Nicotinamide adenine dinucleotide phosphate reduced oxidase 5 (Nox5) regulation by angiotensin II and endothelin-1 is mediated via calcium/calmodulin-dependent, rac-1-independent pathways in human endothelial cells

Circ Res. 2010 Apr 30;106(8):1363-73. doi: 10.1161/CIRCRESAHA.109.216036. Epub 2010 Mar 25.

Abstract

Rationale: Although Nox5 (Nox2 homolog) has been identified in the vasculature, its regulation and functional significance remain unclear.

Objectives: 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).

Methods and results: 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.

Conclusions: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Angiotensin II / metabolism*
  • Calcium / metabolism*
  • Calcium Channel Blockers / pharmacology
  • Calmodulin / antagonists & inhibitors
  • Calmodulin / metabolism*
  • Cells, Cultured
  • Diltiazem / pharmacology
  • Endothelial Cells / drug effects
  • Endothelial Cells / enzymology*
  • Endothelin-1 / metabolism*
  • Enzyme Activation
  • Enzyme Inhibitors / pharmacology
  • Gene Expression Regulation, Enzymologic
  • Humans
  • Imidazoles / pharmacology
  • Inflammation / enzymology
  • JNK Mitogen-Activated Protein Kinases / metabolism
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Mitogen-Activated Protein Kinase 3 / metabolism
  • NADPH Oxidase 5
  • NADPH Oxidases / genetics
  • NADPH Oxidases / metabolism*
  • Phosphorylation
  • Proliferating Cell Nuclear Antigen / metabolism
  • Pyrones / pharmacology
  • Quinolines / pharmacology
  • RNA Interference
  • RNA, Messenger / metabolism
  • Signal Transduction* / drug effects
  • Superoxides / metabolism
  • Time Factors
  • Vascular Cell Adhesion Molecule-1 / metabolism
  • p38 Mitogen-Activated Protein Kinases / metabolism
  • rac1 GTP-Binding Protein / antagonists & inhibitors
  • rac1 GTP-Binding Protein / metabolism*

Substances

  • Calcium Channel Blockers
  • Calmodulin
  • EHT 1864
  • Endothelin-1
  • Enzyme Inhibitors
  • Imidazoles
  • Membrane Proteins
  • Proliferating Cell Nuclear Antigen
  • Pyrones
  • Quinolines
  • RAC1 protein, human
  • RNA, Messenger
  • Vascular Cell Adhesion Molecule-1
  • Superoxides
  • Angiotensin II
  • calmidazolium
  • NADPH Oxidase 5
  • NADPH Oxidases
  • NOX5 protein, human
  • JNK Mitogen-Activated Protein Kinases
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • p38 Mitogen-Activated Protein Kinases
  • rac1 GTP-Binding Protein
  • Diltiazem
  • Calcium