BACKGROUND: The dietary flavanol (-)-epicatechin has been suggested to mediate its vasodilatory effect by increasing nitric oxide levels in endothelial cells. AIM OF THE STUDY: To directly prove the formation of nitric oxide (NO) in human endothelial cells (HUVEC) in vitro by trapping NO to yield a fluorescent nitrosamine. METHODS: HUVEC were treated with (-)-epicatechin; nitrite and NO formation were determined by reductive chemiluminescence detection and the NO-sensitive fluorophore 5-methoxy-2-(1H-naphthol[2,3-d]imidazol-2-yl)-phenol copper complex (MNIP-Cu), respectively. MNIP was synthesized in a rapid and convenient one-step microwave reaction. Endothelial nitric oxide synthase (eNOS) mRNA levels and mRNA stability were measured. RESULTS: Incubation with (-)-epicatechin (0.3-10 μM) led to elevated NO levels in HUVEC measured via reductive chemiluminescence detection and visualized as the fluorescent NO derivative of MNIP. Expression of eNOS mRNA and mRNA stability were not affected by (-)-epicatechin treatment within the time frame studied. CONCLUSION: (-)-Epicatechin augments the level of NO in endothelial cells, a process suggested to be responsible for the vasodilatory properties of the compound.
BACKGROUND: The dietary flavanol (-)-epicatechin has been suggested to mediate its vasodilatory effect by increasing nitric oxide levels in endothelial cells. AIM OF THE STUDY: To directly prove the formation of nitric oxide (NO) in human endothelial cells (HUVEC) in vitro by trapping NO to yield a fluorescent nitrosamine. METHODS: HUVEC were treated with (-)-epicatechin; nitrite and NO formation were determined by reductive chemiluminescence detection and the NO-sensitive fluorophore 5-methoxy-2-(1H-naphthol[2,3-d]imidazol-2-yl)-phenol copper complex (MNIP-Cu), respectively. MNIP was synthesized in a rapid and convenient one-step microwave reaction. Endothelial nitric oxide synthase (eNOS) mRNA levels and mRNA stability were measured. RESULTS: Incubation with (-)-epicatechin (0.3-10 μM) led to elevated NO levels in HUVEC measured via reductive chemiluminescence detection and visualized as the fluorescent NO derivative of MNIP. Expression of eNOS mRNA and mRNA stability were not affected by (-)-epicatechin treatment within the time frame studied. CONCLUSION:(-)-Epicatechin augments the level of NO in endothelial cells, a process suggested to be responsible for the vasodilatory properties of the compound.
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