Tricyclic antidepressant amitriptyline inhibits 5-hydroxytryptamine 3 receptor currents in NCB-20 cells

Abstract

Amitriptyline, a tricyclic antidepressant, is commonly used to treat depression and neuropathic pain, but its mechanism is still unclear. We tested the effect of amitriptyline on 5-hydroxytryptamine 3 (5-HT3) receptor currents and studied its blocking mechanism because the clinical applications of amitriptyline overlapped with 5-HT3 receptor therapeutic potentials. Using a whole-cell voltage clamp method, we recorded the currents of the 5-HT3 receptor when 5-HT was applied alone or co-applied with amitriptyline in cultured NCB-20 neuroblastoma cells known to express 5-HT3 receptors. To elucidate the mechanism of amitriptyline, we simulated the 5-HT3 receptor currents using Berkeley Madonna® software and calculated the rate constants of the agonist binding and receptor transition steps. The 5-HT3 receptor currents were inhibited by amitriptyline in a concentration-dependent, voltage-independent manner, and a competitive mode. Amitriptyline accelerated the desensitization of the 5-HT3 receptor. When amitriptyline was applied before 5-HT treatment, the currents rose slowly until the end of 5-HT treatment. When amitriptyline was co-applied with 5-HT, currents rose and decayed rapidly. Peak current amplitudes were decreased in both applications. All macroscopic currents recorded in whole cell voltage clamping experiments were reproduced by simulation and the changes of rate constants by amitriptyline were correlated with macroscopic current recording data. These results suggest that amitriptyline blocks the 5-HT3 receptor by close and open state blocking mechanisms, in a competitive manner. We could expand an understanding of pharmacological mechanisms of amitriptyline related to the modulation of a 5-HT3 receptor, a potential target of neurologic and psychiatric diseases through this study.

Modulation of nociception by amitriptyline hydrochloride in the Speke’s hinge‐back tortoise (Kiniskys spekii)

“…Tricyclic antidepressants for example are often the first‐line treatment for neuropathic pain in humans (Dworkin et al., 2007; Saarto & Wiffen, 2010; Sindrup et al., 2005). Amitriptyline hydrochloride (tricyclic antidepressant) is commonly used to treat depression and neuropathic pain in humans and animal models (Kremer et al., 2018; Park et al., 2018). In veterinary medicine it has been used to manage neuropathic pain in a prairie falcon (Shaver et al., 2009), idiopathic cystitis in cats (Kraijer et al., 2003), canine separation anxiety (Sargisson, 2014) as well as psychogenic feather picking in companion birds.…”

“…Its primary mechanism of action is through inhibition of serotonin and noradrenaline reuptake on the descending inhibitory pathways (Anser et al., 2018; Kremer et al., 2018; Meshalkina et al., 2018). This drug has considerable affinity for serotonin and norepinephrine reuptake transporters, 5‐HT 1A and 5‐HT 2A receptors, alpha‐adrenergic receptors, muscarinic acetylcholine receptors and histamine receptors (Park et al., 2018). Amitriptyline hydrochloride is also a known voltage‐gated Na + and K + channel blocker (Dick et al., 2007; Li et al., 2018; Song et al., 2000; Wang et al., 2004; Wolff et al., 2016).…”

Gastroprokinetic agent, mosapride inhibits 5-HT3 receptor currents in NCB-20 cells

“…To study the direct effects of mosapride on the 5-HT 3 receptor induced currents, we examined the concentration-dependent effect of mosapride (0.3, 1, 3, 10, 30 µM) on the currents by 3 µM 5-HT, a concentration near the EC 50 of 5-HT 3 receptor currents reported in our previous study [18]. The application of 30 µM mosapride for 5 sec did not induce any current (Fig.…”

“…NCB-20 neuroblastoma cells were provided by Dr. Lovinger (National Institute on Alcohol Abuse and Alcoholism [NIAAA], Bethesda, MD, USA). The NCB-20 cells were maintained under previously described conditions [18]. Frozen cell stocks were maintained in liquid nitrogen and thawed as needed.…”

Selective serotonin reuptake inhibitor escitalopram inhibits 5-HT3 receptor currents in NCB-20 cells

“…First, we evaluated concentration-dependent inhibitory effect of escitalopram on 5-HT 3 receptor (i.e., 1, 3, 10, 30, and 100 µM of escitalopram was co-applied with 3 µM of 5-HT, near the EC 50 value from our previous experiment [33]). Current peak amplitudes were then normalized to 5-HT 3 receptor current induced by 3 µM 5-HT alone.…”