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MS 0105 Received 9 September 1999; accepted after revision 25 November 1999.
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ABSTRACT |
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-methylene ATP (-MeATP) produced fast activating inward currents, which desensitized slowly. The maximum response to -MeATP was 36 ± 23 % (range 0·1-100 %) of that evoked by ATP in the same cell.
-MeATP (300 µM) with ATP (300 µM) produced a response that was 97 ± 1 % of that given by ATP alone. Following desensitization with -MeATP, the decrease in response to ATP was equal to the absolute reduction in response to -MeATP in the same cell.
-MeATP had an EC50 of 42 µM and a Hill coefficient of 1·17. For cells where the ratio of -MeATP/ATP currents at 100 µM was < 0·1, the ATP concentration-response curve had an EC50 of 56 µM and a Hill coefficient of 1·95. However, in cells where the ratio was > 0·7, the curve had an EC50 of 60 µM and a Hill coefficient of 0·97.
-MeATP was inhibited by 2' (or 3')-O-trinitrophenyl-ATP (TNP-ATP) with an IC50 of 70 nM. However, on cells where the ratio of -MeATP/ATP currents was < 0·1, ATP was inhibited by TNP-ATP with an IC50 of 522 nM.
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INTRODUCTION |
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It is now generally accepted that ATP can act as a fast excitatory neurotransmitter at the autonomic neuromuscular junction (for review, see Burnstock, 1997), in the central nervous system (Edwards et al. 1992; Bardoni et al. 1997; Nieber et al. 1997), myenteric neurons (LePard et al. 1997) and cultured coeliac ganglion neurons (Evans et al. 1992; Silinsky & Gerzanich, 1993), where it activates a class of ligand-gated cation channels, the P2X receptors. ATP also plays a role in presynaptic modulation of transmitter release (Gu & MacDermott, 1997; Khakh & Henderson, 1998).
To date seven P2X receptor subunits (P2X1-7) have been cloned. The functional homo-oligomeric receptors formed have different, but overlapping, biophysical and pharmacological properties (Brake et al. 1994; Valera et al. 1994; Bo et al. 1995; Chen et al. 1995; Collo et al. 1996; Surprenant et al. 1996; for review, see North & Barnard, 1997). Thus, P2X1 and P2X3 receptors are activated by ,-methyleneATP (-MeATP) and desensitize rapidly, whereas P2X2 receptors do not respond to this ligand and desensitize very slowly (Brake et al. 1994; Valera et al. 1994; Chen et al. 1995). In addition, some subunits can combine together to form hetero-oligomeric receptors with novel pharmacological and biophysical profiles (Lewis et al. 1995; Lê et al. 1998; Torres et al. 1998). For example, although homomeric P2X3 receptors give rise to fast desensitizing responses, hetero-multimeric P2X2/3 receptors respond to -MeATP (a property of P2X3 receptors), but desensitize slowly (a property of P2X2 receptors). Furthermore, alternatively spliced variants of P2X receptor subunits have been reported for rat P2X2 receptors (Brändle et al. 1997; Simon et al. 1997), guinea-pig P2X2 receptors (Parker et al. 1998), mouse P2X4 receptors (Simon et al. 1999; Townsend-Nicholson et al. 1999) and human P2X4 receptors (Carpenter et al. 1999). The heterologously expressed rat P2X2(b) splice variant desensitized faster than P2X2(a) receptors. The presence of splice variants may thus increase the variety of endogenous P2X receptors.
In the rat, sensory neurons from dorsal root (Robertson et al. 1996; Rae et al. 1998), nodose (Lewis et al. 1995) and trigeminal ganglia (Cook et al. 1997), all exhibit -MeATP sensitivity. Pharmacological studies suggest that the P2X receptors present on these sensory neurons are mainly P2X3 and/or P2X2/3 subtypes (Lewis et al. 1995; Cook et al. 1997; Grubb & Evans, 1999; Ueno et al. 1999; Li et al. 1999). In contrast, the pharmacology of P2X receptors in rat superior cervical ganglion (SCG) neurons (Nakazawa, 1994) and the molecular and pharmacological properties of P2X receptors in rat pelvic ganglion neurons (Zhong et al. 1998) suggest them to be of the P2X2 subtype. Interestingly, -MeATP evoked responses from neurons in intact guinea-pig SCG (Reekie & Burnstock, 1994), and acted as a full agonist in guinea-pig coeliac ganglion neurons and rat nodose ganglion neurons (Khakh et al. 1995). This raises the possibility that, in the guinea-pig, the P2X3 subunit may play a significant role in neurons other than sensory neurons. An alternative explanation for sensitivity to -MeATP might be the expression of the P2X1 subunit in these sympathetic neurons. In this study we have used electrophysiological recording and immunohistochemical techniques to characterize the P2X receptors present in neurons of the guinea-pig SCG. Part of the work has appeared in the form of an abstract (Zhong et al. 1999).
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METHODS |
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Cell culture
Single neurons from the SCG of male guinea-pigs (200 g) were enzymatically isolated as described previously (Zhong et al. 1998). Briefly, guinea-pigs were killed by inhalation of a rising concentration of CO2 and death was confirmed by cardiac haemorrhage. The SCG were rapidly dissected out, and placed in Leibovitz L-15 medium (Life Technologies, Paisley, UK). The ganglia were then desheathed, cut and incubated in 4 ml Ca2+- and Mg2+-free Hanks' balanced salt solution with 10 mM Hepes buffer (pH 7·4) (HBSS; Life Technologies) containing 1·5 mg ml-1 collagenase (Class II, Worthington Biochemical Corporation, Reading, UK) and 6 mg ml-1 bovine serum albumin (Sigma, Poole, UK) at 37°C for 60 min. The ganglia were then incubated in 4 ml HBSS containing 1 mg ml-1 trypsin (Sigma) at 37°C for 20 min. The solution was replaced with 3 ml growth medium comprised of L-15 medium supplemented with 10 % bovine serum, 50 ng ml-1 nerve growth factor, 0·2 % NaHCO3, 5·5 mg ml-1 glucose, 200 i.u. ml-1 penicillin and 200 µg ml-1 streptomycin. The ganglia were dissociated into single neurons by gentle trituration. The cells were then centrifuged at 160 g for 5 min, resuspended in 1 ml growth medium and plated onto 35 mm Petri dishes coated with 10 µg ml-1 laminin (Sigma). Cells were maintained at 37°C in a humidified atmosphere containing 5 % CO2, and used on the following day.
Electrophysiology
Whole-cell voltage-clamp recording was carried out at room temperature using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA). Membrane potential was held at -70 mV. External solution contained (mM): NaCl 154, KCl 4·7, MgCl2 1·2, CaCl2 2·5, Hepes 10 and glucose 5·6; the pH was adjusted to 7·4 using NaOH. Recording electrodes (resistance 2-4 M
) were filled with internal solution which contained (mM): KCl 120, Hepes 10 and tripotassium citrate 10; the pH was adjusted to 7·2 using KOH, or a similar solution in which K+ was replaced by Cs+. No difference in response was observed between the two internal solutions. Series resistance compensation of 72-75 % was used in all recordings. Data were acquired using pCLAMP software (Axon Instruments). Signals were filtered at 2 kHz (-3 dB frequency, Bessel filter, 80 dB per decade).
Drugs were applied rapidly through a 7-barrel manifold comprising fused glass capillaries inserted into a common outlet tube (tip diameter of 
200 µm) which was placed about 200 µm from the cell (Dunn et al. 1996). Solutions were delivered by gravity flow from independent reservoirs. One barrel was used to apply drug-free solution to enable rapid termination of drug application. Solution exchange measured by changes in open tip current was complete in 200 ms; however, complete exchange of solution around an intact cell was considerably slower (
1 s).
Data analysis
All responses were normalized to that evoked by 100 µM ATP in the same cell, unless otherwise stated. Except where indicated to the contrary, all data are expressed as the means ± S.E.M. Statistical analysis (Student's t test, F test and Pearson's correlation test) was performed using Prism v2 (Graphpad, San Diego, CA, USA).
Concentration-response data were fitted with the Hill equation: Y = A/[1 + (K/X)nH], where A is the maximum effect, K is the EC50 and nH is the Hill coefficient, using Prism v2. The combined data from the given number of cells were fitted, and the results are presented as values ± s.e., determined by the fitting routine.
Traces were acquired using Fetchex (pCLAMP software) and plotted using Origin (Microcal, Northampton, MA, USA). The desensitization traces were fitted using Clampfit (pCLAMP) software, to both the first and second order exponential decay. However, for the 2 min application of agonists, a significantly better fit was consistently found using the second order exponential decay (F test, P < 0·0001).
In the series of experiments where -MeATP and ATP were co-applied, the predicted curve assuming -MeATP to be a partial agonist was calculated as follows. The decline of the current in the presence of 300 µM ATP was fitted using the first order exponential decay. -MeATP and ATP were assumed to act on a single population of receptors, where -MeATP was a partial agonist with similar affinity to ATP but lower efficacy (E, given by the ratio of the peak amplitude of the responses to the same near-maximal concentration of -MeATP and ATP from the same neuron). Then, in the presence of -MeATP (300 µM) and ATP (300 µM), half of the receptors would be occupied by -MeATP and half by ATP. The fitted curve was then scaled by a factor of 0·5 (1 + E) to yield the predicted response.
Immunohistochemistry
Male guinea-pigs (200 g) were killed as described above and the nodose, pelvic and superior cervical ganglia were dissected out. These ganglia were rapidly frozen by immersion in isopentane at -70°C for 2 min, cut into 10 µm sections using a cryostat, thaw-mounted on gelatin-coated slides and air-dried at room temperature. The slides were stored at -20°C.
Sequence analysis has revealed that the carboxyl terminal region is one of the least conserved amongst members of the rat P2X purinoceptor family. Peptides corresponding to 15 amino acid residues of the C-terminal region have been used to generate subtype-selective antibodies (Roche Bioscience). These peptides were covalently linked to keyhole limpet haemocyanin, and rabbits were immunized with the conjugated peptide by multiple monthly injection (performed by Research Genetics, Inc., Huntsville, AL, USA). The sequences of the synthetic peptides were: P2X2, QQDSTSTDPKGLAQL; and P2X3, VEKQSTDSGAYSIGH (see Xiang et al. 1998, for peptide sequences for other P2X receptor subtypes). The specificity of the antisera was verified by immunoblotting with the membrane preparation from CHO-K1 cells expressing the cloned P2X1 to P2X6 receptors (Oglesby et al. 1999). Immunoglobulin G (IgG) fractions were isolated from the pre-immune and immune sera following the method of Harboe & Ingild (1973). The protein concentration was determined at 280 nm using an extinction factor of 1·43 for 1 mg ml-1.
Antibodies against rat P2X1-6 receptors have been used in this study, using the avidin-biotin (ABC) technique (Llewellyn-Smith et al. 1993; Zhong et al. 1998). Briefly, the sections were fixed in 4 % formaldehyde (in 0·1 M phosphate buffer) containing 0·03 % picric acid (pH 7·4) for 10 min. Endogenous peroxidase was blocked with 50 % methanol containing 0·4 % hydrogen peroxide (H2O2) for 10 min. Non-specific binding sites were blocked by a 20 min incubation with 10 % normal horse serum (NHS) (Life Technologies) in phosphate-buffered saline (PBS) containing 0·05 % merthiolate (Sigma). The sections were incubated with the primary antibodies diluted to 2·5 µg ml-1 (determined as optimal by previous titrations) with 10 % NHS in PBS containing 0·05 % merthiolate overnight. Subsequently the sections were incubated with biotinylated donkey anti-rabbit IgG (Jackson Immunoresearch, PA, USA) diluted 1:500 in 1 % NHS in PBS containing 0·05 % merthiolate for 1 h, followed by incubation with ExtrAvidin-horseradish peroxidase (Sigma) diluted 1:1500 in PBS containing 0·05 % merthiolate for 1 h. All incubations were held at room temperature and separated by three 5 min washes in PBS. Finally, a freshly prepared colour reaction mixture containing 0·5 % 3,3'-diaminobenzidine, 0·1 M sodium phosphate, 0·004 % NH4Cl, 0·2 % glucose, 0·04 % nickel ammonium sulphate and 0·1 % glucose oxidase was applied to the sections for 5-10 min or until colour product appeared. The sections were then washed, dehydrated, cleared in xylene and mounted using Eukitt (BDH, Poole, UK). Control experiments were performed using an excess of the appropriate homologue peptide antigen to absorb the primary antibodies and thus confirm a specific immunoreaction.
Drugs
ATP and -MeATP were obtained from Sigma Chemical Co. (Poole, UK). ,
-Methylene-L-ATP was obtained from Tocris Cookson (Bristol, UK). 2' (or 3')-O-trinitrophenyl-ATP was obtained from Molecular Probes (Leiden, Netherlands). Solutions (10-100 mM) of ATP and other drugs were prepared using deionized water and stored frozen. All drugs were then diluted in extracellular bathing solution to the final concentration.
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RESULTS |
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Responses to agonists
Fast application of ATP (3-300 µM) onto isolated guinea-pig SCG neurons, voltage clamped at -70 mV, evoked a rapidly activating inward current in all cells tested (> 400 cells). The response to ATP desensitized slowly, with the current at the end of the 5 s application being 80 ± 2 % (n = 5) of the peak amplitude. The mean peak amplitude of the response to 100 µM ATP was 7·9 ± 4·2 nA (mean ± S.D., n = 371).
A response to -MeATP (100 µM) was seen in > 95 % of cells tested, but was always less than that to ATP (100 µM) (Fig. 1A). The mean peak amplitude of the currents evoked by 100 µM -MeATP was 2·8 ± 3·4 nA (mean ± S.D., n = 371). When the two agonists were tested on the same cells, the current elicited by 100 µM -MeATP was 36 ± 23 % (range 0·1-100 %) of that evoked by 100 µM ATP, although the EC50 values for these two agonists were similar (see Figs 1 and 4). The ratio of currents to -MeATP and ATP at 100 µM from the same neuron (-MeATP/ATP ratio) varied considerably from cell to cell. The frequency distribution of the -MeATP/ATP ratio for each of 367 cells was clearly non-Gaussian (Fig. 1B), with the probability of cells having a ratio between 0 and 0·5 being quite uniform. However, there were very few cells showing a ratio greater than 0·8.
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A, representative traces of the inward currents activated by 100 µM | ||
When the -MeATP response was normalized with respect to that produced by 100 µM ATP from the same cell, the individual concentration-response curves yielded variable maximum responses (Fig. 1C). However, when the response to -MeATP was normalized with respect to that produced by 100 µM -MeATP from the same cell, the data were tight, with small error bars (Fig. 1D). Fitting the Hill equation to individual dose-response curves for -MeATP gave a mean EC50 of 42 µM (logEC50 = -4·38 ± 0·05) and a Hill coefficient of 1·17 ± 0·07 (n = 13).
,-Methylene-L-ATP (-Me-L-ATP) was reported to be a selective agonist on P2X1 receptors, with little activity on P2X3 receptors (Trezise et al. 1995). On guinea-pig SCG neurons, -Me-L-ATP was much less potent than -MeATP, producing no response at concentrations less than 100 µM (Fig. 1D).
A larger maximum response to ATP than to -MeATP might indicate that the latter is a partial agonist. Alternatively, this could result from the presence of a mixed population of receptors. To discriminate between these possibilities, we investigated the interaction between ATP and -MeATP at a near-maximum concentration (300 µM), and the effect of cross-desensitization.
Co-application of ATP and -MeATP
If -MeATP is a partial agonist binding to the same sites as ATP, then co-application of both agonists at near-maximum concentrations will result in a reduction in the response to ATP. This is because a significant percentage of the receptors will be occupied by the partial agonist. As illustrated in Fig. 2A and B, co-application of -MeATP (300 µM) with ATP (300 µM) produced a response that was very close to that given by ATP alone. Furthermore, the response in the presence of both agonists was very different from that predicted if -MeATP was a partial agonist (see Methods). In nine cells tested in this series of experiments, co-application of -MeATP (300 µM) with ATP (300 µM) produced responses which were 97 ± 1 % of those given by ATP alone. On the other hand, when ATP (300 µM) was applied in the presence of -MeATP (300 µM), the response was similar to that evoked by ATP on its own (95 ± 4 %, n = 5; Fig. 2C).
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-MeATP to guinea-pig superior cervical ganglion neurons
A, response of one cell to | ||
Cross-desensitization
The effect of long application of 100 µM -MeATP is illustrated in Fig. 3A and B. The time course of the decline in the -MeATP-induced current fitted well to the sum of two exponentials, with time constants (
1 and 2) of 5·2 ± 0·8 and 32·1 ± 2·6 s (n = 8). For 10 cells examined in this series of experiments, the peak response evoked by 100 µM -MeATP was 45 ± 6 % of that to 100 µM ATP. After a 2 min application of 100 µM -MeATP, the response to -MeATP had declined to 14 ± 3 % of the peak (i.e. to 7 ± 2 % of the peak ATP responses), while the response to 100 µM ATP was only reduced to 62 ± 6 % (n = 10) of control. Therefore, the fractional reduction in the -MeATP response was much greater than that of the ATP response (P < 0·001). Further examination revealed that the absolute reduction in -MeATP response (d ') following desensitization was similar to the reduction in the ATP response (d) (d '/d = 99 ± 3 %, n = 10), while the absolute difference between the responses evoked by ATP and -MeATP before (
) and after (') the desensitization remained unchanged ('/ = 99 ± 4 %) (neither d '/d nor '/ was significantly different from 100 %, P > 0·1).
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-MeATP or ATP on guinea-pig superior cervical ganglion neurons
Traces of the membrane current recorded from three cells in response to a prolonged application of 100 µM | ||
On the other hand, when the cells were desensitized by a prolonged exposure to 100 µM ATP, the responses to -MeATP and ATP were reduced proportionally (Fig. 3C). The responses to -MeATP and ATP at the end of the 2 min desensitization were 18 ± 5 and 15 ± 3 % (n = 10) of the control, respectively. The time course of the decline in the ATP-induced current also fitted well to the sum of two exponentials, with time constants (1 and 2) of 5·0 ± 0·4 and 33·3 ± 3·8 s (n = 10).
These results support the presence of two populations of P2X receptors, both sensitive to ATP but only one activated by -MeATP.
Concentration-response curve for ATP
Although preliminary studies have shown that the concentration-response curve for ATP appeared to be monophasic, it must be assumed in the light of the foregoing results that there are in fact two components present (data not shown). We therefore attempted to look specifically for cells showing a small or large -MeATP/ATP ratio, i.e. to select cells with predominantly one population of P2X receptors on them, and to examine the concentration- response relationships for ATP on each population separately (Fig. 4). In eight cells showing an -MeATP/ATP ratio < 0·1 (mean ratio = 0·07 ± 0·01, mean capacitance = 31·2 ± 5·7 pF), the ATP current could be regarded as due to the activation of a single population of -MeATP-insensitive receptors. Fitting the Hill equation to the data gave an EC50 of 56 µM (logEC50 = -4·25 ± 0·11, data from 8 cells) and a Hill coefficient of 1·95.
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Concentration-response curves for ATP were constructed separately for guinea-pig SCG neurons with a small or large | ||
Cells with a large -MeATP/ATP ratio were encountered less frequently. From a total of 83 cells, we obtained 7 cells that had an -MeATP/ATP ratio > 0·7 (mean ratio = 0·8 ± 0·02, mean capacitance = 49·6 ± 5·4 pF). For this group of cells, the concentration-response relationship for ATP could be regarded as being dominated by the -MeATP-sensitive receptors. Fitting the Hill equation to the data gave an EC50 of 60 µM (logEC50 = -4·22 ± 0·26, data from 7 cells) and a Hill coefficient of 0·97.
Effects of TNP-ATP
Recently, the P2X antagonist TNP-ATP (Mockett et al. 1994; King et al. 1997) has been described as a selective antagonist on P2X1, P2X3 and P2X2/3 forms relative to P2X2 receptors (Virginio et al. 1998). We sought to determine whether TNP-ATP would show different affinity for the two types of P2X receptor present on guinea-pig SCG neurons.
TNP-ATP (0·001-10 µM) reversibly attenuated the response activated by 100 µM -MeATP. This inhibition was fitted well by a single site model, giving an IC50 of 70 nM (logIC50 = -7·16 ± 0·08, data from 8 cells), and a Hill coefficient of 0·92 (Fig. 5). The inhibition by TNP-ATP of the response to 100 µM ATP varied substantially from cell to cell. Hence, we specifically looked for cells showing an -MeATP/ATP ratio < 0·1, in which the ATP current was largely due to the activation of -MeATP-insensitive receptors. A total of 25 such cells were studied in this series of experiments. Using 100 µM ATP as the agonist, the inhibition by TNP-ATP fitted well to a single component curve, yielding an IC50 of 522 nM (logIC50 = -6·28 ± 0·13, n = 3-6 for each data point), and a Hill coefficient of 0·79. This was significantly different from the IC50 value of TNP-ATP on the -MeATP response (P < 0·01). We also examined cells showing an -MeATP/ATP ratio of 0·3-0·6. For these cells, the inhibition by TNP-ATP of the response to 100 µM ATP could be fitted with a single component curve, with an IC50 of 195 nM (logIC50 = -6·71 ± 0·06), and a Hill coefficient of 0·86 (n = 3-6 for each data point, pooled data from 33 cells; Fig. 5). Although these data could also be well fitted by a two-component curve using the IC50 values previously determined (70 and 522 nM), with equal proportions of high and low affinity binding sites (see Fig. 5), the fit was not significantly better than that for the single site model (F test, P > 0·1).
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Inhibition by TNP-ATP was studied using 100 µM | ||
Variation of the -MeATP/ATP ratio
As mentioned above, the maximum response of guinea-pig SCG neurons to -MeATP was always less than that to ATP, but the -MeATP/ATP ratio varied greatly from cell to cell. When we selected cells with very large or very small -MeATP/ATP ratios, there was a marked difference in membrane capacitance (see above). However, among the 367 cells, the correlation between cell size (as determined by membrane capacitance) and the -MeATP/ATP ratio was weak yet significant (Pearson's r = 0·22, P < 0·0001, n = 367, data not shown).
Immunohistochemical evidence
To characterize further the P2X receptors on guinea-pig SCG, we carried out immunohistochemistry using the currently available antibodies raised against rat P2X1-6 (rP2X1-6) receptors. So far, the P2X2 receptor is the only member of this family cloned from the guinea-pig (Parker et al. 1998). For this subunit, there are three splice variants which all share a common C-terminal peptide sequence, which differs by only one amino acid from that of the rat. Therefore, it is likely that the antibodies raised against rat P2X receptors are also able to recognize the P2X receptors expressed in guinea-pigs.
To check this, we tested antibodies specific for rP2X2 and rP2X3 receptors on guinea-pig nodose ganglion sections (Fig. 6A and B). The small-diameter neurons showed strong P2X3 immunoreactivity, while medium-diameter neurons showed less intense P2X3 staining, with some large-diameter neurons being negative. In contrast, specific and strong P2X2 immunoreactivity was only detected in a sub-population of neurons. The staining pattern was similar to that observed on rat nodose ganglion for these two receptor subtypes (Xiang et al. 1998).
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P2X2 (A) and P2X3 (B) immunoreactivity in guinea-pig nodose ganglion. P2X2 (C) and P2X3 (D) immunoreactivity in guinea-pig pelvic ganglion. In the same pelvic ganglion, the immunoreactivity is abolished after absorption of rP2X2 antibody with P2X2 peptide (E) and absorption of rP2X3 antibody with P2X3 peptide (F). P2X2 (G) and P2X3 (H) immunoreactivity in guinea-pig superior cervical ganglion. Scale bar, 100 µm. | ||
We then applied antibodies against rP2X1-6 to guinea-pig SCG and pelvic ganglion sections. As shown in Fig. 6C and D, neurons in the guinea-pig pelvic ganglion showed specific immunoreactivity to both P2X2 and P2X3 antibodies. The specificity of the immunoreaction was ascertained with the peptide pre-absorption. Thus, incubation of the antibodies with an excess of corresponding peptides used for immunization abolished the immunoreactivity (Fig. 6E and F). In sections of guinea-pig SCG (Fig. 6G and H), immunoreactivity for P2X2 and P2X3 was clearly present. The staining for P2X2 appeared to be cell membrane-associated, and was present in most neurons, while specific P2X3 immunoreactivity was detected in a sub-population of neurons. Antibodies to rP2X1 and rP2X4-6 receptors failed to detect any immunoreactivity in guinea-pig SCG and pelvic ganglion neurons (data not shown). However, these antibodies did produce specific staining in guinea-pig blood vessel smooth muscle (P2X1), spinous and granular cell layers of epithelium (P2X5) and cerebellar Purkinje cells (P2X4 and P2X6) (data not shown), suggesting that they do recognize the corresponding guinea-pig receptors.
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DISCUSSION |
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Co-expression of two P2X receptors in the same guinea-pig SCG neuron
The major finding in this study is the demonstration of the presence of mixed populations of P2X receptors on guinea-pig SCG neurons. Several lines of evidence are in support of this. (1) The response to -MeATP (100 µM) was always smaller than that to ATP (100 µM) from the same neuron, and the ratio of the -MeATP/ATP currents varied greatly from cell to cell. This phenomenon could not be explained by the assumption that -MeATP behaved as a partial agonist. (2) Co-application of the near-maximum concentration of -MeATP with ATP did not significantly inhibit the response to ATP as would be predicted if -MeATP was a partial agonist. (3) On desensitizing these neurons with -MeATP, the response to ATP was not reduced proportionally, but by the same absolute amount as the response to -MeATP. (4) Responses to -MeATP and ATP had different sensitivity to the antagonist TNP-ATP. Furthermore, when ATP was the agonist, the sensitivity to TNP-ATP depended on the -MeATP/ATP ratio. Thus, with -MeATP as the agonist, the IC50 for TNP-ATP was 70 nM, while for cells expressing predominantly -MeATP-insensitive receptors, the IC50 against ATP was 522 nM. All this evidence strongly suggests that there are two distinct populations of P2X receptors co-existing on the same guinea-pig SCG neuron, one is sensitive to -MeATP whilst the other one is not, with the proportion of each receptor subtype varying from cell to cell. This is very similar to the situation in rat nodose neurons (Thomas et al. 1998).
Is -MeATP a partial agonist?
-MeATP has been reported to be a full agonist on guinea-pig coeliac ganglion, rat nodose and rat dorsal root ganglion (DRG) neurons (Khakh et al. 1995; Lewis et al. 1995; Robertson et al. 1996). On recombinant heteromeric P2X4/6 receptors (Lê et al. 1998), guinea-pig intracardiac neurons (Allen & Burnstock, 1990) and rat cardiac neurons (Fieber & Adams, 1991), -MeATP has been proposed to act as a partial agonist since it was less potent than ATP and produced a smaller maximum response. However, on guinea-pig myenteric neurons (Zhou & Galligan, 1996), -MeATP clearly evoked variable maximum responses in different cells. We found that co-application of near-maximal concentrations of -MeATP and ATP to guinea-pig SCG neurons did not affect the ATP-induced inward currents. A similar effect has been reported on guinea-pig myenteric neurons (Barajas-López et al. 1996). Thus, our results are not consistent with the suggestion that -MeATP is a partial agonist, but rather that both -MeATP-sensitive and -insensitive receptors are co-expressed on the same cell, with -MeATP being a full agonist at the former. In the light of our findings and the work of Thomas et al. (1998), it is clear that -MeATP can produce a reduced maximum response compared with ATP, with the relative amplitudes varying from cell to cell, if mixed P2X receptor populations coexist on the same neuron. Analysis at the single channel level may be required to confirm or refute the suggestion that -MeATP is a partial agonist on other neurons.
Possible identity of the P2X receptors on guinea-pig SCG neurons
The slowly desensitizing response to -MeATP indicates that the -MeATP-sensitive receptors on these neurons may be of P2X2/3 phenotype, like those found in rat nodose ganglion (Lewis et al. 1995), trigeminal ganglion (Cook et al. 1997) and capsaicin-insensitive DRG neurons (Ueno et al. 1999). We demonstrated, using the polyclonal antibodies raised against rat P2X receptors, the presence of the P2X2 and P2X3 immunoreactivity in SCG and pelvic neurons of the guinea-pig. This is in contrast to the situation in the rat pelvic ganglion, where only the P2X2 immunoreactivity was identified (Zhong et al. 1998). Apart from P2X2 (Parker et al. 1998), all the other guinea-pig P2X receptor subtypes have yet to be cloned, so the degree of homology between rat and guinea-pig P2X receptors is at present unknown, and some caution must be used in the interpretation of these results. Nevertheless, the C-terminal peptide sequences used to raise these antibodies are well conserved between P2X2 receptors from rat and guinea-pig, and between other P2X receptors from rat and human. Furthermore, although we cannot completely exclude the possibility that our antibodies raised against the rat sequences do not recognize the corresponding guinea-pig receptors, a similar staining pattern was observed for P2X1-6 antibodies in appropriate guinea-pig tissues, compared with that in rat tissues. This suggests that these antibodies may correctly recognize guinea-pig P2X receptors and that the receptors on guinea-pig SCG neurons, like those on rat nodose neurons, may be P2X2 and P2X2/3.
The presence of varying proportions of two types of P2X receptors on guinea-pig SCG neurons greatly complicated their pharmacological characterization. For -MeATP, presumed to be acting on a single population of receptors, we obtained an EC50 value of 42 µM. This is lower than the EC50 values found on recombinant rP2X2/3 receptors (Lewis et al. 1995), rat nodose neurons and guinea-pig coeliac neurons (Khakh et al. 1995). However, the EC50 value we obtained was similar to those found on capsaicin-insensitive rat DRG neurons (Ueno et al. 1999), and on rP2X2 + 3 receptors co-expressed in C6BU-1 glioma cells (Ueno et al. 1998). It is possible that the coexistence of P2X2 and P2X3 subunits could result in several subsets of heteromeric receptors of unknown stoichiometry, with different affinity for agonists. Furthermore, we cannot exclude the possibility of the involvement of other P2X subunits, or of splice variants. Of the recombinant homomeric receptors, only P2X3 and P2X1 receptors respond to -MeATP. However, the P2X1-selective agonist -Me-L-ATP (Trezise et al. 1995) was much less potent than -MeATP on guinea-pig SCG neurons, suggesting that the P2X1 subunit is unlikely to be involved.
There is at present no selective agonist for the -MeATP-insensitive receptor. We therefore selected cells with a -MeATP/ATP ratio < 0·1, where there were predominantly -MeATP-insensitive receptors and obtained an EC50 value for ATP of 56 µM and a Hill coefficient of 1·95. As cells with a very large -MeATP/ATP ratio (> 0·9) were encountered very rarely (1 % of cells), it was not possible to obtain a precise value for the affinity of ATP at the -MeATP-sensitive receptor. However, analysis of cells where the -MeATP/ATP ratio was > 0·7 revealed an EC50 value for ATP similar to that at -MeATP-insensitive receptors. Interestingly, the Hill coefficient was considerably less (0·97). This might indicate the presence of multiple receptor subtypes, or that there is positive co-operativity at the -MeATP-insensitive receptor, but not at the -MeATP-sensitive ones. Irrespective of the reason, a similar difference in Hill coefficients was observed between -MeATP-sensitive receptors in rat nodose neurons and -MeATP-insensitive receptors in rat SCG neurons (Khakh et al. 1995).
Recently, the coexistence of homomeric P2X2 and heteromeric P2X2/3 receptors has been revealed on rat nodose ganglion neurons using a selective antagonist TNP-ATP (Thomas et al. 1998), which has 1000-fold higher potency on recombinant rP2X2/3 than rP2X2 receptors. On guinea-pig SCG neurons, the IC50 values for TNP-ATP on -MeATP-sensitive and -MeATP-insensitive receptors were 70 and 522 nM, respectively. This 8-fold difference was smaller than that reported between rP2X2 and rP2X2/3 receptors, and the IC50 value for TNP-ATP against -MeATP is greater than that reported for the heterologously expressed rP2X2/3 receptors (Virginio et al. 1998). One possible explanation for this is that TNP-ATP may be unstable in aqueous solution. However, the greater than expected potency of the -MeATP-insensitive receptors would argue against this. Furthermore, we have observed inhibition by TNP-ATP of a rapidly desensitizing ATP response in rat DRG neurons, with an IC50 of approximately 1 nM under identical conditions (P. Dunn, Y. Zhong & G. Burnstock, unpublished observations). In a study on rat nodose neurons, the inhibition of -MeATP by TNP-ATP was biphasic, with the lower affinity component having an IC50 of 50 nM (Thomas et al. 1998), which is quite similar to the value we obtained. Because of the small difference in affinity of TNP-ATP for -MeATP-sensitive and -insensitive receptors, the inhibition curve for cells having approximately equal numbers of both receptors was not clearly biphasic. Although we cannot rule out the possibility that guinea-pig SCG neurons possess a novel P2X receptor, a more likely explanation is that guinea-pig P2X2 and P2X2/3 receptors may exhibit slightly different pharmacological properties compared with those of the rat.
Inter-species variation
Another finding in this study is the species difference in the expression of P2X receptors between rat and guinea-pig. Previous work by Khakh et al. (1995) demonstrated that neurons in the guinea-pig coeliac ganglion, like those in the rat nodose ganglion, respond to -MeATP, while those in the rat SCG do not. On the basis of those observations the authors suggested that neurons of the SCG may be anomalous. An alternative explanation, and one which we favour, is that expression of P2X receptor subtypes is different in rat and guinea-pig. Thus, neurons from guinea-pig SCG (this study), coeliac (Khakh et al. 1995) and pelvic ganglia (Y. Zhong, P. M. Dunn & G. Burnstock, unpublished observations) all respond to -MeATP, while those in the rat SCG (Nakazawa, 1994), coeliac (Zhong et al. 2000) and pelvic ganglia (Zhong et al. 1998) do not. In addition, the properties of the P2X receptors are different in the outer hair cells of rat and guinea-pig (Chen et al. 1997). There seems, therefore, to be inter-ganglion (e.g. autonomic vs. sensory ganglion) as well as inter-species (e.g. rat vs. guinea-pig) differences in the expression of P2X receptors.
In the rat, high levels of P2X3 subunit expression appear to be localized exclusively in sensory neurons (Buell et al. 1996), with a low level expression in sympathetic neurons detectable by immunohistochemistry and in situ hybridization (Xiang et al. 1998). However, immunohistochemical and pharmacological data indicate that the expression of P2X3 subunits may be more widespread in the guinea-pig.
The presence of multiple receptor subtypes occurring in the same neuron has been observed previously for nicotinic acetylcholine receptors (Connolly et al. 1995; Poth et al. 1997), and more recently for P2X receptors (Thomas et al. 1998; Grubb & Evans, 1999; this study). At present, it is not clear what factors may control the expression of these mixed populations of P2X receptors. Whether the proportions of them are determined simply by the relative amounts of the subunits synthesized and remains constant in individual cells, or whether the proportions can change in developmental or pathological conditions remains to be determined.
In conclusion, in the present study, we have characterized P2X receptors on single neurons of guinea-pig SCG, using subtype-selective agonists, antagonists and immunohistochemistry. Our results suggest that varying proportions of two distinct P2X receptors coexist on the same neuron, which may correspond to homomeric P2X2 and heteromeric P2X2/3 receptors. Thus, there is an inter-species difference in the expression of P2X receptors in sympathetic neurons.
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The authors are grateful to E. W. Moules, M. Bardini and T. Robson for their excellent technical support, to Mr R. Jordan for the editorial assistance with the manuscript. Y.Z. and P.M.D. were supported by Roche Bioscience (Palo Alto, CA, USA).
Corresponding author
Y. Zhong: Autonomic Neuroscience Institute, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK.
Email: y.zhong{at}ucl.ac.uk
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) and 
), with responses normalized to that obtained with 100 µM 
), fitting the data to the Hill equation gave an EC50 of 60 µM and a Hill coefficient of 0·97. Responses were normalized with respect to that obtained with 100 µM ATP on the same cell.
, 
) as the agonist on cells with an