Adenosinetriphosphate, calcium and temperature requirements for the final steps of exocytosis in Paramecium cells

In Paramecium cells a synchronized discharge of trichocysts (which involves only the final exocytosis steps of membrane fusion, content discharge and membrane resealing) was achieved with ATPase-blockers, Ca2+-ionophores, lipid solvents (including lysolecithin), polyethyleneglycol, anaesthetics (Dibucain) and cationic detergents (cetyltrimethylammonium bromide (CTMAB) and cetylpyridinium chloride (CPC). Only Dibucain--and to some extent cationic detergents--can trigger exocytosis independently of extracellular Ca2+, possibly by mobilizing intracellular Ca2+. The internal free [Ca2+] necessary for exocytosis can be estimated to be greater than 10(-6) to 10(-4) M. Membrane-free trichocyst contents were isolated by density gradient centrifugation; they are converted from the contracted to the expanded state by Dibucain, CTMAB and CPC, and also by exogenous ATPase (Apyrase). Thus, it is possible to de-couple the discharge (stretching) process from membrane-related phenomena. Since only the latter are inhibited by low temperature (0 degrees C), membrane lipids probably have to be in a fluid state for exocytosis to occur. At least 2 steps appear to be involved: when membrane fusion is initiated, an independent matrix-bound system is activated for the synchronized stretching process. The energy requirement for one discharge event is estimated to be about 14 X 10(6) ATP molecules.