The mechanism of nitrogen monoxide (NO)-mediated iron mobilization from cells. NO intercepts iron before incorporation into ferritin and indirectly mobilizes iron from ferritin in a glutathione-dependent manner.
Nitrogen monoxide (NO) is a cytotoxic effector molecule produced by macrophages that results in Fe mobilization from tumour target cells which inhibits DNA synthesis and mitochondrial respiration. It is well known that NO has a high affinity for Fe, and we showed that NO-mediated Fe mobilization is markedly potentiated by glutathione (GSH) generated by the hexose monophosphate shunt [Watts, R.N. & Richardson, D.R. (2001) J. Biol. Chem. 276, 4724-4732]. We hypothesized that GSH completes the coordination shell of an NO[bond]Fe complex that is released from the cell. In this report we have extended our studies to further characterize the mechanism of NO-mediated Fe mobilization. Native PAGE 59Fe-autoradiography shows that NO decreased ferritin-59Fe levels in cells prelabelled with [59Fe]transferrin. In prelabelled cells, ferritin-59Fe levels increased 3.5-fold when cells were reincubated with control media between 30 and 240 min. In contrast, when cells were reincubated with NO, ferritin-59Fe levels decreased 10-fold compared with control cells after a 240-min reincubation. However, NO could not remove Fe from ferritin in cell lysates. Our data suggest that NO intercepts 59Fe on route to ferritin, and indirectly facilitates removal of 59Fe from the protein. Studies using the GSH-depleting agent, L-buthionine-(S,R)-sulphoximine, indicated that the reduction in ferritin-59Fe levels via NO was GSH-dependent. Competition experiments with NO and permeable chelators demonstrated that both bind a similar Fe pool. We suggest that NO requires cellular metabolism in order to effect Fe mobilization and this does not occur via passive diffusion down a concentration gradient. Based on our results, we propose a model of glucose-dependent NO-mediated Fe mobilization.
The Iron Metabolism and Chelation Group, The Heart Research Institute, Camperdown, Sydney, New South Wales, Australia.
Ralph N
RN
Watts
Des R
DR
Richardson
D000230:Adenocarcinoma / Q000473:pathology; D000818:Animals; D001943:Breast Neoplasms / Q000473:pathology; D002463:Cell Membrane Permeability; D002474:Cell-Free System; D003600:Cytosol / Q000378:metabolism; D003676:Deferoxamine / Q000494:pharmacology; D005260:Female; D005293:Ferritins / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D007501:Iron / Q000378:metabolism*; D007502:Iron Chelating Agents / Q000494:pharmacology; D008262:Macrophage Activation; D051379:Mice; D009447:Neuroblastoma / Q000473:pathology; D018241:Neuroectodermal Tumors, Primitive, Peripheral / Q000473:pathology; D009569:Nitric Oxide / Q000494:pharmacology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009589:Nitrogen Oxides; D010084:Oxidation-Reduction; D010396:Penicillamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D026422:S-Nitrosoglutathione / Q000494:pharmacology; D013096:Spermine / Q000031:analogs & derivatives* / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D007502:Iron Chelating Agents; D020030:Nitric Oxide Donors; D009589:Nitrogen Oxides; C110881:S-nitro-N-acetylpenicillamine; C091861:spermine nitric oxide complex; D013096:Spermine; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D005293:Ferritins; D007501:Iron; D005978:Glutathione; D010396:Penicillamine; D003676:Deferoxamine