Calcium signaling in cultured human and rat duodenal enterocytes

Abstract

Vagal stimuli increase duodenal mucosal[Formula: see text] 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+]ireturned 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.

Melatonin-induced calcium signaling in clusters of human and rat duodenal enterocytes

“…The segment was opened along the antemesenterial axis and the luminal surface was rinsed with a normal respiratory medium (NRM) containing (in mM) 114. 4 (8). The pH was adjusted to 7.40 immediately before use, and temperature was maintained at 37°C.…”

Short fasting dramatically decreases rat duodenal secretory responsiveness to orexin A but not to VIP or melatonin

“…If secretion stimulated by orexins (in fed animals) is controlled via cholinergic mechanisms similar to those for contraction, reduction in cholinergic responsiveness by fasting could induce a secondary attenuation of responses to orexins. Muscarinic stimulation (carbachol) has been shown to directly activate intracellular Ca 2ϩ mobilization in cultured (8) and actutely isolated (43) duodenal enterocytes.…”

“…One explanation for the difference seen here could be different signal pathways used for stimulation of HCO 3 Ϫ transport. Orexins and muscarinic M 3 agonists couple to intracellular Ca 2ϩ mobilization and phospholipase C activation (8,24). VIP is known to stimulate the secretion via cAMPdependent mechanisms (35,36), whereas the signal pathway used by melatonin may be different.…”

“…VIP is a potent stimulant of intestinal secretion, and infusion of VIP increases the HCO 3 Ϫ transport by the duodenal mucosa in all species tested (10,13,47). The peptide does not affect [Ca 2ϩ ] i signaling in human enterocytes in primary culture (8) but causes a marked increase cAMP production in rat and guinea pig isolated duodenal crypt and villus enterocytes (35,36).…”

“…Stimuli mediated by the vagal nerves or parenteral administration of cholinergic agonists, including the predominantly muscarinergic agonist bethanechol, increase the duodenal HCO 3 Ϫ secretion in all species tested, including humans (1,10,15,37). Use of subtype-selective ligands demonstrated that carbachol-induced [Ca 2ϩ ] i signaling in enterocytes isolated from human and rat duodenum is mediated by muscarinic M 3 receptors (8). VIP is a potent stimulant of intestinal secretion, and infusion of VIP increases the HCO 3 Ϫ transport by the duodenal mucosa in all species tested (10,13,47).…”