Calcium signaling in cultured human and rat duodenal enterocytes

Vagal stimuli increase duodenal mucosal HCO3- secretion and may provide anticipatory protection against acid injury, but duodenal enterocyte (duodenocyte) responses and cholinoceptor selectivity have not been defined. We therefore developed a stable primary culture model of duodenocytes from rats and humans. Brief digestion of scraped rat duodenal mucosa or human biopsies with collagenase/dispase yielded cells that attached to the extracellular matrix Matrigel within a few hours of plating. Columnar cells with villus enterocyte morphology that exhibited spontaneous active movement were evident between 1 and 3 days of culture. Rat duadenocytes loaded with fura 2 responded to carbachol with a transient increase in intracellular calcium concentration ([Ca2+](i)), with an apparent EC50 of similar to 3 mu M. In a first type of signaling pattern, [Ca2+](i) returned to basal or near basal values within 3-5 min. In a second type, observed in cells with enlarged vacuoles characteristic of crypt cell morphology, the initial transient increase was followed by rhythmic oscillations. Human duodenocytes responded with a more sustained increase in [Ca2+](i), and oscillations were not observed. Rat as well as human duodenocytes also responded to CCK-octapeptide but not to vasoactive intestinal polypeptide. Equimolar concentrations (100 nM) of the subtype-independent muscarinic antagonist atropine and the M-3 antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide prevented the response to 10 mu M carbachol, whereas the M-1 antagonist pirenzepine and the M-2 antagonists methoctramine and AF-DX 116BS had no effect at similar concentrations. Responses in rat and human duodenocytes were similar. A new agonist-sensitive primary culture model for rat and human duodenocytes has thus been established and the presence of enterocyte CCK and muscarinic M-3 receptors demonstrated.