Figure 2. Membrane currents evoked by ATP, α,β-meATP, ADP-β-S and UTP in HEK293-hP2X3 cells and no interaction of these agonists with each other. The whole-cell variant of the patch-clamp method was used. In the first series of experiments, ATP (0.1–100 μm) and α,β-meATP (0.1–100 μm) were pressure-ejected for 1 s and once to each cell (T1 only). In the second series of experiments, ATP, α,β-meATP, ADP-β-S and UTP were pressure-ejected for 1 s with 5-min intervals between applications at a holding potential of − 80 mV three-times in total (T1, T2, T3). α,β-meATP (3 μm) was ejected at T1 and T3; between these two ejections, increasing concentrations of ADP-β-S (3–100 μm) or UTP (10–1000 μm) were applied at T2. In a third series of experiments, ATP (30 μm)-induced currents were recorded at T1, T2 and T3, either with a pipette filled with standard solution containing GTP (300 μm), or with a pipette filled with solution in which GTP was replaced by GDP-β-S (300 μm). (a) Representative tracings. Neither ADP-β-S (i) nor UTP (ii) interfered with the α,β-meATP-induced currents. (b) Representative tracings. The response to ATP decreased from T1 to T2/T3 under standard conditions (i), while it was stable and much larger in amplitude in the presence of internal GDP-β-S (ii). Note the different scale bars in (i) and (ii). (c) Concentration–response curves of α,β-meATP (▴; n = 5–10), ATP (●; n = 4–9), ADP-β-S (▪; n = 5–10) and UTP (▾; n = 5–14). Calculated Imax and EC50 values at T1 were 464.8 ± 76.1 pA and 1.4 ± 0.7 μm, respectively, for ATP (degrees of freedom, d.f. = 6), 1025.4 ± 101.7 pA and 1.3 ± 0.1 μm, respectively, for α,β-meATP (d.f. = 6), or 638.8 ± 110.6 pA and 10.1 ± 5.5 μm, respectively, for ADP-β-S (d.f. = 3). The corresponding values for UTP could not be calculated, as there was no clear maximum of the curve at 1000 μm of UTP.

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In the following experiments, the concentration–response curves of ATP and α,β-meATP were compared with the concentration–response curves of ADP-β-S and UTP, two agonists which are known to have only minor effects at recombinant P2X receptors (Fig. 2; Ralevic and Burnstock 1998). ADP-β-S (3–100 μm) and UTP (10–1000 μm) were applied at T2; α,β-meATP (3 μm) was applied at T1 and T3 in order to detect possible changes by ADP-β-S and UTP in the sensitivities of P2X3 receptors (Fig. 2a). Both ADP-β-S and UTP had considerably lower potencies than ATP and α,β-meATP (Fig. 2c). It is noteworthy that no apparent maximum response was reached with UTP even at the highest concentration (1000 μm) tested. The rank order of agonist potency was α,β-meATP ≈ ATP > ADP-β-S > UTP. In addition, neither ADP-β-S nor UTP decreased the subsequent α,β-meATP-induced current (Fig. 2a).

As α,β-meATP is a relatively selective agonist at P2X1,3 receptors, it was chosen for further electrophysiological experiments. In these cases, α,β-meATP (3 μm) was applied six times with the already described protocol. The percentage inhibitory effects of various P2 receptor antagonists applied immediately after T2 for 10 min were evaluated at T4 as a percentage of the control current measured at T2. Application of the P2X1,3 receptor antagonist TNP-ATP (10 nm) and the P2X1,2,3 preferential antagonist PPADS (10 μm) inhibited the α,β-meATP (3 μm)-induced current by 80.0 ± 7.6% (n = 9; p < 0.05) and 75.6 ± 4.9% (n = 6; p < 0.05), respectively. When, subsequently, an antagonist-free medium was superfused for another 10 min, a partial recovery was observed at T6 (not shown).

Effects of P2 receptor agonists on the intracellular Ca2+ concentration and interaction with the respective antagonists

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Culture and transfection procedures
  5. Electrophysiology
  6. [Ca2+]i measurement
  7. Reverse transcription and polymerase chain reaction
  8. Immunocytochemistry
  9. Drugs
  10. Statistics
  11. Results
  12. Presence of P2X3 receptors
  13. Electrophysiological effects of P2 receptor agonists and interaction with the respective antagonists
  14. Effects of P2 receptor agonists on the intracellular Ca2+ concentration and interaction with the respective antagonists
  15. Effects of ethanol and TCE on α,β-meATP-induced inward currents
  16. Effects of ethanol and TCE on ATP-, ADP-β-S- and UTP-induced rises of intracellular Ca2+
  17. Discussion
  18. Acknowledgements
  19. References

Superfusion with ATP (30 μm) for 30 s every 30 min caused a reproducible increase of [Ca2+]i in HEK293-hP2X3 cells. Cells not transfected with hP2X3 receptors responded with comparable raises of the intracellular Ca2+ concentration (not shown). Figure 3a shows stable responses to ATP (30 μm) and no effect of α,β-meATP (10, 100 μm; n = 7) in HEK293-hP2X3 cells. The concentration–response relationship for ATP (1–1000 μm) reached a maximum at 1000 μm (2.9 ± 0.1; n = 6; Fig. 3b).

image

Figure 3. Effects of ATP and α,β-meATP on the intracellular Ca2+ concentration of HEK293-hP2X3 cells. Ratios of the fluorescence intensities at 340 versus 380 nm were measured by using fura-2 microfluorimetry. ATP (1–1000 μm) or α,β-meATP (10–100 μm) was superfused for 30 s with a 30-min interval between applications. (a) Representative tracings. (b) Concentration–response curve of ATP (n = 6–30). The calculated Δmax and EC50 values were 2.9 ± 0.2 and 15.9 ± 2.5 μm, respectively (d.f. = 6).

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All subsequent experiments were performed by applying ATP at a submaximal concentration of 30 μm four times according to the above time-schedule (T1-T4). ATP (30 μm) evoked a Δ fluorescence ratio of 2.2 ± 0.1 at T2 which corresponds to an increase of somatic [Ca2+]i from basal 55.8 ± 5.2 nm to 1008.7 ± 163.4 nm (n = 12). ADP-β-S and UTP (both 30 μm) evoked Δ fluorescence ratios of 2.6 ± 0.3 (n = 9) and 2.1 ± 0.2 (n = 7), respectively. Hence, ATP, ADP-β-S and UTP caused a comparable increase of [Ca2+]i at the identical concentration of 30 μm. After establishing two stable responses to ATP, ADP-β-S or UTP (T1 and T2), Ca2+-free solution or various drugs (cyclopiazonic acid, TNP-ATP, PPADS, MRS2179, ethanol, TCE) were superfused for 10 min before and during the third agonist-application (T3), followed by washout for another 30 min (T4). Drug effects were evaluated as the percentage change of the ATP-induced signal at T3 versus the control signal at T2.

In a Ca2+-free medium, the response to ATP, ADP-β-S and UTP, in an equipotent concentration (30 μm) was unchanged during T3, but was greatly reduced following depletion of intracellular Ca2+ stores by cyclopiazonic acid (10 μm) (Fig. 4ai and b). These results suggest the existence of one or more G protein-coupled P2Y receptor-types in HEK293 cells. In the presence of PPADS (10 μm), which blocks both human P2Y1 and P2Y4 receptors, the increase in [Ca2+]i by either agonist was markedly inhibited, although with a clear preference for the ADP-β-S effect (Fig. 4b). In contrast, the selective P2Y1 receptor antagonist MRS2179 (30 μm) abolished the response to ADP-β-S, decreased the response to ATP and did not alter the response to UTP (Fig. 4b). The P2X1,3 receptor antagonist TNP-ATP (10 nm) failed to interfere with ATP. This latter finding clearly contrasts with the already documented antagonism by TNP-ATP of the α,β-meATP-induced current response in the same population of cells (see above).

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Figure 4. Effects of ATP, ADP-β-S and UTP on the intracellular Ca2+ concentration of HEK293-hP2X3 cells in a Ca2+-free medium, or after the depletion of intracellular Ca2+ stores by cyclopiazonic acid (CPA); interaction with PPADS and TNP-ATP. Ratios of the fluorescence intensities at 340 versus 380 nm were measured by using fura-2 microfluorimetry. ATP, ADP-β-S or UTP (30 μm each) were superfused four-times for 30 s each with a 30-min interval (T1–T4). After establishing two stable responses to the agonists (T1 and T2), Ca2+-free solution or various drugs (CPA, PPADS, TNP-ATP) were superfused for 10 min before and during the third agonist-application (T3) followed by washout for another 30 min (T4). Drug effects were evaluated as the percentage change of the agonist-induced signal at T3 versus the control signal at T2. (a) Representative tracings for ATP-evoked Ca2+ transients. (i) 10 min before and during T3, the superfusion medium was changed to a Ca2+-free solution (plus 1 mm EGTA) for 10 min. (ii) 10 min before and during T3, 10 μm cyclopiazonic acid was added to the superfusion medium for 10 min to deplete intracellular Ca2+ stores. (b) Effects of a Ca2+-free medium (plus 1 mm EGTA), cyclopiazonic acid, PPADS (10 μm each), MRS2179 (30 μm), and TNP-ATP (10 nm) on the Ca2+ signals evoked by ATP, ADP-β-S and UTP (30 μm each). Means ± SEM of 6–11 experiments. The Δ fluorescence ratios evoked by these agonists were at T2, 2.1 ± 0.2 for ATP (n = 12), 2.6 ± 0.3 (n = 9) for ADP-β-S and 2.1 ± 0.2 (n = 7) for UTP. *p < 0.05; significant differences from the control response at T2.

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In the previous experiments, all cells in the tissue chamber were superfused with α,β-meATP for 30 s, every 30 min at a slow rate. Under these conditions, α,β-meATP (10–100 μm) failed to induce Ca2+ transients in single cells (Fig. 3). However, when α,β-meATP (1–100 μm) was applied by a fast pressurized system to a few cells for only 10 s, every 15 min, a clear increase of [Ca2+]i was observed in a single cell (Fig. 5). This increase was concentration–dependent within the range of 1–10 μm of α,β-meATP. The response to α,β-meATP (10 μm) was markedly depressed by TNP-ATP (10 nm; Fig. 5a) (90.5 ± 1.2% inhibition; n = 4; p < 0.05) but was not altered following depletion of intracellular Ca2+ stores by cyclopiazonic acid (10 μm; Fig. 5b) (9.8 ± 2.7% inhibition; n = 4; p > 0.05). In contrast, HEK293-hP2X3 cells did not react to the pressure application of a high K+ (50 mm) solution, thereby refuting the existence of voltage-sensitive Ca2+ channels (Fig. 5a).

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Figure 5. Effects of α,β-meATP on the intracellular Ca2+ concentration of HEK293-hP2X3 cells; inhibition by TNP-ATP and influence of the depletion of intracellular Ca2+ stores by cyclopiazonic acid. In contrast to the experiments shown in Figs 3 and 4, all test solutions were pressure-ejected for 10 s each, every 15 min. (a) Representative experiment. Ca2+ signals induced by the application of a high K+ (50 mm) solution and of α,β-meATP (1–100 μm) onto a single cell. The effect of α,β-meATP (10 μm) was inhibited by TNP-ATP (10 nm) superfused for 10 min before and during the application of α,β-meATP (10 μm). This inhibitory action was completely reversed by washout. (b) Representative experiment. Failure of cyclopiazonic acid (CPA) to alter the Ca2+ signals induced by α,β-meATP. CPA (10 μm) itself induced a slow increase in the baseline level of Ca2+. The effect of α,β-meATP (10 μm) was not changed by CPA (10 μm) superfused for 10 min before and during the application of α,β-meATP (10 μm).

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Effects of ethanol and TCE on α,β-meATP-induced inward currents

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Culture and transfection procedures
  5. Electrophysiology
  6. [Ca2+]i measurement
  7. Reverse transcription and polymerase chain reaction
  8. Immunocytochemistry
  9. Drugs
  10. Statistics
  11. Results
  12. Presence of P2X3 receptors
  13. Electrophysiological effects of P2 receptor agonists and interaction with the respective antagonists
  14. Effects of P2 receptor agonists on the intracellular Ca2+ concentration and interaction with the respective antagonists
  15. Effects of ethanol and TCE on α,β-meATP-induced inward currents
  16. Effects of ethanol and TCE on ATP-, ADP-β-S- and UTP-induced rises of intracellular Ca2+
  17. Discussion
  18. Acknowledgements
  19. References

In the following patch-clamp experiments, α,β-meATP (10 μm) was pressure-ejected at a holding potential of − 80 mV to HEK293-hP2X3 cells for 1 s with an interval of 5 min between consecutive applications. Two control responses (T1, T2) were recorded before the application of ethanol or TCE started 2 min before T3 for 7 min. In total, four additional responses to α,β-meATP were recorded, two in the presence (T3, T4) and two in the absence (T5, T6) of ethanol or TCE.

Application of ethanol (100 mm) for 7 min failed to alter the current induced by α,β-meATP (10 µm) at T4 in a statistically significant manner, when compared with the responses recorded at T2 before drug application (2.1 ± 12.5% potentiation; n = 6; p > 0.05). Concentrations higher than 100 mm ethanol were considered to be pharmacologically irrelevant and were therefore not investigated. When cells were held at − 80 mV, α,β-meATP (10 μm) induced an inward current, which was inhibited by TCE (3 mm) at T4 by 64.6 ± 7.9% (n = 14; p < 0.05) (Fig. 6ai). At a holding potential of + 20 mV, α,β-meATP caused an outward current; however, the inhibitory effect of TCE (3 mm) (55.8 ± 12.3%; n = 12; p < 0.05) did not change under these conditions (Fig. 6aii).

image

Figure 6. Inhibition by trichloroethanol of the α,β-meATP-induced current response at two different holding potentials. The whole-cell variant of the patch-clamp method was used. α,β-meATP (10 μm) was pressure-ejected six-times (T1-T6) for 1 s with 5-min intervals between applications at holding potentials of − 80 or + 20 mV. Two minutes before T3, trichloroethanol (TCE; 3 mm) was applied for 7 min in total. (a) Representative tracings showing the effect of TCE (3 mm) at − 80 mV (i) or + 20 mV (ii). Note that the inhibition by TCE was voltage independent. (b) Concentration–response relationship for the inhibitory effect of trichloroethanol (TCE) on α,β-meATP (3 μm)-induced currents. The results were expressed as percentage inhibition of the current recorded at T4, when compared with that recorded at T2 (n = 6–8). The IC50 value of TCE was 71.5 ± 0.2 μm (d.f. = 3). *p < 0.05; significant differences from the control response at T2.

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The effect of TCE (3 mm) was investigated on the inward current evoked by increasing concentrations of α,β-meATP (1–100 μm) at − 80 mV (Fig. 7). A short application protocol (only three ejections of α,β-meATP; T1-T3) was used (see legend to Fig. 7). In these series of experiments, the concentration–response curve of α,β-meATP was considerably steeper than previously observed (see Fig. 2c); the reason for this variability is unknown. TCE (3 mm) shifted the curve in a non-parallel manner to the right with a corresponding decrease in slope but no apparent change in maximum (Fig. 7b). The washout of TCE resulted in a complete recovery of the original sensitivity to α,β-meATP.

image

Figure 7. Inhibition by trichloroethanol of membrane currents induced by increasing concentrations of α,β-meATP. The whole-cell variant of the patch-clamp method was used. α,β-meATP (1, 3, 10, 30 or 100 μm) was pressure-ejected three-times (T1–T3) for 1 s with 5-min intervals between applications at a holding potential of − 80 mV. Trichloroethanol (TCE; 3 mm) was applied 5 min before and together with T2. (a) Representative tracings showing the effect of TCE (3 mm) on currents induced by 10 μm (i) or 30 μmα,β-meATP (ii). Note that the inhibitory effect of TCE depended on the concentration of α,β-meATP. (b) Concentration–response relationship for the α,β-meATP-induced current normalized with respect to the effect of α,β-meATP (30 μm) at T1. Current responses before (●), during (▪) and after (▴) the application of TCE (3 mm). Mean ± SEM of 4–10 experiments. Calculated Imax and EC50 values at T1 were 638.3 ± 110.5 pA and 10.1 ± 5.5 μm, respectively, for ATP (d.f. = 4). *p < 0.05; significant difference from the control response at T1.

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Effects of ethanol and TCE on ATP-, ADP-β-S- and UTP-induced rises of intracellular Ca2+

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Culture and transfection procedures
  5. Electrophysiology
  6. [Ca2+]i measurement
  7. Reverse transcription and polymerase chain reaction
  8. Immunocytochemistry
  9. Drugs
  10. Statistics
  11. Results
  12. Presence of P2X3 receptors
  13. Electrophysiological effects of P2 receptor agonists and interaction with the respective antagonists
  14. Effects of P2 receptor agonists on the intracellular Ca2+ concentration and interaction with the respective antagonists
  15. Effects of ethanol and TCE on α,β-meATP-induced inward currents
  16. Effects of ethanol and TCE on ATP-, ADP-β-S- and UTP-induced rises of intracellular Ca2+
  17. Discussion
  18. Acknowledgements
  19. References

The present experiments have demonstrated by using fura-2 microfluorimetry and a slow superfusion system that application of ATP, ADP-β-S and UTP, but not of α,β-meATP to HEK293-hP2X3 cells increased [Ca2+]i (see above). In view of the patch-clamp recordings, it was of interest to investigate whether the rise of [Ca2+]i induced by these agonists is altered by ethanol or TCE. When ATP (30 μm) was applied every 30 min for 30 s, reproducible control responses (T1, T2) were obtained. Subsequent superfusion with ethanol (100 mm) or TCE (1–10 mm) for 10 min diminished the effect of ATP at T3(Fig. 8). The inhibitory effect of TCE was concentration–dependent and more marked than that of ethanol. After washout with drug-free solution for 30 min, the [Ca2+]i signals fully recovered at T4. Identical results were obtained when the inhibitory effects of ethanol (100 mm) and TCE (1–10 mm) were investigated on the increase by ADP-β-S and UTP (30 μm each) of [Ca2+]i (Figs 8b and c). Hence, the potencies of ethanol and TCE appeared to be the same, irrespective of the type of P2Y receptor agonist used.

image

Figure 8. Inhibition by ethanol or trichloroethanol of ATP-, ADP-β-S or UTP-induced increases of the intracellular calcium concentration in HEK293-hP2X3 cells. Ratios of the fluorescence intensities at 340 versus 380 nm were measured by using fura-2 microfluorimetry. ATP, ADP-β-S or UTP (30 μm each) were superfused four-times for 30 s each with 30-min intervals (T1-T4). The second response to the agonist at T2 was set as a control. Ethanol (ETOH; 100 mm; n = 7–8) or trichloroethanol (TCE; 1, 3, 10 mm, n = 6–8 each) were applied for 30 min before and during T3. The effect of the antagonists was reversed by washout for another 10 min at T4. Percentage change of the Δ fluorescence ratio for ATP (a), ADP-β-S (b) and UTP (c). Means ± SEM are shown. *p < 0.05; significant differences from the control response at T2.

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Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Culture and transfection procedures
  5. Electrophysiology
  6. [Ca2+]i measurement
  7. Reverse transcription and polymerase chain reaction
  8. Immunocytochemistry
  9. Drugs
  10. Statistics
  11. Results
  12. Presence of P2X3 receptors
  13. Electrophysiological effects of P2 receptor agonists and interaction with the respective antagonists
  14. Effects of P2 receptor agonists on the intracellular Ca2+ concentration and interaction with the respective antagonists
  15. Effects of ethanol and TCE on α,β-meATP-induced inward currents
  16. Effects of ethanol and TCE on ATP-, ADP-β-S- and UTP-induced rises of intracellular Ca2+
  17. Discussion
  18. Acknowledgements
  19. References

P2X receptors form a family of at least seven subunits (P2X1-P2X7) which are all present in the CNS (Khakh 2001). Heteromultimeric assemblies of various subtypes have been described, while P2X7 does not co-assemble with any other subunit (Torres et al. 1999). The P2Y family comprises of 10 cloned and functionally defined subtypes. Seven of them (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13) are expressed in human tissues of which P2Y1, P2Y6, P2Y11, P2Y12 and P2Y13 occur in the CNS (Illes and Ribeiro 2003). P2Y1, P2Y12 and P2Y13 receptors react to adenine nucleotides only, while the P2Y2 receptor reacts to both types of nucleotides; the human (h)P2Y4 and hP2Y6 receptors markedly prefer uridine over adenine nucleotides (Ralevic and Burnstock 1998; Illes and Ribeiro 2003). In addition, ADP, and especially its structural analogues, i.e. ADP-β-S, activate P2Y1, P2Y12, and P2Y13 receptors with a higher potency than ATP itself.

The aim of the present study was to investigate the effects of ethanol and TCE on human P2X3 receptors expressed in HEK293 cells. Immunocytochemical labelling with selective antibodies demonstrated the existence of P2X3 and P2Y1 receptor proteins on the plasmalemma. A very few cells exhibited P2Y4 receptor-immunostaining; P2Y2 receptors, however, appeared to be absent. The poor immunostaining of P2Y4 receptors may be due to the low reactivity of the rabbit anti-P2Y4 receptor antibody with its human counterpart. RT-PCR measurements confirmed the presence of P2Y1 and P2Y4 receptor mRNA; P2Y13 receptor mRNA also occurred.

The intact P2X3 receptor function was confirmed by patch-clamp recordings. Rapidly desensitizing current responses to both ATP and the selective P2X1,3 agonist α,β-meATP (Ralevic and Burnstock 1998) were observed as a characteristic feature of homomeric P2X3 receptors (Lewis et al. 1995). Furthermore, the P2X1,3 receptor antagonist TNP-ATP (Lewis et al. 1998) and the P2X1,2,3 preferential antagonist PPADS (Lambrecht 2000) markedly inhibited the effect of α,β-meATP. A few experiments were aimed at clarifying why the concentration–response curve for α,β-meATP had a higher maximum than that for ATP itself. The replacement of GTP in the pipette solution by GDP-β-S, a manipulation known to interfere with G protein-mediated second-messenger mechanisms (Sternweis and Pang 1990) of, for example, P2Y receptors (Ralevic and Burnstock 1998; Communi et al. 2001), largely increased the effect of ATP and thereby eliminated the difference between the current responses to ATP and α,β-meATP. In addition, the more efficient buffering of the intracellular Ca2+ concentration by replacing EGTA with BAPTA in the pipette solution also resulted in similar current responses to the two agonists. Hence, the findings with GDP-β-S and BAPTA suggest that endogenous P2Y1 or P2Y4 receptors activated by ATP but not by α,β-meATP may increase the intracellular-free Ca2+ concentration, possibly via the G protein/phospholipase C/inositol trisphosphate pathway (Ralevic and Burnstock 1998), and thereby decrease the conductance of P2X3 receptor channels (Virginio et al. 1998). It was not the purpose of the present study to exactly identify the P2Y receptor-type(s) involved in this effect or its (their) mechanism of action, especially as the selective P2X1,3 receptor agonist α,β-meATP rather than ATP itself was used in all further electrophysiological experiments.

When [Ca2+]i was measured microfluorimetrically using a slow superfusion system, about 10-times higher concentrations of ATP than those needed to induce inward currents evoked an increase of [Ca2+]i, while α,β-meATP had no effect. Moreover, the ATP-induced rise of intracellular Ca2+ was not altered by the antagonist TNP-ATP, but was inhibited by PPADS, which in addition to its effects at P2X1,2,3 receptors blocks human P2Y1 and P2Y4 receptors (Ralevic and Burnstock 1998; von Kügelgen and Wetter 2000); the P2Y1 receptor selective antagonist MRS2179 (Boyer et al. 1998) also decreased the response to ATP. Eventually, the increase of [Ca2+]i was abolished after depletion of the intracellular Ca2+ stores by cyclopiazonic acid in accordance with the reported ability of P2Y receptors to mobilize Ca2+ from the endoplasmatic reticulum via a G protein-coupled intracellular pathway (Nicholas et al. 1996). In our experiments, α,β-meATP and ATP failed to cause an increase of [Ca2+]i by opening P2X3 receptor channels and thereby allowing the entry of extracellular Ca2+. This finding may be due to a marked desensitization of the P2X3 receptor during the slow buildup of agonist concentrations in the organ bath (McDonald et al. 2002).

In perfect accordance with this suggestion, the fast pressurized application of α,β-meATP onto single cells induced a clear increase in [Ca2+]i. The Ca2+ signals were inhibited by low concentrations of TNP-ATP but were not altered following depletion of intracellular Ca2+ stores by cyclopiazonic acid. The presence of voltage-sensitive Ca2+ channels in HEK293-hP2X3 cells could be excluded by the failure of 50 mm K+ to increase [Ca2+]i. Hence, extracellular Ca2+ appeared to enter the cells via the P2X3 receptor-channels rather than by subsequently activated voltage-sensitive Ca2+ channels (but see Koshimizu et al. 2000).

It is assumed that the transfected P2X3 receptors co-exist in HEK293 cells with endogenous P2Y receptors activated by either ADP or UTP. ATP and ADP caused inositol phosphate accumulation in these cells; UTP was also an agonist, but with a much lower potency (Schachter et al. 1997). The present experiments only partly confirmed the suggestion of these authors that P2Y1 and P2Y2 receptors are present on HEK293 cells, but agreed with a quantitative RT-PCR study which found P2Y1 and P2Y4 mRNA (Moore et al. 2001). ATP, ADP-β-S (P2Y1) and UTP (P2Y4) all increased [Ca2+]i. Although concentration–response curves were constructed for ATP only, the effects of the investigated agonists at 30 μm (approximately EC80 of ATP) were similar in extent. However, the P2Y receptor antagonists PPADS (P2Y1, P2Y4) and MRS2179 (P2Y1) abolished the responses to ADP-β-S, but only incompletely inhibited (PPADS) or even failed to inhibit (MRS2179) the reponses to UTP. Hence, these functional studies unequivocally demonstrate that HEK293 cells possess both P2Y1 and P2Y4 receptors, although the presence of additional P2Y receptor-types (e.g. P2Y13) cannot be excluded by the agonists and antagonists used.

Hence, HEK293-hP2X3 cells were an adequate model to investigate the effects of ethanol and TCE both on P2X and P2Y receptors. Ethanol (100 mm) produced only negligible inhibition at the hP2X3 receptor, while TCE (3 mm) had a strong inhibitory potency. In fact, ethanol and TCE have been described as modulating the function of various ligand-gated ion channels (Faingold et al. 1998). However, differences between ethanol and TCE in their modulatory potency may exist and TCE, in spite of its higher lipophilicity compared with ethanol, does not inevitably influence ion channels to a larger extent than ethanol. In neurons freshly isolated from bullfrog DRG, ethanol was found to inhibit the function of ATP-gated ion channels, while chlorinated hydrocarbons such as TCE and dichloroethanol failed to influence them (Weight et al. 1999). In contrast, NMDA and AMPA receptors of rat or mice cortical neurons were more sensitive to TCE than to ethanol (Peoples and Weight 1994; Fischer et al. 2000).

Finally, P2X4 receptors which are abundantly expressed in the brain, exhibit a higher sensitivity to ethanol than the P2X3 receptors investigated in this study (Xiong et al. 2000). In addition, P2X4 and P2X3 receptors may be inhibited by different mechanisms, as ethanol shifted the concentration–response curve for ATP at P2X4 receptors in a parallel manner to the right, whereas TCE caused a non-parallel shift to the right of the concentration–response curve of α,β-meATP at P2X3 receptors. However, the effects of ethanol and TCE were at both receptor-types voltage-independent.

The inhibitory effect of ethanol at P2X receptors of DRG neurons isolated from bullfrogs was suggested to be due to an allosteric mechanism (Li et al. 1998). In the present experiments, the non-parallel shift by TCE of the concentration–response curve for α,β-meATP appears to argue against such a mode of action (see Li et al. 1993). In mammalian sensory neurons, P2X3 receptors have been found in a homomeric form, or co-localized with P2X2 receptors yielding P2X2/3 heteromers (Lewis et al. 1995; Chizh and Illes 2001). Both homomeric and heteromeric receptors appear to be involved in pain transmission. A characteristic feature of the homomeric receptor is its rapid desensitization, while heteromeric receptors show non-desensitizing kinetics (Garcia-Guzman et al. 1997; Ralevic and Burnstock 1998; Nörenberg and Illes 2000). As the receptors studied by Li et al. (1998) did not show fast desensitization kinetics, they may belong to the P2X2/3 type.

When P2X3 receptors were desensitised during slow superfusion with ATP, ADP-β-S or UTP, ethanol caused a rather modest inhibition of P2Y receptor function even at the high concentration of 100 mm. However, TCE inhibited the P2X3 receptor-mediated currents at 0.03 mm, while it was able to interfere with the P2Y receptor-mediated increase of [Ca2+]i at a concentration of 3 mm only. Moreover, both ethanol and TCE reduced the rise in [Ca2+]i by ATP, ADP-β-S and UTP to a similar extent, suggesting a comparable inhibitory potency of these compounds at P2Y1 and P2Y4 receptors. It is assumed that the mechanism of action of TCE may be the same at both receptors; effects at similar structural motifs of the two receptors, or at the common signal transduction pathway including G proteins, are equal possibilities.

P2Y1 receptors have been shown to be present in the rat DRG at large-fibre mechanosensitive neurons (Nakamura and Strittmatter 1996; Cook et al. 1997). The stimulation of these receptors evokes high-frequency trains of action potentials that in contrast to those evoked by P2X3 receptor-stimulation did not adapt. Thereby, ATP may be involved in the mediation of mechanosensation and neurogenic inflammation.

TCE is the main metabolite of the widely used oral sedative hypnotic drug chloral hydrate (Pershad et al. 1999). In addition to its anaesthetic action, chloral hydrate has been reported to cause remarkable analgesic effects (Field et al. 1993) which may be due to the modulation of pain transmission in DRG cells. There is evidence that P2X3 receptors, which are present at a subset of DRG neurons, are involved in the molecular mechanisms of pain (Chizh and Illes 2001). Ingestion of high doses (> 2.0 g) of chloral hydrate produces plasma TCE levels in the low millimolar range (Owen and Taberner 1980). Consequently, the inhibition of hP2X3, and to a limited extent also P2Y receptor function by TCE observed in our study, may have clinical relevance.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Culture and transfection procedures
  5. Electrophysiology
  6. [Ca2+]i measurement
  7. Reverse transcription and polymerase chain reaction
  8. Immunocytochemistry
  9. Drugs
  10. Statistics
  11. Results
  12. Presence of P2X3 receptors
  13. Electrophysiological effects of P2 receptor agonists and interaction with the respective antagonists
  14. Effects of P2 receptor agonists on the intracellular Ca2+ concentration and interaction with the respective antagonists
  15. Effects of ethanol and TCE on α,β-meATP-induced inward currents
  16. Effects of ethanol and TCE on ATP-, ADP-β-S- and UTP-induced rises of intracellular Ca2+
  17. Discussion
  18. Acknowledgements
  19. References

The authors are grateful to Mrs M. Henschke for expert technical assistance. The supply of P2X3 cDNA and antibodies by the persons mentioned in the Experimental procedures section are gratefully acknowledged. This work was supported by the Bundesministerium für Bildung, Forschung und Technologie, Leitprojekt ‘Molekulare Medizin’ (01GG981/0) and the Deutsche Forschungsgemeinschaft (IL 20/11–1). Dr L. Köles obtained a fellowship within a Hungarian-German intergovernmental project for bilateral co-operation in science and technology (OTKA-BMBF) and additional financial support (OTKA-32736).

References

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Culture and transfection procedures
  5. Electrophysiology
  6. [Ca2+]i measurement
  7. Reverse transcription and polymerase chain reaction
  8. Immunocytochemistry
  9. Drugs
  10. Statistics
  11. Results
  12. Presence of P2X3 receptors
  13. Electrophysiological effects of P2 receptor agonists and interaction with the respective antagonists
  14. Effects of P2 receptor agonists on the intracellular Ca2+ concentration and interaction with the respective antagonists
  15. Effects of ethanol and TCE on α,β-meATP-induced inward currents
  16. Effects of ethanol and TCE on ATP-, ADP-β-S- and UTP-induced rises of intracellular Ca2+
  17. Discussion
  18. Acknowledgements
  19. References