Butyrylcholinesterase distribution in the mouse gastrointestinal tract: An immunohistochemical study

Abstract

Butyrylcholinesterase (BChE) is a hydrolytic enzyme that together with acetylcholinesterase (AChE) belongs to the cholinesterase family. Whereas AChE has a well-established role in regulating cholinergic neurotransmission in central and peripheral synapses, the physiological role of BChE remains elusive. In this morphological immunohistochemical and double-label confocal microscopy study we investigated the distribution of BChE in the mouse gastrointestinal tract. BChE-positive cells were detected in the liver (both in hepatocytes and cholangiocytes), in the keratinised layers of the squamous epithelium of the oesophagus and forestomach, in the oxyntic mucosa of the stomach, in the mucus-secreting cells of duodenal Brunner glands and the small and large intestinal mucosa. Interestingly, BChE-positive cells were often detected close to gastrointestinal proliferative niches. In the oxyntic mucosa, the close proximity of ghrelin-producing and BChE-positive parietal cells suggests that BChE may be involved in ghrelin hydrolysation through paracrine action. To our knowledge, this is the first comprehensive morphological study performed to gain insight into the physiological role of BChE in the gastrointestinal tract.

Keywords: Ki67; Paneth cells; acetylcholine; acetylcholinesterase; ghrelin; non-neuronal cholinergic system; parietal cells.

FIGURE 1

BChE expression and distribution in the liver from mice fed a normal or a high‐fat diet. (a) Representative immunoblot and BChE quantification in liver protein extract from the Control and the HFD groups. Data from 3 mice are expressed as mean ± SEM (student's t‐test). (b) Representative immunoperoxidase image showing BChE immunoreactivity in a liver section from a Control mouse. Specific BChE staining is visible in hepatocytes, enlarged in (c), and in epithelial cells lining intrahepatic biliary ducts, enlarged in (d). Inset on the bottom right corner in (a) negative control without the primary antibody. Dark arrows indicate epithelial cells lining BChE‐positive intrahepatic biliary ducts (e) Representative immunoperoxidase image showing BChE immunoreactivity in a liver section from an HFD mouse. Inset on bottom right corner: negative control without the primary antibody. (f–h) Representative confocal images from liver sections from a Control mouse showing BChE⁺ cells (red) that also express keratin 17/19, a cholangiocyte marker (green). White arrows indicate bile ducts positive for both keratin 17/19 and BChE.

FIGURE 2

BChE distribution in mouse oesophagus. (a) Representative immunoperoxidase image showing BChE immunoreactivity in an oesophagus tissue section. Specific BChE staining is visible in the oesophageal epithelial layers, in the muscularis mucosa layer and the oesophageal lumen. Inset on the bottom left corner: negative control without the primary antibody. (b) Enlargement of the area framed in (a), showing BChE⁺ cells in the muscularis mucosa layer and scattered positive cells in the muscularis externa.

FIGURE 3

BChE distribution in the mouse stomach. (a–c) Representative immunoperoxidase images showing BChE immunoreactivity in the glandular stomach, specifically the squamocolumnar junction region (a), the corpus (b), and the pyloric region (c). Specific BChE staining is visible in the keratinised epithelial cells of the forestomach (a), in the short glands of the cardiac region (a), in the middle and lower portion of corpus glands (b) and in the pyloric region (c), where immunopositive cells decline markedly. (d–f) Enlargements of BChE⁺ cells located in the framed areas in a, b and c, respectively. (g–i) Negative controls without the primary antibody of the experiments shown in a, b and c.

FIGURE 4

BChE⁺ mouse gastric cells. (a–c) Representative confocal microscopy images of the glandular stomach in the corpus region showing BChE⁺ cells (red) that also express H⁺,K⁺‐ATPase, a widely used marker of mouse gastric parietal cells (green). (d–f) At higher magnification, nearly all BChE⁺ cells (red) are also H⁺,K⁺‐ATPase‐positive (green). (g–i) Representative confocal images from the glandular stomach in the corpus region showing BChE⁺ cells (red) that are negative for, but very close, to cells positive for GIF, a widely used marker of mouse gastric chief cells (green). (j–l) Representative confocal images of the glandular stomach in the corpus region showing BChE⁺ cells (red) that are negative for, but in close proximity to, cells positive for CgA, a widely used marker of mouse gastric neuroendocrine cells (green).

FIGURE 5

BChE distribution in the mouse small and large intestine. (a) Representative immunoperoxidase images showing BChE immunoreactivity in the duodenum. Specific BChE staining is visible along the villi, at the base of the crypts and in Brunner glands in the submucosal layer. Upper right corner in (a) negative control without the primary antibody. (b) Representative immunoperoxidase images showing BChE immunoreactivity in the ileum. Specific BChE staining is visible along the villi and at the base of the crypts. In (a) and (b), BChE staining is also visible near the tip of the villi in the intestinal lumen. Upper right corner in (b): negative control without the primary antibody. (c, d) Enlargements of the areas framed in (b) showing BChE⁺ cells in the intestinal crypts (c) and along the intestinal villi (d). (e) Representative immunoperoxidase images showing BChE immunoreactivity in the large intestine. Specific BChE staining is visible in the epithelial mucosal layer of the colon in clusters of goblet cells. Bottom left corner in (e): negative control without the primary antibody. (f) Enlargements of the area framed in (e) showing BChE⁺ cells resembling goblet mucous cells. (g–i) Representative confocal images of the intestinal crypts showing BChE⁺ cells (red) that also express lysozyme, a widely used marker of mouse Paneth cells (green). (j–l) Representative confocal images of the intestinal crypts showing BChE⁺ cells (red) that are negative for, but in close proximity to, cells positive for Olmf4, a widely used marker of mouse intestinal stem cells (green).

FIGURE 6

Close proximity of BChE⁺ cells to the proliferative niche in the mouse GI tract. (a–c) Representative confocal microscopy images of the liver showing BChE⁺ cells (red) that are negative for, but in close proximity to, cells positive for Ki67, a widely used marker of mouse proliferative cells (green). (d–f) Representative confocal images of the stomach showing BChE⁺ cells (red) negative for, but in close proximity to, Ki67⁺ cells. (g–i) Representative confocal images of the small intestine showing BChE⁺ cells (red) that are negative for, but in close proximity to, Ki67⁺ cells. Upper right corner of (a–i) enlargement of the areas framed in each panel.