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The Journal of Physiology logoLink to The Journal of Physiology
. 2005 Mar 17;565(Pt 1):229–242. doi: 10.1113/jphysiol.2005.084186

Reactive oxygen species contribute to the presynaptic action of extracellular ATP at the frog neuromuscular junction

AR Giniatullin 1, SN Grishin 1, ER Sharifullina 1,2, AM Petrov 1, AL Zefirov 1, RA Giniatullin 1,2
PMCID: PMC1464489  PMID: 15774519

Abstract

During normal cell metabolism the production of intracellular ATP is associated with the generation of reactive oxygen species (ROS), which appear to be important signalling molecules. Both ATP and ROS can be released extracellularly by skeletal muscle during intense activity. Using voltage clamp recording combined with imaging and biochemical assay of ROS, we tested the hypothesis that at the neuromuscular junction extracellular ATP generates ROS to inhibit transmitter release from motor nerve endings. We found that ATP produced the presynaptic inhibitory action on multiquantal end-plate currents. The inhibitory action of ATP (but not that of adenosine) was significantly reduced by several antioxidants or extracellular catalase, which breaks down H2O2. Consistent with these data, the depressant effect of ATP was dramatically potentiated by the pro-oxidant Fe2+. Exogenous H2O2 reproduced the depressant effects of ATP and showed similar sensitivity to anti- and pro-oxidants. While NO also inhibited synaptic transmission, inhibitors of the NO-producing cascade did not prevent the depressant action of ATP. The ferrous oxidation in xylenol orange assay showed the increase of ROS production by ATP and 2-MeSADP but not by adenosine. Suramin, a non-selective antagonist of P2 receptors, and pertussis toxin prevented the action of ATP on ROS production. Likewise, imaging with the ROS-sensitive dye carboxy-2′,7′-dichlorodihydrofluorescein revealed increased production of ROS in the muscle treated with ATP or ADP while UTP or adenosine had no effect. Thus, generation of ROS contributed to the ATP-mediated negative feedback mechanism controlling quantal secretion of ACh from the motor nerve endings.

Living cells continuously produce reactive oxygen species (ROS), either from mitochondrial oxidative phosphorylation or activity of NADPH oxidase (Vignais, 2002). Growing evidence suggests that ROS such as superoxide, H2O2 and the hydroxyl radical (initially assumed to be harmful compounds) could serve as important signalling molecules (Suzuki et al. 1997; Oh et al. 2000; Servitja et al. 2000; Suzukawa et al. 2000; Goldstein et al. 2003; Murakami et al. 2003). In the CNS, H2O2 plays a role as an endogenous modulator of synaptic dopamine release (Chen et al. 2001). In skeletal muscle, superoxide and H2O2 are produced and released during intense activity (Murrant & Reid, 2001). We previously hypothesized that at the neuromuscular junction freely diffusible H2O2 participated in feedback control of quantal acetylcholine (ACh) release from the motor nerve ending (Giniatullin & Giniatullin, 2003).

At the neuromuscular junction another endogenous substance, namely extracellular ATP, inhibits quantal ACh release via activation of metabotropic P2Y receptors (Giniatullin & Sokolova, 1998; Sokolova et al. 2003). Our previous study showed that these P2Y receptors are coupled to distinct intracellular second messenger cascades including phospholipase C, protein kinase C, phospholipase A2 and cyclooxygenase (Sokolova et al. 2003). However, the downstream effector mechanisms of these cascades remained unknown.

There are several examples of coupling of purinergic receptors to redox mechanisms. Thus, ATP can stimulate production of ROS via purinergic receptors in glioma cells (Sauer et al. 2003) and astrocytes (Murakami et al. 2003). Stimulation of ionotropic P2X7 receptors induces neuronal death mediated by superoxide/H2O2 generated by glial NADPH oxidase (Parvathenani et al. 2003). At the neuromuscular junction glial perisynaptic Schwann cells surround the motor nerve terminal and respond to the endogenously released ATP (Robitaille, 1995). Upon activation these cells can produce a number of diffusible messengers such as prostaglandins, NO and glutamate (Auld & Robitaille, 2003).

Thus, their similar use-dependent release during intense activity and subsequent modulation of synaptic transmission via presynaptic sites suggest an interaction between purinergic and ROS mechanisms. However, with the exception of cultured glial cells, no evidence has been reported on the crosstalk between ATP receptors and ROS mechanisms at the level of synaptic transmission from motoneurone to skeletal muscle.

The aim of the current study was to test the relationship between purinergic control of transmitter release and redox state of the synapse. We show here that the inhibitory action of ATP (but not of adenosine) on quantal ACh release from the motor nerve endings involves production of ROS.

Methods

Preparation and solutions

Experiments were carried out on frog (Rana ridibunda) sartorius or cutaneous pectoris muscle preparations in vitro at room temperature (for details see Giniatullin et al. 1997; Giniatullin & Giniatullin, 2003). The experiments were conducted according to the principles and requirements of the European Communities Council Directive (24th November 1986; 86/609/EEC). The experimental protocol has been approved by the Ethical Committee of Kazan State Medical University. Animals were anaesthetized with ether before being stunned and pithed. To prevent muscle contractions and preserve a physiologically high level of transmitter release, the muscle fibres were cut transversely (Barstad & Lilleheil, 1968; Glavinovic, 1979). Prior to recording, the cut muscle was rinsed for at least 40 min with physiological solution. The cutting procedure does not produce significant changes in cable properties and, in combination with the voltage clamp technique, enables long-lasting stable recording of multiquantal synaptic currents (Glavinovic, 1979). Physiological solution contained (mm): NaCl 113, KCl 2.5, CaCl2 1.8, NaHCO3 2.4. The pH of all solution was adjusted to 7.3 with NaOH/HCl. Solutions of Fe2+, S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) were prepared immediately before use to avoid light-induced inactivation. In experiments with pertussis toxin, muscles were treated with this toxin (1 μg ml−1) for 18 h at room temperature, i.e. in conditions which abolished the presynaptic affect of ATP on ACh release (Sokolova et al. 2003). All reagents were from Sigma except 5-(and 6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCF), which was purchased from Molecular Probes.

All drugs were dissolved to a final concentration in the bathing solution and applied to a muscle maintained in the chamber (2.5 ml) via a superfusion system at the rate of 2 ml min−1. Measurements were started after 15 min in the presence of the drug unless stated otherwise.

Electrophysiology

Recording of postsynaptic end-plate currents was performed using the standard two-electrode voltage clamp technique with intracellular electrodes (resistance 3–5 MΩ; filled with 2.5 m KCl). The recorded evoked end-plate currents (EPCs) or spontaneous miniature EPCs (MEPCs) were digitized at 50 kHz, stored in a PC and analysed off-line to calculate mean values of EPC or MEPC amplitudes, rise times (from 10 to 90% of the amplitude), and decay time constants. The holding potential for cut preparations was kept at −40 mV. EPCs were elicited every 15–30 s by a single supramaximal nerve stimulation.

Assays for hydroperoxides

The frog muscle concentration of hydroperoxides was measured with the method of ferrous oxidation in xylenol orange (FOX) as described by Jiang et al. (1992) and Wolff (1994). This highly sensitive method consists of peroxide-mediated oxidation of ferrous ions in an acidic medium containing the dye xylenol orange, which binds the resulting ferric ions to produce a blue–purple complex with absorbance maximum of between 540 and 580 nm. The FOX reagent was prepared as described by Wolff (1994) with slight modification: 50 mm xylenol orange, 500 μm ammonium ferrous sulphate, 200 mm d-sorbitol, and 50 mm sulphuric acid. Reagents were of at least analytic grade.

Control (untreated) or purine-treated muscles after addition of cold (−20°C) acetone as a fixative were homogenized and then centrifuged for 10 min at 12 000 g. The supernatant for assay was mixed with an equal volume of the FOX reagent. Reaction mixtures were incubated at room temperature for 1 h, the time needed for the reaction to reach a stable end-point. After completion of the reaction the tissue extract was centrifuged for 5 min at 10 000 g, and the supernatant was assayed spectrophotometrically (absorbance at 540 nm). Blanks were solutions prepared using acetone mixed with the equal amount of the FOX reagent. FOX reagent was made up a day before the analysis and kept overnight at 4°C in the dark. All procedures were performed under dimmed light. Experiments were carried out at least in triplicate and averaged. The peroxide content of samples was determined with reference to a calibration curve obtained with known concentrations of H2O2 and expressed as micromoles of peroxide per gram of tissue.

ROS imaging

ROS in the muscle were detected also with 5-(and 6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCF; Molecular Probes, Eugene, OR, USA). This dye freely permeates the cells, and becomes trapped inside after hydrolysis to carboxy-dichlorodihydrofluorescein, which upon oxidation by intracellular ROS forms highly fluorescent carboxy-dichlorofluorescein (Murrant & Reid, 2001).

Muscles were incubated with 10 μm DCF for 45 min at room temperature, rinsed with normal Ringer solution, and visualized by fluorescence microscopy (Nikon Diaphot, objective 40×, N.A. = 0.55) combined with either video imaging or fast CCD camera (Coolsnap QR; Roper Scientific, USA). Fluorescence intensity was recorded over the surface of the muscle fibres (Murrant & Reid, 2001). Our microscope field allowed visualization of an area containing three to four muscle fibres. Muscle illumination was limited to 21 frames at 2–3 min intervals (exposure time 300 ms) for 15 min to minimize photo-oxidation of DCF. Similar results were obtained with either method. Fluorescence intensity (excitation 475 nm; emission 525 nm), was measured over three to five distinct muscle areas within the imaged field. The intensity of fluorescence was quantified using image analysis software (Morphostar, Paris) or Metafluor software (Metafluor Imaging Series 6.0, Universal Imaging Corporation, USA) and relative change in fluorescence from baseline (ΔF/F0× 100%) was calculated as previously described (Khiroug et al. 1998). The changes in fluorescence induced by ATP were compared with the fluorescence in muscles not exposed to ATP under similar conditions (control).

Analysis

Usually 120–150 spontaneous miniature end-plate currents (MEPCs) and 10–15 multiquantal EPCs were averaged to obtain mean values. In experiments with MEPCs, these uniquantal currents (digitized at 50 kHz) were repeatedly averaged for at least 15 min before superfusion of various drugs to ensure stable baseline conditions. Spontaneous events were detected using amplitude thresholds set as a multiple (4–5 times) of the noise standard deviation. Each event was also visually inspected to prevent noise disturbance of the analysis.

Spontaneous and elicited currents were analysed off-line using Origin software (OriginLab Corp, version 6.0). Quantal content was determined either as the ratio of averaged amplitudes of EPCs and MEPCs or as the ratio of their areas (Giniatullin & Giniatullin, 2003). The data are presented as the mean ± s.e.m. (n = number of synapses), with statistical significance assessed by Student's paired t test (for parametric data) or the Mann–Whitney test (for non-parametric data). A P value of less than 0.05 was accepted as indicative of a statistically significant difference.

Results

ATP and exogenous ROS reduced the amplitude of EPC

On muscle fibres held at −40 mV motor nerve stimulation (0.05 Hz) evoked multiquantal end-plate currents (EPCs; Fig. 1A) with average amplitude of 138 ± 25 nA (n = 11 synapses) and decay time constant of 1.35 ± 0.11 ms (n = 11). At the submaximal concentration (Sokolova et al. 2003) of 100 μm, ATP reversibly reduced the amplitude of EPC to 66 ± 1% of control (n = 11; P < 0.01; Fig. 1A and B). The amplitude of MEPCs in control was 1.8 ± 0.09 nA (n = 6) and the decay time constant was 1.09 ± 0.15 ms (n = 6). ATP (100 μm) did not change the amplitude of MEPCs (98 ± 3%n = 6; P > 0.05; Fig. 1A and B) indicating pure presynaptic action of nucleotide (see also Giniatullin & Sokolova, 1998; Sokolova et al. 2003). Consistent with this, the frequency of MEPCs was reduced by ATP to 65 ± 5% (n = 6; P < 0.05). ATP did not change significantly the decay time constant of EPCs (94 ± 5%; n = 9; P < 0.05). Therefore, ATP reduced the charge transferred during generation of EPCs (estimated as EPCs area, see Methods) to 68 ± 5% (n = 6; P < 0.05), i.e. to the same degree as the amplitude of EPCs.

Figure 1. Presynaptic depression of multiquantal end-plate currents (EPCs) by ATP or H2O2.

Figure 1

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A, action of 100 μm ATP on multiquantal EPCs and miniature EPCs (MEPCs). B, averaged data for changes in the amplitude of EPCs (n = 11) and MEPCs (n = 6) in the presence of 100 μm ATP. C, action of 300 μm H2O2 on multiquantal EPCs and MEPCs. D, averaged data for changes in the amplitude of EPCs (n = 9) and MEPCs (n = 7) in the presence of 300 μm H2O2. *P < 0.05.

In our previous study we showed that ADP or UTP, agonists of metabotropic ATP receptors, mimicked the depressant action of ATP indicating involvement of P2Y receptors (Sokolova et al. 2003). However, out of several antagonists, namely suramin, pyridoxalphosphate-6-azo-phenyl-2′,4′-disulphonate (PPADS) and Reactive Blue-2, only suramin, a non-selective antagonist of P2 receptors, prevents the inhibitory action of ATP (Sokolova et al. 2003). In order to further explore the receptor mechanism of ATP action on EPCs, in the current study we tested the action of 2-(methylthio)adenosine 5′-diphosphate (2-MeSADP), a potent agonist of P2Y1, P2Y12 and P2Y13 receptors (Ralevic & Burnstock, 1998; Zhang et al. 2002). At a relatively low concentration (10 μm), 2-MeSADP reduced the amplitude of EPCs to 50 ± 7% (n = 6; P < 0.05; see Supplemental material, Supplementary Fig. 1A), suggesting that P2Y1, P2Y12 or P2Y13 receptors widely distributed in various tissues, including the nervous system (Laitinen et al. 2001; Zhang et al. 2002; Cheung et al. 2003; Choi et al. 2003), control transmitter release from the motor nerve endings. Interestingly, a slight initial facilitation of EPCs (3–5 min from the start of agonist application) preceded the depressant effect of 2-MeSADP (not shown). To test the possible involvement of P2Y1 receptors, we studied the action of ATP in the presence of MRS-2179, a specific antagonist of this receptor subtype (Zhang et al. 2002). However, MRS-2179 (10 μm) did not change the action of 100 μm ATP (66 ± 4%, n = 5; P < 0.05; Supplementary Fig. 1B), indicating that the depression of EPCs by ATP on frog muscle was not mediated by P2Y1 receptors. While lack of specific P2Y12 and P2Y13 antagonists precluded further identification of ATP receptor subtypes, our previous and current data suggest control of transmitter release from motor nerve endings by metabotropic P2Y receptors.

To explore the involvement of ROS, the action of ATP was compared with the effect of exogenous H2O2, a cell-permeant ROS. We have previously shown that at concentrations exceeding 30 μm, H2O2 inhibits EPCs without changing postsynaptic nicotinic receptor sensitivity (Giniatullin & Giniatullin, 2003). Consistent with this notion, 300 μm H2O2 reduced the amplitude of EPCs to 75 ± 5% (n = 9; P < 0.01; Fig. 1C and D). As in the case of ATP, this effect had a presynaptic origin since the amplitude of MEPCs was not changed (Fig. 1C and D). In line with the presynaptic site of action, the frequency of spontaneous quantal events was reduced by 300 μm H2O2 to 35 ± 6% (n = 7; P < 0.01).

Antioxidants N-acetylcysteine and pyruvate reduced the depressant action of ATP

Next, we tested the action of ATP in the presence of several antioxidants known to prevent the cellular effects of ROS. First, we used 1 mmN-acetylcysteine (NAC); it increased the amplitude of EPCs to 157 ± 16% (n = 5; P < 0.01; Fig. 2A). Pretreatment of the muscle by NAC diminished the depressant action of 100 μm ATP. Thus, the amplitude of EPCs was reduced only to 90 ± 2% (n = 7; P < 0.05, Fig. 2A and B) instead of control depression to 66% (P < 0.05; Mann–Whitney test). NAC at 1 mm concentration prevented the depressant action of 300 μm H2O2 (93 ± 4%; n = 5; P > 0.05; Fig. 2C and D), indicating high efficiency of this antioxidant. Interestingly, the depressant action of 100 μm adenosine (69 ± 2%; n = 15; P < 0.001) operating via DPCPX-sensitive A1 receptors (Giniatullin & Sokolova, 1998) was not changed by NAC (72 ± 2%; n = 4).

Figure 2. Antioxidant N-acetylcysteine (NAC) reduces the effect of ATP and prevents the depressant action of H2O2.

Figure 2

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A, representative EPCs in control, in the presence of 1 mm NAC and after the action of 100 μm ATP. B, averaged effect of 100 μm ATP on EPCs in control (n = 11) and in the presence of 1 mm NAC (n = 7). Note that the depressant action of ATP was significantly reduced (P < 0.05; Mann–Whitney test) in the presence of antioxidant. C, representative EPCs in control, in the presence of 1 mm NAC and after the action of 300 μm H2O2. D, averaged effect of 300 μm H2O2 on the amplitude of EPCs in control (n = 9) and in the presence of 1 mm NAC (n = 5). *P < 0.05.

The attenuated effect of ATP in the presence of NAC was not related to changes in quantal output since the reduction of the quantal release by 1 mm Mg2+ (76 ± 6%; n = 6; P < 0.05) preserved the antagonistic action of NAC on the depressant action of ATP. These data (as well as the control action of 100 μm ATP in the presence of 1 mm Mg2+) are shown in Supplementary Fig. 2.

Pyruvate is also a powerful antioxidant, which can directly scavenge H2O2 (Gyulkhandanyan et al. 2003; Mallet & Sun, 2003). In our experiments 100 μm pyruvate increased the amplitude of EPCs to 155 ± 6% (n = 5; P < 0.05; Fig. 3A). Testing synaptic transmission with 300 μm H2O2 in the presence of pyruvate did not reveal any significant changes in the amplitude of EPCs (98 ± 6%, n = 4; P > 0.05) consistent with the ROS scavenging properties of pyruvate. Like the results obtained with NAC, the depressant action of 100 μm ATP in the presence of 100 μm pyruvate was much weaker (85 ± 3%, n = 4; P < 0.05; Fig. 3A and B). This action was significantly different (P < 0.05 by Mann–Whitney test) from the inhibitory action of ATP in control. The depressant action of adenosine was not changed by 100 μm pyruvate (70 ± 1%, n = 3; P < 0.05; Fig. 3C).

Figure 3. Antioxidant pyruvate reduces the effect of ATP but does not change the action of adenosine.

Figure 3

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A, representative EPCs in control, in the presence of 100 μm pyruvate and after application of 100 μm ATP. B, time course of 100 μm ATP action (indicated by the bar) in control (•) and in the presence of pyruvate (○). C, time course of 100 μm adenosine action in control (•) and in the presence of pyruvate (○). Note that the action of ATP was significantly reduced (P < 0.05) while the effect of adenosine was unchanged.

Thus, treatment by NAC or pyruvate revealed similarities and differences in the action of ATP and ROS. Since both antioxidants completely abolished the action of H2O2, but only partially reduced the effect of ATP, our data suggested that the action of this purine engaged both ROS-dependent and ROS-independent mechanisms.

Catalase prevents the action of H2O2 and reduces the effect of ATP

In order to explore the nature of the ROS which could be involved in the action of ATP, next we used catalase, an enzyme which specifically destroys H2O2 (Murrant & Reid, 2001). In our experiments 1200 U ml−1 catalase increased the amplitude of EPCs to 141 ± 6% (n = 6; P < 0.05). When the muscle was pretreated with catalase, the action of 300 μm H2O2 was abolished (99 ± 3%; n = 6; P > 0.05) in accordance with the selective activity of this enzyme for this type of ROS. Like NAC and pyruvate, catalase significantly reduced (but did not abolish) the inhibitory action of 100 μm ATP to 82 ± 4% (n = 7; P < 0.05; Fig. 4). This effect was significantly (P= 0.02 by Mann–Whitney test) less than the control action of ATP. It is worth noting that the depressant action of adenosine in the presence of catalase was unchanged (68 ± 4%; n = 4; P < 0.05).

Figure 4. Effect of catalase on the depressant action of ATP.

Figure 4

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The time course of ATP action in control (•; n = 11) or in the presence of 1200 U ml−1 catalase (○; n = 7). The depressant effect of ATP was significantly reduced in the presence of catalase (*P < 0.05 by Mann–Whitney test).

These data are consistent with the involvement of ROS in the depressant action of ATP. Since catalase specifically destroys H2O2 (Murrant & Reid, 2001), and since this enzyme does not penetrate through cell membranes, our results suggest H2O2 to be a potential intercellular signalling molecule. Thus, the actual site of this ROS generation would be expected to be outside motor nerve terminals. It should be stressed, however, that neither NAC nor pyruvate nor catalase provided complete protection against the depressant action of ATP, indicating the coexistence of ROS-dependent and ROS-independent mechanisms.

Pro-oxidant Fe2+ enhances the action of ATP

Another approach to test the involvement of ROS in the action of ATP consisted of conditioning the muscle with the pro-oxidant Fe2+ (Giniatullin & Giniatullin, 2003). It is known that ferrous ions in the presence of H2O2 promote generation of hydroxyl radicals (·OH), the most active ROS (Winterbourn, 1995; Oh et al. 2000).

Application of 100 μm Fe2+ reduced the amplitude of EPCs to 75 ± 5% (n = 10; P < 0.05). Testing synaptic transmission with 300 μm H2O2 in the presence of Fe2+ revealed a much stronger depression of EPCs (29 ± 4%; n = 6; P < 0.001). Similarly, the depressant effect of 100 μm ATP in the presence of Fe2+ was almost doubled (reduction to 32 ± 4%; n = 7; P < 0.001; Fig. 5B and C). Interestingly, the depressant action of ATP in the presence of pro-oxidant was weakly reversible since recovery after 20 min washout of Fe2+ was only to 51 ± 10% of control (n = 5; Fig. 5C). We previously found similarly poor reversibility after combined application of Fe2+ plus H2O2 (Giniatullin & Giniatullin, 2003). The strong inhibition of the EPCs by ATP was accompanied by prolongation of EPCs since their decay time constant was increased to 137 ± 8% (n = 7; P < 0.05). Accordingly, ATP reduced the EPC area (47 ± 6%; n = 7; P < 0.05) less than the EPC amplitude. Importantly, the very slowly reversible depressant action of ATP was promptly reversed by the antioxidant NAC. On average, NAC (1 mm, applied 20 min after washout of ATP) increased the amplitude of EPCs to 94 ± 11% (n = 4) indicating the recovery of synaptic transmission via redox mechanisms.

Figure 5. Pro-oxidant Fe2+ increases the inhibitory effect of ATP but not of adenosine.

Figure 5

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A, inhibitory action of 100 μm ATP on EPCs in control. B, inhibitory action of 100 μm ATP on EPCs in the presence of 100 μm Fe2+. C, time course of ATP action in control (•) and in the presence of 100 μm Fe2+ (○). D–F, the same protocols obtained after application of 100 μm adenosine. Note strong and poorly reversible depressant action of ATP contrasting with the moderate and reversible action of adenosine.

The augmentation of the inhibitory action of ATP and the modest recovery were specific for P2 receptors since the depressant action of 100 μm adenosine was fully developed and reversible in the presence of 100 μm Fe2+ (73 ± 3%; n = 4; P < 0.05; Fig. 5D–C).

Taken together these data obtained in the presence of pro-oxidant were consistent with the action of ATP on transmitter release via redox mechanisms.

Reactive nitrogen species do not participate in ATP action

Application of ATP can induce cultured rat microglia to produce NO as a potential signalling messenger (Ohtani et al. 2000). NO has also been suggested as a diffusible messenger mediating the action of adenosine at the neuromuscular junction (Rochon et al. 2001; Auld & Robitaille, 2003). In our experimental conditions the NO donors sodium nitroprusside (SNP; 100 μm) or S-nitroso-N-acetyl-penicillamine (SNAP; 250 μm) significantly reduced the amplitude of EPCs to 62 ± 7% (n = 4; P < 0.05; Fig. 6A) or to 72 ± 2% (n = 4; P < 0.05; Supplementary Fig. 3), respectively. In analogy with the observations of Robitaille's group (Rochon et al. 2001; Auld & Robitaille, 2003), pretreatment of muscle with 250 μm SNAP significantly (P < 0.05 by Mann–Whitney test) reduced the effect of adenosine, suggesting that generation of NO contributed to the action of adenosine. Details of these experiments are presented as supplemental material (Supplementary Fig. 3).

Figure 6. The role of reactive nitrogen species in the action of ATP.

Figure 6

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A, depressant action of the NO donor sodium nitroprusside (SNP) on peak EPC amplitude. B, time course of ATP action in control (•), and in the presence of the NO synthase inhibitor l-NAME (100 μm; ○) and the NO scavenger haemoglobin (10 μm; ▿). C, depressant effect of 100 μm ATP in control (n = 11; P < 0.05) and in the presence of l-NAME (n = 4; P < 0.05) or haemoglobin (n = 5; P < 0.05). Note that both inhibitors of the NO cascade did not prevent the action of ATP.

In order to test the possible involvement of NO in the action of ATP, we studied the action of this nucleotide in the presence of several agents impairing the generation or action of NO. Nω-nitro-l-arginine methyl ester (l-NAME), an NO synthase inhibitor, at 100 μm, did not modify the effect of ATP on synaptic transmission (depression to 64 ± 7%; n = 4; Fig. 6B and C). This result suggested that the action of ATP was independent of the activation of NO synthase and generation of NO. Consistent with this, the effect of ATP was not prevented, and was actually even slightly potentiated by 10 μm haemoglobin (53 ± 4%; n = 5; P < 0.05; Fig. 6B and C), probably due to the pro-oxidant property of haemoglobin (Rogers et al. 2003).

Thus, our data suggested that nitrogen reactive species such as NO did not participate in the depressant action of ATP on transmitter release, indicating the selectivity of P2 receptors coupling to ROS cascades.

FOX assay and DCF imaging confirmed production of ROS

The role of ATP receptor activation in the generation of peroxides was assessed using control preparations (incubated for 15 min with the control Ringer solution) and contralateral neuromuscular preparations treated for 15 min with ATP or other purinergic compounds. Figure 7A shows the dose dependence of ATP action on ROS production. The EC50 value (28 μm) for this effect was close to the one (56 μm) obtained from the dose dependence of ATP action on EPCs (Sokolova et al. 2003). ATP at 100 μm concentration increased the production of ROS fourfold (n = 12; P < 0.001) while adenosine (100 μm) was ineffective (n = 3; P > 0.05; Fig. 7B). The effect of ATP was mimicked by a relatively low (10 μm) concentration of 2-MeSADP (n = 4; P < 0.01; Fig. 7B) indicating involvement of P2Y receptors in ROS production. The action of ATP on ROS production, like the depressant action of ATP on EPCs (Sokolova et al. 2003), was eliminated by 100 μm suramin, a non-specific antagonist of P2 receptors (n = 4; P > 0.05; Fig. 7B). Furthermore, pretreatment of the muscle with pertussis toxin, a blocker of Gi/o proteins, dramatically reduced the production of ROS by ATP (n = 4; P > 0.05; Fig. 7B). It is worth noting that pertussis toxin abolished the depressant affect of ATP on ACh release (Sokolova et al. 2003).

Figure 7. ATP but not adenosine increases the production of hydroperoxides by the neuromuscular preparation.

Figure 7

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A, dose–response curve for the action of ATP on production of hydroperoxides measured by FOX assay (n = 4–8 muscle preparations). B, level of peroxides on untreated muscles (control; n = 8) and on muscle treated with 100 μm ATP (15 min exposure; n = 12), 100 μm ATP in the presence of 100 μm suramin (n = 4), 100 μm ATP after exposure to 1 μg ml−1 pertussis toxin (PTX; 18 h exposure; n = 4), 10 μm 2-MeSADP (n = 4), and 100 μm adenosine (n = 3). Note that ATP and 2-MeSADP strongly increased the production of hydroperoxides (***P < 0.001; **P < 0.01) while adenosine was ineffective (P > 0.05). C, time course of ATP action on the ROS-sensitive DCF fluorescence of the muscle fibres. D, averaged data for the changes in the muscle fluorescence in control (exposure 15 min without ATP; n = 4) and in the presence of 100 μm ATP (n = 7), 100 μm UTP (n = 4), 100 μm ADP (n = 4), 100 μm ADP after treatment by 10 μm MRS-2179 (n = 4) and 100 μm adenosine (n = 4). Note that only ATP and ADP produced significant increase in fluorescence while UTP or adenosine were ineffective. Data are presented as a percentage change in fluorescence from baseline (ΔF/F0× 100%, see Methods) and were compared using the non-parametric Mann–Whitney test. *P < 0.05.

We next explored the possibility that, in electrophysiological experiments, antioxidants had not acted via inhibition of ROS activity, rather via block of the ATP receptor function. To this end, we measured (with the FOX method) the production of ROS generated by application of 100 μm ATP in the presence of the antioxidant NAC. NAC (1 mm) actually increased the production of ROS after application of 100 μm ATP (1.63 ± 0.2 μmol g−1; n = 5 versus 1.25 ± 0.10 μmol g−1 in control; P < 0.05). This result indicated that the reduced effect of ATP on EPCs in the presence of NAC obtained in electrophysiological experiments was not caused by impairment of ROS production.

ROS imaging using the dye DCF provided further insights into the mechanism of ATP action. In keeping with previous data (Murrant & Reid, 2001), surface fibres into which DCF was loaded exhibited a diffuse pattern of intracellular fluorescence. After application of 100 μm ATP there was diffuse (without clear compartmentalization) increase (42 ± 4%, n = 7; P < 0.05) in the fluorescence of the muscle fibres (Fig. 7B and C). Interestingly, this effect was not reproduced by 100 μm UTP (n = 4; P > 0.05; Fig. 7D) but was mimicked by 100 μm ADP (n = 4; P < 0.05; Fig. 7D) consistent with observations obtained by the FOX method. The antagonist of P2Y1 receptors MRS-2179 (10 μm) did not abolish the action of ADP (n = 4; P > 0.05; Fig. 7D), just as it failed to antagonize the depressant action of ATP on EPCs, suggesting that P2Y1 receptors were involved neither in ROS production nor in control of transmitter release.

Adenosine (100 μm) produced insignificant changes in the fluorescence (9 ± 4%, n = 4; P > 0.05; Fig. 7D) indicating the specificity of ROS production by ATP. Likewise, control muscles loaded with DCF and exposed to similar periods of illumination showed minimal changes in fluorescence during the experimental period (5 ± 6%; n = 5; Fig. 7D). The latter, together with the lack of UTP or adenosine action, indicated negligible light-induced autooxidation and photoconversion of the dye.

Thus, two independent methods showed significant production of ROS after application of ATP, ADP or its analogue 2-MeSADP in the time scale matching the presynaptic depression of ACh release.

Discussion

The main finding of the present study is a redox-dependent action of ATP on transmitter release at the neuromuscular junction, suggesting that the action of this nucleotide involved ROS production. The action of ATP was prevented by several antioxidants operating via distinct mechanism, and by the H2O2 hydrolysing enzyme catalase, and was strongly potentiated by a pro-oxidant. In contrast, the depressant effect of adenosine operating via P1 receptors was insensitive to the anti- or pro-oxidants. Biochemical and imaging technique confirmed the production of ROS via activation of metabotropic P2Y receptors. To sum up, this is the first report that endogenous ROS produced via activation of P2 receptors are involved in modulation of cholinergic transmission.

Action of anti- and pro-oxidants suggested involvement of ROS in the action of ATP

Several lines of evidence suggest that ROS production following the activation of ATP receptors participated in the inhibition of ACh release from the motor nerve endings. First, the presynaptic action of ATP on transmitter release was reduced by the antioxidant NAC, a precursor of glutathione. Second, pyruvate, which directly scavenges H2O2 (Gyulkhandanyan et al. 2003; Mallet & Sun, 2003), diminished the inhibitory potency of ATP. Third, catalase, which specifically breaks down H2O2 to H2O (Murrant & Reid, 2001), significantly attenuated the action of ATP. It should also be noted that NAC, pyruvate and catalase in our experimental conditions completely prevented the depressant effect of exogenous H2O2. Fourth, the pro-oxidant Fe2+ dramatically augmented the depressant effect of ATP along with facilitation of the inhibitory action of H2O2 (Giniatullin & Giniatullin, 2003). Fifth, exogenous H2O2 mimicked the inhibitory effect of ATP with similar presynaptic site of action and Fe2+-dependent prolongation of the EPC decay (Giniatullin & Giniatullin, 2003).

H2O2 is a comparatively weak oxidant (Tretter & AdamVizi, 1996). However, in the presence of divalent iron, H2O2 is readily converted to the strong oxidant ·OH via the Fenton reaction: Fe2++ H2O2→ Fe3++ OH−+·OH (Winterbourn, 1995; Kress et al. 2002). In our experiments, the partial recovery of synaptic transmission after combined application of ATP plus Fe2+ was consistent with the generation of toxic ROS such as ·OH and perhaps with lipid peroxidation of cell membranes (Kress et al. 2002). This persistent and apparently irreversible depressant action of ATP was, however, readily reversed by antioxidant NAC, indicating the important role of redox mechanisms in control of ACh releasing machinery.

In addition, the depressant action of ATP (Sokolova et al. 2003) and exogenous H2O2 (Giniatullin & Giniatullin, 2003) was completely abolished by PKC inhibitors, suggesting common pathways involved in both mechanisms. Finally, our biochemical and imaging studies confirmed generation of ROS following ATP application and this effect was mimicked by the potent agonist of metabotropic P2Y receptors, 2-MeSADP.

Taken together, all these data concur to suggest that the action of ATP on transmitter release involved ROS generation.

P1 receptors are not coupled to ROS production while P2 receptors are not coupled to reactive nitrogen species production

Adenosine is a powerful inhibitor of ACh release from motor nerve endings (Ginsborg & Hirst, 1972; Ribeiro & Sebastiao, 1987; Redman & Silinsky, 1994). Because of rapid ATP degradation to adenosine it is often difficult to distinguish the direct action of ATP via P2 receptors from the indirect one mediated by adenosine via P1 receptors (Giniatullin & Sokolova, 1998). In the present study, to address the specificity of P2 receptor coupling to ROS production, we tested the depressant action of adenosine in the presence of the same redox agents that modified the action of ATP. However, the inhibitory effect of adenosine was unchanged either in the presence of antioxidants or catalase or after action of pro-oxidant indicating that the presynaptic P1 signalling in skeletal muscle was virtually redox independent.

The specificity of P2 receptors coupling to ROS generation was further confirmed by the fact that we found no evidence of ATP action via production of reactive nitrogen species, another class of free radicals. For instance, NO is an important signalling molecule and is known to play many roles in neural transmission. In cultured rat microglia or endothelial cells extracellular ATP produces NO via P2Y receptors (Ohtani et al. 2000; Buvinic et al. 2002). NO synthase is present in frog perisynaptic Schwann cells (Descarries et al. 1998) and NO has been suggested as a diffusible messenger mediating the action of adenosine at the neuromuscular junction (Rochon et al. 2001; Auld & Robitaille, 2003). In our experiments SNP or SNAP, two NO donors, reduced EPCs in accordance with the previous observations (Lindgren & Laird, 1994; Zefirov et al. 2000). In keeping with data obtained previously (Rochon et al. 2001; Auld & Robitaille, 2003) the inhibitory effect of adenosine on EPCs was reduced in the presence of a high dose of SNAP. However, inhibition of NO synthase by l-NAME did not prevent the depressant action of ATP, and neither did haemoglobin, a scavenger of NO. Slight potentiation of ATP action by haemoglobin was probably based on the pro-oxidant property of the latter, similar to the one of Fe2+ (Goldstein et al. 2003; Rogers et al. 2003).

These data show that the depression of ACh release by ATP at the neuromuscular junction was not related to NO generation.

Receptor mechanisms and sources of ROS

Given that ATP inhibits ACh release via production of ROS, key questions are the identity and localization of ATP receptors mediating this effect.

In our previous study we showed that UTP or ADP, agonists of metabotropic P2Y2,4,6 and P2Y1,12,13 receptors, respectively (Ralevic & Burnstock, 1998; Abbracchio et al. 2003), reproduced the depressant action of ATP on EPCs (Sokolova et al. 2003) suggesting that at least two subtypes of P2Y receptor were involved in control of transmitter release. Which of these receptors were engaged in ROS generation? Our current study provided new insights into the nature of these receptors. While efficient in the inhibition EPCs (Sokolova et al. 2003) and presynaptic Ca2+ currents (Grishin et al. 2005), application of UTP did not induce ROS generation at the neuromuscular preparation. On the contrary, ADP (Sokolova et al. 2003) as well as its potent analogue, 2-MeSADP, reduced the amplitude of EPCs and generated ROS as efficiently as ATP itself.

Out of several antagonists tested previously, such as suramin, PPADS, and Reactive Blue-2, only suramin, a non-selective antagonist of P2 receptors, prevented the inhibitory action of ATP on EPCs (Sokolova et al. 2003). Suramin eliminated also the action of ATP on ROS production as did pertussis toxin, a blocker of Gi/o-coupled metabotropic receptors.

MRS-2179, a specific antagonist of P2Y1 receptors, abolished neither the depressant action of ATP on EPCs nor the effect of ADP on ROS production. It is worth noting that P2Y1 receptors are coupled to pertussis toxin-insensitive Gq proteins (Ralevic & Burnstock, 1998), while the action of ATP on EPCs (and ROS production) was mediated by pertussis toxin-sensitive Gi/o-protein-coupled receptors (present study and Sokolova et al. 2003). This result suggested that other metabotropic receptor subtypes such as P2Y12 or P2Y13 receptors coupled to Gi/o proteins (Zhang et al. 2002; Wirkner et al. 2004) could play a functional role. Consistent with this, the non-selective antagonist of P2Y12 receptors 2′,3′-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP; Ding et al. 2003) significantly reduced the depressant action of ATP on EPCs (S. Grishin & A. Giniatullin, unpublished observations). However, lack of more selective antagonists for these receptor subtypes precluded more definite identification.

Since the neuromuscular preparation contains different cell types and because H2O2, is a freely diffusible agent, several locations of ATP receptors coupled to ROS generation may be considered. Glial perisynaptic Schwann cells express a high density of PTX-sensitive P2Y receptors (Robitaille, 1995), and different P2Y receptor subtypes sensitive to extracellular adenine and uracil nucleotides exist on muscle, muscle blood vessels, and cells scattered between fibres (Cheung et al. 2003; Tung et al. 2004). Two observations of the present study are consistent with the view (see model in Fig. 8) that muscle fibres serve as one main source for ATP-induced ROS generation. First, the efficiency of exogenous catalase (which specifically decomposes H2O2 in the extracellular space; Murrant & Reid, 2001) in reducing the effect of ATP implies that ATP-induced ROS (presumably H2O2) must diffuse through the extracellular space to target ACh release. Second, ROS imaging indicated that ATP action was accompanied by increased ROS production throughout the body of the muscle without clear compartmentalization.

Figure 8. Scheme of ROS-dependent and ROS-independent actions of ATP on transmitter release.

Figure 8

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Presynaptically released (together with ACh) or postsynaptically liberated (due to the muscle depolarization) ATP acts on P2Y receptors localized on muscle fibres to produce ROS (including superoxide, O2−, a precursor of H2O2). Diffusible H2O2 is released into the extracellular space and targets the motor nerve ending. The presynaptic depressant action of H2O2 on transmitter releasing machinery can be abolished by catalase or potentiated by Fe2+. The second ROS-independent component of ATP action on quantal release involves activation of PLA2 and generation of arachidonic acid (AA), which directly or indirectly inhibits Ca2+ channels activity. The potential role of Schwann cells in purinergic signalling is not shown for the sake of simplicity. The inhibitory pathways are indicated with black arrows, while release pathways are shown with white arrows.

The idea of a two-component mechanism of ATP action (mimicked by UTP or ADP, respectively) on transmitter release was supported by the fact that all ROS scavengers which fully abolished the action of H2O2 only halved the depressant effect of ATP. Therefore, we suggest that ATP inhibits ACh release via a ROS-dependent mechanism on the one hand, and through a ROS-independent mechanism on the other (Fig. 8). The former mechanism (mimicked also by ADP) is likely to involve H2O2, which could directly impair the ACh releasing machinery (Giniatullin & Giniatullin, 2003), while the latter mechanism (mimicked by UTP) probably engages arachidonic acid, which could reduce the presynaptic Ca2+ influx (Grishin et al. 2005). Thus, it seems likely that in control conditions two components of ATP action (Fig. 8) contributed to depression of ACh release.

In summary, our results suggest that the ROS, perhaps H2O2, acts as an intercellular signal mediator for an important component of the ATP-induced inhibition of synaptic transmission.

Functional implications

The preferential action of ROS generated by ATP on the motor nerve terminals is consistent with the generally accepted view that the motoneurones are particularly vulnerable to ROS (Martin & Liu, 2002; Bergmann & Keller, 2004). The synergistic, harmful action of ATP plus Fe2+ (probably due to significant amounts of hydroxyl radicals produced via the Fenton reaction) could contribute to motoneurone damage in neurogenerative diseases such as amyotrophic lateral sclerosis, which is accompanied by disturbances in iron metabolism and early impairment of neuromuscular transmission (Frey et al. 2000).

Apart from pathological conditions, growing evidence also suggests that ROS could play a role as physiological signalling molecules (Suzuki et al. 1997; Oh et al. 2000; Servitja et al. 2000; Suzukawa et al. 2000; Goldstein et al. 2003; Murakami et al. 2003). Endogenous ROS are produced and released by contracting muscle (for review see Murrant & Reid, 2001) and we previously hypothesized that ROS, in particular H2O2, could serve as feedback modulators of ACh release at the neuromuscular junction (Giniatullin & Giniatullin, 2003). Inhibitory feedback control was proposed also for extracellular ATP (Giniatullin & Sokolova, 1998; Galkin et al. 2001; Sokolova et al. 2003), which is coreleased with ACh from motor nerve terminals (Redman & Silinsky, 1994). We now show a direct link between these two pathways since ATP, via metabotropic P2Y receptors, could use endogenous ROS to diminish transmitter release from the motor nerve endings.

The efficiency of ROS-dependent purinergic modulation in vivo (Fig. 8) would be dependent on several factors, in particular on the sources of extracellular ATP, precise location of P2Y receptors and the distance from the site of ROS generation to the motor nerve ending. Synaptic corelease of ATP could provide a peak concentration in the synaptic cleft of up to 1 mm (Choi et al. 2003). However, ATP could be released also directly from the muscle fibres (Ribeiro et al. 1996). Because of the limited resolution of DCF imaging, we did not solve the issue of precise location of ROS-generating P2Y receptors. However, diffuse DCF signalling along muscle fibres during the action of ATP suggests that the muscle-derived ROS (in particular freely diffusible H2O2) could affect the neighbouring nerve terminals. In addition, the ability of glial cells to respond to ATP by ROS generation (Murakami et al. 2003; Sauer et al. 2003) suggests that in vivo perisynaptic Schwann cells could also participate in ROS synthesis following activation by synaptically released ATP (Robitaille, 1995).

Because of the involvement of many intra- and intercellular mechanisms, ATP-mediated modulation of ACh release should be slower than the fast negative feedback control mediated by adenosine via P1 receptors (Ribeiro et al. 1996; Sokolova et al. 2003). It means that ATP-dependent ROS mechanisms most likely participate in muscle fatigue during periods of prolonged activity rather than during short episodes of neuromuscular transmission.

Assuming that endogenous ATP contributes to muscle fatigue, our findings predict that the extent of muscle fatigue may depend on the efficiency of endogenous antioxidant mechanisms and its dynamic balance with endogenous pro-oxidants. Consistent with this notion, antioxidant NAC is reported to efficiently relieve muscle fatigue in man (Reid et al. 1994). Arachidonic acid, which we suggested as a messenger for the ROS-independent inhibitory mechanism of ATP, perhaps plays a complex role in the control of ACh release since this agent could reduce not only the presynaptic Ca2+ influx but also the endogenously released ATP (Cunha et al. 2000), attenuating therefore the efficiency of feedback purinergic signalling.

Further experiments will clarify whether the ATP modulation of synaptic transmission via ROS is restricted to the neuromuscular junction only.

Acknowledgments

This work was supported by grants from RFBR (02-04-49944 and 03-04-96015), INTAS and FIRB (Italy). The authors are grateful to Andrea Nistri and Vladimir Snetkov for the helpful discussion and Anton Akulov for help with the FOX assay.

Supplemental material

The online version of this paper can be accessed at:

10.1113/jphysiol.2005.084186

http://jp.physoc.org/cgi/content/full/jphysiol.2005.084186/DC1 and contains supplemental material consisting of three figures entitled: The inhibitory effect of 2-MeSADP and the action the P2Y1 antagonist, MRS-2179; The inhibitory effect of ATP is reduced by NAC in the presence of 1 mm Mg2+; and The inhibitory effect of adenosine is reduced in the presence of the NO donor, SNAP.

Supplemental Data
jphysiol_2005.084186_index.html (1,017B, html)

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