CHOLESTEROL DISTRIBUTION IN RENAL EPITHELIAL-CELLS LLC-PK1 AS DETERMINED BY CHOLESTEROL OXIDASE - EVIDENCE THAT GLUTARALDEHYDE FIXATION MASKS PLASMA-MEMBRANE CHOLESTEROL POOLS

Treatment with cholesterol oxidases has shown that cholesterol is hete rogeneously distributed in brush border membranes isolated from the ap ical domain of the renal and intestinal epithelial cells [Bloj, B., an d Zilversmit, D. B. (1982) J. Biol. Chem. 257, 7608-7614; El Yandouzi, E. H., and Le Grimellec, C. (1992) Biochemistry 31, 547-551]. Cholest erol distribution between plasma membrane and intracellular membranes of the corresponding cells remains unexplored. The effects of Brevibac terium sp. cholesterol oxidase on the cholesterol content of LLC-PK1 c ells, an epithelial cell line with multiple differentiated characteris tics of the renal proximal tubule, were investigated. In confluent liv ing cells grown as a monolayer on solid support, a small but significa nt fraction (13%) of the cholesterol was oxidized during the first hou r of the oxidase treatment. Glutaraldehyde fixation prior to treatment resulted in a nearly complete (86.1 +/- 1.8) oxidation of the cellula r cholesterol according to first-order kinetics. Filipin labeling and oxidation at 15 degrees C confirmed that cholesterol was essentially c onfined to the plasma membrane in LLC-PK1 cells. When adding the oxida se either on the apical or on the basolateral side of cells grown on p ermeant support and fixed with glutaraldehyde, a comparable monophasic oxidation of cholesterol was observed, despite the presence of effici ent tight junctions. Adding the oxidase to both sides simultaneously d id not increase the rate of oxidation. Finally, fixation of isolated r enal brush border membranes with glutaraldehyde rendered undiscernible their cholesterol pools. We conclude that glutaraldehyde fixation, a commonly used process in the analysis of cholesterol distribution in c ells, can mask the existence of cholesterol pools in plasma membranes.