Desflurane increased the activity of excitatory amino-acid carrier 1 (EAAC1) expressed in Xenopus oocytes

Abstract

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 V_max but had no effect on the K_m 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.

Introduction

Desflurane is a halogenated volatile anaesthetic that has unique features, such as high vapour pressure and minimal hepatic metabolism (Jakobsson, 2012). Since it was first introduced to human use in 1990, the use of desflurane has increased because its kinetic properties make it suitable for rapid induction and recovery for patients undergoing ambulatory anaesthesia (Jakobsson, 2012). In addition, desflurane has demonstrated neuroprotective effects in the developing animal brain (Dean Kurth et al., 2001, Engelhard et al., 1999, Haelewyn et al., 2003, Kodama et al., 2011). Various mechanisms underlying the neuroprotective effect of desflurane have been suggested, including gamma-aminobutyric acid (GABA) activation, decreased presynaptic glutamate secretion, glutamate-related postsynaptic inotropic receptor inhibition, and a subsequent reduction in sympathetic activity (Engelhard et al., 1999, Sakai et al., 2005).

Excitatory amino-acid carrier 1 (EAAC1), a glutamate transporter, plays a key role in taking up glutamate from the extracellular space into cells and in keeping the extracellular glutamate concentration within non-neurotoxic levels (Danbolt, 2001). EAAC1 also contributes to neuroprotection by cysteine uptake (Shanker et al., 2001). The amount of cysteine functions as the rate-limiting factor in the synthesis of glutathione, the major endogenous antioxidant (Shanker et al., 2001). Consistent with these effects, EAAC1 knockout mice have reduced brain ischaemic tolerance (Li and Zuo, 2010).

Do et al. (2002b) showed that isoflurane enhanced EAAC1 activity, and that intracellular signalling pathways, such as protein kinase C (PKC) or phosphatidylinositol-3-kinase (PI3K), mediated these effects. However, little is known about the effect of desflurane on EAAC1, which could be a mechanism underlying the neuroprotective effect of desflurane. It is hypothesised that desflurane affects EAAC1 activity and that PKC or PI3K mediates this process. In this study, we evaluated the effects of desflurane on EAAC1 expressed in Xenopus laevis oocytes, and the involvement of two signalling molecules, PKC and PI3K, in mediating the effects of desflurane on EAAC1.