Study On The Effects And Electrophysiological Mechanisms Of 3-Butylphthalide On The Tandem Pore-domain K~+ Channels TREK-1 And Relevant Pharmacological Studies In Rat Cerebral Hypoperfusion

Two-pore domain potassium channels (K2P) make up the newest branch of the potassium channel super-family. Their structure motif is quite different from classic potassium channels with 4 transmembrane segments (M1-M4) and 2 pore domains (P1, P2). Currently, there are known to be at least 17 mammalian channels in the K2P channel family and can be divided into TWIK、THIK、TASK、TALK、TREK and TRESK six subfamilies on the basis of structural and functional similarities.TREK-1, one of the TREK subfamilies, is the most thoroughly studied K2P channel which highly expressed in human central nervous system. The channel produces currents with unusual characteristics. They are quasi-instantaneous and noninactivating, and they are active at all membrane potentials and insensitive to the classic K+ channel blockers (such as TEA,4AP, Cs+). Moreover, recent many studies have demonstrated this channel can regulated by a wide range of physiological and pharmacological mediators and has a key role in the mechanisms of cellular neuroprotection. In this light we have investigated the ability of a number of well-characterised neuroprotective agents to modulate the activity of TREK-1 channels.Racemic 3-n-butylphthalide (NBP) was synthesized and developed as an anti-cerebral ischemic agent, which was approved by SFDA of China in November 2002. The previous studies in our group had proved that racemic-NBP was an effective drug for treatment of ischemic stroke with multiple actions. However, whether racemic NBP affect TREK-1 channel is not characterized, is TREK-1 channel a new target for racemic NBP treat ischemic stroke? Little is known about it. Here we first investigated the action of NBP on TREK-1 channel activity and some related mechanisms, and then we have set up rat bilateral carotid artery ligation (BCAL) to investigate the effects of 1-3-n-butylphthalide on expression changes of TREK-1 gene and protein in rat cerebral hypoperfusion.Part I. Investigation of the effects of 3-n-butylphthalide on TREK-1 K+ channel in CHO cells stably expressing TREK-1 and revalent mechanisms studies1. Effects of 3-n-butylphthalide on tandem pore-domain K+ channels TREK-1At the first, the basic properties of TREK-1 channels were characterized. We measured the membrane potential (MP) of TREK-1/CHO cells under physiological conditions. TREK-1/CHO cells had resting membrane potentials of -55±2.36 mV (n= 25). Wild-type CHO cells had resting membrane potentials of -17.6±4 mV (n=7). The difference between the membrane potentials was statistically significant (P<0.001). Moreover, TREK-1/CHO cells displayed a large outward and noninactivating current (0.5-3 nA), which were absent in wild-type CHO cells. Results show that 10μM AA can open TREK-1 channel by 50-70%(n= 6)L-NBP, dL-NBP and d-NBP can produced a concentration-dependent inhibition of transfection TREK-1 channels in Chinese hamster ovary (CHO) cells. The IC50 for dl-NBP and d-NBP block was 3μM and 10μM respectively, L-NBP was a more potent inhibitor of TREK-1 currents with an IC50 of 0.42μM. Block by racemic NBP were voltage-independent; NBP can depolarize MP reduced about 11 mV (n=42).We also checked the effects of three NBP isomers on the resting potential of TREK-1/CHO, L-NBP can depolarize MP from -55.6±2.36 mV to -42.9±2.06 mV (n=25), No similar effects were found in wild-type CHO cells.2. Investigation of the Mechanisms of L-NBP action on TREK-1 K+ channelWe studying the effect of AA and L-NBP on the transfected TREK-1 channel in CHO cells; Effect of intracellular dialysis 10μM L-NBP on the transfected TREK-1 channel in CHO cells. Because TREK-1 is a thermosensitive channel, the effect of temperature changes on TREK-1 currents and membrane potential were also characterized.Our results show that the effects that L-NBP inhibite TREK-1 channel may be not related to AA and the changes of temperature. The results of Intracellular dialysis show that L-NBP inhibite TREK-1 channel at the least from intercellular inhibition.When the temperature of external solution increased from 25℃to 35℃, TREK-1 current and the resting membrane potential of CHO and TREK-1/CHO were also increased significantly. The resting membrane potential of TREK-1/CHO can depolarize after L-NBP applying. No changes were found in CHOPartⅡ. Effects of Chiral 3-n-butylphthalide on expression changes of TREK-1 in rat cerebral hypoperfusion1. Effects of L-NBP on neuronal morphology in rat cerebral hypoperfusionHistological examination was carried out on HE and Nissel-stained slices of rat brain on day 3 after permanent bilateral ligation of common carotid arteries. In vehicle group, typical neurodegenerative changes (such as neuronal loss, shrinkage and dark staining) were observed in the cortex, hippocampus and striatum. Administration of L-NBP at 30 mg/kg can attenuate in part the neurodegenerative changes seen in vehicle group demonstrating their protections on the neurons. There no pathological abnormalities were seen in sham group and L-NBP control group.2. Effects of L-NBP on expression changes of TREK-1 in rat cerebral hypoperfusionIn the present study, Real-time PCR and Western blot analysis were employed to investigate the mRNA and protein expression changes of TREK-1 rat cortex, hippocampus and striatum. Results showed that mRNA and protein expressions of the membrane potassium channels TREK-1 in cortex, hippocampus and striatum increased significantly after permanent bilateral ligation of common carotid arteries (P<0.05). But there are not any mRNA and protein expressions changes between sham group and sham +L-NBP control group after cerebral hypoperfusion. Administration of L-NBP at 30 mg/kg, the mRNA and protein levels of TREK-1 was decreased, especially in striatum.. These results demonstrating that L-NBP might inhibite the mRNA and protein expressions of TREK-1 channels. Conclusion:1. TREK-1 can stabilize the membrane potential of neurons and have a major role in the regulation of membrane excitability.2. L-NBP, dL-NBP and d-NBP can produced a concentration-dependent inhibition of transfection TREK-1 channels in Chinese hamster ovary (CHO) cells. L-NBP are potent inhibitors of TREK-1. Block by racemic NBP were voltage-independent.3. Rracemic NBP can depolarize the resting membrane potential of TREK cells, No similar effects were found in wild-type CHO cells.4. Intracellular dialysis and previous results show that L-NBP inhibite TREK-1 channel from intercellular and extercellular inhibition.5.10μM L-NBP can attenuate in part the effect of AA on the transfected TREK-1.6. When the temperature of external solution increased, TREK-1 current and the membrane potential of two cells were also increased significantly. L-NBP can depolarize the resting membrane potential of TREK-1/CHO, No changes were found in CHO7.30 mg/kg L-NBP can in part attenuate the neurodegenerative changes seen in vehicle group demonstrating their protections on the neurons.8. Real-time PCR and Western blot analysis showed that mRNA and protein expressions of the membrane potassium channels TREK-1 in cortex, hippocampus and striatum increased significantly after permanent bilateral ligation of common carotid arteries. This suggested that TREK-1 channels are closely related with rat cerebral ischemia.9. Results suggested that the mRNA and protein expressions of the membrane potassium channels TREK-1 in cortex and hippocampus, especial in striatum decreased significantly after administerd 30 mg/kg L-NBP. L-NBP inhibite TREK-1 channels which in GABA neurons that might also play an important role in neuroprotection.