Effects of caffeic acid phenethyl ester (CAPE) on membrane potential and intracellular calcium in human endothelial cells.

BACKGROUNG AND OBJECTIVES: Caffeic acid phenethyl ester (CAPE) is an active component of the resin propolis obtained from beehives. Propolis has a long history of medicinal use and a number of studies have already reported on some of its pharmaceutical properties. This study aimed to explore the effects of CAPE on the cytosolic Ca2+ concentration, cell proliferation, membrane potential and NO levels in human endothelial cells. Isolated human umbilical vein endothelial cells (HUVEC) were incubated with CAPE (1-100 µM) at 37°C for 48 hours. Cell proliferation was estimated by counting cell numbers with use of a Neubauer chamber. The effect of CAPE (1-100 µM) on the membrane potential was measured with the fluorescence dye DIBAC4(3) whereas its effect on the cytosolic Ca2+ concentration was measured by use of the fluorescence probe Fluo-3 AM (Invitrogen, Leiden, Netherlands). NO production was assayed using the flourophore DAF~AM (Invitrogen, Leiden, Netherlands). Changes in fluorescence intensity was determined with the GENios plate reader (Genios, Tecan, Austria). A dose-dependent hyperpolarization of the endothelial cell membrane was observed with CAPE stimulation. The initial increase in the intracellular Ca2+ concentration showed a subsequent decrease over time. CAPE stimulation also resulted in an increase in NO production; however, at higher doses a decrease in NO levels was observed. HUVEC proliferation was inhibited by CAPE. Here we report on the effect of CAPE stimulation on the cytosolic Ca2+ concentration, cell proliferation, membrane potential and NO production in HUVEC in a dose-dependent manner. These findings provide important insights into some potential key roles that both calcium and the membrane potential play in the CAPE activation of endothelial cells in a concentration-dependent manner.