KATP Channel Openers Facilitate Glutamate Uptake by Gluts in Rat Primary Cultured Astrocytes

Home , Channel blocker, Channel opener, Pinacidil, Potassium channel opener

KATP Channel Openers Facilitate Glutamate Uptake by GluTs in Rat Primary Cultured Astrocytes

1,2 1,2 1 1 1 ,1 Xiu-Lan Sun , Xiao-Ning Zeng , Fang Zhou , Cui-Ping Dai , Jian-Hua Ding and Gang Hu*

1 Laboratory of Neuropharmacology, Department of Anatomy, Histology & Pharmacology, Nanjing Medical University, Nanjing, Jiangsu, P.R. China

Increasing evidence, including from our laboratory, has revealed that opening of ATP sensitive potassium channels (KATP channels) plays

the neuronal protective roles both in vivo and in vitro. Thus K channel openers (KCOs) have been proposed as potential ATP neuroprotectants. Our previous studies demonstrated that K channels could regulate glutamate uptake activity in PC12 cells as well as ATP

in synaptosomes of rats. Since glutamate transporters (GluTs) of astrocytes play crucial roles in glutamate uptake and KATP channels are

also expressed in astrocytes, the present study showed whether and how KATP channels regulated the function of GluTs in primary

cultured astrocytes. The results showed that nonselective KCO pinacidil, selective mitochondrial KCO diazoxide, novel, and blood–brain

barrier permeable KCO iptakalim could enhance glutamate uptake, except for the sarcolemmal KCO P1075. Moreover pinacidil, + diazoxide, and iptakalim reversed the inhibition of glutamate uptake induced by 1-methyl-4-phenylpyridinium (MPP ). These potentiated

effects were completely abolished by mitochondrial K blocker 5-hydroxydecanoate. Furthermore, either diazoxide or iptakalim could ATP + inhibit MPP -induced elevation of reactive oxygen species (ROS) and phosphorylation of protein kinases C (PKC). These findings are the

first to demonstrate that activation of KATP channel, especially mitochondrial KATP channel, improves the function of GluTs in astrocytes

due to reducing ROS production and downregulating PKC phosphorylation. Therefore, the present study not only reveals a novel

pharmacological profile of KCOs as regulators of GluTs, but also provides a new strategy for neuroprotection.

Neuropsychopharmacology (2008) 33, 1336–1342; doi:10.1038/sj.npp.1301501; published online 4 July 2007

Keywords: KATP channel opener; glutamate uptake; mitochondria; iptakalim

INTRODUCTION channels causes membrane hyperpolarization, which limits neuronal excess excitation; activation of presynaptic K ATP-sensitive potassium (K ) channels originally were ATP ATP channels can directly modulate neurotransmitter release discovered in heart cardiac tissue. Uptill now, they have from nerve terminals. Therefore, K channel openers been found to be widely expressed in metabolically active ATP (KCOs) serve as efficient tools that can adjust cell tissues, and in many excitable cells including cardiac excitability, and exhibit beneficial effects under pathological myocytes, hormone-secreting cells, skeletal and smooth conditions such as ischemia, stroke, and neurodegenerative muscle, and neurons (Ashcroft, 1988; Seino, 1999; Zawar diseases (Yao and Gross, 1994; Sargent et al, 1993; Liss et al, et al, 1999). K channels play a critical role in coupling ATP 2005). Emerging evidence including from our laboratory cellular metabolism to electrical activity, and have been has revealed that the opening of K channels play shown to participate in some important biological processes ATP dominant protective roles in various models of neurode- such as insulin secretion, synaptic transmission, and generative diseases such as Parkinson’s disease (PD) (Tai excitability of cardiac, vascular, and nonvascular smooth et al, 2003; Wang et al, 2006; Yang et al, 2004). muscle. It is generally agreed that KATP channels in cardiac It is well-known that astrocytic glutamate transporters muscle and neurons have a protective effect during cellular (GluTs) perform the majority of glutamate uptake from the suffering from injury, such as ischemia and anoxia (Zini extracellular space in the brain, and play an important role et al, 1993; Prensa and Parent, 2001). Within the brain, KATP in maintaining extracellular glutamate concentrations below channels exist in both pre- and post-synaptic membrane, as neurotoxic levels (Matute et al, 2006; Schousboe and well as in glial cells. Activation of post-synaptic K ATP Waagepetersen, 2005). The dysfunction of astrocytic GluTs leads to an excessive extracellular glutamate concentration, *Correspondence: Dr G Hu, Laboratory of Neuropharmacology, thereby inducing excitotoxicity and neurodegeneration Department of Anatomy, Histology & Pharmacology, Nanjing Medical University, 140 Hanzhong Road, Nanjing, Jiangsu 210029, P.R. China, (Ferrarese et al, 2000). Thus, it has been proposed that Tel: + 86 25 86863169, Fax: + 86 25 86863108, improvement of glutamate uptake might be a potential E-mail: [email protected] strategy for neuroprotection (Wadiche and Gersdorff, 2006; 2These authors contributed equally to this work. Genoud et al, 2006; Matsugami et al, 2006; Takayasu et al, Received 8 March 2007; revised 6 May 2007; accepted 6 June 2007 2006). Our previous studies have revealed that KCOs could KATP channel openers improve the function of GluTs X-L Sun et al 1337 decrease extracellular glutamate concentrations and reduce Protein level of each sample was detected using dye excitotoxicity in PD animal and cellular models (Tai et al, coomassie brilliant blue G-250. 2003; Yang et al, 2004; Wang et al, 2004; Busija et al, 2004; Liss et al, 1999). We also demonstrated that KCOs could Measurement of ROS generation regulate glutamate uptake in PC12 cells and in synaptosomes from normal and PD rats (Inagaki et al, 1995). Since Formation of reactive oxygen species (ROS) was evaluated 0 0 KATP channels are expressed in astrocytes and astrocytic using 2 ,7 -dichlorofluorescin diacetate (DCFH-DA), a GluTs play key roles in maintaining extracellular glutamate membrane-permeable probe de-esterified intracellularly. levels, whether activation of KATP channels plays a The non-fluorescent dye freely penetrates cells, is then neuroprotective role via regulating the functions of GluTs hydrolyzed by intracellular esterases to 20,70-dichlorodihy- is essential to be elucidated and is hitherto unknown. In drofluororescein (DCFH), and trapped inside the cells. the present study, we examined the effects of KCOs on Upon oxidation by ROS, DCFH yields the highly fluorescent glutamate uptake in primary cultured astrocytes to deter- product dichlorofluorescein (DCF). mine (1) whether activation of KATP channels would Treated with drugs for 48 h, cells were loaded with DCFH- regulate GluTs function, (2) whether KCOs could protect DA (50 mM final concentration) in DMEM for 60 min in the astrocytes against neurotoxin-induced dysfunction of dark and fixed by 4% formaldehyde. After rinsing cells GluTs, and (3) the possible mechanisms by which KATP twice with PBS solution (137 mM NaCl, 2.7 mM KCl, 9.5 mM channels modulate glutamate uptake. Na2HPO4 Á 12H2O, 1.5 mM KH2PO4, pH 7.20), fluorescence was read at the excitation wavelength (Ex) of 488 nm and the emission wavelength (Em) of 530720 nm. DCFH-DA MATERIALS AND METHODS was initially dissolved to 40–50 mM in ethanol and stored at 1 Primary Astrocyte Cultures À20 C. A final concentration of ethanol (0.6% or less) did not alter the fluorescence measurement. Confluent primary astrocyte cultures were prepared from cerebral cortex of newborn Sprague–Dawley rats as described previously (Ivanova and Beyer, 2003), with minor PepTag Assay for Non-Radioactive Detection of PKC modifications as listed below. All animal procedures were Activity performed according to the NIH Guide for Animal Care and Following KCOs treatment for 48 h, the cells were rinsed approved by the institutional animal care and use with ice-cold PBS solution and harvested by scraping on ice committee. Briefly, neonatal rats were killed by rapid into 0.2 ml of cold protein kinases C (PKC) extraction decapitation, the cerebral cortices were removed and buffer. The lysates were subjected to mild sonication and separated from meninges and basal ganglia, and tissue followed by centrifugation at 41C, 14 000 Â g in a micro- was dissociated with 0.25% trypase (Amresco) at 371C and centrifuge for 5 min. Then the supernatant was used for the terminated by Dulbecco’s modified Eagle’s medium PepTag assay, which is generally performed within 24 h. (DMEM, Gibco-BRL Life Technologies) supplemented with According to the manufacturer’s (Promega) protocol, the 10% fetal bovine serum (FBS) and penicillin/streptomycin. cell samples were incubated with PKC reaction mixture After centrifugation at 1500 r.p.m. for 5 min, the cell pellets (25 ml) at 301C for 30 min and the reactions were stopped by were resuspended and seeded on poly-lysine-coated (Sigma) placing the tubes in a boiling water bath. After adding 80% flask. The cultures were maintained at 371C in a humidified glycerol (1 ml), the samples were loaded on a 0.8% agarose 5% CO2–95% air atmosphere. Culture medium was replaced gel at 100 V for 20 min. The bands were visualized and 24 h later and then changed every 2–3 days. Before photographed under UV light. The units of kinase activity experiments, astrocytes were replated on poly-lysine-coated in each slice of agarose were calculated according to the 6- or 24-well plates. Immunocytochemistry showed that manufacturer’s instructions. B98% of the cells stained positively for the astrocytic marker glial fibrillary acid protein (GFAP). Statistical Analysis 7 Assays of Glutamate Uptake All data were presented as the mean SEM. Statistical analysis for multiple comparisons was performed by one- Glutamate uptake determination was performed according way ANOVA test with Bonferroni’s corrections using SPSS to the procedure as described (Yao et al, 2005). Following 10.0 for Windows (SPSS Inc., Chicago, IL, USA). The level of pinacidil (Pina), diazoxide (Dia), P1075 or iptakalim (Ipt) statistical significance is defined as po0.05. administration for 48 h, uptake assays were initiated by 3 adding [ H]D, L-glutamate (190 nM, final concentration) to reaction flasks covered with astrocytes, and incubated for RESULTS 1 15 min at 37 C in 24-well plates. Nonspecific uptake was KCOs Facilitated Glutamate Uptake in Rat Primary determined from samples incubated in the presence of the + Astrocyte Cultures in a Concentration-Dependent Na -free buffer, and these values were subtracted from the Manner total. All the reactions were terminated by rinsing thrice with ice-cold 0.9% NaCl. Then, the cells were lysed Astrocytes in culture exhibited a high-affinity glutamate immediately with 0.3 M NaOH. After centrifugation at uptake after 48 h of incubation in DMEM with Vmax 10 000 r.p.m. for 15 min, supernatant was abstracted and calculated to 10.6670.06 pM mgÀ1 minÀ1 (Figure 1a). As radioactivity was determined by liquid scintillation counter. shown in Figure 1a, the uptake was significantly increased

Neuropsychopharmacology KATP channel openers improve the function of GluTs X-L Sun et al 1338 (117, 126, and 129% of the control) when exposed to (Figure 2h). To examine further the involvement of pinacidil (Pina, 1, 10, and 100 mM respectively). Preincuba- mitoKATP channel in the glutamate uptake, Dia or P1075 tion with 100, 200, or 500 mM diazoxide (Dia), a mitochon- was preincubated with MPP + . Dia (10, 100, 200 or 500 mM) drial KATP (mitoKATP) channel opener, increased glutamate and the novel KCO Ipt (0.1, 1, 10 or 100 mM) could concen- uptake to 120, 136, and 149%, respectively, of the control in tration-dependently attenuated uptake inhibition induced a concentration-dependent manner (Figure 1b). However, by MPP + (Figure 2c and e), P1075 (1, 10, 20, 50 or 100 mM) compared with control group, no significant effect on had no effect on astrocytic glutamate uptake (p40.05, astroglial glutamate uptake was detected in the presence of Figure 2g). The facilitating effects of Dia (100 mM) and Ipt P1075 (p40.05, Figure 1c), a selective sarcolemmal KATP (10 mM) on glutamate uptake were completely abolished by (sarcKATP) channel opener. As a novel KCO, iptakalim (Ipt) 5-HD (250 mM, Figure 2d and f), indicating that the protec- increased glutamate uptake to 118 and 114% at the tive role of Dia and Ipt in astrocitic glutamate clearance was concentration of 1 and 10 mM, respectively (Figure 1d). actually due to the opening of mitoKATP channels. These findings indicated that the KCOs could facilitate glutamate uptake by GluTs in astrocytes. KCOs Inhibited MPP + -Triggered ROS Elevation in Rat Primary Astrocyte Cultures + KCOs Alleviated MPP -Induced Glutamate Uptake After, preincubation with different KCOs for 30 min, then Inhibition in Rat Primary Astrocyte Cultures followed by incubation with 150 mMMPP+ for 48 h, the After incubation with 1-methyl-4-phenylpyridinium production of ROS was measured with the DCFH-DA probe. + (MPP + , 150 mM) for 48 h, astrocytic glutamate uptake was MPP (Figure 3b) increased the ROS output to 472% of remarkably decreased to 5.1370.46 pmol mgÀ1 minÀ1 com- control group (Figure 3a). Either Dia (100 mM, Figure 3c) pared with control group (7.0870.22 pmol mgÀ1 minÀ1, or Ipt (10 mM) (Figure 3e) could inhibit ROS production Figure 2a). As shown in Figure 2a, preincubation with Pina significantly. Furthermore, the effects of Dia and Ipt were concentration-dependently (0.1–100 mM) alleviated gluta- reversed by 5-HD (250 mM) (Figure 3d and f). P1075 failed + mate uptake impairment in MPP + cotreated astrocytes, to reverse MPP -induced ROS accumulation (Figure 3g). which was completely blocked by glibenclamide (Glib, m + 10 M) and mitoKATP channel blocker 5-hydroxydecanoate KCOs Suppressed PKC Activity in MPP -Treated m m (5-HD, 250 M) but not HMR1098 (20 M, a sarcKATP Astrocyte Cultures channel blocker, Figure 2b). These results indicated that the prevention of the uptake improvement with Glib or 5-HD After preincubation with Dia (100 mM), Ipt (10 mM), P1075 was due to the blockade of the mitoKATP channel, because (20 mM), and 5-HD (250 mM) for 30 min followed by Glib or 5-HD alone had no effects on glutamate uptake exposure to MPP + for 48 h, astroglial protein kinases C

Figure 1 Kinetics of glutamate uptake in differentially pretreated astrocyte populations. Values represented as mean7SEM of at least four independent experiments performed in triplicate. C, control; Pina, pinacidil; Dia, diazoxide; Ipt, iptakalim. *po0.05, **po0.01 vs control group.

Neuropsychopharmacology KATP channel openers improve the function of GluTs X-L Sun et al 1339

Figure 2 Protective effects of KCOs on glutamate uptake inhibition induced by MPP + in primary cultured astrocytes. Results are mean7SEM performed at least four independent experiments in triplicate. C, control; MPP + , 1-methyl-4-phenylpyridinium; Pina, pinacidil; Dia, diazoxide; Ipt, iptakalim; Glib, glibenclamide; 5-HD, 5-hydroxydecanoate. *po0.05, **po0.01 vs unteated group; #po0.05, ##po0.01 vs MPP + -treated group.

Figure 3 Effects of KCOs on ROS production in astrocytes incubated with MPP + . Values represented as mean7SEM of four independent experiments performed in triplicate. C, control; M&MPP + , 1-methyl-4-phenylpyridinium; Dia, diazoxide; Ipt, iptakalim; 5-HD, 5-hydroxydecanoate. **po0.01 vs control group; ##po0.01 vs MPP + -treated group.

(PKC) activity was detected with PepTag non-radioactive DISCUSSION protein kinase assays (Promega). As shown in Figure 4, + MPP (150 mM) increased the astroglial PKC activity to In the brain, KATP channels do not open under physiological 23 nM minÀ1 gProÀ1. Pretreatment with Dia and Ipt signi- conditions, but they can be activated by either a specific ficantly inhibited PKC activity to 13 nM minÀ1 gProÀ1 and opener or under many pathological conditions, such as 15 nM minÀ1 gProÀ1 repectively. However, P1075 failed to acute ischemia/hypoxia, ischemic preconditioning or meta- influence the increase of cellular PKC activity induced bolic inhibition, thereby hyperpolarizing the membrane by MPP + . potential (Farkas et al, 2004; Thomzig et al, 2001; Nicholls

Neuropsychopharmacology KATP channel openers improve the function of GluTs X-L Sun et al 1340 functions of astrocytes? Traditionally, astrocytes were thought to play minor roles in neuronal function and in directing overall activities in the brain, serving only a maintenance role in regulating brain homeostasis (Nicholls and Attwell, 1990; Anderson and Swanson, 2000). Notably, recent studies challenge these assumptions and suggest that rather than being an innocuous bystander, astrocytes may play crucial roles in regulating neuronal activity and signal transmission; deficiencies in these functions may contribute to neurodegeneration (Haydon, 2001). One way astrocytes wield their effects on neuronal function is through GluTs to maintain stimulatory, but nontoxic low levels of free intrasynaptic L-glutamate in the area adjacent to neurons (Nicholls and Attwell, 1990; Robinson, 1998; Lipton and Rosenberg, 1994). Abnorma- lities in this process result in the accumulation of extracellular glutamate in synaptic clefts, and subsequently induce overexcitation, and death of neurons (Nicholls and Attwell, 1990; Robinson, 1998; Lipton and Rosenberg, 1994). Therefore, the present study is to show the effects of KATP channel opening on glutamate uptake by GluTs in astrocytes. We demonstrated that administration with Pina, Dia and Figure 4 Effects of various KCOs on MPP + -induced PKC activation of Ipt increased glutamate uptake in rat primary cultured primary cultured astrocytes. Compared with non-preconditioned cells, + astrocytes, but sarcKATP channel opener P1075 failed to MPP remarkably increased intracellular PKC activity, which could be affect astrocytic glutamate uptake. Furthermore, Pina, Dia restrained by Dia and Ipt. There was no significant change detected in 7 and Ipt could also reverse the inhibition of glutamate P1075-treated group. Results are mean SEM of four independent + experiments performed in triplicate. C, control; M&MPP + , 1-methyl-4- uptake induced by MPP , and mitoKATP blocker 5-HD phenylpyridinium; Dia, diazoxide; Ipt, iptakalim. *po0.05 vs control group; inhibited these effects. These findings suggest that activa- # + po0.05 vs MPP -treated group. tion of KATP channels, especially mitoKATP channel, facilitated the function of GluTs in astrocytes. Ipt is a lipophilic para-amino compound with low molecular and Attwell, 1990). KATP channels are regulated by weight, which can freely cross the blood–brain barrier and intracellular concentrations of ATP ([ATP]i), being has been shown to be a novel KCO by pharmacological, opened as [ATP]i decreases, and thus by linking neuronal electrophysiological and biochemical studies, and receptor metabolism to excitability, they are thought to play an binding test (Wang, 2003; Xie et al, 2005). Ipt was initially important role in protecting cells from neuronal death designed and synthesized as a novel antihypertensive drug caused by hypoxia, ischemia or metabolic inhibition (Wang, 1998). In the view of the technical requirement for (Tai et al, 2003; Yang et al, 2004; Busija et al, 2004; Liss novel antihypertensive drug approval, the preclinical et al, 1999). investigation of Ipt has been completed and clinical trials KATP channels are hetero-octameric proteins composed are currently underway (Wang, 2003). Our previous studies of pore-forming Kir6.x subunits (Kir6.1 and Kir6.2) and provided compelling supports for the neuroprotective regulatory sulfonylurea receptor (SUR) subunits (SUR1, effects of Ipt at the dosages not affecting blood pressure, SUR2A, and SUR2B) (Seino, 1999; Inagaki et al, 1995), and via preventing the excitotoxic insults and anti-neuroin- located in various cellular compartments, including the flammation (Wang et al, 2006, 2004; Yang et al, 2004). The surface of the plasmalemmal membrane (sarcKATP channel) present study revealed a new mechanism involved in the and the inner mitochondrial membrane (mitoKATP channel) neuroprotective effects of the novel KCO. (Farkas et al, 2004). In the brain, KATP channels are It has been reported that mitoKATP channel opening could enriched in neurons as well as in glial cells, but their subunit reduce the rate of ATP loss, and decrease the rate of adenine combinations of KATP channels appear not to be homo- nucleotide degradation so that ADP is available for geneous. For example, the subunits of KATP channels in phosphorylation. The results from our previous studies neurons are Kir6.2 and SUR1/SUR2B (Zawar et al, 1999; also showed that activation of mitoKATP channel inhibited Thomzig et al, 2001), and KATP channels in astrocytes are the decrease of ADP-dependent state 3/4 oxygen consump- composed of Kir6.1 and SUR1 (Thomzig et al, 2001). These tion and respiratory control index injured by MPP + in studies have raised the questions: what is the role of KATP astrocytes. Consequently, activation of mitoKATP channel by channels expressed in discrete cellular populations and how KCOs preserves an adequate supply of ATP (Garlid et al, do they work in particular physiological/pathophysiological 2003). Glutamate uptake into astrocytes is driven by the episodes? electrochemical gradients of Na + and K + , with a stoichio- + + + It has been well-known that activation of KATP channels metry of 3 Na , and 1 H in and 1 K out with the uptake can regulate release of neurotransmitters from neurons, of each glutamate anion (Sung et al, 2003). The resulting + + and reduce neuronal excitability thereby protecting increase in [Na ]i might be corrected by a cycle of the Na against excitotoxicity. How do KATP channels regulate the pump and ATP consumption. Therefore, mitoKATP channel

Neuropsychopharmacology KATP channel openers improve the function of GluTs X-L Sun et al 1341 activation by KCOs might increase the supplementation of ACKNOWLEDGEMENTS ATP, and thereby enhancing glutamate uptake by GluTs. + We acknowledge the gift of HMR 1098 by Dr Juergen Activation of mitoK channel induces K influx, which ATP Puenter (Aventis, Frankfurt, Germany) for this research. partially restores the potential back toward basal level These studies were supported in part by grants from the and enhances mitochondrial respiration by mitochondria National Natural Science Foundation of China (Nos. matrix swelling, and subsequently yields a mild uncoupling (Starkov, 1997). Matrix swelling triggered by mitoK 30625038 and 30572172), the Key Project of Natural Science ATP Foundation of Jiangsu Educational Department (Nos. channel activation changes the geometry between the 05KJA31014, 05KJB310075 and 06KJA31029), the Key inner and outer mitochondrial membranes, and thus Project of Jiangsu Health Department (No. K200501). decreases intermembrane space, inducing faster electron transport and decreases in ROS release (Starkov, 1997; Jezek et al, 2004). Thus, mitoKATP channel acts as a potent governor to mitochondrial ROS release (Brookes et al, DISCLOSURE/CONFLICT OF INTEREST 2004). It has been demonstrated that oxidative stress can There is no duality of interest that we should disclose. reduce glutamate transporter activities, and antioxidants can reverse the impairment of glutamate uptake (Jayakumar et al, 2006). The data from our laboratory also REFERENCES showed that antioxidants glutathione and N-acetylcysteine Anderson CM, Swanson RA (2000). 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