ANALYSIS OF ISOLATED ZYMOGEN GRANULES FROM RAT PANCREAS USING FLOW-CYTOMETRY

Rat pancreatic zymogen granules were analyzed using now cytometry to d etermine their heterogeneity with respect to different characteristics such as size (FSC), internal complexity (SSC) and membrane permeabili ty to lipophilic and cationic dyes using rhodamine-123 as probe. Diffe rences in the chemical composition of the membrane were determined usi ng FITC-labeled lectins (concanavalin A, Wheat germ agglutinin (WGA) a nd Tetragonolobus purpureus) displaying specific binding for different carbohydrates (D-mannose, N-acetyl d-glucosamine and L-fucose, respec tively). Finally, the amylase content of zymogen granules was also ana lyzed using an anti-amylase antiserum. Our results show the existence of two populations of zymogen granules that can be identified on the b asis of their FSC and SSC characteristics: a minor population of appro ximately 5% all zymogen granules with larger size (FSC) and internal c omplexity (SSC), and a major population clearly differentiable on the basis of a lower FSC and SSC. Rhodamine-123 uptake was similar in both subpopulations of zymogen granules. By contrast, labeling with fluore sceinated lectins and anti-amylase antiserum showed the existence of a higher content on both amylase and the monosaccharide residues analyz ed for the Z(2) zymogen granules. However, it is shown that those diff erences are strongly dependent on the FSC (size) of the granules sugge sting that although the carbohydrate contents (D-mannose, N-acetyl D-g lucosamine and L-fucose) and the amount of amylase differed from one g ranule to another, their concentration per granule was similar.