Investigations in intracellular drug storage: localization of disobutamide in lysosomal and nonlysosomal vesicles.

In the investigation of the dynamic nature of intracellular drug-induced storage disorder associated with clear cytoplasmic vacuoles (CCV), rat urinary bladder carcinoma cells (RBT CC-8) were cultured with [14C]disobutamide. Cell monolayers were then harvested and analytical cell fractionation techniques were employed to examine the association of disobutamide with the various subcellular fractions. Disobutamide distributed into two modes: as a free, organelle-independent fraction and as a light membrane-associated fraction that overlapped with markers for the plasma membrane, endoplasmic reticulum, and lysosomes. The similarity in buoyant densities of these organelles derived from RBT CC-8 cells precluded resolution of these structures by isopycnic centrifugation. In additional experiments, disobutamide was incubated in vitro with a suspension of isolated rabbit renal proximal tubules. In these cells, analytic fractionation showed that the drug localized predominantly to lysosomes and to a separate light membrane fraction that was clearly resolved from the markers for the endoplasmic reticulum, brush border, mitochondria, and peroxisomes; this fraction overlapped with the most buoyant aspects of the Golgi apparatus and basolateral plasma membrane. The buoyant density of this disobutamide-associated nonlysosomal fraction was 1.11 g/ml. Electron microscopy of the disobutamide-exposed tubules showed a substantial formation of apical vesicles, especially small, smooth-surfaced vesicles, typical of the endocytic apparatus. From these findings and based on the physicochemical properties of the cationic moiety of disobutamide, we conclude that the drug localizes in lysosomal and nonlysosomal acidic vesicles.