Volume 125, Issue 6, Pages 1774-1784 (December 2003) Impaired gastric secretion and lack of trophic responses to hypergastrinemia in M3 muscarinic receptor knockout mice  Takeshi Aihara, Teruaki Fujishita, Keiko Kanatani, Kazuharu Furutani, Eiji Nakamura, Makoto M Taketo, Minoru Matsui, Duan Chen, Susumu Okabe  Gastroenterology  Volume 125, Issue 6, Pages 1774-1784 (December 2003) DOI: 10.1053/j.gastro.2003.09.018

Figure 1 Comparison of basal gastric acid secretion and serum gastrin levels between wild-type and M3 KO mice. M3 KO mice exhibited lower gastric acid secretion, as shown by (A) a significantly higher basal intragastric pH (P < 0.05) and (B) a significantly lower basal gastric acid output (P < 0.05, n = 12). (C) Ultrastructure of parietal cells in M3 KO mice. Nonsecreting parietal cells exhibited cytoplasmic tubulovesicles (∗) without secretory canaliculi (left). Secreting (active) parietal cells displayed secretory canaliculi (∗∗) (right). Both types of parietal cells are indistinguishable from corresponding secreting and nonsecreting parietal cells in wild-type mice. (D) The proportion of active parietal cells in the oxyntic mucosa of wild-type versus M3 KO mice (n = 5). (E) M3 KO mice had significantly elevated serum gastrin levels compared with wild-type mice (n = 11). Data are presented as means ± SEM. Statistical differences were evaluated using the Alternate Welch t test or Student t test. ∗P < 0.05. Gastroenterology 2003 125, 1774-1784DOI: (10.1053/j.gastro.2003.09.018)

Figure 2 Comparison of stimulated gastric acid secretion in (A) vehicle-treated, (B) carbachol-treated (0.1 mg/kg), (C) histamine-treated (10 mg/kg), and (D) gastrin-treated (1 mg/kg) wild-type versus M3 KO mice. Despite a significant increase in acid output induced by each secretagogue observed for both wild-type and M3 KO mice, M3 KO mice exhibited significantly reduced responses to all 3 secretagogue-evoked gastric acid outputs compared with wild-type mice. Data are presented as means ± SEM (n = 10–14). Statistical differences were evaluated with Student t test. ∗P < 0.05. Gastroenterology 2003 125, 1774-1784DOI: (10.1053/j.gastro.2003.09.018)

Figure 3 (A) Effects of pirenzepine and famotidine on carbachol-stimulated gastric acid secretion in M3 KO mice. Pirenzepine (3 mg/kg subcutaneously) and famotidine (3 mg/kg subcutaneously) significantly inhibited carbachol-evoked gastric acid output (n = 7–9). (B) Effect of vagal nerve stimulation with 2-DG (200 mg/kg intraperitoneally × 2) on gastric acid secretion in wild-type versus M3 KO mice. 2-DG significantly stimulated gastric acid secretion in wild-type mice but failed to do so in M3 KO mice (n = 5). Data are presented as means ± SEM. Statistical differences were evaluated using Dunnett’s multiple comparison test; ∗P < 0.05 was regarded as significantly different. Gastroenterology 2003 125, 1774-1784DOI: (10.1053/j.gastro.2003.09.018)

Figure 4 (A) Gastric histamine levels in wild-type and M3 KO mice. M3 KO mice had significantly increased gastric histamine levels compared with wild-type mice. Data are presented as means ± SEM for 6 mice. Statistical differences were evaluated using Student t test; P < 0.05. Ultrastructure of ECL cells in (B) wild-type and (C) M3 KO mice. Note that numerous secretory vesicles (∗) and vacuoles (∗∗) were observed in the cytoplasm of the ECL cell in C. Gastroenterology 2003 125, 1774-1784DOI: (10.1053/j.gastro.2003.09.018)

Figure 5 (A-F) Gastric fundic and (G-J) antral mucosal histology in (A, C, E, G, and I) wild-type and (B, D, F, H, and J) M3 KO mice. Overall appearance of (A and B) gastric mucosa, (C and D) parietal cells (anti-H+,K+-adenosine triphosphatase antibody positive), and (E and F) endocrine cells (ECL cells) (anti-chromogranin A antibody positive) were similar between M3 KO and wild-type mice. Although there was no visible change in the number of (I and J) D cells (anti-somatostatin antibody positive), the number of (G and H) G cells (anti-gastrin antibody positive) was significantly increased in M3 KO mice compared with wild-type mice. Bar = 100 μm. Gastroenterology 2003 125, 1774-1784DOI: (10.1053/j.gastro.2003.09.018)

Figure 6 Gross gastric appearance and histology of (A, C, E, G, I, and K) 1-, 12-, and 18-month-old wild-type and (B, D, F, H, J, and L) M3 KO mice. There was no visible difference in gross appearance, thickness, or fundic mucosal architecture between wild-type and M3 KO (A-D) 1-month-old male mice, (E-H) 12-month-old male mice, and (I-L) 18-month-old female mice. Bar = 1 cm in gross photographs and 100 μm in histologic reproductions. Gastroenterology 2003 125, 1774-1784DOI: (10.1053/j.gastro.2003.09.018)

Figure 7 Comparisons of cell proliferation in the oxyntic mucosa of wild-type, M3 KO, and omeprazole-treated hypergastrinemic wild-type mice. No difference in BrdU-positive cells was observed between (A) wild-type and (B) M3 KO mice. Although immunostaining for PCNA showed a remarkable increase in proliferating cells in (E) hypergastrinemic wild-type mice, no increase in PCNA-positive cells was observed in (D) M3 KO mice and (C) wild-type mice. Bar = 100 μm. Gastroenterology 2003 125, 1774-1784DOI: (10.1053/j.gastro.2003.09.018)

Figure 8 Graphs showing the mean number of (A) BrdU-positive and (B) PCNA-positive cells. Mean numbers of BrdU-positive cells and PCNA-positive cells are presented as cells per gland and percent of gland, respectively. WT, wild-type; KO, M3 KO; Ome WT, omeprazole-treated wild-type mice. Data are presented as means ± SEM (n = 5 mice). Statistical differences were evaluated using Student t test or Dunnett’s multiple comparison test, with comparisons made with wild-type mice. ∗P < 0.05. Gastroenterology 2003 125, 1774-1784DOI: (10.1053/j.gastro.2003.09.018)

Figure 9 (A) Reverse-transcription polymerase chain reaction study for EGF-receptor ligand messenger RNA expression. In wild-type mice, messenger RNA expression of HB-EGF (154 base pairs) and amphiregulin (144 base pairs) was enhanced by hypergastrinemia induced by long-term administration of omeprazole. On the other hand, there was no up-regulation of EGF (121 base pairs), transforming growth factor α (387 base pairs), and EGF receptor (146 base pairs) messenger RNA expression in hypergastrinemic wild-type mice. The ratio of each signal to Gapd (191 base pairs) was measured, and data are presented as percent of wild-type mice ± SEM for 3–5 mice. Statistical differences were evaluated using Dunnett’s multiple comparison test, with comparisons made with wild-type mice; ∗P < 0.05. (B) Western blot analysis showed that gastric HB-EGF expression differed for M3 KO mice and hypergastrinemic wild-type mice. WT, wild-type; KO, M3 KO; Ome WT, omeprazole-treated wild-type mice. Gastroenterology 2003 125, 1774-1784DOI: (10.1053/j.gastro.2003.09.018)