Investigation On The Mechanism Of Neuropharmaceutic Action Based On Voltage-gated Ion Channel Kinetics

In the new century, one of the serious social problems is the growing percentage of the aged population in the world, and most of the elder have been suffered by the age diseases such as Alzheimer's disease (AD), which is a progressive degenerative disease of the brain from which there is no recovery. AD is the most common form of dementia, which primarily affects older people. Dementia is a loss of intellectual function (cognition and memory) so severe that it interferes with an individual's daily activities and eventually results in death. AD is the fourth leading cause of death in adults, after heart disease, cancer and stroke. The causation of Alzheimer's disease are unknown. However, scientific research has begun to point in several directions. Neurotransmitter deficits have been implicated, with a deficiency of the neurotransmitter acetylcholine being a prominent and consistently identified deficit. The cerebral cholinergic neurotransmission has been abundantly targeted for drug development in AD in view of the well-characterized degeneration of the acetylcholine (ACh) synthesizing neurones. Available treatments of AD produce an increase in the ACh levels by inhibiting the enzymes responsible for ACh breakdown (cholinesterases). Acetylcholines-terase inhibitors (AChEI) was approved in clinical AD treatment, some are being studied in clinical trials to find out whether they can slow the progression of the disease or improve memory for a period of time. Tacrine(THA) was approved by FDA in September 1993 for the treatment of mild to moderate Alzheimer's disease. With the collaboration of researchers from China, America, Hong Kong and Taiwan, a novel acetylcholinesterase inhibitor, bis(7)-tacrine was synthesized and applied to clinical trial to treat AD. Bis(7)-tacrine [bis(7)-tetrahydroaminacrine] is a dimeric acetylcho-lineesterase inhibitor (AChEI), in which two tacrine molecules are linked by a heptylene chain. It has been identified that bis(7)-tacrine is more potent (up to 150 fold) and more selective (around 250 fold) in inhibiting AChE than tacrine, the first anticholinesterasic drugs approved by FDA for the palliative treatment of Alzheimer's disease. Bis(7)-tacrine was also proved to effectively reverse AF64A-induced deficits in navigational memory in rats. Bis(7)-tacrine has been considered to be a multi-functional/multi-target AChE inhibitor, which can act on many targets: enzymes, neurotransmitter receptors and also ion channels. Besides its inhibitory effect on AChE, bis(7)-tacrine is capable to exert modulatory actions on several kinds of ligand-gated ion channels, including GABAAR in rat hippocampal neurons, nAChR in Torpedo electric organ, 5-HT3R in rat trigeminal ganglion neurons and NMDAR on rat hipocampal neurons. The present study was aimed to explore whether bis(7)-tacrine is able to modulate voltage-gated potassium and calcium channel as tacrine to do in rat primary sensory neurons, and it does so on the Kv4.2 potassium channel expressed on Xenopus laevis oocyte, too. In the whole-cell patch experiments, it has been identified that on the preparation of dorsal root ganglion (DRG) neurons, the suppression by bis(7)-tacrine of voltage-gated potassium channels including both transient (IA) and sustained (IKD) potassium channels and L-type calcium channel was much more potent than that by tacrine. The two-electrode voltage clamp experiments were carried out with the drugs of bis(7)-tacrine and tacrine, and USB-based oocyte voltage clamp amplifier was designed for the experiment on the expressed Kv4.2 potassium channel on oocyte. The data and results obtained in the present study were not reported elsewhere. (1) In our present investigation, the first domestic oocyte two-electrode voltage clamp amplifier was designed and developed based USB (Universal Serial Bus) interface, which has features of low noise, low drift, and fast response time for recording from high impedance electrolyte-filled glass electrodes. An USB-based fast high-resolution data acquisition and analyzed system was implemented suitable for the amplifier data recording studies in the research lab. It offers 12-bit 8 analog input channels and 8-bit digital to analog output channels, interfaced to PC via USB, so setup is plug-and-play easy. (2) Using the two-electrode oocyte voltage clamp amplifier system, Kv4.2 mRNA was microinjected into defolliculated Xenopus laevis oocytes to express transient potassium channel. Bis(7)-tacrine and tacrine were applied to expressed transient potassium channels, It was observed that both drugs exert inhibitory action on have inhibitive effect on transient potassium channel current IA. Bis(7)-tacrine was found more potent in inhibiting of IA than tacrine, and the inhibition of IA (IC50=1.12 ±0.04 μM(n=7)) by bis(7)-tacrine was almost two orders more potent than that by tacrine (IC50=121.5 ±4.4 μM(n = 7)). (3) The next experiment was performed on neurons acutely isolated from rat dorsalroot ganglion (DRG) to explore the modulation of both drugs on potassium channel. Potassium currents including transient (IA) and sustained (IKD) components were recorded using whole-cell patch clamp technique. Both IA and IKD were suppressed by bis(7)-tacrine, a novel dimeric AChE inhibitor, which are much more potent (IC50 = 21.1 ±2.3 μM for IKD, IC50 = 0.625 ±0.04 μM for IA) than that by tacrine (IC50 = 2.11 ±0.25 mM for IKD, IC50 = 73.5 ±4.4 uM for IA). It was identified that bis(7)-tacrine (1uM) shifts steady state activation curve and inactivation curve of IA and IKD to the hyperpolarizing direction. In the presence of bis(7)-tacrine no change is in time constant of rising phase (τon) but that in decay phase (τdec) of IA, which decays down more slowly evidently, and the recovery curve of IA shifts downwards significantly. These results suggest that the inhibition by bis(7)-tacrine of IA and IKD may be attributed to reduction of potential ranges of steady state activation and inactivation and delay of decay phase as well as recovery from inactivation of IA. (4) The effects of drugs on calcium channel were identified on neurons acutely isolated from rat dorsal root ganglion (DRG). Calcium channel currents including L-type, N-type and P/Q-type components were recorded using whole-cell patch clamp technique. The drug inhibitory effect on L-type is the most potent, while that on other types is weak. L-type was suppressed by bis(7)-tacrine, a novel dimeric AChE inhibitor, which are much more potent (IC50 =1.077 X10-7M) than that by tacrine (IC50 =2.92 X10-5M). These results suggest that the inhibition by bis(7)-tacrine of may be attributed to reduction of potential ranges of steady state activation and inactivation of calcium channel, therefore block associated increases in Ca2+ influx, calcium overload, hyperexcitability of the neurons. From these experimental data and results, it raise the intriguing possibility by bis(7)-tacrine that reduction of outward K+ current and Ca2+ influx may play a pivotal role on alleviation of AD owing to its neuro-protection.