Osmotic properties and internal pH of isolated rat parotid secretory granules

Secretory granules of the rat parotid have been purified rapidly by differential centrifugation in iso-osmotic sucrose. Despite low-level contamination by other organelles, this fraction is predominantly secretory granules, based on biochemical and morphological characterization. Measurements of intragranular water space show that isolated granules swell in hypo-osmotic media, and begin to release alpha-amylase and other content proteins below 250 mosM. Exposure of granules to selected anions also causes lysis, increasing in the order sulfate less than phosphate less than or equal to gluconate less than or equal to isethionate less than or equal to acetate less than or equal to chloride much less than thiocyanate. The internal pH of isolated parotid granules is approximately 6.8 as measured by the distributions of either [3H]acetate or [14C]methylamine. Varying the external medium pH from 5.0 to 8.0 results in little change in the measured internal pH. Parotid granules have a substantial internal buffering capacity, i.e. a decrease of 85-140 mM intragranular H+ is needed to raise the internal pH by 1.0 unit. Finally, results obtained using acridine orange staining suggest that the secretory granule internal pH in situ is similar to that measured in isolated granules.