Degranulation of Rough Endoplasmic Reticulum in M Phase and Adenosine-Triphosphate-Treated Interphase Cells Is Reversible

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Abstract
In the preferential harvesting of rounded mitotic (M phase) cells of human Chang liver monolayer cultures by mechanical agitation in Ca2+-free phosphate-buffered saline, degranulation of endoplasmic reticulum (ER) was observed. Mitotic cells are known to have a series of Ca2+ transients and, without being subjected to Ca2+-free washings, did not have degranulated ER. Quiescent cells incubated with 0.7 mM adenosine 5’-triphosphate (ATP) in Ca2+-free HEPES-buffered saline produced very similar ER degranulations. Confocal argon laser imaging of fluo-3-loaded cells showed a Ca2+ transient peaking at 2 min after ATP treatment. In the absence of extracellular Ca2+ transients of Ca2+ elevation in the cytosol would exit the cell in a down-gradient, draining the ER Ca2+ stores. Substituting ATP with 1 µM brominated A23187 calcium ionophore in the incubation that contained 1-100 mM CaCl2, respectively, did not produce ER degranulation, thereby excluding raised cytosolic Ca2+ per se as the cause of ER degranulation. In fact, incubation with 0.7 mM ATP in the presence of 1-5 mM CaCl2 failed to produce ER degranulation. ER degranulated cells, from treatment with ATP without extracellular Ca2+ as well as from Ca2+-free washings at M phase, could be rescued by subsequent incubation in growth medium that contains Ca2+ whereupon the rounded cells re-flatten (a round-to-flat change) and have well-defined rough ER. It therefore seems possible for Ca + depletion, or at least a reduction, to be causally related to ER degranulation. If that were the case, ER granularity would appear to be a facultative rather than a constitutive state.

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