Calcium signaling in cultured human and rat duodenal enterocytes.
Vagal stimuli increase duodenal mucosal HCO-3 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 duodenocytes loaded with fura 2 responded to carbachol with a transient increase in intracellular calcium concentration ([Ca2+]i), with an apparent EC50 of approximately 3 microM. 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 M3 antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide prevented the response to 10 microM carbachol, whereas the M1 antagonist pirenzepine and the M2 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 M3 receptors demonstrated.
Department of Physiology, Uppsala University, SE-751 23 Uppsala, Sweden.
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Flemström
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology*; D002448:Cell Adhesion; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004386:Duodenum; D005109:Extracellular Matrix / Q000502:physiology; D005456:Fluorescent Dyes; D016257:Fura-2; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D008297:Male; D010276:Parasympatholytics / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D051381:Rats; D011917:Rats, Inbred Lew; D015398:Signal Transduction; D012844:Sincalide / Q000494:pharmacology; D014617:Vacuoles / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure
D005456:Fluorescent Dyes; D010276:Parasympatholytics; D010880:Piperidines; C042375:4-diphenylacetoxy-1,1-dimethylpiperidinium; D002217:Carbachol; D012844:Sincalide; D002118:Calcium; D016257:Fura-2