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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 V max was increased in the progesterone group compared with that in the control group (2.7±0.2 vs. 3.6±0.2 μC for control group vs. progesterone group; n =18–23; P K m was unaltered (46.7±10.2 μM vs. 55.9±10.5 μM for control group vs. progesterone group; n =18–23; P >0.05). P horbol-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.

This study aimed to evaluate the effect of riluzole on the activity of excitatory amino acid transporter type 3 (EAAT3), a neuronal glutamate transporter, and to investigate the role of protein kinase C (PKC) in this effect. EAAT3 expression was induced in Xenopus oocytes by injecting EAAT3 mRNA. Using the two-electrode voltage clamping method, membrane currents were recorded before, during, and after applying l-glutamate (30 μM) in the absence and presence of prior incubation with riluzole (0.3-100 μM). To study the effect of PKC on the riluzole-induced change in EAAT3 activity, oocytes were preincubated with 100 μM phorbol-12-myristate-13-acetate (PMA), a PKC activator, or PKC inhibitors (2 µM staurosporine and 100 µM chelerythrine) before the recording. Responses were quantified by integrating current traces and are reported in microCoulombs (μC). Riluzole reduced EAAT3 activity in a concentration-dependent manner (0.3-100 μM). Treatment of oocytes with PMA significantly increased the baseline and riluzole-reduced EAAT activity (P0.05). In addition, treatment of oocytes with PKC inhibitors reduced basal transporter currents, but did not show a further significant decrease in the riluzole-reduced EAAT3 activity. These results suggest that riluzole reduces EAAT3 activity through PKC inhibition.

Desflurane is a volatile anaesthetic agent with neuroprotective properties. Excitatory amino-acid carrier 1 (EAAC1) may be neuroprotective by taking up glutamate and cysteine. Therefore, the effects of desflurane on EAAC1 activity were investigated in this study. EAAC1 was expressed in Xenopus laevis oocytes. Two-electrode voltage-clamping technique was used to record membrane currents upon exposure to l -glutamate (30 μM) in the presence or absence of desflurane (0.4, 1.0, 2.0, 2.6, or 3.2 mM). Currents were also measured in oocytes pre-exposed to a protein kinase C (PKC) activator (50 nM phorbol-12-myristate-13-acetate, PMA), PKC inhibitors (1 μM staurosporine or 50 μM chelerythrine), or phosphatidylinositol-3-kinase (PI3K) inhibitors (5 μM wortmannin or 10 μM LY294002). Desflurane significantly increased EAAC1 activity. The EC50 of desflurane for increasing the EAAC1 response was 0.75 mM. A kinetic study showed that desflurane significantly increased the Vmax but had no effect on the Km of the EAAC1 response for glutamate. Treatment of oocytes with desflurane plus PMA significantly increased the transporter currents compared to the control, but did not further increase the response compared to either agent alone. Staurosporine attenuated desflurane-enhanced transporter currents without decreasing the basal activity; chelerythrine did not decrease either. In addition, pretreatment of oocytes with two PI3K inhibitors (wortmannin or LY294002) significantly reduced desflurane-enhanced EAAC1 activity without decreasing basal activity. Our results suggest that desflurane increases EAAC1 activity via PKC or PI3K. This enhanced EAAC1 activity may be a mechanism for the neuroprotective effect of desflurane.

Dexmedetomidine, an α2 adrenergic agonist, has neuroprotective and anticonvulsant properties in addition to its sedative and anxiolytic effects. We hypothesized that dexmedetomidine would increase the activity of excitatory amino acid transporter type 3 (EAAT3) and that this effect would involve protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K), two protein kinases known to regulate EAAT3 activity. EAAT3 was expressed in Xenopus oocytes by injecting its mRNA. Two-electrode voltage clamping was used to record membrane currents before, during, and after application of 30 μM l-glutamate in the presence of 0.1-30 nM dexmedetomidine. Dexmedetomidine-treated oocytes were also exposed to a PKC activator (phorbol-12-myristate-13-acetate [PMA]), PKC inhibitors (chelerythrine, staurosporine, and calphostin C), and PI3K inhibitors (wortmannin and LY294002) before current measurement. Dexmedetomidine application resulted in a concentration-dependent increase in the EAAT3 activity in response to l-glutamate. The kinetic study showed that dexmedetomidine significantly increased the Vmax without changing Km. Treatment of oocytes with PMA significantly increased transporter currents compared with controls, but treatment with dexmedetomidine plus PMA did not further increase the response compared with PMA or dexmedetomidine alone. In addition, pre-treatment of oocytes with PKC inhibitors and PI3K inhibitors significantly abolished the dexmedetomidine-enhanced EAAT3 activity. These results suggest that dexmedetomidine increases the activity of EAAT3 expressed in Xenopus oocytes. PKC and PI3K seem to mediate this effect. These findings may explain the neuroprotective and anticonvulsant effects of dexmedetomidine.