Tricyclic antidepressants inhibit hippocampal α7* and α9α10 nicotinic acetylcholine receptors by different mechanisms.

The activity of tricyclic antidepressants (TCAs) at α7 and α9α10 nicotinic acetylcholine receptors (AChRs) as well as at hippocampal α7-containing (i.e., α7*) AChRs is determined by using Ca2+ influx and electrophysiological recordings. To determine the inhibitory mechanisms, additional functional tests and molecular docking experiments are performed. The results established that TCAs (a) inhibit Ca2+ influx in GH3-α7 cells with the following potency (IC50 in µM) rank: amitriptyline (2.7 ±â€¯0.3) > doxepin (5.9 ±â€¯1.1) ∼ imipramine (6.6 ±â€¯1.0). Interestingly, imipramine inhibits hippocampal α7* AChRs (42.2 ±â€¯8.5 µM) in a noncompetitive and voltage-dependent manner, whereas it inhibits α9α10 AChRs (0.53 ±â€¯0.05 µM) in a competitive and voltage-independent manner, and (b) inhibit [3H]imipramine binding to resting α7 AChRs with the following affinity rank (IC50 in µM): imipramine (1.6 ±â€¯0.2) > amitriptyline (2.4 ±â€¯0.3) > doxepin (4.9 ±â€¯0.6), whereas imipramine's affinity was no significantly different to that for the desensitized state. The molecular docking and functional results support the notion that imipramine noncompetitively inhibits α7 AChRs by interacting with two overlapping luminal sites, whereas it competitively inhibits α9α10 AChRs by interacting with the orthosteric sites. Collectively our data indicate that TCAs inhibit α7, α9α10, and hippocampal α7* AChRs at clinically relevant concentrations and by different mechanisms of action.

Effects of the antidepressant mirtazapine and zinc on nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) and zinc are associated with regulation of mood and related disorders. In addition, several antidepressants inhibit muscle and neuronal nAChRs and zinc potentiates inhibitory actions of them. Moreover, mirtazapine (a noradrenergic, serotonergic and histaminergic antidepressant) inhibits muscarinic AChRs and its effects on nAChRs are unknown. Therefore, we studied the modulation of muscle α1ß1γd nAChRs expressed in oocytes and native α7-containing nAChRs in hippocampal interneurons by mirtazapine and/or zinc, using voltage-clamp techniques. The currents elicited by ACh in oocytes (at -60 mV) were similarly inhibited by mirtazapine in the absence and presence of 100 µM zinc (IC50 ∼15 µM); however, the ACh-induced currents were stronger inhibited with 20 and 50 µM mirtazapine in the presence of zinc. Furthermore, the potentiation of ACh-induced current by zinc in the presence of 5 µM mirtazapine was 1.48 ±â€¯0.06, and with 50 µM mirtazapine zinc potentiation did not occur. Interestingly, in stratum radiatum interneurons (at -70 mV), 20 µM mirtazapine showed less inhibition of the current elicited by choline (Ch) than at 10 µM (0.81 ±â€¯0.02 and 0.74 ±â€¯0.02 of the Ch-induced current, respectively). Finally, the inhibitory effects of mirtazapine depended on membrane potential: 0.81 ±â€¯0.02 and 0.56 ±â€¯0.05 of the control Ch-induced current at -70 and -20 mV, respectively. These results indicate that mirtazapine interacts with muscle and neuronal nAChRs, possibly into the ion channel; that zinc may increase the sensitivity of nAChRs to mirtazapine; and that mirtazapine decreases the sensitivity of nAChRs to zinc.

Bupropion-induced inhibition of α7 nicotinic acetylcholine receptors expressed in heterologous cells and neurons from dorsal raphe nucleus and hippocampus.

The pharmacological activity of bupropion was compared between α7 nicotinic acetylcholine receptors expressed in heterologous cells and hippocampal and dorsal raphe nucleus neurons. The inhibitory activity of bupropion was studied on GH3-α7 cells by Ca2+ influx, as well as on neurons from the dorsal raphe nucleus and interneurons from the stratum radiatum of the hippocampal CA1 region by using a whole-cell voltage-clamp technique. In addition, the interaction of bupropion with the α7 nicotinic acetylcholine receptor was determined by [3H]imipramine competition binding assays and molecular docking. The fast component of acetylcholine- and choline-induced currents from both brain regions was inhibited by methyllycaconitine, indicating the participation of α7-containing nicotinic acetylcholine receptors. Choline-induced currents in hippocampal interneurons were partially inhibited by 10 µM bupropion, a concentration that could be reached in the brain during clinical administration. Additionally, both agonist-induced currents were reversibly inhibited by bupropion at concentrations that coincide with its inhibitory potency (IC50=54 µM) and binding affinity (Ki=63 µM) for α7 nicotinic acetylcholine receptors from heterologous cells. The [3H]imipramine competition binding and molecular docking results support a luminal location for the bupropion binding site(s). This study may help to understand the mechanisms of actions of bupropion at neuronal and molecular levels related with its therapeutic actions on depression and for smoking cessation.

Interaction of bupropion and zinc with neuronal nicotinic acetylcholine receptors.

Zinc is known to exert antidepressant-like actions and to make the effects of some antidepressants more efficient in animal models of depression. Both zinc and bupropion interact with nicotinic acetylcholine receptors (nAChRs) which are related with depression. Here we examined the effects of bupropion, in the absence and presence of zinc, on the ion current elicited by acetylcholine (ACh-current) in Xenopus oocytes expressing neuronal α4ß4 nAChRs. We found that bupropion-inhibited ACh-currents depending on ACh and bupropion concentrations. Thus, the IC(50) of bupropion was lower with a higher ACh concentration: 3.51 and 2.27 µM for the current elicited with 0.5 and 2 µM ACh, respectively. The inhibitory effect of bupropion was more potent in the presence of zinc, e.g. the IC(50) was 0.81 µM in the presence of 100 µM zinc and 2 µM ACh. Furthermore, the zinc-potentiated ACh-current decreased with increasing bupropion concentration. Thus, zinc potentiation was 5.05 and 1.25 fold of the ACh-current inhibited by 10 nM and 5 µM bupropion, respectively. The ACh-current inhibited by 3 µM bupropion was voltage-independent, decreasing to 0.48 of the ACh-current at all voltages. Zinc potentiation of the bupropion-inhibited ACh-current was slight and voltage-independent. In addition, the zinc-potentiated ACh-current was slightly voltage-dependent: 1.8 fold of the ACh-current at -120 mV and 2.3 at -40 mV. Bupropion inhibition of the zinc-potentiated ACh-current was strong and voltage-independent, decreasing to 0.15 of the zinc-potentiated ACh-current at all voltages. Accordingly, zinc may interact within the ion channel, whereas bupropion, and bupropion in the presence of zinc (which causes greater inhibition) interact in an external region of the receptor-channel complex. These results suggest that bupropion interacts with α4ß4 nAChRs in a non-competitive manner, that zinc increases the sensitivity of nAChRs to bupropion, and that bupropion decreases the sensitivity of nAChRs to zinc.

Neuronal nicotinic acetylcholine receptors are modulated by zinc.

It is known that zinc modulates nicotinic acetylcholine receptors (nAChRs). Here, we studied the effects of zinc on neuronal alpha4beta4 nAChRs, expressed in Xenopus oocytes and activated by nicotine. Membrane ion currents elicited by nicotine (10 nM to 100 microM) were enhanced by zinc (100 microM). Maximal zinc potentiation of the nicotine-activated current (2530%) occurred at 50 nM nicotine, and potentiation gradually decreased as the nicotine concentration increased. The EC(50) and IC(50) for the nicotine-activated current were 639 nM and 14.7 microM nicotine, respectively. Both parameters decreased in the presence of zinc to 160 nM and 4.6 microM, respectively, probably due to an increase of sensitivity of nAChRs for nicotine. We used different concentrations and durations of exposure to nicotine, due to desensitization of nAChRs directly depends on both these factors. With 500 nM nicotine and 20 min washing periods between nicotine applications, zinc potentiation remained constant, 901% for 2 min and 813% for 20 min of nicotine exposure. With continuous application of nicotine, zinc potentiation decreased as the time of nicotine exposure increased, 721% for 2 min and 254% for 48 min of nicotine exposure. Our results indicate that zinc-potentiating effects on alpha4beta4 nAChRs strongly depend on both concentration and time of exposure to nicotine, suggesting that zinc potentiation depends on the degree of desensitization.