ABSTRACT
The purpose of this study was to evaluate the effect of ondansetron on excitatory amino acid transporter type 3 (EAAT3) and to elucidate the roles of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) in the effect. EAAT3 was expressed in Xenopus oocytes following the injection of rat EAAT3 mRNAs. Using the two-electrode voltage clamping method, the inward currents induced by L-glutamate were measured for 1 min in the presence and absence of ondansetron (1-1000 µM). Different concentrations of L-glutamate (3-300 µM) were used to determine the kinetic characteristics of EAAT3. To identify the involvement of PKC and PI3K in the effect, oocytes were exposed to a PKC activator and to PKC inhibitors and PI3K inhibitors, and L-glutamate-induced currents were recorded. Ondansetron decreased EAAT3 activity in a dose-dependent manner. In a kinetic study, ondansetron (10 µM for 3 min) reduced Vmax, but not Km compared with the control group. The PKC activator abolished the ondansetron-induced decrease in EAAT3 activity. The PKC inhibitors (staurosporine and chelerythrine) and ondansetron had not additive or synergistic effects on EAAT3 activity. The PI3K inhibitors (wortmannin and LY294002) decreased the EAAT3 response, although there were no differences among the groups comprising ondansetron, PI3K inhibitors, and PI3K inhibitors plus ondansetron. Our results demonstrate that ondansetron attenuates EAAT3 activity and this effect seems to be mediated by PKC and PI3K.
Subject(s)
Excitatory Amino Acid Antagonists/pharmacology , Excitatory Amino Acid Transporter 3/antagonists & inhibitors , Ondansetron/pharmacology , Animals , Dose-Response Relationship, Drug , Female , Glutamic Acid/metabolism , Glutamic Acid/pharmacology , Humans , Membrane Potentials/drug effects , Microelectrodes , Oocytes , Patch-Clamp Techniques , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Protein Kinase Inhibitors/pharmacology , Xenopus laevisABSTRACT
Estrogen, a neuroactive sex hormone in the brain, enhances neuronal excitability and increases seizures. Glutamate transporters help in limiting the excitatory neurotransmission by uptaking glutamate from the synapses. We investigated the effects of 17ß-estradiol on the activity of a glutamate transporter, excitatory amino acid transporter 3 (EAAT3), in Xenopus oocytes. EAAT3 was expressed in Xenopus oocytes by injection of rat EAAT3 mRNA. l-Glutamate (30 µM)-induced membrane currents mediated by EAAT3 were measured using the two-electrode voltage clamp technique. 17ß-Estradiol reduced EAAT3 activity in a concentration- and time-dependent manner. 17ß-Estradiol (10nM for 72h) significantly decreased V(max) but had no effect on K(m) of EAAT3 for glutamate. When 17ß-estradiol treated oocytes were incubated with phorbol-12-myrisate-13-acetate, a protein kinase C (PKC) activator, 17ß-estradiol-induced decrease in EAAT3 activity was abolished. Furthermore, in pretreatment of oocytes with chelerythrine or staurosporine, two PKC inhibitors, EAAT3 activity was significantly decreased. However, there was no statistical difference among the 17ß-estradiol, PKC inhibitor, or 17ß-estradiol plus PKC inhibitor groups. Likewise, wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, significantly reduced basal EAAT3 activity, but the activity did not differ among the 17ß-estradiol, wortmannin, or 17ß-estradiol plus wortmannin groups. Estradiol receptor inhibitor, fulvestrant, did not change the reduced EAAT3 activity by 17ß-estradiol. Our results suggest that 17ß-estradiol decreases EAAT3 activity. PKC and PI3K seem to be involved in this effect, possibly not via estradiol receptors.
