Pharmacologic interventions that combine eNOS up-regulation and reversal of eNOS uncoupling can markedly increase bioactive NO in the vasculature and produce beneficial hemodynamic effects such as a reduction of blood pressure.

Nitric oxide (NO) produced by endothelial nitric-oxide synthase (eNOS) represents an antithrombotic and anti-atherosclerotic principle in the vasculature. Hence, an enhanced expression of eNOS in response to pharmacological interventions could provide protection against cardiovascular diseases. In EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells (HUVECs), an artichoke leaf extract (ALE) increased the activity of the human eNOS promoter (determined by luciferase reporter gene assay). An organic subfraction from ALE was more potent in this respect than the crude extract, whereas an aqueous subfraction of ALE was without effect. ALE and the organic subfraction thereof also increased eNOS mRNA expression (measured by an RNase protection assay) and eNOS protein expression (determined by Western blot) both in EA.hy 926 cells and in native HUVECs. NO production (measured by NO-ozone chemiluminescence) was increased by both extracts. In organ chamber experiments, ex vivo incubation (18 h) of rat aortic rings with the organic subfraction of ALE enhanced the NO-mediated vasodilator response to acetylcholine, indicating that the up-regulated eNOS remained functional. Caffeoylquinic acids and flavonoids are two major groups of constituents of ALE. Interestingly, the flavonoids luteolin and cynaroside increased eNOS promoter activity and eNOS mRNA expression, whereas the caffeoylquinic acids cynarin and chlorogenic acid were without effect. Thus, in addition to the lipidlowering and antioxidant properties of artichoke, an increase in eNOS gene transcription may also contribute to its beneficial cardiovascular profile. Artichoke flavonoids are likely to represent the active ingredients mediating eNOS up-regulation.

Background-Histamine has a short-term, transient, stimulating effect on endothelial nitric oxide synthase (eNOS) activity; however, long-term effects on eNOS have not been described yet. In addition, the vascular effect of histamine seems to depend critically on eNOS functionality. Therefore, we studied the effects of histamine on eNOS gene expression and function. Methods and Results-In human umbilical vein endothelial cells (HUVECs) and HUVEC-derived EA.hy 926 cells, histamine upregulated eNOS mRNA (RNase protection assay) and protein (electron microscopic immunocytochemistry) expression. The upregulation of eNOS could be prevented by mepyramine, a selective antagonist at the H 1 receptor, but not by H 2 and H 3 receptor antagonists. Incubation of EA.hy 926 cells with histamine led to the activation of calcium/calmodulin-dependent protein kinase II (CaMK II; in vitro phosphorylation assay). The histamine-induced eNOS expression was completely prevented by KN-93, an inhibitor of CaMK II. Histamine increased the activity of a 1.6-kb human eNOS promoter fragment (luciferase reporter gene assay), an effect that was also blocked by mepyramine. Under normal conditions, eNOS upregulation by histamine resulted in increased nitric oxide production (measured by nitric oxide chemiluminescence and RFL-6 reporter cell assay). Under conditions of oxidative stress, however, the eNOS upregulated by histamine produced reactive oxygen species (CM-H 2 DCFDA oxidation-based fluorescence assay). Conclusions-Stimulation of the H 1 receptor increases eNOS transcription in endothelial cells by a signaling pathway involving CaMK II. This eNOS upregulation may be protective under normal conditions, but it may become harmful under conditions of oxidative stress when eNOS produces reactive oxygen species at the expense of nitric oxide.