Histamine Upregulates Gene Expression of Endothelial Nitric Oxide Synthase in Human Vascular Endothelial Cells

Abstract

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.

Histamine Induces Egr-1 Expression in Human Aortic Endothelial Cells via the H1 Receptor-mediated Protein Kinase Cδ-dependent ERK Activation Pathway

“…Although both the H1 and H2 histamine receptors are reportedly expressed in endothelial cells (5,19), our data indicate that the H1 receptor, but not the H2 receptor, mediates the histamine signal leading to Egr-1 expression. Related to our finding, previous studies have shown that histamine-induced expression of p-selectin (9), ICAM1, VCAM1 (10), IL6, IL8 (8), cyclooxygenase-2 (27), and tissue factor (11) is mediated by the H1 receptor in vascular endothelial cells.…”

“…An early study indicates that H1 receptor is expressed in human endothelial cells (5), and a recent report suggests that histamine receptor 2 (H2) is also expressed in human endothelial cells (19). To determine which of the receptors indeed mediates histamine-induced Egr-1 expression in HAECs, we pretreated HAECs with one of the H1 receptor-specific antagonists, either mepyramine or chlorpheniramine, or with the H2 receptor-specific antagonist cimetidine for 40 min.…”

Methylene Blue for Distributive Shock: A Potential New Use of an Old Antidote

“…Further evidence to support the role of the NO-cGMP pathway in anaphylaxis is demonstrated by the finding that increased histamine results in upregulation of eNOS gene expression with increased production of NO leading to vasodilation through increased activity of GC. Inhibition of GC reduces vasodilation induced by histamine [53][54][55]. This evidence provides support that histamine-induced vasodilation is at least partly mediated through the NO-cGMP pathway and that inhibition of this pathway reverses the vasodilation.…”

Pro-inflammatory cytokines increase reactive oxygen species through mitochondria and NADPH oxidase in cultured RPE cells

“…Intracellular ROS Production-Intracellular ROS production by human RPE cells in response to TNF-α, IL-1β, or IFN-γ treatment was measured by a cell-based fluorometric assay based on deacetylation and oxidation of non-fluorescent reduced CM-H 2 DCFDA (Molecular Probes) into fluorescent CM-DCF. The reliability and specificity of the probe have been established in various cells including cultured human RPE cells (Deshpande et al, 2000;Nishikawa et al, 2000;Touyz et al, 2001;Yamagishi et al, 2001;Higgins et al, 2003;Li et al, 2003;Fukami et al, 2004;Hermann et al, 2004;Hwang et al, 2004;Kannan et al, 2004;Shanker et al, 2004;Kim et al, 2005). RPE cells were seeded into 96-well plates pre-coated with poly-D-lysine (Sigma-Aldrich) and cultured for 1 day or 7days.…”