PEPSINOGEN SYNTHESIS DURING LONG-TERM CULTURE OF PORCINE CHIEF CELLS

The purpose of this study was to characterize time-dependent changes i n pepsinogen (PG) synthesis of porcine gastric chief cells during long -term monolayer culture. Porcine chief cells were isolated by pronase/ collagenase treatment of fundic mucosa and enriched by density gradien t and counterflow centrifugation. PG isoenzymes were identified in [L- S-35]methionine-labelled cultured chief cells by native polyacrylamide gel electrophoresis followed by phosphor imager analysis, protease de tection and immunoblots with specific PG A and C antibodies. The obtai ned results suggest that porcine chief cell cultures, after an initial settling period, reached an approximate steady state in total protein content and synthesis as well as in PG content and isoenzyme pattern from days 3 to 9 of culture. The latter was characterized by the prese nce of at least two PG A and two PG C isoenzymes. During the supposed steady-state total PG synthesis averaged out at 34 +/- 2% of total pro tein synthesis, as detected by [L-S-35]methionine incorporation, due t o the synthesis of, mainly, PG A2 and, to a much lesser extent, PG C a nd Al. In line with an active secretion, PG A2 proportion was on avera ge significantly higher in released (44 +/- 3%) than in intracellular labelled proteins (19 +/- 2%). In addition, PG release from chief cell s cultured for 6 and 9 days could be stimulated by cholecystokinin-oct apeptide. These data suggest that porcine chief cells in monolayer cul ture are a model well suited for the quantitative and qualitative char acterization of PG isoenzyme synthesis and release during long-term in vestigations, for which an establishment of a culture steady state app ears to be a useful prerequisite. (C) 1997 Elsevier Science B.V.