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Intermediate steps in cellular iron uptake from transferrin. II. A cytoplasmic pool of iron is released from cultured cells via temperature-dependent mechanical wounding
Authors:D. R. Richardson  L. Dickson  E. Baker
Affiliation:(1) Lady Davis Institute for Medical Research of the Sir Mortimer B. Davis-Jewish General Hospital, 3755 Chemin de la Cote-Ste-Catherine, H3T 1E2 Montréal, Québec, Canada;(2) Department of Medicine, McGill University, 6009 Perth, Western Australia;(3) Department of Physiology, University of Western Australia, 6009 Perth, Western Australia
Abstract:Summary A previous study described a cytoplasmic, transferrin (Tf)-free, iron (Fe) pool that was detected only when cells were mechanically detached from the culture substratum at 4°C, after initial incubation with59Fe-125I-Tf at 37°C (Richardson and Baker, 1992a). The release of this internalized59Fe could be markedly reduced if the cells were treated with proteases or incubated at 37°C prior to detachment. The present study was designed to characterize this Fe pool and understand the mechanism of its release. The results show that cellular59Fe release increased linearly as a function of preincubation time with59Fe-Tf subsequent to mechanical detachment at 4°C using a spatula. These data suggest that the59Fe released was largely composed of end product(s) and was not an “intermediate Fe pool.” When the Fe(II) chelator, dipyridyl (DP), was incubated with59Fe-Tf and the cells, it prevented the accumulation of59Fe that was released following mechanical detachment at 4°C. Other chelators had much less effect on the proportion of59Fe released. Examination of the59Fe released showed that after a 4-h preincubation with59Fe-Tf, approximately 50% of the59Fe was present in ferritin. These data indicate that mechanical detachment of cells at 4°C resulted in membrane disruptions that allow the release of high M, molecules. Moreover, electron microscopy studies showed that detachment of cells from the substratum at 4°C resulted in pronounced membrane damage. In contrast, when cells were detached at 37°C, or at 4°C after treatment with pronase, membrane damage was minimal or not apparent. These results may imply that temperature-dependent processes prevent the release of intracellular contents on membrane wounding, or alternatively, prevent wounding at 37°C. The evidence also indicates that caution is required when interpreting data from expriments where cells have been mechanically detached at 4°C.
Keywords:mechanical wounding  intracellular iron pools  ferritin  transferrin
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