Deletion of P2X3 receptors blunts gastro-oesophageal sensation in mice

Abstract

Prior studies have demonstrated P2X receptor expression in the majority of nodose neurons. Immunoreactivity for P2X receptors has also been seen in putative gastric mechanoreceptors, the intraganglionic laminar endings. We therefore hypothesized that deletion of P2X3 receptors will blunt responses to gastric distension in vagal sensory neurons. Using wildtype and P2X3(-/-) mice, we examined responses to purinergic agonists in retrogradely labelled gastric sensory neurons with patch-clamp techniques. Activation of gastro-oesophageal neurons by fluid distension was studied with intracellular electrodes. Distension-evoked ATP release into the gastric lumen was determined with the luciferase assay and intake and gastric emptying of a solid meal was assessed. ATP triggered inward currents in 80% of gastric nodose neurons. In P2X3(-/-) mice, the peak current density was lower compared to controls. Ten of 14 controls but none of 30 neurons from P2X3(-/-) mice responded to alpha,beta-metATP. Gastro-oesophageal sensory neurons of P2X3(-/-) mice showed a blunted response to fluid distension of oesophagus and stomach. This difference was not explained by differences in distension-evoked ATP release, which did not differ between knockout mice and controls. Food intake during a 3-h period was lower in P2X3(-/-) mice. Gastric emptying of a solid meal was slightly faster in knockout mice after 1.5 h, but did not differ between groups at 3 h. Our data support a role of purinergic signalling in gastric vagal afferents. Considering the role of vagal input in sensations of fullness or nausea, P2X receptors may be interesting treatment targets for dyspeptic symptoms.

Figure 1

Stimulation of dissociated gastric sensory neurons with ATP and α,β-metATP. (A) Current responses during superfusion with 30 μmol L⁻¹ of ATP (left) and 30 μmol L⁻¹ of α,β-metATP (right) from a wildtype animal. (B) The corresponding responses from a gastric neuron of a P2X3^−/− mouse. The peak current density (C) and time to peak (D) in response to 30 μmol L⁻¹ of ATP are summarized for wildtype (black bars; n = 15) and P2X3^−/− mice (white bars; n = 22). *P < 0.05; P < 0.01.

Figure 2

Representative voltage tracings demonstrate the effects of gastro-oesophageal distension on action potential firing. The upper tracings demonstrate little baseline activity for wildtype (left) and P2X3^−/− mice (right). Stepwise distension led to an increase in action potential frequency as shown in the lower tracings. Data were obtained 10 s after initiation of the stimulus. The calibration bars represent 20 mV and 1 s respectively.

Figure 3

Response to gastric distension. The upper panel (A) shows the average spike frequency in gastric nodose neurons during stepwise fluid distension, measured during a 20-s interval in wildtype (filled circles) and P2X3^−/− mice (open circles). Differences between the groups were significant (F = 6.2; P < 0.02). The lower panels show a more detailed time course for the distension pressures of (B) 10 cmH₂O (F = 10.2; P < 0.01), (C) 20 cmH₂O (F = 35.5; P < 0.01), (D) 30 cmH₂O (F = 49.8; P < 0.001) and (E) 40 cmH₂O (F = 31.8; P < 0.001). The bar represents the time of luminal pressure application.

Figure 4

Effect of luminal acidification. (A) Representative voltage tracings with action potential firing after luminal perfusion at pH 7 (upper tracings) and pH 4 (lower tracings) for wildtype (left) and P2X3^−/− mice (right). The calibration bars represent 20 mV and 1 s respectively. The data are summarized in the bar graph. (B) The average spike frequency at baseline (left side) and 3 min after decreasing the pH to 4 in wildtype animals (black bars) and P2X3^−/− mice (white bars). While acidification triggered a significant increase in controls (P < 0.05), spike frequency did not change after exposure to pH 4 knockout mice.

Figure 5

Gastric compliance is higher in P2X3^−/− mice. The stomach was stepwise distended to a maximal volume of 1000 μL. Compared to wildtype mice (filled circles), P2X3^−/− (open circles) mice tended to have a slightly higher compliance (F = 3.58; P = 0.062). A post hoc analysis showed significant differences between the groups at 900 and 1000 μL distending volume (P < 0.05).

Figure 6

Mechanical stimulation induces release of ATP into the lumen of the stomach. The plot shows the mean release of ATP in nmol L⁻¹ per sample in response to mechanical stimulation with an ascending series of distension stimuli from 10 to 40 cmH₂O. Increases in pressure resulted in increased release of ATP in both wildtype controls (WT; circles; n = 8) and P2X3^−/− animals (open circles; n = 6). Pressure significantly affected ATP release (F = 5.51; P < 0.01). There was no significant difference between WT and P2X3^−/− mice (F = 0.39; P = 0.53).