Ingress and reactive chemistry of nitroxyl-derived species within human cells.
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.
Radiation Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD20892, USA. SP@nih.gov
Michael Graham
MG
Espey
Katrina M
KM
Miranda
Douglas D
DD
Thomas
David A
DA
Wink
D002474:Cell-Free System; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009569:Nitric Oxide / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000737:chemistry* / Q000378:metabolism; D013053:Spectrophotometry; D013997:Time Factors; D014407:Tumor Cells, Cultured
D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D009584:Nitrogen; D010100:Oxygen