The Mode of Action at the Mouse Neuromuscular Junction of the Phospholipase A‐crotapotin Complex Isolated From Venom of the South American Rattlesnake

Abstract

I 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 370C. 2 A similar concentration of the phospholipase A2 (10 jig/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,g/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 (Qlo (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 iin the neurotoxicity of the complex. 4 A myotoxic action was shown by 30 gg/ml P-C complex and 30 gg/ml phospholipase A2.S P-C complex (150 jg) was injected into the tail vein of mice and the intoxicated hemidiaphragm preparation removed for intracellular recording at 250 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 ofthe Ca-ionophore A23 187. 8 E.p.ps were recorded in partially intoxicated hemidiaphragms with (+)-tubocurarine (0.5-1.0 gg/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.

The pharmacological effect of Bothrops neuwiedii pauloensis (jararaca-pintada) snake venom on avian neuromuscular transmission

“…pauloensis venom is consistent with the available data about phospholipase A 2 neurotoxins. Crotoxin, the main neurotoxin of Crotalus durissus terrificus venom, causes a triphasic change (depression, facilitation and final blockade) of acetylcholine release by the nerve terminals (12)(13)(14)(15), similar to that observed with other snake ß-neurotoxins, such as ß-bungarotoxin (16)(17)(18), notexin (19), taipoxin (20,21), and textilotoxin (22,23). In mouse hemi-diaphragm nerve-muscle preparations, by reducing the external Ca 2+ concentration, ß-bungarotoxin classically produces an initial transient inhibition of twitches (phase 1) followed by a prolonged facilitatory phase (phase 2) and finally a blocking phase (phase 3).…”

A STUDY ON THE INTERACTION OF CROTAPOTIN WITH CROTOXIN PHOSPHOLIPASE A2, NOTEXIN AND OTHER PRESYNAPTIC NEUROTOXINS

“…These single unit phospholipase A toxins such as notexin (Halpert & Eaker, 1975;Cull-Candy et al, 1976) and notechis-5 (Halpert & Eaker, 1976) are highly active on the nerve terminal structure when given alone, whereas the basic phospholipase As of complex neurotoxins such as crotoxin (Hendon & Fraenkel-Conrat, 1971;Rubsamen et al, 1971;Breithaupt, 1976;Hawgood & Smith, 1977) and taipoxin (Fohlman, Eaker, Karlsson & Thesleff, 1976) are much less active on the nerve terminal when given alone and need the help of the other subunits in the complex to be fully active.…”

Sites of Action of Mojave Toxin Isolated From the Venom of the Mojave Rattlesnake

“…The crotoxin complex was reconstituted from its subunits (Hawgood & Smith, 1977) animals in each group were used. The toxin (2LD50) was mixed with 0.1 ml of crude antiserum or antiserum of doubling dilution, and incubated at 37°C for 30 min; 0.2 ml of the solution was injected into the tail vein and the number of mice surviving at 24 h was observed.…”

“…It was important to make a detailed examination of the sites of action of Mojave toxin, particularly in relation to the cause of death as (a) crotoxin blocks the release of transmitter from motor nerve terminals (Chang & Lee, 1977;Hawgood & Smith, 1977); (b) some respiratory impairment was present in Mojave intoxication of the anaesthetized animal (Bieber et al, 1975); (c) Mojave rattlesnake venom is poorly neutralized by the commercially available polyvalent antivenin (Wyeth) in comparison with venoms from other North American rattlesnakes (Glenn & Straight, 1977;.…”