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NEUROPHARMACOLOGY

Competitive Antagonism between the Nicotinic Allosteric Potentiating Ligand Galantamine and Kynurenic Acid at 7* Nicotinic Receptors

Department of Pharmacology and Experimental Therapeutics (C.L., E.F.R.P., P.P., V.N., R.S., E.X.A.), Maryland Psychiatric Research Center (H.-Q.W., R.S.), University of Maryland School of Medicine, Baltimore, Maryland

Received March 19, 2007; accepted April 18, 2007.

	Abstract

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Galantamine, a drug used to treat Alzheimer's disease, is a nicotinic allosteric potentiating ligand, and kynurenic acid (KYNA), a neuroactive metabolite of the kynurenine pathway, is an endogenous noncompetitive inhibitor of 7* nicotinic receptors (nAChRs) [the asterisk next to the nAChR subunit is intended to indicate that the exact subunit composition of the receptor is not known (Pharmacol Rev 51:397–401, 1999)]. Here, possible interactions between KYNA and galantamine at 7* nAChRs were examined in vitro and in vivo. In the presence of tetrodotoxin (TTX), approximately 85% of cultured hippocampal neurons responded to choline (0.3–30 mM) with 7* nAChR-subserved whole-cell (type IA) currents. In the absence of TTX and in the presence of glutamate receptor antagonists, choline triggered inhibitory postsynaptic currents (IPSCs) by activating 7* nAChRs on GABAergic neurons synapsing onto the neurons under study. Galantamine (1–10 µM) potentiated, whereas KYNA (10 nM-1 mM) inhibited, choline-triggered responses. Galantamine (1 µM), applied before KYNA, shifted to the right the concentration-response relationship for KYNA to inhibit type IA currents, increasing the IC₅₀ of KYNA from 13.9 ± 8.3 to 271 ± 131 µM. Galantamine, applied before or after KYNA, antagonized inhibition of choline-triggered IPSCs by KYNA. Local infusion of KYNA (100 nM) in the rat striatum reduced extracellular dopamine levels in vivo. This effect resulted from 7* nAChR inhibition and was blocked by coapplied galantamine (1–5 µM). It is concluded that galantamine competitively antagonizes the actions of KYNA on 7* nAChRs. Reducing 7* nAChR inhibition by endogenous KYNA may be an important determinant of the effectiveness of galantamine in neurological and psychiatric disorders associated with decreased 7* nAChR activity in the brain.

Reduced 7* nAChR function/expression in the brain has been associated with the pathophysiology of catastrophic disorders, including Alzheimer's disease (AD) and schizophrenia (Lindstrom, 2003; Singh et al., 2004). Thus, the 7 nAChR gene is linked to the sensory gating deficit that contributes to attentional deficits in patients with schizophrenia (Freedman et al., 2001), and the degree of 7* nAChR loss correlates well with the magnitude of progressive cognitive decline in mild-to-moderate AD patients (Auld et al., 2002). Therefore, these receptors have become attractive targets for drug development (Dani et al., 2004).

Nicotinic allosteric potentiating ligands (APLs), including galantamine, physostigmine, and codeine, effectively increase 7* nAChR activation at subsaturating agonist concentrations (Pereira et al., 2002). An alkaloid originally isolated from snowdrop flowers, galantamine is currently used to treat mild-to-moderate AD (Corey-Bloom, 2003) and has recently also been tested as an adjuvant therapy to improve cognitive function in schizophrenia (Norén et al., 2006; Schubert et al., 2006). Although galantamine is also a weak reversible cholinesterase inhibitor, its nicotinic APL action seems to be an important determinant of its clinical effectiveness (reviewed in Corey-Bloom, 2003; Maelicke and Albuquerque, 1996; Pereira et al., 2002). Acting primarily as a nicotinic APL, galantamine improves synaptic transmission and decreases neurodegeneration—two effects essential for its cognition-enhancing properties (Santos et al., 2002; Dajas-Bailador et al., 2003; Arias et al., 2004; Kihara et al., 2004; Zhang et al., 2004).

Enhancement of nAChR activity by galantamine results from its interaction with a binding region that is close to but distinct from the agonist-binding region on the extracellular domain of nAChR subunits. The region including and surrounding the Lys-125 residue on the nAChR subunits contains important elements of the APL-binding site on nAChRs (see Pereira et al., 2002 and references therein). Initial molecular modeling studies indicated that the nicotinic APL activity of galantamine, physostigmine, and other alkaloids resides in their aromatic rings, with a tertiary nitrogen that is positively charged at physiological pH located at a fixed distance from a phenolic hydroxyl group (Maelicke and Albuquerque, 1996). The aromatic rings seem to confer the lipophilicity needed for these ligands to access the highly hydrophobic APL-binding region on the nAChRs, whereas the other two pharmacophores may be essential for interactions of the ligands with specific amino acids within the binding pocket.

Kynurenic acid (KYNA), an astrocyte-derived kynurenine pathway metabolite whose levels are elevated in the brain of patients with AD (Baran et al., 1999) and schizophrenia (Schwarcz et al., 2001), is a potent, noncompetitive 7* nAChR antagonist (Hilmas et al., 2001; Alkondon et al., 2004; Rassoulpour et al., 2005; Grilli et al., 2006). This action, which may play a role in the ability of KYNA to disrupt cognitive processes (Shepard et al., 2003; Chess and Bucci, 2006), has been suggested to be mediated by its binding to sites located on the N-terminal domain of the 7 nAChR subunit (Pereira et al., 2002). This raised the intriguing possibility that endogenous KYNA and galantamine may have functionally opposite effects by interacting with the APL site. Therefore, we designed experiments to examine potential interactions between KYNA and galantamine in the APL region of 7* nAChRs. Our results, which were obtained in vitro and in vivo, demonstrate that galantamine, acting as a nicotinic APL, competitively antagonizes the inhibition by KYNA of 7* nAChRs.

	Materials and Methods

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Cultured Hippocampal Neurons. Primary cultures of cells dissociated from the hippocampus of 17 to 19-day-old rat fetuses (Sprague-Dawley; Zivic Laboratories Inc., Pittsburgh, PA) were prepared according to the procedure described elsewhere (Alkondon et al., 1994). Hippocampal cells were plated onto collagen-coated 35-mm Petri dishes. Electrophysiological experiments were performed on neurons cultured for 15 to 30 days.

Electrophysiological Recordings. Recordings were obtained from cultured hippocampal neurons by means of the conventional whole-cell mode or the perforated patch configuration of the patch-clamp technique. The external solution was composed of 165 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 5 mM HEPES, and 10 mM dextrose, pH 7.3 adjusted with 340 mOsm NaOH. In experiments designed to record whole-cell currents, TTX (final concentration, 150 nM) was added to the external solution. IPSCs were recorded from neurons perfused with TTX-free external solution containing the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM), the N-methyl-D-aspartate receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV, 100 µM), and the muscarinic receptor antagonist atropine (1 µM). In conventional whole-cell recordings, the solution used to fill the recording pipettes had 60 mM CsCl, 60 mM CsF, 10 mM EGTA, 10 mM HEPES, 20 mM Tris-phosphocreatine, and 5 mM Tris-ATP, in addition to creatine phosphokinase (50 U/ml), pH 7.3 adjusted with 340 mOsm CsOH (Alkondon et al., 1994). In perforated patch recordings, the pipette solution was composed of 80 mM CsCl, 80 mM CsF, 10 mM EGTA, and 10 mM HEPES, in addition to 10 µg/ml gramicidin-A. Whole-cell currents or IPSCs were induced by U-tube application of choline (0.3–10 mM) alone or in an admixture with KYNA (1 nM-1 mM) and/or galantamine (0.1–100 µM) to the neurons. The U-tube was positioned 50 µm from the neurons. KYNA was also present in the bathing solution at the same concentration as in the U-tube. Unless otherwise stated, galantamine was applied to the neurons only in admixture with the agonist. Currents were recorded using an LM-EPC-7 patch-clamp system (List Electronics, Darmstadt, Germany). Patch pipettes were pulled from borosilicate glass capillaries yielding resistances of 5 M. Signals were filtered at 3 kHz and sampled at 200 µs using the pCLAMP9 software (Molecular Devices, Sunnyvale, CA). No compensation for access resistance was made during the whole-cell recordings. Results obtained from cells in which access resistance changed by >15% were discarded.

Analysis of Electrophysiological Data. Whole-cell currents were analyzed using the Clampfit module of the pCLAMP software (version 9.0). In conventional whole-cell recordings, rundown of the whole-cell current amplitudes triggered by 7* nAChR activation was corrected according to the following procedure. Starting at 5 min after achievement of the G seal, at least 10 pulses of agonist (1-s duration, 0.5–2-min intervals) were applied to the neurons before and after their exposure to KYNA and/or galantamine. During exposure of the neurons to the test compounds, 10 to 20 pulses of agonist-plus-test compound (1-s duration, 0.5–2-min interval) were delivered to the neurons. Amplitudes of currents evoked by the agonist alone before the exposure of the neurons to KYNA and/or galantamine were plotted against recording time, and the resulting graphs were fitted with the exponential function y = a + b x exp(–cx), where y was the current amplitude, x was the recording time, and a was taken as 1. Amplitudes of agonist-evoked currents at any given time were estimated and used to normalize the actual amplitudes of currents recorded in the presence of KYNA and/or galantamine and during washing phases. Concentration-response curves were fitted by the Hill equation: I = (I_max x [A]^nH)/([A]^nH + IC₅₀^nH), where I and I_max are the measured and the maximal current amplitudes, respectively, [A] is the agonist concentration, n_H is the Hill coefficient, and IC₅₀ is the antagonist concentration producing 50% reduction of the response. A correction for rundown was necessary, because in most neurons, it took at least 20 to 30 min for rundown of the choline-evoked currents to be minimized. Given the long duration of the protocols (up to 60 min between drug applications and washouts), waiting for such stabilization of the control responses would make it very difficult to maintain the integrity of the seals and to obtain sufficient reliable data for statistical analysis.

The Mini Analysis software (Synaptosoft, Leonia, NY), which employs a threshold-based event-detection algorithm, was used to analyze pharmacologically isolated IPSCs triggered by application to the neurons of 6-s pulses of choline alone or in admixture with the test compounds. Frequencies and amplitudes of IPSCs were measured during the agonist pulse. Area and amplitude thresholds were set above the noise level and kept constant in each experiment. Events that did not show a typical synaptic waveform were rejected manually. Histograms of IPSC amplitude distributions were made from measurements of amplitude versus number of events. In the histograms, the amplitudes were grouped in 20-pA bins.

In Vivo Microdialysis. Microdialysis was performed as described by Rassoulpour et al. (2005). In brief, adult male Sprague-Dawley rats (200–250 g; Charles River Laboratories, Kingston, NY) were anesthetized with chloral hydrate (360 mg/kg i.p.) and mounted in a stereotaxic frame (David Kopf Instruments, Tujunga, CA). A guide cannula (0.65-mm outer diameter) was positioned over the striatum (coordinates: AP, 1 mm anterior to bregma; L, 2.5 mm from midline; and V, 3.5 mm below the dura) and secured to the skull with anchor screws and acrylic dental cement. The next day, a microdialysis probe (CMA/10, 2-mm long; Carnegie Medicin, Stockholm, Sweden) was inserted through the guide cannula and connected to a microinfusion pump set to a speed of 1 µl/min. Freely moving animals were perfused with Ringer solution containing 144 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO₄, and 1.7 mM CaCl₂, pH 6.7. After establishing a stable baseline, test compounds, including KYNA, galantamine, and -bungarotoxin (-BGT), were applied individually or in admixture to the tissue for 2 h by reverse dialysis. Dialysate samples were collected every 30 min for up to 9 h. Levels of dopamine in 15 µl of microdialysate were determined by high-performance liquid chromatography coupled with electrochemical detection (Rassoulpour et al., 2005). Data were not corrected for recovery from the microdialysis probe. Probe placement was confirmed histologically as described in Wu et al. (1992). Only data collected from rats with proper dialysis probe placement were analyzed.

Drugs. Choline chloride, KYNA (4-hydroxyquinoline-2-carboxylic acid), TTX, CNQX, APV, -BGT, picrotoxin, and gramicidin-A were purchased from Sigma-Aldrich (St. Louis, MO). Galantamine.HBr [(4aS,6R,8aS)-4a,5,9,10,11,12-hexahydro-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-ef][2]benzazepin-6-ol] was a generous gift from Dr. Alfred Maelicke (Galantos Pharma, Inc., Mainz, Germany). For the electrophysiological experiments, stock solutions of all chemicals, with the exception of KYNA and picrotoxin, were prepared using double-distilled water. Stock solutions of KYNA and picrotoxin were prepared using dimethyl sulfoxide. Final test concentrations of any chemical were obtained by diluting the stock solutions in external solution. Whenever needed, appropriate concentrations of dimethyl sulfoxide were added to the external solution used as control. For the microdialysis experiments, KYNA was dissolved directly in Ringer solution.

Molecular Modeling. Molecular modeling studies were conducted to superimpose the structures of galantamine and KYNA by compare/fit options available in the Catalyst 4.11 software (Accelrys, San Diego, CA). Analyses were performed using the software installed on a Silicon Graphic O2 workstation equipped with a 300 MHz Microprocessor without Interlocked Pipeline Stages R5000 processor (128 MB RAM) running the Irix 6.5 operating system (Accelrys). All structures were generated using two-/three-dimensional editor sketcher and minimized to the closest minimum using the CHARMm-like force field implemented in the program. "Undefined" chirality was arbitrarily assigned to asymmetric centers of galantamine, allowing the comparison fit to choose which configuration of the asymmetric carbon atoms was the most appropriate. A stochastic research coupled to a pooling method was applied to generate conformers for each compound by using the "best conformer generation" with a 20 kcal/mol energy cutoff (20 kcal/mol maximum compared with the most stable conformer). While performing the compare/fit for the two compounds, the aromatic atoms of each molecule were selected to superimpose with the "best fit" option.

Statistical Analysis. Results are presented as mean ± S.E.M. Electrophysiological data were assessed by paired Student's t test or ANOVA followed by Dunnett's test for multiple comparisons. Microdialysis data were assessed by repeated measures ANOVA followed by Bonferroni's post hoc test for multiple comparisons.

	Results

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7* nAChRs Mediate Choline-Evoked Whole-Cell Currents Recorded from Cultured Hippocampal Neurons in the Absence of Atropine. Complex interactions have been observed between the muscarinic receptor antagonist atropine and different nAChR subtypes, including 7 nAChRs expressed in heterologous systems (Zwart and Vijverberg, 1997). Yet, most studies of nAChR-mediated responses recorded from hippocampal neurons have included atropine in the extracellular solutions to block muscarinic receptors (Alkondon et al., 1994; Mike et al., 2000; Hilmas et al., 2001; Pereira et al., 2002).

In the present study, cultured hippocampal neurons that were continuously perfused with TTX (150 nM)-containing atropine-free physiological solution responded to choline (0.1–30 mM) with whole-cell currents whose amplitudes increased and decay phases accelerated with increasing choline concentrations (Fig. 1A). To determine the EC₅₀ for choline to evoke these responses, the rundown-corrected amplitudes of currents evoked by 10 mM choline were taken as 100% and used to normalize the amplitudes of currents evoked by any other concentration of choline (0.1, 0.3, 1, 3, or 30 mM) in a particular neuron. The average normalized amplitudes were then plotted against the concentration of choline, and the resulting plot was fitted with the Hill equation, yielding an EC₅₀ of 1.2 ± 0.6 mM and an n_H of 1.0 ± 0.4 (Fig. 1A). The decay phases of whole-cell currents evoked by 1 and 10 mM choline were fitted by single exponential functions with decay-time constants (d) of 640 ± 31 and 153.1 ± 8.9 ms, respectively, at –60 mV (n = 9 neurons).

View larger version (15K): [in this window] [in a new window]   Fig. 1. Characterization of whole-cell currents evoked by application of choline to hippocampal neurons in the absence of atropine. A, concentration-response relationship for choline to evoke whole-cell currents in hippocampal neurons in the absence of atropine. The rundown-corrected amplitudes of currents evoked by 10 mM choline were taken as 100% and used to normalize the amplitudes of currents by any other concentration of choline. Each data point and error bar correspond to the average and S.E.M., respectively, of results obtained from five to six neurons. Inset, sample recordings of whole-cell currents evoked by choline. Horizontal bars on top of the traces represent the duration of the agonist pulses. B, effect of -BGT (100 nM) on whole-cell currents evoked by application of 10 mM choline to hippocampal neurons in the absence of atropine. Data points and error bars correspond to the average and S.E.M., respectively, of results obtained from three neurons. In each cell, the amplitude of the first response was taken as 100% and used to normalize the amplitude of the currents evoked by each subsequent pulse of choline. The horizontal bar indicates the time during which the neurons were perfused with physiological solution containing 100 nM -BGT. Inset, line graph shows the exponential fit of the time course of -BGT-induced inhibition of choline-evoked currents recorded from hippocampal neurons in the absence of atropine. All recordings were obtained from neurons voltage clamped at –60 mV and continuously perfused with physiological solution containing TTX (150 nM).

Galantamine Has a Dual Effect on Whole-Cell Currents Mediated by 7* nAChRs in Cultured Hippocampal Neurons. In the absence of atropine, galantamine had a bimodal effect on choline-induced activation of somatodendritic 7* nAChRs in hippocampal neurons (Fig. 2); i.e., 7* nAChR activation by a subsaturating concentration of choline was enhanced by 1 to 10 µM galantamine and inhibited by 30 µM galantamine. The amplitudes of type IA currents evoked by an admixture of choline (1 mM) and galantamine (1–10 µM) were significantly larger than those induced by choline alone (Fig. 2, A and B). The magnitude of the potentiating effect of any given concentration was the same regardless of whether galantamine was applied to the neurons via the U-tube only (in admixture with the agonist) or via both bath perfusion and U-tube (data not shown). At 1 to 10 µM, galantamine also prolonged the decay phase of choline-evoked currents. For instance, at –60 mV, the d of type IA currents evoked by choline (1 mM) and choline (1 mM)-plus galantamine (1 µM) were 640 ± 31.0 and 705 ± 31.3 ms, respectively (n = 9 neurons, p < 0.001, paired Student's t test). At concentrations 30 µM, galantamine caused a significant reduction in the peak amplitudes of choline-evoked currents (Fig. 2, A and B). Both the potentiating and the inhibitory effects of galantamine were promptly reversed upon washing of the neurons with a galantamine-free physiological solution (Fig. 2A).

View larger version (22K): [in this window] [in a new window]   Fig. 2. Effects of galantamine on choline-evoked type IA currents in cultured hippocampal neurons. A, sample recordings of whole-cell currents evoked by choline (1 mM) before, during, and after exposure of the neurons to different concentrations of galantamine. Galantamine was delivered only via the U-tube in admixture with the agonist. Horizontal bars on top of the traces represent the duration of the agonist pulses. B, concentration-response relationship for galantamine-induced potentiation and inhibition of choline-evoked currents. For any given neuron, the rundown-corrected amplitudes of type IA currents evoked by choline (1 mM, 1-s pulses) were taken as 100% and used to normalize the amplitudes of currents evoked by choline plus galantamine. Graph and error bars represent mean and S.E.M., respectively, of results obtained from three to five neurons. * indicates that galantamine caused a significant change in the amplitude of choline-evoked currents (p < 0.05 according to ANOVA with Dunnett's post hoc test). All recordings were obtained from neurons voltage clamped at –60 mV and continuously perfused with physiological solution containing TTX (150 nM).

To examine the voltage dependence of galantamine-induced potentiation and inhibition of 7* nAChRs, type IA currents were evoked by application of choline (1 mM) alone or in admixture with galantamine (1 or 100 µM) to neurons held at several membrane potentials (Fig. 3A). The extrapolated reversal potential of choline-evoked currents in the absence or in the presence of each test concentration of galantamine was 0 mV. At all membrane potentials, 1 µM galantamine increased the amplitudes of choline (1 mM)-evoked currents by 15% (Fig. 3, A and B). Thus, galantamine-induced potentiation of 7* nAChR activity may result from the interactions of the drug with a receptor site that is not sensitive to the electrical field of the membrane. In contrast, the magnitude of the inhibitory effect of 100 µM galantamine on type IA currents increased as the membrane potentials were made more negative (Fig. 3A). The angular coefficient of the linear regression of the plot of normalized current amplitudes versus membrane potentials revealed that 100 µM galantamine decreased 7* nAChR activity at a rate of 2.4 ± 0.5% per 10-mV step (Fig. 3B). The interaction of the drug with a receptor site that is sensitive to the electrical field of the membrane could explain the voltage sensitivity of galantamine-induced 7* nAChR inhibition.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Voltage dependence of the effects of galantamine on choline-activated type IA currents in hippocampal neurons. A, current-voltage relationships for responses evoked by choline (1 mM) in the absence or presence of galantamine (1 or 100 µM). Under control condition, rundown-corrected amplitudes of choline-evoked currents recorded from neurons voltage clamped at –60 mV were taken as 100% and used to normalize the amplitudes of currents recorded at all membrane potentials. The plot of the normalized current amplitude versus membrane potential was fitted by linear regression. The extrapolated reversal potential was 0 mV. Rundown-corrected amplitudes of type IA currents evoked by choline (1 mM) at any membrane potential were taken as 100% and used to normalize the amplitudes of type IA currents evoked by pulses of choline-plus-galantamine (1 or 100 µM) at that membrane potential. B, plots of the ratio of the amplitudes of currents evoked by pulses of choline-plus-galantamine (1 or 100 µM) and the amplitudes of currents evoked by choline alone versus membrane potential. Data points in each plot were fitted by linear regression. Symbols and error bars represent mean and S.E.M., respectively, of results obtained from three to five neurons. All experiments were performed in the presence of TTX (150 nM).

Doses of galantamine recommended for AD treatment range from 8 to 24 mg/day. Peak plasma concentrations ranging from 0.2 to 3 µM have been detected in healthy human subjects treated, orally or subcutaneously, with a single dose of 10 mg of galantamine (Mihailova et al., 1989; Jann et al., 2002). In rats treated with a dose of galantamine, sufficient to generate a peak plasma concentration of 2 µM, brain concentrations of galantamine peak at around 913 ng/g (Bores et al., 1996) or 2.5 µM (considering 80% brain weight as water and the molecular weight of galantamine as 278). Thus, clinically relevant doses of galantamine are likely to generate brain concentrations of the drug that favor its nicotinic APL actions.

In the Absence of Atropine, KYNA Noncompetitively Inhibits Choline-Evoked Whole-Cell Currents. Exposure of hippocampal neurons to KYNA in the absence of atropine caused a reduction of the peak amplitude of choline-evoked currents (Fig. 4, A and B). Analysis of the concentration-response relationship for KYNA-induced 7* nAChR inhibition revealed an IC₅₀ of 13.9 ± 8.3 µM and an n_H of 0.27 ± 0.06 (Fig. 4B). The effect was not reversed within the time of washing of the neurons with a KYNA-free physiological solution (Fig. 4A).

View larger version (23K): [in this window] [in a new window]   Fig. 4. Galantamine, acting as an APL, prevents KYNA-induced inhibition of type IA currents in cultured hippocampal neurons. A, top row, sample recordings of choline (1 mM)-evoked type IA currents obtained from a hippocampal neuron before its exposure to KYNA (left trace), after 20-min perfusion with KYNA-containing external solution (middle trace), and after 10-min washing with KYNA-free physiological solution (right traces). KYNA was also applied in admixture with the agonist via the U-tube. Bottom row, sample recordings of choline-evoked type IA currents obtained from another hippocampal neuron in the continuous presence of galantamine before its exposure to KYNA (left trace), after 20-min perfusion with a KYNA-containing external solution (middle trace), and after 10-min washing with a KYNA-free physiological solution (right traces). KYNA and galantamine, alone or in association, were delivered to the neurons via U-tube (in admixture with the agonist) and via the bath perfusion. B, concentration-response relationship for KYNA-induced blockade of choline-evoked type IA currents in the presence or absence of galantamine. In the absence of galantamine, the rundown-corrected amplitudes of choline-evoked currents recorded from a given neuron at –60 mV were taken as 100% and used to normalize the amplitudes of currents evoked by choline-plus-KYNA after 10 to 20-min perfusion of that neuron with KYNA-containing physiological solution. In neurons that were first exposed to galantamine and subsequently to KYNA, rundown-corrected amplitudes of currents evoked by choline in the continuous presence of galantamine were taken as 100% and used to normalize the amplitudes of currents evoked by choline-plus-galantamine-plus-KYNA after 10 to 20-min perfusion of the neurons with solution containing galantamine-plus-KYNA. Symbols and error bars represent the mean and S.E.M. of results from three to five neurons. C, concentration-response relationship for choline-evoked currents recorded from hippocampal neurons in the absence (control) and in the presence of KYNA (30 µM). Under control conditions, choline (1-s pulses; 300 µM to 30 mM) was applied to cultured hippocampal neurons. Rundown-corrected amplitudes of currents evoked by 10 mM choline were taken as 100% and used to normalize the amplitudes of currents evoked by other choline concentrations. In another set of experiments, after the control responses evoked by a given concentration of choline were recorded, neurons were exposed for 10 to 15 min to KYNA (30 µM) and tested for their responsiveness to pulses of choline-plus-KYNA. Rundown-corrected amplitudes of currents evoked by a given choline concentration under control condition were then taken as 100% and used to normalize the amplitudes of currents evoked by choline in the presence of KYNA. Symbols and error bars represent mean and S.E.M., respectively, of results obtained from five to six neurons. Inset, double-reciprocal plots of the concentration-response relationships for choline in evoking type IA currents in the absence and presence of KYNA (30 µM). All solutions contained TTX (150 nM). Membrane potential, –60 mV.

Galantamine, Acting as a Nicotinic APL, Competes with KYNA at 7* nAChRs. To examine potential interactions between KYNA and the APL region on 7* nAChRs, we analyzed the effects of galantamine on KYNA-induced inhibition of type IA currents recorded by means of conventional patch clamp from hippocampal neurons. Choline (1 mM)-evoked type IA currents were recorded from neurons in the following sequence of treatments: i) in the absence of test compounds; ii) in the presence of galantamine; and iii) in the presence of galantamine-plus-KYNA. Galantamine and/or KYNA were applied to the neurons both via the U-tube (together with the agonist choline) and the bath solution. The magnitude of the effect of KYNA on the amplitude of type IA currents in the presence of galantamine was smaller than that observed in the absence of the drug (Fig. 4A). Galantamine (1 µM) caused a rightward shift in the concentration-response relationship for KYNA to inhibit type IA currents, increasing the IC₅₀ of KYNA to 271 ± 131 µM without changing the n_H (0.31 ± 0.05; Fig. 4B). These results demonstrated a competitive antagonism between KYNA and galantamine at the 7* nAChR (Fig. 4C).

Galantamine Antagonizes KYNA-Induced Inhibition of Choline-Induced GABA Release from Cultured Hippocampal Neurons. The next set of experiments was designed to analyze the effects of KYNA and galantamine, separately and in association, on IPSCs triggered by activation of 7* nAChRs present on GABAergic neurons synapsing onto whole-cell, voltage-clamped neurons in primary hippocampal cultures. For these experiments, the concentrations of galantamine and KYNA were fixed at 1 and 100 µM, respectively. As depicted in Fig. 4B, the largest reduction by galantamine of the effect of KYNA on type IA currents occurred in neurons that were exposed to 1 µM galantamine and subsequently to 100 µM KYNA. The magnitude of the effect of 100 µM KYNA on type IA currents was approximately 70% smaller in the presence than in the absence of 1 µM galantamine (Fig. 4B).

In the absence of TTX and in the continuous presence of the glutamate receptor antagonists APV (100 µM) and CNQX (10 µM) and of atropine (1 µM), application of choline (300 µM, 6-s pulses) to cultured hippocampal neurons triggered postsynaptic currents (Fig. 5A) that could be blocked by the GABA_A receptor antagonist picrotoxin (100 µM) and by the 7* nAChR antagonist -BGT (100 nM) (data not shown). Therefore, as reported earlier (Hilmas et al., 2001; Pereira et al., 2002), these choline-evoked IPSCs resulted from activation of 7* nAChRs on GABAergic neurons synapsing onto the neurons under study.

View larger version (35K): [in this window] [in a new window]   Fig. 5. Galantamine blocks KYNA-induced inhibition of choline-induced IPSCs in neurons in primary cultures of the rat hippocampus. A, sample recordings of choline (300 µM)-induced IPSCs obtained from cultured neurons at +40 mV in the continuous presence of glutamate receptor antagonists (CNQX, 10 µM; APV, 100 µM). Bars under the traces indicate the time of the agonist pulse application to the neuron. The interval between two pulses was 5 min; during this interval, the cell was perfused with a physiological solution containing galantamine and/or KYNA as indicated. B, the net charge (left graph) and frequency (right graph) of IPSCs evoked by choline alone was taken as 100% and used to normalize the net charge and frequency of IPSCs induced by choline-plus-galantamine or choline-plus-KYNA. Likewise, the net charge and frequency of IPSCs triggered by choline-plus-galantamine (1 µM) was taken as 100% and used to normalize the net charge and frequency of IPSCs triggered by choline-plus-galantamine-plus-KYNA. Asterisks indicate that results obtained in the presence of KYNA and/or galantamine differ significantly from those obtained under corresponding control conditions (*, p < 0.05; **, p < 0.01, according to ANOVA with Dunnett's post hoc test). The effect of KYNA on choline-triggered IPSCs in the absence of galantamine was significantly larger than that observed in the presence of galantamine (p < 0.05 according to ANOVA with Dunnett's post hoc test). Graph and error bars are mean and S.E.M. of results obtained from eight hippocampal neurons.

The magnitude of the effect of KYNA on choline-induced IPSCs was significantly decreased by galantamine. In the absence of galantamine, the magnitude of responses triggered by choline (1 mM)-plus-KYNA (100 µM) was compared with those evoked by choline alone. To offset the potentiating effect of galantamine, the magnitude of responses triggered by choline (1 mM)-plus-galantamine (1 µM)-plus-KYNA (100 µM) was compared with that of responses evoked by choline-plus-galantamine. In the absence and presence of 1 µM galantamine, 100 µM KYNA reduced by approximately 80 and 45%, respectively, the net charge of IPSCs triggered by choline (Fig. 5B). Similar results were obtained from the analysis of the frequency of events (Fig. 5B). Thus, the effect of KYNA on GABA release triggered by 7* nAChR activation was nearly 70% smaller in the presence than in the absence of galantamine.

Neither KYNA nor galantamine caused significant change in the amplitude of choline-triggered IPSCs (Fig. 6). The histogram distribution of the amplitudes of IPSCs induced by choline revealed two major populations; most events had amplitudes <750 pA, and only a few had amplitudes >750 pA. No assumptions were made regarding the transmitter release process, and the histograms were not fit with any specific function. However, the overall distribution of IPSC amplitudes triggered by choline did not seem to be altered by galantamine, KYNA, or the admixture of both (Fig. 6). Potentiation by galantamine and inhibition by KYNA of 7* nAChR activation in GABAergic neurons synapsing onto the neurons under study can explain the effects of each test compound on choline-induced IPSCs.

View larger version (17K): [in this window] [in a new window]   Fig. 6. Histogram distribution of the amplitudes of IPSCs triggered by choline in the presence of galantamine and/or KYNA. Distributions of the amplitudes of all choline (300 µM)-induced IPSCs recorded from hippocampal neurons each under the following experimental conditions: i) control; ii) in the presence of galantamine (1 µM); iii) in the presence of galantamine (1 µM)-plus-KYNA (100 µM); and iv) in the presence of KYNA (100 µM). Neurons were voltage-clamped at +40 mV, and the physiological solutions contained CNQX (10 µM) and APV (100 µM).

Inhibition of choline (300 µM)-induced IPSCs by KYNA was promptly reversed by 5 to 10-min washing of the neurons with a KYNA-free external solution (Fig. 7). This result is in apparent contrast with the finding that KYNA-induced inhibition of choline-evoked whole-cell currents recorded by conventional patch clamp could not be easily reversed within 20 to 30-min washing of the neurons (Fig. 4A). As mentioned above, choline-induced IPSCs result from activation of 7* nAChRs in GABAergic neurons that synapse onto the whole-cell, voltage-clamped neurons. On the other hand, choline-evoked type IA currents result from activation of 7* nAChRs in the somatodendritic region of the whole-cell, voltage-clamped neurons. Thus, loss of diffusible intracellular components in conventional whole-cell patch-clamp recordings could explain the long-lasting effect of KYNA on type IA currents.

View larger version (22K): [in this window] [in a new window]   Fig. 7. KYNA-induced inhibition of choline-evoked IPSCs and choline-evoked whole-cell currents in perforated patches can be promptly reversed upon washing of the neurons with KYNA-free external solution. Graph of normalized choline-evoked responses was recorded from cultured hippocampal neurons before (control), during, and after (wash) their exposure to KYNA. Choline-evoked IPSCs were recorded from hippocampal neurons before, during, and after their exposure to KYNA (100 µM). After recording three control responses, the neurons were perfused for 10 to 20 min with KYNA (100 µM)-containing physiological solution, and choline-plus-KYNA was applied via the U-tube. After a 3-min washing with a KYNA-free external solution, choline alone was applied to the neurons. A similar protocol was used to record choline-evoked whole-cell currents from perforated patches or conventional whole-cell patches. Ten to 20 control whole-cell currents were recorded before the neurons were exposed to KYNA. Furthermore, the washing phase in conventional whole-cell patch-clamp recordings lasted 20 min. Membrane potentials were +40 and –60 mV for IPSC and whole-cell current recordings, respectively. Recordings of IPSCs were obtained in the continuous presence of CNQX (10 µM), APV (100 µM), and atropine (1 µM). Recordings of whole-cell currents were obtained in the presence of TTX (150 nM). The net charge of IPSCs or the amplitude of whole-cell currents triggered by choline before exposure of the neuron to KYNA was taken as 1 and used to normalize the magnitude of the responses recorded in the presence of KYNA and after washing of the neurons with KYNA-free solution. In conventional whole-cell recordings, current amplitudes were corrected for rundown during recording time. Choline-evoked currents recorded from perforated patches did not show any significant rundown. Graph and error bars represent mean and S.E.M., respectively, of results from three to five neurons. **, indicates that results are significantly different from control (p < 0.01 according to paired Student's t test).

To examine whether maintenance of the intracellular content favors the reversibility of KYNA-induced inhibition of type IA currents, choline was applied to hippocampal neurons under the perforated-patch configuration before, during, and after exposure to KYNA (1 µM). The amplitudes of choline-evoked type IA currents recorded from hippocampal neurons under perforated-patch configuration did not show significant rundown. After 2-min exposure of the neurons to 1 µM KYNA, there was an approximate 50% reduction of the amplitudes of choline-evoked whole-cell currents, which was promptly reversed after a 2-min washing of the neurons with KYNA-free physiological solution (Fig. 7).

Functional Interaction between Galantamine and KYNA on 7* nAChR-Mediated Striatal Dopamine Release in Vivo. Application of the 7 nAChR antagonist -BGT (100 nM) by reverse dialysis into the rat striatum caused a substantial and persistent reduction in extracellular dopamine levels (Fig. 8A). Likewise, KYNA (100 nM) reduced the extracellular levels of dopamine in the rat striatum (Fig. 8B) (see Rassoulpour et al., 2005). However, this effect was promptly reversed once; KYNA was removed from the perfusate. Coperfusion with KYNA (100 nM) did not enhance or otherwise influence the effect of -BGT (100 nM; Fig. 8A).

View larger version (14K): [in this window] [in a new window]   Fig. 8. KYNA-induced reduction of extracellular dopamine levels in the rat striatum results from inhibition of 7* nAChRs and is antagonized by galantamine. A, dopamine levels in perfusate samples obtained from the striatum of rats before, during, and after local infusion of -BGT (100 nM) or -BGT-plus-KYNA (100 nM) through the microdialysis probe. B, dopamine levels in perfusate samples obtained from the striatum of rats before, during, and after local infusion of KYNA (100 nM) alone or in association with galantamine (1 or 5 µM). In both graphs, symbols and error bars represent mean and S.E.M., respectively, of results from five animals. Horizontal bars in both graphs indicate the period of local perfusion of test compounds. *, p < 0.05 versus baseline (two-way repeated measures ANOVA with Bonferroni's post hoc test).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The present study demonstrates that galantamine, acting primarily as a nicotinic APL, competitively antagonizes KYNA-induced 7* nAChR inhibition and provides evidence to support the functional relevance of the antagonistic interactions between the two ligands at 7* nAChRs in vivo. These findings suggest a molecular mechanism that may contribute to the therapeutic effectiveness of galantamine in AD and schizophrenia.

Dual Effects of Galantamine on 7* nAChRs. Galantamine had a bimodal effect on somatodendritic 7* nAChRs in hippocampal neurons. At clinically relevant concentrations ( 10 µM) and consistent with an action at the APL site, the drug caused a voltage-independent increase in the amplitude of type IA currents evoked by subsaturating concentrations of choline. At a concentration favoring its nicotinic APL action, galantamine also increased GABA release triggered by 7* nAChR activation. Essentially, galantamine enhanced the net charge of choline-evoked IPSCs by increasing the frequency without affecting the peak amplitude of events. At concentrations 30 µM, galantamine caused a voltage-dependent inhibition of 7* nAChRs that could be explained by its interactions with (a) receptor site(s) sensitive to the electrical field of the membrane.

The enhancement of the amplitude of 7* nAChR-mediated currents observed with increasing agonist concentrations is generally accompanied by acceleration of their decay phase. This phenomenon occurs because the d of type IA currents is proportional to the rate of receptor desensitization, which is accelerated by increasing agonist concentrations, and the rate of channel closure (Mike et al., 2000). Galantamine increases 7* nAChR activity without triggering receptor desensitization, because it enhances the amplitude and prolongs the decay phase of type IA currents evoked by subsaturating agonist concentrations.

In Vitro and in Vivo Inhibition of 7* nAChRs by Physiologically Relevant Concentrations of KYNA. We had previously demonstrated that, in the presence of atropine, KYNA is a noncompetitive antagonist at 7* nAChRs in hippocampal neurons (Hilmas et al., 2001). Reports that complex interactions occur between atropine and different nAChR subtypes, including 7 nAChRs in heterologous systems (Zwart and Vijverberg, 1997), now led us to examine the effects of KYNA on 7* nAChRs in hippocampal neurons in the absence of atropine. Concentration dependently and noncompetitively, KYNA inhibited choline-evoked whole-cell currents. The IC₅₀ for KYNA to inhibit 7* nAChRs in these experiments was found to be similar to that reported earlier in the presence of atropine (Hilmas et al., 2001). Furthermore, as also observed previously in the presence of atropine, the concentration-response relationship for KYNA-induced inhibition of choline-evoked currents was shallower than expected for a simple mass interaction with a single site. The nH of 0.3 suggested negative cooperativity between the binding sites for KYNA on the nAChRs. The slow onset and long-lasting inhibition by KYNA of 7* nAChR-mediated whole-cell currents is not due to an intracellular action of the metabolite, because, as demonstrated earlier (Hilmas et al., 2001), KYNA added to the recording pipette solution is devoid of any significant effect on type IA currents.

KYNA also inhibited GABA release triggered by 7* nAChR activation in hippocampal cultures. Inhibition by KYNA of choline-induced IPSCs, but not type IA currents in conventional patch-clamp recordings was promptly reversed upon washing of the neurons with a KYNA-free physiological solution. The finding that KYNA-induced inhibition of choline-evoked whole-cell currents in perforated patches could be reversed after washing of the neurons supported the concept that loss of diffusible intracellular components contributes to the long-lasting effect of KYNA on type IA currents in conventional patch-clamp recordings. Diffusion of ATP-regenerating compounds in conventional patch-clamp recordings is known to stabilize a nonconducting state of the 7* nAChRs in hippocampal neurons (Alkondon et al., 1994), and small ligands are known to interact differently with the various conformational states of muscle nAChRs (Krauss et al., 2000). Thus, one could speculate that the interactions of KYNA with its sites on 7* nAChRs are dependent on the conformational state of the receptors. The apparent discrepancy between our previous report that KYNA-induced inhibition of type IA currents in hippocampal neurons could be reversed within 8–10 min (Hilmas et al., 2001) and the present findings could be accounted for by the fact that rates of exchange between the intracellular milieu and the pipette contents are largely controlled by the overall shape of the recording pipettes (Hume and Leblanc, 1988).

View larger version (24K): [in this window] [in a new window]   Fig. 9. Superimposition of the lowest energy conformers of galantamine and KYNA showing the best fit. Left, superimposed structures of the lowest energy conformers with color-coded atoms (red, oxygen; blue, nitrogen; gray, hydrogen; black, carbon). Right, the structures of galantamine and KYNA are shown in blue and green, respectively.

Galantamine, as a Nicotinic APL, Competitively Antagonizes KYNA-Induced Inhibition of 7* nAChRs. Acting as a nicotinic APL, galantamine decreased the effect of KYNA on choline-evoked whole-cell currents and on choline-induced IPSCs. The finding that galantamine (1 µM) shifted to the right the concentration-response relationship for KYNA to inhibit type IA currents, increasing the average IC₅₀ of KYNA from 13 to 230 µM, demonstrates that galantamine and KYNA interact competitively at 7* nAChRs. It is, thus, tempting to speculate that the nicotinic APL binding region contains important elements for KYNA recognition by 7* nAChRs.

Superimposition of the lowest energy conformers of galantamine and KYNA revealed a very good fit (Fig. 9) and shed some light on structural differences that could explain the opposite actions that result from the interactions of the two compounds with the APL-binding region on 7* nAChRs. Like galantamine, KYNA has an aromatic ring with a phenolic hydroxyl group. This group, which bears the same spatial orientation as the phenol group in galantamine, is located at a fixed distance from a pyridinic nitrogen. However, this nitrogen is largely un-ionized at physiological pH (Stone, 1993) and is at a shorter distance from the phenolic group than the tertiary nitrogen is from the corresponding phenolic group in galantamine.

The previous report that 7-chloro-KYNA does not inhibit 7* nAChRs (Hilmas et al., 2001) suggests that the carboxyl group contributes to interactions of KYNA with specific residues in the APL-binding region. The introduction of the electron-withdrawing chlorine in position 7 of the phenolic ring creates a dipole in the molecule that can weaken its potential interactions with positively charged residues in the APL region. The nAChR 7 subunit is the only mammalian nAChR subunit that has a positively charged residue within the segment 118–140 of the putative APL-binding region. It is, therefore, tempting to speculate that the selectivity of KYNA for 7* nAChRs is encoded in the carboxyl group in position 2 of the pyridine ring.

The finding that galantamine, acting primarily as a nicotinic APL, caused a concentration-dependent reduction of the effect of exogenously applied KYNA on extracellular levels of dopamine in the striatum is in line with the competitive interactions between the ligands at 7* nAChRs. These interactions could have increased functional significance in humans, because physiological levels of KYNA in the human brain range from 0.1–1.5 µM (Turski et al., 1988; Ogawa et al., 1992). Thus, the antagonism between galantamine and KYNA at 7* nAChRs is likely to be therapeutically relevant.

Clinical Considerations. Levels of KYNA are increased in the brain of patients with schizophrenia and AD (Baran et al., 1999; Schwarcz et al., 2001), whereas the expression/function of different nAChR subtypes, including 7* nAChRs, is decreased in specific areas of the brain of these patients (Nordberg, 2001; Leonard et al., 2002). Furthermore, 7* nAChR antagonists and KYNA impair memory, learning and sensory gating (Luntz-Leybman et al., 1992; Shepard et al., 2003; Chess and Bucci, 2006; Levin et al., 2006). Thereby, increased 7* nAChR inhibition by elevated brain levels of KYNA may contribute to the cognitive impairments observed in patients with AD and schizophrenia and to the sensory gating deficits seen in individuals with schizophrenia.

Drugs currently approved to treat mild-to-moderate AD, including galantamine, donepezil, and rivastigmine, all inhibit acetylcholinesterase, the enzyme that hydrolyzes acetylcholine (Tariot, 2006). Galantamine is unique in that it also acts as a nicotinic APL. Recently, these drugs have been evaluated as adjuvant therapies to decrease the cognitive impairment and negative symptoms of patients with schizophrenia. Data are still sparse and, so far, derived from small samples in open uncontrolled studies. However, a small randomized, double-blind trial showed positive outcomes when galantamine was administered as an add-on therapy to antipsychotics (Schubert et al., 2006). To date, no positive outcomes have been observed with donepezil or rivastigmine (Mazeh et al., 2006; Sharma et al., 2006). In light of the present results, we conclude that the antagonism of KYNA-induced inhibition of 7* nAChRs may be causally related to the effectiveness of galantamine in schizophrenia and AD.

	Acknowledgements

 
We are indebted to Mabel A. Zelle for technical assistance.

	Footnotes

 
This work was supported by the United States Public Health Service Grant NS25296. Part of this work was presented: Lopes C, Pereira EFR, Schwarcz R, Burt DR, and Albuquerque EX (2006) Interactions between galantamine and kynurenic acid on 7 nicotinic receptors in hippocampal and striatal neurons. 2006 Annual Meeting of the Society for Neuroscience; 2006 Oct 14–18; Atlanta, GA. Program number 524.4/C67. Society for Neuroscience, Washington, DC.

doi:10,1124/jpet.107.123109

ABBREVIATIONS: AD, Alzheimer's disease; -BGT, -bungarotoxin; APL, allosteric potentiating ligand; APV, DL-2-amino-5-phosphonovaleric acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; IPSCs, inhibitory postsynaptic currents; KYNA, kynurenic acid; MLA, methyllycaconitine; nAChRs, nicotinic receptors; d, decay-time constant; TTX, tetrodotoxin; ANOVA, analysis of variance.

¹ The asterisk next to the nAChR subunit is intended to indicate that the exact subunit composition of the receptor is not known (Lukas et al., 1999).

Address correspondence to: Dr. Edson X. Albuquerque; 655 W. Baltimore St.; Baltimore, MD, 21201. E-mail: ealbuque{at}umaryland.edu

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