Figure 3.

Lipid raft profiles of blood–brain barrier endothelial cells. a, Characterization of BBB-EC sucrose density fractions with regards to cholesterol concentration (filled triangle; left axis; in μg/ml), phospholipid content (filled diamond; right axis; in values of absorbance), and protein concentration (open square; far right axis; in mg/ml). Lipid rafts are concentrated in fractions 4 and 5 (rectangular outline) where peak values of cholesterol and phospholipids are seen. Fractions 4 and 5 also show a high concentration of lipid raft markers GM1 ganglioside and CD59 but no evidence of transferrin receptor (TfR), a marker of nonlipid raft membrane. b, Western blots for the TJ protein JAM-1 in sucrose density fractions show a similar concentration of JAM-1 in lipid raft fractions under all conditions tested (untreated, 20%; ACM, 23%; AngII, 21% of total occludin). The TJ molecule occludin is weakly expressed in lipid rafts when cells are grown under basal conditions (untreated) but is enriched in fractions 4 and 5 when BBB-ECs are grown in the presence of ACM or AngII (10 nm) (untreated, 12%; ACM, 23%; AngII, 28% of total occludin). c, When BBB-ECs grown in the presence of ACM are treated for 1 h before the isolation of lipid rafts with the raft disrupting agent MβCD, lipid rafts are dissolved as evidenced by the sharp decrease in cholesterol concentration in fractions 4 and 5 as well as the absence of occludin. Similar data were obtained with filipin and nystatin (data not shown). d, Permeability of BBB-EC monolayers grown in ACM, 72 h after a 1 h treatment with the raft disrupting drug MβCD (17.79 ± 5.31%), filipin (20.05 ± 5.2%), or nystatin (17.03 ± 4.9%) is strongly increased when compared with cells grown in ACM alone (−11.99 ± 2.91%). Percentage values are expressed compared with permeability of cells grown under basal conditions (*p < 0.05; n = 3, in duplicate). e, Immunoprecipitation experiments using antibodies specific to phosphorylated forms of threonine (P-Thr) or tyrosine (P-Tyr): whole-cell lysates of BBB-ECs grown in the presence of ACM or AngII show an upregulation of P-Thr occludin (169 and 228% of untreated, respectively) and a downregulation of P-Tyr occludin (37 and 11% of untreated, respectively) compared with cells grown under basal conditions. f, When raft (R, fractions 4 and 5) and soluble (S, fractions 11 and 12) fractions are immunoprecipitated separately, only the raft fractions display a decrease in P-Tyr occludin (6 and 7% of occludin, respectively), whereas P-Thr occludin upregulation (254 and 351% of untreated, respectively) is evident in both raft and soluble fractions, particularly after AngII treatment.