LYS49 phospholipase A2 myotoxins lyse cell cultures by two distinct mechanisms

Three Lys49 phospholipase A2 (PLA2) myotoxins from Agkistrodon contortrix laticinctus (ACLMT), Bothrops jararacussu (bothropstoxin-I) and Bothrops asper (myotoxin II) snake venoms are enzymatically inactive on artificial substrates, yet addition of these toxins to cell cultures causes the release of fatty acids derived from the hydrolysis of membrane phospholipids. Bothropstoxin-I treated with p-bromophenacyl bromide is no longer enzymatically active on cell cultures, suggesting the toxin, not tissue PLA2, may hydrolyze the phospholipids. The NB41A3 cell line is sensitive to lysis by ACLMT by two separate mechanisms. The first mechanism is predominant at lower concentrations of ACLMT (0.1-0.5 microM) and over long incubation periods (24 h) with toxin. This mechanism is antagonized by methylprednisolone (MePDN). The second is predominant at higher concentrations of toxin (1-5 microM) incubated over a short period (1 h) and is not antagonized by MePDN. There is no correlation between enzymatic activity and toxicity at the higher concentrations (5 microM; 1 h) when the enzymatic activity of ACLMT is compared with a noncytolytic PLA2 from Naja naja atra venom (1 microM). However, over a 24 h period, triglyceride formation relative to fatty acids remaining free is about 10-fold greater for ACLMT (ratio about 40:1) than for the PLA2 from Naja naja atra venom (ratio about 4:1), suggesting the two enzymes act on substrates associated with different cellular compartments under this condition. Therefore, two mechanisms of Lys49 PLA2-induced myonecrosis exist and these are dependent on toxin concentration. The MePDN-sensitive mechanism associated with triglyceride accumulation correlates with myotoxicity.