Ingress and reactive chemistry of nitroxyl-derived species within human cells

Michael Graham Espey, Katrina M. Miranda, Douglas D. Thomas, David A. Wink

Research output: Contribution to journalArticle

72 Scopus citations

Abstract

The mechanisms that control the biological signaling and toxicological properties of the nitrogen oxide species nitroxyl (HNO) are largely unknown. The ingress and intracellular reactivity of nitroxyl-derived species were examined using Angeli's salt (AS), which decomposes initially to HNO and nitrite at physiologic pH. Exposure of 4,5-diaminofluorescein (DAF) to AS resulted in fluorescent product formation only in the presence of molecular oxygen. Kinetic analysis and the lack of signal from a nitric oxide (NO)-sensitive electrode suggested that these processes did not involve conversion of HNO to NO. On an equimolar basis, bolus peroxynitrite (ONOO-) exposure generated only 15% of fluorescent product formation observed from AS decomposition. Moreover, infusion of synthetic ONOO- at a rate comparable to AS decomposition resulted in only 4% of the signal. Quenching of AS-mediated product formation within intact human MCF-7 breast carcinoma cells containing DAF by addition of urate to buffer suggested involvement of an oxidized intermediate formed from reaction between HNO and oxygen. Conversely, intact cells competitively sequestered the HNO-derived species from reaction with DAF in solution. These data show this intermediate to be a long-lived diffusible species. Relative product yield from intracellular DAF was decreased 5- to 8-fold when cells were lysed immediately prior to AS addition, consistent with the partitioning of HNO and/or derived species into the cellular membrane, thereby shielding these reactive intermediates from either hydrolysis or cytoplasmic scavenger pools. These findings establish that oxygen-derived species of nitroxyl can readily penetrate and engage the intracellular milieu of cells and suggest this process to be independent of NO and ONOO- intermediacy. The substantial facilitation of oxygen-dependent nitroxyl chemistry by intact lipid bilayers supports a focusing role for the membrane in modulation of cellular constituents proteins by this unique species.

Original languageEnglish (US)
Pages (from-to)827-834
Number of pages8
JournalFree Radical Biology and Medicine
Volume33
Issue number6
DOIs
StatePublished - Sep 15 2002
Externally publishedYes

Keywords

  • Angeli's salt
  • Diaminofluorescein
  • Free radicals
  • MCF-7
  • Nitric oxide

ASJC Scopus subject areas

  • Biochemistry
  • Physiology (medical)

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