LYSOSOMOTROPIC AGENTS ACTIVATE THE CAPACITY FOR CALCIUM-DEPENDENT PINOCYTOSIS IN STARVED AMEBA-PROTEUS - EVIDENCE FOR A MECHANISM INVOLVINGPHOSPHOLIPASE-A(2)

Pinocytosis induced by Na+ was assayed by phase contrast microscopy in 8-12 days starved Amoeba proteus. These cultures were inactive with r espect to calcium-dependent Na+-induced pinocytosis, but treatment wit h amino acid methyl and ethyl esters increased their capacity for pino cytosis. Besides promoting pinocytosis these compounds also stimulated calcium-sensitive secretion of lysosomal enzymes from normal, 2-3 day s starved, cells. Only uncharged 1-forms of the amino acid esters were effective. Also other lysosomotropic compounds including monodansylca daverine, glycine-phenylalanine-2-naphthylamide, NH4Cl, and the ionoph ores monensin and A23187 activated starved cells. The effect of these agents (except A23187) was inhibited by the drug dantrolene suggesting that activation is a consequence of release of Ca2+ from intracellula r cellular stores. Several of the lysosomotropic agents also lost thei r activating effect in the presence of phospholipase A(2) (PLA(2)) inh ibitors. To investigate whether or not PLA(2) activity in the cell cul ture could imitate the effect of the lysosomotropic agents, we incubat ed starved cells with snake venom PLA(2)s. These enzymes caused rapid, dantrolene-sensitive activation of the cells. Measurement of endogeno us PLA(2) in ''normal'' cells revealed significant cellular activity b ut no significant secretion of the enzyme into the culture medium was observed. Together the studies with enzyme inhibitors and dantrolene s uggest that the process by which lysosomotropic agents affect pinocyto sis involves activation of PLA(2) and release of Ca2+ from intracellul ar stores.