Deletion of the acid-sensing ion channel ASIC3 prevents gastritis-induced acid hyperresponsiveness of the stomach-brainstem axis

Abstract

Gastric acid challenge of the rat and mouse stomach is signalled to the brainstem as revealed by expression of c-Fos. The molecular sensors relevant to the detection of gastric mucosal acidosis are not known. Since the acid-sensing ion channels ASIC2 and ASIC3 are expressed by primary afferent neurons, we examined whether knockout of the ASIC2 or ASIC3 gene modifies afferent signalling of a gastric acid insult in the normal and inflamed stomach. The stomach of conscious mice (C57BL/6) was challenged with intragastric HCl; two hours later the activation of neurons in the nucleus tractus solitarii (NTS) of the brainstem was visualized by c-Fos immunocytochemistry. Mild gastritis was induced by addition of iodoacetamide (0.1%) to the drinking water for 7 days. Exposure of the gastric mucosa to HCl (0.25M) caused a 3-fold increase in the number of c-Fos-positive neurons in the NTS. This afferent input to the NTS remained unchanged by ASIC3 knockout, whereas ASIC2 knockout augmented the c-Fos response to gastric HCl challenge by 33% (P<0.01). Pretreatment of wild-type mice with iodoacetamide induced mild gastritis, as revealed by increased myeloperoxidase activity, and enhanced the number of NTS neurons responding to gastric HCl challenge by 41% (P<0.01). This gastric acid hyperresponsiveness was absent in ASIC3 knockout mice but fully preserved in ASIC2 knockout mice. The current data indicate that ASIC3 plays a major role in the acid hyperresponsiveness associated with experimental gastritis. In contrast, ASIC2 appears to dampen acid-evoked input from the stomach to the NTS.

Figure 1. Generation of ASIC3 null mice

(A) ASIC3 gene and flanking sequences. ASIC3 exons are indicated by gray boxes. (B) In the ASIC3 targeting vector a SfiI / XhoI fragment of the ASIC3 gene was replaced by a neomycin resistance expression cassette flanked by two loxp sites. (C) Targeted ASIC3 allele which codes for a 43 amino acid protein truncated before the first transmembrane domain.

Figure 2. Effect of gastric acid challenge on c-Fos expression in the nucleus tractus solitarii (NTS) of wild-type mice

The graph shows the concentration-related effect of IG administered HCl, relative to NaCl, to increase the expression of c-Fos in the unilateral NTS of wild-type mice. NaCl (0.15 M; depicted as 0 M HCl) and HCl (0.25, 0.35 and 0.5 M) were administered IG 2 h before immunocytochemistry. Since this experiment was a pilot experiment, each concentration of HCl was tested in 2 mice only. The values shown represent the results of the individual experiments (black dots) and their means (horizontal line).

Figure 3. Expression of c-Fos in the nucleus tractus solitarii (NTS) and area postrema (AP) of wild-type (A,B), ASIC2−/− (C,D) and ASIC3−/− (E,F) mice

The animals were treated IG with 0.25 M HCl 2 h before immunocytochemical visualization of c-Fos-positive cells in the brainstem. The animals had either received normal drinking water (A,C,E) or drinking water containing 0.1 % iodoacetamide (B,D,F) for 7 days before the gastric acid challenge experiment. HCl induced many cells in the medial and subpostremal nuclei of the NTS and some cells in the AP to express c-Fos. CC, central canal. Calibration bar: 0.1 mm.

Figure 4. Expression of c-Fos in the nucleus tractus solitarii (NTS) of wild-type, ASIC2−/− and ASIC3−/− mice without gastritis

Expression of c-Fos in the unilateral NTS was visualized 2 h after IG administration of NaCl (0.15 M) and HCl (0.25 M). The values represent means ± SEM, n as indicated. **P < 0.01 versus HCl-treated wild-type animals (two-way ANOVA followed by Games-Howell test).

Figure 5. Expression of c-Fos in the nucleus tractus solitarii (NTS) of wild-type, ASIC2−/− and ASIC3−/− mice with gastritis

Expression of c-Fos in the unilateral NTS was visualized 2 h after IG administration of HCl (0.25 M) to wild-type, ASIC2−/− and ASIC3−/− mice that had been pretreated with iodoacetamide (0.1%) added to the drinking water for 7 days or were allowed to drink normal water (control). The values represent means ± SEM, n as indicated. ** P < 0.01 versus control animals of the same genotype (two-way ANOVA followed by Games-Howell test). ASIC2−/− control mice, but not ASIC3−/− control mice, were significantly (P < 0.01) different from wild-type control mice.