Contribution of NADPH oxidase to membrane CD38 internalization and activation in coronary arterial myocytes.
The CD38-ADP-ribosylcyclase-mediated Ca(2+) signaling pathway importantly contributes to the vasomotor response in different arteries. Although there is evidence indicating that the activation of CD38-ADP-ribosylcyclase is associated with CD38 internalization, the molecular mechanism mediating CD38 internalization and consequent activation in response to a variety of physiological and pathological stimuli remains poorly understood. Recent studies have shown that CD38 may sense redox signals and is thereby activated to produce cellular response and that the NADPH oxidase isoform, NOX1, is a major resource to produce superoxide (O2·-)) in coronary arterial myocytes (CAMs) in response to muscarinic receptor agonist, which uses CD38-ADP-ribosylcyclase signaling pathway to exert its action in these CAMs. These findings led us hypothesize that NOX1-derived O2·- serves in an autocrine fashion to enhance CD38 internalization, leading to redox activation of CD38-ADP-ribosylcyclase activity in mouse CAMs. To test this hypothesis, confocal microscopy, flow cytometry and a membrane protein biotinylation assay were used in the present study. We first demonstrated that CD38 internalization induced by endothelin-1 (ET-1) was inhibited by silencing of NOX1 gene, but not NOX4 gene. Correspondingly, NOX1 gene silencing abolished ET-1-induced O2·- production and increased CD38-ADP-ribosylcyclase activity in CAMs, while activation of NOX1 by overexpression of Rac1 or Vav2 or administration of exogenous O2·- significantly increased CD38 internalization in CAMs. Lastly, ET-1 was found to markedly increase membrane raft clustering as shown by increased colocalization of cholera toxin-B with CD38 and NOX1. Taken together, these results provide direct evidence that Rac1-NOX1-dependent O2·- production mediates CD38 internalization in CAMs, which may represent an important mechanism linking receptor activation with CD38 activity in these cells.
Department of Pharmacology and Toxicology, Virginia Commonwealth University School of Medicine, Richmond, Virginia, United States of America.
Ming
M
Xu
Xiao-Xue
XX
Li
Joseph K
JK
Ritter
Justine M
JM
Abais
Yang
Y
Zhang
Pin-Lan
PL
Li
D051997:ADP-ribosyl Cyclase 1 / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology; D019332:Endothelin-1 / Q000378:metabolism; D004789:Enzyme Activation; D005260:Female; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D021962:Membrane Microdomains / Q000201:enzymology; D051379:Mice; D018345:Mice, Knockout; D032389:Myocytes, Smooth Muscle / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000378:metabolism; D009479:Neuropeptides / Q000378:metabolism; D021381:Protein Transport; D051567:Proto-Oncogene Proteins c-vav / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
D019332:Endothelin-1; D008562:Membrane Glycoproteins; D009479:Neuropeptides; D051567:Proto-Oncogene Proteins c-vav; C484602:Rac1 protein, mouse; C490565:Vav2 protein, mouse; D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C000614711:NOX1 protein, mouse; C422943:Nox4 protein, mouse; C498155:Cd38 protein, mouse; D051997:ADP-ribosyl Cyclase 1; D020830:rac1 GTP-Binding Protein