摘要
通过催化抑制性G蛋白α亚基(Gαi／o)的ADP核糖基化,百日咳毒素(PTX)使Gαi／o不能活化,继而抑制Gαi／o-腺苷酸环化酶(AC)-cAMP-蛋白激酶A(PKA)-L型电压门控钙(Ca(L)2+)通道．Ca(L)2+通道可能是拟除虫菊酯类杀虫剂的靶标．本实验观察了PTX对三氟氯氰菊酯(Cy)抗性及敏感棉铃虫神经细胞Ca(L)2+通道的影响, 以期从Ca(L)2+通道动力学的角度探讨棉铃虫对拟除虫菊酯类杀虫剂产生抗性的机制．急性分离并经12-16 h无菌培养3-4龄棉铃虫神经细胞．实验分4组:①Cy敏感组(Cy-S组);②Cy抗性组(Cy-R组);③Cy-S-PTX组; ④Cy-R-PTX组．400 ng／mL PTX加入培养基中．采用全细胞膜片钳技术记录各组,ICa(L)．结果显示:(1)各组 Ca(L)2+通道的激活电压均是-35--30 mV,最大激活电压是0 mV,但Cy-S-PTX组的最大激活电压是 -10 mV;(2)分别与Cy-S组和Cy-R-PTX组峰值电流密度相比,Cy-S-PTX组峰值电流密度分别增高52．06 ％和44．81％(P<0．01),提示Cy-S-PTX组Ca(L)2+通道电导可能增大．Cy-S组和Cy-R组间的峰值电流密度无统计学差异;(3)与Cy-S组相比,Cy-S-PTX组神经细胞Ca(L)2+通道的半数激活电压向超极化方向移动5．1 mV (P<0．05),半数失活电压向超极化方向移动5．93 mV(P<0．05),提示PTX使敏感组神经细胞的Ca(L)2+通道更容易激活和失活;(4)与Cy-R组相比,Cy-R-PTX神经细胞Ca(L)2+通道的半数激活电压几乎未变,半数失活电压向去极化方向移动2．42 mV(P>0．05),提示PTX对抗性组神经细胞的影响不明显;(5)Cy-R组与Cy-S组神经细胞Ca(L)2+通道的半数激活电压和半数失活电压无显著性差异．结果提示:(1)棉铃虫神经细胞存在(Gαi／o)-AC- cAMP-PKA-Ca(L)2+通道信号传导途径;(2)PTX敏感的抑制性Gi／o蛋白的基础活性对棉铃虫神经细胞Ca(L)2+通道有影响;(3)PTX对敏感虫神经细胞的Ca(L)2+通道的影响较对抗性棉铃虫神经细胞Ca(L)2+通道的影响明显,提示抗性棉铃虫对拟除虫菊酯类杀虫剂产生抗性的机理可能与其Ca(L)2+通道对Gαi／o-AC-cAMP-PKA-Ca(L)2+通道调控不敏感有关．
The L-type Ca^2＋ Ca（L）^2＋ channels may be one of the primary targets for pyrethroid insecticides. We did the experiment to analyze the pyrethroid-resistance mechanism concerning with the signal transduction pathway of Gαi/o-AC-cAMP-PKA-L-type Ca^2＋ Ca（L）^2＋ channels and the kinetics of Ca（L）^2＋ channels. TheCa（L）^2＋ channels in the central neurons being a cutely isolated from the 3rd～4th instar larvae of the cyhalothrin-resistant （Cy-R） and cy- halothrin-susceptible （Cy-S） cotton bollworm （Hellcoverpa arrnigera, Hiibner） and incubated sterilely for 12～16 hours were studied by using the whole-cell patch-clamp techniques. Four groups, the Cy-S group, the Cy-R group, the Cy-S-PTX group and the Cy R PTX group, were studied. PTX （400 ng/mL）, a disrupting toxin of the signal transduction pathway of Gαi/o
-AC-cAMP-PKA-L-type Ca^2＋ Ca（L）^2＋ channels by catalyzing ADP ribosylatlon of the a- subunit of Gi/Go-type G proteins, was in or not in the culture medium. Results showed： （1） All of the neurons in the four groups were activated at -35～--30 mV and showed the peak Idensity at 0 mV, but the Cy-S-PTX neurons exhibited the peak Idensity at -10 mV; （2） Compared with the value of the maximum peak Idensity in the Cy-S and Cy R PTX groups, the value in the Cy-S-PTX group increased 52.06 % and 44.81% （P〈0. 01）, respectively, which means the Ca（L）^2＋ channels of the Cy-S-PTX neurons might expressed larger conductance than the others; （3） PTX significantly caused a 5.1 mV hyperpolarizing shift of Cy-S neurons in the voltage dependence of the steady-state activation and a 5.93 mV hyperpolarlzing shift of Cy-S neurons in the voltage dependence of the steady-state inactivation. Those means Cy-S-PTX neurons could be activated and inactivated more easily than Cy-S neurons; （4） PTX showed no significant effects on the Cy-R neurons;（5）No significantly difference of parameters mentioned above in Ca（L）^2＋ were detected between the Cy-R and Cy-S neurons. Our results suggested： （1） The signal transduction pathway of Gαi/o protein-AC-cAMP-PKA-Ca（L）^2＋ channels might exists in the central neurons of the cotton bollworm;（2） The basic activity of the PTX-sensitive Gαi/o protein might has effects on the Ca（L）^2＋ channels; （3） Cy-S neurons might be modulated more easily by PTX than Cy-R neurons; （4） One of the pyrethroid-resistance mechanisms might be related to some changes of the signal transduction pathway of Gαi/o protein-AC cAMP-PKA-Ca（L）^2＋or some alternations of Ca（L）^2＋ channels in Cy-R neurons.
出处
《南开大学学报（自然科学版）》
CAS
CSCD
北大核心
2006年第1期100-106,共7页
Acta Scientiarum Naturalium Universitatis Nankaiensis
基金
国家自然科学基金(30270884)
关键词
百日咳毒素
L型钙通道
Gαi/o蛋白
全细胞膜片钳技术
棉铃虫
抗性
pertussis toxin, L-type calcium channels
Gαi/o proteins
the whole cell patch clamp techniques
Helicoverpa armigera
resistance
