Introduction
The bicarbonate secretion originating from the duodenal mucosa is important in protection against acid discharged from the stomach and duodenal enterocytes (duodenocytes) provide an interesting model of HCO3- secreting epithelia. Duodenocytes export HCO3- by Cl-/ HCO3- exchange and via an apical anion channel, recently indicated to be the CFTR-channel, but duodenocyte intracellular and duodenal mucosal intercellular signaling have not been clarified.
Methods
Duodenocyte intracellular and mucosal intercellular (cell-to-cell) signaling were studied in acutely isolated duodenal enterocytes. Scraped rat duodenal mucosa was cut into approximately 1 mm in diameter pieces followed by trituration and mild digestion (collagenase/dispase) to yield aggregates (10-200 cells) of duodenocytes. These aggregates were loaded with fura-2, mounted in a perfusion chamber and duodenocyte cytoplasmic Ca2+ concentration ([Ca2+]i) measured with dual wavelength fluororescence imaging (InCytlmc2 software). Presence of HCO 3- (* 1 mM) in the preparation and bathing solutions increased the viability of the duodenocytes, possibly reflecting an important role of HCO3-uptake (NaHCO3 cotransport) in maintaining intracellular pH in these cells.
Results
Superfusion with cholecystokinin octapeptide (CCK-8, 1-50 nM), carbachol (10-100 yM) or galanin (100 nM) induced a transient rise in [Ca2+]i. One or two cells in an inter connecting aggregate were consistently the first to respond to CCK-8 (or carbachol) with a rise in [Ca2+]i and this was followed by spread of the [Ca2+]i signal to most cells within the aggregate. Duodenocytes with spontaneous oscillations in [Ca2+]i were observed in some aggregates and this signal , like the secretagogue-induced signal, spread to the other cells. Responses to CCK-8 (1-10 nM), but not those to carbachol, were prevented by CCKA-receptor antagonists, suggesting an action at this receptor type. The peptides orexin A (1-100 nM) and gastrin releasing peptide (100 nM) caused a small and slowly developing rise in [Ca2+]i but, interestingly, pretreatment with these peptides markedly potentiated the [Ca2+]i responses to carbachol and CCK-8, making them more sustained and larger.
Conclusions
Aggregates of interconnecting duodenocytes respond to secretagogues increasing [Ca2+]i, as a syncytium and orexin-induced variation of enterocyte [Ca2+]i may modulate the intestinal secretory response to secretagogues. These phenomena may be important in the neurohumoral control and overall function of the small intestinal mucosa and might provide an approach in treatment of and duodenal ulcer disease and diarrhea.
References