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. 2003 Aug 22;552(Pt 3):809–821. doi: 10.1113/jphysiol.2003.047944

P2X2 subunits contribute to fast synaptic excitation in myenteric neurons of the mouse small intestine

Jianhua Ren †, Xiaochun Bian *,†, Matthew DeVries *, Birthe Schnegelsberg ‡, Debra A Cockayne ‡, Anthony P D W Ford ‡, James J Galligan *,†
PMCID: PMC2343442  PMID: 12937291

Abstract

P2X receptors are ATP-gated cation channels composed of one or more of seven different subunits. ATP acts at P2X receptors to contribute to fast excitatory postsynaptic potentials (fEPSPs) in myenteric neurons but the subunit composition of enteric P2X receptors is unknown. These studies used tissues from P2X2 wild-type (P2X2+/+) and P2X2 gene knockout (P2X2-/-) mice to investigate the role of this subunit in enteric neurotransmission. Intracellular electrophysiological methods were used to record synaptic and drug-induced responses from ileal myenteric neurons in vitro. Drug-induced longitudinal muscle contractions and peristaltic contractions of ileal segments were also studied in vitro. Gastrointestinal transit was measured as the progression in 30 min of a liquid radioactive marker administered by gavage to fasted mice. RT-PCR analysis of mRNA from intestinal tissues and data from immunohistochemical studies verified P2X2 gene deletion. The fEPSPs recorded from S neurons in tissues from P2X2+/+ mice were reduced by mecamylamine (nicotinic cholinergic receptor antagonist) and PPADS (P2X receptor antagonist). The fEPSPs recorded from S neurons from P2X2−/− mice were unaffected by PPADS but were blocked by mecamylamine. ATP depolarized S and AH neurons from P2X2+/+ mice. ATP depolarized AH but not S neurons from P2X2-/- mice. α,β-Methylene ATP (α,β-mATP)(an agonist at P2X3 subunit-containing receptors) did not depolarize S neurons but it did depolarize AH neurons in P2X2+/+ and P2X2-/- mice. Peristalsis was inhibited in ileal segments from P2X2-/- mice but longitudinal muscle contractions caused by nicotine and bethanechol were similar in segments from P2X2+/+ and P2X2-/- mice. Gastrointestinal transit was similar in P2X2+/+ and P2X2-/- mice. It is concluded that P2X2 homomeric receptors contribute to fEPSPs in neural pathways underlying peristalsis studied in vitro.

Many peripheral neurons express P2X receptors which are ligand-gated cation channels activated by extracellular ATP (Dunn et al. 2001). Seven genes encoding P2X receptor subunits (P2X1-P2X7) have been cloned from mammalian tissues (Buell et al. 1996). P2X subunits have two membrane-spanning domains, intracellular N-and C-terminal regions and an extracellular loop (North, 2002). The stoichiometry of functional P2X receptors is not certain but the subunits may form trimers, tetramers (Kim et al. 1997; Stoop et al. 1999) or di-trimers (Khakh et al. 2001). Functional P2X receptors can be homomers or heteromers (Khakh et al. 2001; North, 2002).

Studies of P2X subtypes expressed in heterologous systems have demonstrated that the subunit composition of P2X receptors determines their pharmacological and physiological properties (North & Surprenant, 2000). For example, the stable ATP analogue α,β-methylene ATP (α,β-mATP) activates homomeric P2X receptors composed of P2X1 or P2X3 subunits, but does not activate P2X2 homomeric receptors (North & Surprenant, 2000). α,β-mATP does activate P2X2/3 heteromeric receptors (Lewis et al. 1995). In addition, P2X1 and P2X3 receptors desensitize rapidly (< 1 s) during ATP or α,β-mATP application, while P2X2 receptors desensitize slowly (> 10 s) when activated by ATP (Dunn et al. 2001). It has been shown that P2X3 homomeric receptors mediate an α,β-mATP-sensitive inward current in neonatal rat DRG neurons while P2X2/3 heteromeric receptors mediate a biphasic current response to α,β-mATP in these neurons (Lewis et al. 1995).

Previous studies have shown that P2X receptors are expressed in myenteric neurons of the guinea pig gastrointestinal tract where they contribute to fast excitatory synaptic transmission (Galligan & Bertrand, 1994; LePard et al. 1997; Johnson et al. 1999; Galligan et al. 2000; Nurgali et al. 2003). Myenteric neurons are classified electrophysiologically into two types: S and AH neurons (Hirst et al. 1974; Bornstein et al. 1994). S-type neurons are interneurons and motorneurons in the myenteric plexus (Costa et al. 1996; Brookes, 2001). In isolated longitudinal muscle myenteric plexus preparations, single stimuli applied to interganglionic connectives elicit fast excitatory postsynaptic potentials (fEPSPs) in S neurons (Hirst et al. 1974; Bornstein et al. 1994). Acetylcholine, acting at nicotinic acetylcholine receptors, and ATP, acting at P2X receptors, mediate fEPSPs in 67 % of myenteric S neurons in the guinea pig ileum (Galligan et al. 2000). P2X-mediated, synaptic transmission participates in descending motor pathways in the guinea pig intestine (Smith et al. 1990; LePard & Galligan, 1999; Johnson et al. 1999; Bian et al. 2000; Spencer et al. 2000; Monro et al. 2002).

AH neurons have an action potential that is followed by a long-lasting (1–20 s) afterhyperpolarization (Hirst et al. 1974; Furness et al. 1998). In guinea pig intestine, AH neurons receive slow synaptic input and they are likely to be intrinsic sensory neurons (Kunze et al. 1998; Furness et al. 1998). P2X receptors are expressed on the nerve terminals and cell bodies of AH neurons in guinea pig ileum (Bertrand & Bornstein, 2002). AH neurons have processes in the mucosal villi where they are in a position to respond to lumenal stimuli. In vitro studies have shown that local appliction of ATP to mucosal villi can elicit antidromic action potentials in AH neurons in nearby myenteric ganglia (Bertrand & Bornstein, 2002). These investigators suggested that ATP released in the mucosa could initiate or modify intestinal motor reflexes by activating P2X receptors on the nerve terminals of myenteric AH neurons.

The subunit composition of P2X receptors expressed by myenteric neurons is not known. Previous studies have suggested that most myenteric neurons in primary culture express predominatly homomeric P2X2 receptors (Zhou & Galligan, 1996). However, in a small subset (10 %) of myenteric neurons, α,β-mATP caused a rapidly developing and desensitizing inward current, suggesting that P2X1 or P2X3 subunits may contribute to P2X receptors in some myenteric neurons (Zhou & Galligan, 1996). Phamacological studies have indicated there are α,β-mATP-sensitive P2X receptor subtypes (P2X1 or P2X3) and P2X2 subunit containing receptors in S neurons of guinea pig intestine (LePard et al. 1997). In addition, immunohistochemical data have shown the P2X2 receptors are expressed by some motorneurons, non-cholinergic secretomotor neurons and intrinsic sensory neurons (Castelucci et al. 2002) in the guinea pig gut. Immunohistochemical studies have also shown that P2X3 receptor subunits are expressed by excitatory and inhibitory motorneurons, ascending interneurons and cholinergic secretomotor neurons (Poole et al. 2002; VanNassauw et al. 2002). However, the current lack of subunit selective agonists or antagonists has made characterization of the subunit composition of functional P2X receptors difficult.

An alternative to the pharmacological approach for characterizing P2X receptor subtypes is to use a gene-deletion strategy in mice. We used this strategy in the present study to evaluate the contribution of P2X2 subunits to P2X receptors mediating fEPSPs in the mouse small intestine. In addition, we evaluated the effects of P2X2 subunit gene deletion on peristalsis studied in vitro in segments of small intestine, and on gastrointestinal transit in conscious, fasted mice.

METHODS

All animal use protocols were approved by the All University Committee on Animal Use and Care at Michigan State University. P2X2−/− mice did not exhibit any observable behavioural abnormalities and had an overall healthy appearance. There was no difference in body weights between P2X2+/+ (n = 44; 30.1 ± 0.1 g) and P2X2−/− mice (n = 48; 30.2 ± 0.8 g)(P > 0.05) at the time of these studies (8–16 weeks).

P2X2 gene deletion

P2X2−/− mice were generated as described previously (Cockayne et al. 2002). All mice analysed in this study have the genetic background 129Ola × C57BL/6J, and were derived from homozygous F2 crosses maintained at Roche Palo Alto. Genotype confirmation of all animals was carried out by Southern blot analysis.

Reverse transcriptase PCR analysis of RNA from mouse intestinal tissues

Total RNA was isolated from whole ileum, ileal longitudinal muscle-myenteric plexus preparations, whole colon and colonic circular muscle-myenteric plexus using a Trizol (Invitrogen, Carlsbad, CA, USA) isolation method, and RQ1 RNAse-free DNAse I (Promega, Keene, USA) digestion. To perform semi-quantitative RT-PCR analysis, cDNA was reverse transcribed from 1 μg of total RNA, in a reaction volume of 22 μl, using 1 μl (2.5 U) Superscript RNAseH reverse transcriptase (Invitrogen), 2 μl 10 × PCR buffer II (Perkin Elmer, Fremont, CA, USA), 2 μl (25 mm) MgCl2 (Perkin Elmer), 1 μl (1.1 mm) dNTP mix (Invitrogen), 2 μl (9.1 mm) DTT (Invitrogen), 1 μl RNAsin (Invitrogen) and 1 μl (0.7 μm) random hexamers (Amersham Biosciences, NJ, USA). RT reactions were carried out at 42°C for 50 min, followed by a 15 min elongation at 70°C, and a 20 min RNAse H digestion (2 U, Invitrogen) at 37°C. PCR analysis was done in a 50 μl reaction volume using 230 ng of reverse transcribed cDNA, 1 μl each of 10 μm 5′ or 3′ primers (Amersham Biosciences or Sigma Genosys, The Woodlands, TX, USA), 0.5 μl Failsafe PCR enzyme mix (1.25 U, Epicentre, Monterrey, Mexico) and 25 μl Premix D (Epicentre). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers (BD Biosciences, Palo Alto, CA, USA) were used as a control for all RNA samples, and amplified a 450 bp DNA fragment. P2X2 amplification was carried out using the following primers: sense primer 5′ ACG TTC ATG AAC AAA AAC AAG 3′, and antisense primer 5′ TCA AAG TTG GGC CAA ACC TTT GG 3′. These primers amplify a 360 bp cDNA fragment corresponding to the P2X2 carboxy terminus in exon 11. The mouse P2X2 primers used in this study were derived from a mouse P2X2 cDNA that was cloned at Roche Palo Alto, and which aligns with the GenBank mouse P2X2 sequence NM153400. P2X3 amplification was carried out using the following primers: sense primer 5′ CTG TAT ATC AGA CTT CTT CAC CTA CGA 3′, and antisense primer 5′ TTA TGT CCT TGT CGG TGA GGT TAG 3′. These primers amplify a 596 bp cDNA fragment corresponding to exons 1 to exon 7 of P2X3. PCR products were electrophoresed through 1 % agarose, and the gels were transferred to Nytran N (Schleicher and Schuell, Dassel, Germany) membrane, followed by formamide hybridization at 42°C with probes specific for P2X3 and P2X2 subunits and for GAPDH. Blots were exposed to Kodak Biomax MR film (Kodak).

Intracellular electrophysiological recording

Mice were killed by cervical dislocation after halothane anaesthesia. The small intestine was removed and placed in oxygenated (95 % O2 and 5 % CO2) Krebs solution of the following composition (mm): NaCl, 117; KCl, 4.7; CaCl2, 2.5; MgCl2, 1.2; NaHCO3, 25; NaH2PO4, 1.2; glucose, 11. The Krebs solution contained nifedipine (1 μm) to block longitudunal muscle contractions and scopolamine (1 μm) to block muscarinic cholinergic receptors. A 1.5 cm segment of intestine was cut open along the mesenteric border and pinned out flat with the mucosal surface up in a Petri dish lined with silastic elastomer. A longitudinal muscle-myenteric plexus preparation was made by peeling away the mucosal, submucosal, and circular muscle layers using fine forceps and scissors. A 5 mm2 piece of longitudinal muscle myenteric plexus was transferred to a small (2 ml volume) recording chamber lined with silastic elastomer. The preparation was stretched lightly and pinned to the chamber bottom using stainless steel pins (50 μm diameter). The preparation was superfused with 36°C oxygenated Krebs solution at a flow rate of 4 ml min−1.

Individual myenteric ganglia were visualized at ×200 magnification using an inverted microscope (Olympus CK-2) with differential interference contrast optics. Intracelluar recordings were obtained from single neurons using glass microelectrodes filled with 2 m KCl and a tip resistance of 80–120 MΩ. An amplifier with an active bridge circuit (Axoclamp 2A, Axon Instruments, Foster City, CA, USA) was used to record membrane potential. In most experiments, the membrane potential was adjusted to −70 mV using constant DC current to avoid action potentials when evoking fEPSPs. The amplified signals then were filtered at 1 kHz using a four-pole, low-pass Bessel filter (Warner Instruments, Hamden, CT, USA), and digitized at 2 kHz using a Digitdata 1200 analog/digital converter (Axon Instruments). Data were acquired and stored using Axotape 2.02 software (Axon Instruments).

Neurons were classified as ‘S’ type neurons if the action potential was blocked by tetrodotoxin (0.3 μm) and if a single electrical stimulus applied to an interganglionic connective elicited a fEPSP (Bornstein et al. 1994). Neurons were classified as ‘AH’ cells, if the action potential was only partly inhibited by tetrodotoxin and if the action potential was followed by an afterhyperpolarization that lasted longer than 1 s (Bornstein et al. 1994). There were no neurons encountered in this study that did not fit into either of these two classes of myenteric neuron.

All fEPSPs were elicited using a glass pipette (tip diameter 40–60 μm) filled with Krebs solution as a focal stimulating electrode. The stimulating electrode was positioned over an interganglionic nerve strand. Nerve fibres were stimulated electrically using single stimuli of 0.5 ms duration at a rate of 0.1 Hz. A digital average of eight individual fEPSPs was used as a measurement of fEPSP amplitude in the absence or presence of drug treatments.

Antagonists were applied by superfusion in a known concentration by addition to the superfusing Krebs solution. Antagonists were applied for 10 min prior to measuring the amplitude of evoked responses. Local application of ATP or α,β-mATP was accomplished by ejection from the tip of a micropipette (≈20 μm tip diameter) placed within 150 μm of the impaled neuron. Agonists were applied using short pulses of nitrogen gas (3–35 ms, 10 p.s.i.) using a Picospritzer II (General Valve, Fairfield, NJ, USA).

Assessment of peristalsis in vitro

Intestinal segments (≈6 cm in length) were mounted in a Plexiglass organ bath (30 ml volume) using a technique similar to that described by others (Huizinga et al. 1998; Abdu et al. 2002; Bian et al. 2003). Tissues were equilibrated in Krebs solution at 37°C for 10 min before experiments started. Changes in intraluminal pressure were generated by raising the level of a buffer-containing reservoir connected to the ileal segment via a plastic tube. The filling tube was connected to a pressure transducer (TRN 050; Kent Scientific Co.) via a t-connector so that intraluminal pressure increases caused by raising the reservoir could be recorded. The pressure transducer was connected to a chart recorder (7DAE; Grass Instruments, Quincy, MA, USA). Responses to pressure increases of 1.25, 2.5, 3.75 and 5 mmHg were evaluated. Ileal responses to each pressure increase were recorded for 30 s and then the intraluminal pressure was returned to the basal level. Intraluminal pressure increases caused a series of phasic contractions. Pressure-induced contractile responses were quantitated by measuring: (1) the amplitude of the peak contraction and (2) the frequency of contractions occurring during the 30 s stimulation period.

Drug-induced longitudinal muscle contractions

Whole segments (3 cm) of ileum were suspended between stationary hooks and isometric force transducers (FT03C; Grass Instruments) in jacketed baths (20 ml volume) containing Krebs solution at 37°C. Tissues were placed under 10 mN of initial tension and were allowed to equilibrate for 30 min before the start of experimental protocols. The mechanical activity of the strips was recorded using a chart recorder (7DAE; Grass Instruments). Drugs were added to the baths in volumes of 2–20 μl. Agonist concentration-response curves were constructed in a non-cumulative manner with a 15 min interval between successive doses.

Measurement of gastrointestinal transit

Approximately 0.5 μCi of 51Cr as Na51CrO4 in 0.2 ml of saline was administered to each mouse by gavage using a flexible plastic feeding tube. Thirty minutes later the mice were killed following halothane anaesthesia and cervical dislocation. The stomach, heart and small intestine were carefully removed. The small intestine was placed on a pre-measured template and then divided into ten equal segments. The stomach, heart and each intestinal segment were placed in individual borosilicate glass tubes and the amount of radioactivity in each tube was counted directly using a gamma counter. No radioactivity was found in the heart from any mice indicating that the 51Cr was not absorbed from the gastrointestinal tract. The amount of radioactivity found in the stomach and each intestinal segment was expressed as a fraction of the total radioactivity counted in each animal. Gastrointestinal transit was then quantified as the geometric centre (Miller et al. 1981) of the distribution of radioactivity using the following relationship:

graphic file with name tjp0552-0809-mu1.jpg

where the stomach was taken as segment no. 1 and the ten intestinal segments were numbered consecutively as 2 to 11. Percentage gastric emptying was determined by subtracting the fraction of counts remaining in the stomach from the total counts in the stomach and small intestinal and multiplying this by 100.

Immunohistochemsitry

Segments of ileum were fixed overnight at 4°C in Zamboni's fixative (2 % (v/v) formaldehyde and 0.2 % (v/v) picric acid in 0.1 M sodium phosphate buffer, pH 7.0). The fixative was removed using three washes of dimethyl sulfoxide at 10 min intervals. Tissues were washed three times with phosphate-buffered saline (PBS)(0.01 m; pH 7.2) at 10 min intervals, and subsequently incubated overnight with primary antibody at room temperature. Tissues were then incubated with an antibody (1:200 dilution) raised in rabbits against amino acids 457–472 located on the intracellular C-terminal region of the rat P2X2 receptor (Alomone Laboratories, Jerusalem, Israel). In control experiments, antibodies raised against the P2X2 subunit were preincubated with the antigen peptide provided by the supplier. For these experiments, diluted antibody was incubated with antigen peptide in a 1:1 (μg) ratio for 1 h at room temperature, and then applied to preparations as described above. After primary antibody incubation, tissues were washed three times at 10 min intervals with PBS. Tissues were then incubated (1.5 h at 23°C) with goat anti-rabbitt IgG (1:40 dilution in PBS; Jackson Immunoresearch Laboratories, West Grove, PA, USA) conjugated to fluorescein isothiocyanate. Tissues were washed three times with PBS and mounted in buffered glycerol for fluorescence microscopy. Images were obtained using Leica TCS-SL confocal scanning system (Leica Microsystems, Heidelberg, Germany) and a DMLFSA upright microscope with an HCX PL APO 63 × 1.32 numerical aperture oil immersion objective lens.

Drugs

All drugs and chemicals were purchased from Sigma Chemical (St Louis, MO, USA). All reagents and drugs were diluted in distilled, deionized water except for nifedipine, which was dissolved in 95 % ethanol to make a 10 mm concentrated stock solution. The final working concentration of all drugs was made daily by diluting concentrated stock solutions with Krebs solution.

Statistics

All data are expressed as the mean ±s.e.m. When describing electrophysiological data, n values refer to the number of neurons. For studies of peristalsis or drug-induced longitudinal muscle contractions, n values refer to the number of animals from which tissues were obtained for each type of experiment. In the gastrointestinal transit studies n refers to the number of animals from which the data were obtained. Statistical analysis was performed using Student's t test for unpaired data or analysis of variance when applicable. A P level < 0.05 was considered statistically significant.

RESULTS

RT-PCR and immunohistochemical localization of the P2X2 subunit mRNA and protein in the intestine

In order to determine whether P2X2 receptor transcripts were present in the mouse intestine, we performed RT-PCR on total RNA from whole ileal segments (I), ileal myenteric plexus (IMP), whole colon segments (C) and colonic myenteric plexus (CMP)(Fig. 1). P2X2 transcripts were present in all tissues analysed from P2X2+/+ mice, but were not detected in tissues from P2X2−/− mice. P2X3 transcripts were also detected in intestinal tissues from both P2X2+/+ and P2X2−/− mice, as were transcripts for the internal control, GAPDH. None of the control reactions performed without reverse transcriptase showed amplification of DNA, indicating that the amplicons detected were derived from reverse transcribed cDNA and not contaminating genomic DNA.

Figure 1. Representative RT-PCR data showing the presence of P2X2 subunit transcripts in P2X2+/+ tissues but not in P2X2−/− tissues.

Figure 1

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RT-PCR was performed on whole ileum segments (I), ileal myenteric plexus (IMP), whole colon segments (C) and colonic myenteric plexus (CMP) from 2 separate P2X2+/+ and P2X2−/− mice (data shown from one each). GAPDH, P2X2 and P2X3 autoradiograms were exposed for 15 min, 15 min and 5 h, respectively.

Immunohistochemical studies revealed that P2X2 subunit-ir was expressed by many neurons in the myenteric plexus of the small intestine from P2X2+/+ mice (Fig. 2A). Staining for the P2X2 subunit was blocked when the antibody was pre-incubated with the antigen peptide and staining was also absent when the primary antibody was omitted from the incubation protocol (not shown). P2X2-ir was absent in tissues from P2X2−/− mice (Fig. 2B).

Figure 2. Immunohistochemical localization of the P2X2 subunit in the mouse small intestinal myenteric plexus.

Figure 2

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A, P2X2 immunoreactivity is present in myenteric neurons in a whole mount preparation taken from the ileum of a P2X2+/+ mouse. Most neurons express P2X2 subunit immunoreactivity. B, P2X2 immunoreactivity is absent from the ileal myenteric plexus of P2X2−/− mice. Scale bar applies to both panels.

Electrophysiological properties of myenteric neurons from P2X2+/+ and P2X2−/− mice

Intracellular electrophysiological recordings were obtained from 93 myenteric neurons from P2X2+/+ and P2X2−/− mice. Recordings were obtained from 12 AH neurons (3 from P2X2+/+, 9 from P2X2−/−) and 81 S neurons (36 from P2X2+/+ mice, 45 from P2X2−/− mice).

The action potentials recorded from AH neurons in tissues from from P2X2+/+ and P2X2−/− mice were similar (Fig. 3). The action potential durations at half-amplitude were 2.1 ± 0.1 and 2.3 ± 0.1 ms (P > 0.05) in tissues from P2X2+/+ and P2X2−/− mice, respectively. The peak amplitudes of the action potential slow afterhyperpolarization were 5.9 ± 1.1 and 5.8 ± 0.7 mV in P2X2+/+ and P2X2−/− mice (P > 0.05), respectively. The duration of the afterhyperpolarization in AH neurons from P2X2+/+ mice was 6.4 ± 0.9 s; in AH neurons from P2X2−/− mice it was 5.7 ± 0.4 s (P > 0.05). Finally, the resting membrane potential and input resistance of AH neurons from P2X2+/+ and P2X2−/− mice were not different (Table 1).

Figure 3. P2X2 gene deletion does not alter the action potential or the action potential afterhyperpolarization in myenteric AH neurons.

Figure 3

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Upper traces are recordings obtained from an AH neuron in the myenteric plexus from a P2X2+/+ mouse. The traces show a long-lasting afterhyperpolarization (left) that follows a single action potential shown on the right on an expanded time scale. Lower traces traces show similar recordings obtained from an AH neuron in the myenteric plexus from a P2X2−/− mouse. There were no differences in the afterhyperpolarization or the action potential recorded from tissues in the two types of mice (see text for details).

Table 1.

Resting membrane potential (RMP) and input resistance (Rinput) of AH and S neurons in tissues from P2X2+/+ and P2X2−/−

AH neurons S neurons
P2X2+/+ P2X2−/− P2X2+/+ P2X2−/−
(n = 3) (n = 7) (n = 17) (n = 21)
RMP (mV) Mean 69 ± 5 62 ± 4 63 ± 2 58 ± 2
Range 61–80 48–81 50–74 45–80
Rinput (MΩ) Mean 136 ± 35 132 ± 21 122 ± 13 112 ± 9
Range 96–207 85–221 65–241 54–167
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S neurons received fast excitatory postsynaptic input (see below), and the action potential in these neurons was blocked by tetrodotoxin (Fig. 4). The action potential in S neurons had durations at half-amplitude of 1.7 ± 0.1 and 1.7 ± 0.1 ms (P > 0.05) in tissues from P2X2+/+ and P2X2−/− mice, respectively. The action potential in S neurons was followed by a brief (< 100 ms) afterhyperpolarization (Fig. 4). The resting membrane potential and input resistance of S neurons from P2X2+/+ and P2X2−/− mice were not different (Table 1).

Figure 4. P2X2 gene deletion does not alter the action potential in S neurons.

Figure 4

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Upper traces show that the action potential recorded from S neurons is followed by a brief afterhyperpolarization (arrows) in the myenteric plexus from P2X2+/+ (left) and P2X2−/− (right) mice. Middle traces show that P2X2 subunit gene deletion does not alter the action potential in S neurons. The bottom traces show that the action potential in S neurons is blocked by tetrodotoxin.

P2X2 subunits compose P2X receptors in S neurons

There were no differences in the peak amplitude or time course of fEPSPs recorded from S neurons in tissues from P2X2+/+ and P2X2−/− mice (Table 2). This result suggests that P2X2 subunits may not contribute to the P2X receptors mediating fEPSPs in S neurons. However, in tissues from P2X2+/+ mice, nearly all fEPSPs were reduced in amplitude by the P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2′, 4′-disulphonic acid (PPADS, 10 μm)(Fig. 5). All fEPSPs were blocked by the combined application of PPADS and the nicotinic acetylcholine receptor antagonist mecamylamine (10 μm). PPADs had no effect on fEPSPs recorded from S neurons in tissues from P2X2−/− mice (Fig. 5). Mecamylamine reduced the amplitude of fEPSPs recorded from S neurons in P2X2+/+ tissues, but blocked fEPSPs recorded from S neurons in tissues from P2X2−/− mice (Fig. 6).

Table 2.

Amplitude and time course of fEPSPs recorded from S neurons in the ileal myenteric plexus from P2X2+/+ and P2X2−/− mice

P2X2+/+ P2X2−/−
(n = 10) (n = 15)
Amplitude (mV) 20.3 ± 1.3 18.3 ± 1.0
10–90% rise time (ms) 3.4 ± 0.3 3.3 ± 0.2
Time to half-decay (ms) 9.5 ± 0.8 8.2 ± 1.2
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Figure 5. PPADS inhibits fEPSPs recorded from S neurons in tissues from P2X2+/+ but not P2X2−/− mice.

Figure 5

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A, representative fEPSPs recorded from S neurons in P2X2+/+ (left) and P2X2−/− (right) tissues in the absence or presence of PPADS (10 μm). The fEPSPs recorded from neurons in tissues from P2X2+/+ mice were inhibited by PPADS (left) while fEPSPs recorded from neurons in P2X2−/− tissues were unaffected by PPADS (right). B, mean data showing that the amplitudes of fEPSPs recorded from S neurons in P2X2+/+ (n = 5) and P2X2−/− (n = 6) tissues were similar. PPADS did not alter fEPSP amplitude recorded from P2X2−/− S neurons but it did inhibit the fEPSP in S neurons from P2X2+/+ tissues. * Significantly different from P2X2+/+ control (P < 0.05).

Figure 6. Mecamylamine reduced fEPSPs in S neurons from P2X2+/+ mice but completely blocked fEPSPs in S neurons from P2X2−/− mice.

Figure 6

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A, representative fEPSPs recorded from S neurons in P2X2+/+ (left) and P2X2−/− (right) tissues. The fEPSP in P2X2+/+ neurons is only partly inhibited by mecamylamine (10 μm), but is blocked by mecamylamine in neurons from P2X2−/− mice. B, mean data showing that mecamylamine caused a partial, but significant, reduction in the fEPSP amplitude in P2X2+/+ neurons (n = 5). Mecamylamine completely inhibited the fEPSP in P2X2−/− neurons (n = 9). * Significantly different from P2X2+/+ or P2X2−/− control amplitude (P < 0.05); # Significantly different from the amplitude of the P2X2+/+ fEPSP in the presence of mecamylamine (P < 0.05).

Focal application of nicotine caused a depolarization of S neurons from P2X2−/− mice and this response was similar in amplitude to that recorded from S neurons in P2X2+/+ tissues (Fig. 7). Focal application of ATP, but not α,β-mATP, depolarized S neurons in tissues from P2X2+/+ mice (Fig. 7). Neither ATP or α,β-mATP caused a response in S neurons from P2X2−/− mice (Fig. 7).

Figure 7. Agonist-induced depolarizations of S neurons in the myenteric plexus from P2X2+/+ or P2X2−/− mice.

Figure 7

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A, nicotine caused similar amplitude depolarizations of S neurons from P2X2+/+ and P2X2−/− mice. ATP depolarized S neurons from P2X2+/+ but not P2X2−/− mice, while α,β-mATP did not depolarize any S neuron. Drugs were applied (at the arrows) by pressure ejection from a pipette positioned near the impaled neurons. The concentration of each drug in the pipette was 1 mm. B, mean data from experiments similar to those shown in A. There was no difference in the amplitude of the nicotine-induced depolarization recorded from S neurons in tissues from P2X2+/+ (n = 15) and P2X2−/− (n = 12) mice. * The ATP-induced depolarization was significantly smaller in P2X2−/− neurons compared to those in recorded from P2X2+/+ neurons. α,β-mATP did not elicit a response in S neurons from either type of mouse.

α,β-mATP depolarizes AH neurons from P2X2+/+ and P2X2−/− mice

Focal application of α,β-mATP (1 mm) caused a rapidly developing depolarization that was associated with a decrease in membrane input resistance in AH neurons from P2X2+/+ and P2X2−/− mice (Fig. 8). The α,β-mATP-induced response was mimicked by ATP applied to the same neurons and was blocked by PPADS (data not shown). The amplitudes of α,β-mATP-induced depolarizations were similar in tissues from P2X2+/+ and P2X2−/− mice (Fig. 8).

Figure 8. AH neurons from P2X2+/+ and P2X2−/− mice were depolarized by α,β-mATP.

Figure 8

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A, α,β-mATP was applied by pressure ejection (at the arrows) from a pipette positioned near the impaled neuron. The concentration of α,β-mATP in the pipette was 1 mm. B, data showing that the amplitude of the α,β-mATP-induced depolarization was similar in tissues from P2X2+/+ (n = 3) and P2X2−/− (n = 4) mice.

Peristalsis is impaired in the ileum of P2X2−/− mice

Raising intraluminal pressure (1.25–5 mmHg) increased the frequency and amplitude of phasic contractions in ileal segments from P2X2+/+ and P2X2−/− mice (Fig. 9). Visual inspection revealed that these contractions propagated in an oral to aboral direction.

Figure 9. Peristalsis is impaired in ileal segments from P2X2−/− mice.

Figure 9

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A, pressure-related increases in the peak amplitude of peristaltic contractions. The peak amplitude in tissues from P2X2−/− mice (n = 7) was smaller than that in P2X2+/+ mice (n = 7) (*P < 0.05). Mecamylamine (10 μm) reduced the peak contractions in tissues from P2X2+/+ and P2X2−/− mice (#P < 0.05). B, pressure-related increases in contraction frequency were reduced in tissues from P2X2−/− mice (*P < 0.05). Mecamylamine reduced contraction frequency in tissues from P2X2+/+ and P2X2−/− mice (#P < 0.05).

The propagated contractions required nicotinic cholinergic neurotransmission as mecamylamine (10 μm) reduced the frequency and amplitude of peristaltic contractions in tissues from P2X2+/+ and P2X2−/− mice (Fig. 9A and B). P2 receptor-dependent mechanisms also contribute to peristalsis in tissues from P2X2+/+ mice, as PPADS (10 μm) inhibited pressure-induced contractions in these tissues (Fig. 10). The frequency and amplitude of peristaltic contractions were reduced in tissues from P2X2−/− mice compared to that measured in P2X2+/+ mice (Fig. 9 and Fig. 10). PPADS did not reduce peristaltic contractions in tissues from P2X2−/− mice (Fig. 10A and B).

Figure 10. PPADS inhibits peristalsis in ileal segments from P2X2+/+ but not P2X2−/− mice.

Figure 10

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A, the peak amplitude of phasic contractions was smaller in tissues from P2X2−/− mice compared to that in P2X2+/+ mice (*P < 0.05) but PPADS (10 μm) did not reduce contraction amplitude in P2X2−/− tissues. PPADS reduced the amplitude of phasic contractions in tissues from P2X2+/+ mice (#P < 0.05). B, the frequency of pressure-induced phasic contractions was lower in P2X2−/− tissues (*P < 0.05) and PPADS did not reduce further the contraction frequency in P2X2−/− tissues. PPADS reduced contraction frequency in tissues from P2X2+/+ mice (#P < 0.05).

Drug-induced longitudinal muscle contractions

Disruption of peristalsis in tissues from P2X2−/− mice could be due to alterations in contractile mechanisms in intestinal smooth muscle. To test this possibility, longitudinal muscle contractions caused by the muscarinic receptor agonist, bethanechol (BeCh) and by nicotine were studied in vitro. BeCh activates muscarinic receptors on longitudinal smooth muscle to cause muscle contractions and BeCh concentration-response curves obtained in P2X2+/+ (n = 6) and P2X2−/− (n = 12) mice were similar (Fig. 11A). In order to assess the function of excitatory motorneuron input to the longitudinal muscle, nicotine was used to stimulate motorneurons. Nicotine caused concentration-dependent contractions of the muscle layer, and there were no differences between curves obtained in P2X2+/+ and P2X2−/− tissues (Fig. 11B). ATP (0.01–1 mm) did not cause contractions of the longitudinal muscle in tissues from either P2X2+/+ (n = 5) or P2X2−/− (n = 6) mice.

Figure 11. Bethanechol- and nicotine-induced longitudinal muscle contractions were not different in ileal segments from P2X2+/+ and P2X2−/− mice.

Figure 11

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A, bethanechol caused concentration-dependent contractions of the longitudinal muscle layer. There were no differences in curves obtained from P2X2+/+ and P2X2−/− mice. B, nicotine caused concentration-dependent longitudinal muscle contractions and there were no differences in nicotine responses obtained in tissues from P2X2+/+ and P2X2−/− tissues.

Gastrointestinal transit is not altered by P2X2 gene deletion

The distribution of radioactive marker in the stomach and small intestine of P2X2+/+ and P2X2−/− mice is shown in Fig. 12. These data show that the leading edge of marker in the small intestine of P2X2+/+ mice reached the tenth intestinal segment while in P2X2−/− mice the marker progressed only as far as the seventh segment. However, when the geometric centre of the marker distribution was calculated there were no differences in gastrointestinal transit between P2X2+/+ and P2X2−/− mice. The geometric centre in P2X2+/+ mice was 3.0 ± 0.4 (n = 8), while in P2X2−/− mice it was 3.1 ± 0.3 (n = 8, P > 0.05). There was no difference in gastric emptying of the liquid marker between the two groups of mice. Gastric emptying in P2X2+/+ mice was 45 ± 9 %, while in P2X2−/− mice it was 49 ± 7 % (P > 0.05).

Figure 12. P2X2 gene deletion does not alter gastrointestinal transit of a liquid marker.

Figure 12

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Mean distribution of Na51CrO4 in the stomach and small intestine of P2X2+/+ mice (n = 8) (A) and P2X2−/− mice (n = 8)(B). For both figures, data are the mean ±s.e.m. of the percentage of total marker in the stomach and intestinal segments 30 min after gavage administration of the marker to each mouse. There were no differences in the distribution of marker in the two groups of mice (see text for details).

DISCUSSION

ATP acting at P2X receptors contributes to fast synaptic excitation in myenteric S neurons (Galligan & Bertrand, 1994; Johnson et al. 1999; Galligan et al. 2000; Nurgali et al. 2003). ATP released in the mucosal epithelium may also mediate chemo- or mechanosensory input to AH neurons via an action at a P2X receptor (Bertrand & Bornstein, 2002). However, the specific subunit composition of enteric P2X receptors mediating synaptic or sensory responses is not known. Most of our knowledge about the functional and pharmacological properties of P2X receptors has come from studies of defined expression of P2X subunits in heterologous systems (North & Suprenant; 2000; North, 2002). The lack of P2X subtype selective agonists and antagonists has impaired studies of the subunit composition of P2X receptors in native cell types, including neurons. In the present study, we used mice in which the P2X2 subunit gene had been deleted to investigate the role of this subunit in neurotransmission in the myenteric plexus. Immunohistochemical and RT-PCR studies revealed that the P2X2 subunit was present in the myenteric plexus of P2X2+/+ mice, and that this subunit was absent from the myenteric plexus of P2X2−/− mice.

P2X2 gene deletion does not alter the passive properties or action potentials recorded from murine myenteric neurons

Our previous work (Bian et al. 2003) has shown that the basic electrophysiological properties of S and AH type neurons in mouse small intestinal myenteric neurons are similar to those identified in the guinea pig ileum (Hirst et al. 1974) and colon (Lomax et al. 1999), and rat (Browning & Lees, 1996), mouse (Furukawa et al. 1986) and human (Brookes et al. 1987) colon. These observations about the basic properties of mouse small intestinal myenteric neurons were confirmed in the present study. Our new data show that P2X2 gene deletion does not alter the resting membrane potential, input resistance or action potential duration in S or AH neurons from the mouse small intestine.

In our study, AH type neurons were encountered infrequently using a random impalement strategy with intracellular microelectrodes. This observation is similar to that reported previously in a study of myenteric neurons in the mouse colon (Furukawa et al. 1986). If AH neurons are sensory neurons in the mouse intestine, as they are in the guinea pig intestine (Furness et al. 1998), then normal intestinal reflex behaviours may require only a small number of sensory neurons in the mouse. Alternatively, non-AH neurons may be sensory neurons in the mouse intestine.

Fast synaptic transmission and agonist induced-depolarizations in S neurons from P2X2+/+ and P2X2−/− mice

The fEPSPs recorded from S neurons in tissues from P2X2+/+ mice were partly inhibited by the nicotinic cholinergic receptor antagonist mecamylamine and by the P2 receptor antagonist PPADS. These results indicate that ACh and ATP mediate fast synaptic responses in the murine myenteric plexus as also occurs in the guinea pig intestine (Galligan & Bertand, 1994; LePard et al. 1997; Johnson et al. 1999) and colon (Nurgali et al. 2003). However, the fEPSP in P2X2−/− mice was blocked by mecamylamine while PPADS had no effect on fEPSPs in P2X2−/− tissues. These data indicate that P2X2 subunits are a critical component of the P2X receptor expressed by S neurons.

Although P2X2 subunits mediate the purinergic component of fEPSPs, the average fEPSP amplitude recorded from S neurons in P2X2−/− and P2X2+/+ tissues was not different. This result may be related to the findings from previous studies that showed a functional interaction between P2X and nicotinic acetylcholine receptors (Nakazawa, 1994; Zhou & Galligan, 1998; Searl et al. 1998; Barajas-Lopez et al. 1998; Khakh et al. 2000). In these studies it was found that simultaneous activation of nicotinic and P2X receptors produces a response that is smaller in amplitude than the predicted sum of responses caused by individual activation of each receptor. It was concluded that there is a functional link between P2X and nicotinic receptors that results in cross-inhibition of responses mediated by these receptors (Nakazawa, 1994; Zhou & Galligan, 1998; Searl et al. 1998; Barajas-Lopez et al. 1998; Khakh et al. 2001). Deletion of the P2X2 subunit gene would remove cross-inhibition and synaptic responses mediated by nicotinic receptors in P2X2−/− mice would be larger in amplitude than those occurring in neurons co-expressing the P2X2 subunit. Lack of P2X2-nicotinic receptor cross-inhibition could account for the maintained fEPSP amplitude in P2X2−/− tissues.

S neurons in tissues from P2X2+/+ mice were depolarized by ATP but not by α,β-mATP. This result suggests that S neurons express P2X2 homomeric receptors as α,β-mATP does not activate P2X2 homomers, but does activate P2X3 homomeric and P2X2/3 heteromeric receptors (Lewis et al. 1995; North & Surprenant, 2000). In addition, ATP failed to elicit a depolarization in S neurons from P2X2−/−mice. Based on these results, we conclude that the P2X receptor mediating fEPSPs in murine S neurons is a P2X2 homomeric receptor. In the guinea pig intestine, P2X2 subunit immunoreactivity has been localized to nitric oxide synthase (NOS)-containing neurons (Castelucci et al. 2002). NOS-containing neurons are inhibitory motorneurons and descending interneurons, and both of these classes of neuron would have S-type electrophysiologial properties (Brookes, 2001). If the chemical coding of murine myenteric neurons is similar to that in the guinea pig intestine, these data would indicate that inhibitory motorneurons (Johnson et al. 1999) and some descending interneurons (LePard & Galligan, 1999; Bian et al. 2000; Monro et al. 2002) receive fast excitatory synaptic input mediated in part by P2X2 homomeric receptors.

Agonist induced-depolarization in AH neurons

In the guinea pig intestine, myenteric AH neurons send a process to the mucosal epithelium and these nerve endings can be excited by ATP acting at a P2X receptor (Bertrand & Bornstein, 2002). AH neurons also express P2X receptors on the nerve cell body (Bertrand & Bornstein, 2002). Therefore, we tested the effect of ATP on AH neurons in tissues from P2X2+/+ and P2X2−/− mice to determine if P2X2 gene deletion altered P2X-mediated responses in these neurons. α,β-mATP caused similar amplitude depolarizations in AH neurons from both types of mice. As α,β-mATP activates P2X receptors containing P2X1 or P2X3 subunits (North & Surprenant, 2000), it is possible that AH neurons express P2X receptors composed of P2X1 or/and P2X3 subunits. However, our previous work has shown that α,β-mATP does not depolarize AH neurons in the myenteric plexus of P2X3−/− mice while ATP depolarized the same neurons (Bian et al. 2003). Therefore, the P2X receptors in murine AH neurons are likely to be P2X2/3 heteromers. The conclusion that AH neurons express receptors containing P2X2 subunits is supported by data from immunohistochemical studies done in the guinea pig ileum where it was shown that P2X2 subunits were found in calbindin-containing neurons (Castelucci et al. 2002). Calbindin is a marker for most AH type neurons (Furness et al. 1998; Brookes, 2001). Although our pharmacological data indicate that AH neurons express a receptor containing P2X3 subunits, these subunits are not found in calbindin-containing neurons in the guinea pig intestine myenteric plexus (VanNassauw et al. 2002; Poole et al. 2002). These data may indicate that there is a difference in P2X subunit expression by different subsets of myenteric neurons in the mouse and guinea pig gut.

Peristalsis is impaired in the small intestine of P2X2−/− mice

Instillation of small volumes of saline into the lumen of an ileal segment in vitro evokes peristaltic contractions, and the frequency and amplitude of these contractions are pressure related. Peristaltic contractions in ileal segments from P2X2+/+ and P2X2−/− mice are attenuated by mecamylamine. These data are similar to those reported previously where it was shown that synaptic transmission mediated at nicotinic cholinergic receptors is critical for reflex-evoked contractions in the mouse intestine (Huizinga et al. 1998; Abdu et al. 2002; Bian et al. 2003). Our data also show that P2X2 gene deletion does not alter contractile responses caused by muscarinic receptor stimulation in the longitudinal muscle layer, as BeCh concentration-response curves were similar in P2X2+/+ and P2X2−/− tissues. In addition, nicotinic receptor function on myenteric neurons is not altered by P2X2 gene deletion, as nicotine concentration-response curves are similar in tissues obtained from the two types of mice. Changes in longitudinal muscle muscarinic receptor function, or neuronal nicotinic receptor function cannot account for the impairment of peristalsis caused by P2X2 gene deletion.

We observed that the amplitude and frequency of distention-evoked contractions are reduced in preparations from P2X2−/− mice, indicating that P2X2 subunits contribute to the neural mechanisms mediating peristalsis. The P2 receptor antagonist PPADS inhibited peristalsis in tissues from P2X2+/+ mice to a level comparable to that recorded from P2X2−/− tissues, suggesting that pharmacological inhibition of P2X-mediated mechanisms mimics the effects of P2X2 subunit gene deletion. Indeed, PPADS did not inhibit peristalsis in tissues from P2X2−/− mice, suggesting that a P2X2 subunit-containing receptor is a target of PPADS inhibition. However, peristalsis is impaired in the small intestine of P2X3−/− mice, and PPADS does not inhibit peristalsis in small intestinal preparations from these mice (Bian et al. 2003). These previous studies showed that P2X receptors on myenteric AH neurons contained P2X3 subunits (Bian et al. 2003). Therefore, there are potentially two sites at which PPADS can act to inhibit peristalsis: P2X2 receptors on S neurons (this study) and P2X3 receptors on AH/sensory neurons (Bian et al. 2003). If this were the case then deletion of one P2X subunit should preserve, at least in part, the inhibitory action of PPADS on peristalsis. However, this was not observed in our studies. This discrepancy can be resolved if it is assumed that AH neurons are mechanically sensitive sensory neurons in the mouse intestine, as they are in the guinea pig intestine, (Kunze et al. 1998). It must also be assumed that S neurons are motorneurons and interneurons in the mouse intestine, as they are in the guinea pig intestine (Brookes, 2001). If these assumptions are correct, then P2X3-(localized to sensory neurons) and P2X2-subunit containing receptors (localized to interneurons and motorneurons downstream from sensory neurons) would be localized to in-series neurons in the neural pathway mediating peristalsis. Deletion of P2X receptors at any point along this pathway would occlude the inhibitory effects of PPADS at other points in the same pathway.

Gastrointestinal transit is not changed in P2X2−/− mice

As ganglionic transmission and peristalsis are altered in tissues from P2X2−/− mice, we tested the impact of P2X2 gene deletion on gastrointestinal propulsion of saline in fasted mice. Although peristalsis studied in vitro was impaired, we did not find any changes in gastrointestinal transit of a radioactive marker or in gastric emptying of the marker into the small intestine. The differential effects of P2X2 gene deletion on the results of the in vitro and in vivo motility assays could be attributed to several factors. Changes in distention-evoked reflexes studied in vitro may not be reflected in alterations in propulsive motility studied in vivo. It is also possible that the gastrointestinal transit assay we used is not sensitive enough to detect in vivo motility alterations associated with P2X2 subunit gene deletion. Finally, it may be necessary to study gastrointestinal transit in the freely fed state using a solid marker for assessment of gastrointestinal propulsion in order to reveal motility changes associated with P2X2 gene deletion.

Summary and conclusions

P2X2 gene deletion does not alter the resting electrophysiological properties or action potentials recorded from murine myenteric neurons. However, P2X2 gene deletion eliminates the P2X-mediated component of fEPSPs and ATP-induced depolarizations recorded from S-type neurons. ATP responses recorded from AH-type neurons are not altered by P2X2 gene deletion. Distension-evoked peristalsis is impaired in the ileum of P2X2−/− mice, and this effect is likely due to a loss of P2X receptor-mediated synaptic transmission in the myenteric plexus. The inhibition of fast synaptic transmission and peristalsis that occurs in P2X2−/− mice does not result in changes in the propulsion of a liquid marker through the stomach and small intestine of fasted mice. The data presented here are consistent with the hypothesis that the P2X receptor mediating fast synaptic excitation of S neurons is a P2X2 homomeric receptor.

Acknowledgments

This work was supported by grant number DK57039 from the National Institutes of Health.

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