DISOBUTAMIDE-INDUCED CYTOPLASMIC VACUOLES IN CULTURED DOG CORONARY-ARTERY MUSCLE-CELLS

Smooth muscle cells released by protease from the coronary artery of dogs were cultured in M199 supplemented with 10% fetal calf serum. Cells were grown on glass coverslips to semiconfluence and exposed to disobutamide at 0, 1, 2, 3, 6, 8 and 10 .times. 10-4 M for 24, 48 and 72 h, examined in situ by light microscopy, then fixed in 100% methanol, stained with May-Grunwald Giemsa and examined by light microscopy. Cells were also exposed to the drug for 24 h, pelleted, fixed and prepared routinely for EM. Control cells and cells exposed to 6 .times. 10-4 M disobutamide were examined. Vacuole formation was dose and time dependent between 2 .times. 10-4 and 10-3 M. Light microscopy showed that morphologic alterations induced by the drug were clear cytoplasmic vacuoles in live cells, vacuoles in dead cells and dead cells without vacuoles. Small round vacuoles were an early change. Dark granules were dispersed among the vacuoles. The vacuoles increased in size at higher doses or longer times. Eventually all the cytoplasm was occupied by vacuoles and the cells were enlarged. EM showed that the vacuoles were round, primarily membrane-bound, contained mostly electron-lucent material and occasionally small flocculent bodies. There was vacuolar coalescence. The dose response of vacuole induction in confluent and semiconfluent cells was similar. Cytoplasmic vacuoles without cell death were induced by disobutamide at 2 or 4 .times. 10-4 M during a 3 day exposure. Cell culture was a suitable biological system for studying cytoplasmic vacuoles of the type induced by disobutamide.