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NEUROPHARMACOLOGY
Departments of Electrophysiology (K.K.F., M.M., W.Y.) and Biochemistry and Molecular Biology (J.E.K.), Neurogen Corporation, Branford, Connecticut
Received April 15, 2005; accepted July 11, 2005.
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Abstract |
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-methylene ATP (-meATP)-evoked P2X3 responses in a concentration-dependent manner (IC50 = 0.6 ± 0.1 µM). IP5I effectively inhibited P2X3 currents when pre-exposed to desensitized but not unbound receptors. Furthermore, IP5I equally blocked 1 and 10 µM -meATP-evoked currents and had no effect on the desensitization rate constant of these currents. This supports the action of IP5I as a noncompetitive antagonist that interacts with the desensitized state of the P2X3 receptor. In contrast, TNP-ATP inhibited the current evoked by 1 µM -meATP significantly more than the one evoked by 10 µM -meATP. It also significantly slowed down the desensitization rate constant of the current. These results suggest that TNP-ATP acts as a competitive antagonist and competes with -meATP at the P2X3 agonist binding site. These findings may help to explain why IP5I acts selectively at the fast-desensitizing P2X1 and P2X3 subtypes of the P2X purinoceptor, while having much less potency at slow-desensitizing P2X2 and P2X2/3 subtypes that lack the fast desensitized conformational state.
; Hamilton et al., 2000). P2X3 receptor mRNA studies have revealed that it is expressed in nociceptive neurons, linking the receptor to involvement in pain responses (Chen et al., 1995; Lewis et al., 1995). The P2X3 receptor has also been localized to other areas, including cardiac tissue and autonomic neurons (Garcia-Guzman et al., 1997; Dunn et al., 2001). P2X3 was specifically found to be highly expressed in the dorsal root ganglion (DRG) neurons where pain modulation first occurs (Vulchanova et al., 1998). Later knockout studies strongly linked P2X3 to inflammatory pain and volume reflexes of the urinary bladder (Cockayne et al., 2000; Souslova et al., 2000). Therefore, specific P2X3 antagonists might have clinical utility.
The P2X receptor family is comprised of seven receptor subunits (P2X1-7) (Collo et al., 1996; Surprenant et al., 1996). Three subunits are thought to structurally combine to form ionotropic homomeric or heteromeric complexes (Lewis et al., 1995; Nicke et al., 1998; Stoop et al., 1999). The major subunits localized to rat DRG neurons are P2X2 and P2X3 (Chen et al., 1995; Vulchanova et al., 1997; Rae et al., 1998). In rat DRG recordings, the homomeric P2X3 receptor is the most prevalent subtype identified, whereas heteromeric P2X2/3 is expressed to a lesser extent and homomeric P2X2 is rarely identified (Grubb and Evans, 1999). To specifically study P2X3 for the purpose of developing analgesic medications, it is necessary to identify agonists and antagonists that selectively target the receptor.
Although the agonist ATP activates all P2X receptors, -meATP has a relatively higher affinity for P2X1 and P2X3 homomers and heteromeric receptors containing these subunits (Lewis et al., 1995; Collo et al., 1996). For this reason, -meATP is often used to specifically study P2X3 and P2X2/3 receptors. The activities of -meATP on P2X3 or P2X2/3 receptors can then be differentiated based on receptor kinetics. P2X1 and P2X3 are the only P2X receptors with responses characterized by a rapid onset and fast desensitization (Chen et al., 1995; Lewis et al., 1995; Longhurst et al., 1996; Rettinger and Schmalzing, 2003). At P2X1 or P2X3, desensitization is due to the rapid transition from the open to the desensitized receptor conformation (North, 2002; Rettinger and Schmalzing, 2003). In contrast, P2X2 and P2X2/3 receptors have a rapid onset but desensitize much slower than P2X3 receptors. This slow-desensitizing property of P2X2 and P2X2/3 receptors has been attributed to the interaction between two membrane-spanning hydrophobic receptor domains at the P2X2 subunit (Werner et al., 1996).
Competitive and noncompetitive antagonists have been identified that act at P2X3 with some selectivity. For example, both trinitrophenyl-ATP (TNP-ATP) and A-317491 are competitive antagonists. TNP-ATP is selective for P2X1, P2X2/3, and P2X3 receptors, whereas A-317491 specifically acts at P2X3 and P2X2/3 receptors (Virginio et al., 1998; Burgard et al., 2000; Jarvis et al., 2004). Competitive antagonists interact with the receptor at the agonist binding site, which resides in the extracellular domain of P2X purinoceptors and can be accessed in the unbound state (North, 2002).
The dinucleotide derivative P1, P5-di[inosine-5'] pentaphosphate (IP5I) was identified as a selective antagonist for the P2X1 and P2X3 receptors (King et al., 1999; Dunn et al., 2000). Although a nucleotide such as IP5I may not have a future therapeutic use, as in vivo it may be broken down by ectonucleotidases, it is a good tool for the study of P2X receptors. IP5I is a selective noncompetitive antagonist at the fast-desensitizing P2X1 and P2X3 receptors (King et al., 1999; Dunn et al., 2000). Noncompetitive antagonists do not typically bind to the agonist site of the receptor; however, at present, the location of the IP5I binding site at the P2X3 receptor is not known.
In this study, we used a whole-cell recording technique to examine the activity of IP5I at native P2X3 receptors. We also compared the effect of TNP-ATP versus IP5I as a means of further elucidating the mechanism of IP5I antagonism. Our results suggest that IP5I likely binds to the desensitized state of the P2X3 receptor. This finding appears to explain noncompetitive activity of IP5I at the P2X3 receptor. It may also help explain the selectivity of IP5I for P2X3 versus P2X2/3, because the P2X2/3 receptor lacks a desensitized state. Therefore, as a noncompetitive antagonist, IP5I can be used as a tool to learn more about the fast-desensitizing response of the P2X3 receptor.
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Materials and Methods |
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DRG neurons were then transferred to a 10-ml dissociation solution, which was freshly made from DMEM medium with 25 mM HEPES, 50,000 units/ml trypsin, 1 mg/ml collagenase, and 0.1 mg/ml DNase and sterilized by filtration. The enzymatic dissociation was carried out at 35°C for 1.5 h by shaking DRGs on a physical shaker (BD Biosciences, Franklin Lakes, NJ) at approximately 30 rotations/min. The digestion was stopped by adding 5 ml of trypsin inhibitor solution containing 0.05 g of trypsin inhibitor, 25 mM HEPES, and 10% fetal bovine serum in DMEM. Single DRG cell suspensions were obtained by gentle trituration. Dissociated cells were then sedimented by gentle spinning at 1500 rpm for 5 min. The supernatant was discarded by aspiration, and the pellet was resuspended into a culture medium containing 25 mM HEPES, 10% fetal bovine serum, 100 units/ml penicillin G, 0.25 µg/ml amphotericin B, and 100 µg/ml streptomycin in DMEM. A viable cell count was obtained thereafter, and DRG cells were plated onto poly-D-lysine-coated coverslips in 35-mm dishes with a final cell density of 300,000/dish. Cells were incubated at 5% CO2 and 37°C in the culture medium mentioned above until use, with added 50 ng/ml nerve growth factor (NGF-7S). Electrophysiological experiments were performed 48 to 96 h after dissociation. All reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.
Electrophysiology. Whole-cell voltage-clamp recordings were made using an Axopatch 200B amplifier (Axon Instruments Inc., Union City, CA). Recording electrodes were pulled from 1.5 mM borosilicate pipettes (World Precision Instruments, Inc., Sarasota, FL) using a horizontal puller (Model P-87; Sutter Instrument Company, Novato, CA). Electrodes had resistances between 1 and 3 M
when filled with intracellular solution containing 120 mM K+-aspartate, 15 mM KCl, 1 mM MgCl2, 5 mM NaCl, 5 mM Mg-ATP, 1 mM Na-GTP, 10 mM HEPES, and 5 mM EGTA. The pH of the intracellular solution was adjusted to 7.4 with KOH. The coverslip containing DRG cells was placed in a dish constantly perfused with extracellular solution. The extracellular solution contained 140 mM NaCl, 2 mM CaCl2, 5 mM KCl, 2 mM MgCl2,10 mM HEPES, and 10 mM glucose. The pH was adjusted to 7.4 with NaOH. Both intracellular and extracellular solutions had osmolarities of approximately 300 mOsm.
An in-house-designed drug delivery system was used to apply compounds to the DRG neurons. The manifold contained 13 gravity-driven perfusion lines with a single outlet directly placed approximately 0.1 mm in front of the recorded cell. The cell was continuously perfused with the control solution at a rate of approximately 1 ml/min. The exchange between solutions took approximately 0.5 s, which was not fast enough to resolve the onset kinetics of the P2X3 receptor. Therefore, experiments were focused on the desensitization rate of the response.
Individual DRG cells were voltage-clamped. The pipette potential was zeroed before seal formation. Upon seal formation and the following rupture of the cellular membrane, the series resistance was compensated greater than 80%, and the membrane potential was clamped to -80 mV. Cells with a series resistance of greater than 10 M or a leak current greater than 300 pA were discarded. The capacitance of cells used was less than 60 pF. The voltage protocols were generated using pClamp 8, digitized through Digidata 1320A at 200 Hz (Axon Instruments, Inc.), and then recorded to a personal computer.
The P2X3 receptor was activated by the agonist -meATP, and the amplitude of the current response to repetitive applications of -meATP was monitored. Experiments were conducted after four consecutive stable control responses were obtained. The interval between each agonist application varied depending on agonist concentrations. At 1 µM -meATP, a 2-min interval between applications was needed to obtain stable control responses. When a higher concentration of -meATP (10 µM) was used, 3 min were needed between applications to obtain stable responses.
Statistical Analysis. Data were analyzed using Clampfit (version 8.2; Axon Instruments Inc.), Microsoft Excel, and Origin 7 (OriginLab Corp., Northampton, MA). Data are shown as mean ± S.E.M. Statistical analyses were made using Student's t tests, with P < 0.05 indicating statistical significance. For the dose-response calculation of IP5I shown in Fig. 1B, the percent inhibition at each dose was calculated as follows: Percent inhibition = 100 - [100 x (peak current in IP5I/control peak current)]. Data were then fitted with the following logistic equation (eq. 1),
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) of desensitization of the P2X3 response was calculated by exponentially fitting the decay phase of the -meATP-evoked current. For control 1 or 10 µM -meATP-evoked responses and in the presence of IP5I, the decay phase was best fit with the following double exponential equation (eq. 2),
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1 and 2 are the fast and slow desensitization time constants, and A1 and A2 are the corresponding amplitudes. However, in the presence of TNP-ATP, the decay current was slowed significantly and it could no longer be fit with a double exponential equation while also maintaining its biophysical meaning. Therefore, it was fit with the following single exponential equation (eq. 3),
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Only the fast components of double exponential fitting were analyzed. The peak responses in the presence or absence of antagonists were also compared.
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Results |
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-meATP (Fig. 1A). -meATP-evoked currents in DRGs with these characteristics are attributed to activation of the P2X3 receptor (Chen et al., 1995; Lewis et al., 1995; Virginio et al., 1998). Whole-cell voltage-clamp experiments were conducted on dissociated rat DRG neurons as in Fig. 1A. Full recovery of the P2X3 receptor from its desensitized state could be obtained 3 min after each 10 µM -meATP application. After four stable -meATP responses, the cell was immediately exposed to 3 µM IP5I. In the presence of 3 µM IP5I, the 10 µM -meATP-evoked response was significantly reduced. Recovery from the IP5I blockade was obtained after washing out the drug (Fig. 1A).
An IP5I dose response at the P2X3 receptor in rat DRG neurons was conducted using a similar experimental format. Five IP5I doses between 0.1 and 10 µM produced a concentration-dependent inhibition of the peak P2X3 response (Fig. 1B). When the data were fitted with a logistic equation (eq. 1 under Materials and Methods), an estimated IC50 value of 0.6 ± 0.1 µM with an nH of 0.7 ± 0.1 was obtained (Fig. 1B). This is within a log unit of the IC50 value of 0.1 µM IP5I at the P2X3 receptor in rat DRGs (Dunn et al., 2000).
One possible explanation for IP5I's antagonism of the P2X3 response is that it acts as a weak agonist, desensitizing the receptor and appearing to be an antagonist. McDonald et al. (2002) showed that an agonist, such as -meATP or AP5A, can sufficiently desensitize the P2X3 receptor and therefore manifest as an antagonist. To test whether IP5I or a potential contaminant in its solution could activate P2X3 receptors directly, we carried out experiments to address this possibility. Because the response evoked by IP5I or its contaminant could be very small, we changed our holding potential from -80 to -110 mV to increase the driving force for a potential response. A series of IP5I concentrations from 1 to 100 µM was tested. An example of such an experiment is shown in Fig. 2A. In this cell, 100 µM IP5I failed to evoke any visible current, whereas 1 µM -meATP activated an unequivocal inward current both before and after washing out the IP5I application. Figure 2B summarizes these results. In all of the cells studied under this protocol, 1, 10, or 100 µM IP5I failed to evoke a measurable current. Therefore, it is unlikely that IP5I or a potential contaminant in its solution acted as an agonist and desensitized the P2X3 receptor.
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-meATP-evoked currents were inhibited on average by 76.3 ± 2.4% (mean ± S.E.M., n = 7).
IP5I Binds P2X3 at the Desensitized State in Rat DRG Neurons. To study the interaction of IP5I with P2X3 receptors, we assume that P2X3 has at least three conformational states: unbound, open, and desensitized (Fig. 3A). Upon binding an agonist, a conformational change in the receptor is manifest as a transient inward current (Fig. 3A). The recovery from the desensitized to the unbound state is both time- and agonist concentration-dependent. At a concentration of 10 µM -meATP, agonist application every 3 min results in a stable P2X3 response (Fig. 1A). At 1 µM -meATP, however, a 2-min separation between agonist applications is sufficient to maintain response stability (Fig. 4A).
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IP5I, a noncompetitive P2X receptor antagonist, has potent inhibitory effects at both cloned and native P2X1 and P2X3 receptors yet has no effect at either P2X2 or P2X2/3 receptors (King et al., 1999; Dunn et al., 2000). Because P2X1 and P2X3 are fast-desensitizing P2X subtypes, whereas P2X2 and P2X2/3 are nondesensitizing, IP5I may bind to the desensitized receptor state. If this is the case, IP5I may lock the receptor in the desensitized conformation, preventing its return to the unbound state and reducing the agonist response. Experiments were designed to test this hypothesis.
If IP5I binds to the unbound receptor state, whether it is first pre-exposed when most channels are desensitized (short protocol) or unbound (long protocol) should have no effect on its inhibition of the P2X3 current. This is indicated by the two open bars in Fig. 3B. However, if IP5I acts at the desensitized state, it can only adequately bind the receptor and inhibit the agonist response using the short protocol. Use of the long protocol will result in minimal inhibition of the agonist response at the dose indicated by an open bar (Fig. 3B).
Representative traces illustrating the results of experiments done using the short and long protocols are shown in Fig. 4A. For the purpose of analysis, the agonist response in the presence of IP5I was normalized according to the initial control response. In the short protocol experiment, the first -meATP-evoked current in the presence of 3 µM IP5I is reduced to 22.3 ± 3.9% (n = 6) of the original response (Fig. 4B). This is consistent with our own dose-response data showing that 3 µM IP5I blocks a 10 µM -meATP-evoked current by approximately 76% (Fig. 1B). In contrast, the first -meATP-evoked current in the presence of 3 µM IP5I was 80.3 ± 6.5% (n = 9) of the original response under the long protocol. Full inhibition was observed only at the second application of -meATP in the presence of IP5I (data not shown; Fig. 4A). A control for the long experiment, illustrated in the bottom traces in Fig. 4A, demonstrates that the skipped agonist dose in the long protocol does not significantly alter a stable P2X3 current. The resulting first -meATP-evoked current was 95.1 ± 12.0% (n = 5) of the original response, which is not significantly different from the original response (P = 0.35, paired one-tailed Student's t test; Fig. 4B). The significant difference between the first -meATP-evoked responses in the presence of IP5I for the long and short experimental protocols indicates that IP5I likely binds to the desensitized conformation at the P2X3 receptor (P < 0.001, one-tailed Student's t test, unequal variance; Fig. 4B).
Competitive and Noncompetitive Antagonists Have Different Effects on the Peak Current and the Rate of Desensitization at the P2X3 Receptor. Because of the fast-desensitizing kinetics at P2X3, a competitive antagonist can appear to be noncompetitive if only peak current is measured, as is the case of TNP-ATP at the P2X3 receptor (Virginio et al., 1998; Burgard et al., 2000). If receptor kinetics are considered, however, it can be demonstrated that competitive antagonists tend to slow down the receptor desensitization rate, as seen at the nicotinic acetylcholine and P2X1 receptors (Demazumder and Dilger, 2001; Wenningmann and Dilger, 2001; Hülsmann et al., 2003). Competitive antagonists should also have a lesser effect when challenged with higher concentrations of agonist. If the noncompetitive antagonist IP5I only binds to the desensitized state of the P2X3 receptor, as our data suggest, it should not only block 1 and 10 µM -meATP-evoked currents to the same extent but also have no effect on their desensitization rate constant. Therefore, we studied the inhibitory effects of IP5I on both 1 and 10 µM -meATP-evoked currents while using TNP-ATP, a competitive P2X3 receptor antagonist, as a control (Burgard et al., 2000).
Representative traces comparing the effect of TNP-ATP and IP5I on the P2X3 response are illustrated in Fig. 5. IP5I at a 3 µM concentration (IC80 value) significantly inhibited both 1 and 10 µM -meATP-evoked currents to a similar extent but did not noticeably affect the rate of desensitization (Fig. 5, A and B). TNP-ATP was used at a concentration of 10 nM, an approximate IC80 value based on our internal (data not shown) and published (Virginio et al., 1998) data. TNP-ATP significantly inhibited both 1 and 10 µM -meATP-evoked currents, although the inhibition was greater at 1 µM agonist (Fig. 5, C and D). Most importantly, TNP-ATP dramatically slowed the rate of P2X3 desensitization for both 1 and 10 µM -meATP-evoked currents (Fig. 5, C and D). To quantify the change of desensitization rate, the decay phase of the -meATP-evoked current was fitted with a double exponential equation (eq. 2 under Materials and Methods), which reflects the existence of P2X3 and P2X2/3 receptors in DRGs. The fast component, which likely reflects the kinetics of the P2X3 receptor, was analyzed and discussed below. On the other hand, in the presence of TNP-ATP, the decay current was slowed markedly and could no longer be fit with a double exponential equation while maintaining its biophysical meaning. Therefore, it was fit with a single exponential equation.
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-meATP-evoked P2X3 current was reduced to 27.4 ± 3.0% of control by 3 µM IP5I (Fig. 6A). Similarly, 26.7 ± 3.0% of the P2X3 current evoked by 10 µM -meATP remained after application with 3 µM IP5I, supportive of IP5I as a noncompetitive P2X3 antagonist (P = 0.44, one-tailed Student's t test; Fig. 6A). Figure 6B shows a significant difference in P2X3 inhibition by 10 nM TNP-ATP at both 1 and 10 µM -meATP-evoked responses, which supports the action of TNP-ATP as a competitive P2X3 antagonist (11.5 ± 0.7% versus 28.8 ± 5.2% of control, respectively; P < 0.01, one-tailed Student's t test, equal variance).
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-meATP (n = 12) and 76 ± 9 and 89 ± 10 ms, respectively, for 10 µM -meATP (n = 7; Fig. 6C). IP5I had no significant effect on P2X3 receptor ion kinetics at 1 or 10 µM -meATP (P = 0.43 and 0.11, respectively; paired one-tailed Student's t test). This is consistent with the idea that IP5I binds to the desensitized state of the P2X3 receptor and acts as a noncompetitive P2X3 antagonist. However, TNP-ATP increased the fast desensitization rate constant of 1 µM -meATP-evoked responses from 194 ± 21 to 1751 ± 403 ms (n = 5) and increased 10 µM -meATP-evoked responses from 81 ± 12 to 434 ± 128 ms (n = 5; Fig. 6D). Therefore, TNP-ATP significantly slowed the rate of desensitization of the P2X3 response at both 1 µM -meATP and 10 µM -meATP (P < 0.01 and P < 0.05, respectively; paired one-tailed Student's t test; Fig. 6D). This further supports TNP-ATP as a noncompetitive antagonist at P2X3. |
Discussion |
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). P2X3 is of interest because of its presence on nociceptive sensory neurons (Chen et al., 1995; Lewis et al., 1995). Knockout studies have demonstrated its involvement in inflammatory pain and urinary bladder hyporeflexia (Cockayne et al., 2000; Souslova et al., 2000). Therefore, specific P2X3 antagonists are desirable clinically.
In this study, we used the agonist -meATP to elicit P2X3 responses in native rat DRG cells. Published data indicate an IC50 value of 0.1 ± 0.03 µM for IP5I in rat DRGs and an IC50 value of 2.8 ± 0.7 µM in Xenopus laevis oocytes (King et al., 1999; Dunn et al., 2000). Our IC50 value of 0.6 ± 0.1 µM for IP5I in rat DRGs is within the range of a log unit to the reported data. The quantitative difference between the reported data and ours presumably reflects differences between the preparations and methods used. Heteromeric P2X2/3 responses were differentiated from P2X3 responses by their response kinetics. P2X3 responses were identified based on their fast desensitization kinetics. In most of our recorded cells, the -meATP-evoked current desensitized in less than 2 s, which supports research identifying the homomeric P2X3 receptor as the predominant P2 receptor subtype in rat DRG neurons (Chen et al., 1995; Lewis et al., 1995; Grubb and Evans, 1999).
Radioligand binding studies have shown that competitive antagonists such as A-317491 bind the same site as the agonist -meATP at P2X3 and P2X2/3 receptors (Jarvis et al., 2004). IP5I is a noncompetitive antagonist at the P2X3 receptor, and its binding site has not been determined. Because IP5I potently blocks the P2X3 receptor but has almost no activity at the P2X2/3 receptor (King et al., 1999; Dunn et al., 2000), it may act at an allosteric site at the desensitized state of the P2X3 receptor. If IP5I binds to the desensitized state, pre-exposure of IP5I after most P2X3 channels have returned to the unbound state should block the agonist response less than expected (long protocol, Fig. 3B). If different pre-exposure protocols (long and short protocols) do not affect the amount of block seen, then it is likely that IP5I binds the unbound state of the receptor. Approximately an IC80 dose of IP5I was used to achieve an effective block while avoiding maximal inhibition, which might obscure the resolution of the response.
Our data indicate that if IP5I is pre-exposed after most P2X3 channels have returned to the unbound state, minimal inhibition of the next -meATP response occurs; approximately 20% inhibition was observed (Fig. 4B). This is in sharp contrast to the 80% inhibition expected from the IC80 dose used. After continued exposure of the same cell to IP5I, however, the -meATP response was inhibited by approximately 80% (data not shown; Fig. 4A). This indicates that IP5I has a different effect if it is initially exposed to desensitized channels as opposed to unbound channels. Indeed, if IP5I is initially applied immediately after agonist when the highest percentage of channels is presumed to be desensitized, nearly an 80% reduction in the first agonist response occurs (short protocol, Fig. 4). The difference between these responses is significant (P < 0.001). In the absence of antagonist, skipping a dose of -meATP has no effect on the magnitude of a stable agonist response, ruling out the possibility of the block being an artifact of the experimental design (Fig. 4). Therefore, this suggests that IP5I antagonizes the P2X3 response through interaction with a desensitized receptor state. Similar inhibitory mechanisms have been found in other drug interactions with ion channels. For example, sodium (Na+) channel antagonists lidocaine and phenytoin preferentially bind to the inactivated state of the Na+ channel to produce their inhibitory effects (Ragsdale et al., 1991, 1996; Kuo, 1998).
In contrast, another possible explanation for IP5I's antagonism of the P2X3 response is that it acts as a weak agonist, desensitizing the receptor and appearing to be an antagonist. McDonald et al. (2002) showed that an agonist, such as -meATP or AP5A, can sufficiently desensitize the P2X3 receptor and therefore manifest as an antagonist. However, we do not believe that this is the case here. First, if IP5I acted as an agonist and desensitized the P2X3 receptor, it could explain why the P2X3 current immediately became much smaller upon IP5I application in the short protocol experiment. However, this could not explain why IP5I did not initially desensitize the P2X3 receptor and reduce the current in the long protocol experiment (Fig. 4). The same concentration of IP5I and the same time of exposure were carried out in both short and long protocol experiments. If IP5I acted as an agonist and desensitized the P2X3 receptor, similar inhibitions should be expected in both experiments. Second, we carried out an experiment to test whether IP5I could act as an agonist. Even at a high negative holding potential (-110 mV), IP5I at concentrations from 1 to 100 µM failed to evoke any measurable current in any of the cells tested, whereas 1 µM -meATP activated an unequivocal inward current. Therefore, it is unlikely that IP5I acted as an agonist and desensitized the P2X3 receptor.
Our studies on the P2X3 peak response and the rate of desensitization also provide evidence that IP5I acts as a noncompetitive antagonist at the P2X3 receptor. IP5I equally blocks the P2X3 responses evoked by 1 and 10 µM -meATP and has no effect on the rate of desensitization at either agonist concentration. This further supports the action of IP5I as a noncompetitive P2X3 antagonist and is consistent with our conclusion that IP5I acts at the desensitized conformation of the P2X3 receptor. In addition, it may suggest that the inactivity of IP5I at the P2X2/3 receptor is due to the absence of desensitization at this receptor and therefore the lack of a desensitized receptor state. The selectivity of IP5I for P2X1 and P2X3, the only fast-desensitizing receptors in the P2X family, is probably due to the availability of an inactive state binding site.
Even though TNP-ATP was first reported as a noncompetitive P2X3 receptor antagonist (Virginio et al., 1998), a later study suggested that it acted as a competitive antagonist (Burgard et al., 2000). Evidence presented here supports TNP-ATP as a competitive antagonist. There was a decrease in the block of the -meATP-evoked response by TNP-ATP at a higher concentration of agonist (Fig. 6B). Even though the difference is small, it is significant (P < 0.01) and suggests that the inhibitory effect of TNP-ATP is surmountable by increasing concentrations of -meATP. This trend is expected for a competitive antagonist, although it is not always well resolved because of the fast desensitization of the P2X3 receptor. In addition, TNP-ATP significantly slowed the desensitization rate constants for both 1 and 10 µM -meATP-evoked responses. It has been shown that the activity of competitive compounds can reduce the desensitization rate of agonist-evoked responses. For example, at nicotinic acetylcholine receptors, competitive antagonists reduce the apparent rate of receptor desensitization (Demazumder and Dilger, 2001; Wenningmann and Dilger, 2001). This trend has also been shown at P2X1, where the competitive antagonist NF449 slows receptor desensitization (Hülsmann et al., 2003). The competitive antagonist protects some of the agonist binding sites from immediate access by agonist. Competition between the agonist and antagonist for the same binding site eventually allows the agonist to reach sites previously occupied by the antagonist and to activate receptor channels. This delayed activation, in addition to the desensitization kinetics of the channel, creates an appearance of prolonged channel opening. Our results suggest that TNP-ATP is competing with -meATP for the same binding site so that competition for occupancy of this site prolongs P2X3 desensitization. It is noteworthy that TNP-ATP reduces the rate of P2X3 desensitization almost twice as much in the presence of 1 µM agonist versus 10 µM agonist, supporting the proposal of Hülsmann et al. (2003) that the reduction in desensitization reflects fluctuations in receptor occupancy. If the ratio between agonist and antagonist favors the agonist, there is less of a change in the desensitization rate constant (Hülsmann et al., 2003). However, caution is needed in our interpretation of this data, because it is very likely that our kinetic study was contaminated by the presence of a P2X2/3 component in DRGs. In the presence of TNP-ATP, which dramatically slowed P2X3 desensitization, it is difficult to differentiate the contribution of P2X3 and P2X2/3. However, this does not change the fact that the fast component of desensitization was no longer observed in the presence of TNP-ATP. Because 10 nM TNP-ATP only inhibited approximately 80% of the P2X3 component, it was concluded that the fast kinetics of the residual P2X3 response were slowed by TNP-ATP.
We conclude that the noncompetitive antagonist IP5I binds to a site at the desensitized conformation and also confirm that the P2X3 antagonist TNP-ATP competes with -meATP to bind the unbound state of the receptor. These further observations of the receptor kinetics at P2X3 can assist in the pharmaceutical development of P2X3 antagonists and help characterize the mechanism of action of potential therapeutic compounds for pain indications acting at this target.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: P2X3, purinergic receptor 2X3; DRG, dorsal root ganglion; -meATP, -methylene ATP; TNP-ATP, trinitrophenyl-ATP; A-317491, (5-([(3-phenoxybenzyl)[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]carbonyl)-1,2,4-benzenetricarboxylic acid); IP5I, P5-di[inosine-5'] pentaphosphate; P1, IP5I; DMEM, Dulbecco's modified Eagle's medium.
Address correspondence to: Dr. Weifeng Yu, Neurogen Corporation, 35 Northeast Industrial Road, Branford, CT 06405. E-mail: wyu{at}nrgn.com
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References |
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