G-蛋白偶联受体与信号通路对紫贻贝幼体附着变态的调控作用

Primary study on the regulation of G-protein coupled receptors and signal pathways in the larval settlement and metamorphosis of the blue mussel Mytilus galloprovincialis

摘要:

为了阐明紫贻贝幼体附着变态的内在机理,在天然诱导物-微生物膜的诱导参照下,通过使用G蛋白偶联受体(GPCRs)和信号通路的活化剂与抑制剂初步研究了其对该物种幼体附着变态过程的调控作用。结果显示,GPCRs活化剂Gpp[NH]p与抑制剂GDP-β-S没有诱导和抑制活性,表明可能非GPCRs的其他细胞反应过程参与了紫贻贝幼体的附着变态。腺苷酸环化酶/环腺苷酸(AC/cAMP)信号通路相关、理论上具有诱导活性的6种神经性试剂,除了磷酸二脂酶抑制剂(IBMX)外,其他在10^-8~10^-3 M浓度下48、72 h后都没有活性,并且高浓度下表现出较高的神经性药物毒性。IBMX仅在10^-4M 浓度72 h后呈现出一定的诱导活性,但是与微生物膜48 h就具有非常高的活性相比显著性较低且时间滞后。磷脂酰肌醇/二酰基甘油/蛋白激酶(PI/DAG/PKC)信号通路相关的2种活化剂48、72 h均无诱导活性,但是其抑制剂H-7在10^-6 M的低浓度下48 h后表现出显著性的抑制效果,72 h后效果丧失,抑制浓度上升到10^-5M。以上活性浓度与作用时间2个方面的结果表明了PI/DAG/PKC信号通路可能参与调控紫贻贝幼体的附着变态,而AC/cAMP信号通路可能作为次要通路,或是由于所用神经性试剂的毒性造成的一种实验假象。本研究有助于推进该品种的发育生物学研究以及通过切断信号通路实现生物污损防治技术的开发。

Abstract:

To investigate the internal mechanism of the larval settlement and metamorphosis of the blue mussel Mytilus galloprovincialis,the roles of G-protein coupled receptors (GPCRs) and signal pathways were primarily studied by using their activators and inhibitors together with the inducing control of natural inducer-biofilms.The results showed that the activator of GPCRs (Gpp[NH]p) and the inhibitor (GDP-β-S) did not have inductive and inhibitive activities,respectively.This indicated that other cellular action or processes not related to GPCRs may be involved in the larval settlement and metamorphosis of the blue mussel M.galloprovincialis.Six kinds of neuro-pharmaceutical agents except IBMX,which are related to the AC/cAMP pathways and theoretically have inductive activities,did not have active effects at the concentrations of 10^-8-10^-3 M after 48 and 72 h and even presented relatively high neuro-pharmaceutical toxicities.IBMX presented certain inductive activity at the concentration of 10^-4M after 72 h,whereas this was significantly lower and hysteretic compared to that of biofilms which had very high activity after 48 h.Two kinds of activators related to PI/DAG/PKC pathway did not have inductive activity after 48 h and 72 h,but the inhibitor,H-7 showed significant inhibitive effect at the low concentration of 10^-6 M.This inhibitive effect of H-7 disappeared after 72 h and the inhibitive concentration increased to 10^-5 M.By comparing the effective concentrations and time,PI/DAG/PKC signal pathway was confirmed to involve in and regulated the larval settlement and metamorphosis of M.galloprovincialis,whereas AC/cAMP signal pathway may be the minor pathway or just the experimental false result due to the toxicity of the agents.Moreover,the present study utilized biofilms but not artificial inducers which can demonstrate the internal mechanism of larval settlement and metamorphosis under natural conditions.This can also benefit the study of the developmental biology of this species and realize the technique development of anti-biofouling by cutting the signal pathway.