Regulator of G Protein Signaling Protein Suppression of Gα_o Protein-Mediated α_2A Adrenergic Receptor Inhibition of Mouse Hippocampal CA3 Epileptiform Activity

Activation of G protein-coupled 2 adrenergic receptors (ARs) inhibits epileptiform activity in the hippocampal CA3 region. The specific mechanism underlying this action is unclear. This study investigated which subtype(s) of 2ARs and G proteins (G o or G i) are involved in this response using recordings of mouse hippocampal CA3 epileptiform bursts. Application of epinephrine (EPI) or norepinephrine (NE) reduced the frequency of bursts in a concentration-dependent manner: ( )EPI ( )NE ( )NE. To identify the 2AR subtype involved, equilibrium dissociation constants (pKb) were determined for the selective AR antagonists atipamezole (8.79), rauwolscine (7.75), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride (WB-4101; 6.87), and prazosin (5.71). Calculated pKb values correlated best with affinities determined previously for the mouse 2AAR subtype (r 0.98, slope 1.07). Furthermore, the inhibitory effects of EPI were lost in hippocampal slices from 2AARbut not 2CAR-knockout mice. Pretreatment with pertussis toxin also reduced the EPImediated inhibition of epileptiform bursts. Finally, using knock-in mice with point mutations that disrupt regulator of G protein signaling (RGS) binding to G subunits to enhance signaling by that G protein, the EPI-mediated inhibition of bursts was significantly more potent in slices from RGS-insensitive G o G184S heterozygous (G o /GS) mice compared with either G i2 G184S heterozygous (G i2 /GS) or control mice (EC50 2.5 versus 19 and 23 nM, respectively). Together, these findings indicate that the inhibitory effect of EPI on hippocampal CA3 epileptiform activity uses an 2AAR/G o protein-mediated pathway under strong inhibitory control by RGS proteins. This suggests a possible role for RGS inhibitors or selective 2AAR agonists as a novel antiepileptic drug therapy. The noradrenergic system modulates many physiological and pathological processes within the central nervous system (CNS). Noradrenergic neurons regulate attention and arousal, sleep, and learning and memory (Pupo and Minneman, 2001) and seem to attenuate epileptic activity (Giorgi et al., 2004). The hippocampus receives substantial noradrenergic innervation in all regions, including the cornu ammonis 3 (CA3), a region essential for many cognitive functions such as spatial pattern recognition, novelty detection, and shortterm memory (Kesner et al., 2004). The CA3 region possesses a dense recurrent network of excitatory axons between the pyramidal neurons that may be crucial for performing these This work was supported by the North Dakota Experimental Program to Stimulate Competitive Research through the National Science Foundation (NSF) [Grant EPS-0447679]; NSF Faculty Early Career Development Award [Grant 0347259]; NSF Research Experience for Undergraduates Site [Grant 0639227]; NSF Research Experience for Teachers [Grant 0639227]; National Institutes of Health National Institute on Drug Abuse [Grant 5-R01DA17963]; National Institutes of Health National Institute of General Medical Sciences [Grant 5-R01-GM039561]; and National Institutes of Health National Center for Research Resources INBRE Program [Grant P20-RR016741]. Preliminary reports of these findings were presented at the 2007 annual meeting of the American Society for Biochemistry and Molecular Biology (ASBMB) Northwest Regional Undergraduate Affiliate Network; 2007 October 26–27; Moorhead, MN; and the 2008 annual meetings of the ASBMB and the American Society for Pharmacology and Experimental Therapeutics, 2008 April 5–9, San Diego, CA. B.L.G. and B.W.N. contributed equally to this work. 1 Current affiliation: Schroeder Middle School, Grand Forks, North Dakota. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.108.054296. ABBREVIATIONS: CNS, central nervous system; ACSF, artificial cerebral spinal fluid; AR, adrenergic receptor; CA3, cornu ammonis 3; EPI, epinephrine; GPCR, G-protein coupled receptor; KO, knockout; NE, norepinephrine; RGS, regulator of G-protein signaling; WB-4101, 2-(2,6dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride; WT, wild type; PTX, pertussis toxin; JP-1302, N-[4-(4-methyl-1-piperazinyl)phenyl]-9-acridinamine dihydrochloride. 0026-895X/09/7505-1222–1230$20.00 MOLECULAR PHARMACOLOGY Vol. 75, No. 5 Copyright © 2009 The American Society for Pharmacology and Experimental Therapeutics 54296/3464534 Mol Pharmacol 75:1222–1230, 2009 Printed in U.S.A. 1222 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from cognitive functions but also makes the region vulnerable to overexcitation (Schwartzkroin, 1986). This region has one of the lowest seizure thresholds and is often involved in temporal lobe epilepsy, the most common human epileptic syndrome. It is clear that thoroughly delineating the inhibitory and excitatory aspects of this region is critical to understanding CNS function and dysfunction and to designing targeted therapeutic approaches. Norepinephrine (NE) is the major neurotransmitter released by noradrenergic neurons and modulates several CA3 processes. NE has been shown to facilitate long-term potentiation, which is involved in memory formation, and antiepileptic activity (Giorgi et al., 2004) in the hippocampal CA3 region. Increased NE release in the brain has been shown to inhibit epileptiform activity, whereas reduced NE levels seem to increase seizure susceptibility (Weinshenker and Szot, 2002). Although the mechanism by which NE mediates these effects is still unclear, NE may both potentiate memory and inhibit the overexcitation associated with seizures (Jurgens et al., 2005) through the distinct and diverse expression of postsynaptic receptor subtypes (Hillman et al., 2005). Adrenergic receptors (ARs) are divided into three major classes, each of which has a unique G protein pairing resulting in diverse physiological actions (Pupo and Minneman, 2001). Studies have suggested that ARs mediate the enhancement of long-term potentiation (Hopkins and Johnston, 1988) and memory (Devauges and Sara, 1991), whereas the antiepileptogenic actions of NE may involve 2AR activation (Giorgi et al., 2004). Pharmacological and molecular cloning studies have revealed the existence of three 2AR subtypes denoted 2A, 2B, and 2C (Bylund et al., 1994). We recently showed that NE inhibits rat hippocampal CA3 epileptiform bursts through 2AAR activation (Jurgens et al., 2007). Furthermore, specific activation of 2AARs attenuates seizures in mice elicited by chemoconvulsants (Szot et al., 2004). ARs are part of a large and diverse family of GTP-binding (G) protein-coupled receptors (GPCRs). The extracellular signals received by GPCRs are relayed by heterotrimeric G proteins (G ) to effector enzymes and channels within the cell (Gilman, 1987). The conversion of GDP-bound inactive G heterotrimer into activated G -GTP and Gsubunits is achieved by catalyzing nucleotide exchange on G subunits via GPCR activation. Once released, the subunits interact with a variety of downstream effectors in an intracellular signaling cascade (Offermanns, 2003). Deactivation of the G protein is achieved by hydrolysis of the G -bound GTP, a step that controls the duration of the signal. The GDP-bound G subunit will then reform with the Gheterodimer, forming an inactive trimer once again. For some G families (Gi/o and Gq), the rate of GTP hydrolysis can be enhanced by regulator of G protein signaling (RGS) proteins (Berman et al., 1996; Watson et al., 1996). Consequently, RGS proteins are negative modulators of signaling through receptors coupled to the Gi/o and Gq family of G proteins (Clark et al., 2008) and enhance intrinsic GTPase activity of the GTP-bound G subunits. This GTPase acceleration attenuates G protein signaling by resetting the G subunit to its inactive conformation (Hollinger and Hepler, 2002). Interfering with the activity of RGS proteins allows the G subunit to remain active for a longer time, effectively enhancing the signal (Lan et al., 1998; Clark et al., 2003). Therapeutic agents targeting RGS proteins could be used to enhance the effect of current GPCR-mediated drug therapies by reducing the required therapeutic dose while increasing the regional agonist specificity, thereby decreasing the possibility of side effects (Zhong and Neubig, 2001; Neubig and Siderovski, 2002). This study investigated the role of 2ARs and RGS proteins in the antiepileptic actions of NE using field recordings of hippocampal CA3 epileptiform burst activity and a combination of selective blockers for the AR and G protein subtypes, transgenic 2AR knockout, and RGS-insensitive G subunit knock-in mice. Delineating which 2AR and G protein subtypes are involved in attenuating hippocampal epileptiform activity will help to further elucidate the mechanism by which NE inhibits epileptogenesis and may suggest potential targets for antiepileptic drug therapy. Materials and Methods