Pharmacological Characterization of cGMP Regulation by the Biarylpropylsulfonamide Class of Positive, Allosteric Modulators of -Amino-3-hydroxy-5-methyl-4- isoxazolepropionic Acid Receptors

The biarylpropylsulfonamide class of -amino-3-hydroxy-5methyl-4-isoxazolepropionic acid (AMPA) potentiators represented by N-2-(4-(4-cyanophenol)phenol)propyl-2-propanesulfonamide (LY404187) and (R)-4 -[1-fluoro-1-methyl-2-(propane-2-sulfonylamino)-ethyl]-biphenyl-4-carboxylic acid methylamide (LY503430) are positive, allosteric AMPA receptor activators, which enhance AMPA receptor-mediated neurotransmission by reducing desensitization of the ion channel. Although these compounds have efficacy in in vivo rodent models of cognition, depression, and Parkinson’s disease, little is known about biochemical pathways activated by these agents. Given the well established regulation of the nitric oxide/ cGMP pathway by excitatory neurotransmission, the current study characterized AMPA receptor potentiator-mediated cGMP response in mouse cerebellum. Acute treatment by both LY404187 and LY503430 [2.0, 5.0, or 10 mg/kg subcutaneously (s.c.)] elevated basal cerebellar cGMP levels in a dosedependent manner. Pretreatment with the noncompetitive, allosteric AMPA receptor-selective antagonist 7H-1,3-dioxolo[4,5-h][2,3]benzodiazepine-7-carboxamide, 5-(4-aminophenyl)-8,9-dihydro-N,8-dimethyl-monohydrochloride-(9CI) (GYKI 53655) [3.0 mg/kg intraperitoneally (i.p.)], completely blocked the effect of LY404187, demonstrating that activation of AMPA receptors induces cGMP levels. Interestingly, pretreatment with the N-methyl-D-aspartate (NMDA) open channel blocker dizocilpine (0.3 and 1.0 mg/kg i.p.) also abolished the AMPA receptor potentiator-mediated cGMP accumulation, indicating that activation of AMPA receptors leads to NMDA receptor-mediated transmission involved in cGMP regulation. Pharmacological augmentation of the endogenous glutamate tone via the alkaloid harmaline (20–60 mg/kg i.p.) synergized with AMPA potentiator activity and provided further direct evidence of in vivo allosteric activation of AMPA receptors by LY404187. The synergism between harmaline and LY404187 was specific, since cGMP accumulation induced by foot-shock stress was not augmented by the AMPA receptor potentiator. Taken together, these data indicate that the cGMP system may play an important role in pharmacological efficacy of the biarylpropylsulfonamide class of AMPA receptor potentiators. Glutamate is the major excitatory neurotransmitter in the brain acting at ionotropic and metabotropic receptors. Glutamate controls fast synaptic transmission and also plays a key role in synaptic plasticity (Parsons et al., 2002). Ionotropic glutamate receptors are ligand-gated ion channels that mediate the majority of fast synaptic transmission in the brain. There are three classes of ionotropic glutamate receptors: N-methyl-D-aspartate (NMDA) receptors, -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, and kainite receptors (Hollmann and Heinemann, 1994). Of these, AMPA receptors have received significant attention as a result of their dominant role in direct excitatory neurotransmission and neuromodulation contributing to preand postsynaptic plasticity phenomena. Indeed, the wide range of AMPA receptor pharmacology seems to contribute to the therapeutic potential of AMPA receptor activators for a variety of central nervous system disorders, such as major Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.105734. ABBREVIATIONS: NMDA, N-methyl-D-aspartate; AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; GluR, glutamate receptor; LY404187, N-2-(4-(4-cyanophenol)phenol)propyl-2-propanesulfonamide; LY503430, (R)-4 -[1-fluoro-1-methyl-2-(propane-2-sulfonylamino)-ethyl]-biphenyl-4-carboxylic acid methylamide; GYKI 53655, 7H-1,3-dioxolo[4,5-h][2,3]benzodiazepine-7-carboxamide, 5-(4-aminophenyl)-8,9-dihydro-N,8-dimethyl-monohydrochloride-(9CI); FS, foot-shock; ANOVA, analysis of variance; s.c., subcutaneous; i.p., intraperitoneal; MK-801, dizocilpine maleate. 0022-3565/06/3191-293–298$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 319, No. 1 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 105734/3137643 JPET 319:293–298, 2006 Printed in U.S.A. 293 at A PE T Jornals on Sptem er 7, 2017 jpet.asjournals.org D ow nladed from depression, anxiety disorders, cognition, schizophrenia, and Parkinson’s disease (Danysz, 2002; O’Neill et al., 2004; Black, 2005; Alt et al., 2006). AMPA receptors are cation-selective tetrameric complexes composed of homomeric and heteromeric combinations of subunits termed GluR1, GluR2, GluR3, or GluR4 (Hollmann and Heinemann, 1994). Like other ligand-gated ion channels, AMPA receptors possess multiple allosteric modulatory sites. The positive allosteric modulators do not possess direct agonist activity at the channel but markedly potentiate the effects of the endogenous transmitter glutamate or exogenously applied AMPA, by reducing the desensitization (e.g., cyclothiazide) and/or deactivation (e.g., aniracetam) of the agonist/channel complex (Yamada, 1998). These data suggest that multiple allosteric sites regulate the function of AMPA receptors. This property has been taken advantage of by several research groups who have generated a number of small molecule chemical classes of positive AMPA receptor modulators termed AMPA receptor potentiators or AMPAkines (Arai et al., 1994; Bleakman and Lodge, 1998; Borges and Dingledine, 1998; Arai et al., 2002; Danysz, 2002; Lynch, 2004; O’Neill et al., 2004). Among these, a new class of biarylpropylsulfonamides, represented by LY404187 and LY503430 act as potent and selective AMPA receptor potentiators (Ornstein et al., 2000, Miu et al., 2001; Quirk and Nisenbaum, 2002; Murray et al., 2003). These compounds augment AMPA-mediated neurotransmission by reducing desensitization of the channel and have been shown to have efficacy in a variety of cell-based and in vivo models indicative of potential therapeutic efficacy in cognitive disorders, major depression, and Parkinson’s disease (O’Neill et al., 2004). Despite the well characterized pharmacology of the biarylpropylsulfonamide AMPA receptor potentiators on behavioral endpoints in several rodent models, little is known about the downstream biochemical signaling pathways activated by these agents. The primary objective of the present set of studies was to characterize the activation of synthesis of cGMP in response to the AMPA receptor potentiators. The focus on cGMP stems from its well documented coupling to glutamate-mediated excitatory neurotransmission via NO (Fedele and Raiteri, 1999). Thus, cGMP is a second messenger produced by soluble guanylyl cyclase (Miki et al., 1977), an enzyme directly activated by NO (Bredt and Snyder, 1990; Dinerman et al., 1994). Additionally, the role of cGMP in regulation of excitatory neurotransmission, neuroplasticity phenomena such as long-term potentiation/depression, and neuroprotection is well established (Calabresi et al., 1999; Fedele and Raiteri, 1999; Puzzo et al., 2005). We hypothesized that the antidepressant, cognitive and anti-parkinsonian effects of the AMPA receptor potentiators are mediated, at least in part, by the cGMP signaling cascade. Hence, the objective of the present set of studies was to characterize effects of the AMPA receptor potentiators LY404187 and LY503430 on cGMP accumulation in the mouse cerebellum. Cerebellum was selected since cGMP is the major second messenger in this structure with highest tissue levels among the different brain regions (Wood, 1991) and the interactions between glutamate receptors, NO and cGMP are well established (Fedele and Raiteri, 1999). Using an ex vivo assay for cGMP tissue content, we evaluated three aspects of the pharmacology of the cGMP response induced by the AMPA receptor potentiators: 1) basal effects of AMPA receptor potentiation, 2) assessment of AMPA and NMDA receptor involvement in the AMPA receptor potentiator-induced response, and 3) effects of altering glutamatergic tone on AMPA receptor potentiator-mediated cGMP accumulation. Materials and Methods Drugs. AMPA receptor potentiators LY404187 and LY503430 and the noncompetitive AMPA receptor antagonist GYKI 53655 (Bleakman et al., 1996), representing the 2,3-benzodiazepine class, were synthesized at our institution. Harmaline was obtained from SigmaAldrich (St. Louis, MO). The AMPA receptor potentiators, the AMPA antagonist, and harmaline were dissolved in 12.5% 2-hydroxypropyl-cyclodextran vehicle (Sigma-Aldrich). The noncompetitive NMDA receptor antagonist dizocilpine (dizocilpine maleate, MK-801) was purchased from Alexis Laboratories (San Diego, CA) and was dissolved in sterile distilled H2O. Animals and Treatment. Adult male CF-1 mice (25–30 g) were used in all studies presented in this report. Mice were obtained from Harlan (Indianapolis, IN) and housed under a 12-h light/dark cycle in a temperatureand humidity-controlled environment and acclimated for at least 3 to 5 days before use. Studies described here were approved by the Eli Lilly & Co. Animal Care and Use Committee. Mice received a single s.c. injection of AMPA receptor potentiator LY404187 or LY503430 at 2.0, 5.0, or 10 mg/kg and were sacrificed 30 to 60 min after dosing. To establish that the AMPA receptor potentiators acted specifically via AMPA receptors, mice were treated with the noncompetitive AMPA receptor-selective antagonist GYKI 53655 (3.0 mg/kg i.p.) for 5 min, followed by administration of the AMPA receptor potentiator. To investigate the role of the NMDA receptors, mice were pretreated, with the noncompetitive, selective NMDA open channel blocker dizocilpine (0.1 or 1.0 mg/kg i.p.) for 30 min, followed by AMPA receptor potentiator administration. To assess the effects of endogenous glutamate tone on AMPA receptor potentiator-mediated cGMP accumulation, mice were treated with harmaline (10–60 mg/kg i.p.), an alkaloid that releases glutamate from the climbing fibers, 10 min before AMPA receptor potentiator treatment. As a means to establish the specificity of interactions between the glutamate system and AMPA receptor potentiators, we undertook a nonpharmacologic approach by evaluating whether footshock (FS) stress, a paradigm known to induce cerebellar cGMP response, modulates allosteric AMPA receptor potentiator-mediated cGMP accumulation. Mice were euthanized at 30 min to 1.0 h after the treatment with the AMPA receptor potentiator (as specified in figure legends) using a beam of microwave radiation (microwave fixation system model GA5013; Gerling Applied Engineering, Modesto, CA) focused on the skull for 0.5 s at high power setting. This method was used to preserve tissue cGMP content. All studies were carried out between 9:00 AM and 12:00 PM to reduce the effects of diurnal fluctuations. Tissue Dissection and cGMP Radioimmunoassay. After microwaving, a small piece (10–20 mg) of cerebellar cortex was quickly removed from the skull, tissue weight was recorded, and the tissue was homogenized in 2 ml of 1.0% perchloric acid (Sigma-Aldrich). Tissue homogenate was kept on ice for 30 min and then placed in a boiling water bath for 5 min followed by centrifugation at 11,700g for 20 min. One milliliter of supernatant was then acetylated with 40 l of triethylamine (Sigma-Aldrich) and 20 l of acetic anhydride (Sigma-Aldrich), vortexed, and centrifuged at 13,000g for 20 min at 4°C. Acetylated mouse cerebellum samples were stored at 4°C until cGMP analysis by radioimmunoassay. Cerebellar cGMP content in the acetylated samples was determined using the cGMP I Flash Plate radioimmunoassay (PerkinElmer Life and Analytical Sciences, Boston, MA) on duplicate samples from each animal as per manufacturer’s instructions. 294 Ryder et al. at A PE T Jornals on Sptem er 7, 2017 jpet.asjournals.org D ow nladed from Foot-Shock Stress. For mouse foot-shock stress studies, the Habitest Modular Test System was used (Coulbourn Instruments, Allentown, PA). Mice were subjected to either a 0.5or 1.0-mA foot-shock for 10 or 30 s in the Habitest Operant cage equipped with a modular shock floor using an automated remote timer. As with drug treatments, all foot-shock studies were carried out between 9:00 AM and 12:00 PM to reduce the effects of diurnal fluctuations. Data Presentation and Statistical Analysis. For each animal, cGMP levels were normalized to wet tissue weight and used to compute group averages and S.E.M. All data were analyzed on the log scale to correct problems with heterogeneity of variance. However, the figures represent data in raw picomoles per gram. For Figs. 1, 4, and 5A, statistical analyses were performed using ANOVA followed by Dunnett’s method to compare each treatment group to the vehicle group. For Figs. 2 and 3, statistical analyses were performed using ANOVA followed by multiple comparisons using Bonferroni’s method to compare selected treatment groups to each other. For Fig. 5B, statistical analysis was performed using ANOVA followed by Fisher’s least significant difference method to test for treatment differences from FS alone.