Analysis of membrane structure in the transitional epithelium of rat urinary bladder. 3. Localization of cholesterol using filipin and digitonin

Abstract

Filipin (a polyene antibiotic) and digitonin (a saponin) interact with cholesterol in membranes, producing characteristic lesions detectable by electron microscopy. Using these agents in conjunction with the techniques of freeze-fracture, negative staining, and thin-sectioning, we have investigated the distribution of cholesterol in the membrane systems of superficial urothelial cells, with particular reference to the luminal plasma membrane. This membrane is structurally differentiated into plaques (composed of arrays of subunits) and interplaque regions, which possess markedly different physical properties. Plaques were sensitive to digitonin but not to filipin whereas interplaque regions responded to both agents. These results are interpreted with reference to the factors that influence lesion formation, and to data on the structure and composition of the luminal membrane. It is concluded that both interplaque regions and plaques are cholesterol rich, and the significance of this in relation to the physical properties of the luminal membrane is discussed. Intra- and intermembrane variations in response of the other membrane systems of the cell, and the nature of the lesions induced by the agents are examined. Technical aspects of the application of filipin and digitonin to the localization of membrane cholesterol are also considered. The results obtained with bladder luminal membrane plaques emphasise that caution should be exercised in the interpretation of a negative response to filipin, particularly in membrane domains containing closely packed protein subunits.