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Fig. 2. Both CO gas and GSNO reduce: (A) 59Fe uptake from 59Fe-transferrin (59Fe-Tf) and (B) 59Fe incorporation into ferritin by LMTK- fibroblasts. (A) Cells were labelled with 59Fe-Tf (0.75 mM) for 5-180 min at 37°C in the presence or absence of 2% CO gas or GSNO (0.5 mM). The cells were then washed four times on ice and incubated for 30 min with Pronase (1 mg/ml) at 4°C to separate internalized from membrane-bound 59Fe. (B) The LMTK- cells were labelled with 59Fe-Tf (0.75 mM) for 60-180 min at 37°C, washed, and native-PAGE 59Fe-autoradiography performed. Densitometric analysis is presented below the autoradiograph. The results in (A) are expressed as mean of duplicate determinations in a typical experiment of 5 performed, while the results in (B) are representative of 3 separate experiments. (Taken with permission from Ref. [66].)

THE MECHANISM OF NITROGEN MONOXIDE-MEDIATED Fe RELEASE FROM CELLS

Regarding the effect of NO on cellular Fe release, initial studies using NO+ generators (e.g. sodium nitroprusside) or ONOO- donors (e.g. SIN-1) [77] demonstrated that these agents did not result in appreciable Fe mobilization from cells [73]. In contrast, NO^-releasing agents [e.g. S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO) or spermine-NONOate (SperNO)] showed high efficacy [25,65,78]. The effect of these agents in inducing Fe release was due to their ability to generate NO, as their precursor compounds which do not bear the NO-moiety had no effect [25,65]. Hence, it was the NO^ redox state which was important for forming intracellular Fe complexes, probably because this form of NO is capable of generating coordination complexes [10]. While NO could markedly induce intracellular release, other similar diatomic molecules such as CO had little effect (Fig. 3A) [66]. Further, the ability of NO to mobilize cellular Fe could not be augmented by CO, despite their similar chemistry and the potential of CO to directly ligate Fe pools (Fig. 3B) [10,66,79]. Like synthetic high-affinity Fe chelators [80], the efficacy of NO in mobilizing Fe from cells decreased as the labelling time with 59Fe-transferrin (59Fe-Tf)

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