Intermediate steps in cellular iron uptake from transferrin. II. A cytoplasmic pool of iron is released from cultured cells via temperature-dependent mechanical wounding.

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 degrees C, after initial incubation with 59Fe-125I-Tf at 37 degrees C (Richardson and Baker, 1992a). The release of this internalized 59Fe could be markedly reduced if the cells were treated with proteases or incubated at 37 degrees 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 cellular 59Fe release increased linearly as a function of preincubation time with 59Fe-Tf subsequent to mechanical detachment at 4 degrees C using a spatula. These data suggest that the 59Fe release was largely composed of end product(s) and was not an "intermediate Fe pool." When the Fe(II) chelator, dipyridyl (DP), was incubated with 59Fe-Tf and the cells, it prevented the accumulation of 59Fe that was released following mechanical detachment at 4 degrees C. Other chelators had much less effect on the proportion of 59Fe released. Examination of the 59Fe released showed that after a 4-h preincubation with 59Fe-Tf, approximately 50% of the 59Fe was present in ferritin. These data indicate that mechanical detachment of cells at 4 degrees C resulted in membrane disruptions that allow the release of high M(r), molecules. Moreover, electron microscopy studies showed that detachment of cells from the substratum at 4 degrees C resulted in pronounced membrane damage. In contrast, when cells were detached at 37 degrees C, or at 4 degrees 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 degrees C. The evidence also indicates that caution is required when interpreting data from experiments where cells have been mechanically detached at 4 degrees C.