The interaction between human low density lipoproteins and bovine aortic endothelial cells. Measurements of membrane fluidity.

Bovine aortic endothelial cells in culture have been incubated with human low density lipoproteins (LDL) characterized in their cholesterol content. The incubation was done at different time intervals up to 72 h and various LDL concentrations. It began after endothelial cells had been starved for 24 h in lipoprotein deficient serum. The transfer of some LDL-components to endothelial cells plasmalemma was monitored by measurements of membrane fluidity. Namely, the fluorescent probe trimethylamonio-diphenyl hexatriene was inserted in the cell membrane and fluorescence anisotropy was determined; a higher fluorescence anisotropy means a higher rigidity of the plasmalemma. The results show that the rigidity of the endothelial cell plasmalemma increased progressively with the time of incubation (+11% to +19.5% after 24 h and 72 h, respectively for the concentration of 200 micrograms. LDL-cholesterol/dish) and with the greater amount of cholesterol in LDL (+10.9%) for 200 micrograms LDL-cholesterol/dish to +15% for 800 micrograms LDL-cholesterol/dish after 24 h incubation). In order to see if the LDL material transfer proceeded by receptor-mediated endocytosis of LDL and/or directly through aqueous solution a lysosomal inhibitor, chloroquine, was used at the concentration of 20 microM for preventing the lysosomal hydrolase activity. In the presence of this inhibitor the fluorescence anisotropy in treated endothelial cells increased by a lesser amount, suggesting an approx. 30% participation of intracellular route. Therefore, the transfer of material (probably cholesterol) from LDL to endothelial plasmalemma could take place both by receptor-mediated endocytosis and directly through the aqueous solution.

Evidence that some membrane ligands modulate the plasmalemma fluidity of endothelial cells in culture.

The effect of some membrane ligands on the plasmalemmal fluidity of endothelial cells from bovine aorta in culture was investigated. The ligands used were: cationic ferritin (pI 8.5), soybean agglutinin, concanavalin A, wheat germ agglutinin, as well as glutaraldehyde at different concentrations. The fluidity probe employed was 1,6-diphenyl-1, 3, 5-hexatriene (DPH) and the parameter determined to quantify the fluidity was fluorescence steady-state anisotropy. The optimum time interval required by the insertion of the fluorescent probe in plasmalemma and the appropriate density of cells in the sample were determined. Rigidisation of plasmalemma was detected following its interaction with glutaraldehyde at concentrations ranging from 0.5% to 2% (+8% to +14% relative to the controls). After exposing endothelial cells to wheat germ agglutinin, concanavalin A and cationic ferritin pI 8.5, no modifications in the steady-state DPH fluorescence anisotropy were noticed. However, plasmalemmal rigidisation of +10% to +14% relative to the controls was obtained when endothelial cells were treated with 1 mg/ml and 2 mg/ml of soybean agglutinin, respectively. The possible mechanism of membrane fluidity modulation by membrane ligands and the usefulness of such investigations are discussed.