endothelin-1 and calmidazolium

Although Nox5 (Nox2 homolog) has been identified in the vasculature, its regulation and functional significance remain unclear.. We sought to test whether vasoactive agents regulate Nox5 through Ca(2+)/calmodulin-dependent processes and whether Ca(2+)-sensitive Nox5, associated with Rac-1, generates superoxide (O(2)(*-)) and activates growth and inflammatory responses via mitogen-activated protein kinases in human endothelial cells (ECs).. Cultured ECs, exposed to angiotensin II (Ang II) and endothelin (ET)-1 in the absence and presence of diltiazem (Ca(2+) channel blocker), calmidazolium (calmodulin inhibitor), and EHT1864 (Rac-1 inhibitor), were studied. Nox5 was downregulated with small interfering RNA. Ang II and ET-1 increased Nox5 expression (mRNA and protein). Effects were inhibited by actinomycin D and cycloheximide and blunted by diltiazem, calmidazolium and low extracellular Ca(2+) ([Ca(2+)](e)). Ang II and ET-1 activated NADPH oxidase, an effect blocked by low [Ca(2+)](e), but not by EHT1864. Nox5 knockdown abrogated agonist-stimulated O(2)(*-) production and inhibited phosphorylation of extracellular signal-regulated kinase (ERK)1/2, but not p38 MAPK (mitogen-activated protein kinase) or SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase). Nox5 small interfering RNA blunted Ang II-induced, but not ET-1-induced, upregulation of proliferating-cell nuclear antigen and vascular cell adhesion molecule-1, important in growth and inflammation.. Human ECs possess functionally active Nox5, regulated by Ang II and ET-1 through Ca(2+)/calmodulin-dependent, Rac-1-independent mechanisms. Nox5 activation by Ang II and ET-1 induces ROS generation and ERK1/2 phosphorylation. Nox5 is involved in ERK1/2-regulated growth and inflammatory signaling by Ang II but not by ET-1. We elucidate novel mechanisms whereby vasoactive peptides regulate Nox5 in human ECs and demonstrate differential Nox5-mediated functional responses by Ang II and ET-1. Such phenomena link Ca(2+)/calmodulin to Nox5 signaling, potentially important in the regulation of endothelial function by Ang II and ET-1.

Topics: Angiotensin II; Calcium; Calcium Channel Blockers; Calmodulin; Cells, Cultured; Diltiazem; Endothelial Cells; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Humans; Imidazoles; Inflammation; JNK Mitogen-Activated Protein Kinases; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NADPH Oxidase 5; NADPH Oxidases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Proliferating Cell Nuclear Antigen; Pyrones; Quinolines; rac1 GTP-Binding Protein; RNA Interference; RNA, Messenger; Signal Transduction; Superoxides; Time Factors; Vascular Cell Adhesion Molecule-1