Establishment Of Cell Models Stably Overexpressing HOCT2and The Role Of OCT2in The Potential Mechanism Of Antidepressant Action

Depression, that is major depressive disorder (MDD), is a calamity for individuals and society with a lifetime prevalence of about15-20%. MDD gains its popularity with life pressure and work intensity increasing at a staggering rate. It is true that antidepressant treatment has made breakthrough in the past decades. Yet novel antidepressants or treatment strategies that may offer a more rapid onset of action, improved efficacy, and greater tolerability are in desperate need. Because current clinically utilized antidepressants, which target high-affinity transporters for serotonin and norepinephrine, fail to provide satisfactory treatment outcomes for quite a portion of patients.In recent investigations, a low-affinity but high-capacity transporter organic cation transporter2(OCT2, SLC22A2) has been proposed as an important postsynaptic determinant of aminergic tonus and mood-related behaviors, a complementary system to the high-affinity transporters. In order to evaluate whether OCT2inhibition may at least in part contribute to the pharmacological effects of antidepressants, we first of all established cell models overexpressing hOCT2, and then examined the models from the aspects of mRNA, protein and translocaing function to ensure authenticity. The uptake assay was composed of rough screen by fluorescent substrate ASP+, and metformin and MPP+uptake as further verification.Several typical antidepressant compounds of various mechanism categories were employed to inhibit OCT2activity in the best performing cell model. The tested antidepressant agents included selective serotonin reuptake inhibitors (SSRIs, fluoxetine, sertraline and paroxetine), tricyclic antidepressants (TCAs, amitriptyline, imipramine, desipramine), monoamine oxidase inhibitor (MAOI, moclobemide), and serotonin-norepinephrine reuptake inhibitor (SNRI, venlafaxine). All of the eight antidepressants showed moderate inhibitory effects on OCT2-mediated metformin, serotonin and/or norepinephrine uptake, implying a crucial role of hOCT2in antidepressant treatment. The order of inhibitory potency for the tested antidepressants at hOCT2was obtained:SSRIs (IC50range was1-10μM)≈TCAs (IC50range was about100nM-1μM)> moclobemide (IC50range for metformin and serotonin was10-100μM, and>100μM for norepinephrine)> venlafaxine (IC50>100μM). Sertraline and desipramine tended to inhibit OCT2activity via a competitive mechanism. The fact could be easily belied, since passive diffusion predominated the influx process. Therefore, although it remains to be seen whether OCT2inhibition plays a role to the overall therapeutic effects in clinical practice, the part of OCT2inhibition can never be ignored in the pharmacological facet.Natural compounds are usually superior in price, safety and acquisition over common prescribed antidepressant medications. Three hOCT2inhibitors pipeline, berberine and jatrorrhizine were discovered among natural antidepressant compounds reported in literatures. The alkaloids were examined on stably transfected cell models to calculate the inhibitory potencies of OCT2and organic cation transporter3(OCT3, SLC22A3)-mediated metformin, serotonin and/or norepinephrine uptake. Based on data derived from cellular accumulation, synaptosomes uptake assays were conducted to identify OCT pathway as one mechanism of antidepressant-like activity of pipeline, berberine and jatrorrhizine. Not surprisingly, significant inhibitory effects were observed on both OCT and results from synaptosomes uptake assays revealed that OCT played an indispensable role in serotonin modulation without the interference of monoamine oxidase.