Journal article
NADPH diaphorase detects S-nitrosylated proteins in aldehyde-treated biological tissues
Scientific reports, Vol.10(1), pp.21088-21088
12/03/2020
DOI: 10.1038/s41598-020-78107-6
PMCID: PMC7713249
PMID: 33273578
Abstract
NADPH diaphorase is used as a histochemical marker of nitric oxide synthase (NOS) in aldehyde-treated tissues. It is thought that the catalytic activity of NOS promotes NADPH-dependent reduction of nitro-blue tetrazolium (NBT) to diformazan. However, it has been argued that a proteinaceous factor other than NOS is responsible for producing diformazan in aldehyde-treated tissues. We propose this is a NO-containing factor such as an S-nitrosothiol and/or a dinitrosyl-iron (II) cysteine complex or nitrosated proteins including NOS. We now report that (1) S-nitrosothiols covalently modify both NBT and TNBT, but only change the reduction potential of NBT after modification, (2) addition of S-nitrosothiols or beta- or alpha-NADPH to solutions of NBT did not elicit diformazan, (3) addition of S-nitrosothiols to solutions of NBT plus beta- or alpha-NADPH elicited rapid formation of diformazan in the absence or presence of paraformaldehyde, (4) addition of S-nitrosothiols to solutions of NBT plus beta- or alpha-NADP did not produce diformazan, (5) S-nitrosothiols did not promote NADPH-dependent reduction of tetra-nitro-blue tetrazolium (TNBT) in which all four phenolic rings are nitrated, (6) cytoplasmic vesicles in vascular endothelial cells known to stain for NADPH diaphorase were rich in S-nitrosothiols, and (7) procedures that accelerate decomposition of S-nitrosothiols, markedly reduced NADPH diaphorase staining in tissue sections subsequently subjected to paraformaldehyde fixation. Our results suggest that NADPH diaphorase in aldehyde-fixed tissues is not enzymatic but is due to the presence of NO-containing factors (free SNOs or nitrosated proteins such as NOS), which promote NADPH-dependent reduction of NBT to diformazan.
Details
- Title: Subtitle
- NADPH diaphorase detects S-nitrosylated proteins in aldehyde-treated biological tissues
- Creators
- James M. Seckler - Case Western Reserve UniversityJinshan Shen - University of IowaTristan H. J. Lewis - University of GeorgiaMohammed A. Abdulameer - Case Western Reserve UniversityKhalequz Zaman - Case Western Reserve UniversityLisa A. Palmer - University of VirginiaJames N. Bates - University of Iowa, AnesthesiaMichael W. Jenkins - Case Western Reserve UniversityStephen J. Lewis - Case Western Reserve University
- Resource Type
- Journal article
- Publication Details
- Scientific reports, Vol.10(1), pp.21088-21088
- DOI
- 10.1038/s41598-020-78107-6
- PMID
- 33273578
- PMCID
- PMC7713249
- NLM abbreviation
- Sci Rep
- ISSN
- 2045-2322
- eISSN
- 2045-2322
- Publisher
- Springer Nature
- Number of pages
- 14
- Grant note
- 0051596Z / National American Heart Association; American Heart Association PO1HL101871 / National Institutes of Health; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA
- Language
- English
- Date published
- 12/03/2020
- Academic Unit
- Anesthesia
- Record Identifier
- 9984425351202771
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