Mn 2+ ions reduce the enzymatic and pharmacological activities of bothropstoxin-I, a myotoxic Lys49 phospholipase A 2 homologue from Bothrops jararacussu snake venom

Abstract

Bothropstoxin-I (BthTX-I), a myotoxic Lys49 phospholipase A 2 (PLA 2) homologue isolated from Bothrops jararacussu snake venom, causes a range of biological effects, including myonecrosis, mouse paw edema, irreversible neuromuscular blockade and lysis of cell cultures. Among eight divalent cations assayed, Mn 2+ was the most effective in reducing mouse paw edema induced by BthTX-I (25 μg). Preincubating BthTX-I with Mn 2+ (1.0 mM) reduced mouse paw edema by 70% and myotoxicity by 60% in mice injected i.m. with 50 μg toxin. Mn 2+ (50 μl of a 1 mM solution) administered within 1 min at the site of toxin injection was still but less effective in antagonising BthTX-I-induced myotoxicity. Mn 2+ (1.0 mM) completely prevented BthTX-I (1.4 μM)-induced neuromuscular blockade in the mouse phrenic-nerve diaphragm preparation. Mn 2+ (0.25 mM) protected about 85% of NB41A3 cells from lysis when coincubated with BthTX-I (1.0 μM) for 25 h. Preincubation with 0.25 mM Mn 2+ increased the sensitivity of the cells to subsequent lysis by BthTX-I in the absence of Mn 2+. BthTX-I (1 μM) caused extensive fatty acid release (from 0.8% of the total radiolabeled lipid in control cells to 56% with toxin) when incubated with the NB41A3 cell line for 25 h. PLA 2 activity observed in cell cultures after addition of BthTX-I was considerably reduced by 0.25 mM Mn 2+. Mn 2+ ions constitute a promising agent to assess the action mechanism and the effects of enzymatic inhibition on the pharmacological activity of Lys49 PLA 2 homologues.