Iptakalim Confers Protection Against Glutamate Transporters Downregulation Induced By Dopaminergic Neurotoxins: Therapeutic Implications For Parkinson's Disease

ATP sensitive potassium (KATP) channels are unique in their molecular makeup and their cellular function: functional channels are octameric proteins formed by two different types of subunits, members of the Kir6 inwardly rectifying potassium channel family and sulphonylurea receptor (SUR) subunits. As the open probability of KATP channels directly depends on the intracellular ATP/ADP levels, the channels directly couple the metabolic state of a cell to its electrical activity. The neuronal KAtp channels are not only relevant for acute metabolic challenges, but also in the more chronic settings of neurodegenerative disorders like AD or PD. SUR1 expression and activation of KAtpchannels can affect the process of dopaminergic neurodegeneration. Katp channel openers (KCOs) have been shown to exert strong neuroprotective effects when injected shortly prior to severe hypoxia/ischemia or epileptic insult. The initiation and execution of hypoxic/ischemic neuronal death is believed to critically depend on excessive glutamate release and subsequent excitotoxicity. However, it was never determined whether KCOs exhibit neuroprotective effects in enhancement of glutamate uptake.Katp channels are attractive targets in the pathophysiological scenario of PD. Iptakalim (Ipt), a novel KCO, was designed and synthesized in Wang's laboratory (1998). In the present study, we examined: (1) whether Ipt is involved in neuroprotection; (2) the effects of 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-2,3-dihydropyridinium ion (MPP+), and rotenone (Rot) on glutamate uptake, and GluTs protein expression, in attempt to determine whether the loss of GluTs function and protein expression is related to PD; and (3) whether Ipt protects against GluTs impairment in PD models and has the potential to treat PD in which the dysfunction of GluTs is implicated.Part I The effects of Iptakalim on 6-OHDA-induced apoptosis and glutamate-induced cytotoxicity.AIM: To investigate the effects of IPT on astrocytic apoptosis and glutamate-induced cytotoxicity.METHODS: The primary astrocytes prepared from rat cortex were cultured. The cell apoptosis was measured with Hoechst 33342 staining. PC 12 cells viability was measured using MTT assay.RESULTS: 100 uM 6-OHDA-induced cell death presented some features of apoptosis, such as cell body shrinkage, the increase in light scattering, cell process membrane blebbing, and a coronary chromatin condensation close to the nuclear envelope. IPT or Pin protected astrocytes from apoptosis induced by 6-OHDA. IPT, at the concentrations of 1-100 uM, markedly mitigated glutamate-inducedneurotoxicity. The protective effect of IPT was blocked by pretreatment with Gli (10 uM), a selective KATp channels blocker.CONCLUSION: IPT plays an important neuroprotective role by the mechanism of anti-apoptosis and cytotoxicity. IPT may serve as a novel and effective drug for PD therapy.Part II The relationship between altered GluTs function and PD, and the effect of IPT on glutamate uptakeAIM: To examine the relationship between GluTs function and PD, and the effect of IPT on glutamate uptake.METHODS: Rats were received stereotactic injection with 6-OHDA into substantia nigra pars compacta (SNpc). The synaptosomes from normal and PD rats were isolated. [3H]-D,L-Glutamate uptake of synaptosomes or PC 12 cells was measured by using liquid scintillation counting.RESULTS: [3H]-D,L-glutamate uptake by synaptosomes from striatum and cortex of PD rats decreased. In cultured PC 12 cells, chronically exposed to neurotoxins such as 6-OHDA, MPP+ or Rot decreased overall glutamate uptake in a concentration-dependent manner. The decreased glutamate uptake in striatum and cortex synaptosomes of 6-OHDA-treated rats was restored by administration with IPT (10, 50, 100 uM). IPT (5 and 10 uM) or Pin (10 uM) completely prevented 6-OHDA (50 uM) and MPP+ (400 uM)-induced GluTs dysfunction; partly protected Rot (10 nM)-induced the loss of GluTs activity. The protective effects of IPT and Pin were blocked by pretreatment with...