Reversibility of disobutamide-induced clear cytoplasmic vacoules in cultured cells following withdrawal of drug

When cultured rat urinary bladder carcinoma cells are incubated with medium containing disobutamide (D), they take up D intracellularly and form clear cytoplasmic vacoules (CCV) which apparently are distended lysosomes containing D. To determine whether CCV are reversible, the authors incubated cells with 10/sup -3/M /sup 14/C-labelled D for 25 hr and cells became full of CCV. Then, cells were incubated in medium free of D and examined at 0,2,4,6,24,48,72 and 144 hr of D withdrawal for radioactivity (600 DPM = 1 ..mu..D), and in situ by phase light microscopy for presence of CCV. The DPM counts at these time points were: 1832, 1539, 1564, 1443, 1073, 738, 481 and 84, respectively. This gradual decrease in cellular content of D parallelled a diminution in size and number of CCV. By 72 hr (481 DPM = 0.93 up D) only few small CCB had remained, and by 144 hr (84 DPM = 0.16 ..mu..g D) CCV had entirely disappeared. Mass spectrometric analysis of the culture medium at 144 hr showed presence of D. These results indicate that (1) the induced CCV are reversible (2) reversibility is associated is associated with release of D from cells (3) the principle compound released frommore » cells is D and not its metabolite(s). The results support the interpretation that CCV are formed as a result of intracellular storage of D.« less