Control of Instant Membrane Resealing by Lysosomal Acid Ceramidase in Endothelial Cells

Abstract

Previous studies have shown that lysosome exocytosis and ceramide platform formation in the outer leaflet of the lipid bilayer are essential for plasma membrane repair. As a lysosomal enzyme catalyzing ceramide hydrolysis, the role of acid ceramidase (AC) in the control of cell membrane repair remains unknown. In the present study, we tested a hypothesis that lysosomal AC‐mediating signaling plays a crucial role in instant membrane resealing of endothelial cells (ECs), which help maintain the integrity of EC membrane and related functional activity. By Western blot analysis, it was found that carmofur, an AC inhibitor, decreased the AC expression in murine ECs. On the contrary, Genistein, an AC activator, elevated the level of AC in these cells. Using wavelength‐switching fluorescent microscopy with propidium iodide (PI), a fluorescent membrane‐impermeant dye as membrane resealing marker and fura‐2 as intracellular Ca2+ indicator, we monitored instant membrane resealing following saponin‐induced membrane disruption simultaneously with Ca2+ influx recording in murine ECs. The speed of PI fluorescence increase (df/dt) within ECs and time to a peak of PI fluorescence elevation were used to reflect the instant membrane resealing. Under Ca2+‐free condition, we found that membrane resealing in ECs was enhanced by carmofur as shown by df/dt reduction, but inhibited by genistein (increase in df/dt). In Hanks’ buffer containing Ca2+, however, carmofur and genistein failed to alter membrane resealing of murine ECs when Ca2+ influx was increased. In isolated coronary ECs from WT/WT and Asah1fl/fl/Endocre mice, we demonstrated that AC gene knockout specifically in ECs remarkably enhanced membrane resealing following saponin‐induced membrane interruption, which also mainly occurred under Ca2+‐free condition. These results suggest that lysosomal AC expression and activity are critical for the regulation of instant membrane resealing in ECs after acute injury, which represents a Ca2+‐independent membrane repairing mechanism.Support or Funding InformationThis study was supported by NIH grants DK54927 and DK102539.