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Pharmacological comparison of P2X receptors on rat coeliac, mouse coeliac and mouse pelvic ganglion neurons
Neuropharmacology 39 (2000) 172–180 www.elsevier.com/locate/neuropharm
Pharmacological comparison of P2X receptors on rat coeliac, mouse coeliac and mouse pelvic ganglion neurons Yu Zhong *, Philip M. Dunn, Geoffrey Burnstock Autonomic Neuroscience Institute, Department of Anatomy and Developmental Biology, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK Accepted 29 June 1999
Abstract Characteristics of P2X receptors on neurons of the rat coeliac, mouse coeliac and mouse pelvic ganglia have been studied using the whole cell voltage-clamp technique. Fast application of ATP (100 µM) on to isolated neurons voltage clamped at ⫺70 mV induced a slowly desensitising inward current in 96% of the cells tested. Concentration–response curves for ATP yielded EC50 values of 86 µM, 64 µM and 123 µM, for rat coeliac, mouse coeliac and mouse pelvic ganglion neurons, respectively, while α,βmethylene ATP was inactive. The response to ATP was antagonised by suramin, Cibacron blue and pyridoxalphosphate-6-azophenyl2⬘,4⬘-disulphonic acid (PPADS). The potency of ATP was increased by extracellular acidification and by co-application of micromolar concentrations of Zn2+, while raising pH decreased it. On rat coeliac ganglion neurons, the EC50 values for ATP were 35 µM and 253 µM at pH 6.8 and 8.0, respectively. On mouse coeliac and pelvic ganglion neurons, altering the pH produced comparable changes. In conclusion, our results indicate that, in contrast to the guinea-pig coeliac ganglion, the characteristics of the P2X receptors present on rat coeliac, mouse coeliac and mouse pelvic ganglia are all identical to those present on rat pelvic ganglion, i.e. they are homomeric P2X2 receptors, or heteromultimers with P2X2 being the dominant subunit. 2000 Elsevier Science Ltd. All rights reserved. Keywords: P2X receptor; Coeliac ganglion; Pelvic ganglion; ATP; pH; Zn2+
1. Introduction ATP acts as a fast excitatory neurotransmitter (for review, see Surprenant et al., 1995; Burnstock, 1997), where it activates a class of ligand-gated ion channels, the P2X receptors. To date, seven P2X receptor subunits have been cloned (P2X1–7), all of which have been reported to form functional homo-oligomeric receptors with different though overlapping biophysical and pharmacological properties, including agonist profiles, desensitisation and sensitivities to antagonists (Brake et al., 1994; Valera et al., 1994; Bo et al., 1995; Chen et al., 1995; Collo et al., 1996; Surprenant et al., 1996). In addition, these P2X receptor subtypes are differentially modulated by pH (King et al., 1996; Stoop et al., 1997) and Zn2+ (Wildman et al. 1998, 1999). The pharmaco-
* Corresponding author. Tel.: +44-171-794-0500, ext. 6754; fax: +44-171 830 2949. E-mail address: [email protected] (Y. Zhong)
logical profiles of the recombinant P2X receptors do not always match those of the endogenous P2X receptors; thus, it is plausible that some native P2X receptors are hetero-multimeric channels composed of different P2X subunits (Lewis et al., 1995; Leˆ et al., 1998). In autonomic and sensory neurons, two broad groups of native P2X receptors can be distinguished by their sensitivity to α,β-methylene ATP (α,β-meATP) (Evans and Surprenant, 1996). Thus, neurons from rat dorsal root (DRG) (Robertson et al., 1996; Rae et al., 1998), nodose (Lewis et al., 1995) and trigeminal ganglion neurons (Cook et al., 1997) are activated by α,β-meATP. The involvement of P2X3 and P2X2/3 receptors has been suggested. In another group of neurons, such as those of the rat superior cervical ganglion (SCG) (Nakazawa, 1994) and rat pelvic ganglion (Zhong et al., 1998), α,βmeATP is inactive or very weak as an agonist. The molecular and pharmacological properties of P2X receptors on those neurons suggest them to be of the P2X2 subtype. This seems to suggest that in the rat, sensory neurons demonstrate α,β-meATP sensitivity, while the auto-
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nomic neurons demonstrate α,β-meATP insensitivity. However, neurons from guinea-pig SCG (Reekie and Burnstock, 1994) and guinea-pig coeliac ganglion (Khakh et al., 1995) respond to α,β-meATP and the receptors present on guinea-pig coeliac ganglion neurons resemble those of rat nodose ganglion (Khakh et al., 1995). Therefore, in this study, we sought to determine the pharmacological properties of the P2X receptors on rat coeliac ganglion neurons. We were interested in finding whether P2X receptors on rat coeliac (sympathetic) ganglion neurons are similar to those on rat SCG (also sympathetic) and pelvic neurons, or whether they exhibit similar properties to those on guinea-pig coeliac ganglion neurons, i.e. are similar to those on rat nodose (sensory) ganglion neurons. We have also characterised the P2X receptors on mouse coeliac and pelvic ganglia, to explore further the existence of inter-species and interganglion variation, and to provide background information for studies of “knock-out” mice lacking specific P2X subunits. A preliminary report on part of the work has appeared in the form of an abstract (Zhong et al., 1999).
2. Methods 2.1. Isolation of neurons Single neurons of the coeliac ganglion from 17-dayold male Sprague-Dawley rats and coeliac and pelvic ganglia from adult male mice were enzymatically isolated as described previously (Zhong et al., 1998). The animals were killed by a rising concentration of CO2 and death was confirmed by cervical dislocation. The coeliac and pelvic ganglia were rapidly dissected out and placed in Leibovitz’s L-15 medium (Life Technologies, Paisley, UK). The ganglia were desheathed and three to four deep cuts were made in each before incubation in 4 ml Ca2+/Mg2+-free Hank’s Balanced Salt Solution with 10 mM HEPES pH 7.4 buffer (HBSS) (Life Technologies) containing 1.5 mg/ml collagenase (Class-II; Worthington Biochemical Corporation, Reading, UK) and 6 mg/ml bovine serum albumin (Sigma Chemical Co., Poole, UK) at 37°C for 40 min. The ganglia were then incubated with 4 ml HBSS containing 1 mg/ml trypsin (Sigma) at 37°C for 15 min. The solution was replaced with 3 ml growth medium comprising L-15 medium supplemented with 50 ng/ml nerve growth factor, 10% bovine serum, 2 mg/ml NaHCO3, 5.5 mg/ml glucose, 200 IU/ml penicillin and 200 µg/ml streptomycin. The ganglia were dissociated into single neurons by gentle trituration with two fire-polished glass pipettes of decreasing diameter. The cells were then centrifuged at 900 rev/min for 5 min, resuspended in 1 ml growth medium and plated onto 35 mm Petri dishes coated with 10 µg/ml laminin (Sigma).
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Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2, and used between 3 and 48 h after plating.
2.2. Whole cell voltage-clamp recording
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 voltage clamped at ⫺70 mV. External solution contained (mM): NaCl 154, KCl 4.7, MgCl2 1.2, CaCl2 2.5, HEPES 10, glucose 5.6, the pH being adjusted to 7.4 using NaOH. Recording electrodes were made from thin-walled glass capillary tubes with fine filament (GC 150TF; Clark Electromedical Instruments, Reading, UK) using a two-stage puller (PP-830, Narishige, Tokyo, Japan), fire-polished, and had a resistance of 2–4 M⍀ when filled with an internal solution containing (mM): citric acid 56, MgCl2 3, CsCl 10, NaCl 10, HEPES 40, EGTA 0.1, tetraethylammonium chloride 10, and the pH adjusted to 7.2 using CsOH (total Cs+ concentration 170 mM). Series resistance compensation was not used. However, only those cells with a maximum voltage clamp error of ⱕ8 mV, due to the series resistance, were used for pharmacological studies. The liquid– liquid junction potential, measured at 6 mV, was not corrected for. Data were acquired using pCLAMP software (Axon Instruments). Signals were filtered at 2 kHz (⫺3 dB frequency, Bessel filter, 80 dB/decade). Drugs were applied rapidly through a seven-barrel manifold composed of fused glass capillaries inserted into a common outlet tube with a tip diameter of 苲200 µm, which was placed very close to the cell (Dunn et al., 1996). Solutions were delivered by gravity flow from independent reservoirs placed above the preparation. One barrel was used to apply drug-free solution to enable rapid termination of drug application. Applications of agonists (5 s) were separated by intervals of 2 min, which were sufficient for responses to be reproducible. Antagonists were present for 2 min before and during the reapplication of agonists. All responses were normalised to that evoked by ATP (1 mM) under control conditions in the same cell, and expressed as the mean±SEM, unless otherwise indicated. The concentration–response data were fitted with the Hill equation: Y=A/[1+(K/X)n], where A is the maximum effect, K is the EC50, and n is the Hill coefficient, using Prism v2 (GraphPad, San Diego, CA, USA). The combined data from the number of treated cells were fitted, and the parameters are presented as fitted values ±SEM, determined by the fitting routine. Traces were acquired using pCLAMP and plotted using Origin (Microcal, Northampton, MA, USA). Statistical analysis (Student’s ttest) was performed using Prism v2.
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2.3. Drugs and materials ATP, α,β-meATP and other chemicals were obtained from Sigma Chemical Co. Suramin was a gift from Bayer plc (Newbury, UK), and pyridoxalphosphate-6azophenyl-2⬘,4⬘-disulphonic acid (PPADS) was from Tocris Cookson (Bristol, UK). Stock solutions (10–100 mM) of ATP and other drugs were prepared using deionised water and stored frozen. For application all drugs were diluted in extracellular bathing solution to the required concentration.
3. Results 3.1. Response to agonists Fast application of ATP (100 µM) to isolated rat coeliac, mouse coeliac and mouse pelvic ganglion neurons voltage-clamped at ⫺70 mV induced a rapidly activating inward current in 96% of cells tested (Fig. 1). The peak amplitudes (means±SD) of response to ATP (100 µM) were 0.73±0.89 nA (n=94; range 0.025–5.6 nA) for rat coeliac ganglion neurons, 0.66±0.63 nA (n=70; range 0.02–3.2 nA) for mouse coeliac ganglion neurons, and 0.71±0.60 nA (n=61; range 0.025–3.2 nA) for mouse pelvic ganglion neurons. Despite the large variability in response, for neurons from each ganglia, the amplitude distribution revealed a single skewed population, with 80% of cells giving small to moderate responses (⬍1 nA) (data not shown). We have previously observed a similar distribution of the amplitude of inward currents evoked by ATP in rat major pelvic ganglion neurons (Zhong et al., 1998). The response to ATP (100 µM) desensitised slowly, with the evoked current declining to 61±3% (n=8), 66±5% (n=5) and 70±2% (n=4) of control at the end of 30 s application for rat coeliac, mouse coeliac and mouse pelvic ganglion neurons, respectively. The decay of the current was fitted well by a single exponential decay yielding time constants of 19.2±1.5 s (n=8), 21.4±1.3 s (n=5) and 21.0±3.1 s (n=4), for rat coeliac, mouse coeliac and mouse pelvic ganglion neurons, respectively. The concentration–response curves for ATP on these neurons are shown in Fig. 2. Fitting the Hill equation to the data gave EC50 values of 86 µM (log EC50=⫺4.067±0.043, data from seven cells), 64 µM (log EC50=⫺4.191±0.101, data from nine cells) and 123 µM (log EC50=⫺3.910±0.087, data from ten cells), for rat coeliac, mouse coeliac and mouse pelvic ganglion neurons, respectively, with Hill coefficients of 1.6, 1.5 and 1.4. When α,β-meATP was tested, it failed to evoke any response at concentrations up to 100 µM in any cell tested (at least six neurons for each of these three ganglia).
Fig. 1. Representative traces of the inward currents activated by prolonged (30 s) application of ATP (100 µM) to isolated rat coeliac (a), mouse coeliac (b) and mouse pelvic (c) ganglion neurons, voltage clamped at ⫺70 mV.
3.2. Effects of antagonists The P2 receptor antagonists Cibacron blue (Cibacron Blue 3GA, 65% pure) (Manzini et al., 1986), suramin (Dunn and Blakeley, 1988) and PPADS (Lambrecht et
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Fig. 2. Concentration–response curves for ATP on rat coeliac, mouse coeliac and mouse pelvic ganglion neurons. Cells were voltage clamped at ⫺70 mV and various concentrations of ATP were applied for 5 s every 2 min. Responses were normalised with respect to that obtained with ATP (1 mM) on the same cell. Each point represents the mean±SEM from seven to ten cells. The Hill equation was fitted to the data, yielding EC50 values of 87 µM, 64 µM and 123 µM, for rat coeliac, mouse coeliac and mouse pelvic ganglion neurons, respectively.
al., 1992) were tested on neurons from these three ganglia (Fig. 3). On rat coeliac ganglion neurons (Fig. 3a), 2-min incubation with Cibacron blue (10 µM) abolished the response to ATP (100 µM; n=7). The inhibition was readily reversible, with the response being 71±4% of control (n=7) after a 2-min wash-out. Suramin was less effective than Cibacron blue in inhibiting the response to ATP. With a 2-min preincubation, 100 µM suramin reduced the response to ATP (100 µM) to 16±3% of control (n=5). The response recovered rapidly and was 86±4% of control (n=5) 2 min after washing out suramin. PPADS was also able to inhibit the response to ATP on rat coeliac ganglion neurons. After a 4-min preincubation, PPADS (10 µM) reduced the response to ATP (100 µM) to 9±1% of control (n=5). Recovery from the inhibition of PPADS was slow, with the response to ATP (100 µM) being only 19±3% of control (n=5) 4 min after washing out the antagonist. The kinetics of the inhibition by PPADS was concentration-dependent, and considerably slower than those of Cibacron blue and suramin. When fitting the time profile of the inhibition by PPADS of the response to ATP (100 µM) by the single exponential decay, the time constants yielded were 7.5 min and 1.3 min, for 1 and 10 µM PPADS, respectively (n=6, data not shown). This is similar to that seen on rat major pelvic ganglion neurons (Zhong et al., 1998). The effect of antagonists on mouse coeliac and pelvic ganglion neurons were similar to those on rat coeliac ganglion neurons (Fig. 3a and c). Thus, after 2-min preincubation with Cibacron blue (10 µM), the response to
Fig. 3. Effect of P2 receptor antagonists, Cibacron blue (CB, 10 µM), suramin (SUR, 100 µM) and PPADS (10 µM) on responses to ATP on neurons of rat coeliac (a), mouse coeliac (b) and mouse pelvic (c) ganglion neurons. The histogram shows the peak amplitudes of the inward currents induced by ATP (100 µM) alone (control, open bars), in the presence of the antagonists (solid bars) and after washing out the antagonists (recovery, hatched bars). Responses were normalised with respect to that obtained with ATP (100 µM) in the absence of antagonists on the same cell. Each bar represents the mean±SEM from five to seven cells. In the cases of Cibacron blue and suramin, the antagonists were present 2 min before and during the re-application of ATP, with the recovery responses measured 2 min after washing out the antagonists. PPADS was present 4 min before and during the re-application of ATP, and the recovery responses were measured 4 min after washing out.
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ATP (100 µM) was reduced to 18±2% (n=6) and 27±2% of control (n=6) on mouse coeliac and pelvic ganglion neurons, respectively, with rapid and full recovery. A 2 min preincubation with 100 µM suramin reversibly reduced the response to ATP (100 µM) to 41±3% of control (n=6) on mouse coeliac ganglion neurons and 28±1% of control (n=6) on mouse pelvic ganglion neurons, respectively. Again, the recovery was also rapid and full. PPADS, similar to its action on rat coeliac ganglion neurons, produced inhibition of the ATP response with much slower kinetics. After a 4-min preincubation, PPADS (10 µM) reduced the response to ATP (100 µM) to 3±1% of control (n=6) on mouse coeliac ganglion neurons and 2±1% of control (n=6) on mouse pelvic ganglion neurons. The responses recovered to 15±2% and 10±1% of control (n=6), respectively, 4 min after washing out PPADS. 3.3. Modulation by pH The response to ATP on neurons from all three ganglia was very sensitive to changes in extracellular pH. On rat coeliac ganglion neurons, the amplitude of the inward current induced by ATP (100 µM) was increased greatly when pH was reduced from 7.4 to 6.8, and was markedly attenuated when pH was elevated from 7.4 to 8.0 (Fig. 4a). The effect of protons on currents activated by ATP was rapid and reversible. On average, the current activated by ATP (100 µM) was enhanced at pH 6.8 to 156±6% (n=9) of that at pH 7.4 (control), while raising pH to 8.0 suppressed the response to 36±3% of control (n=10). On rat coeliac ganglion neurons, changing the pH did not alter the slope of the concentration–response curve for ATP, but rather changed the affinity of ATP (Fig. 4a). The EC50 of ATP was changed from 86 µM at pH 7.4 to 35 µM (log EC50=⫺4.451±0.057, n=10) at pH 6.8, and 253 µM (log EC50=⫺3.597±0.045, n=10) at pH 8.0. The EC50 values of ATP at pH 6.8 and 8.0 were significantly different from that at pH 7.4 (P⬍0.001 and P⬍0.05, respectively). The Hill coefficients (1.7 and 1.3 at pH 6.8 and 8.0, respectively) were not significantly different from that at pH 7.4. On mouse coeliac and pelvic ganglion neurons, altering extracellular pH produced comparable changes in the ATP response (trace not shown). For these neurons, the effects of pH 6.8 and pH 8.0 were studied on the concentration–response relationship of ATP at 30–100 µM and 100–300 µM, respectively. These concentrations were chosen because they fell on the most linear part of the ATP concentration–response curve. On mouse coeliac ganglion neurons, lowering the pH from 7.4 to 6.8 potentiated the response to 100 µM ATP (to 154±7% of control, n=7), and shifted the ATP concentration– response curve significantly to the left by 0.272±0.025 log units (P⬍0.001, n=6). Raising the pH to 8.0 attenu-
ated the response to ATP (100 µM) (to 36±5% of control, n=9), and shifted the ATP concentration–response curve significantly to the right by 0.547±0.048 log units (P⬍0.001, n=6) (Fig. 4b). On mouse pelvic ganglion neurons, lowering the pH from 7.4 to 6.8 potentiated the response to 100 µM ATP (to 201±9% of control, n=7), and shifted the ATP concentration–response curve significantly to the left by 0.407±0.029 log units (P⬍0.001, n=6). Raising the pH to 8.0 attenuated the responses to 100 µM ATP (to 25±2% of control, n=7), and shifted the ATP concentration–response curve significantly to the right by 0.537±0.063 log units (P⬍0.001, n=6) (Fig. 4c). 3.4. Modulation by Zn2+ In a previous study, we have shown that Zn2+ could enhance the response to ATP (30 µM) on rat major pelvic ganglion neurons with an EC50 of 9.6 µM, and increase the affinity of ATP for the receptor (Zhong et al., 1998). Therefore, we investigated the effect of Zn2+ (10 µM) on neurons from rat coeliac, mouse coeliac and mouse pelvic ganglia. Similar to its effect on rat major pelvic ganglion, coapplication of Zn2+ (10 µM) with ATP on rat coeliac ganglion neurons shifted the concentration–response curve of ATP to the left in a parallel fashion (Fig. 5a). The EC50 of ATP was decreased significantly from the control value of 86 µM to 23 µM (log EC50=⫺4.632±0.099, P⬍0.0001, n=7), while Emax and the Hill coefficient (1.3) were not significantly altered. On mouse coeliac and pelvic ganglion neurons, we studied the effect of Zn2+ on the concentration–response relationship between ATP (30 and 100 µM; Figs. 5b and c). Co-application of Zn2+ (10 µM) with ATP shifted the concentration–response curve to ATP to the left in a parallel fashion by 0.368±0.066 log units (n=7), and 0.529±0.048 log units (n=5), respectively. Both shifts were significantly greater than zero (P⬍0.001).
4. Discussion In the present study, the pharmacological properties of the P2X receptors on neurons from rat coeliac, mouse coeliac and mouse pelvic ganglia were compared. 4.1. Involvement of P2X2 subunits Almost all neurons from the three ganglia studied responded to ATP (100 µM), with a slowly desensitising inward current. The potency of ATP was similar on three ganglia. In contrast, α,β-meATP was inactive at concentrations up to 100 µM in all three ganglia. The three purine receptor antagonists tested (suramin, Cibacron blue and PPADS) all inhibited the response to ATP with
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Fig. 4. Effect of pH on ATP induced currents. (a) Rat coeliac ganglion neurons concentration–response curves for ATP at pH 6.8 and 8.0. Each point represents the mean±SEM from ten cells. The Hill equation was fitted to the data assuming the same maximum, yielding EC50 values of 35 µM and 253 µM at pH 6.8 and 8.0, respectively. The traces (top panel) show representative responses to ATP (100 µM) recorded from one rat coeliac ganglion neuron at different pH. The horizontal bars represent the application period of ATP. (b) Mouse coeliac and (c) mouse pelvic ganglion neurons concentration–response relationship for ATP at pH 6.8, 7.4 and 8.0. Each point represents the mean±SEM from six cells. All responses were normalised with respect to that evoked by ATP (1 mM) at pH 7.4 on the same cell. For each cell type, the appropriate control concentration–response curve (pH 7.4) taken from Fig. 2 has been superimposed (dotted curve) for comparison.
comparable potencies to those observed previously on rat pelvic ganglion neurons (Zhong et al., 1998). The effect of ATP was potentiated by acidification and by co-application of micromolar concentrations of Zn2+. Thus the receptors present on neurons of the rat coeliac, mouse coeliac and mouse pelvic ganglia would appear to be identical with those found on rat pelvic ganglion neurons, which are thought to be of the P2X2 subtype, or hetero-multimeric receptors in which P2X2 is the dominant subunit (for further discussion see Zhong et al., 1998).
4.2. Expression of hetero-multimeric receptors? Recently, biochemical evidence has suggested that among the seven P2X receptor subtypes, six can form homo-oligomeric complexes, the exception being P2X6 (Torres et al., 1999). In addition, all, apart from P2X7, are able to form hetero-oligomeric assemblies with P2X2 combining with P2X1, P2X3, P2X5 and P2X6 subunits. Although the functional properties of most of these combinations have yet to be determined, the coexpression of P2X2 and P2X3 receptor subunits are required to produce
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ATP-gated currents with the properties seen in rat nodose neurons (Lewis et al., 1995), while it has also been suggested that P2X4 and P2X6 subunits coassemble to form multimeric channels with unique functional properties that resemble those found on native neurons in the central nervous system (Leˆ et al., 1998). The unusual pharmacological properties of the P2X receptors present on guinea-pig myenteric neurons might also result from the formation of heteromeric receptors (Barajas-Lo´pez et al., 1996). Therefore, although the neurons investigated in this study demonstrate a pharmacological profile of the P2X2 phenotype, it is still possible that these P2X receptors are hetero-multimers, with P2X2 being the dominant component. The presence of alternatively spliced variants of P2X receptor subunits may impose additional complexity to the characterisation of endogenous P2X receptors. In the rat, four splice variants of the P2X2 receptor have been isolated: P2X2(a), P2X2(b), P2X2(c) and P2X2(d) (Bra¨ndle et al., 1997; Simon et al., 1997). In guinea-pig, three splice variants of the P2X2 receptor have been isolated, with P2X2-1 and P2X2-2 being the homologues of rat P2X2(a) and P2X2(b), while P2X2-3 is novel (Parker et al., 1998). In mouse, four splice variants of the mouse P2X4 receptor, P2X4(a), P2X4(b), P2X4(c) and P2X4(d), have been reported (Simon et al., 1999; Townsend-Nicholson et al., 1999). It is possible that splice variants of the P2X2 receptor also exist in mouse. This may increase the number of subunits available for hetero-multimeric assembly. 4.3. Multiple P2X receptors in the same ganglion, and the co-existence of multiple receptors on the same neuron?
Fig. 5. Enhancement of the potency of ATP by Zn2+ on rat coeliac, mouse coeliac and mouse pelvic ganglion neurons. (a) Concentration– response curves for ATP in the presence of Zn2+ (10 µM) on neurons from rat coeliac ganglia. The Hill equation was fitted to the data, yielding an EC50 of 23 µM. Each point represents the mean±SEM from seven cells. (b) and (c) Concentration–response relationship for ATP in the presence of Zn2+ (10 µM) on neurons from mouse coeliac (b) and mouse pelvic (c) ganglia. Each point represents the mean±SEM from five to seven cells. Responses were normalised with respect to that evoked by ATP (1 mM) in the absence of Zn2+ on the same cell. For each cell type, the appropriate control concentration–response curve taken from Fig. 2 has been superimposed (dashed curve) for comparison.
Evidence from recent studies has suggested that the expression of P2X receptors in native tissue is more complicated than previously thought. Both fast-desensitising and slow-desensitising ATP responses have been identified on rat trigeminal nociceptive neurons (Cook et al., 1997), adult rat DRG neurons (Ueno et al., 1999) and guinea-pig myenteric neurons (Zhou and Galligan, 1996). Moreover, two P2X receptors have been shown to co-exist on the same cell from rat nodose ganglion (Thomas et al., 1998), rat DRG (Grubb and Evans, 1999) and guinea-pig SCG (Y. Zhong and P. Dunn, unpublished observations). The proportion of each receptor type varies from cell to cell. On neurons examined in this study, there was no evidence to suggest that more than one form of P2X receptors exist in the same cell. However, better pharmacological tools or novel techniques may in the future clarify the situation. 4.4. Inter-species and inter-ganglion variation Nakazawa (1994) has suggested that the properties of P2X receptors may depend on both species and types of
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tissues, since there is difference in the sensitivity to α,βmeATP between rat SCG neurons and guinea-pig coeliac ganglion neurons (Evans et al., 1992) or rat DRG neurons (Bean, 1990). However, Khakh et al. (1995) suggested that P2X receptors in guinea-pig coeliac ganglion neurons were similar to those in rat nodose ganglion neurons, while those expressed in rat SCG neurons were anomalous. In this study, we have characterised the pharmacological properties of P2X receptors on rat coeliac ganglion neurons, and found them to be P2X2-like, and similar to those present on rat SCG and pelvic ganglion neurons. Therefore, rat SCG neurons are not unique in the type of P2X receptors they express. Thus, available evidence indicates that rat autonomic neurons (from SCG, pelvic, cardiac and coeliac ganglia) all possess the same P2X2-like receptor (Fieber and Adams, 1991; Nakazawa, 1994; Zhong et al., 1998; this study). In contrast, rat sensory neurons (from DRG, nodose and trigeminal ganglia) express P2X2 and P2X3 subunits, leading to the formation of varying proportions of homomeric and heteromeric receptors (Lewis et al., 1995; Cook et al., 1997; Thomas et al., 1998; Grubb and Evans, 1999; Ueno et al., 1999). Therefore, in the rat, there is inter-ganglion difference for the expression of P2X receptors, e.g. between autonomic and sensory ganglia, but not between neurons in different autonomic ganglia. Interestingly, all guinea-pig autonomic neurons examined (SCG, coeliac and pelvic ganglia) possess slowly desensitising α,βmeATP sensitive receptors which resemble the P2X2/3 heteromeric receptor (Reekie and Burnstock, 1994; Khakh et al., 1995; Y. Zhong and P. Dunn, unpublished observations). Hence, inter-species variation does exist. The mouse P2X3 protein shows 99% identity with rat P2X3 (Souslova et al., 1997), and the mouse P2X4 protein shares 94% identity with rat P2X4 receptor (Townsend-Nicholson et al., 1999). Hence it is possible that the mouse P2X2 receptor will show a similar degree of homology to the rat P2X2 receptor. In this study, we investigated the pharmacological properties of the P2X receptors in mouse coeliac and pelvic ganglia, and found their pharmacology to be similar to that of the heterologously expressed rat P2X2 receptor. Therefore, it is likely that the P2X2 subtype is the dominant component in the P2X receptors in mouse coeliac and pelvic ganglion neurons, just as it is in the rat. Another example of interspecies variation is demonstrated by the effect of Zn2+ on sensory neurons. On the P2X receptors expressed in rat nodose ganglion neurons, Zn2+ enhances the potency of ATP (Li et al., 1993), while on bullfrog DRG neurons, Zn2+ decreases the affinity of the P2X receptor for ATP (Li et al., 1997). The mechanism for the differences seen between these two species is not clear. To conclude, we have demonstrated that on rat coeliac, mouse coeliac and mouse pelvic ganglion neurons, the pharmacological characteristics of P2X receptors are
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indistinguishable, and are consistent with them being of the P2X2 subtype, or hetero-multimers with P2X2 being the dominant component. Thus, while the distribution of P2X receptors in ganglion neurons may be similar in mouse and rat, it differs from that observed in guineapig. Although there may be little variation in the expression of P2X receptors between neurons from different autonomic ganglia, there are differences between species. This must be considered when extrapolating from experimental animals to man.
Acknowledgements The authors are grateful to E.W. Moules for excellent technical support, to R. Jordan for help in the preparation of the manuscript. YZ and PMD were supported by Roche Bioscience.
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Pharmacological comparison of P2X receptors on rat coeliac, mouse coeliac and mouse pelvic ganglion neurons Yu Zhong *, Philip M. Dunn, Geoffrey Burnstock Autonomic Neuroscience Institute, Department of Anatomy and Developmental Biology, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK Accepted 29 June 1999
Abstract Characteristics of P2X receptors on neurons of the rat coeliac, mouse coeliac and mouse pelvic ganglia have been studied using the whole cell voltage-clamp technique. Fast application of ATP (100 µM) on to isolated neurons voltage clamped at ⫺70 mV induced a slowly desensitising inward current in 96% of the cells tested. Concentration–response curves for ATP yielded EC50 values of 86 µM, 64 µM and 123 µM, for rat coeliac, mouse coeliac and mouse pelvic ganglion neurons, respectively, while α,βmethylene ATP was inactive. The response to ATP was antagonised by suramin, Cibacron blue and pyridoxalphosphate-6-azophenyl2⬘,4⬘-disulphonic acid (PPADS). The potency of ATP was increased by extracellular acidification and by co-application of micromolar concentrations of Zn2+, while raising pH decreased it. On rat coeliac ganglion neurons, the EC50 values for ATP were 35 µM and 253 µM at pH 6.8 and 8.0, respectively. On mouse coeliac and pelvic ganglion neurons, altering the pH produced comparable changes. In conclusion, our results indicate that, in contrast to the guinea-pig coeliac ganglion, the characteristics of the P2X receptors present on rat coeliac, mouse coeliac and mouse pelvic ganglia are all identical to those present on rat pelvic ganglion, i.e. they are homomeric P2X2 receptors, or heteromultimers with P2X2 being the dominant subunit. 2000 Elsevier Science Ltd. All rights reserved. Keywords: P2X receptor; Coeliac ganglion; Pelvic ganglion; ATP; pH; Zn2+
1. Introduction ATP acts as a fast excitatory neurotransmitter (for review, see Surprenant et al., 1995; Burnstock, 1997), where it activates a class of ligand-gated ion channels, the P2X receptors. To date, seven P2X receptor subunits have been cloned (P2X1–7), all of which have been reported to form functional homo-oligomeric receptors with different though overlapping biophysical and pharmacological properties, including agonist profiles, desensitisation and sensitivities to antagonists (Brake et al., 1994; Valera et al., 1994; Bo et al., 1995; Chen et al., 1995; Collo et al., 1996; Surprenant et al., 1996). In addition, these P2X receptor subtypes are differentially modulated by pH (King et al., 1996; Stoop et al., 1997) and Zn2+ (Wildman et al. 1998, 1999). The pharmaco-
* Corresponding author. Tel.: +44-171-794-0500, ext. 6754; fax: +44-171 830 2949. E-mail address: [email protected] (Y. Zhong)
logical profiles of the recombinant P2X receptors do not always match those of the endogenous P2X receptors; thus, it is plausible that some native P2X receptors are hetero-multimeric channels composed of different P2X subunits (Lewis et al., 1995; Leˆ et al., 1998). In autonomic and sensory neurons, two broad groups of native P2X receptors can be distinguished by their sensitivity to α,β-methylene ATP (α,β-meATP) (Evans and Surprenant, 1996). Thus, neurons from rat dorsal root (DRG) (Robertson et al., 1996; Rae et al., 1998), nodose (Lewis et al., 1995) and trigeminal ganglion neurons (Cook et al., 1997) are activated by α,β-meATP. The involvement of P2X3 and P2X2/3 receptors has been suggested. In another group of neurons, such as those of the rat superior cervical ganglion (SCG) (Nakazawa, 1994) and rat pelvic ganglion (Zhong et al., 1998), α,βmeATP is inactive or very weak as an agonist. The molecular and pharmacological properties of P2X receptors on those neurons suggest them to be of the P2X2 subtype. This seems to suggest that in the rat, sensory neurons demonstrate α,β-meATP sensitivity, while the auto-
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nomic neurons demonstrate α,β-meATP insensitivity. However, neurons from guinea-pig SCG (Reekie and Burnstock, 1994) and guinea-pig coeliac ganglion (Khakh et al., 1995) respond to α,β-meATP and the receptors present on guinea-pig coeliac ganglion neurons resemble those of rat nodose ganglion (Khakh et al., 1995). Therefore, in this study, we sought to determine the pharmacological properties of the P2X receptors on rat coeliac ganglion neurons. We were interested in finding whether P2X receptors on rat coeliac (sympathetic) ganglion neurons are similar to those on rat SCG (also sympathetic) and pelvic neurons, or whether they exhibit similar properties to those on guinea-pig coeliac ganglion neurons, i.e. are similar to those on rat nodose (sensory) ganglion neurons. We have also characterised the P2X receptors on mouse coeliac and pelvic ganglia, to explore further the existence of inter-species and interganglion variation, and to provide background information for studies of “knock-out” mice lacking specific P2X subunits. A preliminary report on part of the work has appeared in the form of an abstract (Zhong et al., 1999).
2. Methods 2.1. Isolation of neurons Single neurons of the coeliac ganglion from 17-dayold male Sprague-Dawley rats and coeliac and pelvic ganglia from adult male mice were enzymatically isolated as described previously (Zhong et al., 1998). The animals were killed by a rising concentration of CO2 and death was confirmed by cervical dislocation. The coeliac and pelvic ganglia were rapidly dissected out and placed in Leibovitz’s L-15 medium (Life Technologies, Paisley, UK). The ganglia were desheathed and three to four deep cuts were made in each before incubation in 4 ml Ca2+/Mg2+-free Hank’s Balanced Salt Solution with 10 mM HEPES pH 7.4 buffer (HBSS) (Life Technologies) containing 1.5 mg/ml collagenase (Class-II; Worthington Biochemical Corporation, Reading, UK) and 6 mg/ml bovine serum albumin (Sigma Chemical Co., Poole, UK) at 37°C for 40 min. The ganglia were then incubated with 4 ml HBSS containing 1 mg/ml trypsin (Sigma) at 37°C for 15 min. The solution was replaced with 3 ml growth medium comprising L-15 medium supplemented with 50 ng/ml nerve growth factor, 10% bovine serum, 2 mg/ml NaHCO3, 5.5 mg/ml glucose, 200 IU/ml penicillin and 200 µg/ml streptomycin. The ganglia were dissociated into single neurons by gentle trituration with two fire-polished glass pipettes of decreasing diameter. The cells were then centrifuged at 900 rev/min for 5 min, resuspended in 1 ml growth medium and plated onto 35 mm Petri dishes coated with 10 µg/ml laminin (Sigma).
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Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2, and used between 3 and 48 h after plating.
2.2. Whole cell voltage-clamp recording
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 voltage clamped at ⫺70 mV. External solution contained (mM): NaCl 154, KCl 4.7, MgCl2 1.2, CaCl2 2.5, HEPES 10, glucose 5.6, the pH being adjusted to 7.4 using NaOH. Recording electrodes were made from thin-walled glass capillary tubes with fine filament (GC 150TF; Clark Electromedical Instruments, Reading, UK) using a two-stage puller (PP-830, Narishige, Tokyo, Japan), fire-polished, and had a resistance of 2–4 M⍀ when filled with an internal solution containing (mM): citric acid 56, MgCl2 3, CsCl 10, NaCl 10, HEPES 40, EGTA 0.1, tetraethylammonium chloride 10, and the pH adjusted to 7.2 using CsOH (total Cs+ concentration 170 mM). Series resistance compensation was not used. However, only those cells with a maximum voltage clamp error of ⱕ8 mV, due to the series resistance, were used for pharmacological studies. The liquid– liquid junction potential, measured at 6 mV, was not corrected for. Data were acquired using pCLAMP software (Axon Instruments). Signals were filtered at 2 kHz (⫺3 dB frequency, Bessel filter, 80 dB/decade). Drugs were applied rapidly through a seven-barrel manifold composed of fused glass capillaries inserted into a common outlet tube with a tip diameter of 苲200 µm, which was placed very close to the cell (Dunn et al., 1996). Solutions were delivered by gravity flow from independent reservoirs placed above the preparation. One barrel was used to apply drug-free solution to enable rapid termination of drug application. Applications of agonists (5 s) were separated by intervals of 2 min, which were sufficient for responses to be reproducible. Antagonists were present for 2 min before and during the reapplication of agonists. All responses were normalised to that evoked by ATP (1 mM) under control conditions in the same cell, and expressed as the mean±SEM, unless otherwise indicated. The concentration–response data were fitted with the Hill equation: Y=A/[1+(K/X)n], where A is the maximum effect, K is the EC50, and n is the Hill coefficient, using Prism v2 (GraphPad, San Diego, CA, USA). The combined data from the number of treated cells were fitted, and the parameters are presented as fitted values ±SEM, determined by the fitting routine. Traces were acquired using pCLAMP and plotted using Origin (Microcal, Northampton, MA, USA). Statistical analysis (Student’s ttest) was performed using Prism v2.
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2.3. Drugs and materials ATP, α,β-meATP and other chemicals were obtained from Sigma Chemical Co. Suramin was a gift from Bayer plc (Newbury, UK), and pyridoxalphosphate-6azophenyl-2⬘,4⬘-disulphonic acid (PPADS) was from Tocris Cookson (Bristol, UK). Stock solutions (10–100 mM) of ATP and other drugs were prepared using deionised water and stored frozen. For application all drugs were diluted in extracellular bathing solution to the required concentration.
3. Results 3.1. Response to agonists Fast application of ATP (100 µM) to isolated rat coeliac, mouse coeliac and mouse pelvic ganglion neurons voltage-clamped at ⫺70 mV induced a rapidly activating inward current in 96% of cells tested (Fig. 1). The peak amplitudes (means±SD) of response to ATP (100 µM) were 0.73±0.89 nA (n=94; range 0.025–5.6 nA) for rat coeliac ganglion neurons, 0.66±0.63 nA (n=70; range 0.02–3.2 nA) for mouse coeliac ganglion neurons, and 0.71±0.60 nA (n=61; range 0.025–3.2 nA) for mouse pelvic ganglion neurons. Despite the large variability in response, for neurons from each ganglia, the amplitude distribution revealed a single skewed population, with 80% of cells giving small to moderate responses (⬍1 nA) (data not shown). We have previously observed a similar distribution of the amplitude of inward currents evoked by ATP in rat major pelvic ganglion neurons (Zhong et al., 1998). The response to ATP (100 µM) desensitised slowly, with the evoked current declining to 61±3% (n=8), 66±5% (n=5) and 70±2% (n=4) of control at the end of 30 s application for rat coeliac, mouse coeliac and mouse pelvic ganglion neurons, respectively. The decay of the current was fitted well by a single exponential decay yielding time constants of 19.2±1.5 s (n=8), 21.4±1.3 s (n=5) and 21.0±3.1 s (n=4), for rat coeliac, mouse coeliac and mouse pelvic ganglion neurons, respectively. The concentration–response curves for ATP on these neurons are shown in Fig. 2. Fitting the Hill equation to the data gave EC50 values of 86 µM (log EC50=⫺4.067±0.043, data from seven cells), 64 µM (log EC50=⫺4.191±0.101, data from nine cells) and 123 µM (log EC50=⫺3.910±0.087, data from ten cells), for rat coeliac, mouse coeliac and mouse pelvic ganglion neurons, respectively, with Hill coefficients of 1.6, 1.5 and 1.4. When α,β-meATP was tested, it failed to evoke any response at concentrations up to 100 µM in any cell tested (at least six neurons for each of these three ganglia).
Fig. 1. Representative traces of the inward currents activated by prolonged (30 s) application of ATP (100 µM) to isolated rat coeliac (a), mouse coeliac (b) and mouse pelvic (c) ganglion neurons, voltage clamped at ⫺70 mV.
3.2. Effects of antagonists The P2 receptor antagonists Cibacron blue (Cibacron Blue 3GA, 65% pure) (Manzini et al., 1986), suramin (Dunn and Blakeley, 1988) and PPADS (Lambrecht et
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Fig. 2. Concentration–response curves for ATP on rat coeliac, mouse coeliac and mouse pelvic ganglion neurons. Cells were voltage clamped at ⫺70 mV and various concentrations of ATP were applied for 5 s every 2 min. Responses were normalised with respect to that obtained with ATP (1 mM) on the same cell. Each point represents the mean±SEM from seven to ten cells. The Hill equation was fitted to the data, yielding EC50 values of 87 µM, 64 µM and 123 µM, for rat coeliac, mouse coeliac and mouse pelvic ganglion neurons, respectively.
al., 1992) were tested on neurons from these three ganglia (Fig. 3). On rat coeliac ganglion neurons (Fig. 3a), 2-min incubation with Cibacron blue (10 µM) abolished the response to ATP (100 µM; n=7). The inhibition was readily reversible, with the response being 71±4% of control (n=7) after a 2-min wash-out. Suramin was less effective than Cibacron blue in inhibiting the response to ATP. With a 2-min preincubation, 100 µM suramin reduced the response to ATP (100 µM) to 16±3% of control (n=5). The response recovered rapidly and was 86±4% of control (n=5) 2 min after washing out suramin. PPADS was also able to inhibit the response to ATP on rat coeliac ganglion neurons. After a 4-min preincubation, PPADS (10 µM) reduced the response to ATP (100 µM) to 9±1% of control (n=5). Recovery from the inhibition of PPADS was slow, with the response to ATP (100 µM) being only 19±3% of control (n=5) 4 min after washing out the antagonist. The kinetics of the inhibition by PPADS was concentration-dependent, and considerably slower than those of Cibacron blue and suramin. When fitting the time profile of the inhibition by PPADS of the response to ATP (100 µM) by the single exponential decay, the time constants yielded were 7.5 min and 1.3 min, for 1 and 10 µM PPADS, respectively (n=6, data not shown). This is similar to that seen on rat major pelvic ganglion neurons (Zhong et al., 1998). The effect of antagonists on mouse coeliac and pelvic ganglion neurons were similar to those on rat coeliac ganglion neurons (Fig. 3a and c). Thus, after 2-min preincubation with Cibacron blue (10 µM), the response to
Fig. 3. Effect of P2 receptor antagonists, Cibacron blue (CB, 10 µM), suramin (SUR, 100 µM) and PPADS (10 µM) on responses to ATP on neurons of rat coeliac (a), mouse coeliac (b) and mouse pelvic (c) ganglion neurons. The histogram shows the peak amplitudes of the inward currents induced by ATP (100 µM) alone (control, open bars), in the presence of the antagonists (solid bars) and after washing out the antagonists (recovery, hatched bars). Responses were normalised with respect to that obtained with ATP (100 µM) in the absence of antagonists on the same cell. Each bar represents the mean±SEM from five to seven cells. In the cases of Cibacron blue and suramin, the antagonists were present 2 min before and during the re-application of ATP, with the recovery responses measured 2 min after washing out the antagonists. PPADS was present 4 min before and during the re-application of ATP, and the recovery responses were measured 4 min after washing out.
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ATP (100 µM) was reduced to 18±2% (n=6) and 27±2% of control (n=6) on mouse coeliac and pelvic ganglion neurons, respectively, with rapid and full recovery. A 2 min preincubation with 100 µM suramin reversibly reduced the response to ATP (100 µM) to 41±3% of control (n=6) on mouse coeliac ganglion neurons and 28±1% of control (n=6) on mouse pelvic ganglion neurons, respectively. Again, the recovery was also rapid and full. PPADS, similar to its action on rat coeliac ganglion neurons, produced inhibition of the ATP response with much slower kinetics. After a 4-min preincubation, PPADS (10 µM) reduced the response to ATP (100 µM) to 3±1% of control (n=6) on mouse coeliac ganglion neurons and 2±1% of control (n=6) on mouse pelvic ganglion neurons. The responses recovered to 15±2% and 10±1% of control (n=6), respectively, 4 min after washing out PPADS. 3.3. Modulation by pH The response to ATP on neurons from all three ganglia was very sensitive to changes in extracellular pH. On rat coeliac ganglion neurons, the amplitude of the inward current induced by ATP (100 µM) was increased greatly when pH was reduced from 7.4 to 6.8, and was markedly attenuated when pH was elevated from 7.4 to 8.0 (Fig. 4a). The effect of protons on currents activated by ATP was rapid and reversible. On average, the current activated by ATP (100 µM) was enhanced at pH 6.8 to 156±6% (n=9) of that at pH 7.4 (control), while raising pH to 8.0 suppressed the response to 36±3% of control (n=10). On rat coeliac ganglion neurons, changing the pH did not alter the slope of the concentration–response curve for ATP, but rather changed the affinity of ATP (Fig. 4a). The EC50 of ATP was changed from 86 µM at pH 7.4 to 35 µM (log EC50=⫺4.451±0.057, n=10) at pH 6.8, and 253 µM (log EC50=⫺3.597±0.045, n=10) at pH 8.0. The EC50 values of ATP at pH 6.8 and 8.0 were significantly different from that at pH 7.4 (P⬍0.001 and P⬍0.05, respectively). The Hill coefficients (1.7 and 1.3 at pH 6.8 and 8.0, respectively) were not significantly different from that at pH 7.4. On mouse coeliac and pelvic ganglion neurons, altering extracellular pH produced comparable changes in the ATP response (trace not shown). For these neurons, the effects of pH 6.8 and pH 8.0 were studied on the concentration–response relationship of ATP at 30–100 µM and 100–300 µM, respectively. These concentrations were chosen because they fell on the most linear part of the ATP concentration–response curve. On mouse coeliac ganglion neurons, lowering the pH from 7.4 to 6.8 potentiated the response to 100 µM ATP (to 154±7% of control, n=7), and shifted the ATP concentration– response curve significantly to the left by 0.272±0.025 log units (P⬍0.001, n=6). Raising the pH to 8.0 attenu-
ated the response to ATP (100 µM) (to 36±5% of control, n=9), and shifted the ATP concentration–response curve significantly to the right by 0.547±0.048 log units (P⬍0.001, n=6) (Fig. 4b). On mouse pelvic ganglion neurons, lowering the pH from 7.4 to 6.8 potentiated the response to 100 µM ATP (to 201±9% of control, n=7), and shifted the ATP concentration–response curve significantly to the left by 0.407±0.029 log units (P⬍0.001, n=6). Raising the pH to 8.0 attenuated the responses to 100 µM ATP (to 25±2% of control, n=7), and shifted the ATP concentration–response curve significantly to the right by 0.537±0.063 log units (P⬍0.001, n=6) (Fig. 4c). 3.4. Modulation by Zn2+ In a previous study, we have shown that Zn2+ could enhance the response to ATP (30 µM) on rat major pelvic ganglion neurons with an EC50 of 9.6 µM, and increase the affinity of ATP for the receptor (Zhong et al., 1998). Therefore, we investigated the effect of Zn2+ (10 µM) on neurons from rat coeliac, mouse coeliac and mouse pelvic ganglia. Similar to its effect on rat major pelvic ganglion, coapplication of Zn2+ (10 µM) with ATP on rat coeliac ganglion neurons shifted the concentration–response curve of ATP to the left in a parallel fashion (Fig. 5a). The EC50 of ATP was decreased significantly from the control value of 86 µM to 23 µM (log EC50=⫺4.632±0.099, P⬍0.0001, n=7), while Emax and the Hill coefficient (1.3) were not significantly altered. On mouse coeliac and pelvic ganglion neurons, we studied the effect of Zn2+ on the concentration–response relationship between ATP (30 and 100 µM; Figs. 5b and c). Co-application of Zn2+ (10 µM) with ATP shifted the concentration–response curve to ATP to the left in a parallel fashion by 0.368±0.066 log units (n=7), and 0.529±0.048 log units (n=5), respectively. Both shifts were significantly greater than zero (P⬍0.001).
4. Discussion In the present study, the pharmacological properties of the P2X receptors on neurons from rat coeliac, mouse coeliac and mouse pelvic ganglia were compared. 4.1. Involvement of P2X2 subunits Almost all neurons from the three ganglia studied responded to ATP (100 µM), with a slowly desensitising inward current. The potency of ATP was similar on three ganglia. In contrast, α,β-meATP was inactive at concentrations up to 100 µM in all three ganglia. The three purine receptor antagonists tested (suramin, Cibacron blue and PPADS) all inhibited the response to ATP with
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Fig. 4. Effect of pH on ATP induced currents. (a) Rat coeliac ganglion neurons concentration–response curves for ATP at pH 6.8 and 8.0. Each point represents the mean±SEM from ten cells. The Hill equation was fitted to the data assuming the same maximum, yielding EC50 values of 35 µM and 253 µM at pH 6.8 and 8.0, respectively. The traces (top panel) show representative responses to ATP (100 µM) recorded from one rat coeliac ganglion neuron at different pH. The horizontal bars represent the application period of ATP. (b) Mouse coeliac and (c) mouse pelvic ganglion neurons concentration–response relationship for ATP at pH 6.8, 7.4 and 8.0. Each point represents the mean±SEM from six cells. All responses were normalised with respect to that evoked by ATP (1 mM) at pH 7.4 on the same cell. For each cell type, the appropriate control concentration–response curve (pH 7.4) taken from Fig. 2 has been superimposed (dotted curve) for comparison.
comparable potencies to those observed previously on rat pelvic ganglion neurons (Zhong et al., 1998). The effect of ATP was potentiated by acidification and by co-application of micromolar concentrations of Zn2+. Thus the receptors present on neurons of the rat coeliac, mouse coeliac and mouse pelvic ganglia would appear to be identical with those found on rat pelvic ganglion neurons, which are thought to be of the P2X2 subtype, or hetero-multimeric receptors in which P2X2 is the dominant subunit (for further discussion see Zhong et al., 1998).
4.2. Expression of hetero-multimeric receptors? Recently, biochemical evidence has suggested that among the seven P2X receptor subtypes, six can form homo-oligomeric complexes, the exception being P2X6 (Torres et al., 1999). In addition, all, apart from P2X7, are able to form hetero-oligomeric assemblies with P2X2 combining with P2X1, P2X3, P2X5 and P2X6 subunits. Although the functional properties of most of these combinations have yet to be determined, the coexpression of P2X2 and P2X3 receptor subunits are required to produce
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ATP-gated currents with the properties seen in rat nodose neurons (Lewis et al., 1995), while it has also been suggested that P2X4 and P2X6 subunits coassemble to form multimeric channels with unique functional properties that resemble those found on native neurons in the central nervous system (Leˆ et al., 1998). The unusual pharmacological properties of the P2X receptors present on guinea-pig myenteric neurons might also result from the formation of heteromeric receptors (Barajas-Lo´pez et al., 1996). Therefore, although the neurons investigated in this study demonstrate a pharmacological profile of the P2X2 phenotype, it is still possible that these P2X receptors are hetero-multimers, with P2X2 being the dominant component. The presence of alternatively spliced variants of P2X receptor subunits may impose additional complexity to the characterisation of endogenous P2X receptors. In the rat, four splice variants of the P2X2 receptor have been isolated: P2X2(a), P2X2(b), P2X2(c) and P2X2(d) (Bra¨ndle et al., 1997; Simon et al., 1997). In guinea-pig, three splice variants of the P2X2 receptor have been isolated, with P2X2-1 and P2X2-2 being the homologues of rat P2X2(a) and P2X2(b), while P2X2-3 is novel (Parker et al., 1998). In mouse, four splice variants of the mouse P2X4 receptor, P2X4(a), P2X4(b), P2X4(c) and P2X4(d), have been reported (Simon et al., 1999; Townsend-Nicholson et al., 1999). It is possible that splice variants of the P2X2 receptor also exist in mouse. This may increase the number of subunits available for hetero-multimeric assembly. 4.3. Multiple P2X receptors in the same ganglion, and the co-existence of multiple receptors on the same neuron?
Fig. 5. Enhancement of the potency of ATP by Zn2+ on rat coeliac, mouse coeliac and mouse pelvic ganglion neurons. (a) Concentration– response curves for ATP in the presence of Zn2+ (10 µM) on neurons from rat coeliac ganglia. The Hill equation was fitted to the data, yielding an EC50 of 23 µM. Each point represents the mean±SEM from seven cells. (b) and (c) Concentration–response relationship for ATP in the presence of Zn2+ (10 µM) on neurons from mouse coeliac (b) and mouse pelvic (c) ganglia. Each point represents the mean±SEM from five to seven cells. Responses were normalised with respect to that evoked by ATP (1 mM) in the absence of Zn2+ on the same cell. For each cell type, the appropriate control concentration–response curve taken from Fig. 2 has been superimposed (dashed curve) for comparison.
Evidence from recent studies has suggested that the expression of P2X receptors in native tissue is more complicated than previously thought. Both fast-desensitising and slow-desensitising ATP responses have been identified on rat trigeminal nociceptive neurons (Cook et al., 1997), adult rat DRG neurons (Ueno et al., 1999) and guinea-pig myenteric neurons (Zhou and Galligan, 1996). Moreover, two P2X receptors have been shown to co-exist on the same cell from rat nodose ganglion (Thomas et al., 1998), rat DRG (Grubb and Evans, 1999) and guinea-pig SCG (Y. Zhong and P. Dunn, unpublished observations). The proportion of each receptor type varies from cell to cell. On neurons examined in this study, there was no evidence to suggest that more than one form of P2X receptors exist in the same cell. However, better pharmacological tools or novel techniques may in the future clarify the situation. 4.4. Inter-species and inter-ganglion variation Nakazawa (1994) has suggested that the properties of P2X receptors may depend on both species and types of
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tissues, since there is difference in the sensitivity to α,βmeATP between rat SCG neurons and guinea-pig coeliac ganglion neurons (Evans et al., 1992) or rat DRG neurons (Bean, 1990). However, Khakh et al. (1995) suggested that P2X receptors in guinea-pig coeliac ganglion neurons were similar to those in rat nodose ganglion neurons, while those expressed in rat SCG neurons were anomalous. In this study, we have characterised the pharmacological properties of P2X receptors on rat coeliac ganglion neurons, and found them to be P2X2-like, and similar to those present on rat SCG and pelvic ganglion neurons. Therefore, rat SCG neurons are not unique in the type of P2X receptors they express. Thus, available evidence indicates that rat autonomic neurons (from SCG, pelvic, cardiac and coeliac ganglia) all possess the same P2X2-like receptor (Fieber and Adams, 1991; Nakazawa, 1994; Zhong et al., 1998; this study). In contrast, rat sensory neurons (from DRG, nodose and trigeminal ganglia) express P2X2 and P2X3 subunits, leading to the formation of varying proportions of homomeric and heteromeric receptors (Lewis et al., 1995; Cook et al., 1997; Thomas et al., 1998; Grubb and Evans, 1999; Ueno et al., 1999). Therefore, in the rat, there is inter-ganglion difference for the expression of P2X receptors, e.g. between autonomic and sensory ganglia, but not between neurons in different autonomic ganglia. Interestingly, all guinea-pig autonomic neurons examined (SCG, coeliac and pelvic ganglia) possess slowly desensitising α,βmeATP sensitive receptors which resemble the P2X2/3 heteromeric receptor (Reekie and Burnstock, 1994; Khakh et al., 1995; Y. Zhong and P. Dunn, unpublished observations). Hence, inter-species variation does exist. The mouse P2X3 protein shows 99% identity with rat P2X3 (Souslova et al., 1997), and the mouse P2X4 protein shares 94% identity with rat P2X4 receptor (Townsend-Nicholson et al., 1999). Hence it is possible that the mouse P2X2 receptor will show a similar degree of homology to the rat P2X2 receptor. In this study, we investigated the pharmacological properties of the P2X receptors in mouse coeliac and pelvic ganglia, and found their pharmacology to be similar to that of the heterologously expressed rat P2X2 receptor. Therefore, it is likely that the P2X2 subtype is the dominant component in the P2X receptors in mouse coeliac and pelvic ganglion neurons, just as it is in the rat. Another example of interspecies variation is demonstrated by the effect of Zn2+ on sensory neurons. On the P2X receptors expressed in rat nodose ganglion neurons, Zn2+ enhances the potency of ATP (Li et al., 1993), while on bullfrog DRG neurons, Zn2+ decreases the affinity of the P2X receptor for ATP (Li et al., 1997). The mechanism for the differences seen between these two species is not clear. To conclude, we have demonstrated that on rat coeliac, mouse coeliac and mouse pelvic ganglion neurons, the pharmacological characteristics of P2X receptors are
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indistinguishable, and are consistent with them being of the P2X2 subtype, or hetero-multimers with P2X2 being the dominant component. Thus, while the distribution of P2X receptors in ganglion neurons may be similar in mouse and rat, it differs from that observed in guineapig. Although there may be little variation in the expression of P2X receptors between neurons from different autonomic ganglia, there are differences between species. This must be considered when extrapolating from experimental animals to man.
Acknowledgements The authors are grateful to E.W. Moules for excellent technical support, to R. Jordan for help in the preparation of the manuscript. YZ and PMD were supported by Roche Bioscience.
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