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Selective Modulation of Ligand-Gated P2X Purinoceptor Channels by Acute Hypoxia Is Mediated by Reactive Oxygen Species

School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom

Received March 30, 2004; accepted August 24, 2004

	Abstract

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Purinergic excitatory synapses use ATP to mediate fast synaptic transmission via activation of P2X receptor cation channels, and this response can be altered by acute hypoxia. This study examined the effect of acute hypoxia on cloned homo- and heteromeric P2X₂ and P2X₃ receptors expressed in human embryonic kidney 293 cells. In cells expressing homomeric P2X₂ receptors, perfusion of 5 µM ATP (EC₂₅) induced an inward whole-cell current that showed little desensitization during repeated exposures under continuously normoxic conditions. Exposure to a hypoxic ATP solution (pO₂, 25-40 mm Hg) significantly reduced the whole-cell current to 49% of normoxic control. This hypoxic inhibition of P2X₂-mediated inward current was maintained across all potentials when a voltage-step protocol was applied. In contrast, currents mediated by homomeric P2X₃ receptors or heteromeric P2X_2/3 receptors were insensitive to an acute hypoxic challenge. One mechanism whereby hypoxia may modulate P2X₂ channels is via the production of reactive oxygen species (ROS). H₂O₂ (1.8 mM) reversibly reduced homomeric P2X₂ whole-cell currents to 38% of control. Furthermore, H₂O₂ attenuated the effect of hypoxia on homomeric P2X₂ whole-cell currents. Inhibitors of the mitochondrial electron transport chain that reduce (rotenone and myxothiazol) or increase (antimycin A) the production of ROS altered the magnitude of P2X₂-mediated currents. In summary, this is the first report indicating that acute hypoxia is able to regulate the activity of any ligand-gated ion channel. Furthermore, our data show that acute hypoxia selectively modulates the P2X₂ receptor and that the response of P2X₂ receptor subunits to hypoxia is mediated through the mitochondrial production of ROS.

Purinoceptors are classified as P1 (adenosine receptors, A₁-A₃) or P2 (ATP receptors) on the basis of their pharmacological, biochemical, and molecular properties. The P2 family of purinoceptors can be further divided into two subfamilies, the G protein-coupled P2Y receptor family and the ligand-gated P2X receptor family. Activation of P2X receptors results in the influx of cations such as Na⁺, K⁺, and Ca²⁺ across the plasma membrane, which increases the intracellular Ca²⁺ concentration. To date, seven members of the P2X family (P2X_1-7) have been cloned (North, 2002) whose structural properties differ from that of other members of the ligand-gated ion channel superfamily. The primary structure of the P2X receptor subunit consists of two potential transmembrane domains (M1 and M2), intracellular N- and C-terminal domains, and a large cysteine-rich extracellular domain that contains an ATP binding motif and sites for glycosylation (Valera et al., 1994; Brake et al., 1994; Surprenant et al., 1995). The second transmembrane domain (M2) and a hydrophobic H5 sequence immediately before the M2 domain is thought to form the ion pore and binding sites of the P2X receptor (Brake et al., 1994; Valera et al., 1994). Furthermore, there is increasing evidence that three P2X subunits are required to form a functional receptor (Nicke et al., 1998; Stoop et al., 1999).

P2X receptor subtypes are widely expressed in the brain and nervous system and are associated with autonomic sensory motor reflexes, sensory afferents, and olfactory and visual systems (North, 2002). Furthermore, P2X receptors are expressed in cells that respond to hypoxia, such as the carotid body, pulmonary smooth muscle, and PC12 cells (Kobayashi et al., 1998; Prasad et al., 2001; Chootip et al., 2002). Indeed, in the carotid body, hypoxia leads to an increased afferent discharge rate, and this is mediated via P2X₂ and P2X₃ receptors (Zhang et al., 2000; Prasad et al., 2001). However, the response of cloned P2X receptor cation channels to hypoxia is unknown. Therefore, the primary purpose of this study was to evaluate the effect of hypoxia on cloned P2X₂ and P2X₃ homo- and heteromeric receptors expressed in human embryonic kidney (HEK) 293 cells.

	Materials and Methods

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Expression Systems. HEK293 cells that stably express either homomeric rat P2X₂ receptors, homomeric human P2X₃ receptors, or rat P2X₂ and P2X₃ receptors in a bicistronic vector, thus giving rise to heteromeric P2X_2/3 receptor channels, were used in this study. These cell lines have been previously characterized (Brake et al., 1994; Evans et al., 1995; Valera et al., 1995; Kawashima et al., 1998). All the P2X cell lines were maintained in Dulbecco's modified Eagle's media supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1% antibiotic antimycotic, 100 µg/ml gentamicin, and 300 µg/ml Geneticin (G418) (all purchased from Invitrogen Ltd., Renfrew, Renfrewshire, UK) in a humidified incubator gassed with 5% CO₂/95% air. Cells were passaged every 7 days using Ca²⁺- and Mg²⁺-free phosphate-buffered saline (Invitrogen Ltd.). For the electrophysiological experiments described herein, HEK293 cells were plated onto glass coverslips and cultured at 37°C for 1 to 5 days.

Reagents. All compounds were of analytical grade and obtained from BDH Laboratory Supplies (Poole, Dorset, UK). Disodium ATP, ,-methylene ATP (,-MeATP), H₂O₂, rotenone, antimycin A, myxothiazol, EGTA, and HEPES were obtained from Sigma Chemical (Poole, Dorset, UK). ATP and ,-MeATP were dissolved at 100 mM in distilled H₂O, divided into aliquots, and stored at -20°C. For recording, ATP and ,-MeATP were diluted to the desired concentration in bath solution. Stock solutions of the mitochondrial inhibitors were prepared in either ethanol (50 µg/ml antimycin A and 10 mM myxothiazol) or dimethyl sulfoxide (100 mM rotenone), and aliquots were stored at -20°C. Oxygen-free nitrogen gas and medical air (21% O₂) were obtained from BOC Ltd. (Guildford, Surrey, UK). All tubing was gas-impermeant (Tygon tubing; BDH).

Electrophysiology. Whole-cell currents were recorded at room temperature. Recording pipettes pulled from borosilicate glass had resistances of 4 to 6 M when filled with pipette solution that contained 117 mM KCl, 10 mM NaCl, 2 mM MgCl₂, 1 mM CaCl₂, 11 mM EGTA, 2 mM Na-ATP, and 11 mM HEPES, with the pH adjusted to 7.2 with KOH. The bath solution contained 135 mM NaCl, 5 mM KCl, 1.2 mM MgCl₂, 2.5 mM CaCl₂, 5 mM HEPES, and 10 mM glucose, with the pH adjusted to 7.4 with NaOH. Hypoxic solutions were bubbled with N₂ gas for at least 30 min before perfusion of cells, which produced no shift in pH. Normoxic solutions were either equilibrated with room air or bubbled with medical air (21% O₂). pO₂ was measured (at the cell) using a polarized (-800-mV) carbon fiber electrode (Mojet et al., 1997). For the experiments reported herein, the pO₂ values were 150 (normoxia) and 25 to 40 (hypoxia) mm Hg.

Resistive feedback voltage clamp was achieved using an Axopatch 200B amplifier (Axon Instruments Inc., Union City, CA). Voltage protocols were generated and currents recorded using pCLAMP 8.0 software employing a Digidata 1322 A/D convertor (Axon Instruments Inc.). Data were filtered (4-pole Bessel) at 2 kHz and digitized at 5 kHz. After transition to the whole-cell configuration capacitance, transients were compensated and measured.

To monitor the response of P2X-mediated currents to repeated exposures of ATP under normoxic or hypoxic conditions, the following protocol was used. Having gained the whole-cell configuration, the cells were voltage-clamped at a potential of -70 mV. Control recordings were made by exposing cells to three exposures of ATP under normoxic conditions. The bathing solution was then switched to either a normoxic or hypoxic solution for 2 min before examining the response to three subsequent exposures of ATP under normoxic or hypoxic conditions. Finally, the bathing solution was switched back to normoxic conditions for 2 min, and the subsequent responses to the final three exposures of ATP under normoxic conditions were monitored. In some experiments, cells were held at a potential of 0 mV, and 200-ms voltage steps were applied from -100 to +100 mV in 20-mV increments were applied under normoxic and hypoxic conditions. To create concentration-response curves for P2X-mediated currents under normoxic and hypoxic conditions, cells were held in the whole-cell configuration at a potential of -70 mV and perfused with either normoxic or hypoxic bathing solution for 2 min. The cells were then exposed to ATP under either normoxic or hypoxic conditions. Each cell was only exposed to one concentration of ATP under either normoxic or hypoxic conditions. The P2X-mediated currents generated in response to the first application of ATP were then plotted, with each point representing at least five cells.

Data Analysis. Data analyses were performed using the pCLAMP 8.0 suite of software (Axon Instruments Inc.). Data are reported as mean ± S.E.M. values. Statistical comparisons were made using the paired or unpaired Student's t test as appropriate, with P < 0.05 regarded as significant. Concentration-response curves for ATP and H₂O₂ were fitted to the Hill equation using GraphPad Prism software (GraphPad Software Inc., San Diego, CA).

	Results

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Hypoxia Modulates Homomeric P2X₂-Mediated Currents. To evaluate the effect of hypoxia on P2X₂-mediated currents expressed in HEK293 cells, a concentration of ATP was required that would elicit responses of similar magnitude after repeated exposures to ATP. Extracellular ATP activated P2X₂-mediated currents in a concentration-dependent manner, with an EC₅₀ of 9.59 µM and a Hill slope of 2.75 (n = 28, data not shown). At high concentrations of ATP (100 µM), the magnitude of the P2X₂-mediated current elicited in response to ATP decreased with repeated exposures caused by desensitization of the P2X₂ receptor (data not shown). However, at a concentration of 5 µM ATP (which equates to the EC₂₅ value), P2X₂-mediated currents of similar magnitude could be evoked repeatedly, even in the presence of the divalent cations Ca²⁺ and Mg²⁺. As a result of these observations, ATP concentrations of 5 µM were used in the following experiments that evaluated the effect of hypoxia on P2X₂-mediated currents.

Figure 1A shows exemplar ATP-induced whole-cell homomeric P2X₂-mediated currents recorded under normoxic and hypoxic conditions at a potential of -70 mV in the presence of external Ca²⁺ and Mg²⁺. After exposing the cells to a hypoxic solution for 2 min, the magnitude of the P2X₂-mediated current activated in response to 5 µM ATP was reduced compared with that under normoxic conditions. This reduction in the magnitude of the P2X₂-mediated current was partially relieved upon returning to normoxic conditions. Figure 1B summarizes the response of P2X₂-expressing cells when exposed to repeated exposures of 5 µM ATP under either continuously normoxic conditions or exposed to an acute hypoxic challenge. Under continuously normoxic conditions, there was no difference in the magnitude of the P2X₂-mediated current over time (the magnitude of the last exposure was 0.96 ± 0.09 that of the first exposure, n = 5). In contrast, exposure to a hypoxic challenge significantly reduced the magnitude of the P2X₂-mediated current activated in response to 5 µM ATP [0.51 ± 0.09 that of the current under normoxic conditions (P < 0.01, n = 5)]. The reduction in the magnitude of the P2X₂-mediated whole-cell current was partially reversible upon washout (0.68 ± 0.16 compared with control, n = 5).

View larger version (17K): [in this window] [in a new window]   Fig. 1. Hypoxia attenuates ATP-induced currents mediated by the homomeric P2X2 receptor. HEK293 cells expressing homomeric P2X2 receptors were held at a potential of -70 mV in the presence of external Ca2+ and Mg2+ and repeatedly exposed to extracellular ATP. A, illustrative trace of a cell exposed to repeated exposures to 5 µM ATP under normoxic and hypoxic conditions. The stars represent the application of ATP for 10 s; the arrow indicates zero current. B, summarized data showing that exposure to an acute hypoxic challenge attenuates the magnitude of P2X2-mediated currents evoked in response to 5 µM ATP () compared with cells held under continuously normoxic conditions ().

The effect of hypoxia on the magnitude of the P2X₂-mediated current was also examined using a voltage-step protocol. Figure 2 shows illustrative traces recorded sequentially in response to 200-ms voltage steps from -100 mV to +100 mV in 20-mV increments from a holding potential of 0 mV under control conditions in the absence of ATP (Fig. 2A) and in the presence of 5 µM ATP under hypoxic (Fig. 2B), normoxic (Fig. 2C), and hypoxic (Fig. 2D) conditions. In the absence of ATP, the voltage-step protocol elicited small endogenous currents (Fig. 2A). Exposure to ATP under hypoxic conditions activated an inwardly rectifying P2X₂-mediated current (Fig. 2B). Switching the perfusate from a hypoxic to normoxic solution increased the magnitude of the P2X₂-mediated current (Fig. 2C), and this increase in current was fully reversible upon returning to hypoxic conditions (Fig. 2D). Figure 2E shows the mean control-subtracted data from six cells. Currents are normalized to the current evoked in response to 5 µM ATP at a potential of -100 mV under hypoxic conditions. Under hypoxic conditions, the P2X₂-mediated currents reversed at a potential of -8.3 mV, whereas under normoxic conditions, the currents were 38 ± 10% larger (P < 0.05, n = 7) and reversed at a potential of -4.2 mV.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Normoxia relieves the hypoxic inhibition of the ATP-induced current in cells expressing homomeric P2X2 receptors. Cells expressing the P2X2 receptor were held at a potential of 0 mV, and 200-ms voltage steps were applied from -100 to +100 mV in 20-mV increments. A-D, illustrative P2X2-mediated currents evoked in response to the voltage-step protocol. Cells were initially exposed to 5 µM ATP in the presence of external Ca2+ and Mg2+ under hypoxic conditions and then normoxic conditions before returning to hypoxic conditions. E, summarized current-voltage relationship from six cells. Currents are normalized to the current recorded at a potential of -100 mV under hypoxic conditions.

A similar modulation of P2X₂-mediated currents by hypoxia was obtained when the reverse protocol was applied, i.e., when the cells expressing the P2X₂ receptor were sequentially exposed to control conditions in the absence of ATP and in the presence of 5 µM ATP under normoxic, hypoxic, and normoxic conditions. In this instance, hypoxia reduced the magnitude of P2X₂-mediated currents at all potentials less than -20 mV by 20% (P < 0.05, n = 7), and this was fully reversible upon returning to a normoxic solution (data not shown).

Hypoxia Does Not Modulate Homomeric P2X₃ Channels. The P2X-mediated current in sensory neurons is mediated principally through homomeric P2X₃ and heteromeric P2X_2/3 channels (Dunn et al., 2001). Therefore, the responses of homomeric P2X₃ and heteromeric P2X_2/3 channels to hypoxia were also examined, with the results summarized in Figs. 3 and 4. As shown in Fig. 3A, exposing cells expressing homomeric P2X₃ receptors to 10 µM ATP under normoxic conditions in the presence of external Ca²⁺ and Mg²⁺ activated an inward current that rapidly inactivated (peak normoxic mean P2X₃-mediated current, -124.1 ± 53.7 pA/pF, n = 9). The inactivation of the P2X₃-mediated current under normoxic conditions was best fit with two time constants (₁ = 1473 ± 234 and ₂ = 214 ± 37.5 ms, n = 9; Fig. 3D). Because a second exposure to ATP significantly reduced the magnitude of the homomeric P2X₃ whole-cell current (data not shown), we could not perform repeated exposures to ATP on the same cell. Therefore, to examine the response to hypoxia, we used separate cell populations, and immediately after gaining the whole-cell configuration, the cells were exposed to a hypoxic bathing solution for 2 min before exposing the cells to ATP. Under hypoxic conditions, 10 µM ATP activated a P2X₃-mediated current whose magnitude was comparable with that activated under normoxic conditions (mean peak hypoxic P2X₃-mediated current, -110.4 ± 24.5 pA/pF, n = 7; Fig. 3C). The inactivation of the P2X₃-mediated current under hypoxic conditions was best fit with two time constants (₁ = 1059 ± 119 and ₂ = 163 ± 31.7 ms; Fig. 3D).

View larger version (19K): [in this window] [in a new window]   Fig. 3. Currents mediated by the homomeric P2X3 receptor are insensitive to hypoxia. Illustrative P2X3-mediated currents from cells held at a potential of -70 mV in the presence of external Ca2+ and Mg2+ and exposed to extracellular ATP (10 µM) under either normoxic (A) or hypoxic (B) conditions. The stars represent the application of ATP for 10 s; the arrows indicate zero current. C-D, summarized data showing that hypoxia has no effect on either (C) the magnitude of the peak ATP-induced P2X3-mediated currents or (D) the inactivation time constants.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Heteromeric P2X2/3-mediated currents are insensitive to hypoxia. HEK293 cells expressing heteromeric P2X2/3 receptors were held at a potential of -70 mV in the presence of external Ca2+ and Mg2+ and repeatedly exposed to extracellular ATP. A, illustrative trace of a cell exposed to repeated exposures to 10 µM ATP under normoxic and hypoxic conditions. The stars represent the application of ATP for 10 s; the arrow indicates zero current. B, summarized data showing that exposure to an acute hypoxic challenge has no affect on the magnitude of P2X2/3-mediated currents () compared with cells held under continuously normoxic conditions ().

Similar results were obtained when the cells were exposed to ,-MeATP, a P2X₁- and P2X₃-selective agonist that differentiates between P2X₂ and P2X₃ receptor subunits (data not shown). Exposure to 10 µM ,-MeATP activated inactivating P2X₃-mediated currents under either normoxic or hypoxic conditions. The magnitudes of the peak P2X₃-mediated currents were similar under normoxic and hypoxic conditions (normoxia, -129 ± 35.6 pA/pF, n = 8; hypoxia, -80.6 ± 21.7 pA/pF, n = 8). The inactivation of the P2X₃-mediated currents under normoxic and hypoxic conditions was best fit with two time constants (normoxia, ₁ = 1790 ± 322 and ₂ = 390 ± 138 ms; hypoxia, ₁ = 912 ± 109 and ₂ = 172 ± 18.6 ms).

Hypoxia Does Not Modulate Heteromeric P2X_2/3 Channels. The observation that the peak activation of homomeric P2X₂ receptors was sensitive to hypoxia whereas that of homomeric P2X₃ receptors was not was intriguing. Because the sustained component of the P2X-mediated current in sensory neurons is primarily mediated through P2X_2/3 heteromeric channels (Dunn et al., 2001), we examined the response to hypoxia of P2X_2/3 heteromeric channels expressed in HEK293 cells. Figure 4A shows exemplar ATP-induced whole-cell heteromeric P2X_2/3-mediated currents recorded under normoxic and hypoxic conditions at a potential of -70 mV in the presence of external Ca²⁺ and Mg²⁺. Figure 4B summarizes the response of P2X_2/3-expressing cells when exposed to repeated exposures of 10 µM ATP under either continuously normoxic conditions or exposed to an acute hypoxic challenge. Under continuously normoxic conditions, P2X_2/3-mediated currents reduced in magnitude over time (the magnitude of the last exposure was 0.77 ± 0.10 that of the first exposure, n = 3). Although exposure to a hypoxic challenge reduced the magnitude of the P2X_2/3-mediated current activated in response to 10 µM ATP (0.70 ± 0.06 that of the first exposure to ATP, P < 0.01, n = 5), this reduction was not different from that observed in cells held under continuously normoxic conditions. These data suggest that hypoxia does not modulate the activation of heteromeric P2X_2/3 receptor channels by ATP in the presence of external Ca²⁺ and Mg²⁺.

Hypoxia Selectively Alters the Concentration-Response Curves of P2X Receptors. To further examine the effect of hypoxia on the P2X receptors used in this study, full concentration-response curves were generated under either normoxic or hypoxic conditions. Figure 5A shows the concentration-response curve of P2X₂-mediated currents evoked in response to the first exposure to ATP under either normoxic or hypoxic conditions. Under normoxic conditions, extracellular ATP activated P2X₂-mediated currents in a concentration-dependent manner with an EC₅₀ of 3.73 µM. In contrast, under hypoxic conditions, the concentration-response curve was shifted to the right, with an EC₅₀ of 6.34 µM. However, the magnitude of the P2X₂-mediated currents evoked in response to a maximal concentration of ATP (100 µM) in either normoxia or hypoxia was not significantly different.

View larger version (13K): [in this window] [in a new window]   Fig. 5. The effect of hypoxia on the concentration-response curves of homomeric P2X2, homomeric P2X3, and heteromeric P2X2/3 receptors. HEK293 cells expressing homomeric P2X2, homomeric P2X3, or heteromeric P2X2/3 were held at a potential of -70 mV and perfused with either normoxic or hypoxic bathing solution for 2 min. The cells were then exposed to ATP in normoxic or hypoxic solution. Each cell was only exposed to one concentration of ATP under either normoxic or hypoxic conditions. A, exposure to a hypoxic solution () shifts the concentration-response curve of homomeric P2X2 receptors to the right relative to cells exposed to normoxic solutions (). B, concentration-response curve of heteromeric P2X2 receptors under normoxic () and hypoxic () conditions. C, concentration-response curve of homomeric P2X3 receptors under normoxic () and hypoxic () conditions.

It was possible that the concentration of ATP (10 µM, which equates to the EC₉₀ value) used to examine the effect of hypoxia on the homomeric P2X₃ and heteromeric P2X_2/3 receptors could have masked any effect of hypoxia on these P2X receptors. Therefore, full concentration-response curves were also generated under normoxic and hypoxic conditions for these receptors. Figure 5, B and C, shows the concentration-response curves for heteromeric P2X_2/3- and homomeric P2X₃-mediated currents, respectively. Hypoxia had no effect on either heteromeric P2X_2/3- and homomeric P2X₃-mediated currents. Thus, in agreement with our initial findings, hypoxia selectively modulates homomeric P2X₂ receptors.

Hydrogen Peroxide Mimics the Effect of Hypoxia on Homomeric P2X₂-Mediated Currents. One of the mechanisms whereby hypoxia may modulate channel function is through the production of reactive oxygen species (ROS). We examined whether the hypoxic modulation of homomeric P2X₂ receptors was mediated by ROS by examining the response of P2X₂-mediated currents to H₂O₂ under normoxic and hypoxic conditions. Figure 6A shows the effect of perfusing 1.8 mM H₂O₂ on the magnitude of ATP-induced whole-cell P2X₂-mediated currents under continuously normoxic conditions at a potential of -70 mV in the presence of external Ca²⁺ and Mg²⁺. After exposing the cells to 1.8 mM H₂O₂ solution for 2 min, the magnitude of the P2X₂-mediated current activated in response to 5 µM ATP was significantly reduced compared with that under control conditions (to 0.61 ± 0.15 that of the first exposure to ATP, P < 0.05, n = 5). This reduction in the magnitude of the P2X₂-mediated current was partially relieved upon returning to control conditions (0.76 ± 0.20 that of the first exposure to ATP, n = 3). The H₂O₂-induced reduction in the magnitude of the P2X₂-mediated current was concentration-dependent with an IC₅₀ of 0.73 mM (Fig. 6B).

View larger version (14K): [in this window] [in a new window]   Fig. 6. H2O2 mimics the effect of hypoxia on homomeric P2X2-mediated currents and attenuates the effect of hypoxia. A, summarized data showing that the perfusion of 1.8 mM H2O2 significantly reduces the magnitude of P2X2-mediated whole-cell currents evoked in response to 5 µM ATP. The inset shows an illustrative trace of P2X2-mediated currents in the absence and presence of 1.8 mM H2O2 at a holding potential of -70 mV. The stars represent the application of ATP for 10 s; the arrow indicates zero current. B, H2O2 concentration-response curve of P2X2-mediated currents. The IC50 value is 0.74 mM. C, summarized data showing that pre-exposure to 1.8 mM H2O2 attenuates the effect of hypoxia on P2X2-mediated currents evoked in response to 5 µM ATP. For comparison, the open symbols are in the absence of H2O2 and taken from Fig. 1B.

If hypoxia reduces the magnitude of P2X₂-mediated currents through the production of ROS, then pre-exposure to H₂O₂ should abolish this effect. Figure 6C shows the mean data recorded from six cells. In the presence of 1.8 mM H₂O₂ and hypoxia, the magnitude of the P2X₂-mediated current activated in response to 5 µM ATP in the presence of external Ca²⁺ and Mg²⁺ was 0.77 ± 0.07 that of the current under normoxic conditions (P < 0.05, n = 6). This hypoxic-induced reduction in the magnitude of the P2X₂-mediated current in the presence of 1.8 mM H₂O₂ was attenuated compared with that in the absence of H₂0₂ (0.51 ± 0.09, P < 0.05, the open symbols for comparison are taken from Fig. 1B). Thus, H₂O₂ itself mimics the effect of hypoxia on P2X₂-mediated currents, and the presence of H₂O₂ attenuates the effect of a subsequent hypoxic challenge.

Mitochondrial Inhibitors That Modulate the Production of ROS Mimic the Effect of Hypoxia on Homomeric P2X₂-Mediated Currents. To examine the source of the ROS that might underlie the hypoxic inhibition of P2X₂ currents, mitochondrial inhibitors of the electron transport chain (ETC) were employed; these ETC inhibitors were rotenone, antimycin A, and myxothiazol, and each block the mitochondrial ETC at a different site of the ETC. Figure 7A shows that the perfusion of cells expressing P2X₂ receptors with rotenone (100 nM), a complex I inhibitor that causes a reduction in ROS, significantly increased the magnitude of P2X₂-mediated currents in response to the first exposure to 5 µM ATP after the application of rotenone (1.22 ± 0.016 that of the first exposure to ATP, P < 0.05, n = 6). The magnitude of P2X₂-mediated currents in response to subsequent exposures to ATP in the presence of rotenone declined over time (sixth exposure was 0.78 ± 0.14 that of the first exposure to ATP, n = 6) and was irreversible upon washout (0.62 ± 0.11 that of the first exposure to ATP, n = 5). This was a common phenomenon with all the mitochondrial inhibitors and is presumably caused by metabolic poisoning of the cell.

View larger version (16K): [in this window] [in a new window]   Fig. 7. Inhibitors of the mitochondrial ETC modulate P2X2-mediated currents. HEK293 cells expressing homomeric P2X2 receptors were held at a potential of -70 mV in the presence of external Ca2+ and Mg2+ and repeatedly exposed to extracellular ATP (5 µM). A, perfusion of 100 nM rotenone, a complex I inhibitor that decreases the production of ROS, caused a transient increase in the magnitude of P2X2-mediated currents evoked in response to 5 µM ATP (). The presence of 5 mM succinate, a complex II substrate, abolished the increase in the P2X2-mediated currents (). B, perfusion of 10 µg/ml antimycin A, a complex III inhibitor that increases the production of ROS, caused a reduction in the magnitude of P2X2-mediated currents evoked in response to 5 µM ATP. C, perfusion of 100 nM myxothiazol, a complex III inhibitor that decreases the production of ROS, caused a transient increase in the magnitude of P2X2-mediated currents evoked in response to 5 µM ATP.

If the transient increase in the P2X₂-mediated current is caused by the reduction in the production of ROS, then this should be overcome by providing succinate, a complex II substrate. As shown in Fig. 7A, rotenone failed to induce an increase in the magnitude of P2X₂-mediated currents in response to the first exposure to 5 µM ATP in the presence of 5 mM succinate. Rather, P2X₂-mediated currents declined over time (to 0.54 ± 0.08 that of the first exposure to ATP, n = 3), and this was irreversible upon washout (0.52 ± 0.03 that of the first exposure to ATP, n = 3). This reduction in the magnitude of the P2X₂-mediated currents was similar to those observed in the absence of succinate (see above).

To further elucidate the role of ROS in the modulation of P2X₂-mediated currents, we examined the effect of two inhibitors of complex III in the mitochondrial ETC, antimycin A and myxothiazol. These inhibitors act at different sites within complex III to increase or decrease ROS, respectively. Perfusion of antimycin A (10 µg/ml) caused a reduction in the magnitude of P2X₂-mediated currents over time (to 0.46 ± 0.12 that of the first exposure to ATP, P < 0.01, n = 5), and this was irreversible upon washout (0.41 ± 0.11 that of the first exposure to ATP, n = 3). In contrast, myxothiazol (100 nM) caused a transient increase in P2X₂-mediated currents similar to that observed with rotenone. The magnitude of P2X₂-mediated current in response to the first exposure to 5 µM ATP in the presence of myxothiazol was 1.28 ± 0.08 that of the first exposure to ATP (P < 0.05, n = 4). The magnitude of P2X₂-mediated currents in response to subsequent exposures to ATP in the presence of myxothiazol declined over time (sixth exposure was 0.70 ± 0.04 that of the first exposure to ATP, P < 0.05, n = 4) and was irreversible upon washout (0.58 ± 0.22 that of the first exposure to ATP, n = 3). Thus, together these data suggest that changes in the mitochondrial production of ROS can modulate P2X₂ currents.

Mitochondrial Inhibitors That Modulate the Production of ROS Do Not Affect Homomeric P2X₃- or Heteromeric P2X_2/3-Mediated Currents. The findings thus far suggest that hypoxia selectively modulates homomeric P2X₂-mediated currents and that this is mediated, in part, via the mitochondrial production of ROS. If this is true, then H₂O₂ and the mitochondria inhibitors should have no effect on either homomeric P2X₃ or heteromeric P2X_2/3 receptors. Figure 8A shows the magnitude of peak and sustained currents mediated by heteromeric P2X_2/3 receptors evoked in response to the first exposure to 0.5 µM ATP (which equates to the EC₂₅ value) in the presence or absence of the ROS mediators. The magnitudes of the peak and sustained P2X_2/3-mediated currents in the presence of 1.8 mM H₂O₂ (85.3 ± 10.7 pA/pF and 19.9 ± 2.5 pA/pF, n = 10), 100 nM rotenone (88.2 ± 15.1 pA/pF and 22.7 ± 3.8 pA/pF, n = 11), 100 nM myxothiazol (105.2 ± 12.1 pA/pF and 22.7 ± 5.3 pA/pF, n = 12), or 10 µg/ml antimycin A (70.5 ± 9.9 pA/pF and 11.6 ± 2.2 pA/pF, n = 9) were not found to be statistically significant from control-treated cells (97.2 ± 9.93 pA/pF and 22.0 ± 4.2 pA/pF, n = 15). Likewise, Fig. 8B shows the magnitude of peak and sustained currents mediated by homomeric P2X₃ receptors evoked in response to the first exposure to 0.5 µM ATP (which equates to the EC₂₅ value) in the presence or absence of the ROS mediators. The magnitudes of the peak and sustained P2X₃-mediated currents in the presence of H₂O₂ (44.4 ± 9.6 pA/pF and 1.7 ± 0.8 pA/pF, n = 10), rotenone (39.9 ± 9.3 pA/pF and 2.0 ± 0.4 pA/pF, n = 11), myxothiazol (41.9 ± 10.4 pA/pF and 2.0 ± 0.5 pA/pF, n = 10), or antimycin A (89.2 ± 36.4 pA/pF and 1.9 ± 0.6 pA/pF, n = 9) were not found to be statistically significant from control-treated cells (61.4 ± 20.0 pA/pF and 2.6 ± 0.4 pA/pF, n = 15). Thus, these findings support the hypothesis that hypoxia and ROS selectively modulate homomeric P2X₂ receptors.

View larger version (28K): [in this window] [in a new window]   Fig. 8. Currents mediated by heteromeric P2X2/3 or homomeric P2X3 receptors are not affected by ROS. HEK293 cells expressing heteromeric P2X2/3 or homomeric P2X3 receptors were held at a potential of -70 mV in the presence of external Ca2+ and Mg2+ and perfused with 1.8 mM H2O2, 100 nM rotenone, 100 nM myxothiazol, or 10 µg/ml antimycin A for 2 min before being exposed to extracellular ATP (0.5 µM). A, peak and sustained P2X2/3-mediated currents evoked by the first exposure to 0.5 µM ATP in the absence or presence of ROS generators. B, peak and sustained P2X3-mediated currents evoked by the first exposure to 0.5 µM ATP in the absence or presence of ROS generators.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The purpose of this study was to evaluate whether hypoxia could affect the activation of homo- and heteromeric P2X₂ and P2X₃ receptor cation channels and thus be a possible mechanism contributing to the ventilatory response during a hypoxic challenge. The present findings show that hypoxia attenuates homomeric P2X₂-mediated currents but has no effect on homomeric P2X₃ or heteromeric P2X_2/3 currents. This is the first direct demonstration of acute hypoxic modulation of a ligand-gated ion channel. Furthermore, the selective modulation of the P2X₂ receptor subunit is consistent with the recent report by Rong et al. (2003), who found that mice deficient in the P2X₂ receptor subunit showed an attenuated ventilatory response to hypoxia, whereas mice deficient in the P2X₃ receptor subunit were comparable with wild type. Previous studies have shown that under hypoxic conditions, ATP is coreleased with ACh from type I glomus cells and activates P2X₂ and P2X₃ receptors expressed on petrosal ganglia, which leads to an increased afferent discharge rate (Zhang et al., 2000; Prasad et al., 2001). Therefore, the combined results from this current study and that of Rong et al. (2003) suggest that the expression of homomeric P2X₂ receptors may play a critical role in mediating the ventilatory response to an acute hypoxic challenge within the carotid body.

A similar modulatory role of P2X₂ receptors may be involved in the regulation of the secretory response of PC12 to a hypoxic stimulus. In PC12 cells, exposure to extracellular ATP activates an inwardly rectifying current that stimulates Ca²⁺ entry through L-type Ca²⁺ channels and promotes nor-adrenaline secretion, and this secretion is potentiated under hypoxic conditions (Inoue et al., 1989; Nakazawa et al., 1990; Taylor and Peers, 1999; Hur et al., 2001). Further studies showed that the channel mediating the ATP-sensitive current was encoded by the P2X₂ receptor (Brake et al., 1994). Therefore, the attenuation of the P2X₂-mediated currents under hypoxia shown in this study may be a general mechanism to limit catecholamine secretion.

Although numerous types of ion channels are modulated by hypoxia (for review, see Lopez-Barneo et al., 2001), the exact mechanism whereby cells sense changes in oxygen is largely unknown. Several mechanisms have been proposed in the literature (for reviews, see Chandel and Schumacker, 2000; Sham, 2002). The predominant theory is that hypoxia results in either a decrease (Mohazzab and Wolin, 1994) or an increase (Leach et al., 2001; Waypa et al., 2001) in the production of ROS, which shifts the ratio of redox couples (i.e., glutathione disulfide/glutathione and NAD⁺/NADH) to a more reduced state and hence alters channel function. The results from this study suggest that the hypoxic modulation of the P2X₂ receptor is mediated via an increase in the production of ROS, because exposure to H₂O₂ mimicked the effect of hypoxia on the P2X₂-mediated currents and attenuated the reduction in P2X₂-mediated currents when exposed to a hypoxic challenge. Although the extracellular concentration of H₂O₂ (1.8 mM) that was used in this study seems to be outside the expected physiological concentration of H₂O₂, there is increasing evidence in the literature that a H₂O₂ concentration gradient is established across biological membranes, such that the intracellular concentration of H₂O₂ may be 10-fold less than the extracellular concentration (Antunes and Cadenas, 2000; Seaver and Imlay, 2001). In this study, we have shown that currents mediated by homomeric P2X₂ receptors were inhibited by H₂O₂ in a concentration-dependent manner, and that the P2X₂-mediated currents were inhibited by extracellular H₂O₂ concentrations of 100 µM. Therefore, the perceived intracellular concentration of H₂O₂ would be in the micromolar range, which is within physiological range of H₂O₂ concentrations reported in the literature (Halliwell et al., 2000).

We further investigated the source of ROS that modulates the P2X₂ receptor by using mitochondrial inhibitors that either increase or decrease the production of ROS. In the presence of either rotenone or myxothiazol, which decreases the production of ROS, a transient increase in the magnitude of the P2X₂-mediated currents is observed. Furthermore, the transient increase in the P2X₂ current observed in the presence of rotenone could be abolished by the presence of succinate, which bypasses the inhibition at complex I. In contrast, antimycin A, which increases ROS production, reduced the magnitude of the P2X₂-mediated currents. These observations support the theory that ROS can modulate the P2X₂ receptor. In addition, the observation that H₂O₂ and the mitochondrial inhibitors did not affect homomeric P2X₃ and heteromeric P2X_2/3 receptors supports the observation that hypoxia and ROS selectively modulate homomeric P2X₂-mediated currents. However, it must be noted that H₂O₂ did not entirely inhibit the hypoxia-induced reduction in P2X₂-mediated currents; therefore, some additional mechanism must also contribute to the modulation of the P2X₂ receptor in response to a hypoxic challenge. Potential additional mediators of the hypoxic modulation of P2X₂ receptors include nonoxidase iron proteins and/or a direct effect on the channel protein itself (Lopez-Barneo et al., 2001).

The ability of hypoxia and H₂O₂ to reduce the magnitude of P2X₂-mediated currents was only partially reversible upon washout, and the extent of recovery varied between individual cells (13-100%). The reason for this is unclear, but one possibility is that exposure to a hypoxic challenge may lead to a permanent modification of the P2X₂ receptor. Thus, exposure to a hypoxic challenge results in an increase in the mitochondrial production of ROS. The ROS subsequently passes from the mitochondria into the cytosol, where they may modify cysteine residues on the P2X₂ receptor subunit and hence lead to a permanent modification in the function of the P2X₂ receptor. Because all P2X receptors have a conserved cysteine-rich extracellular domain (Brake et al., 1994; Valera et al., 1994; Surprenant et al., 1995), the hypoxia and ROS-sensitive residue on the P2X₂ receptor must lie on one or more of the nonconserved residues. On the other hand, the limited reversibility of the response to hypoxia and ROS may be caused by cell-to-cell variation in the ability of individual cells to remove ROS via the action of catalases, peroxidases, and thioredoxin-linked systems. In summary, we have shown that that the magnitude of currents mediated by homomeric P2X₂ receptors are attenuated under hypoxic conditions and that this modulation of P2X₂ receptors is dependent on the production of ROS at the level of mitochondrial electron transport.

	Acknowledgements

 
We thank Professor R. Alan North (Institute of Molecular Physiology, University of Sheffield, Sheffield, UK) for providing the P2X stable cell lines used in this study.

	Footnotes

 
The Wellcome Trust and the British Heart Foundation provided funding for this study.

ABBREVIATIONS: HEK, human embryonic kidney; ,-MeATP, ,-methylene ATP; ROS, reactive oxygen species; ETC, electron transport chain.

¹ Current address: Cardiff School of Biosciences, University of Cardiff, Cardiff, UK.

Address correspondence to: Dr. H. S. Mason, Cardiff School of Biosciences, University of Cardiff, Biomedical Sciences Bldg., Museum Ave., Cardiff, CF10 3US, UK. E-mail: dentyh{at}netscape.net

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