Effect And Mechanism Of The Inhibition Of Mitochondrial Respiratory Chain Complex Ⅲ On High Voltage Gated Calcium Channels In Rat Cortical Neurons

Mitochondria are the most important organelles in neurons to generate ATP, and the production of ATP relys on the function of the respiratory chain complexes. Under hypoxia, the function of mitochondria and the respiratory chain is inhibited. The inhibition of the function of mitochondria and the respiratory chain leads to the decrease in the production of ATP and the increase in the production of the reactive oxygen species (ROS). Such changes, especially the production of ROS further leads to the changes in gene transcription, changes in the function of ion channels, changes in synaptic transmission and changes in intracellular environments. Among these changes, we are interested in the function of votage-gated calcium channels after the inhibition of respiratory chain complex III.Researches show that the respiratory chain complex III is the major site of ROS production during hypoxia. What is more, it is also indicated that complex III acts as an oxygen sensor during hypoxia. All these results indicate the important role of complex III during hypoxia. Our previous work showed that the inhibition of complex III exerted regulatory effect on the voltage-gated sodium channel. As the voltage-gated sodium channel is related to the excitibality of the neurons, the regulation of sodium channel by the complex III became one pathway of its regulatory effect on the cell viability during hypoxia.Another important question is whether the inhibition of the complex III may affect the function of the voltage-gated calcium channels. This is a question of significant importance because it is widely accepted that the voltage-gated calcium channels can directly influence the intracellular calcium concentration in neurons during hypoxia. Considering the important role of intracellular calcium concentration in determining the neuronal function and survival during hypoxia, it is of great significance to study the relationship between calcium channels and the inhibition of the complex III and the underlying mechanisms.In this study, we used the selective inhibitor of the Qi center and the Q。center■—Antimycin A and Myxothiazol——to mimic the inhibition of the complexⅢunder hypoxia. And we further used the whole cell patch-clamp method to investigate the functional change of the voltage gated calcium channel under such conditions. The results showed that the selective inhibitor against the Qi center——Antimycin depressed the function of voltage-gated calcium channel while Myxothiazol enhanced it. Our study also showed that scavenger of hydrogen peroxide and the inhibitor of hydroxyl radical formation could abolish the inhibitory effect of Antimycin A on calcium channel, while the effect of Myxothiazol was abolished by superoxide scavenger. Further study showed that wide spectrum PKC inhibitor Chelerythrine could abolish the effect of Antimycin A and Myxothiazol. Further mechanism study about Antimcyin A showed that the classic PKC inhibitor Ro318220 could abolish the effect of Antimycin on calcium channel and CGP53353 4μM (inhibitor of PKCβsubtype) could also abolish the effect of Antimycin A, but CGP 53353 0.5μM (inhibitor of PKCβⅡsubtype) couldn't abolish the effect of Antimycin A. In addition, activator of classic PKC Thymeleatoxin could mimic the effect of Antimycin A on calcium channel. All these results indicated that Antimycin A inhibited calcium currents via H2O2-hydroxyl radicals/cPKC (mainly PKCβⅠ) pathway. As for the mechanism study about Myxothiazol, the results showed that the inhibitor of PKCδsubtype could abolish the effect of Myxothiazol on calcium channel. In addition, the activator of PKCδsubtype could mimic the effect of Myxothiazol on calcium channel. All these results indicated that Myxothiazol increased calcium currents via superoxide anion/nPKC (mainly PKCδ) pathway.Calcium overload has always been thought to lead to the cell death during hypoxia. As showed by our study, Antimycin A could inhibit the calcium currents and this would alleviate the calcium overload. Therefore it is likely that Antimycin A may have protective effect on neurons during hypoxia. On the other hand, Myxothiazol enhanced the calcium currents and this would increase calcium overlad. Therefore Myxothiazol might have injurious effect on neurons during hypoxia. To test this possibility, we studied the effect of Antimycin A and Myxothiazol on neurons in the oxygen and glucose deprivation (OGD) model by using the cell survival ratio and LDH release as indexes. The results showed that Antimycin A could protect the neurons against OGD while Myxothiazol increased neurons susceptibility to OGD.