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1 Department of Physiology, Uppsala University, SE-751 23 Uppsala, Sweden; and 2 Institute for Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 31902
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 ~3
µM. 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 µM 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.
cholecystokinin; calcium oscillations; muscarinic M3 receptors; primary culture of duodenal enterocytes
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