The Protective Roles And Mechanisms Of Novel ATP-sensitive Potassium Channel Opener Iptakalim On MPP～+-induced Cytotoxicity In SH-SY5Y Cells

Parkinson's disease (PD) is one of the most common neurodegenerative movement disorders, affecting approximately 2% of the population older than 55 years old, among which 95% of PD cases are sporadic. The pathological hallmarks and biochemical changes of PD are the loss of nigrostriatal dopaminergic neurons, the reduction of dopamine (DA) content in the striatum and the presence of intraneuronal proteinacious cytoplasmic inclusions, termed "Lewy Bodies". Up till now, the underlying mechanisms for the loss of dopaminergic neurons remain unclear. Lines of evidence have revealed that the pathogenesis of cell death in PD is associated with excitotoxicity, energy defects, and oxidative damage processes. Therapeutic strategies derived from these, such as L-DOPA administration, are essentially symptomatical or accompanied with serious side effects, and cannot stop the progressive loss of dopaminergic neurons. Hence, many studies are focused on searching for new potential therapeutic targets and agents for the treatment of PD. Multiple studies, including several from our lab, have revealed that ATP-sensitive potassium channel (K_ATP channel) may be a promising target for neuroprotection.K_Atp channels are activated rapidly in response to the decrease ofintracellular ATP/ADP ration, coupling cell metabonism and its electrical activity. Our recent research has revealed that iptakalim (IPT) exhibits significant neuroprotection—not only in promoting behavioral recovery but also in protecting neurons against necrosis and apoptosis in different animal models of stroke, such as Parkinson's disease, as well as in cultured cells. IPT is a lipophilic para-amino compound with low molecular weight, which can freely cross the blood-brain barrier and has been demonstrated to be a novel KAtp channel opener (KCO) by pharmacological, electrophysiological and biochemical studies, and receptor binding test. Our previous studies found that both novel KAtp channel opener IPT and diaxozide were able to alleviate the symptoms, such as paramyotonia, hypolocomotion of PD model rats induced by several neurotoxins including 6-hydroxydopa (6-OHDA) and rotenone. However, the underlying mechanisms are undefined.The present study is undertaken to investigate whether IPT could protect human dopaminergic neuroblastoma SH-SY5Y cells against MPP+-induced cytotoxicity, and if so, the underlying mechanisms for this beneficial effects. Simultaneously, we aimed to accumulate theoretical and experimental evidence for developing IPT as a drug for the treatment ofPD.We firstly tried to explore exact molecular formation of KAtp channels in SH-SY5Y cells by RT-PCR and western blot, which may offer useful information on the study of KATP channels. The results confirmed that SH-SY5Y cells expressed the mRNA and proteins for Kir6.1, Kir6.2, SUR1 and SUR2. We also found that MPP+ down-regulated mRNA of SUR1 and up-regulated mRNA of Kir6.2 inSH-SY5Y cells, while not affecting the mRNA expression of either Kir6.1 or SUR2. Pre-incubation with IPT prevented the downregulation of SUR1 mRNA, but did not influence the upregulation of Kir6.2 induced by MPP+. Moreover, we found that both pinacidil and IPT could alleviate the cell injury induced by MPP+ by MTT method. The KAtp channel blocker either glibenclamide or 5-HD prevented the protective roles of KCOs. Additionally, pretreatment with NMDA receptor antagonist MK-801 could also protect against MPP+ induced cell.injury, suggesting that glutamate mediated excitotoxicity might be actively participated in the neurotoxicity of MPP+. Then, it was further investigated whether the beneficial effects of KCOs was related to the anti-excitotoxicity. The data indicated that either pinacidil or IPT could decrease the extracellular glutamate level of SH-SY5Y cells exposure to MPP+, which could be antagonized by glibenclamide and 5-HD. These results revealed that the roles of decreasing extracellular glutamate level of KCOs might be mediated by either hyperpolorizating of pre-synaptic membrane, limiting calcium influx, and then inhibiting...