Reversal of SIN-1-induced eNOS dysfunction by the spin trap, DMPO, in bovine aortic endothelial cells via eNOS phosphorylation

Amlan Das, Bhavani Gopalakrishnan, Lawrence J. Druhan, Tse Yao Wang, Francesco De Pascali, Antal Rockenbauer, Ira Racoma, Saradhadevi Varadharaj, Jay L. Zweier, Arturo J. Cardounel, Frederick A. Villamena

Research output: Contribution to journalArticlepeer-review

Abstract

Background and Purpose Nitric oxide (NO) derived from eNOS is mostly responsible for the maintenance of vascular homeostasis and its decreased bioavailability is characteristic of reactive oxygen species (ROS)-induced endothelial dysfunction (ED). Because 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), a commonly used spin trap, can control intracellular nitroso-redox balance by scavenging ROS and donating NO, it was employed as a cardioprotective agent against ED but the mechanism of its protection is still not clear. This study elucidated the mechanism of protection by DMPO against SIN-1-induced oxidative injury to bovine aortic endothelial cells (BAEC). Experimental Approach BAEC were treated with SIN-1, as a source of peroxynitrite anion (ONOO-), and then incubated with DMPO. Cytotoxicity following SIN-1 alone and cytoprotection by adding DMPO was assessed by MTT assay. Levels of ROS and NO generation from HEK293 cells transfected with wild-type and mutant eNOS cDNAs, tetrahydrobiopterin bioavailability, eNOS activity, eNOS and Akt kinase phosphorylation were measured. Key Results Post-treatment of cells with DMPO attenuated SIN-1-mediated cytotoxicity and ROS generation, restoration of NO levels via increased in eNOS activity and phospho-eNOS levels. Treatment with DMPO alone significantly increased NO levels and induced phosphorylation of eNOS Ser1179 via Akt kinase. Transfection studies with wild-type and mutant human eNOS confirmed the dual role of eNOS as a producer of superoxide anion (O2-) with SIN-1 treatment, and a producer of NO in the presence of DMPO. Conclusion and Implications Post-treatment with DMPO of oxidatively challenged cells reversed eNOS dysfunction and could have pharmacological implications in the treatment of cardiovascular diseases.

Original languageEnglish (US)
Pages (from-to)2321-2334
Number of pages14
JournalBritish Journal of Pharmacology
Volume171
Issue number9
DOIs
StatePublished - 2014
Externally publishedYes

Keywords

  • cardiovascular diseases
  • DMPO
  • endothelial cells
  • endothelial dysfunction
  • eNOS
  • nitric oxide
  • oxidative stress
  • peroxynitrite
  • reactive oxygen species
  • spin trapping

ASJC Scopus subject areas

  • Pharmacology
  • Medicine(all)

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