Rat pancreatic zymogen granules. An actively acidified compartment.

In this study, we looked for acidification in pancreatic zymogen granules as recently reported for other secretory vesicles. In intact dispersed acinar cells, acidic intracellular compartments identified by fluorescence microscopy using acridine orange corresponded exactly to the distribution of zymogen granules visualized by light microscopy. Acridine orange fluorescence in zymogen granules was reversibly dissipated by protonophores (carbonyl cyanide m-chlorophenylhydrazone, monensin) and NH4Cl; and the percentages of cytoplasmic area occupied by the acidic compartments and by zymogen granules were identical under fasting conditions and decreased in parallel after in vivo cholinergic stimulation. Zymogen granules released acutely from hypotonically disrupted cells without homogenization also accumulated acridine orange. Red-orange fluorescence in released granules was also abolished by protonophores and NH4Cl; and it reappeared after washout of protonophores in the presence, but not absence of adenosine triphosphate. Dicyclohexylcarbodiimide, which inhibits all proton pumps, and N-ethylmaleimide, which inhibits the proton pump of endocytic vesicles and lysosomes, but not mitochondria, prevented this adenosine triphosphate-dependent reappearance of acridine orange fluorescence, whereas vanadate did not. In contrast to these observations with zymogen granules in situ or acutely released from disrupted cells, granules isolated by conventional multistep homogenization/centrifugation procedures did not exhibit adenosine triphosphate-dependent acidification or development of a positive membrane potential as measured by quenching of acridine orange or Oxonol V, respectively. The latter findings may indicate release of inhibitors or granule damage during isolation. Collectively, the present results provide direct evidence that zymogen granules contain an active acidification mechanism which appears similar to that of other secretory vesicles and endosomes. This acidification process may have important implications for the storage, stabilization, and secretion of intragranular proteins including proenzymes.