ABSTRACT

Endothelium-derived hyperpolarizing factor (EDHF) is, at least in some blood vessels, assumed to be identical with 11,12-epoxyeicosatrienoic acid (11,12-EET), a product of endothelial CYP450 2C metabolism. It is investigated whether 11,12-EETs affect membrane potential not only of vascular smooth muscle cells but also of platelets. Platelet membrane potential was assessed by flow cytometry using the membrane potential sensitive fluorescent dye DiBac4(3). Calibration was performed using increasing extracellular K

concentrations in the presence of valinomycin. Resting membrane potential of washed human platelets was calculated to 58 9 mV. The Na/K-ionophor gramicidin induced a membrane depolarization of platelets, while the K-ionophor valinomycin decreased fluorescence indicating hyperpolarization. 11,12-EET also hyperpolarized platelets in a dose-dependent manner. This effect was abolished by charybdotoxin indicating involvement of calcium-gated K (KCa) channels. In contrast, valinomycin-induced hyperpolarization could not be prevented by charybdotoxin. Depolarization of platelets caused a strong release of superoxide, which was scavenged by superoxide dismutase. This amount of superoxide anions released could also be decreased by 11,12-EETs. In conclusion, 11,12-EETs that have been reported to be released from endothelial cells, hyperpolarize platelets, which inhibits the depolarization-induced platelet release of superoxide anions.