Protective Roles Of Iptakalim On Parkinsion's Disease Models In Rats Via Anti-excitotoxicity Mechansims

Parkinson's disease (PD) is a common neurodegenerative disorder ofunknown etiology, the cardinal features of which include tremor, rigidity,slowness of movement and postural instability. Current understanding ofbasal ganglia circuitry and the pathophysiology of Parkinson's disease (PD)has led to major breakthroughs in the treatment of this debilitatingmovement disorder. Unfortunately, there are significant problems with thecurrent available pharmacological therapies, which mainly focus ondopamine replacement (L-3-4-dihydroxyphenylalanine, L-DOPA) ordopaminergic agonists. There are no successful therapies to prevent celldeath ('neuroprotection') and/or restore damaged neurons to a normal state('neurorestoration') in PD. Consequently, considerable amounts of effortshave focused on developing novel targets for the treatment of PD.ATP-sensitive potassium channel (K_ATP) has been proved as an importantnovel strategy for neuroprotection. Our recent research has revealed thatiptakalim (IPT), a novel K_ATP channel opener, exhibits significantneuroprotection—not only in promoting behavioral recovery but also inprotecting neurons against necrosis and apoptosis in different animal modelsof stroke, as well as in cultured cells. To investigate the potential pharmaceutical benefit of ATP-sensitive potassium channel openers onneurodegenerative diseases, such as Parkinson's disease, we studied theeffects of IPT on rotenone and 6-OHDA-induced nigro-striatal degenerationin rats and investigated its effects on the gluatmatergic transmission.Partâ… Effects of iptakalim on Parkinson's disease model in ratsand the extracelluar glutamate levels in the stratiumAIM: To investigate the potential benefit of IPT on rotenone and6-OHDA induced motor and neurochemiacl alterations in rats.METHODES: 1) IPT (1.5 mg·kg^-1·day^-1, p.o.) or diazoxide (1.5mg·kg^-1·day^-1, p.o.) alone was administered to rats for 3 days, and then for 4weeks was used daily with an injection of rotenone (2.5 mg·kg^-1·day^-1, s.c.) 1h later each time. The catalepsy test was chosen for the assessment of theeffects of drugs on rotenone-induced parkinsonian symptoms. The rats wereplaced with both forepaws on bars 9 cm above and parallel from the baseand were in a half-rearing position. Latency time of the removal of the pawwas recorded. 2) Establishing unilateral 6-OHDA lesioning of SNc PDmodel rats: Injection of 6-OHDA into fight SNc of rats employingstereotaxic apparatus. Three weeks later, apomorphine-induced rotationability was tested to obtain successful PD model rats. 2) Successful modelrats were divided into five groups: model group, L-DOPA group, IPT 0.75mg·kg^-1·day^-1 group, IPT 1.5 mg·kg^-1·day^-1 group, IPT 3.0 mg·kg^-1·day^-1group. IPT (0.75 mg·kg^-1·day^-1, 1.5 mg·kg^-1·day^-1, 3.0 mg·kg^-1·day^-1, p.o.) orL-DOPA (10 mg·kg^-1·day^-1) alone was administered to rats for 21 days, ratsin model and sham-operated groups were treated with the same volume of saline. At the end of each week, apomorphine (0.05 mg·kg^-1, s.c.) wasadministrated to test change in the rotation behavioral of 6-OHDA rats. 3) Atthe end of the second week, stainless steel dialysis guide cannula wasimplanted in both sides of the striatum. 7 days later, microdialysisexperiments were carried out to collect dialysate sample and HPLCcombining with ECD detector were used to assay dopamine and glutamatelevels. 4) Immunohistochemistry was carried out to deterimine the numberof TH positive and GFAP positive cells in SN prepared from the abovementioned rats that treated with drugs for three weeks.RESULTS: 1) Rotenone-infused rats exhibited parkinsonian symptomsand had dopamine depletion in the striatum and substantia nigra.Pretreatment with IPT or diazoxide prevented rotenone-induced catalepsyand the reduction of striatum dopamine contents. L-DOPA treated ratsexhibited significant alleviation of rotation behavior induced byapomorphine; all the three groups treated with IPT had a tendency todecrease the rotation behavior. 2) IPT increase extracellular dopamine levelsin the 6-OHDA lesioned side of the striatum and decrease dopamine levelsin the intact side of the striatum. Meanwhile, IPT had no influence onglutamate levels in the intact side, but it did decrease glutamate levels in thelesioned side of the striatum of PD rats. 3) The number of TH positive cellsin lesioned side of SN in model rats decreased significantly compared to thatin intact side and sham-operated rats, the number of TH positive cells inlesioned side of SN in IPT treated rats did not increased significantlycompared to model rats; The number of GFAP positive cells in lesioned sideof SN in model rats increased significantly compared to that in intact sideand sham-operated rats, the number of GFAP positive cells in lesioned side of SN in IPT treated rats decreased significantly compared to model rats.CONCLUSION: 1) Rats treated with 2.5 mg·kg^-1·day^-1 rotenone for 4weeks can mimic the model of early PD. 2) IPT can improve the behavioraland neurochemical abnormalities of the PD model rats, suggesting thatselective activation of K_ATP may provide a new therapeutic strategy forneurodegenerative disorders such as PD. The regulation of glutamanergictransmission might be a neuroprotective target of IPT against rotenone and6-OHDA toxicity.Partâ…¡Effects of iptakalim on glutamate release from PC12 cellsinduced by KCIAIM: To investigate the potential pharmaceutical benefit of IPT onglutamate-induced neurotoxicity in PC12 cells and the effects of IPT onglutamate release from PC 12 cells.METHODES: Cells in DMEM were exposed to different concentrationof IPT with or without glibenclamide (Tocris Cookson, St. Louis, MO) for15 min at 37℃. Then medium were added with 10 mmol/L glutamate. Cellviability was then determined by Thiazolyl blue (MTT) assay 24h later.Glutamate released from PC12 cells were determined by HPLC. Intracellularcalcium was measured by Fluo-3 AM with A Bio-Rad Radiance 2100TMconfocal system in conjunction with a Nikon TE300 microscope. Thepercentage of apoptotic PC12 cells was detected using flow cytometry.RESULTS: 10 mmol·L^-1 glutamate decreased cell viability significantlyafter 24 h exposure. 10μmol·L^-1 IPT significantly alleviated rotenone-induced PC12 cells apoptosis and reduced extracellular glutamatelevels induced by 80 mmol·L^-1 KC1. IPT also reduced the [Ca²⁺]iincreases. The effects of IPT were abolished by glibenclamide, a K_ATPblocker.CONCLUSION: These results suggest that IPT may open K_ATP tomodulate [Ca²⁺]i and reduce extracellular glutamate levels, thereby itprotecting PC 12 cells against glutamate-induced injury.Partâ…¢Effects of iptakalim on glutamate uptake in synaptosome ofPD model rats and the primary cultured astrocyteAIM: To determine whether IPT could regulate the glutatmate uptakeactivities of synapotsome and primary cultured astrocyte.METHODES: Rats were stereotaxically injected with 6-OHDA in SNpc.The synaptosomes from normal and PD rats were isolated.[3H]-D,L-Glutamate uptake was measured by using liquid scintillationcounting.RESULTS: [³H]-glutamate uptake by synaptosomes from striatum andcortex of PD rats decreased. The decreased glutamate uptake in striatum andcortex synaptosomes of 6-OHDA-treated rats was recovered byadministration with Ipt (10, 50, 100μmol/L). Ipt (5, and 10μmol/L)improved glutamate uptake activity of primary cultured astrocyte inhibitedby 6-OHDA (50μmol/L)-and MPP⁺ (400μmol/L); The effects of Pina weresimilar to Ipt. The protective effects of Ipt and Pina were blocked byco-adiministration with Glib (20μmol/L). CONCLUSION: IPT could enhance the glutamated uptake activities ofsynapotsome and primary cultured astrocyte induced by 6-OHDA and MPP⁺.