CAJ | 학술논문

霍乱毒素(CTX)可激活兴奋性异三聚体G蛋白(Gαs)的α-亚基和刺激电压门控L-型钙通道,而昆虫的L-型钙通道可能是拟除虫菊酯类杀虫剂的作用靶点.为进一步探讨农业害虫对拟除虫菊酯类杀虫剂产生抗药性的作用机理,我们检测了CTX对三氟氯氰菊酯抗性及敏感棉铃虫Helicoverpa armigera中枢神经细胞电压门控L-型钙通道的调节作用.分别急性分离三氟氯氰菊酯抗性及敏感的3～4龄棉铃虫幼虫胸腹神经节细胞,并在改良的L15培养基(加入或未加入700 ng/mL的CTX)中培养12～16 h.钡离子为载流子,应用全细胞膜片钳技术记录电压门控L-型钙通道电流.结果显示,CTX可使敏感组棉铃虫神经细胞L-型钙通道的峰值电流密度增大36%、峰值电压左移5 mV,但对抗性组棉铃虫神经细胞L-型钙通道无上述作用.并且,CTX对敏感组及抗性组棉铃虫神经细胞L-型钙通道的激活电位、翻转电位、激活曲线和失活曲线等其他一些参数的影响也不明显.在无CTX作用时,所检测到的抗性组与敏感组棉铃虫神经细胞L-型钙通道的上述参数值间差异不显著.结果提示,棉铃虫神经细胞内存在Gs-腺苷酸环化酶(AC)-cAMP-蛋白激酶A (PKA)-L-型钙通道信号调节系统;与敏感棉铃虫神经细胞L-型钙通道相比,三氟氯氰菊酯抗性棉铃虫神经细胞L-型钙通道的活性相对不易受到CTX调节,这可能与昆虫对拟除虫菊酯产生抗药性的机理有关.
곽란독소(CTX)가격활흥강성이삼취체G단백(Gαs)적α-아기화자격전압문공L-형개통도,이곤충적L-형개통도가능시의제충국지류살충제적작용파점.위진일보탐토농업해충대의제충국지류살충제산생항약성적작용궤리,아문검측료CTX대삼불록청국지항성급민감면령충Helicoverpa armigera중추신경세포전압문공L-형개통도적조절작용.분별급성분리삼불록청국지항성급민감적3～4령면령충유충흉복신경절세포,병재개량적L15배양기(가입혹미가입700 ng/mL적CTX)중배양12～16 h.패리자위재류자,응용전세포막편겸기술기록전압문공L-형개통도전류.결과현시,CTX가사민감조면령충신경세포L-형개통도적봉치전류밀도증대36%、봉치전압좌이5 mV,단대항성조면령충신경세포L-형개통도무상술작용.병차,CTX대민감조급항성조면령충신경세포L-형개통도적격활전위、번전전위、격활곡선화실활곡선등기타일사삼수적영향야불명현.재무CTX작용시,소검측도적항성조여민감조면령충신경세포L-형개통도적상술삼수치간차이불현저.결과제시,면령충신경세포내존재Gs-선감산배화매(AC)-cAMP-단백격매A (PKA)-L-형개통도신호조절계통;여민감면령충신경세포L-형개통도상비,삼불록청국지항성면령충신경세포L-형개통도적활성상대불역수도CTX조절,저가능여곤충대의제충국지산생항약성적궤리유관.
Cholera toxin (CTX) activates the α-subunit of stimulatory heterotrimeric G-proteins (Gαs) and stimulates voltage-gated L-type (Ca2+(L)) channels, which may be primary targets of pyrethroids. To investigate the potential mechanisms underlying the resistance to pyrethroids in agriculturally important insect pests, we examined the modulations by CTX of Ca2+(L) channels in the central neurons of the cyhalothrin-resistant (Cy-R) and cyhalothrin-susceptible (Cy-S) cotton bollworms (Helicoverpa armigera). Neurons were isolated from the 3rd-4th instar larvae of the Cy-R and Cy-S cotton bollworms, respectively. The isolated neurons from each group were cultured for 12-16 h in an improved L15 insect culture medium with or without CTX (700 ng/mL). Barium currents (IBa) through Ca2+(L)channels were recorded by the whole-cell patch-clamp technique. The results showed that CTX increased the IBa peak current density by 36.1 % and caused a hyperpolarizing shift by 5 mV in the I-V curve in the Cy-S neurons, but had no such effect in the Cy-R neurons. Moreover, CTX exerted little effects on other parameters such as the activation potential, reverse potential, activation and inactivation curves in either Cy-S or Cy-R neurons. No significant differences of the parameters mentioned above in the Ca2+(L) channels were detected between the Cy-S and Cy-R neurons cultured without CTX. The results suggest that the Gs-adenylyl cyclase (AC)-cAMP-protein kinase A (PKA)-Ca2+(L) channel signal transduction pathway may exist in cotton bollworm neurons, and the reduced sensitivity of Ca2+(L)channels to the CTX modulation in the Cy-R neurons (but not in the Cy-S neurons) may account for the reduced nerve sensitivity in the pyrethroids-resistant insects.