THE MODE OF ACTION AT THE MOUSE NEUROMUSCULAR JUNCTION OF THE PHOSPHOLIPASE A‐CROTAPOTIN COMPLEX ISOLATED FROM VENOM OF THE SOUTH AMERICAN RATTLESNAKE

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Abstract
1 Phospholipase A2-crotapotin complex (P-C complex) isolated from the venom of Crotalus durissus terrificus induced an irreversible blockade of neuromuscular transmission when twitch tension was measured in the mouse phrenic nerve-hemidiaphragm preparation in vitro at 37°C. 2 A similar concentration of the phospholipase A2 (10 μg/ml) alone did not affect neuromuscular transmission and no priming action was detected on later addition of crotapotin. 3 The rate of neuromuscular blockade induced by P-C complex (15 μg/ml) was not altered by raising the frequency of nerve stimulation. Lower temperatures markedly increased the time of onset and reduced the rate of blockade (Q10 (27-37°C) of 4.4) whilst replacement of Ca by Sr in the medium prevented this activity. These latter results suggest that enzymatic activity is important in the neurotoxicity of the complex. 4 A myotoxic action was shown by 30 μg/ml P-C complex and 30 μg/ml phospholipase A2. 5 P-C complex (150 μg) was injected into the tail vein of mice and the intoxicated hemidiaphragm preparation removed for intracellular recording at 25°C. 6 In fully intoxicated hemidiaphragms, resting membrane potentials were unaltered and endplate potentials (e.p.ps) varied in average amplitude from zero to less than 3 mV. 7 Miniature endplate potential (m.e.p.p.) frequency was lower at fully poisoned endplates than at controls; the frequency rose during a 50 Hz tetanus but was unaffected by either raising external K or the application of the Ca-ionophore A23187. 8 E.p.ps were recorded in partially intoxicated hemidiaphragms with (+)-tubocurarine (0.5-1.0 μg/ml) added to prevent contraction. Evoked release was abnormal as 50 Hz tetanus elicited e.p.ps of very variable amplitude, no facilitation of response was shown to paired stimuli, and tetraethylammonium (0.5 mM) failed to increase e.p.p. amplitudes. 9 M.e.p.ps and e.p.ps were recorded at partially poisoned endplates in low Ca-high Mg solution. A reduction in the quantal content of evoked transmitter release was observed in comparison with controls. 10 M.e.p.ps recorded at partially and at fully intoxicated endplates showed an altered amplitude distribution with a higher proportion of large potentials. 11 It is concluded that P-C complex has a presynaptic site of action and may interfere with depolarization - secretion coupling at the motor nerve terminals.