INTERMEDIATE STEPS IN CELLULAR IRON UPTAKE FROM TRANSFERRIN .2. 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 Fe-59-I-125-Tf at 37 degrees C (Richardson and Baker, 1992a). The release of this internalized Fe-59 could be markedly reduced if the c ells were treated with proteases or incubated at 37 degrees C prior to detachment. The present study was designed to characterize this Fe po ol and understand the mechanism of its release. The results show that cellular Fe-59 release increased linearly as a function of preincubati on time with Fe-59-Tf subsequent to mechanical detachment at 4 degrees C using a spatula. These data suggest that the Fe-59 released was lar gely composed of end product(s) and was not an ''intermediate Fe pool. '' When the Fe(II) chelator, dipyridyl (DP), was incubated with Fe-59- Tf and the cells, it prevented the accumulation of Fe-59 that was rele ased following mechanical detachment at 4 degrees C. Other chelators h ad much less effect on the proportion of Fe-59 released. Examination o f the Fe-59 released showed that after a 4-h preincubation with Fe-59- Tf, approximately 50% of the Fe-59 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, molecules. M oreover, electron microscopy studies showed that detachment of cells f rom the substratum at 4 degrees C resulted in pronounced membrane dama ge. In contrast, when cells were detached at 37 degrees C, or at 4 deg rees C after treatment with pronase, membrane damage was minimal or no t apparent. These results may imply that temperature-dependent process es 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 experi ments where cells have been mechanically detached at 4 degrees C.