Cell surface complexes (‘cortices’) isolated from Paramecium tetraurelia cells as a model system for analysing exocytosis in vitro in conjunction with microinjection studies

Abstract

Cortex preparations isolated from Paramecium tetraurelia cells consist of surface membranes with secretory organelles (trichocysts) still attached. In the absence of nucleotides, in media with a pCa of 5-5.5 and a pH of .gtoreq. 6.5, maximal exocytosis occurred when the Mg2+ concentration was lowered from 10 to 0.5 mM. ATP, as well as its non-hydrolysable analogues adenosine 5''-[.gamma.-thio]triphosphate (ATP[S]) and adenosine 5''[.beta..gamma.-imido]triphosphate (App[NH]p), inhibited exocytosis at a concentration equivalent to that occurring in vivo (as determined by h.p.l.c.), but preincubation with ATP augmented the exocytotic response. GTP and its analogues only slightly stimulated exocytosis in vitro, but sensitivity to Ca2+ was increased significantly, in particular with GTP. These effects of nucleotides were rapidly reversible. Intracellular GTP concentrations (0.35 mM) would suffice for full activation with the pCai values assumed to occur in these cells during activation. On microinjection, ATP inhibited the secretagogue response in intact cells. Whereas microinjected GTP stimulated exocytosis (membrane fusion) without a secretagogue added, Gpp[NH]p remained without any effect; GTP[S] permanently abolished any triggered secretory response. Concomitantly, h.p.l.c. analysis of triggered and untriggered cells showed that GTP hydrolysis occurs immediately after synchronous (1 s) exocytosis in vivo. The precise site(s) of action of GTP during signal transduction in Paramecium cells remain to be determined.