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

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. alpha,beta-Methylene ATP (alpha,beta-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.

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

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.

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

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.

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

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).

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

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.

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

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).

Figure 5. PPADS inhibits fEPSPs recorded from S neurons in tissues from P2X₂^+/+ but not P2X₂^−/− mice

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

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.

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

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.

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

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).

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

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).

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

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.

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

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).