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Progesterone increases the activity of glutamate transporter type 3 expressed in Xenopus oocytes
European Journal of Pharmacology 715 (2013) 414–419
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Progesterone increases the activity of glutamate transporter type 3 expressed in Xenopus oocytes Ilsoon Son a,b, Hyun-Jung Shin c, Jung-Hee Ryu b,c, Hae-Kyoung Kim a, Sang-Hwan Do b,c,(n), Zhiyi Zuo d a
Department of Anesthesiology & Pain Medicine, Konkuk University School of Medicine, Seoul, South Korea Department of Anesthesiology & Pain Medicine, Seoul National University College of Medicine, Seoul, South Korea c Department of Anesthesiology & Pain Medicine, Seoul National University Bundang Hospital, Gyeonggi-do, South Korea d Department of Anesthesiology, University of Virginia Health System, Charlottesville, VA, USA b
art ic l e i nf o
a b s t r a c t
Article history: Received 13 January 2013 Received in revised form 25 March 2013 Accepted 28 March 2013 Available online 16 April 2013
Progesterone is an important sex hormone for pregnancy and also has neuroprotective and anticonvulsant effects. It is well-known that full-term parturients become more susceptible to volatile anesthetics. Glutamate transporters are important for preventing neurotoxicity and anesthetic action in the central nervous system. We investigated the effects of progesterone on the activity of glutamate transporter type 3 (EAAT3), the major neuronal EAAT. EAAT3 was expressed in Xenopus laevis oocytes by injecting its mRNA. Oocytes were incubated with diluted progesterone for 72 h. Two-electrode voltage clamping was used to measure membrane currents before, during, and after applying 30 μM L-glutamate. Progesterone (1–100 nM) significantly increased EAAT3 activity in a dose-dependent manner. Our kinetic study showed that the Vmax was increased in the progesterone group compared with that in the control group (2.7 70.2 vs. 3.6 70.2 μC for control group vs. progesterone group; n ¼18–23; P<0.05), however, Km was unaltered (46.7 7 10.2 μM vs. 55.9 710.5 μM for control group vs. progesterone group; n ¼18–23; P>0.05). Phorbol-12-myristate-13-acetate, a protein kinase C (PKC) activator, did not change progesterone-enhanced EAAT3 activity. Inhibitors of PKC or phosphatidylinositol 3-kinase (PI3K) abolished the progesterone-induced increases in EAAT3 activity. Our results suggest that progesterone enhances EAAT3 activity and that PKC and PI3K are involved in mediating these effects. These effects of progesterone may contribute to its anticonvulsant and anesthesia-related properties. & 2013 Elsevier B.V. All rights reserved.
Keywords: Excitatory amino acid transporter type 3 Glutamate transporter Phosphatidylinositol 3-kinase Progesterone Protein kinase C Xenopus oocyte
1. Introduction Glutamate transporters, also called excitatory amino acid transporters (EAATs), are located in neurons or glial cells of the central nervous system (CNS). They transport glutamate from the extracellular space into the cell, thereby keeping extracellular glutamate concentrations low enough to prevent glutamateinduced neurotoxicity (excitotoxicity) under physiological conditions (Danbolt, 2001). It has been shown that EAAT dysfunction may result in extracellular glutamate accumulation and lead to glutamate-mediated neuronal damage, which has been implicated in the pathophysiology of ischemic brain damage and other neurodegenerative disorders such as Alzheimer's disease or amyotrophic lateral sclerosis (Rothstein et al., 1995; Tanaka et al., 1997). As the major neuronal EAAT, EAAT3 is abundantly distributed in the hippocampus, cerebral cortex, cerebellum and basal ganglia. (n ) Correspondence to: Department of Anesthesiology & Pain Medicine, 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 addresses: [email protected], [email protected] (S.-H. Do).
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.03.053
Although the overall capacity of EAAT3 for glutamate uptake may be lower than that of glial EAATs (particularly EAAT2) in the whole brain, EAAT3 plays a primary role in glutamate uptake in certain brain areas such as the hippocampus and cerebral cortex (main action sites of progesterone in the brain) where many synapses are not surrounded by astrocytic processes (Bergles et al., 1999). EAAT3 has additional important functions in the CNS. The glutamate taken up by EAAT3 provides the substrate for synthesis of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) in neurons. Dysfunction of EAAT3 has been associated with the development of epilepsy (Gorter et al., 2002; Mathews and Diamond, 2003; Sepkuty et al., 2002). Moreover, EAAT3 mediates the uptake of cysteine, the rate-limiting substrate for the synthesis of glutathione, which is the principal intracellular antioxidant (Dringen et al., 1999; Zerangue and Kavanaugh, 1996). Progesterone is a major hormone participating in sex development, differentiation, metabolism, and reproduction in females. As a neurosteroid hormone, it also has various CNS effects. Progesterone has been shown to have neuroprotective effects against traumatic brain injury (Schumacher et al., 2000) and neuronal death/injury in a genetic mouse model of spinal cord motor
I. Son et al. / European Journal of Pharmacology 715 (2013) 414–419
neuron disease (Gonzalez Deniselle et al., 2002). In addition, progesterone has anticonvulsant effects, and a low progesterone concentration was related to catamenial epilepsy (Verrotti et al., 2010). The main target for the CNS action of progesterone is the hippocampus where EAAT3 is distributed in high concentrations. However, the involvement of EAAT3 in the effects of progesterone has not yet been studied. In our previous reports, various anesthetics (volatile, local, and intravenous) were shown to influence EAAT3 activity (Do et al., 2002a, 2002b, 2003; Ryu et al., 2009; Yun et al., 2006). Considering that progesterone renders parturient women undergoing a Cesarean section more susceptible to volatile or local anesthetics (Miller, 2010), we hypothesized that progesterone can affect EAAT3 activity. In the present study, we investigated the effects of progesterone on the activity of EAAT3 expressed in Xenopus oocytes, using two-electrode voltage clamping, and the involvement of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K), two signaling molecules, in the effects of progesterone on EAAT3.
2. Material and methods The study protocol adopted was approved by the Institutional Animal Care and Use Committee at Seoul National University College of Medicine. 2.1. Preparation of Xenopus oocytes Xenopus oocytes were isolated and microinjected as described (Do et al., 2002b). Mature female Xenopus laevis frogs were purchased and fed with regular frog brittle twice weekly. To harvest oocytes, frogs were anesthetized in 500 ml of 0.2% 3-aminobenzoic acid ethyl ester (Sigma, St. Louis, MO) in water until unresponsive to painful stimuli (toe pinching). Operations were performed on ice. A 5 mm long incision was made in the lower lateral abdominal quadrant, and then a lobule of ovarian tissue, containing approximately 150 oocytes, was removed and placed immediately in calcium-free OR-2 solution (containing in mM: NaCl 82.5, KCl 2, MgCl2 1, HEPES 5, collagenase type Ia 0.1%, pH 7.5) to remove vitelline membrane surrounding oocytes. They were defolliculated by gentle shaking for approximately 2 h and then kept at 18 1C 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, pH adjusted to 7.6). Fully grown stage V or VI Xenopus oocytes were chosen for the following experiments. 2.2. Expression of EAAT3 The rat EAAT3 complementary DNA (cDNA) construct used was provided by Dr. M.A. Hediger (Brigham and Women's Hospital, Harvard Institute of Medicine, Boston, MA). The cDNA was subcloned in a commercial vector. Plasmid DNA was linearized using a restriction enzyme, and messenger RNA (mRNA) was synthesized in vitro using a commercially available kit (Ambion, Austin, TX). The resulting mRNA was quantified and diluted in sterile water to be injected directly into oocyte cytoplasm at 30 ng per 30 nl using an automated microinjector (Nanoject; Drummond Scientific Co., Broomall, PA) via a glass micropipette whose tip diameter was 17–20 μm. Oocytes were then incubated for 3 days at 18 1C before recording currents. To study the dose-response effect of progesterone on EAAT3 activity, oocytes were exposed for 72 h to 0 (control), 0.3, 1, 3, 10, 30 or 100 nM progesterone which is naturally micronized (P3972, Sigma, St. Louis, MO), respectively. Control group was incubated in Barth solution, and progesterone
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group were incubated in Barth's solution containing progesterone from 0.3 to 100 nM. 2.3. Electrophysiologic recording We performed electrophysiological recordings at room temperature (approximately 21–23 1C). Microelectrodes were pulled from 10 μl capillary glass (Drummond Scientific Co.) on a micropipette puller. Tips were broken at a diameter of approximately 10 μm. A single defolliculated oocyte was placed in a recording chamber (0.5 ml volume) and perfused with Tyrode's solution (containing in mM: NaCl 150, KCl 5, CaCl2 2, MgSO4 1, dextrose 10, and HEPES 10 at pH 7.5) at a flow rate of 3 ml/min for 4 min before recording currents. Two recording electrodes (1–5 MΩ) filled with 3 M KCl were inserted into individual oocytes. A Warner Oocyteclamp OC 725-C (Warner, Hamden, CT) was used to voltage clamp each oocyte at -70 mV. Data acquisition and the analysis were performed by using a personal computer running OoClamp software. Oocytes that did not show a stable holding current of less than 1 μA were excluded from the analysis. L-glutamate which was diluted in Tyrode's solution to the concentration 30 μM was superfused over clamped oocytes for 25 s at 3 ml/min. L-glutamate-induced inward currents were recorded at 125 Hz for 1 min, i.e., 5 s at baseline, 25 s of agonist application, and 30 s of washing period (conducted with L-glutamate free Tyrode's solution). Responses were quantified by integrating current traces and are reported as microCoulombs (μC), which reflect the total amount of glutamate transported because EAATs are sodium co-transporters. Each experiment was performed using oocytes from at least four different frogs. 2.4. Administration of experimental chemicals To determine the effects of progesterone on the Km and Vmax values of EAAT3 for glutamate, serial concentrations of L-glutamate (3, 10, 30, 100, and 300 μM) were used. In other experiments, 30 μM L-glutamate was used to induce glutamate transporter currents. To study the role of PKC on EAAT3 activity, oocytes were preincubated with 100 nM phorbol-12-myristate-13acetate (PMA) for 10 min before recording. To study the effects of PKC inhibitors on EAAT3 activity, oocytes were exposed to the PKC inhibitors, staurosporine (1 μM for 1 h), chelerythrine (50 μM for 1 h), or calphostin C (3 μM for 2 h). The PI3K inhibitors LY294002 (2-(4-morpholinyl)-8-phenyl-1(4 H)-benzopyran-4-one, 10 μM and 50 μM) or wortmannin (5 μM and 10 μM) were added to the control and progesterone-treated oocytes 1 h prior to current recording in order to investigate the role of PI3K in regulating EAAT3 activity. To determine the temporal effects of progesterone on EAAT3 activity, oocytes were incubated with 1 μM progesterone for 12, 24, 48, and 72 h before the L-glutamate-induced currents were measured. Finally, to determine whether the progesterone effects on EAAT3 was mediated by intracellular progesterone receptors, oocytes were preincubated with mifepristone (10 and 50 μM for 6 h), a progesterone receptor antagonist, before current measurements. Molecular biology reagents were obtained from Ambion (Austin, TX), and other chemicals from Sigma (St. Louis, MO), unless otherwise specified. 2.5. Data analysis Data are summarized as means 7S.E.M. We normalized each response by the results of the same-day controls to compensate batch-to-batch variability in oocyte response, which is common due to different expression levels. Statistical analysis was performed using the Student's t-test or ANOVA. Data were analyzed
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using SPSS 17.0 (Chicago, IL) and Prism version 4.0 (GraphPad, San Diego, CA). P values of <0.05 were considered significant.
3. Results Oocytes without injection of EAAT3 mRNA showed no response (data not shown), whereas oocytes injected with EAAT3 mRNA showed inward currents in response to L-glutamate. This finding suggests that EAAT3 expressed on the oocyte membrane produces inward currents with L-glutamate. Previous studies have revealed that the Km of EAAT3 for L-glutamate is about 30 μM (Do et al., 2003; Kim et al., 2005). Thus, we used 30 μM L-glutamate in the present study. When oocytes injected with EAAT3 mRNA were exposed to progesterone (0.3, 1, 3, 10, 30 or 100 nM) for 72 h, the response to L-glutamate increased in a concentration-dependent manner (Fig. 1). We could not obtain reliable results at 300 or 1000 nM progesterone because the resting currents of the oocytes incubated with 300 or 1000 nM progesterone became unstable 1–2 min after superfusion. The responses in the presence of 1 nM or higher concentrations of progesterone were significantly increased compared with control values (n¼ 13–37 in each group, P<0.05) (Fig. 1). The EC50 of this enhancement by progesterone was 0.8 nM, and thus 1 nM progesterone was used for subsequent experiments. Oocytes exposed to 1 nM progesterone for 72 h showed an increased response to not only 30 μM L-glutamate but also 10, 100, and 300 μM L-glutamate (Fig. 2). Further analysis of the data showed that 1 nM progesterone significantly increased Vmax (2.77 0.2 vs. 3.6 7 0.2 μC for control group vs. progesterone group; n ¼18–23 in each group; P<0.05), but did not cause a significant change in Km (46.7 710.2 vs. 55.9 710.5 μM for control group vs. progesterone group; n ¼18–23 in each group; P>0.05). Oocytes pre-incubated with PMA (100 nM) for 10 min showed greater EAAT3 activity than controls (1.070.1 vs. 1.3 70.1 μC; n ¼14–16 in each group; P <0.05), which agrees with our previous findings (Fig. 3) (Do et al., 2002a, 2002b; Ryu et al., 2009; Yun et al., 2007). When progesterone-treated oocytes (1 nM for 72 h) were exposed to PMA (100 nM for 10 min), no additive or synergistic interaction was observed among groups (Fig. 3). Additionally, basal EAAT3 activity was not decreased significantly by pre-incubation with a PKC inhibitor (staurosporine, 1 μM) for 1 h, which is in accordance with our previous results (Do et al., 2002a;
Fig. 1. Concentration-response of progesterone on the activity of excitatory amino acid transporter type 3. Oocytes were exposed to various concentrations (control, 0.3 nM, 1 nM, 3 nM, 10 nM, 30 nM or 100 nM) of progesterone for 72 h. The EAAT3 response was then induced by 30 μM L-glutamate. Oocytes injected with EAAT3 mRNA showed increased responses to L-glutamate in a progesterone concentration-dependent manner. Data are means 7S.E.M. (n¼ 13–37 in each group). *P<0.05 compared with control.
Fig. 2. Concentration-response curve of EAAT3 to glutamate in the presence or absence of 1 nM progesterone for 72 h. In addition to enhancing the responses induced by 30 μM L-glutamate, 1 nM progesterone also significantly increased the responses induced by 10, 100 or 300 μM L-glutamate. Data are means 7S.E.M. (n ¼18–23 in each group). * P<0.05 compared to the corresponding control.
Fig. 3. Effects of protein kinase C (PKC) activation on EAAT3 activity in the presence or absence of 1 nM progesterone for 72 h. Oocytes exposed to progesterone, PMA, or both showed a significant increase in EAAT3 activity compared with control; whereas there was no statistical difference among the progesterone, PMA, or PMA plus progesterone groups. PMA; phorbol-12-myristate-13-acetate. Data are means 7S.E.M. (n¼ 14–16 in each group). * P<0.05 compared with control.
Kim et al., 2003). However, when progesterone-treated oocytes were exposed to staurosporine (1 μM for 1 h), progesteroneinduced enhancement of EAAT3 activity was abolished (Fig. 4). Similarly, two other PKC inhibitors (chelerythrine at 50 μM for 1 h or calphostin C at 3 μM for 2 h) did not inhibit basal EAAT3 activity but abolished progesterone-enhanced EAAT3 activity (n ¼13–22 in each group, Fig. 4). Pretreatment with LY294002 (50 μM) or wortmannin (10 μM) for 1 h significantly decreased basal EAAT3 activity, although 5 μM wortmannin did not significantly decrease basal EAAT3 activity. Progesterone-induced enhancement of EAAT3 activity was also abolished by LY294002 or wortmannin at each of the two concentrations (n ¼14–33 in each group Fig. 5). In oocytes injected with EAAT3 mRNA and exposed to 1 nM progesterone for 12, 24, 48, or 72 h, the response to L-glutamate showed a time-dependent increase. EAAT3 activity was significantly increased compared with control activity only after 72 h of exposure to progesterone (control, 1.00 70.09; 12 h, 1.06 70.08;
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Fig. 4. Effects of protein kinase C (PKC) inhibition on EAAT3 activity in the presence or absence of 1 nM progesterone for 72 h. Staurosporine, chelerythrine and calphostin C abolished the 1 nM progesterone-enhanced EAAT3 activity. Data are mean 7 S.E.M. (n ¼13–22 in each group). * P<0.05 compared with control.
Fig. 5. Effects of phosphatidylinositol 3-kinase (PI3K) inhibition on EAAT3 activity in the presence or absence of progesterone for 72 h. Pretreatment of the oocytes with LY294002 or wortmannin decreased the progesterone-enhanced EAAT3 activity. Data are mean 7 S.E.M. (n¼ 14–33 in each group). * P<0.05 compared with control.
24 h, 0.99 70.10; 48 h, 1.177 0.08; 72 h, 1.30 70.09 μC; n ¼18–30 in each group, P<0.05). Pretreatment of the oocytes with mifepristone (10 or 50 μM for 6 h) produced no significant difference in EAAT3 activity compared with the activity in control oocytes. Compared with the activity in control oocytes, EAAT3 activity was increased significantly in oocytes treated with progesterone in the absence or presence of mifepristone (n ¼18–22 in each group Fig. 6).
4. Discussion Progesterone plays a critical role for the establishment and maintenance of pregnancy. The levels of progesterone are low, usually below 2 nM, during the pre-ovulatory phase of the menstrual cycle, rise above 15 nM after ovulation, and are elevated throughout the luteal phase. When a woman becomes pregnant, her progesterone levels are initially maintained at luteal levels. With the onset of the luteal-placental shift associated with progesterone support of the pregnancy, progesterone levels start to increase further and may reach 300–600 nM at full term (Arck et al., 2007). In the present study, progesterone levels up to
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Fig. 6. Effects of 1 nM progesterone on EAAT3 activity in the presence or absence of progesterone receptor antagonist, mifepristone. Incubation of oocytes with the progesterone receptor antagonist did not affect the 1 nM progesterone-enhanced EAAT3 activity. Data are mean 7 S.E.M. (n¼ 18–22 in each group). *P<0.05 compared with control.
100 nM were tested because oocytes exposed to 300 or 1000 nM progesterone became unstable and were not appropriate for measuring currents. High glutamate concentrations can induce neurotoxicity and seizure and it is well known that the prevention of extracellular glutamate accumulation in the CNS by EAATs is an important mechanism for neuroprotection (Sheldon and Robinson, 2007). In the present study, EAAT3 activity was increased by progesterone at concentrations of 1 nM and higher, which correspond to physiological ovulatory concentrations of progesterone. This enhanced EAAT3 activity should lead to tight control of extracellular glutamate concentrations, which may be related to the anticonvulsant, neuroprotective, and anesthesia-potentiating effects of progesterone (Hughes, 2002; Rogawski, 2003; Stein et al., 2008). In catamenial epilepsy, seizure susceptibility changes during the menstrual cycle: when the estrogen/progesterone ratio decreases (mid-luteal phase), seizures subside, and when the ratio increases (premenstrual and pre-ovulatory phase), seizures are exacerbated (Verrotti et al., 2010). Thus, progesterone appears to act as an anticonvulsant; and estrogen, as a proconvulsant. Recently, we have reported that 17β‐estradiol, an active form of estrogen, decreased EAAT3 activity (Na et al., 2012). The present kinetic study showed a significant increase in Vmax with 1 nM progesterone, but no significant change in Km, suggesting that enhanced EAAT3 activity may be attributable to an increased availability or turnover rate of EAAT3, rather than an increased affinity of EAAT3 for glutamate. Glutamate transport activity via EAATs is regulated by PKC (Casado et al., 1993; Do et al., 2002b). PMA, a PKC activator, increases EAAT3 activity, as was also observed in our previous studies (Do et al., 2002a, Kim et al., 2003). However, in the present study, PMA had no additive or synergistic effect on progesteroneenhanced EAAT3 activity, suggesting that PMA and progesterone may increase EAAT3 activity via the same mechanism. Additionally, all three PKC inhibitors tested in the present study attenuated the progesterone-induced increase in EAAT3 activity. These results indicate that PKC may mediate the effect of progesterone on EAAT3 activity. LY294002 and wortmannin, PI3K inhibitors, also attenuated the progesterone-induced increase in EAAT3 activity, suggesting that PI3K may also be involved in mediating the effect of progesterone on EAAT3 activity.
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It has been reported that EAAT3 activity/expression is highly regulated by intracellular signaling pathways involving PKC and PI3K (Nieoullon et al., 2006), as had been previously shown for the non-classical mechanism of action of progesterone in the brain (Balasubramanian et al., 2008; Ishrat et al., 2012). In the present study, inhibition of the progesterone receptor with mifepristone did not affect the enhancement of EAAT3 activity by progesterone, suggesting that progesterone acts to increase EAAT3 activity by a non-classical (non-genomic) mechanism. It is not clear how progesterone can lead to activation of PKC. One possible candidate is G protein-coupled receptors that are expressed in the oocytes and have been shown to act as progesterone receptors on the plasma membrane (Lutz et al., 2000). Also, it is known that G protein-coupled receptors can be linked to PKC and PI3K (Callaghan et al., 2004; Banday et al., 2007). Our results support that both PKC and PI3K are involved in the effects of progesterone on EAAT3. A question can be raised about why PKC and PI3K that are members of different signaling pathways are both involved in the progesterone's effects. Although PKC and PI3K are independent signaling molecules, the signaling pathways involving PKC and PI3K can interact with each other and PKC can work as a molecule downstream of PI3K (Frey et al., 2006). Pregnancy has been observed to decrease anesthetic requirements. Compared with non-pregnant women, pregnant women required less isoflurane (Gin and Chan, 1994), and the minimum alveolar concentration was reduced in postpartum women until 3 days after delivery (Chan and Gin, 1995). Furthermore, pregnant women had more heat-pain tolerance (Carvalho et al., 2006). In a rat study, administration of exogenous progesterone significantly decreased the anesthetic requirement (Shimizu et al., 2010). In neuraxial anesthesia, the requirement for local anesthetics was also reduced during pregnancy. This was thought to be attributable mainly to increased intra-abdominal pressure and decreased cerebrospinal fluid volume. However, a reduction of anesthetic requirement was also shown during early pregnancy (Fagraeus et al., 1983). When the effects of bupivacaine on the isolated vagus nerve were compared between pregnant and nonpregnant rabbits, the onset of the block was significantly faster in nerves from pregnant rabbits (Flanagan et al., 1987). Thus, nerve fibers may become more susceptible to local anesthetics during pregnancy. In addition to their effects on anesthetic requirements, steroid hormones themselves also have anesthetic actions. For example, progesterone has been shown to have analgesic and sedative properties (Frye et al., 2004; Van Broekhoven et al., 2006). Although an effect of progesterone on GABA receptors or opioid receptors has been proposed as the underlying mechanism (Dawson-Basoa and Gintzler, 1996; Pluchino et al., 2006), progesterone effects on glutamate signaling have recently received attention (Coronel et al., 2011; Ren et al., 2000). Additionally, progesterone and its metabolites have shown neuroprotective properties against traumatic brain injury (Stein, 2011). Allopregnanolone (10 μM), a metabolite of progesterone, attenuated N-methyl D-aspartate-induced excitotoxicity and apoptosis in human cell cultures (Lockhart et al., 2002), and the administration of progesterone after acute traumatic injury limited CNS damage and improved recovery (Stein et al., 2008). In the present study, progesterone enhanced the activity of EAAT3, a finding that could explain its effects on analgesia, sedation, neuroprotection, excitotoxicity, and GABA signaling. Our study has some limitations. We used an oocyte expression system to investigate EAAT in vitro. Although Xenopus oocytes have been used for studies of EAAT activity (Do et al., 2002b; Xia et al., 2006), Xenopus oocytes provide an artificial environment for EAATs. Nevertheless, is well established that membrane proteins function similarly in Xenopus oocytes and human cells (Wagner et al., 2000). Our experiments were performed at room
temperature, whereas the membrane protein was from a homeothermic animal (rat). However, there is no evidence to indicate that such difference in temperature would result in significant changes in the kinetics and function of this type of membrane protein. In conclusion, our results suggest that progesterone enhances EAAT3 activity and that PKC and PI3K mediate the effect. This progesterone effect may contribute to the neuroprotective and antinociceptive actions of progesterone.
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Ryu, J., Cheong, I.Y., Do, S.H., Zuo, Z., 2009. Alphaxalone, a neurosteroid anaesthetic, increases the activity of the glutamate transporter type 3 expressed in Xenopus oocytes. Eur. J. Pharmacol. 602, 23–27. Schumacher, M., Akwa, Y., Guennoun, R., Robert, F., Labombarda, F., Desarnaud, F., Robel, P., De Nicola, A.F., Baulieu, E.E., 2000. Steroid synthesis and metabolism in the nervous system: trophic and protective effects. J. Neurocytol. 29, 307–326. Sepkuty, J.P., Cohen, A.S., Eccles, C., Rafiq, A., Behar, K., Ganel, R., Coulter, D.A., Rothstein, J.D., 2002. A neuronal glutamate transporter contributes to neurotransmitter GABA synthesis and epilepsy. J. Neurosci. 22, 6372–6379. Sheldon, A.L., Robinson, M.B., 2007. The role of glutamate transporters in neurodegenerative diseases and potential opportunities for intervention. Neurochem. Int. 51, 333–355. Shimizu, T., Inomata, S., Tanaka, M., 2010. Progesterone decreases sevoflurane requirement in male mice: a dose-response study. Br. J. Anaesth. 104, 603–605. Stein, D.G., 2011. Progesterone in the treatment of acute traumatic brain injury: a clinical perspective and update. Neuroscience 191, 101–106. Stein, D.G., Wright, D.W., Kellermann, A.L., 2008. Does progesterone have neuroprotective properties? Ann. Emerg. Med. 51, 164–172. Tanaka, K., Watase, K., Manabe, T., Yamada, K., Watanabe, M., Takahashi, K., Iwama, H., Nishikawa, T., Ichihara, N., Kikuchi, T., Okuyama, S., Kawashima, N., Hori, S., Takimoto, M., Wada, K., 1997. Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science 276, 1699–1702. Van Broekhoven, F., Backstrom, T., Verkes, R.J., 2006. Oral progesterone decreases saccadic eye velocity and increases sedation in women. Psychoneuroendocrinology 31, 1190–1199. Verrotti, A., Laus, M., Coppola, G., Parisi, P., Mohn, A., Chiarelli, F., 2010. Catamenial epilepsy: hormonal aspects. Gynecol. Endocrinol. 26, 783–790. Wagner, C.A., Friedrich, B., Setiawan, I., Lang, F., Broer, S., 2000. The use of Xenopus laevis oocytes for the functional characterization of heterologously expressed membrane proteins. Cell Physiol. Biochem. 10, 1–12. Xia, P., Pei, G., Schwarz, W., 2006. Regulation of the glutamate transporter EAAC1 by expression and activation of delta-opioid receptor. Eur. J. Neurosci. 24, 87–93. Yun, J.Y., Kim, J.H., Kim, H.K., Lim, Y.J., Do, S.H., Zuo, Z., 2006. Effects of intravenous anesthetics on the activity of glutamate transporter EAAT3 expressed in Xenopus oocytes: evidence for protein kinase C involvement. Eur. J. Pharmacol. 531, 133–139. Yun, J.Y., Park, K.S., Kim, J.H., Do, S.H., Zuo, Z., 2007. Propofol reverses oxidative stress-attenuated glutamate transporter EAAT3 activity: evidence of protein kinase C involvement. Eur. J. Pharmacol. 565, 83–88. Zerangue, N., Kavanaugh, M.P., 1996. Interaction of L-cysteine with a human excitatory amino acid transporter. J. Physiol. 493 (Pt 2), 419–423.
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Progesterone increases the activity of glutamate transporter type 3 expressed in Xenopus oocytes Ilsoon Son a,b, Hyun-Jung Shin c, Jung-Hee Ryu b,c, Hae-Kyoung Kim a, Sang-Hwan Do b,c,(n), Zhiyi Zuo d a
Department of Anesthesiology & Pain Medicine, Konkuk University School of Medicine, Seoul, South Korea Department of Anesthesiology & Pain Medicine, Seoul National University College of Medicine, Seoul, South Korea c Department of Anesthesiology & Pain Medicine, Seoul National University Bundang Hospital, Gyeonggi-do, South Korea d Department of Anesthesiology, University of Virginia Health System, Charlottesville, VA, USA b
art ic l e i nf o
a b s t r a c t
Article history: Received 13 January 2013 Received in revised form 25 March 2013 Accepted 28 March 2013 Available online 16 April 2013
Progesterone is an important sex hormone for pregnancy and also has neuroprotective and anticonvulsant effects. It is well-known that full-term parturients become more susceptible to volatile anesthetics. Glutamate transporters are important for preventing neurotoxicity and anesthetic action in the central nervous system. We investigated the effects of progesterone on the activity of glutamate transporter type 3 (EAAT3), the major neuronal EAAT. EAAT3 was expressed in Xenopus laevis oocytes by injecting its mRNA. Oocytes were incubated with diluted progesterone for 72 h. Two-electrode voltage clamping was used to measure membrane currents before, during, and after applying 30 μM L-glutamate. Progesterone (1–100 nM) significantly increased EAAT3 activity in a dose-dependent manner. Our kinetic study showed that the Vmax was increased in the progesterone group compared with that in the control group (2.7 70.2 vs. 3.6 70.2 μC for control group vs. progesterone group; n ¼18–23; P<0.05), however, Km was unaltered (46.7 7 10.2 μM vs. 55.9 710.5 μM for control group vs. progesterone group; n ¼18–23; P>0.05). Phorbol-12-myristate-13-acetate, a protein kinase C (PKC) activator, did not change progesterone-enhanced EAAT3 activity. Inhibitors of PKC or phosphatidylinositol 3-kinase (PI3K) abolished the progesterone-induced increases in EAAT3 activity. Our results suggest that progesterone enhances EAAT3 activity and that PKC and PI3K are involved in mediating these effects. These effects of progesterone may contribute to its anticonvulsant and anesthesia-related properties. & 2013 Elsevier B.V. All rights reserved.
Keywords: Excitatory amino acid transporter type 3 Glutamate transporter Phosphatidylinositol 3-kinase Progesterone Protein kinase C Xenopus oocyte
1. Introduction Glutamate transporters, also called excitatory amino acid transporters (EAATs), are located in neurons or glial cells of the central nervous system (CNS). They transport glutamate from the extracellular space into the cell, thereby keeping extracellular glutamate concentrations low enough to prevent glutamateinduced neurotoxicity (excitotoxicity) under physiological conditions (Danbolt, 2001). It has been shown that EAAT dysfunction may result in extracellular glutamate accumulation and lead to glutamate-mediated neuronal damage, which has been implicated in the pathophysiology of ischemic brain damage and other neurodegenerative disorders such as Alzheimer's disease or amyotrophic lateral sclerosis (Rothstein et al., 1995; Tanaka et al., 1997). As the major neuronal EAAT, EAAT3 is abundantly distributed in the hippocampus, cerebral cortex, cerebellum and basal ganglia. (n ) Correspondence to: Department of Anesthesiology & Pain Medicine, 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 addresses: [email protected], [email protected] (S.-H. Do).
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.03.053
Although the overall capacity of EAAT3 for glutamate uptake may be lower than that of glial EAATs (particularly EAAT2) in the whole brain, EAAT3 plays a primary role in glutamate uptake in certain brain areas such as the hippocampus and cerebral cortex (main action sites of progesterone in the brain) where many synapses are not surrounded by astrocytic processes (Bergles et al., 1999). EAAT3 has additional important functions in the CNS. The glutamate taken up by EAAT3 provides the substrate for synthesis of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) in neurons. Dysfunction of EAAT3 has been associated with the development of epilepsy (Gorter et al., 2002; Mathews and Diamond, 2003; Sepkuty et al., 2002). Moreover, EAAT3 mediates the uptake of cysteine, the rate-limiting substrate for the synthesis of glutathione, which is the principal intracellular antioxidant (Dringen et al., 1999; Zerangue and Kavanaugh, 1996). Progesterone is a major hormone participating in sex development, differentiation, metabolism, and reproduction in females. As a neurosteroid hormone, it also has various CNS effects. Progesterone has been shown to have neuroprotective effects against traumatic brain injury (Schumacher et al., 2000) and neuronal death/injury in a genetic mouse model of spinal cord motor
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neuron disease (Gonzalez Deniselle et al., 2002). In addition, progesterone has anticonvulsant effects, and a low progesterone concentration was related to catamenial epilepsy (Verrotti et al., 2010). The main target for the CNS action of progesterone is the hippocampus where EAAT3 is distributed in high concentrations. However, the involvement of EAAT3 in the effects of progesterone has not yet been studied. In our previous reports, various anesthetics (volatile, local, and intravenous) were shown to influence EAAT3 activity (Do et al., 2002a, 2002b, 2003; Ryu et al., 2009; Yun et al., 2006). Considering that progesterone renders parturient women undergoing a Cesarean section more susceptible to volatile or local anesthetics (Miller, 2010), we hypothesized that progesterone can affect EAAT3 activity. In the present study, we investigated the effects of progesterone on the activity of EAAT3 expressed in Xenopus oocytes, using two-electrode voltage clamping, and the involvement of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K), two signaling molecules, in the effects of progesterone on EAAT3.
2. Material and methods The study protocol adopted was approved by the Institutional Animal Care and Use Committee at Seoul National University College of Medicine. 2.1. Preparation of Xenopus oocytes Xenopus oocytes were isolated and microinjected as described (Do et al., 2002b). Mature female Xenopus laevis frogs were purchased and fed with regular frog brittle twice weekly. To harvest oocytes, frogs were anesthetized in 500 ml of 0.2% 3-aminobenzoic acid ethyl ester (Sigma, St. Louis, MO) in water until unresponsive to painful stimuli (toe pinching). Operations were performed on ice. A 5 mm long incision was made in the lower lateral abdominal quadrant, and then a lobule of ovarian tissue, containing approximately 150 oocytes, was removed and placed immediately in calcium-free OR-2 solution (containing in mM: NaCl 82.5, KCl 2, MgCl2 1, HEPES 5, collagenase type Ia 0.1%, pH 7.5) to remove vitelline membrane surrounding oocytes. They were defolliculated by gentle shaking for approximately 2 h and then kept at 18 1C 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, pH adjusted to 7.6). Fully grown stage V or VI Xenopus oocytes were chosen for the following experiments. 2.2. Expression of EAAT3 The rat EAAT3 complementary DNA (cDNA) construct used was provided by Dr. M.A. Hediger (Brigham and Women's Hospital, Harvard Institute of Medicine, Boston, MA). The cDNA was subcloned in a commercial vector. Plasmid DNA was linearized using a restriction enzyme, and messenger RNA (mRNA) was synthesized in vitro using a commercially available kit (Ambion, Austin, TX). The resulting mRNA was quantified and diluted in sterile water to be injected directly into oocyte cytoplasm at 30 ng per 30 nl using an automated microinjector (Nanoject; Drummond Scientific Co., Broomall, PA) via a glass micropipette whose tip diameter was 17–20 μm. Oocytes were then incubated for 3 days at 18 1C before recording currents. To study the dose-response effect of progesterone on EAAT3 activity, oocytes were exposed for 72 h to 0 (control), 0.3, 1, 3, 10, 30 or 100 nM progesterone which is naturally micronized (P3972, Sigma, St. Louis, MO), respectively. Control group was incubated in Barth solution, and progesterone
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group were incubated in Barth's solution containing progesterone from 0.3 to 100 nM. 2.3. Electrophysiologic recording We performed electrophysiological recordings at room temperature (approximately 21–23 1C). Microelectrodes were pulled from 10 μl capillary glass (Drummond Scientific Co.) on a micropipette puller. Tips were broken at a diameter of approximately 10 μm. A single defolliculated oocyte was placed in a recording chamber (0.5 ml volume) and perfused with Tyrode's solution (containing in mM: NaCl 150, KCl 5, CaCl2 2, MgSO4 1, dextrose 10, and HEPES 10 at pH 7.5) at a flow rate of 3 ml/min for 4 min before recording currents. Two recording electrodes (1–5 MΩ) filled with 3 M KCl were inserted into individual oocytes. A Warner Oocyteclamp OC 725-C (Warner, Hamden, CT) was used to voltage clamp each oocyte at -70 mV. Data acquisition and the analysis were performed by using a personal computer running OoClamp software. Oocytes that did not show a stable holding current of less than 1 μA were excluded from the analysis. L-glutamate which was diluted in Tyrode's solution to the concentration 30 μM was superfused over clamped oocytes for 25 s at 3 ml/min. L-glutamate-induced inward currents were recorded at 125 Hz for 1 min, i.e., 5 s at baseline, 25 s of agonist application, and 30 s of washing period (conducted with L-glutamate free Tyrode's solution). Responses were quantified by integrating current traces and are reported as microCoulombs (μC), which reflect the total amount of glutamate transported because EAATs are sodium co-transporters. Each experiment was performed using oocytes from at least four different frogs. 2.4. Administration of experimental chemicals To determine the effects of progesterone on the Km and Vmax values of EAAT3 for glutamate, serial concentrations of L-glutamate (3, 10, 30, 100, and 300 μM) were used. In other experiments, 30 μM L-glutamate was used to induce glutamate transporter currents. To study the role of PKC on EAAT3 activity, oocytes were preincubated with 100 nM phorbol-12-myristate-13acetate (PMA) for 10 min before recording. To study the effects of PKC inhibitors on EAAT3 activity, oocytes were exposed to the PKC inhibitors, staurosporine (1 μM for 1 h), chelerythrine (50 μM for 1 h), or calphostin C (3 μM for 2 h). The PI3K inhibitors LY294002 (2-(4-morpholinyl)-8-phenyl-1(4 H)-benzopyran-4-one, 10 μM and 50 μM) or wortmannin (5 μM and 10 μM) were added to the control and progesterone-treated oocytes 1 h prior to current recording in order to investigate the role of PI3K in regulating EAAT3 activity. To determine the temporal effects of progesterone on EAAT3 activity, oocytes were incubated with 1 μM progesterone for 12, 24, 48, and 72 h before the L-glutamate-induced currents were measured. Finally, to determine whether the progesterone effects on EAAT3 was mediated by intracellular progesterone receptors, oocytes were preincubated with mifepristone (10 and 50 μM for 6 h), a progesterone receptor antagonist, before current measurements. Molecular biology reagents were obtained from Ambion (Austin, TX), and other chemicals from Sigma (St. Louis, MO), unless otherwise specified. 2.5. Data analysis Data are summarized as means 7S.E.M. We normalized each response by the results of the same-day controls to compensate batch-to-batch variability in oocyte response, which is common due to different expression levels. Statistical analysis was performed using the Student's t-test or ANOVA. Data were analyzed
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using SPSS 17.0 (Chicago, IL) and Prism version 4.0 (GraphPad, San Diego, CA). P values of <0.05 were considered significant.
3. Results Oocytes without injection of EAAT3 mRNA showed no response (data not shown), whereas oocytes injected with EAAT3 mRNA showed inward currents in response to L-glutamate. This finding suggests that EAAT3 expressed on the oocyte membrane produces inward currents with L-glutamate. Previous studies have revealed that the Km of EAAT3 for L-glutamate is about 30 μM (Do et al., 2003; Kim et al., 2005). Thus, we used 30 μM L-glutamate in the present study. When oocytes injected with EAAT3 mRNA were exposed to progesterone (0.3, 1, 3, 10, 30 or 100 nM) for 72 h, the response to L-glutamate increased in a concentration-dependent manner (Fig. 1). We could not obtain reliable results at 300 or 1000 nM progesterone because the resting currents of the oocytes incubated with 300 or 1000 nM progesterone became unstable 1–2 min after superfusion. The responses in the presence of 1 nM or higher concentrations of progesterone were significantly increased compared with control values (n¼ 13–37 in each group, P<0.05) (Fig. 1). The EC50 of this enhancement by progesterone was 0.8 nM, and thus 1 nM progesterone was used for subsequent experiments. Oocytes exposed to 1 nM progesterone for 72 h showed an increased response to not only 30 μM L-glutamate but also 10, 100, and 300 μM L-glutamate (Fig. 2). Further analysis of the data showed that 1 nM progesterone significantly increased Vmax (2.77 0.2 vs. 3.6 7 0.2 μC for control group vs. progesterone group; n ¼18–23 in each group; P<0.05), but did not cause a significant change in Km (46.7 710.2 vs. 55.9 710.5 μM for control group vs. progesterone group; n ¼18–23 in each group; P>0.05). Oocytes pre-incubated with PMA (100 nM) for 10 min showed greater EAAT3 activity than controls (1.070.1 vs. 1.3 70.1 μC; n ¼14–16 in each group; P <0.05), which agrees with our previous findings (Fig. 3) (Do et al., 2002a, 2002b; Ryu et al., 2009; Yun et al., 2007). When progesterone-treated oocytes (1 nM for 72 h) were exposed to PMA (100 nM for 10 min), no additive or synergistic interaction was observed among groups (Fig. 3). Additionally, basal EAAT3 activity was not decreased significantly by pre-incubation with a PKC inhibitor (staurosporine, 1 μM) for 1 h, which is in accordance with our previous results (Do et al., 2002a;
Fig. 1. Concentration-response of progesterone on the activity of excitatory amino acid transporter type 3. Oocytes were exposed to various concentrations (control, 0.3 nM, 1 nM, 3 nM, 10 nM, 30 nM or 100 nM) of progesterone for 72 h. The EAAT3 response was then induced by 30 μM L-glutamate. Oocytes injected with EAAT3 mRNA showed increased responses to L-glutamate in a progesterone concentration-dependent manner. Data are means 7S.E.M. (n¼ 13–37 in each group). *P<0.05 compared with control.
Fig. 2. Concentration-response curve of EAAT3 to glutamate in the presence or absence of 1 nM progesterone for 72 h. In addition to enhancing the responses induced by 30 μM L-glutamate, 1 nM progesterone also significantly increased the responses induced by 10, 100 or 300 μM L-glutamate. Data are means 7S.E.M. (n ¼18–23 in each group). * P<0.05 compared to the corresponding control.
Fig. 3. Effects of protein kinase C (PKC) activation on EAAT3 activity in the presence or absence of 1 nM progesterone for 72 h. Oocytes exposed to progesterone, PMA, or both showed a significant increase in EAAT3 activity compared with control; whereas there was no statistical difference among the progesterone, PMA, or PMA plus progesterone groups. PMA; phorbol-12-myristate-13-acetate. Data are means 7S.E.M. (n¼ 14–16 in each group). * P<0.05 compared with control.
Kim et al., 2003). However, when progesterone-treated oocytes were exposed to staurosporine (1 μM for 1 h), progesteroneinduced enhancement of EAAT3 activity was abolished (Fig. 4). Similarly, two other PKC inhibitors (chelerythrine at 50 μM for 1 h or calphostin C at 3 μM for 2 h) did not inhibit basal EAAT3 activity but abolished progesterone-enhanced EAAT3 activity (n ¼13–22 in each group, Fig. 4). Pretreatment with LY294002 (50 μM) or wortmannin (10 μM) for 1 h significantly decreased basal EAAT3 activity, although 5 μM wortmannin did not significantly decrease basal EAAT3 activity. Progesterone-induced enhancement of EAAT3 activity was also abolished by LY294002 or wortmannin at each of the two concentrations (n ¼14–33 in each group Fig. 5). In oocytes injected with EAAT3 mRNA and exposed to 1 nM progesterone for 12, 24, 48, or 72 h, the response to L-glutamate showed a time-dependent increase. EAAT3 activity was significantly increased compared with control activity only after 72 h of exposure to progesterone (control, 1.00 70.09; 12 h, 1.06 70.08;
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Fig. 4. Effects of protein kinase C (PKC) inhibition on EAAT3 activity in the presence or absence of 1 nM progesterone for 72 h. Staurosporine, chelerythrine and calphostin C abolished the 1 nM progesterone-enhanced EAAT3 activity. Data are mean 7 S.E.M. (n ¼13–22 in each group). * P<0.05 compared with control.
Fig. 5. Effects of phosphatidylinositol 3-kinase (PI3K) inhibition on EAAT3 activity in the presence or absence of progesterone for 72 h. Pretreatment of the oocytes with LY294002 or wortmannin decreased the progesterone-enhanced EAAT3 activity. Data are mean 7 S.E.M. (n¼ 14–33 in each group). * P<0.05 compared with control.
24 h, 0.99 70.10; 48 h, 1.177 0.08; 72 h, 1.30 70.09 μC; n ¼18–30 in each group, P<0.05). Pretreatment of the oocytes with mifepristone (10 or 50 μM for 6 h) produced no significant difference in EAAT3 activity compared with the activity in control oocytes. Compared with the activity in control oocytes, EAAT3 activity was increased significantly in oocytes treated with progesterone in the absence or presence of mifepristone (n ¼18–22 in each group Fig. 6).
4. Discussion Progesterone plays a critical role for the establishment and maintenance of pregnancy. The levels of progesterone are low, usually below 2 nM, during the pre-ovulatory phase of the menstrual cycle, rise above 15 nM after ovulation, and are elevated throughout the luteal phase. When a woman becomes pregnant, her progesterone levels are initially maintained at luteal levels. With the onset of the luteal-placental shift associated with progesterone support of the pregnancy, progesterone levels start to increase further and may reach 300–600 nM at full term (Arck et al., 2007). In the present study, progesterone levels up to
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Fig. 6. Effects of 1 nM progesterone on EAAT3 activity in the presence or absence of progesterone receptor antagonist, mifepristone. Incubation of oocytes with the progesterone receptor antagonist did not affect the 1 nM progesterone-enhanced EAAT3 activity. Data are mean 7 S.E.M. (n¼ 18–22 in each group). *P<0.05 compared with control.
100 nM were tested because oocytes exposed to 300 or 1000 nM progesterone became unstable and were not appropriate for measuring currents. High glutamate concentrations can induce neurotoxicity and seizure and it is well known that the prevention of extracellular glutamate accumulation in the CNS by EAATs is an important mechanism for neuroprotection (Sheldon and Robinson, 2007). In the present study, EAAT3 activity was increased by progesterone at concentrations of 1 nM and higher, which correspond to physiological ovulatory concentrations of progesterone. This enhanced EAAT3 activity should lead to tight control of extracellular glutamate concentrations, which may be related to the anticonvulsant, neuroprotective, and anesthesia-potentiating effects of progesterone (Hughes, 2002; Rogawski, 2003; Stein et al., 2008). In catamenial epilepsy, seizure susceptibility changes during the menstrual cycle: when the estrogen/progesterone ratio decreases (mid-luteal phase), seizures subside, and when the ratio increases (premenstrual and pre-ovulatory phase), seizures are exacerbated (Verrotti et al., 2010). Thus, progesterone appears to act as an anticonvulsant; and estrogen, as a proconvulsant. Recently, we have reported that 17β‐estradiol, an active form of estrogen, decreased EAAT3 activity (Na et al., 2012). The present kinetic study showed a significant increase in Vmax with 1 nM progesterone, but no significant change in Km, suggesting that enhanced EAAT3 activity may be attributable to an increased availability or turnover rate of EAAT3, rather than an increased affinity of EAAT3 for glutamate. Glutamate transport activity via EAATs is regulated by PKC (Casado et al., 1993; Do et al., 2002b). PMA, a PKC activator, increases EAAT3 activity, as was also observed in our previous studies (Do et al., 2002a, Kim et al., 2003). However, in the present study, PMA had no additive or synergistic effect on progesteroneenhanced EAAT3 activity, suggesting that PMA and progesterone may increase EAAT3 activity via the same mechanism. Additionally, all three PKC inhibitors tested in the present study attenuated the progesterone-induced increase in EAAT3 activity. These results indicate that PKC may mediate the effect of progesterone on EAAT3 activity. LY294002 and wortmannin, PI3K inhibitors, also attenuated the progesterone-induced increase in EAAT3 activity, suggesting that PI3K may also be involved in mediating the effect of progesterone on EAAT3 activity.
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It has been reported that EAAT3 activity/expression is highly regulated by intracellular signaling pathways involving PKC and PI3K (Nieoullon et al., 2006), as had been previously shown for the non-classical mechanism of action of progesterone in the brain (Balasubramanian et al., 2008; Ishrat et al., 2012). In the present study, inhibition of the progesterone receptor with mifepristone did not affect the enhancement of EAAT3 activity by progesterone, suggesting that progesterone acts to increase EAAT3 activity by a non-classical (non-genomic) mechanism. It is not clear how progesterone can lead to activation of PKC. One possible candidate is G protein-coupled receptors that are expressed in the oocytes and have been shown to act as progesterone receptors on the plasma membrane (Lutz et al., 2000). Also, it is known that G protein-coupled receptors can be linked to PKC and PI3K (Callaghan et al., 2004; Banday et al., 2007). Our results support that both PKC and PI3K are involved in the effects of progesterone on EAAT3. A question can be raised about why PKC and PI3K that are members of different signaling pathways are both involved in the progesterone's effects. Although PKC and PI3K are independent signaling molecules, the signaling pathways involving PKC and PI3K can interact with each other and PKC can work as a molecule downstream of PI3K (Frey et al., 2006). Pregnancy has been observed to decrease anesthetic requirements. Compared with non-pregnant women, pregnant women required less isoflurane (Gin and Chan, 1994), and the minimum alveolar concentration was reduced in postpartum women until 3 days after delivery (Chan and Gin, 1995). Furthermore, pregnant women had more heat-pain tolerance (Carvalho et al., 2006). In a rat study, administration of exogenous progesterone significantly decreased the anesthetic requirement (Shimizu et al., 2010). In neuraxial anesthesia, the requirement for local anesthetics was also reduced during pregnancy. This was thought to be attributable mainly to increased intra-abdominal pressure and decreased cerebrospinal fluid volume. However, a reduction of anesthetic requirement was also shown during early pregnancy (Fagraeus et al., 1983). When the effects of bupivacaine on the isolated vagus nerve were compared between pregnant and nonpregnant rabbits, the onset of the block was significantly faster in nerves from pregnant rabbits (Flanagan et al., 1987). Thus, nerve fibers may become more susceptible to local anesthetics during pregnancy. In addition to their effects on anesthetic requirements, steroid hormones themselves also have anesthetic actions. For example, progesterone has been shown to have analgesic and sedative properties (Frye et al., 2004; Van Broekhoven et al., 2006). Although an effect of progesterone on GABA receptors or opioid receptors has been proposed as the underlying mechanism (Dawson-Basoa and Gintzler, 1996; Pluchino et al., 2006), progesterone effects on glutamate signaling have recently received attention (Coronel et al., 2011; Ren et al., 2000). Additionally, progesterone and its metabolites have shown neuroprotective properties against traumatic brain injury (Stein, 2011). Allopregnanolone (10 μM), a metabolite of progesterone, attenuated N-methyl D-aspartate-induced excitotoxicity and apoptosis in human cell cultures (Lockhart et al., 2002), and the administration of progesterone after acute traumatic injury limited CNS damage and improved recovery (Stein et al., 2008). In the present study, progesterone enhanced the activity of EAAT3, a finding that could explain its effects on analgesia, sedation, neuroprotection, excitotoxicity, and GABA signaling. Our study has some limitations. We used an oocyte expression system to investigate EAAT in vitro. Although Xenopus oocytes have been used for studies of EAAT activity (Do et al., 2002b; Xia et al., 2006), Xenopus oocytes provide an artificial environment for EAATs. Nevertheless, is well established that membrane proteins function similarly in Xenopus oocytes and human cells (Wagner et al., 2000). Our experiments were performed at room
temperature, whereas the membrane protein was from a homeothermic animal (rat). However, there is no evidence to indicate that such difference in temperature would result in significant changes in the kinetics and function of this type of membrane protein. In conclusion, our results suggest that progesterone enhances EAAT3 activity and that PKC and PI3K mediate the effect. This progesterone effect may contribute to the neuroprotective and antinociceptive actions of progesterone.
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