Hong-Hong Yao

Enhancement of glutamate uptake mediates the neuroprotection exerted by activating group II or III metabotropic glutamate receptors on astrocytes

We investigated whether the activation of astroglial group II and III metabotropic glutamate receptors (mGluRs) could exert neuroprotective effects and whether the neuroprotection was related to glutamate uptake. Our results showed that the activation of astroglial group II or III mGluRs exerted neuroprotection against 1‐methyl‐4‐phenylpyridinium (MPP+) astroglial conditioned medium‐induced neurotoxicity in midbrain neuron cultures. Furthermore, MPP+ decreased glutamate uptake of primary astrocytes and C6 glioma cells, which was recovered by activating group II or III mGluRs. Specific group II or III mGluRs antagonists completely abolished the neuroprotective effects and the enhancement of glutamate uptake of their respective agonists. Our results showed that the primary cultured rat astrocytes and C6 glioma cells expressed receptor proteins for group II mGluR2/3, group III mGluR4, mGluR6 and mGluR7. C6 glioma cells expressed mRNA for group II mGluR3, group III mGluR4, mGluR6, mGluR7 and mGluR8. In conclusion, we confirmed that the activation of astroglial mGluRs exerted neuroprotection, and demonstrated that the mechanism underlying this protective role was at least partially related to the enhancement of glutamate uptake.

Iptakalim alleviates rotenone-induced degeneration of dopaminergic neurons through inhibiting microglia-mediated neuroinflammation.

Inhibition of microglia-mediated neuroinflammation has been regarded as a prospective strategy for treating neurodegenerative disorders, such as Parkinsons disease (PD). In the present study, we demonstrated that systematic administration with iptakalim (IPT), an adenosine triphosphate (ATP)-sensitive potassium channel (KATP) opener, could alleviate rotenone-induced degeneration of dopaminergic neurons in rat substantia nigra along with the downregulation of microglial activation and mRNA levels of tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2). In rat primary cultured microglia, pretreatment with IPT suppressed rotenone-induced microglial activation evidenced by inhibition of microglial amoeboid morphological alteration, declined expression of ED1 (a marker for activated microglia), and decreased production of TNF-α and prostaglandin E2 (PGE2). These inhibitory effects of IPT could be reversed by selective mitochondrial KATP (mitoKATP) channel blocker 5-hydroxydecanoate (5-HD). Furthermore, pretreatment with IPT prevented rotenone-induced mitochondrial membrane potential loss and p38/c-jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) activation in microglia, which might in turn regulate microglial activation and subsequent production of TNF-α and PGE2. These data strongly suggest that the KATP opener IPT may be a novel and promising neuroprotective drug via inhibiting microglia-mediated neuroinflammation.

ATP‐sensitive potassium channel opener iptakalim protected against the cytotoxicity of MPP+ on SH‐SY5Y cells by decreasing extracellular glutamate level

Mounting evidence reveals that ATP‐sensitive potassium (KATP) channel openers (KCOs) exert significant neuroprotection in vivo and in vitro in several models of Parkinsons disease (PD). However, the mechanisms are not well understood. In this study, we demonstrated that SH‐SY5Y cells expressed mRNA and proteins for Kir6.1, Kir6.2, SUR1 and SUR2 subunits of KATP channels. Moreover, our results showed that 1‐methyl‐4‐phenyl‐pyridinium ion (MPP+) induced up‐regulation of mRNA for the Kir6.2 subunit and down‐regulation of SUR1. It was further found that pretreatment with iptakalim, a novel KATP channel opener, could attenuate increased extracellular glutamate level and decreased cell survival in SH‐SY5Y cell culture after exposure to MPP+. Trans‐pyrrolidine‐2, 4‐dicarboxylic acid (t‐PDC), a glutamate transporter inhibitor, partially blocked the effect of iptakalim decreasing extracellular glutamate level. Additionally, iptakalim prevented MPP+‐induced inhibition of glutamate uptake in primary cultured astrocytes. The beneficial effects of iptakalim on glutamate uptake of astrocytes were abolished by selective mitochondrial KATP (mitoKATP) channel blocker 5‐HD. These results suggest (i) KATP channel dysfunction may be involved in the mechanisms of MPP+‐induced cytotoxicity and (ii) iptakalim may modulate glutamate transporters and subsequently alleviate the increase of extracellular glutamate levels induced by MPP+ through opening mitoKATP channels, thereby protecting SH‐SY5Y cells against MPP+‐induced cytotoxicity.

Activation of group II/III metabotropic glutamate receptors attenuates LPS-induced astroglial neurotoxicity via promoting glutamate uptake

Altered glial function that leads to oxidative stress and excitotoxicity may contribute to the initiation or progression of neuronal death in neurodegenerative diseases. We report the pivotal role of astroglial Group II and III metabotropic glutamate receptors (mGluR) against neurotoxicity. Activation of Group II or III mGluR on astrocytes with selective agonists DCG‐IV or L‐AP4 respectively inhibited astroglial lipopolysaccharide (LPS)‐conditioned medium induced apoptosis of primary cultured mesencephalic neurons. Specific Group II or III mGluR antagonists APICA or MSOP completely abolished the neuroprotective effects of DCG‐IV and L‐AP4. Morphologic analysis showed that DCG‐IV or L‐AP4 could also attenuate the astroglial neurotoxicity to dopaminergic neurons. Measurement of extracellular glutamate concentration and [3H]‐glutamate uptake showed that the restoration of glutamate uptake capability in LPS‐treated astrocytes might be involved in the neuroprotective effects of activating astroglial Group II or III mGluR. Furthermore, we found that the repression of astroglial uptake function could be revived by GSH, and both Group II and III mGluR agonists could recover the endogenous reduced glutathione (GSH) level in LPS‐treated astrocytes. These results suggested that the possible mechanisms of neuroprotection by either Type II or Type III mGluR activation may involve restoration of endogenous GSH, in turn affording recovery of astroglial capability to take up glutamate.

Systematic administration of iptakalim, an ATP-sensitive potassium channel opener, prevents rotenone-induced motor and neurochemical alterations in rats

Our previous studies revealed that iptakalim, a novel ATP‐sensitive potassium channel opener, has a significant neuroprotective function against ischemia in vivo or rotenone‐induced neurotoxicity in vitro. To investigate the potential pharmaceutical benefit of ATP‐sensitive potassium channel openers on neurodegenerative diseases, we studied the effects of iptakalim and diazoxide, a selective mitochondrial ATP‐sensitive potassium channel opener, on the rotenone‐induced nigrostriatal degeneration in rats. Iptakalim (1.5 mg/kg/day, orally) or diazoxide (1.5 mg/kg/day, orally) 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/day, subcutaneously) 1 hr later each time. The results showed that rotenone‐infused rats exhibited parkinsonian symptoms and had dopamine depletion in the striatum and substantia nigra. Pretreatment with iptakalim or diazoxide prevented rotenone‐induced catalepsy and the reduction of striatum dopamine contents. Moreover, iptakalim and diazoxide reduced the enzymatic activities and mRNA levels of inducible nitric oxide synthase elicited by chronic administration of rotenone. These neuroprotective effects of iptakalim and diazoxide were abolished by 5‐hydroxydecanoate, a selective mitochondrial ATP‐sensitive potassium channel blocker. In conclusion, our data suggested that mitochondrial ATP‐sensitive potassium channels might play a key role in preventing both parkinsonian symptoms and neurochemistry alterations induced by rotenone in rats. The selective activation of mitochondrial ATP‐sensitive potassium channels may provide a new therapeutic strategy for prevention and treatment of neurodegenerative disorders such as Parkinsons disease.

Effects of iptakalim on rotenone-induced cytotoxicity and dopamine release from PC12 cells

Parkinsons disease is characterized by an extensive loss of dopaminergic neurons in the substantia nigra pars compacta. The final common pathway in the demise of these cells may involve dopamine-dependent oxidative stress. Previous studies revealed a new neuronal protective role of ATP-sensitive potassium channel openers. But the exact mechanism is still unknown. In the present study, the neuroprotective effect of iptakalim, a novel ATP-sensitive potassium channel opener, was studied against rotenone-induced cytotoxicity in rat dopaminergic PC12 cells. Rotenone decreased cell viability significantly after 48 h exposure and induced dopamine release from PC12 cells concentration-dependently. Iptakalim significantly enhanced dopamine uptake and alleviated rotenone-induced PC12 cells death and reduced dopamine release induced by rotenone or GBR-12909, a classical dopamine transporter inhibitor. These results suggest that iptakalim may open mitochondrial K(ATP) channels to modulate dopamine transporter and reduce extracellular dopamine levels, thereby it protecting PC12 cells against rotenone-induced injury.

Activation of mitochondrial ATP-sensitive potassium channels improves rotenone-related motor and neurochemical alterations in rats.

Our previous studies revealed that activation of mitochondrial ATP-sensitive potassium channels exerted protective effects on rotenone-treated rats and cultured cells. The aim of the present study is to examine the potential therapeutic effects of iptakalim, an ATP-sensitive potassium-channel opener, and diazoxide, a selective mitochondrial ATP-sensitive potassium-channel opener, on Parkinsonian symptoms in rats induced by rotenone. Rats were treated with rotenone (2.5 mg/kg s.c.) daily for 4 wk. This treatment caused a depletion of dopamine in the striatum and substantia nigra. Behaviourally, rotenone-infused rats exhibit Parkinsonian symptoms. Catalepsy was estimated by a 9-cm bar test. Treatment with L-dopa (10 mg/kg.d p.o.), iptakalim (0.75, 1.5, 3.0 mg/kg.d p.o.) and diazoxide (3.0 mg/kg.d p.o.) for 2 wk improved behavioural dysfunction and elevated dopamine contents in the striatum and substantia nigra of rotenone-treated rats. Studies also found that iptakalim and diazoxide could reduce the enzymic activities and mRNA levels of inducible nitric oxide synthase elicited by chronic administration of rotenone. All neurorestorative effects by both iptakalim and diazoxide were abolished by 5-hydroxydecanoate, a selective mitochondrial ATP-sensitive potassium-channel blocker. Collectively, the data suggested that mitochondrial ATP-sensitive potassium channels play a key role in improving both Parkinsonian symptoms and neurochemistry alterations of rotenone model rats, and selective activation of mitochondrial ATP-sensitive potassium channels may provide a new therapeutic strategy for treatment of early Parkinsons disease.

The regulation of rotenone-induced inflammatory factor production by ATP-sensitive potassium channel expressed in BV-2 cells

Our previous studies have demonstrated that activating ATP-sensitive potassium channel (K(ATP) channel), not only improved Parkinsonian behavior and neurochemical symptoms, but also reduced iNOS activity and mRNA levels in striatum and nigra of rotenone rat model of Parkinsons disease (PD). In this study, it was first shown that the subunits of K(ATP) channels are expressed in BV-2 cells, and then it was investigated whether K(ATP) channel was involved in regulating inflammatory factor production from BV-2 cells activated by rotenone. It was found that K(ATP) channel was expressed in BV-2 cell and formed by the combination of Kir 6.1 and SUR 2A/2B. K(ATP) channel openers (KCOs) including pinacidil, diazoxide and iptakalim (Ipt) exerted beneficial effects on rotenone-induced morphological alterations of BV-2 cells, decreased tumor necrosis factor alpha (TNF-alpha) production and the expression and activity of inducible isoform of nitric oxide synthase (iNOS). Either glibenclamide or 5-hydroxydecanoate acid (a selective mitochondrial K(ATP) channel blocker) could abolish the effects of KCOs, suggesting that K(ATP) channels, especially mitochondrial ATP-sensitive potassium channels (mitoK(ATP) channels), played a crucial role in preventing the activation of BV-2 cells, and subsequently the production of a variety of proinflammatory factors. Therefore, activation of K(ATP) channel might be a new therapeutic strategy for treating neuroinflammatory and neurodegenerative disorders.

6-Hydroxydopamine-induced glutathione alteration occurs via glutathione enzyme system in primary cultured astrocytes

AbstractAim:To define the role of enzymes involved in glutathione metabolism in 6-hydroxydopamine (6-OHDA)-induced glutathione alteration in primary cultured astrocytes.Methods:Total glutathione (GSx) levels were determined using the modified enzymatic microtiter plate assay. The mRNA levels of γ-glutamylcysteine synthetase (γGCS), γ-glutamyltransferase (γGT), glutathione peroxidase (GPx), GR (glutathione reductase), and glutathione transferases (GST) were determined using RT-PCR. γGT activity was determined using γGT assay kits.Results:In primary cultured astrocytes, 6-OHDA induced a significant elevation of cellular GSx levels after treatment for 24 h. However, the GSx levels decreased after 24 h and the values were even lower than the value in the control group without 6-OHDA at 48 h. RT-PCR data showed that the mRNA levels of γGCS, the rate-limiting enzyme of γ-L-glutamyl-L-cysteinylglycine (GSH) synthesis, were increased by 6-OHDA after treatment for 24 h and 48 h; the mRNA levels of GPx, GR, and GST did not alter in 6-OHDA-treated astrocytes after treatment for 24 h and 48 h; and 6-OHDA increased the mRNA levels and the activity of γGT after treatment for 48 h, which induced a decrease in GSx levels, despite the up-regulation of γGCS after exposure to 6-OHDA for 48 h.Conclusion:The change in γGCS correlated with the increase in GSH levels induced by 6-OHDA after treatment for 24 h. GSx levels decreased because of increased γGT mRNA levels and γGT activity induced by 6-OHDA after treatment for 48 h.

Protective effects of iptakalim, a novel ATP-sensitive potassium channel opener, on global cerebral ischemia-evoked insult in gerbils

AbstractAim:To investigate the protective role of iptakalim, a novel ATP sensitive potassium channel opener, on global cerebral ischemia-evoked insult in gerbils and glutamate-induced PC12 cell injury.Methods:Global cerebral ischemia was induced by occluding the bilateral common carotid arteries in gerbils for 5 min. The open field maze and T-maze were employed to investigate the experimental therapeutic value of iptakalim on ischemic brain insult (n=8). The pyramidal cells in the hippocampal CA1 regions were counted to assess the protective effects of iptakalim. Glutamate released from the gerbil hippocampus and PC12 cells were determined by HPLC. Intracellular calcium was measured by Fluo-3 AM with A Bio-Rad Radiance 2100TM confocal system in conjunction with a Nikon TE300 microscope. Astrocyte glutamate uptake measurements were determined by liquid scintillation counting.Results:Iptakalim (0.5–4.0 mg/kg per day, ip) could reduce the high locomotor activity evoked by ischemia and improve global cerebral ischemia-induced working memory impairments. Histological studies revealed that iptakalim could increase the survival neuron in the hippocampus CA1 zone in a dose-dependent manner. Moreover, iptakalim could reverse ischemia-evoked increases of glutamate in the hippocampus of gerbils. In an in vitro study, iptakalim protected PC12 cells against glutamate-induced excitotoxicity, reduced the [Ca2+]i increases, and enhanced the glutamate uptake activity of primary cultured astrocytes.Conclusions:Iptakalim plays a key role in preventing global cerebral ischemia-evoked insults in gerbils and glutamate-induced PC12 cell injury by anti-excitotoxicity. Iptakalim might be a promising novel candidate for the prevention and/or treatment of stroke.