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Bioactivation of nitroprusside by porcine endothelial cells.

  • Rochelle, L G
  • Kruszyna, H
  • Kruszyna, R
  • Barchowsky, A
  • Wilcox, D E
  • Smith, R P
Published Article
Toxicology and applied pharmacology
Publication Date
Sep 01, 1994
PMID: 8079345


Sodium nitroprusside (Na2[(CN)5FeNO], SNP), which is stable, diamagnetic, and not detectable by electron paramagnetic resonance (EPR) spectroscopy, can be activated by one-electron reduction. The initial product, which retains the five cyanides and is here called penta, has a distinctive EPR signal. Penta spontaneously dissociates the trans-cyanide ligand resulting in a second paramagnetic species called tetra, which has a different and distinctive EPR signal. Tetra is able to transfer its NO ligand to a suitable acceptor, and all four equatorial cyanides subsequently dissociate. However, excess free cyanide shifts the tetra-penta equilibrium in the direction of penta and prevents NO release. This study was an attempt to extend the above results on SNP reduction, which were obtained in a model hemoglobin system, to intact porcine cells by characterizing all EPR-detectable intermediates. When porcine aortic endothelial or smooth muscle cells in culture were incubated under anaerobic conditions with SNP, an EPR spectrum was obtained, which could be resolved into the signal for penta and a signal previously described as a nonheme iron-nitrosyl-sulfur complex, Fe-NOSR. Tetra was not detected. This FeNOSR has some differences in its stability and location from that described by others in activated macrophages. When incubations were carried out under air, penta could not be detected, but a somewhat diminished signal for FeNOSR was still detectable. When incubations were carried out in the presence of excess free cyanide, conditions under which reduced SNP does not nitrosylate hemoglobin, the penta signal became stronger and the FeNOSR signal, though decreased, was still observed. Depletion (95%) of intracellular reduced glutathione in endothelial cells had no effect on the FeNOSR signal strength. We conclude that SNP is activated in porcine endothelial cells by a one-electron reduction to penta, which apparently dissociates its trans-cyanide to form tetra which then goes on to form FeNOSR upon reaction with a membrane-bound thiol. Glutathione is not involved in any of these reactions.

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