Nitrogen monoxide (NO) and glucose - Unexpected links between energy metabolism and NO-mediated iron mobilization from cells

Nitrogen monoxide (NO) affects cellular iron metabolism due to its high affinity for this metal ion. Indeed, NO has been shown to increase the mRNA binding activity of the iron-regulatory protein 1, which is a major regulator of iron homeostasis, Recently, we have shown that NO generators increase Fe-59 efflux from cells pre-labeled with Fe-59-transferrin (Wardrop, S. L., Watts, R. N., and Richardson, D. R. (2000) Biochemistry 39, 2748-2758), The mechanism involved in this process remains unknown, and in this investigation we demonstrate that it is potentiated upon adding D-glucose (D-Glc) to the reincubation medium. In D-Glc-free or D-Glc-containing media, 5.6 and 16.5% of cellular 59Fe was released, respectively, in the presence of S-nitrosoglutathione. This difference in 59Fe release was observed with a variety of NO generators and cell types and was not due to a change in cell viability. Kinetic studies showed that D-Glc had no effect on the rate of NO production by NO generators. Moreover, only the metabolizable monosaccharides D-Glc and D-mannose could stimulate NO-mediated Fe-59 mobilization, whereas other sugars not easily metabolized by fibroblasts had no effect. Hence, metabolism of the monosaccharides was essential to increase NO-mediated Fe-59 release. Incubation of cells with the citric acid cycle intermediates, citrate and pyruvate, did not enhance NO-mediated Fe-59 release. Significantly, preincubation with the GSH-depleting agents, L-buthionine-[S,R]-sulfoximine or diethyl maleate, prevented NO-mediated Fe-59 mobilization, This effect was reversed by incubating cells with N-acetyl-L-cysteine that reconstitutes GSH. These results indicate that GSH levels are essential for NO-mediated Fe-59 efflux. Hence, D-Glc metabolism via the hexose monophosphate shunt resulting in the generation of GSH may be essential for NO-mediated Fe-59 release. These results have important implications for intracellular signaling by NO and also NO-mediated cytotoxicity of activated macrophages that is due, in part, to iron release from tumor target cells.