Effects of Actinomyces Amphiphile on the Fluidity of Endothelial Cells: A Spin Label Study

Abstract

Actinomyces amphiphile (AcA) is an amphipathic molecule produced by Actinomyces viscosus that exhibits several biological activities. The effect of AcA on the fluidity and permeability of the plasma membrane in human umbilical vein endothelial cells was analyzed by a spin label method with 5- and 16-stearic acid nitroxide labels (SAL). These labels help to visualize the fluidity at the shallow (5-SAL) and deep (16-SAL) portions of the lipid bilayer. Cells were incubated with and without AcA (control) at 37°C for 6 hours, and membrane fluidity was periodically measured. Another spin label, 4-(N, N-dimethyl-N-hexadecyl) ammonium-2, 2, 6, 6-tetramethyl-piperidine-l-oxyliodine (CAT-16), was also used to assess the physical state of the cell surface. The order parameter of 5-SAL was significantly lower in the cells incubated with AcA than in control cells after the six-hour incubation. The motion parameter of 16-SAL was significantly lower in AcA-treated cells than in controls after 4 and 6 hours of incubation. These findings indicated that the AcA increased the fluidity. There were no significant differences between the AcA-treated and control cells incubated for only 2 hours. In addition, there were no differences in CAT-16 measurements between AcA-treated and control cells. The release of endoplasmic lactate dehydrogenase (LDH) into the medium tended to increase in the AcA-treated vs. the control cells. LDH release increased in both a dose- and time-dependent manner, indicating that AcA increased the permeability of plasma membranes. These findings suggest that AcA alters the biophysical properties of the plasma membranes of endothelial cells, affecting membrane function.