title: Critical role of NADPH oxidase-derived reactive oxygen species in generating Ca2+ oscillations in human aortic endothelial cells stimulated by histamine.
abstract: There is increasing evidence that intracellular reactive oxygen species (ROS) play a role in cell signaling and that the NADPH oxidase is a major source of ROS in endothelial cells. At low concentrations, agonist stimulation of membrane receptors generates intracellular ROS and repetitive oscillations of intracellular Ca(2+) concentration ([Ca(2+)](i)) in human endothelial cells. The present study was performed to examine whether ROS are important in the generation or maintenance of [Ca(2+)](i) oscillations in human aortic endothelial cells (HAEC) stimulated by histamine. Histamine (1 microm) increased the fluorescence of 2',7'-dihydrodichlorofluorescin diacetate in HAEC, an indicator of ROS production. This was partially inhibited by the NADPH oxidase inhibitor diphenyleneiodonium (DPI, 10 microm), by the farnesyltransferase inhibitor H-Ampamb-Phe-Met-OH (2 microm), and in HAEC transiently expressing Rac1(N17), a dominant negative allele of the protein Rac1, which is essential for NADPH oxidase activity. In indo 1-loaded HAEC, 1 microm histamine triggered [Ca(2+)](i) oscillations that were blocked by DPI or H-Ampamb-Phe-Met-OH. Histamine-stimulated [Ca(2+)](i) oscillations were not observed in HAEC lacking functional Rac1 protein but were observed when transfected cells were simultaneously exposed to a low concentration of hydrogen peroxide (10 microm), which by itself did not alter either [Ca(2+)](i) or levels of inositol 1,4,5-trisphosphate (Ins-1,4,5-P(3)). Thus, histamine generates ROS in HAEC at least partially via NADPH oxidase activation. NADPH oxidase-derived ROS are critical to the generation of [Ca(2+)](i) oscillations in HAEC during histamine stimulation, perhaps by increasing the sensitivity of the endoplasmic reticulum to Ins-1,4,5-P(3).
authors.affiliation: Department of Medicine, Division of Cardiology, Johns Hopkins Bayview Medical Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA. ginghuaa@jhmi.edu
authors.forename: Qinghua
authors.initials: Q
authors.lastname: Hu
authors.forename: Zu-Xi
authors.initials: ZX
authors.lastname: Yu
authors.forename: Victor J
authors.initials: VJ
authors.lastname: Ferrans
authors.forename: Kazuyo
authors.initials: K
authors.lastname: Takeda
authors.forename: Kaikobad
authors.initials: K
authors.lastname: Irani
authors.forename: Roy C
authors.initials: RC
authors.lastname: Ziegelstein
mesh_terms: D001011:Aorta / Q000166:cytology*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005799:Genes, Dominant; D006632:Histamine / Q000494:pharmacology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007700:Kinetics; D019255:NADPH Oxidases / Q000378:metabolism*; D009861:Onium Compounds / Q000494:pharmacology; D017382:Reactive Oxygen Species; D015398:Signal Transduction; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D009861:Onium Compounds; D017382:Reactive Oxygen Species; C007517:diphenyleneiodonium; D006632:Histamine; D015544:Inositol 1,4,5-Trisphosphate; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; D002118:Calcium