Effects of 5 -Alkyl-Benzothiadiazides on (R,S)- -Amino-3- hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Biophysics and Synaptic Responses

Alkyl-substituted benzothiadiazides (BTDs) were tested for their effects on (R,S)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors. In excised patches, the 5 -ethyl derivative “D1” blocked the desensitization of AMPA receptor currents during prolonged application of glutamate (EC50, 36 M), and it slowed deactivation of responses elicited by 1-ms glutamate pulses greater than 10fold. [H]Fluorowillardiine binding to rat synaptic membranes was increased by D1 by a factor of 3.6 (EC50, 17 M) with a Hill coefficient near 2. In hippocampal slices, the compound reversibly increased excitatory postsynaptic currents and field excitatory postsynaptic potentials (EPSPs) with thresholds around 10 M. The size of the alkyl substituent influenced both the potency and nature of the drug effect on synaptic currents: 5 -methyl compounds had a 2-fold greater effect on response amplitude than on response duration, whereas 5 -ethyl compounds like D1 caused greater increases in duration than amplitude. In tests with recombinantly expressed AMPA receptor subunits, D1 preferred the glutamate receptor (GluR) subunit GluR4 flip (0.64 M) over GluR4 flop (5.3 M); similar affinities but with smaller flip-flop differences were obtained for GluR1 through 3. These results show that D1 and congeners are significantly more potent than the parent compound IDRA-21 and that they differ in two fundamental aspects from cyclothiazide, the most widely studied BTD: 1) D1 markedly increases the agonist affinity of AMPA receptors and 2) it has immediate and large effects on field EPSPs. The large gain in potency conferred by alkyl substitution suggests that the 5 substituent is in intimate contact with the receptor, with the size of the substituent determining the way in which receptor kinetics is changed. AMPA-type glutamate receptors are abundant throughout the brain and account for much of the transmission occurring at excitatory synapses. Ito et al. (1990) made the seminal observation that the current through these receptors can be enhanced by the nootropic compound aniracetam. The drug did not influence other types of glutamate receptors, and it had no evident effect on AMPA receptors in the absence of glutamate. Other compounds were subsequently discovered that “up-modulate” or “potentiate” AMPA receptor function in a similar manner, including diazoxide (Yamada and Rothman, 1992), cyclothiazide (Yamada and Tang, 1993), IDRA-21 (Bertolino et al., 1993), and PEPA (Sekiguchi et al., 1997). Using the structural leads given by these compounds, several laboratories have developed entire families of potent AMPA receptor modulators beginning with the ampakines (Arai et al., 1994, 1996b,c, 2000; Staubli et al., 1994a,b) and including the pyridothiadiazines (Pirotte et al., 1998) and the biarylpropylsulfonamides (Ornstein et al., 2000). The interest in these compounds has been fostered in part by the possibility that some neurological disorders, such as age-related memory impairment, schizophrenia, and perhaps depression, may be associated with lower than normal excitatory transmission in some brain regions (Masliah et al., 1993; Tamminga, 1998). If so, upmodulation of AMPA receptors could potentially be of therThis work was supported by grants from the Central Research Committee of Southern Illinois University (201-08; to A.C.A.), the Air Force Office of Scientific Research (98-1-03317; to G.L.), and the National Institutes of Health (NS41020 to A.C.A. and NS27600 to R.C.), and by a Graduate Research Fellowship from DuPont Pharmaceuticals (to D.P.). ABBREVIATIONS: AMPA, (R,S)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; FW, fluorowillardiine; GYKI, GYKI 52466 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride]; CNQX, 6-cyano-7-nitro-quinoxaline-2,3-dione; SCN , thiocyanate; DMSO, dimethyl sulfoxide; BTD, benzothiadiazide; EPSP, excitatory postsynaptic potential; EPSC, excitatory postsynaptic current; HEK, human embryonic kidney; IDRA-21, 7-chloro-3-methyl-3–4-dihydro-2H-1,2,4 benzothiadiazine (S,S)-dioxide; PEPA, 4-[2-(phenylsulfonylamino)ethylthio]2,6-difluoro-phenoxyacetamide; ACSF, artificial cerebrospinal fluid; GluR, glutamate receptor; KSCN, potassium thiocyanate; S18986, (S)-2,3dihydro-[3,4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide. 0026-895X/02/6203-566–577$7.00 MOLECULAR PHARMACOLOGY Vol. 62, No. 3 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1537/1001850 Mol Pharmacol 62:566–577, 2002 Printed in U.S.A. 566 at A PE T Jornals on A ril 0, 2016 m oharm .aspeurnals.org D ow nladed from apeutic value. Experimental evidence to support this has been obtained in behavioral studies with ampakines (e.g., Granger et al., 1993, 1996; Staubli et al., 1994b; Larson et al., 1996) and recently also with other modulators (Zivkovic et al., 1995; Lebrun et al., 2000; Li et al., 2001). Several of these compounds have been shown to cross the blood-brain barrier to increase excitatory responses in vivo (Staubli et al., 1994a; Vandergriff et al., 2001), and their ability to facilitate long-term synaptic potentiation (Arai and Lynch, 1992, 1996a; Staubli et al., 1994a) has been suggested to account for the observed improvements in various memory tests. The drugs may have similar actions in humans according to preliminary clinical tests (Lynch et al., 1996; Goff et al., 2001). Most current AMPA receptor modulators belong to one of two structural families, referred to as benzamides (aniracetam and ampakines) and benzothiadiazides (BTDs). PEPA and the more recent biarylpropylsulfonamides fall outside these two categories but share elements with the latter. Behavioral tests have largely involved the benzamides because the first modulators in the BTD family (diazoxide and cyclothiazide) have clinically important peripheral effects and only weakly affect field EPSPs in hippocampal slices, even at concentrations far above their affinities for the AMPA receptor (Larson et al., 1994; Arai and Lynch, 1998; Hjelmstad et al., 1999). Some modulators have substantial effects on extracellular synaptic responses that align well with their affinities for AMPA receptors, whereas others do not; this is thought to be related to how the compounds affect receptor kinetics. Diverse experiments indicate that cyclothiazide acts mainly on desensitization (Johansen et al., 1995; Partin et al., 1996), whereas ampakines also have a strong, even primary, influence on channel gating (Arai and Lynch, 1998). This would explain the observed differences in the effects of the modulators if, as has been argued, the latter process plays a larger role than the former in shaping the size and waveform of undisturbed synaptic responses. IDRA-21, which does not have the peripheral side effects associated with diazoxide and cyclothiazide and unlike the latter rapidly increases synaptic potentials (Arai et al., 1996a), was the first benzothiadiazide examined in behavioral tests (Zivkovic et al., 1995). However, IDRA-21 has modest potency, with concentrations above 200 M typically needed to enhance excitatory transmission in hippocampal slices (Arai et al., 1996a). Our efforts as well as those of others (Desos et al., 1996; Pirotte et al., 1998) have therefore been directed at finding analogs that have higher potency yet maintain effect profiles that are suitable for behavioral applications. In the present project, we systematically modified substituents at various locations around the benzothiadiazide core of IDRA-21 and measured the effects on various aspects of AMPA receptor operation. Some of the modifications resulted in compounds that have much greater potency than IDRA-21 and have effects on AMPA receptor kinetics that differ radically from those of cyclothiazide. A comprehensive description of the compounds that were synthesized and examined in this study is given elsewhere (Phillips et al., 2002), and some data have been presented in abstract form (Arai et al., 1999). Materials and Methods AMPA Receptor Currents in Excised Patches. Patch-clamp studies were carried out with outside-out patches excised from pyramidal neurons in field CA1 of organotypic hippocampal slices (Arai et al., 1996c, 2000). The slice cultures were prepared from 13 to 14-day-old Sprague-Dawley rats and grown for 2 weeks on cellulose membrane inserts (Millipore CM; Millipore Corp., Bedford, MA). Patches were excised in a medium containing 125 mM NaCl, 2.5 mM KCl, 1.25 mM KH2PO4, 2 mM CaCl2, 1 mM MgCl2, 5 mM NaHCO3, 25 mM D-glucose, and 20 mM HEPES, pH 7.3, and relocated to a chamber perfused with recording medium containing 130 mM NaCl, 3.5 mM KCl, 20 mM HEPES, 0.01 mM dizocilpine maleate, and 0.05 mM D-2-amino-5-phosphonopentanoic acid. Patch pipettes had a resistance of 3 to 8 M and were filled with a solution of 65 mM CsF, 65 mM CsCl, 10 mM EGTA, 2 mM MgCl2, 2 mM ATP disodium salt, and 10 mM HEPES, pH 7.3. A piezo device was employed to switch solutions applied to the patch within a fraction of a millisecond (Arai et al., 1996b). In brief, background medium and agonist containing medium were flowing continuously through two lines of a double pipette that was moved by a piezo device across a distance of 50 m in 0.4 ms (Arai et al., 1996b,c). Data were acquired with a patch amplifier (AxoPatch-1D; Axon Instruments, Inc., Foster City, CA) at a filter frequency of 5 kHz and digitized at 10 kHz with PClamp/ Digidata 1200 (Axon Instruments, Inc.). The holding potential was 50 mV. The drugs were applied at the same concentration in both background and glutamate lines, and background flow lines were switched at least 15 s before applying the first glutamate pulse. Typically, five responses were collected and averaged for each condition. Measurement with each patch was alternated repeatedly between control (A: glutamate alone) and test conditions (B: glutamate drug). For data analysis, response B was compared with the average of the responses A taken before and after response B, and peak and steady-state currents recorded in the presence of drug were normalized to those without drug. Deactivation rates were determined by fitting the decay phase of the response to a 1-ms glutamate pulse (10 mM) with a single or double exponential function. Drug solutions were prepared from 1000-fold stock solutions in dimethyl sulfoxide (DMSO); the same final concentrations of DMSO (maximum, 0.1%) were included in all drug and control solutions. Whole-Cell Recording from Pyramidal Cells in Field CA1 of Hippocampal Slices. Male Sprague-Dawley rats of postnatal day 15 to 21 (Harlan, Indianapolis, IN) were decapitated under anesthesia following National Institutes of Health guidelines and an institutionally approved protocol. Transverse hippocampal slices (400 m) were prepared using a vibratome (Leica Microsystems, Deerfield, IL). The slices were submerged in oxygenated artificial cerebrospinal fluid (ACSF) infused at 0.5 ml/min. The experiments were carried out at ambient temperature. The ACSF contained 124 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 2 mM CaCl2, 1 mM MgSO4, 5 mM NaHCO3, 10 mM glucose, and 10 mM HEPES, pH 7.4. The intrapipette solution contained 130 mM CsF, 10 mM EGTA/K, 2 mM ATP disodium salt, 2 mM MgCl2, and 10 mM HEPES, pH 7.4. Pyramidal cells were visualized with an infrared microscope (BXI50; Olympus, Tokyo, Japan) with differential interference contrast configuration. Synaptic responses were recorded using borosilicate glass electrodes (2–5 M ) in response to activation of Schaffer-commissural fibers stimulated by a bipolar nichrome electrode in stratum radiatum. After establishing a stable baseline, the perfusion line was switched to one containing the drug; solution exchange in the recording chamber was complete within 3 min. EPSCs were recorded with AxoPatch 200B and digitized at 10 kHz with Digidata1200/PClamp 7. The holding potential was 70 mV, and the signals were filtered at 5 kHz. Recordings were discarded if the input resistance varied by greater than 10% over the course of the experiment. Whole-Cell Recordings from HEK 293 Cells. Patch-clamp recordings were carried out in whole-cell configuration from human embryonic kidney (HEK) 293 cells that stably express homomeric 5 -Alkyl-Benzothiadiazide Effects on AMPA Receptors 567 at A PE T Jornals on A ril 0, 2016 m oharm .aspeurnals.org D ow nladed from AMPA receptors consisting of GluR3 flop subunits (see Hennegriff et al., 1997). Recordings were made at room temperature in serum-free minimal essential medium (Invitrogen, Carlsbad, CA). Patch pipettes had a resistance of 3 to 7 M and were filled with 130 mM CsF, 10 mM EGTA, 2 mM MgCl2, 2 mM ATP disodium salt, and 10 mM HEPES (pH 7.4). The holding potential typically was 100 mV. Agonist was applied with a fast solution switch system in which cells are exposed to a constant flow of the background solution that is momentarily interrupted during application of glutamate. The drugs were included in both background and agonist lines. Extracellular Recording in Hippocampal Slices. Transverse hippocampal slices (400 m) were prepared as described elsewhere (Arai et al., 1996c) and placed in an interface chamber, which was perfused at 0.5 ml/min with oxygenated ACSF containing 124 mM NaCl, 3 mM KCl, 1.25 mM KH2PO4, 3.4 mM CaCl2, 2.5 mM MgSO4, 26 mM NaHCO3, and 10 mM D-glucose and exposed to humidified 95% O2/5% CO2. Field EPSPs were recorded from the stratum radiatum in response to activation of Schaffer-commissural fibers in the same stratum. The input-output relation of the synaptic response was first established to determine the maximum EPSP amplitude without spike component, and the stimulation intensity was adjusted to 50% of the maximum EPSP amplitude. After establishing a stable baseline, the perfusion line was switched to one containing the