Amitriptyline inhibits the activity of the rat glutamate transporter EAAT3 expressed in Xenopus oocytes

Abstract

Objectives: Evidence suggests that glutamatergic systems may be involved in the pathophysiology of major depression and the mechanism of action of antidepressants. We have investigated the effects of amitriptyline, a tricyclic antidepressant, on the activity of the excitatory amino acid transporter type 3 (EAAT3), a protein that can regulate extracellular glutamate concentrations in the brain.

Methods: EAAT3 was expressed in Xenopus oocytes. Using a two-electrode voltage clamp, membrane currents were recorded after application of 30 microM L-glutamate in the presence or absence of various concentrations of amitriptyline or after application of various concentrations of L-glutamate in the presence or absence of 0.64 microM amitriptyline.

Key findings: Amitriptyline concentration-dependently reduced EAAT3 activity. This inhibition reached statistical significance at 0.38-1.27 microM amitriptyline. Amitriptyline 0.64 microM reduced the pharmacokinetic parameter Vmax, but did not affect the pharmacokinetic parameter Km, of EAAT3 for L-glutamate. The amitriptyline inhibition disappeared after a 4-min washout. Phorbol-12-myristate-13-acetate, a protein kinase C activator, increased EAAT3 activity. However, 0.64 microM amitriptyline induced a similar degree of decrease in EAAT3 activity in the presence or absence of phorbol-12-myristate-13-acetate.

Conclusions: Our results suggested that amitriptyline at clinically relevant concentrations reversibly reduced EAAT3 activity via decreasing its maximal velocity of glutamate transporting function. The effects of amitriptyline on EAAT3 activity may have represented a novel site of action for amitriptyline to increase glutamatergic neurotransmission. Protein kinase C may not have been involved in the effects of amitriptyline on EAAT3.

Fig. 1. Dose-response of amitriptyline inhibition of EAAT3 responses to 30 µM L-glutamate

The IC50 value for this inhibition was 0.11 µM. Three typical current traces are inserted in the figure. The short line above each current trace represents the time of L-glutamate application. Data are means ± S.D. (n = 27 – 48 in each data point). * P < 0.05 compared to control.

Fig. 2. Effects of amitriptyline on EAAT3 activity

The EAAT3 responses to various L-glutamate concentrations were measured before (control group) and immediately after amitriptyline treatment (amitriptyline group) and after a 4-min Tyrode’s perfusion (Tyrode’s washout). The Vmax and Km values of EAAT3 response to L-glutamate are listed in the table above the figure. Data are means ± S.D. (n = 6 in each data point). * P < 0.05 compared to the corresponding values in control group.

Fig. 3. Effects of protein kinase C (PKC) activation on EAAT3 activity in the presence or absence of 0.64 µM amitriptyline

Oocytes were exposed to or were not exposed to 100 nM phorbol-12-myristate-13-acetate (PMA, a PKC activator) for 10 min before they were stimulated by 30 µM L-glutamate in the presence or absence of amitriptyline. Data are means ± S.D. (n = 24 – 45 in each data point). * P < 0.05 compared to control, † P < 0.05 compared to PMA alone, ‡ P < 0.05 compared to amitriptyline alone.