Synchronous exocytosis in Paramecium cells involves the rapid (_<1 s) dephosphorylation of a 65-kD phosphoprotein, which, after a lag phase of ~5 s, is reversed within ~20 s. Exocytosis inhibitors suppress this reaction; stimulatory and inhibitory effects are dose dependent. The dephosphorylation of the 65-kD phosphoprotein occurs only in exocytosiscompetent strains, but not in mutant strains that cannot carry out membrane fusion, or that are devoid of secretory organelles or cannot transport them to the cell membrane. Since under all conditions analyzed the transient dephosphorylation of the 65-kD phosphoprotein strictly parallels the actual amount of exocytosed organelles, this process might be involved in exocytosis performance, perhaps in its initiation.Protein phosphorylation and dephosphorylation processes are now considered to represent a general mechanism for a transient activation of various cell functions (18,19,23,24,38). Protein phosphorylation accompanies exocytotic activity in endocrine (1,7,8,16,37) and exocrine (3, 9, 27, 36) gland cells as well as in mast (39, 41) and nerve (11,23,25) cells. Whereas a dephosphorylation of single proteins was reported to occur only rarely during stimulation (3, 9, 40), in a comparison of different systems the opposite was observed quite frequently with a considerable number of proteins, which were all of different molecular weights. However, because most systems do not allow one to induce exocytosis in a synchronous way that would definitely exclude possible overlaps of phosphorylation/dephosphorylation processes, their possible role in stimulus-secretion coupling remains largely unknown.In this respect Paramecium cells offer a unique advantage, since by exocytosis they can expel most of their secretory organelles (trichocysts), more than 1,000 per cell, when triggered by certain polyamino compounds (29, 31). The actual time required for exocytosis in one cell, as determined by electrophysiological membrane capacitance changes, is only 1 s.' In cell suspensions all processes are accomplished within a few seconds (14). This short reaction time is possible due to the fact that >90% of trichocysts are docked onto the cell membrane, ready for immediate exocytosis (28). This allowed Deitmer, J., and H. Plattner. Manuscript in preparation.us to analyze phosphorylation/dephosphorylation cycles under synchronous conditions. Although the precise mode of action ofpolyamines is not yet known, it may be due to direct effects on protein kinase and phosphoprotein phosphatase activity, as shown with other systems (2, 21, 35). Our analyses clearly show that dephosphorylation may be a crucial step in exocytosis performance. When we began our study Gilligan and Satir (13) had recently found the dephosphorylation of a 65-kD phosphoprotein in Paramecium cells, which discharged trichocysts in response in picric acid; however, because this does not allow the cells to survive, no time sequence or dose-response analyses could be made. With polyamines, however, kinetic analyses ...