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Corticosterone decreases the activity of rat glutamate transporter type 3 expressed in Xenopus oocytes
Steroids 75 (2010) 1113–1118
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Corticosterone decreases the activity of rat glutamate transporter type 3 expressed in Xenopus oocytes Maehwa Kang a , Junghee Ryu b , Jin-Hee Kim a,b , Hyoseok Na b , Zhiyi Zuo c , Sang-Hwan Do a,b,∗ a b c
Department of Anesthesiology, Seoul National University College of Medicine, Seoul, South Korea Department of Anesthesiology, Seoul National University Bundang Hospital, Gyeonggi-do, South Korea Department of Anesthesiology, University of Virginia Health System, Charlottesville, VA, USA
a r t i c l e
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Article history: Received 20 July 2009 Received in revised form 21 June 2010 Accepted 14 July 2010 Available online 21 July 2010 Keywords: Corticosterone Excitatory amino acid transporter type 3 Glutamate transporter Protein kinase C Phosphatidylinositol 3-kinase Xenopus oocyte
a b s t r a c t Glucocorticoids can increase the extracellular concentrations of glutamate, the major excitatory neurotransmitter. We investigated the effects of corticosterone on the activity of a glutamate transporter, excitatory amino acid carrier 1 (EAAC1; also called excitatory amino acid transporter type 3 [EAAT3]), and the roles of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) in regulating these effects. Rat EAAC1 was expressed in Xenopus oocytes by injecting mRNA. l-Glutamate (30 M)-induced membrane currents were measured using the two-electrode voltage clamp technique. Exposure of these oocytes to corticosterone (0.01–1 M) for 72 h decreased EAAC1 activity in a dose-dependent fashion, and this inhibition was incubation time-dependent. Corticosterone (0.01 M for 72 h) significantly decreased the Vmax , but not the Km , of EAAC1 for glutamate. Furthermore, pretreatment of oocytes with staurosporine, a PKC inhibitor, significantly decreased EAAC1 activity (1.00 ± 0.06 to 0.70 ± 0.05 C; P < 0.05). However, no statistical differences were observed between oocytes treated with staurosporine, corticosterone, or corticosterone plus staurosporine. Similar patterns of responses were achieved by chelerythrine or calphostin C, other PKC inhibitors. Phorbol-12-myristate-13-acetate (PMA), a PKC activator, inhibited corticosterone-induced reduction in EAAC1 activity. Pretreating oocytes with wortmannin or LY294002, PI3K inhibitors, also significantly reduced EAAC1 activity, but no difference was observed between oocytes treated with wortmannin, corticosterone, or wortmannin plus corticosterone. The above results suggest that corticosterone exposure reduces EAAC1 activity and this effect is PKC- and PI3K-dependent. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Although glutamate plays critical roles in many physiologic processes, including memory, learning, central pain transduction, and in the control of motor function [1], glutamate at high extracellular concentrations may be neurotoxic. Glutamate transporters (excitatory amino acid transporters [EAATs]) are located in the plasma membranes of neurons and glial cells and contribute to maintain extracellular glutamate concentrations within non-toxic levels by facilitating extracellular glutamate uptake into cells. Thus, dysfunction of EAATs causes extracellular glutamate accumulations which may lead to neuronal cell death [2]. Dysfunction of EAAT3 (also called EAAC1), the major neuronal EAAT, has been associated with the development of epilepsy [3,4],
∗ Corresponding author at: Department of Anesthesiology, Seoul National University Bundang Hospital, 166 Gumi-ro, Bundang-gu, Seongnam-si, Gyeonggi-do 463-707, South Korea. Tel.: +82 31 787 7501; fax: +82 31 787 4063. E-mail address: [email protected] (S.-H. Do). 0039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2010.07.003
ischemic brain damage, and some neurodegenerative diseases [5]. EAAC1 is densely distributed in the hippocampus and basal ganglia where there are high levels of glutamatergic synapses [2]. Hippocampus is also known to regulate stress responses and is a principal neural target site for corticosteroid hormones [6]. Glucocorticoids are one of the major classes of corticosteroid hormones and are secreted in high concentrations during many pathologic conditions, such as alcoholism, Cushing’s syndrome, major depression, and Alzheimer’s disease [7,8]. Prolonged elevations of glucocorticoids contribute to neuronal injury during the course of the above-mentioned diseases, especially the injury in the hippocampus [9]. In addition, prolonged stress or over-exposure to high physiologic concentrations of glucocorticoids can damage the rodent or primate hippocampus [10] and impair hippocampal neuronal capacity to survive during neurologic insults. Glucocorticoids are one of the most widely prescribed medications. However, glucocorticoids have been associated with diverse psychiatric side effects, such as mania, depression, psychosis, memory deficits and cognitive changes [11]. It has been found that short-term exposure to glucocorticoids may enhance neuronal
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damage during excitotoxic events via the modulation of glutamatergic neurotransmission [10,12]. Furthermore, stress-induced glutamate outflow is attenuated in the hippocampus and prefrontal cortex of adrenalectomized rats and glucocorticoid replacement abolishes this attenuation [13]. Chen et al. (1998) suggested that glucocorticoids may aggravate ischemic neuronal damage by causing glutamate to accumulate in the extracellular space [14]. However, the precise effects of glucocorticoids on EAAT subtypes have not been reported. In the present study, we examined the effects of corticosterone on EAAC1 activity in a Xenopus oocyte expression system. We also investigated the involvement of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) in the effects of corticosterone on EAAC1.
2. Experimental The protocol for this study was approved by the Institutional Animal Care and Use Committee at Seoul National University College of Medicine. The isolation and microinjection of Xenopus oocytes were performed as previously described [15]. With the frog placed on ice, a lobule of ovarian tissue containing approximately 200 oocytes was taken and placed immediately in calcium free OR-2 solution (88 mM NaCl, 2 mM KCl, 1 mM MgCl2 , 5 mM HEPES, and 0.1% collagenase type Ia [pH adjusted to 7.5]). Oocytes were defolliculated by gentle shaking for approximately 2 h at 30 ◦ C and then placed in modified Barth’s solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO2 , 0.41 mM CaCl2 , 0.82 mM MgSO4 , 0.3 mM Ca(NO3 )2 , 0.1 mM gentamicin, 15 mM HEPES [pH adjusted to 7.6]) for 1 day at 18 ◦ C. The rat EAAT3 (EAAC1) complementary DNA (cDNA) used in our experiments was provided by Dr. M.A. Hediger (Brigham and Women’s Hospital, Harvard Institute of Medicine, Boston, MA, USA). cDNA was subcloned in a commercial vector (BluscriptSKm) [16] and the plasmid DNA was linearized using a restriction enzyme (Not I), and the mRNA was synthesized in vitro using a commercially available transcription kit (Ambion, Austin, TX, USA). The resulting mRNA was quantified spectrophotometrically, diluted in sterile water and then cytoplasmically injected into oocytes at 30 ng/30 nl using an automated microinjector (Nanoject; Drummond Scientific Co., Broomall, PA, USA). The oocytes were subsequently incubated at 18–20 ◦ C for 3 days before measuring currents. Experiments were performed at room temperature (approximately 21–23 ◦ C). Tips of microelectrodes were broken at a diameter of approximately 10 m to produce microelectrodes with a resistance of 1–5 M when filled with 3 M KCl. A single defolliculated oocyte was placed in a recording chamber (0.5-ml volume) and perfused with 3 ml/min of Tyrode’s solution (150 mM NaCl, 5 mM KCl, 2 mM CaCl2 , 1 mM MgSO4 , 10 mM dextrose and 10 mM HEPES [pH adjusted to 7.5]) for 4 min before measuring currents. Oocytes were voltage-clamped using a two-microelectrode voltage clamp amplifier (OC725-C: Warner Co., New Haven, CT, USA). All measurements were performed at a holding potential of −70 mV. Oocytes without a stable holding current <0.6 A were excluded from the analysis. l-Glutamate was diluted in Tyrode’s solution and superfused over an oocyte for 20 s at a rate of 3 ml/min. lGlutamate-induced inward currents were sampled at 125 Hz for 1 min (5 s at baseline, 20 s during agonist application, and 35 s during the washing period with Tyrode’s solution). Responses were quantified by integrating current traces and reported in microCoulombs (C), reflecting the total amount of glutamate transported. Each experiment was performed with oocytes from at least three different frogs.
Corticosterone was diluted in modified Barth’s solution to the appropriate final concentrations (3 nM, 10 nM, 30 nM, 100 nM, 300 nM, and 1 M). In the corticosterone-treated groups, oocytes were incubated with corticosterone in a modified Barth’s solution for 72 h, whereas in the control group, oocytes were incubated with modified Barth’s solution alone. Because the level of expression reaches maximum after 3–5 days, our experiments were performed after 3 days of incubation. To determine the effects of corticosterone on the Km and Vmax of EAAC1 for l-glutamate, serial concentrations of lglutamate (3, 10, 30, 100, and 300 M) were used. In other experiments, 30 M l-glutamate was used to generate EAAC1induced currents. To evaluate the effects of PKC activation on EAAC1 activity, oocytes were preincubated with the PKC activator phorbol-12-myristate-13-acetate (PMA; 50 and 100 nM for 10 min) before l-glutamate-induced inward currents were recorded. Corticosterone-treated oocytes were exposed to PMA in the same manner. To study the effect of PKC inhibition on EAAC1 activity, oocytes were exposed to the PKC inhibitors: staurosporine (2 and 4 M for 1 h), chelerythrine (100 and 200 M for 1 h), or calphostin C (9 M for 1 h). The PI3K inhibitors wortmannin (1 and 2 M for 1 h) or LY294002 (50 M for 1 h) were added to the control and corticosterone-treated oocytes 1 h prior to recording in order to investigate the effect of PI3K on EAAC1 activity. To determine the temporal effects of corticosterone on EAAC1 activity, oocytes were incubated with 0.03 M corticosterone for 10, 30, and 60 min or with 0.01 M corticosterone for 12, 24, and 72 h before the l-glutamate-induced currents were measured. Finally, to determine whether the corticosterone effects on EAAT3 was through corticosteroid receptors, oocytes were preincubated with RU-486 (MifepristoneTM ; 1 and 5 M for 1 h), a glucocorticoid receptor antagonist, or spironolactone (1 and 5 M for 1 h), a mineralocorticoid receptor antagonist, before the treatment with corticosterone. The molecular biologic reagents were obtained from Promega (Madison, WI, USA). Mature female Xenopus laevis frogs were purchased from Kato S Science (Chiba, Japan), and other chemicals were purchased from Sigma (St. Louis, MO, USA) unless specified in the text. As batch-to-batch expression variability is common, oocyte responses were at times normalized vs. same day controls. The results are reported as the means ± S.E.M. and the differences between the groups were analyzed using the Student’s t-test or one way analysis of variance (ANOVA) followed by the Student–Newman–Keuls correction. P values <0.05 were considered significant.
3. Results Oocytes injected with EAAC1 mRNA showed inward currents after the application of l-glutamate (Fig. 1), whereas oocytes which were not injected were unresponsive to l-glutamate (data not shown). Previously, we found that the response was l-glutamate concentration-dependent and that the median effective concentration (EC50 ) of l-glutamate required to induce EAAC1 responses was 30.1 M [17]. Accordingly, 30 M of l-glutamate was used in the present study. When oocytes injected with EAAC1 mRNA were exposed to corticosterone (3 nM, 10 nM, 30 nM, 100 nM, 300 nM, and 1 M; n = 16–23) for 72 h, the responses to l-glutamate were reduced in a concentration-dependent manner (IC50 = 8 nM; Fig. 1) and, therefore, 10 nM corticosterone was used for further experiments. After 72 h of incubation with 10 nM, 30 nM, 100 nM, 300 nM, or 1 M corticosterone, EAAC1 activities were significantly reduced vs. control (P < 0.05, Fig. 1). In addition to reducing responses induced by 30 M l-glutamate, exposure to 10 nM corticosterone for 72 h also decreased the responses induced by 3, 10, 100, and
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Fig. 1. Effects of corticosterone exposure on the activity of EAAC1. Oocytes were exposed to 0.003, 0.01, 0.03, 0.1, 0.3, and 1 M corticosterone for 72 h before the responses to 30 M of l-glutamate were recorded. Each set of data has been normalized by using the mean value of the control group from the same batch. Inset graphs are representative current traces. Data are expressed as the mean ± S.E.M. (n = 16–23 in each group). *P < 0.05 compared to control. Differences between the control and corticosterone group were analyzed using the Student’s t-test.
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Fig. 3. Effects of protein kinase C (PKC) activation on EAAC1 activity in the presence or absence of 0.01 M corticosterone for 72 h. When corticosterone-treated oocytes (10 nM for 72 h) were exposed to PMA (50 and 100 nM for 10 min), the corticosterone-induced reduction in EAAC1 activity was attenuated. CS, corticosterone; PMA, phorbol-12-myristate-13-acetate. Data are expressed as the mean ± S.E.M. (n = 15–26 in each group). *P < 0.05 compared to control. Differences between the groups were analyzed using one way analysis of variance (ANOVA) followed by the Student–Newman–Keuls correction.
300 M l-glutamate (Fig. 2). A further analysis of the data with Prism version 5.0 (GraphPad, San Diego, CA, USA) showed that corticosterone exposure significantly reduced Vmax (1.31 ± 0.07 C for control vs. 1.02 ± 0.07 C for corticosterone, n = 17–22 in each group, P < 0.05), but did not cause a significant change in the Km (23.3 ± 1.5 M for control vs. 22.0 ± 2.7 M for corticosterone, n = 17–22 in each group, P > 0.05), of EAAC1 for l-glutamate. Preincubation of oocytes with 100 nM PMA, a PKC activator, significantly increased EAAC1 activity (1.00 ± 0.05 for control vs. 1.19 ± 0.09 for PMA, n = 22–26, P < 0.05). When corticosteronetreated oocytes (10 nM for 72 h) were exposed to PMA (100 nM for 10 min), the corticosterone-induced reduction in EAAC1 activity was attenuated (0.56 ± 0.07 for corticosterone vs. 0.86 ± 0.06 for
PMA + corticosterone, n = 15–17, P < 0.05; Fig. 3). Similar response patterns were seen at 50 nM PMA (Fig. 3, left hand panel). In our previous reports, 50 M chelerythrine, and 1 M staurosporine did not significantly affect the basal EAAC1 activity [18,19]. Thus, we used 100 M chelerythrine, and 2 M staurosporine in the current study. Preincubation of oocytes with 2 M staurosporine significantly reduced EAAC1 activity (1.00 ± 0.06 for control vs. 0.70 ± 0.05 for staurosporine, n = 19–23, P < 0.001). Oocytes exposed to staurosporine, corticosterone, or staurosporine plus corticosterone showed a significant decrease in EAAC1 activity as compared with untreated controls. However, the EAAC1 activity among oocytes treated by staurosporine, corticosterone, or staurosporine plus corticosterone was not different significantly (Fig. 4), indicating the possibility of the absence of an additive or synergistic interaction between the effects of staurosporine and corticosterone on EAAC1 activity. These results suggest that these
Fig. 2. Concentration–response curves of EAAC1 to l-glutamate in the presence or absence of 0.01 M corticosterone for 72 h. In addition to enhancing the responses induced by 30 M l-glutamate, 0.01 M corticosterone also significantly decreased the responses induced by 3, 10, 100 or 300 M l-glutamate. Data are expressed as the mean ± S.E.M. (n = 17–22 in each group). *P < 0.05 compared to the corresponding controls. Differences between corresponding data in two groups were analyzed using the Student’s t-test.
Fig. 4. Effects of protein kinase C (PKC) inhibition on EAAC1 activity in the presence or absence of 0.01 M corticosterone for 72 h. Whereas oocytes exposed to PKC inhibitor, corticosterone, or PKC inhibitor plus corticosterone showed a significant decrease in EAAC1 activity compared to control, the EAAC1 activity among oocytes treated by PKC inhibitor, corticosterone, or PKC inhibitor plus corticosterone was not different significantly. CS; corticosterone. Data are expressed as the mean ± S.E.M. (n = 15–26 in each group). *P < 0.05 compared to control. Differences between the groups were analyzed using one way analysis of variance (ANOVA) followed by the Student–Newman–Keuls correction.
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Fig. 5. Effects of phosphatidylinositol 3-kinase (PI3K) inhibition on EAAC1 activity in the presence or absence of 0.01 M corticosterone for 72 h. Whereas preincubation of oocytes with a PI3K inhibitor, wortmannin (1 M for 1 h), significantly reduced basal EAAC1 activity, the activity was not different among the wortmannin, corticosterone, or wortmannin plus corticosterone groups. CS; corticosterone. Data are expressed as the mean ± S.E.M. (n = 21–33 in each group). *P < 0.05 compared to control. Differences between the groups were analyzed using one way analysis of variance (ANOVA) followed by the Student–Newman–Keuls correction.
two agents may decrease EAAC1 activity via the same mechanism. The response at a higher concentration (4 M) of staurosporine was similar to that at 2 M (0.73 ± 0.05 for 2 M vs. 0.64 ± 0.05 for 4 M, n = 15–23, P > 0.05). The EAAC1 activity did not differ among the 4 M staurosporine, corticosterone, or 4 M staurosporine plus corticosterone groups (data not shown). Essentially identical patterns of responses were found for the PKC inhibitors chelerythrine and calphsostin C (Fig. 4), and for the PI3K inhibitors LY294002 and wortmannin (Fig. 5). Oocytes exposed to corticosterone (30 nM) for 10, 30, or 60 min did not cause significant change in EAAC1 activity compared with the control. corticosterone (10 nM) for 12, 24, or 72 h showed significantly lower EAAC1 activity than untreated controls. Furthermore, this inhibitory effect was increased with longer incubation times (n = 20–27 in each group, P < 0.05, Fig. 6).
Fig. 7. Effects of 0.01 M corticosterone for 72 h in the presence or absence of corticosteroid receptor antagonists on EAAC1 activity. RU-486 or spironolactone (5 M) did not affect the basal EAAC1 activity. The EAAC1 activity between oocytes treated by corticosterone and RU-486 (1 and 5 M for 1 h) plus corticosterone was not different significantly. Similarly, The EAAC1 activity between oocytes treated by corticosterone and spironolactone (1 and 5 M for 1 h) plus corticosterone was not different significantly. CS; corticosterone. Data are expressed as the mean ± S.E.M. (n = 18–23 in each group). *P < 0.05 compared to control. Differences between the groups were analyzed using one way analysis of variance (ANOVA) corticosteroid receptor antagonists.
Spironolactone or RU-486 (1 and 5 M, respectively, for 1 h) alone had no effect on current response of oocytes to glutamate (Fig. 7, the data of 1 M spironolactone and RU-486 was not shown). Incubation of the oocytes with corticosterone and RU-486 (1 and 5 M preincubation for 1 h) plus corticosterone significantly decreased EAAC1 activity compared with the control (1.00 ± 0.05 for control vs. 0.71 ± 0.05 for corticosterone vs. 0.72 ± 0.07 for 1 M RU-486 plus corticosterone vs. 0.74 ± 0.06 for 5 M RU-486 plus corticosterone, n = 18–23, P < 0.05). However, there was no significant difference among the last 3 groups (Fig. 7). Likewise, Treatment of the oocytes with corticosterone and spironolactone (1 and 5 M pretreatment for 1 h) plus corticosterone reduced the EAAC1 activity compared with the control (1.00 ± 0.05 for control vs. 0.71 ± 0.05 for corticosterone vs. 0.78 ± 0.07 for 1 M spironolactone plus corticosterone vs. 0.74 ± 0.06 for 5 M spironolactone plus corticosterone, n = 19–23, P < 0.05). There was also no significant difference among the last 3 groups (Fig. 7). 4. Discussion
Fig. 6. Time course of the effects of corticosterone exposure on the activity of EAAC1. Oocytes were exposed to 0.03 M corticosterone for 10, 30, 60 min and 0.01 M corticosterone for 12, 24, or 72 h before the responses to 30 M of l-glutamate were recorded. Oocytes exposed to corticosterone (30 nM) for 10, 30, or 60 min did not cause a significant change in EAAC1 activity compared with the control. Corticosterone (10 nM) incubation for 12, 24, or 72 h showed a significantly lower EAAC1 activity than controls. In addition, this inhibitory effect increased with longer incubation times. CS; corticosterone. Data are expressed as the mean ± S.E.M. (n = 20–27 in each group). *P < 0.05 compared to control. † P < 0.05 compared to 12 h. ‡ P < 0.05 compared to 24 h. Differences between the groups were analyzed using one way analysis of variance (ANOVA) followed by the Student–Newman–Keuls correction.
Xenopus oocyte expression system, pioneered in 1971 [20], has been well documented as a unique tool for studies of protein expression and function analysis of a broad range of proteins including neurotransmitter receptors and ionic channels. While mammalian synaptosomes or cultured cells contain many types of receptors, Xenopus oocytes have no inherent receptors except for lysophosphatidate receptor. In addition, Xenopus oocytes contain components of all major intracellular signaling pathways of mammalian cells [21,22]. Thus, oocyte expression system is often used for studying functions of plasma membrane proteins. Corticosterone is secreted in a circadian pattern. Under basal conditions, only 5–10% of the total corticosterone is in free biologically active form. The plasma concentrations of free corticosterone in rats have been reported to be 9–40 nM [23]. Weber et al. (2006) suggested that brain corticosterone concentrations were slightly lower than, but linearly correlated to, plasma concentrations [24].
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Therefore, we included this concentration range in the present study. Our findings indicate that corticosterone applied continuously at physiologic levels reduces EAAC1 activity. We also investigated the involvement of PKC and PI3K in the effects of corticosterone on EAAC1. Protein kinases, such as PKC and protein kinase A (PKA), are known to modulate the activities of EAATs [2]. It was previously shown that acute stress reduced hippocampal PKCϒ expression in C57BL/6J mice [25]. On the other hand, chronic stress (8 days or more) or chronic dexamethasone treatment increases PKCϒ expression in the rat hippocampus [26,27]. PKC has also been shown to be abnormally expressed in psychiatric illnesses, such as major depression, which often have elevated serum cortisol levels [28]. Although previous studies have suggested the modulation between glucocorticoids and PKC, the PKC modulation in the effects of glucocorticoids on EAAC1 remains to be elucidated. One of the mechanisms of EAAC1 activity regulation involves the modulation of redistribution of EAAC1 between intracellular storage sites and the plasma membrane [29]. This regulation results in the change of Vmax but not Km of EAATs for glutamate [30]. In the present study, the kinetic investigations showed a decreased Vmax and no change in the Km of the effects of corticosterone on EAAC1, which suggests that corticosterone exposure reduces the total EAAC1 levels or the amount of EAAC1 in the plasma membrane, rather than reducing the affinity of EAAC1 for glutamate. Previous studies have shown that PKC activation increases Vmax but not the Km of EAAC1 for glutamate [18,29]. Thus, it is possible that the effects of corticosterone on EAAC1 are mediated by changes in PKC activity. In the present study, inhibition of PKC was found to reduce EAAC1 activity. However, no obvious additive or synergistic interaction was found between PKC inhibition and corticosterone at the selective concentrations, suggesting that corticosterone decreases EAAC1 activity via PKC inhibition. This suggestion is supported by the finding that the effects of corticosterone on EAAC1 activity were attenuated by PMA, a PKC activator. Since the acute application of PKC modulators (inhibitors or activators) in a way similar to that used in this study has been shown to affect EAAC1 activity by changing EAAC1 redistribution to the plasma membrane [29,30], the results herein suggest that corticosterone reduces EAAC1 redistribution to the plasma membrane to decrease EAAC1 activity. It is also possible that prolonged glucocorticoid exposure (1–3 days) may reduce the total EAAC1 protein expression in the cells, which can then proportionally decrease the amount of EAAC1 proteins in the plasma membrane. However, our results using PKC inhibitors do not support this notion because we did not observe additional inhibition of EAAC1 activity by PKC inhibitors in the presence of corticosterone exposure. PI3K is involved in the production of lipid second messenger [31] and several studies have reported that the unique phospholipid products of PI3K activate a number of PKC isoforms. Furthermore, it has been suggested that PI3K interacts directly with several PKC isoforms [32,33]. PI3K, like PKC, has also been suggested to modulate EAAT3 activity via an independent but converging pathway [30,34]. The present study suggests that PI3K, as well as PKC, may be involved in mediating the glucocorticoid effects on EAAC1 activity. Traditionally, corticosterone binding to mineralocorticoid or glucocorticoid receptors is believed to regulate the expressions of a variety of genes via the activations of transcription factors [35]. However, Xenopus oocytes lack endogenous glucocorticoid receptors which are necessary to elicit a glucocorticoid response. Glucocorticoid-induced nuclear uptake was not detected with an immunoblot using anti-glucocorticoid receptor antibodies of the nuclear and cytoplasmic extracts from noninjected oocytes [36]. Our current study showed that the corticosterone-induced decrease in EAAC1 activity was not blocked by the steroid receptor antagonists RU-486 or spironolactone at a 100 and 500-fold excess
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concentration over corticosterone. These results, along with the findings that oocytes lack steroid nuclear receptors, can exclude the role of these steroid receptors in the corticosterone effects observed in this study. Rapid and transient increases in extracellular glutamate induced by administration of corticosterone or dexamethasone were not inhibited by the specific antagonists of the two types of corticoid receptors or by an inhibitor of protein synthesis, anisomycin. These observations suggest that glucocorticoid has non-genomic effects on neuronal constituents that can increase hippocampal glutamate concentrations [37,38]. It is not known whether the effects of corticosterone on EAAC1 as identified in our study is a mechanism for corticosterone to increase extracellular glutamate concentrations as observed in the previous in vivo studies. However, the relative slow time-course of responses for the effects of corticosterone on EAAC1 may preclude such a relationship. Unfortunately, previous studies did not monitor the extracellular glutamate levels for long durations. Thus, it remains unknown whether the slow effects of steroid on EAAC1 will translate into another increase of extracellular glutamate levels under the in vivo condition. Of note, a previous report showed that dexamethasone upregulated GLT1 (EAAT2) expression and activity in cultured astrocytes [39], suggesting the multifold effects of glucocorticoids on glutamate transporter expression and activity, and the consequences of these effects on the extracellular glutamate levels. Glucocorticoid receptors have been proposed to play an important modulatory role in neuronal damage and the development of seizures [40]. In a murine model of seizures, the incidence of seizures was parallel to the plasma corticosteroid levels and the highest incidence of seizures occurred after corticosterone treatment, whereas metyrapone which decreases the production of corticosterone by inhibiting 11-hydroxylase, reduced the seizure incidence. On the other hand, RU38486 (a glucocorticoid receptor antagonist) only partly attenuated the incidence of seizures [41]. Based on these results, it appears that corticosterone causes seizures via mechanisms other than glucocorticoid receptor activation. Since a decrease or lack of EAAC1 function may reduce the threshold of seizures [3,4], the inhibitory effects of corticosterone on EAAC1 activity, as shown in the current study, may contribute to the facilitation of seizure development by corticosterone. In conclusion, our results showed that exposure to corticosterone at clinically-relevant concentrations decreased EAAC1 activity in a concentration-dependent manner. These effects may be mediated by PKC and PI3K. Since EAATs are known to regulate glutamate neurotransmission, our results suggest that the inhibition of glucocorticoids on EAAC1 activity may contribute to their neurotoxic effects on neurons under stressful conditions. Acknowledgements All experiments were done in the Department of Anesthesiology and Clinical Research Institute, Seoul National University Hospital, and supported by a grant (No. 02-2007-018) from the Seoul National University Bundang Hospital Research Fund (Awarded to Dr. Sang-Hwan Do). References [1] Hudspith MJ. Glutamate: a role in normal brain function, anaesthesia, analgesia and CNS injury. Br J Anaesth 1997;78(6):731–47. [2] Danbolt NC. Glutamate uptake. Prog Neurobiol 2001;65(1):1–105. [3] Crino PB, Jin H, Shumate MD, Robinson MB, Coulter DA, Brooks-Kayal AR. Increased expression of the neuronal glutamate transporter (EAAT3/EAAC1) in hippocampal and neocortical epilepsy. Epilepsia 2002;43(3):211–8. [4] Sepkuty JP, Cohen AS, Eccles C, Rafiq A, Behar K, Ganel R, et al. A neuronal glutamate transporter contributes to neurotransmitter GABA synthesis and epilepsy. J Neurosci 2002;22(15):6372–9.
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Corticosterone decreases the activity of rat glutamate transporter type 3 expressed in Xenopus oocytes Maehwa Kang a , Junghee Ryu b , Jin-Hee Kim a,b , Hyoseok Na b , Zhiyi Zuo c , Sang-Hwan Do a,b,∗ a b c
Department of Anesthesiology, Seoul National University College of Medicine, Seoul, South Korea Department of Anesthesiology, Seoul National University Bundang Hospital, Gyeonggi-do, South Korea Department of Anesthesiology, University of Virginia Health System, Charlottesville, VA, USA
a r t i c l e
i n f o
Article history: Received 20 July 2009 Received in revised form 21 June 2010 Accepted 14 July 2010 Available online 21 July 2010 Keywords: Corticosterone Excitatory amino acid transporter type 3 Glutamate transporter Protein kinase C Phosphatidylinositol 3-kinase Xenopus oocyte
a b s t r a c t Glucocorticoids can increase the extracellular concentrations of glutamate, the major excitatory neurotransmitter. We investigated the effects of corticosterone on the activity of a glutamate transporter, excitatory amino acid carrier 1 (EAAC1; also called excitatory amino acid transporter type 3 [EAAT3]), and the roles of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) in regulating these effects. Rat EAAC1 was expressed in Xenopus oocytes by injecting mRNA. l-Glutamate (30 M)-induced membrane currents were measured using the two-electrode voltage clamp technique. Exposure of these oocytes to corticosterone (0.01–1 M) for 72 h decreased EAAC1 activity in a dose-dependent fashion, and this inhibition was incubation time-dependent. Corticosterone (0.01 M for 72 h) significantly decreased the Vmax , but not the Km , of EAAC1 for glutamate. Furthermore, pretreatment of oocytes with staurosporine, a PKC inhibitor, significantly decreased EAAC1 activity (1.00 ± 0.06 to 0.70 ± 0.05 C; P < 0.05). However, no statistical differences were observed between oocytes treated with staurosporine, corticosterone, or corticosterone plus staurosporine. Similar patterns of responses were achieved by chelerythrine or calphostin C, other PKC inhibitors. Phorbol-12-myristate-13-acetate (PMA), a PKC activator, inhibited corticosterone-induced reduction in EAAC1 activity. Pretreating oocytes with wortmannin or LY294002, PI3K inhibitors, also significantly reduced EAAC1 activity, but no difference was observed between oocytes treated with wortmannin, corticosterone, or wortmannin plus corticosterone. The above results suggest that corticosterone exposure reduces EAAC1 activity and this effect is PKC- and PI3K-dependent. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Although glutamate plays critical roles in many physiologic processes, including memory, learning, central pain transduction, and in the control of motor function [1], glutamate at high extracellular concentrations may be neurotoxic. Glutamate transporters (excitatory amino acid transporters [EAATs]) are located in the plasma membranes of neurons and glial cells and contribute to maintain extracellular glutamate concentrations within non-toxic levels by facilitating extracellular glutamate uptake into cells. Thus, dysfunction of EAATs causes extracellular glutamate accumulations which may lead to neuronal cell death [2]. Dysfunction of EAAT3 (also called EAAC1), the major neuronal EAAT, has been associated with the development of epilepsy [3,4],
∗ Corresponding author at: Department of Anesthesiology, Seoul National University Bundang Hospital, 166 Gumi-ro, Bundang-gu, Seongnam-si, Gyeonggi-do 463-707, South Korea. Tel.: +82 31 787 7501; fax: +82 31 787 4063. E-mail address: [email protected] (S.-H. Do). 0039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2010.07.003
ischemic brain damage, and some neurodegenerative diseases [5]. EAAC1 is densely distributed in the hippocampus and basal ganglia where there are high levels of glutamatergic synapses [2]. Hippocampus is also known to regulate stress responses and is a principal neural target site for corticosteroid hormones [6]. Glucocorticoids are one of the major classes of corticosteroid hormones and are secreted in high concentrations during many pathologic conditions, such as alcoholism, Cushing’s syndrome, major depression, and Alzheimer’s disease [7,8]. Prolonged elevations of glucocorticoids contribute to neuronal injury during the course of the above-mentioned diseases, especially the injury in the hippocampus [9]. In addition, prolonged stress or over-exposure to high physiologic concentrations of glucocorticoids can damage the rodent or primate hippocampus [10] and impair hippocampal neuronal capacity to survive during neurologic insults. Glucocorticoids are one of the most widely prescribed medications. However, glucocorticoids have been associated with diverse psychiatric side effects, such as mania, depression, psychosis, memory deficits and cognitive changes [11]. It has been found that short-term exposure to glucocorticoids may enhance neuronal
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damage during excitotoxic events via the modulation of glutamatergic neurotransmission [10,12]. Furthermore, stress-induced glutamate outflow is attenuated in the hippocampus and prefrontal cortex of adrenalectomized rats and glucocorticoid replacement abolishes this attenuation [13]. Chen et al. (1998) suggested that glucocorticoids may aggravate ischemic neuronal damage by causing glutamate to accumulate in the extracellular space [14]. However, the precise effects of glucocorticoids on EAAT subtypes have not been reported. In the present study, we examined the effects of corticosterone on EAAC1 activity in a Xenopus oocyte expression system. We also investigated the involvement of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) in the effects of corticosterone on EAAC1.
2. Experimental The protocol for this study was approved by the Institutional Animal Care and Use Committee at Seoul National University College of Medicine. The isolation and microinjection of Xenopus oocytes were performed as previously described [15]. With the frog placed on ice, a lobule of ovarian tissue containing approximately 200 oocytes was taken and placed immediately in calcium free OR-2 solution (88 mM NaCl, 2 mM KCl, 1 mM MgCl2 , 5 mM HEPES, and 0.1% collagenase type Ia [pH adjusted to 7.5]). Oocytes were defolliculated by gentle shaking for approximately 2 h at 30 ◦ C and then placed in modified Barth’s solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO2 , 0.41 mM CaCl2 , 0.82 mM MgSO4 , 0.3 mM Ca(NO3 )2 , 0.1 mM gentamicin, 15 mM HEPES [pH adjusted to 7.6]) for 1 day at 18 ◦ C. The rat EAAT3 (EAAC1) complementary DNA (cDNA) used in our experiments was provided by Dr. M.A. Hediger (Brigham and Women’s Hospital, Harvard Institute of Medicine, Boston, MA, USA). cDNA was subcloned in a commercial vector (BluscriptSKm) [16] and the plasmid DNA was linearized using a restriction enzyme (Not I), and the mRNA was synthesized in vitro using a commercially available transcription kit (Ambion, Austin, TX, USA). The resulting mRNA was quantified spectrophotometrically, diluted in sterile water and then cytoplasmically injected into oocytes at 30 ng/30 nl using an automated microinjector (Nanoject; Drummond Scientific Co., Broomall, PA, USA). The oocytes were subsequently incubated at 18–20 ◦ C for 3 days before measuring currents. Experiments were performed at room temperature (approximately 21–23 ◦ C). Tips of microelectrodes were broken at a diameter of approximately 10 m to produce microelectrodes with a resistance of 1–5 M when filled with 3 M KCl. A single defolliculated oocyte was placed in a recording chamber (0.5-ml volume) and perfused with 3 ml/min of Tyrode’s solution (150 mM NaCl, 5 mM KCl, 2 mM CaCl2 , 1 mM MgSO4 , 10 mM dextrose and 10 mM HEPES [pH adjusted to 7.5]) for 4 min before measuring currents. Oocytes were voltage-clamped using a two-microelectrode voltage clamp amplifier (OC725-C: Warner Co., New Haven, CT, USA). All measurements were performed at a holding potential of −70 mV. Oocytes without a stable holding current <0.6 A were excluded from the analysis. l-Glutamate was diluted in Tyrode’s solution and superfused over an oocyte for 20 s at a rate of 3 ml/min. lGlutamate-induced inward currents were sampled at 125 Hz for 1 min (5 s at baseline, 20 s during agonist application, and 35 s during the washing period with Tyrode’s solution). Responses were quantified by integrating current traces and reported in microCoulombs (C), reflecting the total amount of glutamate transported. Each experiment was performed with oocytes from at least three different frogs.
Corticosterone was diluted in modified Barth’s solution to the appropriate final concentrations (3 nM, 10 nM, 30 nM, 100 nM, 300 nM, and 1 M). In the corticosterone-treated groups, oocytes were incubated with corticosterone in a modified Barth’s solution for 72 h, whereas in the control group, oocytes were incubated with modified Barth’s solution alone. Because the level of expression reaches maximum after 3–5 days, our experiments were performed after 3 days of incubation. To determine the effects of corticosterone on the Km and Vmax of EAAC1 for l-glutamate, serial concentrations of lglutamate (3, 10, 30, 100, and 300 M) were used. In other experiments, 30 M l-glutamate was used to generate EAAC1induced currents. To evaluate the effects of PKC activation on EAAC1 activity, oocytes were preincubated with the PKC activator phorbol-12-myristate-13-acetate (PMA; 50 and 100 nM for 10 min) before l-glutamate-induced inward currents were recorded. Corticosterone-treated oocytes were exposed to PMA in the same manner. To study the effect of PKC inhibition on EAAC1 activity, oocytes were exposed to the PKC inhibitors: staurosporine (2 and 4 M for 1 h), chelerythrine (100 and 200 M for 1 h), or calphostin C (9 M for 1 h). The PI3K inhibitors wortmannin (1 and 2 M for 1 h) or LY294002 (50 M for 1 h) were added to the control and corticosterone-treated oocytes 1 h prior to recording in order to investigate the effect of PI3K on EAAC1 activity. To determine the temporal effects of corticosterone on EAAC1 activity, oocytes were incubated with 0.03 M corticosterone for 10, 30, and 60 min or with 0.01 M corticosterone for 12, 24, and 72 h before the l-glutamate-induced currents were measured. Finally, to determine whether the corticosterone effects on EAAT3 was through corticosteroid receptors, oocytes were preincubated with RU-486 (MifepristoneTM ; 1 and 5 M for 1 h), a glucocorticoid receptor antagonist, or spironolactone (1 and 5 M for 1 h), a mineralocorticoid receptor antagonist, before the treatment with corticosterone. The molecular biologic reagents were obtained from Promega (Madison, WI, USA). Mature female Xenopus laevis frogs were purchased from Kato S Science (Chiba, Japan), and other chemicals were purchased from Sigma (St. Louis, MO, USA) unless specified in the text. As batch-to-batch expression variability is common, oocyte responses were at times normalized vs. same day controls. The results are reported as the means ± S.E.M. and the differences between the groups were analyzed using the Student’s t-test or one way analysis of variance (ANOVA) followed by the Student–Newman–Keuls correction. P values <0.05 were considered significant.
3. Results Oocytes injected with EAAC1 mRNA showed inward currents after the application of l-glutamate (Fig. 1), whereas oocytes which were not injected were unresponsive to l-glutamate (data not shown). Previously, we found that the response was l-glutamate concentration-dependent and that the median effective concentration (EC50 ) of l-glutamate required to induce EAAC1 responses was 30.1 M [17]. Accordingly, 30 M of l-glutamate was used in the present study. When oocytes injected with EAAC1 mRNA were exposed to corticosterone (3 nM, 10 nM, 30 nM, 100 nM, 300 nM, and 1 M; n = 16–23) for 72 h, the responses to l-glutamate were reduced in a concentration-dependent manner (IC50 = 8 nM; Fig. 1) and, therefore, 10 nM corticosterone was used for further experiments. After 72 h of incubation with 10 nM, 30 nM, 100 nM, 300 nM, or 1 M corticosterone, EAAC1 activities were significantly reduced vs. control (P < 0.05, Fig. 1). In addition to reducing responses induced by 30 M l-glutamate, exposure to 10 nM corticosterone for 72 h also decreased the responses induced by 3, 10, 100, and
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Fig. 1. Effects of corticosterone exposure on the activity of EAAC1. Oocytes were exposed to 0.003, 0.01, 0.03, 0.1, 0.3, and 1 M corticosterone for 72 h before the responses to 30 M of l-glutamate were recorded. Each set of data has been normalized by using the mean value of the control group from the same batch. Inset graphs are representative current traces. Data are expressed as the mean ± S.E.M. (n = 16–23 in each group). *P < 0.05 compared to control. Differences between the control and corticosterone group were analyzed using the Student’s t-test.
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Fig. 3. Effects of protein kinase C (PKC) activation on EAAC1 activity in the presence or absence of 0.01 M corticosterone for 72 h. When corticosterone-treated oocytes (10 nM for 72 h) were exposed to PMA (50 and 100 nM for 10 min), the corticosterone-induced reduction in EAAC1 activity was attenuated. CS, corticosterone; PMA, phorbol-12-myristate-13-acetate. Data are expressed as the mean ± S.E.M. (n = 15–26 in each group). *P < 0.05 compared to control. Differences between the groups were analyzed using one way analysis of variance (ANOVA) followed by the Student–Newman–Keuls correction.
300 M l-glutamate (Fig. 2). A further analysis of the data with Prism version 5.0 (GraphPad, San Diego, CA, USA) showed that corticosterone exposure significantly reduced Vmax (1.31 ± 0.07 C for control vs. 1.02 ± 0.07 C for corticosterone, n = 17–22 in each group, P < 0.05), but did not cause a significant change in the Km (23.3 ± 1.5 M for control vs. 22.0 ± 2.7 M for corticosterone, n = 17–22 in each group, P > 0.05), of EAAC1 for l-glutamate. Preincubation of oocytes with 100 nM PMA, a PKC activator, significantly increased EAAC1 activity (1.00 ± 0.05 for control vs. 1.19 ± 0.09 for PMA, n = 22–26, P < 0.05). When corticosteronetreated oocytes (10 nM for 72 h) were exposed to PMA (100 nM for 10 min), the corticosterone-induced reduction in EAAC1 activity was attenuated (0.56 ± 0.07 for corticosterone vs. 0.86 ± 0.06 for
PMA + corticosterone, n = 15–17, P < 0.05; Fig. 3). Similar response patterns were seen at 50 nM PMA (Fig. 3, left hand panel). In our previous reports, 50 M chelerythrine, and 1 M staurosporine did not significantly affect the basal EAAC1 activity [18,19]. Thus, we used 100 M chelerythrine, and 2 M staurosporine in the current study. Preincubation of oocytes with 2 M staurosporine significantly reduced EAAC1 activity (1.00 ± 0.06 for control vs. 0.70 ± 0.05 for staurosporine, n = 19–23, P < 0.001). Oocytes exposed to staurosporine, corticosterone, or staurosporine plus corticosterone showed a significant decrease in EAAC1 activity as compared with untreated controls. However, the EAAC1 activity among oocytes treated by staurosporine, corticosterone, or staurosporine plus corticosterone was not different significantly (Fig. 4), indicating the possibility of the absence of an additive or synergistic interaction between the effects of staurosporine and corticosterone on EAAC1 activity. These results suggest that these
Fig. 2. Concentration–response curves of EAAC1 to l-glutamate in the presence or absence of 0.01 M corticosterone for 72 h. In addition to enhancing the responses induced by 30 M l-glutamate, 0.01 M corticosterone also significantly decreased the responses induced by 3, 10, 100 or 300 M l-glutamate. Data are expressed as the mean ± S.E.M. (n = 17–22 in each group). *P < 0.05 compared to the corresponding controls. Differences between corresponding data in two groups were analyzed using the Student’s t-test.
Fig. 4. Effects of protein kinase C (PKC) inhibition on EAAC1 activity in the presence or absence of 0.01 M corticosterone for 72 h. Whereas oocytes exposed to PKC inhibitor, corticosterone, or PKC inhibitor plus corticosterone showed a significant decrease in EAAC1 activity compared to control, the EAAC1 activity among oocytes treated by PKC inhibitor, corticosterone, or PKC inhibitor plus corticosterone was not different significantly. CS; corticosterone. Data are expressed as the mean ± S.E.M. (n = 15–26 in each group). *P < 0.05 compared to control. Differences between the groups were analyzed using one way analysis of variance (ANOVA) followed by the Student–Newman–Keuls correction.
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Fig. 5. Effects of phosphatidylinositol 3-kinase (PI3K) inhibition on EAAC1 activity in the presence or absence of 0.01 M corticosterone for 72 h. Whereas preincubation of oocytes with a PI3K inhibitor, wortmannin (1 M for 1 h), significantly reduced basal EAAC1 activity, the activity was not different among the wortmannin, corticosterone, or wortmannin plus corticosterone groups. CS; corticosterone. Data are expressed as the mean ± S.E.M. (n = 21–33 in each group). *P < 0.05 compared to control. Differences between the groups were analyzed using one way analysis of variance (ANOVA) followed by the Student–Newman–Keuls correction.
two agents may decrease EAAC1 activity via the same mechanism. The response at a higher concentration (4 M) of staurosporine was similar to that at 2 M (0.73 ± 0.05 for 2 M vs. 0.64 ± 0.05 for 4 M, n = 15–23, P > 0.05). The EAAC1 activity did not differ among the 4 M staurosporine, corticosterone, or 4 M staurosporine plus corticosterone groups (data not shown). Essentially identical patterns of responses were found for the PKC inhibitors chelerythrine and calphsostin C (Fig. 4), and for the PI3K inhibitors LY294002 and wortmannin (Fig. 5). Oocytes exposed to corticosterone (30 nM) for 10, 30, or 60 min did not cause significant change in EAAC1 activity compared with the control. corticosterone (10 nM) for 12, 24, or 72 h showed significantly lower EAAC1 activity than untreated controls. Furthermore, this inhibitory effect was increased with longer incubation times (n = 20–27 in each group, P < 0.05, Fig. 6).
Fig. 7. Effects of 0.01 M corticosterone for 72 h in the presence or absence of corticosteroid receptor antagonists on EAAC1 activity. RU-486 or spironolactone (5 M) did not affect the basal EAAC1 activity. The EAAC1 activity between oocytes treated by corticosterone and RU-486 (1 and 5 M for 1 h) plus corticosterone was not different significantly. Similarly, The EAAC1 activity between oocytes treated by corticosterone and spironolactone (1 and 5 M for 1 h) plus corticosterone was not different significantly. CS; corticosterone. Data are expressed as the mean ± S.E.M. (n = 18–23 in each group). *P < 0.05 compared to control. Differences between the groups were analyzed using one way analysis of variance (ANOVA) corticosteroid receptor antagonists.
Spironolactone or RU-486 (1 and 5 M, respectively, for 1 h) alone had no effect on current response of oocytes to glutamate (Fig. 7, the data of 1 M spironolactone and RU-486 was not shown). Incubation of the oocytes with corticosterone and RU-486 (1 and 5 M preincubation for 1 h) plus corticosterone significantly decreased EAAC1 activity compared with the control (1.00 ± 0.05 for control vs. 0.71 ± 0.05 for corticosterone vs. 0.72 ± 0.07 for 1 M RU-486 plus corticosterone vs. 0.74 ± 0.06 for 5 M RU-486 plus corticosterone, n = 18–23, P < 0.05). However, there was no significant difference among the last 3 groups (Fig. 7). Likewise, Treatment of the oocytes with corticosterone and spironolactone (1 and 5 M pretreatment for 1 h) plus corticosterone reduced the EAAC1 activity compared with the control (1.00 ± 0.05 for control vs. 0.71 ± 0.05 for corticosterone vs. 0.78 ± 0.07 for 1 M spironolactone plus corticosterone vs. 0.74 ± 0.06 for 5 M spironolactone plus corticosterone, n = 19–23, P < 0.05). There was also no significant difference among the last 3 groups (Fig. 7). 4. Discussion
Fig. 6. Time course of the effects of corticosterone exposure on the activity of EAAC1. Oocytes were exposed to 0.03 M corticosterone for 10, 30, 60 min and 0.01 M corticosterone for 12, 24, or 72 h before the responses to 30 M of l-glutamate were recorded. Oocytes exposed to corticosterone (30 nM) for 10, 30, or 60 min did not cause a significant change in EAAC1 activity compared with the control. Corticosterone (10 nM) incubation for 12, 24, or 72 h showed a significantly lower EAAC1 activity than controls. In addition, this inhibitory effect increased with longer incubation times. CS; corticosterone. Data are expressed as the mean ± S.E.M. (n = 20–27 in each group). *P < 0.05 compared to control. † P < 0.05 compared to 12 h. ‡ P < 0.05 compared to 24 h. Differences between the groups were analyzed using one way analysis of variance (ANOVA) followed by the Student–Newman–Keuls correction.
Xenopus oocyte expression system, pioneered in 1971 [20], has been well documented as a unique tool for studies of protein expression and function analysis of a broad range of proteins including neurotransmitter receptors and ionic channels. While mammalian synaptosomes or cultured cells contain many types of receptors, Xenopus oocytes have no inherent receptors except for lysophosphatidate receptor. In addition, Xenopus oocytes contain components of all major intracellular signaling pathways of mammalian cells [21,22]. Thus, oocyte expression system is often used for studying functions of plasma membrane proteins. Corticosterone is secreted in a circadian pattern. Under basal conditions, only 5–10% of the total corticosterone is in free biologically active form. The plasma concentrations of free corticosterone in rats have been reported to be 9–40 nM [23]. Weber et al. (2006) suggested that brain corticosterone concentrations were slightly lower than, but linearly correlated to, plasma concentrations [24].
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Therefore, we included this concentration range in the present study. Our findings indicate that corticosterone applied continuously at physiologic levels reduces EAAC1 activity. We also investigated the involvement of PKC and PI3K in the effects of corticosterone on EAAC1. Protein kinases, such as PKC and protein kinase A (PKA), are known to modulate the activities of EAATs [2]. It was previously shown that acute stress reduced hippocampal PKCϒ expression in C57BL/6J mice [25]. On the other hand, chronic stress (8 days or more) or chronic dexamethasone treatment increases PKCϒ expression in the rat hippocampus [26,27]. PKC has also been shown to be abnormally expressed in psychiatric illnesses, such as major depression, which often have elevated serum cortisol levels [28]. Although previous studies have suggested the modulation between glucocorticoids and PKC, the PKC modulation in the effects of glucocorticoids on EAAC1 remains to be elucidated. One of the mechanisms of EAAC1 activity regulation involves the modulation of redistribution of EAAC1 between intracellular storage sites and the plasma membrane [29]. This regulation results in the change of Vmax but not Km of EAATs for glutamate [30]. In the present study, the kinetic investigations showed a decreased Vmax and no change in the Km of the effects of corticosterone on EAAC1, which suggests that corticosterone exposure reduces the total EAAC1 levels or the amount of EAAC1 in the plasma membrane, rather than reducing the affinity of EAAC1 for glutamate. Previous studies have shown that PKC activation increases Vmax but not the Km of EAAC1 for glutamate [18,29]. Thus, it is possible that the effects of corticosterone on EAAC1 are mediated by changes in PKC activity. In the present study, inhibition of PKC was found to reduce EAAC1 activity. However, no obvious additive or synergistic interaction was found between PKC inhibition and corticosterone at the selective concentrations, suggesting that corticosterone decreases EAAC1 activity via PKC inhibition. This suggestion is supported by the finding that the effects of corticosterone on EAAC1 activity were attenuated by PMA, a PKC activator. Since the acute application of PKC modulators (inhibitors or activators) in a way similar to that used in this study has been shown to affect EAAC1 activity by changing EAAC1 redistribution to the plasma membrane [29,30], the results herein suggest that corticosterone reduces EAAC1 redistribution to the plasma membrane to decrease EAAC1 activity. It is also possible that prolonged glucocorticoid exposure (1–3 days) may reduce the total EAAC1 protein expression in the cells, which can then proportionally decrease the amount of EAAC1 proteins in the plasma membrane. However, our results using PKC inhibitors do not support this notion because we did not observe additional inhibition of EAAC1 activity by PKC inhibitors in the presence of corticosterone exposure. PI3K is involved in the production of lipid second messenger [31] and several studies have reported that the unique phospholipid products of PI3K activate a number of PKC isoforms. Furthermore, it has been suggested that PI3K interacts directly with several PKC isoforms [32,33]. PI3K, like PKC, has also been suggested to modulate EAAT3 activity via an independent but converging pathway [30,34]. The present study suggests that PI3K, as well as PKC, may be involved in mediating the glucocorticoid effects on EAAC1 activity. Traditionally, corticosterone binding to mineralocorticoid or glucocorticoid receptors is believed to regulate the expressions of a variety of genes via the activations of transcription factors [35]. However, Xenopus oocytes lack endogenous glucocorticoid receptors which are necessary to elicit a glucocorticoid response. Glucocorticoid-induced nuclear uptake was not detected with an immunoblot using anti-glucocorticoid receptor antibodies of the nuclear and cytoplasmic extracts from noninjected oocytes [36]. Our current study showed that the corticosterone-induced decrease in EAAC1 activity was not blocked by the steroid receptor antagonists RU-486 or spironolactone at a 100 and 500-fold excess
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concentration over corticosterone. These results, along with the findings that oocytes lack steroid nuclear receptors, can exclude the role of these steroid receptors in the corticosterone effects observed in this study. Rapid and transient increases in extracellular glutamate induced by administration of corticosterone or dexamethasone were not inhibited by the specific antagonists of the two types of corticoid receptors or by an inhibitor of protein synthesis, anisomycin. These observations suggest that glucocorticoid has non-genomic effects on neuronal constituents that can increase hippocampal glutamate concentrations [37,38]. It is not known whether the effects of corticosterone on EAAC1 as identified in our study is a mechanism for corticosterone to increase extracellular glutamate concentrations as observed in the previous in vivo studies. However, the relative slow time-course of responses for the effects of corticosterone on EAAC1 may preclude such a relationship. Unfortunately, previous studies did not monitor the extracellular glutamate levels for long durations. Thus, it remains unknown whether the slow effects of steroid on EAAC1 will translate into another increase of extracellular glutamate levels under the in vivo condition. Of note, a previous report showed that dexamethasone upregulated GLT1 (EAAT2) expression and activity in cultured astrocytes [39], suggesting the multifold effects of glucocorticoids on glutamate transporter expression and activity, and the consequences of these effects on the extracellular glutamate levels. Glucocorticoid receptors have been proposed to play an important modulatory role in neuronal damage and the development of seizures [40]. In a murine model of seizures, the incidence of seizures was parallel to the plasma corticosteroid levels and the highest incidence of seizures occurred after corticosterone treatment, whereas metyrapone which decreases the production of corticosterone by inhibiting 11-hydroxylase, reduced the seizure incidence. On the other hand, RU38486 (a glucocorticoid receptor antagonist) only partly attenuated the incidence of seizures [41]. Based on these results, it appears that corticosterone causes seizures via mechanisms other than glucocorticoid receptor activation. Since a decrease or lack of EAAC1 function may reduce the threshold of seizures [3,4], the inhibitory effects of corticosterone on EAAC1 activity, as shown in the current study, may contribute to the facilitation of seizure development by corticosterone. In conclusion, our results showed that exposure to corticosterone at clinically-relevant concentrations decreased EAAC1 activity in a concentration-dependent manner. These effects may be mediated by PKC and PI3K. Since EAATs are known to regulate glutamate neurotransmission, our results suggest that the inhibition of glucocorticoids on EAAC1 activity may contribute to their neurotoxic effects on neurons under stressful conditions. Acknowledgements All experiments were done in the Department of Anesthesiology and Clinical Research Institute, Seoul National University Hospital, and supported by a grant (No. 02-2007-018) from the Seoul National University Bundang Hospital Research Fund (Awarded to Dr. Sang-Hwan Do). References [1] Hudspith MJ. Glutamate: a role in normal brain function, anaesthesia, analgesia and CNS injury. Br J Anaesth 1997;78(6):731–47. [2] Danbolt NC. Glutamate uptake. Prog Neurobiol 2001;65(1):1–105. [3] Crino PB, Jin H, Shumate MD, Robinson MB, Coulter DA, Brooks-Kayal AR. Increased expression of the neuronal glutamate transporter (EAAT3/EAAC1) in hippocampal and neocortical epilepsy. Epilepsia 2002;43(3):211–8. [4] Sepkuty JP, Cohen AS, Eccles C, Rafiq A, Behar K, Ganel R, et al. A neuronal glutamate transporter contributes to neurotransmitter GABA synthesis and epilepsy. J Neurosci 2002;22(15):6372–9.
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