Isolated canine ileal mucosal cells in short-term culture: a model for study of neurotensin release.

Abstract

We have developed a new technique for maintaining isolated enteric mucosal cells containing neurotensinlike immunoreactivity (NTLI) in short-term culture to determine the regulation of NTLI release. Collagenase-dispersed ileal mucosal cells, separated by centrifugal elutriation, were enriched for NTLI-containing cells as determined by immunohistochemistry and radioimmunoassay. Bombesin (BBS) rapidly stimulated NTLI release from freshly isolated cells. However, studies with freshly isolated cells were limited by high, unstable basal release measurements and rapid degradation of added [3H]neurotensin-(1-13). A culture system of elutriator-enriched NTLI cells was therefore developed. After 48 h NTLI cells selectively adhered to the collagen substrate and constituted 40% of the viable cells in culture. The morphology of NTLI cells in culture closely resembled neurotensin cells in intestinal tissue sections, with a secretory granule diameter of 292 +/- 14 nm. Bombesin stimulated a dose-dependent increase in NTLI secretion over 120 min. In these cell cultures, degradation of added [3H]-neurotensin-(1-13) was minimal over 120 min. High-pressure liquid chromatographic analysis of culture supernatants characterized neurotensin-(1-13) as the primary molecular form of neurotensin released in response to BBS stimulation. In conclusion, we have established a primary culture of enteric neurotensin cells that provides a model for studying the regulation of peptide release. Bombesin stimulation of NTLI release verified the functional responsiveness of this isolated cell system.