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 There is increasing evidence that intracellular reactive oxygen species (ROS) 1 play an important role in cell signaling(1-4). Both vascular smooth muscle cells and endothelial cells are capable of generating ROS (5). A number of enzyme systems likely contribute to ROS generation in endothelial cells, including arachidonic acid-metabolizing systems (6), the mitochondrial electron transport chain (7), xanthine oxidase (8), nitric-oxide synthase (9), the cytochrome P450 enzyme system (10), and endothelial NADPH oxidase (11). The endothelial NADPH oxidase shares some structural features of the multicomponent NADPH oxidase of phagocytes, most of which have been identified in endothelial cells at the RNA or protein level (12)(13)(14). Among these include the small GTP-binding protein Rac1, which is necessary for enzyme function. Whereas the oxidase of phagocytes generates large quantities of ROS that are necessary to eliminate engulfed microorganisms, the NADPH oxidase of non-phagocytic cells generates low levels of ROS that appear to have a cell signaling function. ROS generation by endothelial cells has been observed after stimulation by acetylcholine (15), interleukin-4 (16), interleukin-1 (17), interferon-␥ (17), bradykinin (18, 19), platelet-activating factor (20), vascular endothelial growth factor (21), tumor necrosis factor (22-25), angiotensin II (26, 27), and thrombin (28). In several circumstances, this ROS production has been shown to result from activation of endothelial NADPH oxidase (24,27,28). We recently showed that activation of endothelial NADPH oxidase increases the sensitivity of endoplasmic reticulum (ER) Ca 2ϩ stores to inositol 1,4,5-trisphosphate (Ins-1,4,5-P 3 ) (29). Activation of the NADPH oxidase in human aortic endothelial cells (HAEC) shifted the Ins-1,4,5-P 3 -Ca 2ϩ release dose-response curve to the left and decreased the threshold concentration of Ins-1,4,5-P 3 required to release intracellularly stored Ca 2ϩ . This effect was blocked by the NADPH oxidase inhibitor diphenyleneiodonium (DPI) and was not observed in cells lacking functional Rac1 protein.At low concentrations, agonists like histamine (30), bradykinin (31), and thrombin (32) stimulate repetitive oscillations of intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) in endothelial cells. The initiation of each Ca 2ϩ spike appears to be due chiefly to the generation of Ins-1,4,5-P 3 (33), and the maintenance of oscillations may be related to repetitive cycles of fast activation and slow inactivation of the Ins-1,4,5-P 3 receptor by Ca 2ϩ (34,35). Thus, the observation, that NADPH oxidase-generated ROS increases the sensitivity of intracellular Ca 2ϩ stores to Ins-1,4,5-P 3 , suggests a potential link between ox...

Ca2+ oscillation frequency regulates agonist-stimulated gene expression in vascular endothelial cells

“…In other words, histamine stimulation increases the efficiency of [Ca 2+ ] i -oscillation-frequency-regulated VCAM1 gene expression. It is also noted that histamine-stimulated VCAM1 mRNA expression is optimized at a [Ca 2+ ] i oscillation frequency of 0.3 oscillations/minute, which is the mean [Ca 2+ ] i oscillation frequency in this type of cells upon 1 μM histamine stimulation (Hu et al, 1999;Hu et al, 2002). The distinct [Ca 2+ ] i -oscillationfrequency dependence of gene expression in the presence and absence of agonist stimulation suggests that, in addition to [Ca 2+ ] i oscillations alone, other intracellular signaling pathway(s) are also involved in agonist-stimulated gene expression.…”

Blockade of NOX2 and STIM1 signaling limits lipopolysaccharide-induced vascular inflammation

“…We and others have previously shown that paracrine-derived superoxide triggers Ca 2+ influx in ECs (16,36,37). To examine whether the loss of gp91 phox impacts endothelial Ca 2+ entry, we pretreated ECs with thapsigargin and measured Ca 2+ entry.…”

NAD(P)H Oscillates in Pollen Tubes and Is Correlated with Tip Growth

“…NAD(P)H is fluorescent and has been visualized by exciting at 340 to 365 nm and detecting emission at 400 to 500 nm (Lakowicz et al, 1992;Paul and Schneckenburger, 1996;Danø et al, 1999;Pogue et al, 2001;Schuchmann et al, 2001;Hu et al, 2002;Brachmanski et al, 2004;Blinova et al, 2005;Kasimova et al, 2006). To visualize the fluorescent signal, we used an inverted wide-field fluorescence microscope (Nikon TE-300), equipped with a CCD camera (CoolSnap HQ; Roper Instruments) mounted on the Kö hler port, and a Xenon excitatory light source (DG-4; Sutter Instruments).…”

“…Their high energy status and reducing power drive many key biosynthetic reactions and ATP production. Because NAD(P)H but not NAD(P) 1 possesses an endogenous fluorescence, it is possible to detect the reduced form in living cells; this property has been widely exploited (Lakowicz et al, 1992;Pogue et al, 2001;Schuchmann et al, 2001;Hu et al, 2002;Brachmanski et al, 2004;Blinova et al, 2005;Kasimova et al, 2006). Particularly pertinent are the oscillations in NADH that have been observed in yeast cells, which serve as a marker for changes in metabolism (Ghosh and Chance, 1964;Goldbeter, 1996;Danø et al, 1999).…”