SYNCHRONOUS EXOCYTOSIS IN PARAMECIUM CELLS .4. POLYAMINO COMPOUNDS AS POTENT TRIGGER AGENTS FOR REPEATABLE TRIGGER-REDOCKING CYCLES

Abstract

Different polyamino compounds trigger the instantaneous and simultaneous release of trichocysts from P. tetraurelia cells (monoxenically bacterized cultures), provided amino groups are spaced in intervals of .apprx. 1.0 nm; in this case even diamines or oligopeptides exert some trigger effect. The most potent trigger agent found was aminoethyldextran (AED, MW=40 kDa[kilodalton]) which was used mainly as a derivative with 40 - NH2/molecule. A maximal response (strain K401) was achieved at 1.38 x 10-6 M, a half maximal response at 1.08 .times. 10-6 M. AED acts by a dual effect, i.e., not only by statistically properly spaced amino groups but also by some additional effect of oligoamines, although it exerts no trigger effect per se. From a total of .apprx. 1120 or .apprx. 1230 trichocysts per cell (strain K401 or 7S) .apprx. 95% are releasable by AED. In these strains the number of non-releasable trichocysts corresponds closely to the number of undocked trichocysts floating in the cytoplasm, so that practically all trichocysts which are docked to the cell membrane, can be released. (Different mutant strains were analyzed for their response to AED.) Massive trichocyst release does not impair cell viability or culture growth, and multiple release-redocking cycles can be performed; up to 5 trigger-docking cycles were tested with individual cells in 12 h intervals. AED-triggered exocytosis requires a free extracellular [Ca2+]of .gtoreq. 10-5 M; it is inhibited by EGTA (ethyleneglycol-bis(.beta.-aminoethyl ether)-N,N''-tetraacetate), by a short pH 5.5 shock or by neomycin at 10-5 M concentration. Several problems of exocytosis performance are more easily approachable now with this type of synchronized exocytotic system (e.g., adaptation phenomena during re-insertion of trichocysts), since paramecia can be rapidly and repeatedly triggered by AED, followed by a long replenishing period, during which the status quo is re-established.