Pepsinogen synthesis during long-term culture of porcine chief cells

Abstract

The purpose of this study was to characterize time-dependent changes in 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 gradient and counterflow centrifugation. PG isoenzymes were identified in [L-35S]methionine-labelled cultured chief cells by native polyacrylamide gel electrophoresis followed by phosphor imager analysis, protease detection and immunoblots with specific PG A and C antibodies. The obtained 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 presence 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 protein synthesis, as detected by [L-35S]methionine incorporation, due to the synthesis of, mainly, PG A2 and, to a much lesser extent, PG C and A1. In line with an active secretion, PG A2 proportion was on average significantly higher in released (44 +/- 3%) than in intracellular labelled proteins (19 +/- 2%). In addition, PG release from chief cells cultured for 6 and 9 days could be stimulated by cholecystokinin-octapeptide. These data suggest that porcine chief cells in monolayer culture are a model well suited for the quantitative and qualitative characterization of PG isoenzyme synthesis and release during long-term investigations, for which an establishment of a culture steady state appears to be a useful prerequisite.

Hepatocyte growth factor regulates the development of highly pure cultured chief cells from rat stomach by stimulating chief cell proliferation in vitro

“…For this, cells isolated by collagenase digestion were purified by centrifugal elutriation to yield cultures with a high transepithelial resistance (TER) and low permeability that maintained differentiated function as shown by agonist-induced pepsinogen secretion (2,26). Since the procedure was developed, short-term cultures have been produced from isolated human and rabbit (10), guinea pig (25), pig (12), and rat (20,30,31) chief cells. Although isolated chief cells and short-term cultured cells from all species secrete pepsinogen (10,12,25,31), it is not currently possible to produce a confluent monolayer of chief cells with a high TER, low permeability, and high rate of agonist-induced pepsinogen secretion from a species other than dog.…”

“…Since the procedure was developed, short-term cultures have been produced from isolated human and rabbit (10), guinea pig (25), pig (12), and rat (20,30,31) chief cells. Although isolated chief cells and short-term cultured cells from all species secrete pepsinogen (10,12,25,31), it is not currently possible to produce a confluent monolayer of chief cells with a high TER, low permeability, and high rate of agonist-induced pepsinogen secretion from a species other than dog. Because probes are now available to facilitate studies concerned with gastric physiology and pathophysiology in rodent species, development of cultured chief cells from the rat or mouse stomach would be timely and important.…”