Low, Physiologically‐relevant, Concentrations of Hydrogen Peroxide Inhibit IP <sub>3</sub> Receptor Activity in Endothelial Cells in Intact Blood Vessels (2018)

The endothelium is the innermost lining of blood vessels and plays a significant role in controlling virtually every cardiovascular function. Constant signalling among endothelial cells and between endothelial cells and blood cells and smooth muscle cells is critical for the control of blood fluidity, vascular tone and angiogenesis. Intracellular Ca 2+ signals act as a messenger system that decodes extracellular signals arriving at the endothelium and encodes intracellular signals to determine cardiovascular activity. Hydrogen peroxide (H 2 O 2 ), a key reactive oxygen species (ROS) generated by the respiratory chain in mitochondria, is also an important regulator of endothelial function. In endothelial cells, H 2 O 2 may also be produced by NADPH oxidase, xanthine oxidase and nitric oxide synthases. Although mitochondria‐derived ROS are usually considered to induce oxidative stress and damage cells, the physiological roles of ROS in mechano‐stress signal transduction, vascular relaxation, permeability of endothelial cells is now acknowledged. In this study, the interactions of low, physiological, concentrations (1 μM) H 2 O 2 and Ca 2+ signals were examined in the endothelium of intact rat mesenteric arteries. The intracellular Ca 2+ was visualized and measured in ~200 endothelial cells using the Ca 2+ indicator Cal‐520 through high‐resolution, high‐speed imaging. Endothelial cells responded to ACh (100 nM) with Ca 2+ signals that initiated in clusters of endothelial cells and propagated within and between cells. ACh‐evoked Ca 2+ signals were largely IP 3 ‐evoked signalling events since the signals persisted after removal of external Ca 2+ and were blocked by the SERCA inhibitor cyclopiazonic acid (10 μM) and channel blocker 2‐APB (100 μM). H 2 O 2 (100 nM – 1 mM) inhibited ACh‐evoked Ca 2+ signals in a concentration‐dependent manner. Low concentrations (e.g. 1 μM) of H 2 O 2 are likely to be most physiologically relevant in signalling and were investigated further. H 2 O 2 suppression of ACh‐evoked Ca 2+ signals appeared as a decrease in the amplitude of the Ca 2+ signal in each activated cell and a reduced number of cells activated. H 2 O 2 inhibition of Ca 2+ signals were reversed by catalase (1000 U/ml). H 2 O 2 may decrease ACh‐evoked Ca 2+ signals by reducing the affinity of the ACh receptor, decreasing the production of IP3 or limiting the activity of the IP3 receptor. To distinguish between these possibilities, the effects of H 2 O 2 on direct activation of the IP 3 receptor was examined using photoactivatable caged‐inositol 1,4,5‐trisphosphate (cIP 3 ). Ca 2+ release from IP 3 receptor, by photolysis of cIP 3 , was inhibited by H 2 O 2 , an effect reversed by catalyse (1000 U/ml). Together, these results suggest low concentrations of H 2 O 2 inhibit intracellular Ca 2+ signalling by desensitizing IP 3 receptors. These findings point an important interaction in low concentrations of ROS in the control of physiological Ca 2+ signalling. Support or Funding Information Supported by the Wellcome Trust and British Heart Foundation This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .