IMMUNOLOCALIZATION OF CYTOPLASMIC AND MYELIN MCALPAIN IN TRANSFECTED SCHWANN-CELLS .2. EFFECT OF WITHDRAWAL OF GROWTH-FACTORS

We have examined the reversal of the regulatory effect of growth facto rs on calpain/calpastatin activity in transfected Schwann cells (tSc) after their subsequent withdrawal, Removal of nerve growth factor (NGF ) or cyclic adenosine monophosphate (cAMP) from tSc resulted in a smal ler loss of mu calpain (37%) and mcalpain (36.5%) activity compared to treated cells from which the growth factors were not withdrawn, The m u calpain activity increased approximately 12% following withdrawal of acidic fibroblast growth factor (aFGF) and basic fibroblast growth fa ctor (bFGF) at 24 hr, while the increased mcalpain activity was more t han 30-40% compared with that of cells that were continuously treated, The activity of both isoforms returned to their normal levels (untrea ted) at 48-72 hr following withdrawal of various growth factors, inclu ding NGF, cAMP, aFGF, bFGF, platelet-derived growth factor aa (PDGFaa) , and PDGFbb. The inhibitory activity of calpastatin was greater than control following withdrawal of NGF, cAMP, PDGFaa, or PDGFbb at 24 hr and this inhibitory activity was less with treatment by aFGF and bFGF, The control activity was restored at 48 hr following withdrawal of th ese factors, The intensity of the cytoplasmic calpain immunoreactivity was significantly decreased in the nuclear and nonnuclear regions of the cytoplasm, respectively, following withdrawal of cAMP at 144 hr. R emoval of bFGF from the medium resulted in an increase of cytoplasmic calpain immunoreactivity in the nuclear regions and cytoplasm, while t here was dramatic loss of myelin calpain immunoreactivity from both th e nuclear region and cytoplasm, The changes in calpain activity and im munoreactivity in tSc following withdrawal of growth factors suggest t hat release of calpain from membrane to cytosol may be regulated by th ese factors. (C) 1997 Wiley-Liss, Inc.