CHOLECYSTOKININ RELEASE FROM ISOLATED CANINE EPITHELIAL-CELLS IN SHORT-TERM CULTURE

Canine jejunal epithelial cells were isolated and maintained in short-term culture to study cholecystokinin (CCK) release. Sequential digestion of jejunal mucosa with collagenase and ethylenediaminetetraacetic acid was followed by counterflow elutriation to enrich CCK-containing cells. After 40 hours in culture on collagen-coated plates, 8.4% of the initially seeded cells were attached; 8.7% of them stained positive with a C-terminal CCK/gastrin antibody and 2.5% stained positive with a gastrin-specific antibody. Basal release of CCK into the culture medium amounted to 1.3% of total cell content over 105 minutes. Receptor-independent stimulation of protein kinase C by the phorbol ester β-phorbol-12-myristate-13-acetate caused significant CCK release. The inactive form, 4α-phorbol-12-myristate-13-acetate, had no effect. Activation of adenylate cyclase by 10-5 mol/L forskolin evoked a 2.5-fold increase in CCK concentrations, which was completely abolished by 10-8 mol/L somatostatin. l-phenylalanine stimulated CCK release at 20 and 50 mmol/L, whereas d-phenylalanine caused significant hormone output only at 50 mmol/L. l-tryptophan had no effect. Cholecystokinin release stimulated by l-phenylalanine was not influenced by the addition of either somatostatin or somatostatin antibody. In conclusion, a system of isolated canine jejunal epithelial cells was developed in short-term culture. This preparation proved suitable for the study of CCK release on a cellular basis. © 1992.