Molecular Psychiatry (2001) 6, 511–519  2001 Nature Publishing Group All rights reserved 1359-4184/01 $15.00 www.nature.com/mp ORIGINAL RESEARCH ARTICLE Antagonism of nicotinic acetylcholine receptors by inhibitors of monoamine uptake HE Lo´pez-Valde´s and J Garcı´a-Colunga

Centro de Neurobiologı´a, Universidad Nacional Auto´noma de Me´xico, Campus Juriquilla, Apartado Postal 1–1141, Juriquilla, Quere´taro 76001, Me´xico

A study was made of the effects of several monoamine-uptake inhibitors on membrane cur- rents elicited by acetylcholine (ACh-currents) generated by rat neuronal ␣2␤4 and mouse mus- cle nicotinic acetylcholine receptors (AChRs) expressed in Xenopus laevis oocytes. For the two types of receptors the monoamine-uptake inhibitors reduced the ACh-currents albeit to different degrees. The order of inhibitory potency was norfluoxetine Ͼ clomipramine Ͼ inda- traline Ͼ fluoxetine Ͼ imipramine Ͼ zimelidine Ͼ 6-nitro-quipazine Ͼ trazodone for neuronal ␣2␤4 AChRs, and norfluoxetine Ͼ fluoxetine Ͼ imipramine Ͼ clomipramine Ͼ indatraline Ͼ zimelidine Ͼ trazodone Ͼ 6-nitro-quipazine for muscle AChRs. Thus, the most potent inhibitor was norfluoxetine, whilst the weakest ones were trazodone, 6-nitro-quipazine and zimelidine. Effects of the tricyclic antidepressant imipramine were studied in more detail. Imipramine inhibited reversibly and non-competitively the ACh-current with a similar inhibiting potency for both neuronal ␣2␤4 and muscle AChRs. The half-inhibitory concentrations of imipramine were 3.65 ± 0.30 ␮M for neuronal ␣2␤4 and 5.57 ± 0.19 ␮M for muscle receptors. The corre- sponding Hill coefficients were 0.73 and 1.2 respectively. The inhibition of imipramine was slightly voltage-dependent, with electric distances of ෂ0.10 and ෂ0.12 for neuronal ␣2␤4 and muscle AChRs respectively. Moreover, imipramine accelerated the rate of decay of ACh- currents of both muscle and neuronal AChRs. The ACh-current inhibition was stronger when oocytes, expressing neuronal ␣2␤4 or muscle receptors, were preincubated with imipramine alone than when it was applied after the ACh-current had been generated, suggesting that imipramine acts also on non-activated or closed AChRs. We conclude that monoamine-uptake inhibitors reduce ACh-currents and that imipramine regulates reversibly and non- competitively neuronal ␣2␤4 and muscle AChRs through similar mechanisms, perhaps by interacting externally on a non-conducting state of the AChR and by blocking the open recep- tor-channel complex close to the vestibule of the channel. These studies may be important for understanding the regulation of AChRs as well as for understanding antidepressant- and side-effects of monoamine-uptake inhibitors. Molecular Psychiatry (2001) 6, 511–519. Keywords: serotonin transporters; antidepressants; cholinergic-serotonergic interaction; nicotinic receptor modulation; Xenopus oocytes

Introduction modified by both endogenous and exogenous sub- stances.4,5 Nicotinic acetylcholine receptors (AChRs) mediate the Although serotonin (5-hydroxytryptamine, 5-HT) transmission of signals across the vertebrate neuro- acts as neurotransmitter on specific receptors,6 it has muscular junction as well as across central and periph- been shown that 5-HT is also able to modulate AChRs. eral synapses.1 The AChR, a ligand-gated ion channel Furthermore, various agonists and antagonists of differ- opened by acetylcholine, is a pentameric structure, ent 5-HT receptors, as well as inhibitors of the 5-HT- composed of four different subunits in muscle recep- transporter alter the function of neuronal and muscle tors (␣1, ␤1, ␥,or⑀, and ␦) with a stoichiometry of AChRs.7–17 The most common action of serotonergic 2:1:1:1; whereas in neuronal ␣2␤4 AChRs the stoichi- compounds on AChRs appears to be a non-competitive ometry is presumably 2:3.2,3 The function of AChRs is inhibitory process through interactions with the open channel.5,7–14 On the other hand, it is known that different classes of inhibitors of monoamine-uptake systems, that are Correspondence: J Garcı´a-Colunga, Centro de Neurobiologı´a, used clinically as antidepressants, interact with differ- Universidad Nacional Auto´noma de Me´xico, Campus Juriquilla, ent membrane proteins at synaptic regions. For Apartado Postal 1–1141, Juriquilla, Quere´taro 76001, Me´xico. E- mail: garciacȰcalli.cnb.unam.mx example: imipramine, trazodone, clomipramine, zimel- Received 6 November 2000; revised 18 January 2001; accepted idine, fluoxetine and norfluoxetine inhibit a wide var- 24 January 2001 iety of receptors including muscarine, histamine, adre- Antidepressants on nicotinic receptors HE Lo´pez-Valde´s and J Garcı´a-Colunga 512 naline, dopamine and serotonin receptors,18 and they ACh plus imipramine were obtained by fitting the data also inhibit biogenic amine transporters.19 In addition, with the Hill equation: fluoxetine, an antidepressant that initially was thought I = I * [ACh]nH/([ACh]nH + EC nH), (2) to be a very high selective inhibitor of the 5-HT uptake ACh max 50 20 system, inhibits nicotinic and serotonin recep- where IACh is the ACh dose-dependent current ampli- 11,13,21,22 tors. Similarly, imipramine, a non-selective tude in the absence or presence of imipramine, Imax is 23 inhibitor of serotonin uptake, blocks nicotinic recep- the amplitude of the maximal ACh-current, EC50 is the tors of frog motor endplate and electric organ in a non- half-excitatory concentration of ACh, and nH is the competitive and voltage-dependent way,24,25 inhibits Hill coefficient. the current evoked by dimethylphenylpiperazinium in To determine if the imipramine-binding site on human neuroblastoma cells and blocks the secretion of AChR is located externally or within the ion channel, ATP mediated by AChRs in chromaffin cells.26,27 a one-site blockage model was used.10,30 It assumes that In this paper, we studied effects of various inhibitors the block caused by the drug is fast. The voltage depen- of monoamine transporters on neuronal ␣2␤4 and mus- dence of the binding of the blocking agent is also cle AChRs, with emphasis on the mechanisms of action related with the electrical distance of its binding site, of imipramine. measured from the external side of the membrane channel, and is given by the equation: Materials and methods = IC50(Vm) IC50(0) * exp(dzFVm/RT), (3) The experiments were performed on Xenopus oocytes where IC (0) is the half-blocking concentration at ␣ ␤ 50 expressing rat neuronal 2 4 or embryonic mouse 0 mV, V is the applied membrane potential, d is the 9,13 m muscle AChRs. Briefly, oocytes were dissected from fraction of the electrical field sensed at a binding site ° the ovary and maintained at 16 C in Barth’s solution of imipramine within the receptor’s ion channel, z is containing: 88 mM NaCl, 1 mM KCl, 0.33 mM the valence of the blocking molecule, F is the Faraday Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, 2.4 mM constant, R is the gas constant, and T is the absolute NaHCO3, 5 mM HEPES (pH 7.4; NaOH), and 0.1 mg temperature. Combining Equation (3) with the Hill equ- −1 ml gentamicin sulfate. The next day individual ation for the blocking, Equation (1), gives: oocytes were injected with 0.5–5 ng of a mixture of − = cRNA subunits in a volume of 50 nl water (␣1, ␤1, ␥ IACh/Iimipramine 1 (4) and ␦ or ␣2 and ␤4). Two days later the oocytes were [imipramine]/IC50(0) * exp(dzFVm/RT), treated with 140 units ml−1 collagenase type I (Sigma, Equation (4) was linearized to fit the data in a voltage St Louis, MO, USA) for 0.5–1 h to remove the ovarian range of −140 to −60 mV. epithelial and follicular cells.28 Membrane currents The time course of the decay of the ACh-current is were recorded 3–9 days after RNA injection using a influenced by several factors including the speed of the voltage-clamp technique with two microelectrodes29 solution exchange, the size of the cell and the receptor with a commercially available amplifier (Warner OC- desensitization31 and can be fitted by the function: 725A; Warner Instruments Corp, Hamden, CT, USA), = − ␶ + − ␶ monitored with an oscilloscope (Nicolet 310; Nicolet I(t) Io + Af exp( t/ f) As exp( t/ s), (5) Instruments Corp, Madison, WI, USA) and stored in discs for subsequent analyses using a program written where I(t) is the current at time t; Io is an asymptotic by Rico Miledi. The oocytes were continuously super- component; Af and As are the amplitudes for a fast and ␶ ␶ fused at room temperature (20–23°C) with frog Ringer’s a slow component; and f and s are their respective solution containing 115 mM NaCl, 2 mM KCl, 1.8 mM time constants.

CaCl2, 5 mM HEPES (pH 7.0; NaOH). Unless otherwise indicated, the oocyte membrane potential was main- Results tained at −60 mV. Monoamine-uptake inhibitors were obtained from RBI (Natick, MA, USA). ACh and other ACh-current decrease by monoamine-transporter drugs were diluted in Ringer and applied onto the inhibitors oocytes by superfusion at a rate of 7–10 ml min−1 Several monoamine-transporter inhibitors were tested (chamber volume ෂ0.1 ml). Membrane current values on neuronal ␣2␤4 and muscle AChRs expressed in are given as the mean ± standard error (SE). Xenopus oocytes. Control ACh-currents were elicited The dose-response relationships for ACh-currents in by low concentrations of ACh and after the peak had the presence of imipramine were obtained by fitting the been reached, 10 ␮M of a monoamine-transporter data with the Hill equation: inhibitor was coapplied with ACh. After ෂ2 min the nH nH inhibitor was removed, to allow recovery of the ACh- I = I *IC /([imipramine]nH + IC ), (1) imip p 50 50 current (Figure 1). The effect of each compound was

where Iimip is the ACh-current amplitude in the pres- estimated as the ratio between the current elicited by ence of imipramine, Ip is the control peak current, IC50 ACh plus the drug over the control ACh-current value is the half-inhibitory concentration of imipramine, and at the end of the drug application, giving the fraction nH is the Hill coefficient. of the ACh-current that remains at this time (Figure 1c). The dose-response relationships for ACh alone or for These experiments disclosed two groups of com-

Molecular Psychiatry Antidepressants on nicotinic receptors HE Lo´pez-Valde´s and J Garc´ıa-Colunga 513

Figure 1 ACh-current inhibition by monoamine-transporter inhibitors. Control ACh-currents (a) and effects of zimelidine and fluoxetine on the ACh-current from neuronal ␣2␤4 and muscle AChRs respectively (b). Bar calibrations: 60 nA (upper traces) and 360 nA (bottom traces). In this and subsequent figures the timing of ACh and drug applications are indicated by bars above the records, and by 10 mV depolarizing pulses to monitor membrane conductance. (c) The height of the columns indicates the fraction of ACh-current remaining at the end of 10 ␮M application of monoamine-uptake inhibitor. The number of oocytes recorded was n = 4–10 for neuronal ␣2␤4 and n = 4–7 for muscle AChRs. pounds. The first group included the weakest ACh-cur- (n = 5–11) for neuronal ␣2␤4 and 5.57 ± 0.19 ␮M rent inhibitors: trazodone, 6-nitro-quipazine, and zime- (n = 4–7) for muscle AChRs. The corresponding Hill lidine (selective inhibitors of the 5-HT-transporter). coefficients were 0.73 ± 0.04 and 1.20 ± 0.03, suggest- The second group was constituted by the strongest ing that imipramine interacts with AChRs at a single ACh-current inhibitors: indatraline (an uptake inhibi- site. tor of 5-HT, dopamine and noradrenaline), clomipram- ine, imipramine (tricyclic antidepressants), fluoxetine Non-competitive inhibition of AChRs by imipramine (a highly selective inhibitor of the 5-HT-transporter) We examined two forms of applying imipramine to and norfluoxetine (a principal metabolite of oocytes expressing either neuronal ␣2␤4 or muscle fluoxetine). AChRs. In one, the oocyte was superfused simul- After this brief screening, we focused on the effects taneously with ACh and imipramine. In the other, the of imipramine, that is used as antidepressant and is oocyte was preincubated with imipramine for 2 min also considered as a reference drug in clinical stud- and then the ACh and imipramine were coapplied. In ies.32 both cases the concentration of imipramine was main- tained constant and the ACh concentration was ACh-current inhibition by imipramine changed to obtain the full dose-response relationship When imipramine was applied alone (at concen- (Figure 3). With both procedures the level of ACh- trations up to 10 ␮M) to injected or non-injected current inhibition was insurmountable by increasing oocytes, no measurable membrane currents were ACh concentration and this inhibition was also inde- observed. However, imipramine, at concentrations pendent of the concentration of ACh. above 100 nM, inhibited rapidly and partially reversi- ACh dose-current response relationships were fitted bly ACh-currents in oocytes expressing AChRs with Equation 2. The half-effective concentrations,

(Figure 2a). The extent of inhibition and recovery of the EC50, of ACh and the Hill coefficients, nH, were ACh-current depended on the dose of imipramine obtained when oocytes were superfused with ACh (Figure 2b); at high imipramine concentrations the alone, simultaneously superfused with ACh plus imip- inhibition is more potent and the recovery is slower. ramine and preincubated with imipramine (Table 1).

The half-inhibitory concentrations, IC50, of imipram- For both types of AChRs the EC50 increased approxi- ine, calculated from Equation 1, were 3.65 ± 0.30 ␮M mately twice in the presence of imipramine without

Molecular Psychiatry Antidepressants on nicotinic receptors HE Lo´pez-Valde´s and J Garc´ıa-Colunga 514

Figure 2 Imipramine concentration dependence of the ACh-current inhibition. (a) Superimposed records of the control ACh- current and ACh-currents in the presence of the indicated concentrations of imipramine on neuronal ␣2␤4 and muscle AChRs. (b) Imipramine dose-response relationships of ACh-current inhibition obtained from records as in (a). The currents inhibited by imipramine were normalized to control ACh-currents. Continuous lines were obtained by fitting the data with Equation 1. The number of oocytes recorded was n = 5–11 for neuronal ␣2␤4 and n = 4–7 for muscle AChRs.

Figure 3 Non-competitive inhibition of AChRs by imipramine. ACh dose-response relationships were obtained with ACh alone and with ACh plus imipramine, on neuronal ␣2␤4 (a) and muscle (b) AChRs. The imipramine was applied simultaneously with or before ACh (ACh plus imip or imip preincubation respectively). Data were normalized to the maximal control ACh- current and fitted by using Equation 2 (continuous lines). Insets: Representative superimposed records of the control ACh- current and that obtained by simultaneous application of drugs. The number of oocytes recorded was n = 4–9 for neuronal ␣2␤4 and n = 5–10 for muscle AChRs (see Table 1).

changing the Hill coefficient, suggesting that the affin- Additional forms of imipramine application ity of neuronal and muscle receptors for ACh is slightly To explore if imipramine interacts with the close reduced by imipramine, while maintaining normal and/or activated states of the AChR, effects of various cooperativity. forms of imipramine application to oocytes expressing

Molecular Psychiatry Antidepressants on nicotinic receptors HE Lo´pez-Valde´s and J Garc´ıa-Colunga 515 Table 1 The half-effective concentrations, EC50, of ACh on Table 2 Percentage of inhibition by different forms of imip- neuronal and muscle AChRs ramine application on neuronal and muscle AChRs

a ␮ ␣ ␤ a EC50 ( M) nH Type of application 2 4 AChRs muscle AChRs

␣2␤4 AChRs imip, ACh 42.6 ± 8.3 (n = 6) 42.8 ± 2.7 (n = 4) ACh alone 26.8 ± 2.4 1.2 ± 0.1 ACh, ACh + imip, 48.9 ± 4.8 (n = 5) 44.2 ± 4.5 (n = 4) (n = 9) ACh ACh plus imip 7 ␮M 53.4 ± 4.1 1.4 ± 0.3 ACh + imip 56.9 ± 7.5 (n = 7) 51.1 ± 6.5 (n = 4) (n = 5) imip, ACh + imip 70.6 ± 3.8 (n = 6) 71.7 ± 1.7 (n = 4) Preincubation with imip 48.2 ± 1.4 0.9 ± 0.2 7 ␮M(n = 4) aThe membrane current was elicited by 0.5 ␮M ACh and inhibited by 4 ␮M imipramine for neuronal ␣2␤4 and 5 ␮M muscle AChRs imipramine for muscle AChRs. n = number of oocytes stud- ACh alone 5.3 ± 0.3 1.1 ± 0.1 ied. (n = 10) ACh plus imip 5 ␮M 13.6 + 1.3 1.3 ± 0.2 = (n 5) ing to note that when imipramine was applied before Preincubation with imip 13.8 ± 1.2 1.6 ± 0.2 5 ␮M(n = 5) ACh (Figure 4a), the ACh-current inhibition was simi- lar to that obtained when imipramine was applied after a activation of the AChRs (cf Figure 4b, c). In contrast, The values of EC50 and nH were obtained from Equation 2. The application of ACh and imipramine (imip) was simul- preincubation with imipramine, followed by coappli- taneous. n = number of oocytes studied. cation with ACh resulted in the strongest inhibition of the ACh-current (Figure 4d). These results (summarized in Table 2) suggest that imipramine inter- either ␣2␤4 or muscle AChRs were tested (Figure 4). acts with both resting and activated AChRs. Following two control applications of ACh the oocyte was preincubated briefly with imipramine and then Voltage-dependence of AChR inhibition by exposed to ACh alone (a); the imipramine was applied imipramine after the ACh-current had reached its peak (b); the To determine if imipramine is interacting within the membrane current was elicited by simultaneous super- ion channel or externally we studied the ACh-current fusion of ACh and imipramine (c); and the oocyte was inhibition as a function of membrane potential. preincubated with imipramine and then exposed Figure 5a shows a typical effect of 10 ␮M imipramine immediately to ACh plus imipramine (d). It is interest- on the membrane current elicited by 0.5 ␮M ACh in an

Figure 4 Different forms of imipramine application on muscle AChRs. Control currents were elicited with 0.5 ␮M ACh alone, then 5 ␮M imipramine was applied in different ways: preincubating the oocyte and then ACh was applied alone (a); in the continuous presence of ACh after the peak control current was reached (b); simultaneously with ACh (c); before the current was elicited by ACh in the presence of imipramine (d). The bar calibration corresponds to 50 nA and 300 s (a, c and d) and 70 nA and 100 s (b). The number of oocytes recorded was n = 5–7 for neuronal ␣2␤4 and n = 4 for muscle AChRs (see Table 2).

Molecular Psychiatry Antidepressants on nicotinic receptors HE Lo´pez-Valde´s and J Garc´ıa-Colunga 516

Figure 5 Voltage dependence of the ACh-current inhibition. (a) Representative current elicited by ACh alone and then inhibited by imipramine on muscle AChRs. The membrane potential was held at −60 mV and 20-mV voltage pulses were applied from −140 to 40 mV, in normal Ringer solution, during ACh superfusion, and during coapplication of ACh and imipramine. (b) ACh-current inhibited by imipramine as a function of membrane potential on muscle and neuronal AChRs. The continuous lines were obtained by fitting a one-site blocking model (Equation 4). (c) I–V relationships are shown in the absence and presence of imipramine for neuronal ␣2␤4 AChRs. (d) I–V relationships are shown for muscle AChRs. ACh-currents were normalized to the control ACh-currents at −140 mV. The number of oocytes recorded was n = 7 for neuronal ␣2␤4 and n = 6 for muscle AChRs.

␮ ␣ ␤ oocyte expressing muscle AChRs. The membrane potential of 0 mV, IC50(0), was 1.21 M for 2 4 potential was held at −60 mV and voltage pulses to dif- AChRs, whilst it was 3.61 ␮M for muscle AChRs. In − − ferent levels (from 140 to 40 mV in 20-mV steps) were contrast, the values of IC50 at 60 mV were similar for applied before and during ACh application, as well as both types of receptors (Figure 2b). Current-voltage in the presence of imipramine plus ACh. The results (I–V) relationships for neuronal ␣2␤4 and muscle were analyzed using a simple one-site blocking AChRs (Figure 5c, d) show an inward rectification that model,10,30 that allows estimation of the ‘electrical dis- decreased slightly by imipramine. tance’ (Equation 4). In oocytes expressing neuronal ␣2␤4 or muscle AChRs, the ACh-current in the pres- Effects of imipramine on the decay of the ACh-

ence of imipramine (Iimipramine) was compared with the current control current (IACh) at each membrane potential To explore the possibility that imipramine modifies the (Figure 5b). For both types of receptors the inhibition desensitization of AChRs, effects of imipramine on the of ACh-current by imipramine was voltage-dependent, decay of the ACh-currents were studied in oocytes being stronger at hyperpolarized holding potentials. In expressing neuronal or muscle AChRs. The control the case of neuronal ␣2␤4 AChRs the calculated electri- membrane current was elicited by 100 ␮Mor5␮M cal distance was 0.10 ± 0.02 (n = 7), whereas for muscle ACh: for neuronal ␣2␤4 or muscle AChRs respectively. AChRs it was 0.12 ± 0.02 (n = 6), indicating that imipra- In both cases the control current reached a peak and mine interacts within the pore of the receptor-channel then declined in the continuous presence of ACh, and complex, close to its extracellular end. The half- the time-course of decay followed an exponential func- inhibitory concentration of imipramine at a membrane tion. This decay is due to several factors such as the

Molecular Psychiatry Antidepressants on nicotinic receptors HE Lo´pez-Valde´s and J Garc´ıa-Colunga 517

Figure 6 Imipramine accelerates the time course of the ACh-current. Records of the control current and that obtained in the presence of imipramine on neuronal ␣2␤4 (a) and muscle AChRs (b) are shown superimposed and normalized. Control current amplitudes were 4931 nA (a) and 54.6 ␮A (b); whereas ACh-current amplitudes in the presence of imipramine were 1280 nA and 11.6 ␮A respectively. The number of oocytes recorded was n = 4 for neuronal ␣2␤4 and n = 6 for muscle AChRs (see Table 3). speed of the superfusion that determines the rate of sol- exponential functions (Equation 5). For both types of ution exchange, the presence of unstirred layer close control ACh-currents the decay was well fitted by a sin- to the surface of the cell, the size and shape of the cell, gle exponential function and a steady-state component. desensitization of receptors. Considering that the first In contrast, in the presence of imipramine the ACh- three of these factors are maintained constant during current decayed biphasically, following the sum of two one experiment, we think that imipramine accelerates exponential functions and a steady-state component; the decay of the ACh-current by blocking AChRs and where the two time constants of the decay were faster accelerating the desensitization of receptors. For than the time constant of the corresponding control instance, when the ACh was simultaneously super- ACh-current (Table 3). These results suggest that imip- fused with 7 ␮Mor10␮M imipramine (for neuronal ramine might accelerate the desensitization of AChRs. ␣2␤4 or muscle AChRs), the peak current was substan- Additionally, with high ACh concentrations, a transi- tially reduced and the rate of decay was accelerated ent tail current was elicited when ACh and imipramine (Figure 6). The averaged peak control current was were simultaneously washed out. This effect was more 2.86 ␮A for neuronal ␣2␤4 and 34.27 ␮A for muscle evident for muscle than for neuronal ␣2␤4 AChRs AChRs; whereas the averaged peak ACh-current in the (Figure 6). However, when ACh was removed leaving presence of imipramine was 0.76 ␮A for neuronal ␣2␤4 the imipramine in the superfusing fluid, the transient and 8.97 ␮A for muscle AChRs. inward current was not observed. This suggests a rever- We compared the rate of ACh-current decay in the sal of the open channel blockage by imipramine, simi- absence and presence of imipramine in two ways. lar to that seen with atropine and ACh itself.9,33

Firstly, we defined Dft as the fraction of the ACh- current that was reduced after 5 min of the application Discussion of ACh alone or ACh plus imipramine, compared with its own peak current. This value was lower for control The present study shows that several substances, used ACh-currents than for ACh-currents in the presence of widely as antidepressants, have an inhibitory effect on imipramine, indicating less degree of decay in control the ACh-current generated by the activation of AChRs ACh-currents and probably less desensitization expressed in Xenopus oocytes. (Table 3). Secondly, the current decay was fitted with Our results show that the imipramine exerts a non-

Table 3 Amplitudes and time constants of the components of the ACh-current decay of neuronal and muscle AChRs

Parametera ␣2␤4 AChRs (n = 4) muscle AChRs (n = 6)

100 ␮M ACh 100 ␮M ACh + 7 ␮M5␮M ACh 5 ␮M ACh + 10 ␮M imip imip

± ± ± ± Dft 0.62 0.03 0.95 0.08 0.36 0.07 0.91 0.03 ± ± ± ± Io 0.18 0.06 0.01 0.00 0.45 0.07 0.05 0.04 ± ± ± ± Af 0.83 0.07 0.60 0.10 0.54 0.08 0.60 0.11 ␶ ± ± ± ± f (s) 220.2 30.8 47.46 20.90 126.9 23.0 52.5 25.0 ± ± As – 0.41 0.09 – 0.39 0.06 ␶ ± ± s (s) – 84.85 28.40 – 95.2 23.0 a Amplitudes of the fast (Af) and slow (As) components and the asymptotic component (Io) were normalized to the total current amplitude. The application of ACh and imipramine was simultaneous. n = number of oocytes studied.

Molecular Psychiatry Antidepressants on nicotinic receptors HE Lo´pez-Valde´s and J Garc´ıa-Colunga 518 competitive inhibition on muscle AChRs. This result lemented previous studies and performed a compara- is in agreement with previous reports carried out on tive electrophysiological characterization of effects of AChRs of the electric organ of Torpedo and frog mus- imipramine on muscle and neuronal AChRs. A new cle.24,25 However, the location of the binding site of finding was that imipramine acts on both types of imipramine on these receptors is not clear. It has been AChRs through similar mechanisms, probably inter- proposed that the imipramine binding site is the same acting within the ionic channel at the same site and as that for phencyclidine and perhydrohistrionico- accelerating the desensitization of AChRs. Also this is toxin, which is located within the channel,24 in con- the first study in which effects of substances such as trast with other studies suggesting that the binding site norfluoxetine, indatraline, zimelidine, 6-nitro-quipa- of imipramine, also located within the channel, is dif- zine and trazodone have been explored on nicotinic ferent.25 On the other hand, the electrical distance for receptors. spiperone on muscle AChRs was d = 0.17,10 similar to It has become increasingly important to determine the value obtained here for imipramine (d = 0.12), sug- the effects of antidepressants on neurotransmitter gesting that both drugs interact at the same or closely receptors and ionic channels, because the role played related site of the muscle AChR. by them on mental depression and other diseases is not In contrast to effects of imipramine that had no effect completely understood. For instance, fluoxetine has an on current rise or decay times in frog motor endplate,25 inhibitory effect on muscle as well as on neuronal 11,13,16,17,22 ␣ ␣ we observed that imipramine accelerates in a concen- AChRs, histamine H1, adrenergic 1 and 2 tration-dependent manner the rate of decay of ACh- and muscarinic receptors,19 and voltage-activated K+ currents generated by both neuronal and muscle and Na+ channels.38 AChRs. For instance, when the concentration of imip- Finally, it is possible that the actions of imipramine, ramine was increased the ACh-current decay, and inhibiting neuronal AChRs, are related with thera- probably its desensitization, became faster. The differ- peutic- and/or side-effects, because the concentration ent results may be due to molecular differences in the of imipramine in blood of depressed patients may M2 region between frog and mouse muscle AChRs,34 reach ෂ2 ␮M,39 and that the concentration in the brain 40 similar to the difference in affinity to phencyclidine is 11–16 fold higher, compared with the IC50 for imip- between AChRs from Torpedo electric organ and ramine (3.65 ␮M) on neuronal ␣2␤4 AChRs studied mouse muscle; which is based on a difference in three here. Additionally, the demethylated metabolite of amino acids within the ionic channel.35 imipramine, desipramine, that is more concentrated in It has been reported that ACh potentials in rat soleus blood and brain than imipramine,40 exerts a similar muscle fibers are potentiated with 1 ␮M imipramine.25 activity in the neuroblastoma cell line SH-SY5Y.26 However, under our experimental conditions even These cells express AChR ␣3, ␣5, ␣7, ␤2, and ␤4 sub- smaller concentrations of imipramine exerted an unit genes.41 Thus, it is very likely that imipramine inhibitory effect on the ACh-current. Further studies interacts with AChRs in the peripheral and central ner- are necessary to clarify these differences, which may vous systems. Further studies are required to clarify be due to the different types of receptors and cells how AChRs are involved in a number of physiological examined. and behavioral processes42 and how these drugs exert The non-competitive inhibition of both neuronal and their action on other AChR subtypes. muscle AChRs by imipramine shows that the drug exerts its effects by a steric mechanism with a similar Acknowledgements affinity. Considering the electric distance (ෂ0.10) for both types of receptors, it seems likely that the binding We are grateful to Dr Ricardo Miledi for initiating these site of imipramine is within the ionic channel, close to experiments and for subsequent invaluable help during its extracellular end, similar to the binding site of many this work. We are also grateful to Drs J Boulter and S non-competitive inhibitors, including chlorpromazine Heinemann (The Salk Institute) for providing the AChR and histrionicotoxin.5 In addition, it appears that in clones, and MSc Marina Herrera Gonza´lez for prepar- both neuronal and muscle AChRs, imipramine binds ing cRNAs. This work was supported by Grants from to the resting receptor and prevents it from the opening Consejo Nacional de Ciencia y Tecnologı´a, Me´xico of the ionic channel. 3717P-N9608 and G25775N (to JGC) and a DGEP Nearly three decades ago it was reported that sero- UNAM scholarship to HELV. tonin inhibits the AChRs of the neuromuscular junc- 36 tion. This effect was subsequently widely confirmed References for both muscle and neuronal AChRs.7,9–11,37 Such a cross-interaction between serotonin and AChRs needs 1 Clementi F, Fornasari D, Gotti C. Neuronal nicotinic receptors, important new players in brain function. Eur J Pharmacol 2000; to be considered in explaining the varied effects of 393:3–10. antidepressant drugs, bearing in mind that two inhibi- 2 Karlin A, Akabas MH. Toward a structural basis for the function tory effects may occur on AChRs, one exerted by the of nicotinic acetylcholine receptors and their cousins. Neuron antidepressant and the another by the non-competitive 1995; 15: 1231–1244. 3 McGhee DS. Molecular diversity of neural acetylcholine receptor. inhibitory effect of serotonin. Ann NY Acad Sci 1999; 868:565–577. Few papers have studied effects of imipramine on 4 Lukas RJ, Bencherif M. Heterogeneity and regulation of nicotinic muscle24,25 and neuronal26,27 AChRs. Here we comp- acetylcholine receptors. Int Rev Neurobiol 1992; 34:25–131.

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Molecular Psychiatry