Effects of cartap on isolated mouse phrenic nerve diaphragm and its related mechanism

Cartap, a nereistoxin analogue pesticide, is reported to have no irritation to eyes in rabbits. However, we have demonstrated recently that cartap could actually cause acute death in rabbits via ocular exposure. Our preliminary study with isolated mouse phrenic nerve diaphragms has shown that instead of neuromuscular blockade, cartap caused muscular contracture. The objective of the study was to examine the effect of cartap on the neuromuscular junction in more detail and to investigate its possible underlying mechanism with isolated mouse phrenic nerve diaphragms and sarcoplasmic reticulum (SR) vesicles. Cartap or nereistoxin at various concentrations was added in the organ bath with isolated mouse phrenic nerve diaphragm and both nerve- and muscle-evoked twitches were recorded. Instead of blocking the neuromuscular transmission as nereistoxin did, cartap caused contracture in stimulated or quiescent isolated mouse phrenic nerve diaphragm. Both the cartap-induced muscular contracture force and the time interval to initiate the contracture were dose-dependent. The contracture induced by cartap was not affected by the pretreatment of the diaphragm with the acetylcholine receptor blocker α-bungarotoxin; the Na+ channel blocker tetrodotoxin; or various Ca²⁺ channel blockers, NiCl₂, verapamil, and nifedipine. On the contrary, the contracture was significantly inhibited when the diaphragm was pretreated with ryanodine or EGTA containing Ca²⁺ -free Krebs solution or in combination. This suggested that both internal and extracellular Ca²⁺ might participate in cartap-induced skeletal muscle contracture. Moreover, cartap inhibited the [3H]-ryanodine binding to the Ca²⁺ release channel of SR in a dose-dependent manner. Additionally, cartap could induce a significant reduction in Ca²⁺-ATPase activity of SR vesicles at a relatively high dose. The results suggested that cartap might cause the influx of extracellular Ca²⁺ and the release of internal Ca²⁺, with subsequent induction of muscular contracture in the isolated mouse phrenic nerve diaphragm. Based on these findings, we propose that the acute death of rabbits following ocular exposure to cartap might have resulted from respiratory failure secondary to diaphragm contracture.