The Insecticidal Mechanism Of Celangulin To The Calcium Channel Of Myocyte Of Armyworm Mythimna Seperata (Walker)

Celangulinâ…£and Celangulinâ…¤, the representatives of insecticidal components from the Chinese bittersweet,Celastrus angulatus (Max), were myotoxic to the armyworm, Mythimna seperata (Walker) and could induce flaccid paralysis and contractual paralysis respectively. Calcium plays an important role in many physiological regulative functions such as muscle contracture and the release of neurotransmitter, et al. The effects of celangulinâ…£and celangulinâ…¤on the intracellular calcium ions concentration ([Ca²⁺]i), ryanodine receptors(RyRs) of SR and L-calcium channels (DHPRs) of cytomembrane and the paths which induced the change of [Ca²⁺]i in muscle cells were studied by means of laser confocal microscope(LSM) and calcium fluorescent indicator. The results are as follows:1. Muscle cell culture of the adult armyworm, Mythimna seperata (Walker) in vitro:To explore the primary culture method of insect muscle cells from the adult armyworm to get high-quality muscle cells in vitro, we established the model of insect muscle cells for toxicological research. The muscle tissues of thorax were dissected and then digested by 0.25% trypsin and 0.1% collagenaseâ…¡. The cells were collected and cultured in Grace′s media with 5-10% fetal bovine serum at 28℃with pH value of 6.5-6.8. The morphologic characteristics of muscle cells were observed with light microscopy. Enough muscle cells were obtained and developed well with rod shape, and muscle satellite cells could develop into muscle cells which could offer more muscle cells for toxicology research.2. Effect of celangulinâ…£on [Ca²⁺]i of muscle cells of the adult armyworm:(1) [Ca²⁺]i was affected by celangulinâ…£concentration. When the concentration was at mill-molar level , [Ca²⁺]i would decrease; when it was at micromolar level, [Ca²⁺]i would have negative correlation with celangulinâ…£; while at nanomolar level, [Ca²⁺]i would have positive correlation.(2) The cytomembrane of muscle cell and the membrane of sarcoplasmic reticulum were the sites of action of celangulinâ…£. DHPRs in the cytomembrane and Ryanodine receptors (RyRs) in the sarcoplasmic reticulum membrane were the target sites of celangulinâ…£.(3) Mechanism of action of celangulinâ…£on [Ca²⁺]i : \â…°. At lower concentration (1nmol/L to 1μmol/L), Celangulinâ…£would activate DHPRs in the cytoplasma of muscle cells and allow Ca²⁺ influx into the cytoplasm. The conformational change of DHPRs would lead to RyRs to open and release Ca²⁺ from the sarcoplasmic reticulum (SR) store; the entry of Ca²⁺ would activate RyRs, allowing Ca²⁺ release from the SR store. The increase of Ca²⁺ influx into the cytoplasm and Ca²⁺ release from the SR store would induce the growth of [Ca²⁺]i and in turn disturb the functions of exciting contraction(E-C) coupling. The intracellular Ca²⁺ overload would result in imbalance of calcium homeostasis, which would cause cellular injuries, apoptosis and necrosis.â…±. At millimole level, celangulinâ…£would inactivate or close DHPRs, The conformational change of DHPRs would lead to RyRs to close or inactivate. The decrease of Ca²⁺ influx into the cytoplasm and Ca²⁺ release from the SR store would cause the decrease of [Ca²⁺]i and in turn disturb the functions of E–C coupling, resulting in the flaccid paralysis and the imbalance of calcium homeostasis, which would cause the cellular injuries, apoptosis and necrosis as well .(4) Celangulinâ…£and nifedipine would competitively combine with DHPRs on same sites, but Calangulinâ…£and caffeine would have different binding sites on RyRs.3. Effect of celangulinâ…¤on [Ca²⁺]i of muscle cells of the adult armyworm:(1) The concentration of celangulinâ…¤(nanomole to millimole) would have positive correlation with [Ca²⁺]i. and negative correlation with time.(2) The cytomembrane of muscle cell and the sarcoplasmic reticulum membrane would be the sites of action of celangulinâ…¤. DHPRs in the cytomembrane and RyRs in the sarcoplasmic reticulum membrane would be the sites of celangulinâ…¤target.(3) Mechanism of action of celangulinâ…¤on [Ca²⁺]i : Celangulinâ…¤would activate the DHPRs, and DHPRs would activate RyRs in the form of mechanical and functional coupling. In turn, Ca²⁺ influx into the cytoplasm and Ca²⁺ exflux from the SR calcium store would increase the total of [Ca²⁺]i. The insect muscles would continuously contract and lead to contractual paralysis, and cause the depletion of energy indirectly. In the end, the imbalance of calcium homeostasis would lead to the killing of insect.(4) Celangulinâ…¤and nifedipine would competitively combine with DHPRs at the same binding sites. Calangulinâ…£and caffeine would have different binding sites on RyRs.