The Therapeutic Effects Of Iptakalim, A Novel ATP-sensitive Potassium Channel Opener, On The Rotenone Model Rats Of Parkinson's Disease

Parkinson's disease (PD) is a common neurodegenerative disorder of unknown etiology, the cardinal features of which include tremor, rigidity, slowness of movement and postural instability. Current understanding of basal ganglia circuitry and the pathophysiology of Parkinson's disease (PD) has led to major breakthroughs in the treatment of this debilitating movement disorder. Unfortunately, there are significant problems with the current available pharmacological therapies, which mainly focus on dopamine replacement (L-3-4-dihydroxyphenylalanine, L-DOPA) or dopaminergic agonists. There are no successful therapies to prevent cell death ('neuroprotection') and/or restore damaged neurons to a normal state ('neurorestoration') in PD. Consequently, considerable amounts of efforts have focused on developing novel targets for the treatment of PD.ATP-sensitive potassium channel (K_ATP) has been proved as an important novel strategy for neuroprotection. Our recent research has revealed that iptakalim (IPT), a novel K_ATP channel opener, exhibits significant neuroprotection—not only in promoting behavioral recovery but also in protecting neurons against necrosis and apoptosis in different animal models of stroke, as well as in cultured cells. To investigate the potential pharmaceutical benefit of ATP-sensitive potassium channelopeners on neurodegenerative diseases, such as Parkinson's disease, we studied the effects of IPT on rotenone-induced cultured PC 12 cells death and on rotenone-induced nigro-striatal degeneration in rats.Part Ⅰ Systematic administration of iptakalim prevents rotenone-induced motor and neurochemical alterations in ratsAIM: To investigate the potential benefit of IPT on rotenone-induced motor and neurochemiacl alterations in rats.METHODES: 1) IPT (1.5 mg·kg^-1·day^-1, p.o.) or diazoxide (1.5 mgkg^-1·day^-1, p.o.) alone was administered to rats for 3 days, and then for 4 weeks was used daily with an injection of rotenone (2.5 mg·kg^-1·day^-1, s.c.) 1 h later each time. The catalepsy test was chosen for the assessment of the effects of drugs on rotenone-induced parkinsonian symptoms. The rats were placed with both forepaws on bars 9 cm above and parallel from the base and were in a half-rearing position. Latency time of the removal of the paw was recorded. 2) Rats were treated with rotenone (2.5 mg·kg^-1, s.c.) daily for four weeks. The catalepsy test was chosen for the assessment of the effects of drugs on rotenone-induced parkinsonian symptoms. The criterion for a successful model was defined as latency time ≥ 15 s, which were selected and divided into eight groups. Then they were treated with IPT, diazoxide or L-DOPA, respectively, for two weeks. Dopamine and related neurotransmitters in the striatum and substantia nigra tissues were measured by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The activities were measured by spectrophotometrically using a commercially available kit(Juli Bioengineering Co., Nanjing, China); The mRNA levels of nitric oxide synthase were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).RESULTS: 1) Rotenone-infused rats exhibited parkinsonian symptoms and had dopamine depletion in the striatum and substantia nigra. Pretreatment with IPT or diazoxide prevented rotenone-induced catalepsy and the reduction of striatum dopamine contents. Moreover, IPT and diazoxide reduced the enzymatic activities and mRNA levels of inducible nitric oxide synthase elicited by chronic administration of rotenone. 2) Treatment with IPT (0.75, 1.5, 3.0 mg·kg^-1·day^-1, p.o.), diazoxide (3.0 mg·kg^-1·day^-1, p.o.) and L-DOPA (10 mgkg·kg^-1·day^-1, p.o.) for two weeks improved behavioral dysfunction and elevated dopamine contents in the striatum and substantia nigra of rotenone-treated rats. IPT and diazoxide could reduce the enzymatic activities and mRNA levels of inducible nitric oxide synthase elicited by chronic administration of rotenone. These neuroprotective and neurorestorative effects of IPT and diazoxide were abolished by 5-hydroxydecanoate, a selective mito-K_ATP blocker.CONCLUSION: 1) Rats treated with 2.5 mg·kg^-1·day^-1 rotenone for 4 weeks can mimic the model of early PD. 2) IPT can improve the behavioral and neurochemical abnormalities of the PD model rats, suggesting that selective activation of mito-K_ATP may provide a new therapeutic strategy for neurodegenerative disorders such as PD. The regulation of dopaminergic neurons apoptosis and glial iNOS activity might be a neuroprotective target of IPT against rotenone toxicity.Part Ⅱ Effects of iptakalim on rotenone-induced PC12 cells deathAIM: To investigate the potential pharmaceutical benefit of IPT on rotenone-induced neurotoxicity in PC 12 cells.METHODES: Cells in DMEM were exposed to different concentration of EPT with or without glibenclamide (Tocris Cookson, St. Louis, MO) for 15 min at 37°C. Then medium were added with different concentrations of rotenone. Cell viability was then determined by Thiazolyl blue (MTT) assay and morphological changes were observed under phase contrast microscopy 48h later. The levels of dopamine were measured by HPLC-ECD.RESULTS: 0.1 umol·L^-1 rotenone decreased cell viability significantly after 48 h exposure and induced dopamine release from PC 12 cells concentration-dependently. 1, 10, 100 umol·L^-1 EPT significantly alleviated rotenone-induced PC 12 cells death and reduced extracellular dopamine levels induced by 0.1 umol·L^-1 rotenone or GBR-12909, a classical dopamine transporter inhibitor. The effects of IPT were abolished by glibenclamide, a K_Atp blocker.CONCLUSION: These results suggest that EPT may open mito-K_ATP to modulate dopamine transporter and reduce extracellular dopamine levels, thereby it protecting PC 12 cells against rotenone-induced injury.Part Ⅲ Effects of iptakalim on the activation and function of K_ATP expressed in primary cultured rat microglial cellsAIM: To determine whether K_ATP channel subunits are expressed in primary cultured microglia and whether IPT exerts effects on microglial activation and proinflammatory factor production from microgliaMETHODES: Rat primary microglial cells were prepared following a previously described protocol. The expressions of mRNA and proteins for the K_ATP subunits were determined by RT-PCR (reverse transcription-polymerase chain reaction), Western-blotting analyses and double immunolabling methods. The activation of microglial cells were determined by double immunolabling of OX-42 and ED-1. The amount of TNF-a in the medium was determined with a rat TNF-a enzyme-linked immunosorbent assay kit (Jingmei Bioengineering Co., Shanghai, China.)RESULTS: The primary cultured microglial cells express both mRNA and proteins for Kir6.1, SUR2A and SUR2B subunits. Application of IPT, pinacidil and diazoxide inhibited ED1 expression and tumor necrosis factor a (TNF-a) production from primary cultured microglial cells treated with rotenone. The inhibitory effects were abolished by the mito-K_ATP channel blocker 5-HD.CONCLUSION: The newly-identified K_ATP subunits on microglia may be involved in regulation of the inflammatory response to microglia activation. In addition, these results indicate that IPT significantly diminishes microglia-mediated inflammatory processes and that mito-K_ATP exert important regulative effects on the activation and function of primary cultured microglial cells, which may be a noveltherapeutic strategy for the treatment of inflammation-related neurodegenerative disorders.