Linked Life Data

17516243

Source:http://linkedlifedata.com/resource/pubmed/id/17516243

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
6
pubmed:dateCreated
2007-5-22
pubmed:abstractText
All methods used for quantitation of superoxide have limitations when it comes to differentiating between extracellular and intracellular sites of superoxide production. In the present study, we monitored dihydroethidium (DHE)-derived fluorescence at 570 nm, which indicates hydroxyethidium derived from reaction with superoxide produced by human leukemia cells (HL-60) and microvascular endothelial cells (HMEC-1). Phorbol-12-myristate 13-acetate (PMA; 100 ng/ml) caused an increase in fluorescence and lucigenin chemiluminescence in HL-60, which was abolished by superoxide dismutase (SOD; 600 U/ml) indicating that DHE detects extracellular superoxide. Furthermore, both HL-60 cells and HMEC-1 generated a fluorescence signal in the presence of DHE under resting conditions, which was unaffected by SOD, but abolished by polyethylene glycosylated-SOD (PEG-SOD) (100 U/ml) and MnTmPyP (25 microM), indicating that DHE also detects superoxide produced intracellularly. In HMEC-1, silencing of either Nox2 or Nox4 components of NADPH oxidase with small interference RNA (siRNA) resulted in a significant reduction in superoxide detected by both DHE fluorescence (Nox2 siRNA; 71 +/- 6% and Nox4 siRNA 83 +/- 7% of control) and lucigenin chemiluminescence (Nox2; 54 +/- 6% and Nox4 74 +/- 4% of control). In conclusion, DHE-derived fluorescence at 570 nm is a convenient method for detection of intracellular and extracellular superoxide produced by phagocytic and vascular NADPH oxidase.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
http://linkedlifedata.com/resource/pubmed/chemical/10,10'-dimethyl-9,9'-biacridinium, http://linkedlifedata.com/resource/pubmed/chemical/Acridines, http://linkedlifedata.com/resource/pubmed/chemical/CYBB protein, human, http://linkedlifedata.com/resource/pubmed/chemical/Ethidium, http://linkedlifedata.com/resource/pubmed/chemical/Luminescent Agents, http://linkedlifedata.com/resource/pubmed/chemical/Membrane Glycoproteins, http://linkedlifedata.com/resource/pubmed/chemical/NADPH Oxidase, http://linkedlifedata.com/resource/pubmed/chemical/NOX4 protein, human, http://linkedlifedata.com/resource/pubmed/chemical/RNA, Small Interfering, http://linkedlifedata.com/resource/pubmed/chemical/Superoxide Dismutase, http://linkedlifedata.com/resource/pubmed/chemical/Superoxides, http://linkedlifedata.com/resource/pubmed/chemical/Xanthine, http://linkedlifedata.com/resource/pubmed/chemical/Xanthine Oxidase, http://linkedlifedata.com/resource/pubmed/chemical/dihydroethidium
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
1071-5762
pubmed:author
pubmed:issnType
Print
pubmed:volume
41
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
699-712
pubmed:dateRevised
2007-11-15
pubmed:meshHeading
pubmed:year
2007
pubmed:articleTitle
Analysis of dihydroethidium fluorescence for the detection of intracellular and extracellular superoxide produced by NADPH oxidase.
pubmed:affiliation
Cytoprotection Pharmacology Laboratory, Bernard O'Brien Institute of Microsurgery, University of Melbourne, Melbourne, Vic., Australia.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't