Effects of Bis(7)-Tacrine on Spontaneous Synaptic Activity and on the Nicotinic ACh Receptor of Torpedo Electric Organ

Esteve Ros,1 Jordi Aleu,1 Inmaculada Gomez De Aranda,1 Carles Cantí,2 Yuan-Ping Pang,3 Jordi Marsal,1 and Carles Solsona1

 1Laboratori de Neurobiologia Cellular i Molecular, Departament de Biologia Cellular i Anatomia Patològica, Facultat de Medicina, Hospital de Bellvitge, Universitat de Barcelona, E-08907 L'Hospitalet de Llobregat, Spain;  2Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom; and  3Mayo Clinic Cancer Center and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota 55905


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

Ros, Esteve, Jordi Aleu, Inmaculada Gomez De Aranda, Carles Cantí, Yuan-Ping Pang, Jordi Marsal, and Carles Solsona. Effects of Bis(7)-Tacrine on Spontaneous Synaptic Activity and on the Nicotinic ACh Receptor of Torpedo Electric Organ. J. Neurophysiol. 86: 183-189, 2001. Bis(7)-tacrine is a potent acetylcholinesterase inhibitor in which two tacrine molecules are linked by a heptylene chain. We tested the effects of bis(7)-tacrine on the spontaneous synaptic activity. Miniature endplate potentials (MEPPs) were recorded extracellularly on slices of electric organ of Torpedo marmorata. Bis(7)-tacrine, at a concentration of 100 nM, increased the magnitudes that describe MEPPs: amplitude, area, rise time, rate of rise, and half-width. We also tested the effect of bis(7)-tacrine on nicotinic acetylcholine receptors by analyzing the currents elicited by acetylcholine (100 µM) in Torpedo electric organ membranes transplanted in Xenopus laevis oocytes. Bis(7)-tacrine inhibited the acetylcholine-induced currents in a reversible manner (IC50 = 162 nM). The inhibition of nicotinic acetylcholine receptors was not voltage dependent, and bis(7)-tacrine increased the desensitization of nicotinic acetylcholine receptors. The Hill coefficient for bis(7)-tacrine was -0.72 ± 0.02, indicating that bis(7)-tacrine binds to the nicotinic acetylcholine receptor in a molecular ratio of 1:1, but does not affect the binding of alpha -bungarotoxin with the nicotinic acetylcholine receptor. In conclusion, bis(7)-tacrine greatly increases the spontaneous quantal release from peripheral cholinergic terminals at a much lower concentration than tacrine. Bis(7)-tacrine also blocks acetylcholine-induced currents of Torpedo electric organ, although the mechanism is different from that of tacrine: bis(7)-tacrine enhances desensitization, whereas tacrine reduces it.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Alzheimer's disease is the most frequent cause of dementia in western populations over the age of 65. The drugs that have been most extensively studied for the palliative treatment of Alzheimer's disease are acetylcholinesterase (AChE) inhibitors (Krall et al. 1999), which, according to the cholinergic hypothesis (Bartus et al. 1982), increase the levels of brain acetylcholine (ACh) and relieve cognitive deficiencies. In the European Union, the only drugs approved for clinical treatment are tacrine, donepezil, and rivastigmine, all of which are AChE inhibitors (Francis et al. 1999). Tacrine was one of the first anticholinesterasic drugs approved by the Food and Drug Administration of the United States in 1993 (Krall et al. 1999). Doses between 80 and 160 mg per day are effective (Farlow et al. 1992; Knapp et al. 1994), but in some cases this concentration is hepatotoxic (Watkins et al. 1994). In addition, it has been demonstrated that tacrine can induce myopathy by the excessive stimulation of nicotinic receptors (Jeyarasasingam et al. 2000).

Recently, it has been shown that tacrine could have other effects on Alzheimer's disease that are not directly related to its AChE inhibitory activity: 1) tacrine inhibits the secretion of amyloid beta-peptides in human neuroblastoma cells (Lahiri et al. 1997, 1998); 2) in murine neuroblastoma cells, acute application of tacrine inhibits monoamine oxidase isoform A and activates the B isoform in the long term (Zatta et al. 1998); and 3) tacrine also attenuates the influx of calcium by inhibiting the L-type calcium channels (Dolezal et al. 1997).

In the early 1990s, a number of second-generation AChE inhibitors were developed, some of them based on the chemical structure of the tacrine molecule. Bis(7)-tetrahydroaminacrine [bis(7)-tacrine; Fig. 1] is a potent and selective inhibitor of AChE, in which two tacrine molecules are linked by a heptylene chain spaced so as to permit simultaneous binding at the catalytic and peripheral sites of AChE (Pang et al. 1996). Bis(7)-tacrine is up to 150-fold more potent and 250-fold more selective in inhibiting AChE than tacrine over butyrylcholinesterase (BChE), and its effects on AChE are reversible (Wang et al. 1999). Recent studies have also shown that bis(7)-tacrine effectively reverses AF64A-induced deficits in navigational memory in rats (Liu et al. 2000) and is a potent GABA(A)-receptor antagonist (Li et al. 1999).



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Fig. 1. A: chemical structure of tacrine (9-amino-1,2,3,4-tetrahydroacridine). B: bis(7)-tetrahydroaminacrine [bis(7)-tacrine].

Here we show the effect of bis(7)-tacrine molecule on the spontaneous synaptic activity and on the current associated with the nicotinic acetylcholine receptor. We used the electric organ of Torpedo marmorata. Synaptic activity was recorded extracellularly on slices of fresh electric organ (Cantí et al. 1994; Ros et al. 2000). The activation of nicotinic acetylcholine receptors was measured on Xenopus laevis oocytes that were transplanted with membranes isolated from the electric organ (Cantí et al. 1998; Marsal et al. 1995; Ros et al. 2000).


    METHODS
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METHODS
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Animals and solutions

Torpedo marmorata specimens were caught off the Catalan Mediterranean coast and kept in artificial seawater. The fish were anesthetized with tricaine (3-aminobenzoic acid ethyl ester methanesulfonate salt; Sigma, St. Louis, MO) at a concentration of 0.03% in seawater, before surgical excision of electric organs.

Torpedo marmorata electric organ fragments were kept in the following saline solution (in mM): 280 NaCl, 3 KCl, 3.4 CaCl2, 1.8 MgCl2, 5.5 glucose, 300 urea, 100 sucrose, and 6.8 HEPES/NaOH-buffer, pH adjusted to 7.0 with NaHCO3. The same solution was used to record the spontaneous synaptic activity.

Mature females of Xenopus laevis were purchased from the Center d'Elevage des Xenopes (Montpellier, France) and were anesthetized by immersion in water containing 0.17% tricaine. A few lobes of ovaries were removed through a small incision in the abdomen.

Solutions for Xenopus oocytes were as follows. Barth's solution contained (in mM) 88 NaCl, 1 KCl, 0.33 Ca(NO3)2, 0.82 MgSO4, 2.40 NaHCO3, and 10 mM HEPES at pH 7.4, supplemented with 100 IU/ml penicillin and 0.1 mg/ml streptomycin. Recording solution contained (in mM) 115 NaCl, 2 KCl, 1.8 CaCl2, and 5 HEPES at pH 7.0. None of the Xenopus female donors used in this study exhibited muscarinic acetylcholine receptors in their oocytes.

Recording of the spontaneous synaptic activity

All recordings were made at room temperature (20-22°C). From 5 to 10 prisms of the electric organ were cut with a scalpel blade, and 1- to 5-mm sections were incubated overnight, in Torpedo saline solution containing bis(7)-tacrine, to ensure complete diffusion throughout the tissue. Measurements were performed in fragments fixed in a Plexiglas chamber with a silicone elastomer (Sylgard)-coated base.

The spontaneous synaptic release of ACh was recorded with focal extracellular low-resistance microelectrodes (Katz and Miledi 1977) as adapted to electric organ by Soria (1983; see Dunant and Muller 1986; Muller and Dunant 1987, for details) and as described elsewhere (Cantí et al. 1994; Ros et al. 2000). The method allows long-term recording with little damage to the cells. Spontaneous miniature endplate potentials (MEPPs) were amplified (Axoclamp-2A, Axon Instruments, Foster City, CA) monitored on a Tektronix (Beaverton, OR) 5110 oscilloscope and recorded in parallel on a VCR (Biologic, Echirolles, France) and on a PC-Computer with a LabView (National Instruments, Austin, TX) program (Quantadat) written in our laboratory with an AT-MIO16X (National Instruments) digitizing interface. Signals were acquired at a frequency of 100 kHz and analyzed using the same Labview program and the Whole Cell Analysis program, kindly provided by Prof. J. Dempster (Strathclyde University, Scotland, UK), and a TL-1 Labmaster digitizing interface. Data in ASCII form were exported to Sigmaplot 4.01.

The following parameters of each MEPP were measured: amplitude, rise time, rate or velocity of rising, the area corresponding to the charge that generated them (measured as the integral of the contour delimited by each MEPP), and the half-width that indicates the rate of the decay phase (see Fig. 2A). Results were obtained from five separate experiments, and they were represented as histogram and cumulative plots (Van der Kloot 1991), comparing all the variables in the different experimental conditions (Fig. 3). The number of MEPPs analyzed was 7,011 for the control condition and 7,290 for bis(7)-tacrine.



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Fig. 2. Effect of bis(7)-tacrine on the spontaneous cholinergic synaptic activity. A: parameters analyzed in each miniature recorded. Diagram of the studied variables in the miniature endplate potentials (MEPPs). A, total area (equivalent to the electrical charge mobilized by an MEPP); P, peak amplitude; RT, rise time; R of R, rate of rise; T50%, half-width. B and C: effect of bis(7)-tacrine on spontaneous MEPPs. Superimposed oscilloscope traces showing spontaneous MEPPs recorded in Torpedo marmorata electric organ. B: the untreated fragments. C: after an incubation with bis(7)-tacrine (100 nM). Bis(7)-tacrine increased the amplitude and prolonged MEPPs.



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Fig. 3. Analysis of MEPPs in bis(7)-tacrine-treated fragments of electric organ of Torpedo. Data are presented as cumulative plots and as bar histograms (inset). In the cumulative plots: the untreated tissue, continuous line; bis(7)-tacrine (100 nM), dotted line. Inset bar histograms are presented in the untreated tissue (A); the bis(7)-tacrine-treated tissue (B; 100 nM). A: effect of bis(7)-tacrine on the amplitude of MEPP (peak). B: electric charge mobilized by spontaneous MEPP. Effect of bis(7)-tacrine on electrical charge mobilized by spontaneous ACh release. The area delimited below an MEPP contour corresponds to the total electrical charge passed through the nicotinic acetylcholine receptors as a consequence of spontaneous quanta. C: rise time of MEPP. D: rate of rise time of MEPP. Bis(7)-tacrine increased the rate of rise. E: half-width of the MEPP at the 50% of the amplitude. Bis(7)-tacrine prolonged the decay phase of MEPPs. The number of MEPPs analyzed for control condition was 7,011, and 7,290 after bis(7)-tacrine treatment. All the data presented were obtained from 5 experiments.

Oocyte preparation, microinjection, and recording

Oocytes at stages V and VI (Dumont 1972) were dissected out and kept at 15-16°C in sterile Barth's solution. One day before injection the oocytes were treated with collagenase type 1A (Sigma; 0.5 mg/ml) for 45-50 min at room temperature to remove the surrounding layers (Miledi and Woodward 1989).

Healthy oocytes were microinjected with 50 nl of thawed suspension (2-8 mg/ml) of electroplaque membranes (Cantí et al. 1998; Marsal et al. 1995; Ros et al. 2000) by means of an injector (World Precision Instruments, Sarasota, FL, model A203XVZ). Samples were sonicated prior to injection.

Eighteen to 48 h after the injection, oocytes were voltage clamped with a two-electrode system (Axoclamp-2A, Axon Instruments). Intracellular electrodes (1-4 MOmega resistance) were filled with 3 M potassium chloride for voltage recording and with 3 M potassium acetate for current injection. The volume of the oocyte recording chamber was 200 µl. Membrane currents were low-pass filtered at 10 Hz and recorded on a PC using Whole Cell Analysis v. 2.1. program after sampling signals by Lab PC+ (National Instruments) at twice the filter frequency. In all recordings currents were elicited by challenges of 100 µM ACh chloride. The interval between consecutive responses was systematically set to 10 min (flow rate 8 ml/min), as we had previously established that this was sufficient to ensure a complete recovery from receptor desensitization. All the oocytes were tested for consistent response amplitudes, with at least three challenges prior to the application of the drug.

125I-alpha -bungarotoxin binding

Binding of 125I-alpha -bungarotoxin (Amersham Life Science, Piscataway, NJ) was undertaken according to the procedure previously described (Schmidt and Raftery 1973). In brief, Torpedo electric organ membranes were solubilized for 30 min in the following solution: 10 mM HEPES, 100 mM NaCl, and 1% Triton X-100 at pH 7.4 and centrifuged 30 min at 100,000 g. The protein concentration of the supernatant was between 0.8 and 1.1 mg/ml. Aliquots of 50 µl of the supernatant were incubated with alpha -bungarotoxin (Sigma) at 10 µg/ml and containing 2.5 µCi. In some samples, bis(7)-tacrine at different concentrations was added. After 2 h of incubation at room temperature, the mixture was placed in Whatman DE-81 of 2.3-cm-diam disk and rinsed three times in 10 ml of 10 mM sodium phosphate buffer, 50 mM Na Cl, and 100 0.1% Triton X at pH 7.4. Dried disks were counted in a 1470 Wizard gamma  counter. Unspecific binding was subtracted.

Calculations and statistics

Differences between distribution functions were evaluated with Sigmastat 3.2 software (SPSS, Chicago, IL) by Mann-Whitney rank sum test. Dose-response data were fitted by a nonlinear regression to a sigmoidal curve using the Sigmaplot 4.01 (SPSS) and Inplot (GraphPad, San Diego, CA) programs. Values are expressed as means ± SE calculated by the program.

Drugs

The stock solution of bis(7)-tacrine (1 mM) was made with deionized water and kept at -80°C before it was diluted to its final concentration in the recording solution.


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ABSTRACT
INTRODUCTION
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DISCUSSION
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Effects on MEPPs

We have tested bis(7)-tacrine at a concentration of 10 nM on fragments of electric organ from Torpedo marmorata, with extracellular recording through a loose patch configuration (Dunant and Muller 1986), and we did not detect any change in the size of spontaneous MEPPs (data not shown). However, at the concentration of 100 nM, bis(7)-tacrine increased all the parameters of MEPPs (Fig. 2, B and C).

The amplitude of MEPPs in the untreated fragments (n = 7,011) was 1.27 ± 0.03 (SE) mV, whereas in the fragments treated with bis(7)-tacrine at the concentration of 100 nM (n = 7,290), it was 1.91 ± 0.02 mV. The distribution amplitude is shown in Fig. 3A.

The area under the profile of each MEPP reflects the electrical charge carried during the release of a single quantum of ACh. The area was 1.05 ± 0.01 mV · ms, in the untreated fragments, and it was 2.30 ± 0.03 mV · ms in the treated fragments. Bis(7)-tacrine increased the area of MEPPs in the untreated fragments by more than twofold. Figure 3B shows the statistical distribution of areas.

The rise time of an MEPP is the result of the addition of time periods from different cellular processes: the release and diffusion of ACh throughout the synaptic cleft and the opening time of the nicotinic acetylcholine receptors. The rise time of MEPP was 0.44 ± 0.01 ms in the untreated fragments and 0.48 ± 0.01 ms in the treated fragments. See Fig. 3C for statistical distribution.

The rate of rise between 10 and 90% of the MEPP amplitude was 3.29 ± 0.05 mV/ms in untreated fragments, while in the fragments treated with bis(7)-tacrine at the concentration of 100 nM it was 4.69 ± 0.08 mV/ms. Figure 3D shows the statistical distribution.

The duration or the width of an MEPP depends on the persistence of ACh within the synaptic cleft. The drugs with AChE inhibitory properties prolonged the action of ACh by decreasing its rate of hydrolysis, and the decay phase of the MEPPs was therefore prolonged, because a single molecule of ACh can interact more than once with nicotinic acetylcholine receptors. Since the decay phase of an MEPP is an exponential function, the end of which is difficult to establish, the half-width of an MEPP is particularly appropriate for estimating the duration of an MEPP and, thus very convenient for investigating the action of AChE inhibitory agents on synaptic activity. The half-width was 0.70 ± 0.01 ms in the untreated fragments and 0.87 ± 0.01 ms in the treated fragments. The half-width distribution is shown in Fig. 3E.

In all the variables analyzed, the differences between the two groups were significant (P < 0.001).

Effects on nicotinic acetylcholine receptors in transplanted Xenopus oocytes

Bis(7)-tacrine reduced the amplitude of currents elicited by 100 µM ACh in a dose-dependent manner (Fig. 4A). The inhibition was fully reversible after a wash time of 10 min. A sigmoidal function of four parameters fitted the curves representing the inhibitory action of bis(7)-tacrine and tacrine. The IC50 for bis(7)-tacrine was 162 ± 5 nM, while for tacrine it was 8.9 ± 1.2 µM (tacrine data were taken from previous studies conducted in our laboratory: Cantí et al. 1998). The Hill coefficient for bis(7)-tacrine was -0.72 ± 0.02. 



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Fig. 4. Interactions of bis(7)-tacrine and tacrine on the nicotinic acetylcholine receptor. Effects of bis(7)-tacrine. A: currents elicited by 100 µM ACh in 1 oocyte in the presence of bis(7)-tacrine at different concentrations. Bars indicate ACh application, and the holding potential was -70 mV. B: effects of bis(7)-tacrine () and tacrine (open circle ) on the current elicited by 100 µM ACh. Data are means ± SE of values obtained from 10 oocytes from 5 different donor frogs, each oocyte injected with 1 of 3 different fish membrane batches, and are represented as percentages of the maximal current elicited by 100 µM ACh at a holding potential of -70 mV.

The effect of bis(7)-tacrine on the amplitude of ACh-activated currents in oocytes was not voltage dependent. In a series of experiments, currents were elicited by 100 µM ACh at different holding potentials (from -70 to +10 mV) in each oocyte. The concentration of bis(7)-tacrine used (100 nM) was close to its IC50 on nicotinic acetylcholine receptors. At negative membrane potentials, the slope conductance of ACh currents in the presence of bis(7)-tacrine was similar to control (peak current reduction of 40 ± 5% at -70 mV compared with 46 ± 6% at -20 mV, n = 8; Fig. 5A). However, the reversal potential of the ACh current was not modified by the treatment with bis(7)-tacrine, showing that the ion selectivity of the channel was maintained.



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Fig. 5. Voltage dependence of the effect of bis(7)-tacrine on the nicotinic acetylcholine receptor and the desensitization time constant of nicotinic responses. A: the effect of bis(7)-tacrine was reduced as the membrane was depolarized, indicating a preferential interaction with the open-channel form on the nicotinic receptor. Every point is the mean of 7 different oocytes. B: currents recorded from an oocyte after sequential application of 100 µM ACh and 100 µM ACh plus 100 nM bis(7)-tacrine during 30 s at a holding potential of -70 mV. The wash time between the application of solutions was 10 min at a flux of 8 ml/min. The time constant of the decay phase was calculated by fitting a simple exponential curve. Bis(7)-tacrine reduced the time constant.

Oocytes were exposed to solutions containing either ACh (100 µM) in the presence or absence of bis(7)-tacrine (100 nM) during long periods of time (30 s; see Fig. 5B). A prolonged exposure of the nicotinic acetylcholine receptor to ACh results in a desensitization or open channel block, which would perhaps be increased by bis(7)-tacrine. The time constant (tau ) was calculated by fitting the decay phase of the ACh currents to a single exponential function. In oocytes stimulated with ACh in the absence of bis(7)-tacrine, the time constant was tau  = 11.3 ± 4.1 s, while in the presence of bis(7)-tacrine the time constant was reduced to tau  = 2.8 ± 0.3 s.

Effects of bis(7)-tacrine on the binding of 125I-alpha -bungarotoxin to solubilized electric organ membranes

The number of molecules of alpha -bungarotoxin that bound to solubilized membranes of Torpedo electric organ was 7.47.1012, which correspond to a concentration about 1 nmol of alpha -bungarotoxin/mg of protein. We have found that bis(7)-tacrine at concentrations of 10, 100, and 1,000 nM did with not interfere the binding of alpha -bungarotoxin to the nicotinic acetylcholine receptor (n = 3; Fig. 6).



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Fig. 6. Effects of different bis(7)-tacrine concentrations on the specific binding of 125I-alpha -bungarotoxin to solubilized membranes of Torpedo electric organ. Vertical bar charts show means ± SE in control conditions and in the presence of different concentrations of bis(7)-tacrine, 10, 100, and 1,000 nM. None of the differences are statistically significant (n = 3).


    DISCUSSION
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The clinical goal of the administration of AChE inhibitors is to achieve high brain ACh levels and relieve the cognitive deficiency symptoms in Alzheimer's disease patients. Bis(7)-tacrine is a highly potent second-generation AChE inhibitor. The lethal toxicity of bis(7)-tacrine determined by intragastric administration to rats is reportedly increased only twofold respect to tacrine (Han et al. 2000), yet the inhibition of AChE by bis(7)-tacrine and the increase of acetylcholine spontaneous synaptic activity by bis(7)-tacrine described here are several order of magnitudes greater than those reported for tacrine. Furthermore, bis(7)-tacrine is 24-fold more potent in reversing ethylcholine mustard aziridinium ion-induced memory impairment in rats (Liu et al. 2000) and has a much higher intragastric efficacy in inhibiting AChE (Wang et al. 1999) than that shown by tacrine. It would therefore appear logical to expect bis(7)-tacrine to be less toxic than tacrine.

Physostigmine, tacrine (Cantí et al. 1994), and the derivative of tacrine CI-1002 (Ros et al. 2000) increased the amplitude and duration of MEPPs recorded from the electric organ of Torpedo. Increased amplitude of MEPPs were also obtained on the neuromuscular junction using tacrine (Braga et al. 1991; Thesleff et al. 1990) and CI-1002 (Ros et al. 2000). The concentrations assayed for these drugs were usually between 1 and 100 µM. In contrast, bis(7)-tacrine increased the size of MEPPs in the electric organ of Torpedo at a much lower concentration (100 nM). This observation is consistent with reports that bis(7)-tacrine is a more potent inhibitor of AChE than tacrine (Pang et al. 1996; Wang et al. 1999).

The prolongation of the decay phase of the MEPPs is directly related to the AChE inhibitory activity. In previous studies (Cantí et al. 1994; Ros et al. 2000) we have found that tacrine (100 µM) or CI-1002 (1 µM) prolonged the half-width and, thus the time course of a single MEPP. However, the concentration of bis(7)-tacrine used in the present study was 1,000- to 10-fold lower than those cited previously. These low concentrations of bis(7)-tacrine produced a modest increase in the decay time in MEPPs with respect to other AChE inhibitors. This effect may be related to a direct interaction of bis(7)-tacrine on nicotinic acetylcholine receptor as discussed below.

The increase in the rise time and the rate of rise of the MEPPs recorded on fragments of electric organ of Torpedo treated with bis(7)-tacrine suggests that, in addition to its AChE inhibitory activity, bis(7)-tacrine may affect the time course of exocytosis of a single synaptic vesicle.

Using membranes from the electric organ of Torpedo transplanted in Xenopus oocytes, we tested the effects of tacrine and bis(7)-tacrine on the native form of the nicotinic acetylcholine receptor. Membrane transplantation preserves the activity of native receptors much better than the receptors translated in oocytes from mRNA (Marsal et al. 1995; Morales et al. 1995). The IC50 for ACh-induced currents of bis(7)-tacrine (162 nM) was much lower than previously reported for tacrine (8.9 µM) (Cantí et al. 1998), which indicates that bis(7)-tacrine is a more potent inhibitor of nicotinic acetylcholine receptor than tacrine. Consequently, bis(7)-tacrine needs to recruit more ACh molecules for release by the exocytosis of a single vesicle to increase the size of an MEPP, thus overcoming the higher inhibition of the nicotinic acetylcholine receptor.

The Hill coefficient suggested that bis(7)-tacrine, like tacrine, binds to the nicotinic acetylcholine receptor in a molecular ratio of 1:1. However, in contrast to tacrine (Cantí et al. 1998), the action of bis(7)-tacrine was not sensitive to membrane voltage, suggesting that the drug did not interact preferably with the open-state receptor. According to the records obtained during long periods of ACh perfusion, the receptor desensitized more quickly in the presence of bis(7)-tacrine, suggesting that the drug-receptor complex closed faster than in the absence of the drug. It might be the case that part of this effect is caused for the open channel block by the drug. Our results indicate that the union of the bis(7)-tacrine to the nicotinic acetylcholine receptor must accupy a different position within the alpha -bungarotoxin site, perhaps interacting with the inner part of the receptor. These results contrast with those obtained with tacrine on nicotinic acetylcholine receptor desensitization (Cantí et al. 1998), in which tacrine slowed down receptor desensitization. These results also suggest that bis(7)-tacrine might act as a slowly dissociating antagonist of the nicotinic acetylcholine receptor, whereas tacrine would be a rapidly dissociating antagonist (due to its lower affinity). If the latter were the case, part of the tacrine molecules unbound from the nicotinic acetylcholine receptors would be replaced by ACh molecules, giving the effect of a slowed desensitization (Clements 1996).

In summary, bis(7)-tacrine produced similar effects to those of tacrine on spontaneous synaptic activity, but at concentrations that were 100-1,000 times lower. The action of bis(7)-tacrine on nicotinic acetylcholine receptors differs from that of tacrine. Even if we consider that the cholinergic synapse of the electric organ is not the same as the central nervous cholinergic synapse, our results suggest that bis(7)-tacrine should have beneficial effects on cholinergic function in the CNS at a much lower concentration than tacrine, and the potency of bis(7)-tacrine in proportion with its toxicity may therefore be less toxic in the palliative treatment of Alzheimer's disease patients (Han et al. 2000). On the basis of the present study, the reported biochemical, pharmacological, and behavioral information (Li et al. 1999; Liu et al. 2000; Pang et al. 1996; Wang et al. 1999), and the low-cost synthesis of bis(7)-tacrine (Pang et al. 1996), it appears that bis(7)-tacrine fits closely with the established criteria for an ideal cholinergic drug for the palliative treatment of Alzheimer's disease.


    ACKNOWLEDGMENTS

We thank Dr. J. Dempster for kindly providing "Whole Cell Analysis Program 2.1." We are also indebted to J. Segura, Universitat Politècnica de Catalunya, for writing the Labview software (Quantadat) to record and analyze MEPPs, and Servei d'Assesorament Lingüístic of the University of Barcelona.

This study was supported by Dirección General de Enseñanza Superior e Investigación Científica from the Spanish Government and Comissió Interdepartamental de Recerca i Innovació Tecnològica from the Generalitat de Catalunya.


    FOOTNOTES

Address for reprint requests: C. Solsona, Laboratori de Neurobiologia Cellular i Molecular, Departament de Biologia Cellular i Anatomia Patològica, Facultat de Medicina, Hospital de Bellvitge, Universitat de Barcelona, Campus de Bellvitge, Pavelló de Govern, Feixa Llarga s/n, E-08907 L'Hospitalet de Llobregat, Spain (E-mail: solsona{at}bellvitge.bvg.ub.es).

Received 5 June 2000; accepted in final form 23 March 2001.


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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society




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