STUDIES ON DISPERSED PANCREATIC EXOCRINE CELLS II . Functional Characteristics of Separated Cells

The functional characteristics of separated guinea pig pancreatic exocrine cells have been examined following dissociation of the gland by a procedure described in the previous paper (J. Cell Biol. 1974. 63:1037). Th e ability of isolated cells to incorporate labeled amino acids into secretory proteins was assessed biochemically and by quantitative electron microscope autoradiography. Incorporation remained linear for up to 4-h incubation at levels equivalent to those of pancreatic slices; over 95% of the exocrine cells in the population were viable, and all appeared to be equally active in incorporating amino acids. The capacity of separated Cells to transport, concentrate, and store exportable proteins was monitored by electron microscope autoradiography on populations pulse labeled with [aH]leucine and chase incubated for 4 h. The same overall pathway previously mapped in pancreatic slices was followed by secretory proteins in separated cells alttaough in quantitative studies a defect was noted in the rate of conversion of condensing vacuoles to zymogen granules. Secretogogue responsiveness was assessed by monitoring discharge of labeled secretory proteins or of amylase in response to carbamylcholine and caerulein to the medium. While the separated cells released secretory proteins linearly for up to 4 h in response to both secretogogues, the net release was ~50% less than previously noted for pancreatic slices and required a ten times higher concentration of stimulant. The defect may represent alteration in receptors due to the protease used for dissociation. Our data indicate, however, that separated exocrine cells retain their ability to process secretory proteins stepwise and vectorially which is consistent with preservation of structural polarity. In the preceding paper we have described a procedure for dissociation of the guinea pig pancreas into a population consisting primarily of isolated exocrine cells (1). The method includes digestion with an enzyme mixture containing pure collagenase, chymotrypsin, and hyaluronidase, an interposed chelation of divalent cations by EDTA, and repeated pipeting. It is simple to perform, reproducible, and results in cell yields of from 50 to 60% based on DNA content of the final preparation. Fine structural studies indicated that these isolated exocrine cells retain the main characteristics of their counterparts in situ both with regard to polarized distribution of cell organelles and to regional differentiation of the bounding plasmalemma. In addition to the use of morphologic criteria, THE JOURNAL OF CELL BIOLOGY . VOLUME 63, 1974 • pages 1 0 5 7 1 0 7 3 1057 we have also used functional parameters to assess the adequacy of the dissociation procedure. In this paper we report on the ability of isolated exocrine cells to synthesize, transport, and concentrate exportable proteins into zymogen granules and to release the stored products in response to secretogogues. These studies were conducted under conditions simulating those previously used to assess exocrine cell function in pancreatic slices (2 5). In this way the functional characteristics of the isolated cells can be related to data previously obtained on cells in situ. M A T E R I A L S A N D M E T H O D S