Characterization of secretin release in secretin cell-enriched preparation isolated from canine duodenal mucosa.

The release of secretin was studied in secretin cell-enriched preparations isolated from canine duodenal mucosa. The crude enterocytes were isolated by treating the duodenal mucosa sequentially with collagenase and ethylenediaminetetraacetic acid. Secretin cell-enriched fraction was prepared by centrifugation of the crude enterocytes in a counterflow elutriation rotor to obtain a final preparation containing 3.2 +/- 0.3 pmol/10(6) cell of immunoreactive secretin, which was 13-fold greater than the crude cell preparation (N = 5). The cells were incubated in Hanks' balanced salt solution for 20 min at 37 degrees C under 95% O2/5% CO2 before adding various agents and further incubated for various periods of time. The amounts of secretin released into the medium and retained by the cells were then determined by a specific radioimmunoassay. The release of immunoreactive secretin was increased dose-dependently over the control by dibutyryl cyclic-3',5'-adenosine monophosphate, forskolin, 4 beta-12-O-tetradecanoylphorbol-13-acetate, the synthetic serine protease inhibitor, camostat, and the calcium ionophore, A23187. The effects of forskolin, the phorbol ester, and A23187 were time-dependent and not observed at 4 degrees C. The release of immunoreactive secretin was also stimulated by KCl in high concentration and by sodium oleate. The effect of A23187 was abolished in a Ca(2+)-free medium, while those of dibutyryl cyclic-3',5'-adenosine monophosphate and forskolin were potentiated by 3-isobutyl-1-methylxanthine, which did not have a significant effect when added alone. These results indicate that the release of secretin is regulated by both Ca(2+)- and cyclic-3',5'-adenosine monophosphate-dependent mechanisms.2+ release.