| Parkinson's disease (PD) is one of the most common neurodegenerative disorders, affecting almost 1% of the population over 65 years old. The pathological hallmarks and biochemical changes of PD are the loss of nigrostriatal dopaminergic neurons, the reduction of dopamine content in the striatum and the presence of intraneuronal proteinacious cytoplasmic inclusions, termed "Lewy's Bodies". Until now, the underlying mechanisms for the loss of dopaminergic neurons remain unclear although both genetic mutations and environmental factors have been identified as contributing to certain forms of this disorder. So there is lack of relatively good symptomatic therapy and medicine for PD. Hence, many studies are focused on searching for new potential therapeutic targets and agents for the treatment of PD. Recent evidence suggests that mitochondrial damage, particular to the respiratory chain of complex I (CXI, NADH/ubiquinone oxido reductase) resulting in oxidative and nitrative stress, underlies the pathology of PD. Katp channels are activated rapidly in response to the decrease of intracellular ATP/ADP ration, coupling cell metabonism and its electrical activity. So many studies revealed that ATP sensitive potassium channels related to PD.We developed a new compound iptakalim, which has beendemonstrated to be a novel Katp channel opener by pharmacological, electrophysiological, and biochemical studies. Notably, IPT is one of the KCOs that can pass through blood-brain-barrier. Our previous research have revealed that IPT could antagonize the toxicity of 6-OHDA, MPP+, Retonone. So in the base of this we identified the expression of subunits of Katp channel on PC12 cells and established the model of oxidative stress to reveal the protction of IPT on oxidative injuried of PC12 cells and investigated possible pharmacological mechanism in the present study.1. The expression of subunits of Katp channel on PC12 cellsTo determine whether SUR and Kir subunits are present in PC 12 cells, we used specific antibodies against SUR1, SUR2, Kir6.1 and Kir6.2 in Western blotting analysis and immunocytochemistry. The mRNA expression of subunits in PC 12 cells were determined by RT-PCR. PC 12 cells expressed mRNA for Kir6.1, SUR2 subunits of Katp channels. Western blotting and immunocytochemistry of PC12 cells revealed a same results, respectively.2. Effects of iptakalim on H2O2 induced cytotoxicity in PC 12 cellsNeurotoxity injury was induced by H2O2 in PC12 cells. The cell viablity was tested by MTT assay. DPT markedly mitigated H2O2-induced decrease of cell survival and morphological alteration in PC12 cells. But 5-HD can abolish part of protective effect of IPT.3. Mechanisms of iptakalim aganinst H2O2 induced neurotoxity.The glutamate release from PC12 cells was measured by HPLCcombined with fluorescent detector analysis. 10 uM IPT or 100 uM diazoxide can inhibit 40 uM H2O2 induced glutamate release increasing. Changes in the intracellular free Ca2+ concentration ([Ca2+]j) were determined in fluo-3 AM loaded PC 12 cells. The increase of [Ca2+]i induced by H2O2 can be prevented by IPT. Hoechst 33342 staining results showed that some of the H2O2-treated cell exhibited highly condensed and fragmented nuclei morphology, which were the typical characteristics of apoptosis. In contrast, the cells in the culture with 10 uM IPT or 100 uM diazoxide showed normal cell nuclei morphology. If the cells were pretreated with 10 uM IPT and 100 uM diazoxide, 40 uM H2O2-induced over-expression of Bax and lowered Bcl-2 levels were attenuated further suggesting a possible link between IPT's anti-apoptotic role and gene modulation. But the cells exposed with 5-HD together can partly abolish the effect of IPT.Conclusions:1. PC12 cells expressed mRNA and proteins for Kir6.1 and SUR2.2. IPT provided neuroprotection against H2O2 induced cytoxixity by activating mitochondrial KAtp channels decreseing the release of glutamate and intracellular free Ca2+ concentration.3. IPT exerted certain protective... |
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