| phencynonate hydrochloride (PCH), a central anticholinergic agent synthesized in our institute, has been developed as a new medicine for treatment of motion sickness. Clinical application has confirmed that PCH has higher efficacy and lower central side effects in preventing motion sickness than other drugs such as Dimenhydrinate and Scopolamine HBr. Besides, previous studies showed that PCH afforded considerable anticonvulsant effect as well as partly blocked the lethal effects in mice and loss in cell viability of rat primary hippocampal neurons induced by NMDA, which strongly suggested a pharmacological profile of both anticholinergic and anti-NMDA properties of PCH. Because of one chiral carbonic atom in the molecular structure, there exist two optical isomers of PCH, the R(-) and S(+) configurations. However, no studies evaluating and comparing the potential neuroprotective effects of PCH racemate and its isomers have been performed so far.N-methyl-D-aspartate (NMDA) receptor is a crucial receptor for the neurotransmitter glutamate, which is the most important excitatory transmitter in the brain. NMDA is the well-known agonist of the NMDA receptors, the latter being named after it. NMDA receptors are vital for brain function. They are central to many of the activity-dependent changes in synaptic strength and connectivity that are thought to underlie the formation of memory and learning. By contrast, NMDA receptors can cause cell death in many neuropathological conditions when intensely or chronically activated, such as stroke, mechanical trauma or seizure. Moreover, NMDA receptor activity is thought to contribute to the aetiology of many chronic neurodegenerative disorders, such as Huntington's disease, HIV-associated dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, lathyrism, acquired immunodeficiency syndrome (AIDS), schizo phrenia and other psychiatric disorders. The toxicity on neurones caused by interactions of excitatory amino acids such as NMDA and glutamate with their receptors is termed as "excitotoxicity". Differential agents perform the anti-excitotoxicity effects through variable approaches. But it still remains unknown by what mechanism PCH achieve neuroprotective effects. The present study is performed to identify eutomer of PCH with higher protective potency and less toxicity so as to provide potential new drug for treatment of NMDA-related diseases. We comparatively investigated the protective effects of PCH racemate and its stereo isomers against NMDA-induced neurotoxicity and reveal the possible mechanisms. Meanwhile, the effects of PCH and its isomers on rats cardiovascular and respiratory systems were explored.1. Comparative effects of PCH and its optical isomers on NMDA-induced neurotoxicityTo clarify whether PCH racemate and its isomers possess anti-NMDA property, a model of NMDA-induced lethality in mice was utilized, lethality proved to be the only pharmacological end-point that was most reliably produced by administration of NMDA and was blocked by phencyclidine-like drugs and specific NMDA-defined receptor antagonists, but not by other CNS drugs. PC12 cells, derived from chromaffin cells of the adrenal medulla, have been widely used as a valuable neural model for toxicological and pharmacological studies. In the presence of nerve growth factor (NGF), PC12 cells differentiate into neuronal cells and express functional NMDA receptor. In the present study, we developed lethality model in mice and damage model in differentiated PC12 cells. Thus, in the present study lethality model of mice was developed and the in vitro part of NMDA-induced toxicity was practiced in differentiated PC12 cells.PCH racemate and its isomers can attenuate NMDA-induced toxicity both in vivo and in vitro. R(-)-PCH pretreatment significantly protected mice against NMDA-induced death, which was more potent than S(+)-PCH. PCH racemate demonstrated an equipotent best protective effects to R(-)-PCH, but required a higher dose than PCH racemate. In in vitro study, R(-)-PCH demonstrated a greater potency as a neuroprotectant than S(+)-PCH, since the viability of PC12 cells pretreated with R(-)-PCH was higher, and the LDH release was less than that pretreated with S(+)-PCH. PCH racemate showed a modest potency between R(-)-PCH and S(+)-PCH. Besides, R(-)-PCH and PCH racemate significantly promoted the cell morphological change induced by NMDA, while S(+)-PCH showed less effects.2. Mechanism of action of PCH racemate and its isomers against NMDA-induced toxicityThe possible mechanism of PCH and its isomers against NMDA-induced toxicity was studied. Activation of NMDA receptors leads to increase in [Ca2+]i, which can activate a number of pathways leading to irreversible alterations, and ultimately to cell death. Mitochondrion, which is responsible for Ca2+ sequestration and generation of reactive oxygen species (ROS), also plays important roles in survival or death of cells in excitotoxicity. Studies revealed a striking correlation between the degree of mitochondrial depolarization and cell failure to restore [Ca2+]i following toxic NMDA chanllenge. Further investigations showed that the above correlation cannot be explained by overproduction of reactive oxygen species (ROS). And the overproduction of ROS became apparent only after the deregulation of homeostasis and mitochondrial depolarization. We hypothesized that the neuroprotective effects of PCH may be closely related to the events above in excitotoxicity.To clarify whether PCH and its isomers perform the protective effects by attenuating the events above in NMDA excitotoxicity, the detection of apoptosis, [Ca2+]i, mitochondrial membrane potential and production of ROS were performed using confocal laser scanning microscope. The results revealed that PCH and its isomers can attenuate NMDA-induced deregulation of cell homeostasis. R(-)-PCH demonstrated more protective potency than S(+)-PCH in abrogating NMDA-induced apoptosis and overproduction of ROS and maintaining Ca2+ homeostasis and mitochondrial membrane potential. More important was that effects of R(-)-PCH were equipotent to MK-801, a noncompetitive antagonist of NMDA receptor. S(+)-PCH only inhibited apoptosis and ROS overproduction significantly. PCH racemate showed a modest protective potency between the two isomers. All the above strongly suggested that abrogation of [Ca2+]i overload and mitochondrial depolarization played important roles in the mechanisms of action, while inhibition of ROS overproduction alone made no sense in promoting cell viability.3. Effects of PCH racemate and its isomers on rats cardiovascular and respiratory systemsTo identify the eutomer of PCH with less toxicity, effects of PCH and its isomers on rats cardiovascular and respiratory systems were explored. Wistar rats were divided into 10 groups at random and were administered different doses of PCH racemate, R(-)-PCH or S(+)PCH, or NS. Then blood pressure, heart rate, respiratory frequency and tidal volume were recorded. Results were expressed as x|-±s, and compared with control. Results revealed that R(-)PCH,S(+)PCH and PCH racemate had no obvious effects on rats respiratory system and heart rate. R(-)PCH,S(+)PCH and PCH racemate all decreased blood pressure, but no obvious dose-effects relations were observed.Taken collectively, PCH racemate and its isomers are effective in attenuating NMDA-induced toxicity both in vivo and in vitro. R(-)-PCH, more potent than S(+)-PCH against NMDA-induced toxicity, shows protective effects equipotent to MK-801. PCH racemate demonstrate a modest protection between the two isomers. And inhibition of [Ca2+]i overload and mitochondrial depolarization might play important roles in the mechanism of protection afforded by PCH. R(-)PCH,S(+)PCH and PCH racemate. PCH and its isomers had no obvious effects on rats respiratory system and Heart rate. R(-)PCH,S(+)PCH and PCH racemate all can decrease blood pressure, but no obvious dose-effects relations were observed. |
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