The Protective Roles And Mechanisms Of ATP-sensitive Potassium Channel Opener Iptakalim

Astrocytes, the most abundant glial cell type in the brain, providemetabolic and trophic support to neurons and modulate synaptic activity.Accordingly, astrocyte dysfunction and even dysregulation ofastrocyte-specific functions can critically influence neuronal survival.Emerging evidences, including from our laboratory, has revealed thatmodulation of astrocyte function may provide a novel therapeutic strategyfor neurodegenerative disorders.Parkinson's disease (PD) is a progressive, age-associated,neurodegenerative disease characterized by bradykinesia, resting tremor,rigidity and gait disturbance. The pathological hallmarks andbiochemical changes of PD are the loss of nigrostriatal dopaminergicneurons, the reduction of dopamine (DA) content in the striatum and thepresence of intraneuronal proteinacious cytoplasmic inclusions, termed"Lewy Bodies". Up to now, its etiology remains unclear. Variousmechanisms, including mitochondrial defects, oxidative stress, glutamatetoxicity, genetic factors and apoptosis, may be involved in thepathogenesis of PD. Hence, many studies focused on searching for newpotential therapeutic targets and agents for the treatment of PD.Increasing evidence demonstrates that astrocyte activation involves in thepathogenesis of Parkinson's disease (PD). Indeed, activated astrocytesproduce broad array of neurotoxic molecules, including pro-inflammatory factors, reactive oxygen species and reactive nitrogen species. Thesefactors-mediated inflammatory processes contribute to PD. Astrocyteapoptosis participates in many neurodegenerative disorders, such as PD,and astrocyte dysfunction may further contribute to neuronal loss and theoverall pathology. Astrocytes surrounding dopaminergic neurons in thebrain may have a close relation with the selective vulnerability of theseneurons by scavenging ROS and releasing CysGly, which is the precursorof GSH synthesis in neurons. Hence, alteration in astrocyte glutathionelevel may be an important contributor to the pathogenesis ofneurodegenerative disease such as PD. Thus, inhibition of astrocytedysfunction may provide therapeutic benefits for neurodegenerativedisorders such as PD.ATP-sensitive potassium (K-ATP) channels, originally discovered inheart, are widely distributed in many tissues and cell types. They couplethe metabolic state of the cell to membrane potential by sensing changesin intracellular adenine nucleotide concentration. K-ATP channels playimportant roles in the cellular responses of various tissues under alteredmetabolic states. Opening the K-ATP channels, the mitoK-ATPchannels especially, in the brain under metabolic stress could protectagainst neuronal damage and neurodegeneration. MitoK-ATP channels,which located in the mitochondrial inner-membrane, activation mayprotect mitochondrial functions. The abnormalities in mitochondrialmetabolism precede some characteristic signs of functional impairment inmany neurodegenerative diseases, such as PD. In addition to theestablished role of the mitochondria in energy metabolism, regulation ofcell death has emerged as a second major function of these organelles.This seems to be intimately linked to their generation of reactive oxygenspecies (ROS), which have been implicated in mtDNA mutations, aging,and cell death. Therefore, K-ATP channel openers that can freely crossthe blood-brain barrier are considered as potential neuroprotectants. IPT is a lipophilic para-amino compound with low molecular weight, whichcan freely cross the blood-brain barrier and has been demonstrated to be anovel K-ATP channel opener (KCO) by pharmacological,electrophysiological and biochemical studies, and receptor binding assay.Therefore, IPT has been employed in various ischemic/hypoxin models invivo and in vitro, as well as chemical stress-induced neurodegenerativemodels. Emerging evidence has shown that IPT exhibited remarkableneuroprotection against several neurotoxicities. Nevertheless, little isknown about the precise pharmacological mechanisms of IPT-inducedneuroprotection, and the role of regulating astrocyte K-ATP channels inneurodegenerative disorders.The neurotoxin MPP~+ (1-methyl-4-phenylpyridinium), a highaffinity inhibitor of mitochondrial complex I, can induce selectivedopaminergic neuronal degeneration in substantia nigra. The aims ofthe present studies are: 1) To investigate the neuroprotective effects ofIPT on MPP~+-induced PD model rats and the glial activity; 2) To illustratethe possible mechanisms underlying IPT-induced the protective effects onastrocyte activity, apoptosis and the decrease of antioxidation; 3) Toinvestigate the protective effects of IPT on MPP~+-induced astrocytemitochondrial dysfunction and its underlying mechanisms.PartⅠ: Neuroprotective effect of iptakalim on the MPP~+induced PD model ratsAIM: To investigate the potential benefit of IPT on MPP~+-inducedmotor deficit and pathological alteration in rats.METHODS: Systematic administration with IPT or Diazoxidealone for 3 days, and then rats were treated with IPT or Diazoxide for 7days following intrastriatal injected with MPP~+. The rotarod test waschosen for the assessment of the effects of drugs on MPP~+-induced parkinsonian symptoms. The rats were placed on the rod andsequentially tested at 5, 10, 15, 20, 25 and 30 rpm, time-on-the-rod ateach speed was recorded. The overall rod performance (ORP) score foreach animal was calculated as the area under the curve in the plot oftime-on-the-rod. The expression of tyrosine hydroxylase (TH), glialfibryllary acidic protein (GFAP) and ED1 were detected byimmunohistochemistry.RESULTS: 1) MPP~+-treated rats exhibited parkinsonian symptoms,dopamine depletion in the striatum, loss of dopaminergic neurons andreduced proliferation of glial cells in the substantia nigra pars compacta(SNpc); 2) Pretreatment with IPT and Diazoxide significantly alleviatedmotor deficit and prevented the dopaminergic neurons damage in theSNpc. Furthermore, IPT and Diazoxide could inhibit the glial activationand the release of TNF-αinduced by MPP~+.CONCLUSION: IPT can improve the behavioral and pathologicalabnormalities of the PD model rats. Inhibiting astrocyte activation andsubsequent release of proinflammatory factors might mediate theneuroprotective effects of iptakalim on MPP~+-induced parkinsoniansymptoms.PartⅡIptakalim inhibits primary cultured rat astrocytesdysfunction induced by MPP~+AIM: To determine the effects and possible mechanism of IPT onMPP~+-induced astrocyte apoptosis, the decrease of antioxidation andproinflammatory factor release from reactive astrocytes.METHODS: Rat primary astrocytes used in the test of cell viabilitywere divided into two groups: control group; MPP~+ treated groups: 50,100, 150, 200, 400, 800μM MPP~+. Astrocyte apoptosis was determinedby staining with Hoechst 33324 (Sigma, USA) and flow cytometry apparatus. Astrocyte mitochondrial membrane potential was assessedwith the fluorescent probe JC-1. We introduced western blotting for theanalyses of AIF, Cytochrome c, p38, pJNK and pERK mitogen-activatedprotein kinase (MAPK) in astrocytes. GSH was assayed as totalglutathione (GSx) by a modified enzymatic recycling method of Tietze.The amount of TNF-αin the medium was determined with a rat TNF-αenzyme-linked immunosorbent assay kit (Jingmei Bioengineering Co.,Shanghai, China.).RESULTS: 1) IPT and Diazoxide protected against MPP~+-inducedastrocytic apoptosis by inhibiting mitochondrial membrane potential(△Ψ_m) loss and subsequent release of pro-apoptotic factors (cytochrome cand AIF), and JNK/MAPK phosphorylation induced by MPP~+. IPT andDiazoxide also activated ERK/MAPK and maintained increasedphospho-ERK1/2 level after MPP~+ exposure. The anti-apoptotic effectsof IPT and Diazoxide were abolished by the mitoK-ATP channel blocker5-HD; 2) IPT and Diazoxide inhibited glutathione (GSH) depletioninduced by MPP~+, but failed to affect theγGT activity; 3) p38/MAPKexpression and TNF-αrelease were significantly increased induced byMPP~+. IPT (10μM) or Diazoxide (100μM) inhibited tumor necrosisfactorα(TNF-α) production and phosphorylation of the p38/MAPK inastrocytes treated with MPP~+, which was abolished by 5-HD (250μM).CONCLUSION: These results indicate that IPT could inhibitMPP~+-induced astrocyte apoptosis, the decrease of antioxidation andproinflammatory factor release from reactive astrocytes, suggesting thatIPT may be a regulator of the astrocyte functions.PartⅢIptakalim ameliorates mitochondrial dysfunction ofprimary cultured rat astrocytes induced by MPP~+AIM: To investigate the protective effect of IPT on MPP~+-induced astrocyte mitochondrial dysfunction and its underlying mechanisms.METHODS: Mitochondrial preparations from rat primary culturedastrocytes. The levels of Cyt c of both intra-and extra-mitochondriawere measured with Western-blotting; the mitochondrial respiration ofastrocytes was determined by oxygen electrode; we applied luciferasereporter assays to measure the content of mitochondrial ATP, andchromatametry to determine the activities of complexⅠandⅣ.RESULTS: 1) Administration with MPP~+ (50μM) to astrocytemitochondrial preparations induced an acute release of Cyt c frommitochondria, enhanced the ROS production, and inhibited mitochondrialrespiration, generation of ATP, as well as the activities of mitochondrialcomplexⅠandⅣ; 2) IPT (10μM) and Diazoxide (10μM) reverseddecreased mitochondrial respiration and ATP production, subsequentlyprevented reduced generation of ROS and the translocation of Cyt cinduced by MPP~+. These effects can be partially blocked by themitoK-ATP channel blocker 5-HD (50μM); 3) IPT could inhibit decreaseof mitochondrial complexⅠandⅣactivities induced by MPP~+.Diazoxide could also inhibit decrease of mitochondrial complexⅠactivityinduced by MPP~+, but had no effect on the activity of complexⅣ.MitoK-ATP channel blocker 5-HD failed to abolish these effects.CONCLUSION: IPT inhibited MPP~+-induced mitochondrialdysfunction by opening mitoK-ATP channel and regulating the activitiesof complexⅠandⅣ.In summary, the results of the present study suggest that IPT, anovel ATP-sensitive potassium channels opener, could significantlyalleviate MPP~+-induced behavioral symptoms in rotarod motorperformance, the loss of dopaminergic neurons and astrocyte activation inthe substantia nigra pars compacta. The regulatory effects of IPT onastrocyte functions may be its neuroprotective mechanism. Moreover, IPT can alleviate mitochondria dysfunction by opening mitoK-ATP andregulating the activities of complexⅠandⅣ. These results suggest thatopening K-ATP channels in astrocytes may be neuroprotection, whichmay provide a novel therapeutic strategy for neurodegenerative diseases,such as PD.