Propofol reverses oxidative stress-attenuated glutamate transporter EAAT3 activity: Evidence of protein kinase C involvement

European Journal of Pharmacology 565 (2007) 83 – 88 www.elsevier.com/locate/ejphar

Propofol reverses oxidative stress-attenuated glutamate transporter EAAT3 activity: Evidence of protein kinase C involvement☆,☆☆,★ Jung-Yeon Yun a , Kum-Suk Park b , Jin-Hee Kim c,d , Sang-Hwan Do b,c,⁎, Zhiyi Zuo e a

Department of Anesthesiology, Research Institute and Hospital, National Cancer Center, Gyeonggi -do, South Korea b Department of Anesthesiology, Seoul National University Hospital, Seoul, South Korea c Department of Anesthesiology, Seoul National University College of Medicine, Seoul, South Korea d Department of Anesthesiology, Seoul National University Bundang Hospital, Gyeonggi-do, South Korea e Department of Anesthesiology, University of Virginia Health System, Charlottesville, VA, USA Received 7 October 2006; received in revised form 10 February 2007; accepted 19 February 2007 Available online 3 March 2007

Abstract The authors investigated the effects of propofol on EAAT3 (excitatory amino acid transporter 3) activity under oxidative stress induced by tertbutyl hydroperoxide (t-BHP), and the mediation of these effects by protein kinase C (PKC). Rat EAAT3 was expressed in Xenopus oocytes and Lglutamate (30 μM)-induced membrane currents were measured using the two-electrode voltage clamp technique. Exposure of these oocytes to tBHP (1–20 mM) for 10 min dose-dependently decreased EAAT3 activity, and t-BHP (5 mM) significantly decreased the Vmax, but not the Km of EAAT3 for glutamate, and propofol (1–100 μM) dose-dependently reversed this t-BHP-attenuated EAAT3 activity. Phorbol-12-myristate-13acetate (a PKC activator), also abolished this t-BHP-induced reduction in EAAT3 activity, whereas staurosporine (a PKC inhibitor), significantly decreased EAAT3 activity. However, as compared with staurosporine or t-BHP alone, t-BHP and staurosporine in combination did not further reduce EAAT3 activity. A similar pattern was observed for chelerythrine (also a PKC inhibitor). In oocytes pretreated with combinations of t-BHP and PMA (or staurosporine), propofol failed to change EAAT3 activity. Our results suggest that propofol restores oxidative stress-reduced EAAT3 activity and that these effects of propofol may be PKC-mediated. © 2007 Elsevier B.V. All rights reserved. Keywords: Excitatory amino acid transporter 3; Glutamate; Neuroprotection; Oxidative stress; Propofol; Protein kinase C

1. Introduction Glutamate is the predominant excitatory amino acid in the central nervous system (CNS) and is neurotoxic when extracellular concentrations are excessive. Glutamate transporters (excitatory amino acid transporters, EAATs) play an important role in maintaining extracellular glutamate concen☆ This study was performed at the Department of Anesthesiology and Clinical Research Institute, Seoul National University Hospital. ☆☆ This study was supported by the Seoul National University Hospital Research Fund (to Dr. Do; grant no. 21–2004–028–0). ★ Presented in part at Euroanaesthesia 2005, Vienna, Austria, May 29, 2005. ⁎ Corresponding author. Department of Anesthesiology, Seoul National University Hospital, 28 Yongon-dong, Chongno-ku, Seoul, 110-744, South Korea. Tel.: +82 2 2072 2466; fax: +82 2 747 5639. E-mail address: [email protected] (S.-H. Do).

0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.02.045

trations within nontoxic levels by facilitating extracellular glutamate uptake into cells. Thus, EAAT dysfunction leads to extracellular glutamate accumulation and excitotoxic neuronal injury (Danbolt, 2001). Five different isoforms of EAATs have been identified (EAAT1–5): EAAT1 and EAAT2 are glial, EAAT3 and EAAT4 neuronal, and EAAT5 is mainly found in the retina. Glial EAATs (EAAT1 and 2) are widely distributed in the CNS and play a dominant role in extracellular glutamate clearance (Danbolt, 2001). Dysfunction of EAAT3, the major neuronal EAAT, has been associated with the development of epilepsy (Crino et al., 2002), which is partly due to inadequate extracellular glutamate clearance and a reduced inhibitory synaptic strength secondary to the reduced synthesis of inhibitory neurotransmitter γ-aminobutyric acid (GABA), because glutamate uptake by EAATs is utilized for GABA synthesis (Mathews and Diamond, 2003).

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Extracellular concentrations of glutamate increase after oxidative stress, a pathological condition that occurs during chronic neurodegenerative diseases and cerebral ischemia, and after traumatic brain injury (Danbolt, 2001; Trotti et al., 1998; Wilson, 1997). Oxidative stress is due to the excessive production of reactive oxygen species (ROS) (Wilson, 1997), and it has been shown that EAATs are vulnerable to oxidative stress (Miralles et al., 2001; Trotti et al., 1998), and that EAAT dysfunction by ROS probably occurs during some neurodegenerative diseases, such as, amyotrophic lateral sclerosis (Rothstein et al., 1995) and Alzheimer's disease (Li et al., 1997). Propofol (2,6-diisopropylphenol) is a widely used intravenous anesthetic/sedative in clinical practice, and has been shown to have neuroprotective effects in animals (Arcadi et al., 1996; Pittman et al., 1997) and humans (Lavine et al., 1997). The reason for this protection may involve a reduction in cerebral metabolism (Kochs et al., 1992), the potentiation of GABA receptors (Ito et al., 1999), a direct antiexcitotoxic property (Hans et al., 1994), and its antioxidative effects (Ergun et al., 2002). However, more recent studies have suggested that propofol increases glutamate uptake, which could explain its neuroprotective properties. Sitar et al. found that propofol preserved the activity of EAATs in primary cultures of rat cerebral cortex under oxidative stress (Sitar et al., 1999), whereas Peters et al. found that clinical levels of propofol and hypothermia attenuated the effects of oxidative stress on astrocytic glutamate uptake (Peters et al., 2001). Moreover, Velly et al. demonstrated that propofol restores EAAT2independent EAAT activity in ischemic rat cortical glial– neuronal co-cultures (Velly et al., 2003). In addition, we found that propofol enhances the activity of EAAT3 and that protein kinase C (PKC) may mediate this effect (Do et al., 2003). Thus, we hypothesized that propofol reverses oxidative stress-reduced EAAT3 activity and that this effect is mediated by PKC. In this study, we investigated whether oxidative stress affects EAAT3 activity and whether propofol reverses this effect of oxidative stress. We also investigated the role of PKC on these effects of propofol. 2. Materials and methods 2.1. Oocyte experiments The study protocol was approved by the Institutional Animal Care and Use Committee at Seoul National University College of Medicine. Isolation and microinjections into Xenopus oocytes were performed as previously described (Do et al., 2002). Rat EAAT3 cDNA construct was provided by Dr. M A. Hediger (Brigham and Women's Hospital, Harvard Institutes of Medicine, Boston, MA). A single defolliculated oocyte was placed in a recording chamber (0.5-ml volume) and perfused with 3 ml/min of Tyrode's solution containing (in mM) 150 NaCl, 5 KCl, 2 CaCl2, 1 MgSO4, 10 dextrose, and 10 HEPES all adjusted to pH 7.5. Microelectrodes were pulled in one stage from 10-μl capillary glass (Drummond Scientific Co., Broomall, PA) using a micropipette

puller. Tips were broken to a diameter of approximately 10 μm. Microelectrodes had a resistance of 1–3 MΩ when filled with 3 M KCl. Oocytes were voltage clamped using a two-microelectrode oocyte voltage clamp amplifier (OC725-A; Warner Corporation, New Haven, CT). All measurements were performed at a holding potential of −70 mV, and oocytes without a stable holding current of b 1 μA were excluded from the analysis. L-glutamate was diluted in Tyrode's solution and perfused over an oocyte for 20 s (3 ml/min). L-glutamate-induced inward currents were sampled from the oocyte at 125 Hz for 1 min, 5 s at baseline and 20 s of agonist application, and then the oocyte was washed with Tyrode's solution for 35 s. Responses were quantified by integrating current traces and are reported in microCoulombs (μC). Each experiment was performed using oocytes from at least three different frogs. Experiments were performed at room temperature (approximately 21–23 °C). 2.2. Drug administration and response measurements Propofol was diluted in Tyrode's solution to final concentrations of 1, 3, 10, 30, and 100 μM. The propofol was a clinical formulation (Diprivan®, AstraZeneca) dissolved in a fat emulsion (Intralipid® 20%, Pharmacia, Clayton, NC). Vehicle control experiments were performed using the same concentrations of Intralipid® as that present in the 30 μM propofol solution. In the control group, oocytes were perfused with Tyrode's solution for 4 min prior to response measurements. In propofol-treated groups, oocytes were perfused with Tyrode's solution for 1 min, and then with Tyrode's solution containing the propofol or Intralipid® for the next 3 min before responses were measured. Oxidative stress was induced by preincubating oocytes for 10 min in modified Barth's solution (containing in mM: NaCl 88, KCl 1, NaHCO3 2.4, CaCl2 0.41, MgSO4 0.82, Ca(NO3)2 0.3, gentamicin 0.1, HEPES 15, all adjusted to pH 7.6) containing various concentrations of t-BHP (1, 2, 3, 5, 10, 20 mM) or hydrogen peroxide (H2O2; 0.03, 0.1, 0.3, 1, 3 mM). In another set of experiments, oocytes were incubated with 5 mM t-BHP for 10 min, and then responses to serial concentrations of L-glutamate (3, 10, 30, 100, and 300 μM) were measured to determine the effects of t-BHP on the Km and Vmax of EAAT3 for glutamate. To study the involvement of PKC activation on the effects of propofol, oocytes were preincubated with 5 mM t-BHP for 10 min, and then incubated PMA (phorbol-12-myristate-13acetate; a PKC activator) at 100 nM for 10 min. These oocytes were perfused with Tyrode's solution for 1 min, and then with Tyrode's solution containing propofol at 30 μM for 3 min before responses were measured. To study the effects of inhibiting PKC on EAAT3 activity after oxidative stress, oocytes were preincubated with 5 mM t-BHP for 10 min, and then incubated with 1 μM staurosporine for 1 h or 100 μM chelerythrine for 1 h (both PKC inhibitors) before responses were measured. Additional experiments were performed by preincubating oocytes with 5 mM t-BHP for 10 min, and treating then with 1 μM staurosporine for 10 min followed by perfusion with propofol at 30 μM prior to response measurements.

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Fig. 1. Dose-dependent effects of tert-butyl hydroperoxide (t-BHP) on EAAT3 activity. Responses were induced by adding L-glutamate at 30 μM (data are means ± S.E.M., n = 13–16). ⁎P b 0.05 versus controls. Each data set was normalized using the mean values of batch-matched controls. Units on the Y-axis are fold values versus controls. (Top) Typical current traces induced by the application of L-glutamate (horizontal bar) are shown. (Inset) Dose–response effect of hydrogen peroxide on EAAT3 activity.

2.3. Materials Biologic reagents were obtained from Promega (Madison, WI). Mature female Xenopus laevis frogs were purchased from Purunamu (Seoul), and other chemicals from Sigma (St. Louis, MO) unless otherwise indicated. 2.4. Data analysis Responses are reported as means ± S.E.M. Moreover, as oocyte responses are commonly batch dependent (due to variable EAAT3 protein levels), responses were normalized versus same-day controls taken from the same batches and are reported as ratios versus these controls. Differences between groups were analyzed using either the Student's t-test or by oneway analysis of variance followed by the Student–Newman– Keuls correction. P values of b0.05 were considered significant.

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Fig. 2. Dose–response effects of EAAT3 activity in the presence or absence of 5 mM tert-butyl hydroperoxide (t-BHP). In the t-BHP treated group, oocytes were exposed to 5 mM t-BHP for 10 min before response measurements were made. Data shown are means ± S.E.M. (n = 9–13). ⁎P b 0.05 versus the corresponding controls.

EAAT3 mRNA thereafter. Incubation of oocytes with various concentrations of hydrogen peroxide (n = 13–16 for each concentration), also caused a dose-dependent decrease in response to L-glutamate (IC50; 164 μM, inset of Fig. 1). Data analysis (Prism version 2.0, Graph Pad, San Diego, CA) showed that 5 mM t-BHP significantly decreased the Vmax of EAAT3 for glutamate (from 2.9 ± 0.3 μC to 1.4 ± 0.1 μC, n = 9–13 in each group, P b 0.05), but not the Km (control: 32.9 ± 10.2 μM, t-BHP group: 13.4 ± 5.2 μM, P N 0.05) (Fig. 2). Propofol dose-dependently reversed 5 mM t-BHP-reduced EAAT3 activity (n = 20–23 in each group). Responses in the presence of 5 mM t-BHP and propofol at ≥ 3 μM were significantly greater than for 5 mM t-BHP only (Fig. 3). However, Intralipid® did not restore the response reduction induced by t-BHP pretreatment (t-BHP: 0.6 ± 0.1 μC, n = 9 versus Intralipid®: 0.6 ± 0.1 μC, n = 7; P N 0.05). Oocytes pretreated with 100 nM PMA for 10 min showed greater EAAT3 activity than controls (control: 1.0 ± 0.1 μC, n =26 versus PMA: 1.3 ± 0.1 μC, n = 23; P b 0.05) (Fig. 4). Oocytes exposed to PMA after 5 mM t-BHP pretreatment showed a

3. Results Whereas oocytes not injected with EAAT3 mRNA were unresponsive to L-glutamate application (data not shown), oocytes injected with EAAT3 mRNA showed inward currents after L-glutamate application (Fig. 1). In vehicle control experiments, Intralipid® did not affect the current responses of EAAT3 expressing oocytes to 30 μM L-glutamate (control: 1.0 ± 0.1 μC, n = 9 versus Intralipid®: 0.9 ± 0.1 μC, n = 7; P N 0.05). When oocytes not injected with EAAT3 mRNA were exposed to t-BHP, no inward or outward currents were recorded, but oocytes injected with EAAT3 mRNA and then exposed to various concentrations of t-BHP showed a dose-dependent decrease in response to 30 μM L-glutamate (n = 14–16 in each group). The IC50 of this inhibition by t-BHP was 4.1 mM (Fig. 1). Experiments were done on oocytes injected with

Fig. 3. Propofol (PPF) dose-dependently reversed 5 mM t-BHP-reduced EAAT3 activity (n = 20–23 in each group). Responses were measured after adding increasing concentrations of PPF to Xenopus oocytes preincubated with 5 mM t-BHP. ⁎P b 0.05 versus controls. +P b 0.05 versus t-BHP. t-BHP; tert-butyl hydroperoxide.

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Fig. 4. Effects of protein kinase C (PKC) activation/inhibition on EAAT3 activity. Oocytes were preexposed to 5 mM tert-butyl hydroperoxide (t-BHP) and then perfused with 30 μM propofol (PPF) in the presence or absence of PKC activator or inhibitor. Data are means ± S.E.M. (n = 18–26). ⁎P b 0.05 versus controls. PMA; phorbol-12-myristate-13-acetate.

significant increase in EAAT3 activity versus t-BHP alone treated oocytes (t-BHP: 0.7 ± 0.1 μC, n =23 versus t-BHP + PMA: 0.9 ± 0.1 μC, n= 21; P b 0.05). To determine whether there was an interaction between the effects of PMA and propofol on EAAT3 activity after exposure to t-BHP, oocytes treated with t-BHP and PMA were exposed to propofol. These oocytes showed a significant increase in EAAT3 activity compared to those treated with t-BHP only (t-BHP: 0.7 ± 0.1 μC, n = 23 versus t-BHP + PMA + propofol: 1.0 ± 0.1 μC, n = 18; P b 0.05) (Fig. 4). However, no statistical difference was observed between the t-BHP + PMA treated oocytes (0.9 ± 0.1 μC, n = 18, P N 0.05) and t-BHP + PMA + propofol treated oocytes (1.0 ± 0.1 μC, n = 18; P N 0.05). Therefore, it appears that no additive or synergistic interaction occurs between PMA and propofol with respect to EAAT3 activity under conditions of oxidative stress. Oocytes pretreated with 1 μM staurosporine for 1 h showed less EAAT3 activity than control oocytes (control: 1.0 ± 0.1 μC, n = 26 versus staurosporine: 0.8 ± 0.1 μC, n = 24; P b 0.05) (Fig. 4), but no statistical difference was observed between the t-BHP + staurosporine treated oocytes (0. = 0, 0.7 ± 0.1 μC, n = 20) and the t-BHP + staurosporine + propofol treated oocytes (0.7 b 0.05, 0.7 ± 0.1 μC, n = 19; P N 0.05), although both showed less EAAT3 activity than controls (Fig. 4). Similarly, pretreating oocytes with 100 μM chelerythrine for 1 h reduced EAAT3 activity versus controls (control: 1.0 ± 0.1 μC, n = 25 versus chelerythrine: 0.7 ± 0.1 μC, n = 19; P b 0.05), and t-BHP + chelerythrine did not reduce response further versus t-BHP alone or chelerythrine alone treated oocytes (t-BHP + chelerythrine: 0.7 ± 0.1 μC, n = 21) (Fig. 4). 4. Discussion Since EAATs are sodium co-transporters, they transport one negatively charged glutamate molecule together with two or three Na+ ions into cells (Danbolt, 2001). Therefore, at least one net positive charge enters the cell per glutamate molecule transported, and thus, glutamate transport is described as being electrogenic. Moreover, the sizes of currents induced by glutamate reflect the amount of glutamate transported, and therefore, measurements of glutamate-induced currents can be used to quantify EAAT activity. Although EAAT4 is another subtype of EAAT, it shows a

different behavior from other EAATs. It was recently demonstrated in a radioactive tracer based study that glutamate-induced EAAT4 currents in Xenopus oocytes are uncoupled from substrate transport (Fang et al., 2006). The main findings of the present study are as follows. (1) Oxidative stress induced by t-BHP or H2O2 reduces EAAT3 activity. (2) Propofol, at clinically relevant concentrations, restores EAAT3 activity reductions by t-BHP. (3) PMA (a PKC activator) abolishes EAAT3 activity reduction by t-BHP, and two PKC inhibitors (staurosporine or chelerythrine) did not further reduce EAAT3 activity in oocytes preincubated with t-BHP. In addition, propofol could not increase EAAT3 activity further in oocytes exposed to combinations of t-BHP plus PMA or t-BHP plus staurosporine. Previous studies have shown that oxidative stress reduces glutamate uptake in cultured neurons and glial cells (Abe and Saito, 1998; Sitar et al., 1999), and since these cells express at least two types of EAATs, the effects of oxidative stress have not been previously studied on single EAAT types. In the present study, EAAT3 was expressed in Xenopus oocytes. The results obtained show that exposure of EAAT3 to t-BHP or H2O2 induces a dose-dependent decrease in EAAT3 activity; because the IC50 of t-BHP for this effect was 4.1 mM, 5 mM t-BHP was used in subsequent experiments. The kinetic study showed that 5 mM t-BHP significantly reduced Vmax, but had no effect on the Km of glutamate uptake by EAAT3, which suggests that t-BHP decreases available EAAT3 or its turnover rate rather than changing the affinity of EAAT3 for glutamate. Previous studies have shown that PKC activation increases Vmax but not the Km of EAAT3 for glutamate (Do et al., 2002; Huang and Zuo, 2005). Thus, it is possible that the effects of t-BHP on EAAT3 are mediated by PKC activity. Consistent with this idea, no additive or synergistic interaction between PKC inhibitors and t-BHP was observed on EAAT3 activity. Moreover, PMA abolished t-BHP-induced EAAT3 activity attenuation. In a previous study, we found that propofol dose-dependently increases EAAT3 activity, which was attributed to an increased Vmax, and that propofol has no effect on the Km of EAAT3 for glutamate. At that time, based on the results of experiments on PKC inhibitors and activator, we suggested that the effect of propofol on EAAT3 is mediated by PKC activation (Do et al., 2003). Thus, it appears that propofol may be able to reverse the effect of t-BHP on EAAT3. Consistent with this belief and with the findings of previous studies (Daskalopoulos et al., 2001; Peters et al., 2001; Sitar et al., 1999), the results of the present study show that propofol (at ≥ 3 μM) dose-dependently reverses EAAT3 activity downregulation by t-BHP. In addition, these concentrations are within the blood levels required for surgical anesthesia when propofol is used in combination with nitrous oxide or opioid (i.e., 2.5–8 μg/ml, which corresponds to 14– 45 μM of propofol) (Miller, 2005). The results of the present study suggest that PKC mediates the effects of t-BHP and propofol on EAAT3 activity, which concurs with the previous findings that t-BHP decreases PKC activity (Nowak, 2003); however, t-BHP has also been reported to increase PKC activity (Gopalakrishna and Jaken, 2000).

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Moreover, another study concluded that propofol activates PKC (Wickley et al., 2006). Interestingly, published data suggest that PKC activation differentially regulates EAAT subtypes (Gonzalez and Robinson, 2004). The activities of glial EAATs have been reported to be inhibited (Conradt and Stoffel, 1997; Ganel and Crosson, 1998), enhanced (Casado et al., 1993), and unaffected (Tan et al., 1999) by PKC activation. On the other hand, PKC activation has been shown to consistently increase EAAT3 activity in C6 glioma cells (Davis et al., 1998), neuron-enriched cultures (Gonzalez et al., 2002) and in Xenopus oocytes expressing EAAT3 (Do et al., 2002), although one study found that PKC activation reduces EAAT3 activity (Trotti et al., 2001). However, increased EAAT3 activity after PKC activation may require upregulated EAAT3 cell surface expression, as is mediated by PKCα activation (Davis et al., 1998; Gonzalez et al., 2002; Huang and Zuo, 2005) or enhanced catalytic efficiency, as mediated by PKCϵ activation (Davis et al., 1998). Xenopus oocytes have been reported to express several PKC isoforms (i.e., α, β1, β2, γ, δ, and ζ), but not PKCϵ (Johnson and Capco, 1997). Thus, increased EAAT3 activity by PKC activation in Xenopus oocytes may be mediated via the upregulation of EAAT3 cell surface expression, which is consistent with the kinetic results reported in our previous paper, i.e., increased Vmax with no change in Km by PMA (Do et al., 2002). Many commonly encountered human diseases, such as, ischemic brain injury and neurodegenerative diseases are associated with oxidative stress (Wilson, 1997), and these diseases are often associated with increased extracellular glutamate concentrations in the brain (Danbolt, 2001; Trotti et al., 1998). Moreover, glutamate excitotoxicity and oxidative stress may synergistically cause brain cell injury and death (Trotti et al., 1998). The results of the present study show that oxidative stress inhibits EAAT3 activity, and thus, provide a potentially important means whereby oxidative stress increases extracellular glutamate concentrations. Our results also show that propofol preserved EAAT3 activity when cells were under oxidative stress. In fact, propofol has been shown to be neuroprotective (Arcadi et al., 1996; Lavine et al., 1997; Pittman et al., 1997) and to reduce extracellular glutamate concentrations after ischemic insult (Velly et al., 2003). Thus, one of the mechanisms underlying these effects of propofol might be via the preservation of EAAT3 activity. Finally, our results suggest that PKC modulates the effects of oxidative stress and propofol on EAAT3. PKC activity has been previously shown to be closely related with the cell death of neurons exposed to oxidative stress, and it has been reported that the loss of PKC activity is critically required for oxidative neuronal death (Durkin et al., 1997); moreover, PKC activation has been shown to protect nerve cells from cell death induced by oxidative stress (Maher, 2001). Acknowledgments The authors thank Mattias A. Hediger, PhD., Professor, Laboratory of Molecular and Cellular Physiology, Brigham and Women's Hospital for donating the rat EAAT3 cDNA construct.

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