The effects of hypoxia and reoxygenation on the lateral mobility of membrane lipids were studied in primary cultures of rat proximal tubule epithelial cells at a subconfluent stage. The lipid lateral diffusion coefficient (D(L)) of the fluorescent probe 1-acyl-2-(N-4-nitrobenzo-2-oxa- 1,3-diazol)-aminocaproyl phosphatidylethanolamine was determined in membranes of attached cells by the technique of fluorescence redistribution after photobleaching, using confocal imaging to differentiate apical from basal surfaces. Fluidity was greater in basolateral membranes than in apical membranes [D(L) (apical) = 3.15 ± 0.8 and D(L) (basolateral) = 6.56 ± 1.2 x 10-10 cm2/s]. In apical membranes, 60 min of hypoxia plus 30 min of reoxygenation increased D(L) to 10.04 ± 2.1. When cells were pretreated with the antioxidants superoxide dismutase, catalase, or α-tocopherol, there was no clear-cut effect on the hypoxia-reoxygenation-induced increase in apical membrane D(L), although t-butyl hydroperoxide treatment of cells did increase D(L). Disruption of the cytoskeleton with cytochalasin D or exposure to Ca2+-free medium markedly increased D(L). However, when cells were pretreated with phallacidin to stabilize cytoskeletal actin microfilaments, the hypoxia-reoxygenation-induced change in lipid lateral mobility was completely prevented. Thus the cytoskeleton is an important regulator of the translational motion of membrane phospholipids. Hypoxia-reoxygenation may have increased D(L) because of cytoskeletal dysfunction.
- cytochalasin D
- membrane fluidity