Calmodulin antagonists inhibit secretion in Paramecium

Secretion in Paramecium is Ca2+-dependent and involves exocytic release of the content of the secretory organelle, known as the trichocyst. The content, called the trichocyst matrix, undergoes a Ca2+-induced reordering of its paracrystalline structure during release, and we have defined three stages in this expansion process. The stage I, or fully condensed trichocyst, is the 4 microns-long membrane-bounded form existing prior to stimulation. Stage II, the partially expanded trichocyst, we define as an intermediate stage in the transition, preceding stage III, the fully expanded extruded form which is a 20-40 microns-long needlelike structure. These stages have been used to assay the effects of trifluoperazine (TFP) and W-7, calmodulin (CaM) antagonists, on trichocyst matrix expansion in vivo. TFP and W-7 are shown to reversibly block matrix release induced by picric acid. Ultra-structural examination reveals that one effect of this inhibition is reflected in the organelles themselves, which are prevented from undergoing the stage I-stage II transition by preincubation in 14 microM TFP or 35 microM W-7 before fixation. This inhibition of expansion by TFP can be moderated but not abolished by high extracellular Ca2+ (5 mM). The moderation by high Ca2+ can be eliminated by raising TFP concentration to 20 microM. A possible explanation for the ability to titrate the inhibition in this manner is that TFP is acting to block expansion by binding to the Ca2+-CaM complex. Brief exposure of cells to the Ca2+ ionophore A23187 and 5 mM Ca2+ following TFP treatment promotes matrix expansion, although in 14 microM TFP a residual level of inhibition remains. These results suggest that, following stimulation, CaM regulates secretion in Paramecium, possibly by controlling the Ca2+-dependent matrix expansion which accompanies exocytosis in these cells.