Stimulation of cell surface phospholipase A₂ and prostaglandin synthesis in 3T3 mouse fibroblasts by phallolysin, a toxin from Amanita phalloides

Phallolysin, a cytotoxic glycoprotein from the poisonous mushroom Amanita phalloides, stimulates high levels of cellular phospholipase A₂ in 3T3 Swiss mouse fibroblasts biosynthetically labeled by incorporation of [5,6,8,9, 11,12,14,15-³H]arachidonic acid. Up to 39.9% of labeled cell lipids were hydrolyzed in 30 min with concomitant synthesis of prostaglandins (up to 5.3 and 1.7% of incorporated label released in 30 min as chromatographically identifiable prostaglandins E₂ and F_2α, respectively). A similar extent of hydrolysis of labeled phosphatidylcholine was observed in cells biosynthetically labeled with [1,2-¹⁴C]choline, with production of an equivalent amount of lysophosphatidylcholine. No detectable hydrolysis of sphingomyelin was observed, indicating (1) that phospholipases B, C and D and sphingomyelinases were not significantly activated, and (2) that the phospholipase A₂ activated did not exhibit a high degree of specificity for arachidonic acid moieties in its substrates. An analysis of the Ca²⁺ requirement for activation suggests that phallolysin activates primarily cell surface, Ca²⁺-requiring phospholipase A₂. Several lines of evidence suggest that stimulation of phospholipase A₂ by phallolysin involves binding to receptor sites which are also sites for wheat germ agglutinin. The extent of cell death, as assessed by failure to exclude trypan blue dye, correlated with the percent of maximal activation of phospholipase A₂ by a range of concentrations of phallolysin, suggesting that the toxin kills cells by inducing hydrolysis of membrane phospholipids.