Development of serotonin transporter reuptake inhibition assays using JAR cells

Abstract

Introduction: The development and validation of serotonin transporter reuptake inhibition assays in 96-well format using commercially available human placental choriocarcinoma JAR cells is described.

Methods: JAR cells were first shown to uptake [³H]serotonin in a saturable fashion with a K_M value of 1 μM as determined by a Michaelis-Menten kinetic analysis. The cells were then utilized to determine the reuptake inhibition potencies of known ligands and the results were compared with results previously generated in the two most commonly used transporter assays (rat brain synaptosomes and transfected HEK293 cells).

Results: Examination of a variety of ligands including selective serotonin reuptake inhibitors, tricyclic antidepressants, piperazine derivatives, and phenyltropane derivatives demonstrated that JAR cells are capable of detecting reuptake inhibition activity of a variety of ligands with potencies that correlate with one or both of the other assays.

Discussion: This study demonstrates a novel pharmacological method of assessing human serotonin transporter reuptake inhibition activity using commercially available JAR cells. Our results show that JAR cells provide an easily available and good alternative to using rat brain tissue and HEK293 cells, with the advantage of studying serotonin transporter reuptake inhibition in a human background.

Keywords: JAR cells; Methods; Reuptake inhibitors; Serotonin transporter.

Figure 1

Michaelis-Menten kinetics of [³H]5-HT uptake by adherent JAR cells. Experiments were carried out at 37°C with multiple incubation times (60, 45, 30, 15, and 0 min) in 96-well plates (51.8 µg of protein/well) as described in the methods. All data shown are mean ± S.E.M. of N=3 conducted with duplicate determinations. JAR cells uptake [³H]5-HT with K_M = 1.0 ± 0.2 µM and V_MAX = 943 ± 72 fmol radioactivity/mg protein/min. (A) Velocity of [³H]5-HT uptake as a function of [³H]5-HT concentration. (B) Plot of specific bound radioactivity (fmol radioactivity/mg protein) as a function of time to determine reaction rate for the Michaelis-Menten plot.

Figure 2

Reuptake inhibition activity at the hSERT endogenously expressed in JAR cells. Experiments were carried out with 1.0 µM final [³H]5-HT at 37°C for 60 minutes in 96-well plates (51.8 µg of protein/well) as described in the methods. All data shown are expressed as % inhibition and each data point is the mean ± S.E.M. of at least N=3 conducted with duplicate determinations. (A) Activity of the selective serotonin reuptake inhibitors fluoxetine (●) and citalopram (○). (B) Activity of the tricyclic antidepressant desipramine (■) and the non-selective monoamine transporter inhibitor indatraline (□). (C) Activity of the piperazine derivatives GBR12935 (◆) and GBR12909 (◊). (D) Activity of the phenyltropane derivatives RTI-55 (▲) and RTI-229 (▼).