Regulator of G protein signaling protein suppression of Galphao protein-mediated alpha2A adrenergic receptor inhibition of mouse hippocampal CA3 epileptiform activity.
ABSTRACT Activation of G protein-coupled alpha(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 alpha(2)ARs and G proteins (Galpha(o) or Galpha(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 alpha(2)AR subtype involved, equilibrium dissociation constants (pK(b)) were determined for the selective alphaAR 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 pK(b) values correlated best with affinities determined previously for the mouse alpha(2A)AR subtype (r = 0.98, slope = 1.07). Furthermore, the inhibitory effects of EPI were lost in hippocampal slices from alpha(2A)AR-but not alpha(2C)AR-knockout mice. Pretreatment with pertussis toxin also reduced the EPI-mediated inhibition of epileptiform bursts. Finally, using knock-in mice with point mutations that disrupt regulator of G protein signaling (RGS) binding to Galpha subunits to enhance signaling by that G protein, the EPI-mediated inhibition of bursts was significantly more potent in slices from RGS-insensitive Galpha(o)(G184S) heterozygous (Galpha(o)+/GS) mice compared with either Galpha(i2)(G184S) heterozygous (Galpha(i2)+/GS) or control mice (EC(50) = 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 alpha(2A)AR/Galpha(o) protein-mediated pathway under strong inhibitory control by RGS proteins. This suggests a possible role for RGS inhibitors or selective alpha(2A)AR agonists as a novel antiepileptic drug therapy.
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ABSTRACT: Opioids (eg morphine) are powerful analgesics that are used clinically to treat a variety of pain conditions. However, chronic use of opioids is associated with the development of adaptations such as tolerance and dependence, which limit their utility as long-term pain ...01/2013;
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ABSTRACT: Heterotrimeric G proteins, composed of α, β, and γ subunits, can transduce a variety of signals from seven-transmembrane-type receptors to intracellular effectors. By whole-exome sequencing and subsequent mutation screening, we identified de novo heterozygous mutations in GNAO1, which encodes a Gαo subunit of heterotrimeric G proteins, in four individuals with epileptic encephalopathy. Two of the affected individuals also showed involuntary movements. Somatic mosaicism (approximately 35% to 50% of cells, distributed across multiple cell types, harbored the mutation) was shown in one individual. By mapping the mutation onto three-dimensional models of the Gα subunit in three different complexed states, we found that the three mutants (c.521A>G [p.Asp174Gly], c.836T>A [p.Ile279Asn], and c.572_592del [p.Thr191_Phe197del]) are predicted to destabilize the Gα subunit fold. A fourth mutant (c.607G>A), in which the Gly203 residue located within the highly conserved switch II region is substituted to Arg, is predicted to impair GTP binding and/or activation of downstream effectors, although the p.Gly203Arg substitution might not interfere with Gα binding to G-protein-coupled receptors. Transient-expression experiments suggested that localization to the plasma membrane was variably impaired in the three putatively destabilized mutants. Electrophysiological analysis showed that Gαo-mediated inhibition of calcium currents by norepinephrine tended to be lower in three of the four Gαo mutants. These data suggest that aberrant Gαo signaling can cause multiple neurodevelopmental phenotypes, including epileptic encephalopathy and involuntary movements.The American Journal of Human Genetics 08/2013; · 11.20 Impact Factor
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ABSTRACT: G protein-coupled receptors strongly modulate neuronal excitability but there has been little evidence for G protein mechanisms in genetic epilepsies. Recently, four patients with epileptic encephalopathy (EIEE17) were found to have mutations in GNAO1, the most abundant G protein in brain, but the mechanism of this effect is not known. The GNAO1 gene product, Gαo, negatively regulates neurotransmitter release. Here, we report a dominant murine model of Gnao1-related seizures and sudden death. We introduced a genomic gain-of-function knock-in mutation (Gnao1 (+/G184S)) that prevents Go turnoff by Regulators of G protein signaling proteins. This results in rare seizures, strain-dependent death between 15 and 40 weeks of age, and a markedly increased frequency of interictal epileptiform discharges. Mutants on a C57BL/6J background also have faster sensitization to pentylenetetrazol (PTZ) kindling. Both premature lethality and PTZ kindling effects are suppressed in the 129SvJ mouse strain. We have mapped a 129S-derived modifier locus on Chromosome 17 (within the region 41-70 MB) as a Modifer of G protein Seizures (Mogs1). Our mouse model suggests a novel gain-of-function mechanism for the newly defined subset of epileptic encephalopathy (EIEE17). Furthermore, it reveals a new epilepsy susceptibility modifier Mogs1 with implications for the complex genetics of human epilepsy as well as sudden death in epilepsy.Mammalian Genome 04/2014; · 2.42 Impact Factor
Regulator of G Protein Signaling Protein Suppression of G?o
Protein-Mediated ?2AAdrenergic Receptor Inhibition of Mouse
Hippocampal CA3 Epileptiform Activity
Brianna L. Goldenstein, Brian W. Nelson, Ke Xu, Elizabeth J. Luger, Jacquline A. Pribula,
Jenna M. Wald, Lorraine A. O’Shea,1David Weinshenker, Raelene A. Charbeneau,
Xinyan Huang, Richard R. Neubig, and Van A. Doze
Department of Pharmacology, Physiology and Therapeutics, University of North Dakota, School of Medicine and Health
Sciences, Grand Forks, North Dakota (B.L.G., B.W.N., K.X., E.J.L., J.A.P., J.M.W., L.A.O., V.A.D.); Department of Human
Genetics, Emory University, Atlanta, Georgia (D.W.); and Department of Pharmacology, University of Michigan, Medical School,
Ann Arbor, Michigan (R.A.C., X.H., R.R.N.)
Received December 18, 2008; accepted February 18, 2009
Activation of G protein-coupled ?2adrenergic 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?oor 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
dioxane hydrochloride (WB-4101; 6.87), and prazosin (5.71).
Calculated pKbvalues 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 ?2AAR- but not ?2CAR-knockout
mice. Pretreatment with pertussis toxin also reduced the EPI-
mediated 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-insen-
(EC50? 2.5 versus 19 and 23 nM, respectively). Together,
these findings indicate that the inhibitory effect of EPI on hip-
pocampal CA3 epileptiform activity uses an ?2AAR/G?opro-
tein-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.
G184Sheterozygous (G?o?/GS) mice compared with
G184Sheterozygous (G?i2?/GS) or control mice
The noradrenergic system modulates many physiological
and pathological processes within the central nervous system
arousal, sleep, and learning and memory (Pupo and Minne-
man, 2001) and seem to attenuate epileptic activity (Giorgi et
al., 2004). The hippocampus receives substantial noradren-
ergic 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 short-
term 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-R01-
DA17963]; 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.
1Current affiliation: Schroeder Middle School, Grand Forks, North Dakota.
Article, publication date, and citation information can be found at
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-(2,6-
dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride; WT, wild type; PTX, pertussis toxin; JP-1302, N-[4-(4-methyl-1-piperazi-
Copyright © 2009 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 75:1222–1230, 2009
Vol. 75, No. 5
Printed in U.S.A.
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 tempo-
ral lobe epilepsy, the most common human epileptic syn-
drome. It is clear that thoroughly delineating the inhibitory
and excitatory aspects of this region is critical to understand-
ing CNS function and dysfunction and to designing targeted
Norepinephrine (NE) is the major neurotransmitter re-
leased by noradrenergic neurons and modulates several CA3
processes. NE has been shown to facilitate long-term poten-
tiation, which is involved in memory formation, and antiepi-
leptic 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 (Jur-
gens 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 result-
ing in diverse physiological actions (Pupo and Minneman,
2001). Studies have suggested that ?ARs mediate the en-
hancement 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). Fur-
thermore, 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 sig-
nals 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 G-?? subunits
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 G-?? heterodimer, forming
an inactive trimer once again.
For some G? families (Gi/oand Gq), the rate of GTP hydro-
lysis 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 sig-
naling through receptors coupled to the Gi/oand Gqfamily of
G proteins (Clark et al., 2008) and enhance intrinsic GTPase
activity of the GTP-bound G? subunits. This GTPase accel-
eration 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 pos-
sibility of side effects (Zhong and Neubig, 2001; Neubig and
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 combina-
tion 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 sub-
types 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
Atipamezole was made by Orion Corporation (Espoo, Finland).
rine (?)-bitartrate, D-(?)-norepinephrine (?)-bitartrate, oxymetazo-
line hydrochloride, pertussis toxin, picrotoxin, pindolol, and timolol
maleate were obtained from Sigma-Aldrich (St. Louis, MO). Prazosin
hydrochloride, rauwolscine hydrochloride, and WB-4101 were ac-
quired from Tocris Cookson Inc. (Ellisville, MO). All chemical re-
agents used to make the artificial cerebrospinal fluid (ACSF) were of
biological grade from J. T. Baker, Inc. (Phillipsburg, NJ) or Thermo
Fisher Scientific (Waltham, MA). Isoflurane was purchased from
Abbott Diagnostics (Chicago, IL).
C57BL/6J mice of both genders were used in the present study.
Mice were housed two to four per cage (size 11.5 ? 7 inches) under
standard laboratory conditions on a 12-h light/dark cycle (lights on at
7:00 AM) in rooms maintained at a temperature of ?22°C with a
relative humidity of ?55%. Water and dried laboratory food (Teklad
Global 18% Protein Rodent Diet; Harlan Teklad, Madison, WI) were
provided ad libitum. Mice were allowed to acclimate for at least 4
days after arrival (see below). All protocols described were approved
by the Institutional Animal Care and Use Committee of Emory
University (Atlanta, GA), the University of Michigan (Ann Arbor,
MI), and the University of North Dakota (Grand Forks, ND) in
accordance with National Institute of Health guidelines (Institute of
Laboratory Animal Resources, 1996) and meet the guidelines of the
American Association for Accreditation of Laboratory Animal Care.
Generation of ?2AAR- and ?2CAR-Knockout Mice. ?2A(?/?)
[?2A/?2C; (?/?)/(?/?)] and ?2C(?/?) [?2A/?2C; (?/?)/(?/?)] mice,
maintained on a pure C57BL/6J background, were generated at
Emory University using heterozygous ?2C(?/?) and ?2AC(?/?) mice
obtained from Brian K. Kobilka (Stanford University, Stanford, CA).
Genotypes were confirmed by polymerase chain reaction. All mice
were reared in a specific pathogen-free facility at Emory University
with a 12-h light/dark cycle (lights on at 7:00 AM) and were shipped
to the University of North Dakota at age 2 to 5 months. Control
animals used in these studies were wild-type (WT) C57BL/6J [?2A/
?2C; (?/?)/(?/?)] mice purchased from The Jackson Laboratory (Bar
Generation of G?o
Mice. The original G?o
2006), was developed in a 129-D3 ES cell background, which never
went germline. Consequently, the G?o
structed from a 129-CJ7 ES line using methods similar to those
previously reported for the G?i2
G184SHeterozygous (G?o?/GS) Knock-In
G184SES cell line, described in Fu et al. (2004,
G184Smouse strain was con-
G184Sstrain (Fu et al., 2006; Huang
G?o/RGS Regulation of ?2AAdrenergic Receptor Inhibition
et al., 2006). Specifically, we prepared a targeting construct by re-
striction digestion to obtain DNA fragments of the mouse Gnao gene
from a Bac clone derived from 129-CJ7 DNA Bac library (ResGen;
Invitrogen, Carlsbad, CA). Using those fragments, a targeting con-
struct was prepared in the TKLNL vector (Mortensen et al., 1992).
First the mutant G?oexon 5 was produced by mutating the sequence
AAAACAACTGGCATCGTAGAAA to AAAACAACTAGTATCGTA-
GAAA. The bases in boldface type indicate the changed codon (Gly184
to Ser184), and the underline portion designates the location of the
resulting diagnostic SpeI restriction site. This mutated exon 5 and
additional 5? genomic sequence to form the “left” homology arm was
cloned into TKLNL to introduce the loxP-flanked neo marker after
exon 5, then the right arm genomic fragment from exons 6 to 8 was
cloned 3? of the loxP cassette in a manner similar to that for prepar-
ing the G?i2
2006). CJ7 ES cells were electroporated with the targeting vector,
and homologous recombinants were isolated. Targeted CJ7 ES cells
were microinjected into C57BL/6NCrl ? (C57BL/6J ? DBA/2J)F1
mouse blastocysts to generate ES cell-mouse chimeras. After identi-
fication of chimeric offspring, the mice were backcrossed onto a
CJ7BL/6J background for at least four generations. Only heterozy-
gous offspring of crosses between G?o(?/G184S) male and C57BL/6J
female mice (N4-N5) were used in these studies because homozygous
G?o(G184S/G184S) offspring from heterozygous ? heterozygous
crosses were not viable. Control animals used in these studies were
WT littermates [(?/?)] of the G?o
Generation of G?i2
on a pure C57BL/6J background (?10 generations), were generated
at the University of Michigan, Ann Arbor, as described previously
(Fu et al., 2006). All genotypes were confirmed by polymerase chain
reaction. Control animals used in these studies were WT C57BL/6J
mice from The Jackson Laboratory. Both the G?o
heterozygous (?/GS) knock-in mice were reared at the University of
Michigan and were confirmed to be pathogen-free before their ship-
ping to the University of North Dakota at age 1 to 3 months.
G184Stargeting vector (Fu et al., 2006; Huang et al.,
G184SHeterozygous (G?i2?/GS) Knock-In
G184Sheterozygous (G?i2?/GS) knock-in mice, maintained
Hippocampal Slice Preparation
After being deeply anesthetized with isoflurane, mice weighing 16
to 27 g were decapitated, and their brains were rapidly removed. The
hippocampi were then quickly dissected from each hemisphere and
placed into an ice-cold Ringer solution containing 110 mM choline
chloride, 2.5 mM KCl, 7 mM MgSO4, 0.5 mM CaCl2, 1.25 mM
NaH2PO4, 25 mM NaHCO3, 25 mM D-glucose, 11.6 mM sodium
ascorbate, and 3.1 mM sodium pyruvate, saturated with 95% O2/5%
CO2. Using a conventional tissue sectioning apparatus (Stoelting,
Wood Dale, IL), the hippocampi were sliced transversely into 500-?m
thick sections and transferred to ACSF consisting of 119 mM NaCl,
5 mM KCl, 1.3 mM MgSO4, 2.5 mM CaCl2, 1 mM NaH2PO4, 26.2 mM
NaHCO3, and 11 mM D-glucose, which was continually aerated with
95% O2/5% CO2. The slices were incubated at 32 ? 1°C for 30 min,
then transferred to room temperature (22 ? 1°C) and allowed to
recover for at least 30 min before experimentation.
A single slice was transferred to the recording chamber, where it
was submerged and superfused continuously at a rate of at least 4
ml/min with ACSF at room temperature. Glass microelectrodes were
made using a vertical two-stage puller (PP-830; Narishige, Tokyo,
Japan). Extracellular field potentials were recorded using microelec-
trodes filled with 3 M NaCl placed in the stratum pyramidale of the
CA3 region of the hippocampus using an SZ-61 stereo microscope
(Olympus, Melville, NY). Potentials were detected using either an
Axoclamp 2B (Molecular Devices, Sunnyvale, CA) or BVC-700A (Da-
gan, Minneapolis, MN) microelectrode amplifier, amplified using a
Brownlee 440 signal conditioner (Brownlee Precision, San Jose, CA),
digitized with a Digidata 1322A analog-to-digital converter (Molec-
ular Devices), and recorded using Axoscope 9.0 software (Molecular
Generation of Epileptiform Activity
Hippocampal CA3 pyramidal neurons are prone to spontaneously
firing epileptiform bursts partly as a result of their extensive asso-
ciational connections (Schwartzkroin, 1986). This activity was easily
generated by superfusing the slice with ACSF containing 100 ?M
picrotoxin, a GABAAreceptor blocker, to attenuate synaptic inhibi-
tion. If no burst discharges were seen after 30 min of superfusion, the
slice was determined to be unresponsive and discarded. Once con-
tinuous spontaneous epileptiform burst discharges were evident, 30
min of baseline data were recorded before any exposure to an AR
agonist. The ACSF also contained 0.5 ?M desipramine to block NE
transporters [i.e., potential reuptake of the catecholamines epineph-
rine (EPI) and NE] and 30 ?M timolol to block any ?AR-mediated
excitatory effects (Jurgens et al., 2005), as well as any applicable
?AR antagonist. Before being used, each AR antagonist was tested to
ensure that it possessed no independent effects. Preliminary exper-
iments also confirmed that each AR agonist concentration caused its
maximal effect during an 8-min application (data not shown). Be-
cause the ?2AR antagonist rauwolscine also has potent serotonergic
5-hydroxytryptamine1A receptor-mediated agonist activity (New-
man-Tancredi et al., 1998), we substituted 3 ?M pindolol (which
blocks both ?AR and 5-hydroxytryptamine1Areceptors) for timolol
(which only blocks ?ARs) when using this particular ?2AR
Epileptiform burst discharge frequencies were visualized in real
time (Fig. 1A) while being recorded for subsequent analysis. Postex-
periment analysis was completed using Mini Analysis 6.0 software
(Synaptosoft, Decatur, GA). The last interval correlating to each
agonist concentration was noted, the baseline frequency was sub-
tracted, and that value was used to plot a concentration-response
expressed as a percentage of maximal response. Frequency versus
agonist concentration data were then entered into Prism 5.0 soft-
ware (GraphPad Software Inc., San Diego, CA), and concentration-
response curves were constructed using a nonlinear least-squares
curve-fitting method. Each curve was fitted with a standard (slope ?
unity) or variable slope, and the best fit was determined using an F
test with a value of p ? 0.05. The calculated EC50value was used as
a measurement of agonist potency. Significance between groups was
compared statistically using the Student’s t test (p ? 0.05).
Schild analysis was used to determine the apparent equilibrium
dissociation constants (pKb) for selective ?AR antagonists (Arunlak-
shana and Schild, 1959). For each experiment, cumulative concen-
tration-response curves were performed in adjacent slices from the
same mouse (one dose-response curve per slice). Dose ratios of EC50
values were calculated in the presence and absence of a selective
?2AR antagonist and Schild plots constructed by graphing the log of
the dose ratio ? 1 versus the log of the antagonist concentration.
Linear regression analysis of these points was used to determine the
slope and x-intercept. Schild regression slopes are given as mean ?
S.E. and were considered to be nonunity if the 95% confidence inter-
val did not include the value of 1. The pKbvalues of ?AR antagonists
causing competitive inhibition of the EPI-mediated reduction in
burst frequencies were calculated from Schild regression x-inter-
cepts. Differences in pKbvalues and Schild regression slopes were
determined by analysis of covariance with a p ? 0.05 level of prob-
ability accepted as significant. EC50and pKbvalues are expressed as
the mean ? S.E. for n experiments.
Effects of EPI and NE on Mouse CA3 Epileptiform
Activity. We first examined the effects of EPI on mouse CA3
Goldenstein et al.
epileptiform burst discharges in the presence of timolol (?AR
blockade) to elucidate the action of ?AR activation on hip-
pocampal activity. Picrotoxin-induced epileptiform burst dis-
charges are shown in Fig. 1, and their frequency is reduced
by application of EPI in a concentration-dependent manner.
For this particular experiment, the EC50value calculated
from nonlinear regression analysis was 48 nM. Our previous
work in rats has shown that this effect is most likely medi-
ated by an ?2AR in the CA3 region of the hippocampus
(Jurgens et al., 2007). As illustrated in Fig. 2, the rank order
of potency of the three AR agonists tested in this manner
revealed that (?)EPI (31 ? 8.1 nM, n ? 45 slices) ? (?)NE
(150 ? 45 nM, n ? 15 slices) ??? (?)NE (4700 ? 3300 nM,
n ? 10 slices), which is consistent with our previous results
(Jurgens et al., 2007) and the order expected for ?ARs.
Effects of the Selective ?2AR Antagonist Atipam-
ezole and Subtype-Selective ?2AR Antagonists on the
EPI-Mediated Decrease in Burst Discharge Frequen-
cies. Functional determination of equilibrium dissociation
constant (Kb) value for selective ?AR antagonists was used to
characterize the type of ?AR mediating decreased burst fre-
quency in the hippocampal CA3 region. Pretreatment of hip-
pocampal slices with 3, 10, and 30 nM atipamezole produced
2-, 6-, and 22-fold parallel rightward shifts of the fitted EPI
concentration-response curve (Fig. 3A). The pKbof 8.79 (n ?
5) for atipamezole (Fig. 3B) was similar to previously pub-
lished binding pKivalues for the mouse ?2ARs (Link et al.,
1992; Chruscinski et al., 1992; see also Table 1). This result
suggests that the response is mediated by an ?2AR.
Subtype-selective antagonists were then used to determine
the specific subtype of ?2AR mediating burst frequency re-
duction in the mouse hippocampal CA3 region. Apparent pKb
values of subtype-selective ?2AR competitive antagonists
were determined using Schild regression analysis. Slices pre-
treated with either prazosin (?2BAR-selective), rauwolscine
(?2CAR-selective), or WB-4101 (?2CAR-selective) produced
parallel rightward shifts of the fitted EPI concentration-
response curve in all instances (data not shown). For each of
these selective ?2AR antagonists, the slope of the regression
line was close to the value of unity. The logs of the equilib-
rium dissociation constants (pKb) calculated for these ?2AR
antagonists were as follows: rauwolscine (7.75, n ? 3), WB-
4101 (6.87, n ? 3), and prazosin (5.71, n ? 4) (Table 1).
?2AR Antagonist Functional pKbEstimates Corre-
late to ?2AAR pKiValues. A method often used to compare
equilibrium dissociation constants of many receptor antago-
nists is to correlate pKbvalues with previously published pKi
values (Bylund, 1988). Both the correlation coefficient and
slope of the correlation line should be close to unity if the
calculated functional values correspond to the published
binding constants for a specific receptor. Illustrated in Fig. 4
are the correlations between the pKbvalues determined for
the selective ?AR antagonists used in this study and the
previously published pKivalues of these AR antagonists for
each mouse ?2AR subtype (Table 1). For the mouse ?2AAR
subtype, a very high correlation coefficient (r ? 0.98) along
with a slope similar to unity (slope ? 1.07) were calculated
for our experimental pKbvalues compared with published
binding affinity values (Fig. 4A). In contrast, for the mouse
?2BAR, a poor correlation coefficient (r ? 0.88) and low slope
Fig. 1. Effects of EPI on mouse hippocampal CA3 epileptiform activity. A, continuous 150-s long chart recordings of burst discharges recorded in the
hippocampal CA3 region of brain slices from WT mice. Epileptiform burst discharges were elicited by including 100 ?M concentration of the GABAA
receptor blocker picrotoxin in the perfusing ACSF containing 30 ?M timolol and 0.5 ?M desipramine. Under these conditions, bath application of EPI
reduced burst frequency in a concentration-dependent manner from 10 bursts (0.067 Hz) in control Ringer solution to 7 (0.047 Hz) in 30 nM EPI, 3
(0.020 Hz) in 300 nM EPI, and 1 (0.007 Hz) in 3 ?M EPI. B, frequency histogram of the number of burst discharges versus time of EPI application.
Each bin represents the frequency averaged over an approximately 150-s epoch. Increasing concentrations of EPI were applied to the bath for the
8-min periods indicated. Inset, concentration-response curve derived from the frequency histogram. Data points were plotted as the percentage of
maximal inhibition (decrease in epileptiform burst frequency), and the curve was constructed using a nonlinear least-squares curve-fitting method.
For this experiment, the concentration-response curve was fit best by a nonvariable sigmoidal model with a calculated EC50value for EPI of 48 nM.
G?o/RGS Regulation of ?2AAdrenergic Receptor Inhibition
(slope ? 0.40) were observed when comparing our experimen-
tal pKbvalues with previously published pKivalues (Fig. 4B).
Likewise for the mouse ?2CAR, a poor correlation coefficient
(r ? 0.89) and low slope (slope ? 0.63) were seen (Fig. 4C).
These results suggest that the ?2AAR is the predominant
subtype mediating the antiepileptic action of EPI in mouse
Effects of EPI on Epileptiform Activity in Slices from
?2AAR- and ?2CAR-Knockout Mice. To confirm our phar-
macological results, we examined the effects of EPI on hip-
pocampal CA3 epileptiform activity in ?2AAR- and ?2CAR-
knockout (KO) mice. As illustrated in Fig. 5, EPI was applied
in increasing concentrations to hippocampal brain slices pre-
pared from either ?2AAR- or ?2CAR-KO mice. The potency of
EPI in the ?2CAR-KO mouse line (37 ? 12 nM, n ? 15) fit best
with a unity-slope sigmoidal model and was not significantly
different from the WT mice (see also Fig. 2). In contrast, the
effects of EPI were largely abolished in brain slices from
?2AAR-KO mice with a maximum effect of less than 10%
inhibition. These results demonstrate that the ?2AAR is the
predominant receptor subtype mediating the inhibitory ef-
fects of EPI in the mouse hippocampus.
Oxymetazoline on the EPI-Mediated Decrease in Burst
Discharge Frequencies in ?2CAR-KO Mice. To further
evaluate a potential role for ?2BARs and confirm that the
response was primarily an ?2AAR response, the selective
?2AAR antagonist oxymetazoline was used in brain slices
made from ?2CAR-KO mice. ?2CAR-KO mouse slices that had
been pretreated with 100, 300, and 1000 nM oxymetazoline
produced 6-, 22-, and 70-fold parallel rightward shifts of the
fitted EPI concentration-response curve (Fig. 6A). The Schild
regression slope was 1.16 ? 0.12 and the x-intercept corre-
lating to a pKbvalue of 7.50 (n ? 7 animals) (Fig. 6B). The
mouse ?2AAR reported a pKivalue of 7.49 matched closely to
our pKbvalue, whereas the ?2BAR and ?2CAR reported pKi
Fig. 2. Potency for EPI and NE inhibiting hippocampal CA3 epileptiform
burst activity. Extracellular field potential recordings were used to gen-
erate concentration-response curves using increasing amounts of (?)EPI
(f), (?)NE (F), and (?)NE (E) in the presence of 100 ?M picrotoxin, 30
?M timolol, and 0.5 ?M desipramine. There was a significant difference
in the potencies (EC50values) calculated for (?)EPI (31 ? 8.1 nM, n ? 45
slices from 18 animals), (?)NE (150 ? 45 nM, n ? 15 slices from 7
animals), and (?)NE (E) (4700 ? 3300 nM, n ? 10 slices from 4 animals).
Concentration-response curves for each agonist were plotted as a percent-
age of decrease (reduction) in epileptiform burst frequency. Each individ-
ual experiment best fit to a nonvariable sigmoidal curve. There was no
significant difference in the efficacy of (?)EPI (68 ? 2.6%), (?)NE (67 ?
3.7%), and (?)NE (58 ? 7.2%) at reducing epileptiform activity.
Fig. 3. Schild regression analysis using the
selective ?2AR antagonist atipamezole. A, con-
secutive EPI concentration-response curves
demonstrate a concentration-dependent effect
of the selective ?2AR antagonist, atipamezole,
on the EPI-mediated inhibition of hippocampal
CA3 epileptiform activity in brain slices from
WT mice. Pretreatment with 3 nM (E), 10 nM
(f), and 30 nM (?) concentrations of this an-
tagonist produced consecutive parallel right-
ward shifts of the EPI curve that were signifi-
cantly different from control (F) (EC50? 76 ?
34 ? 12 nM for control). B, using dose ratios
calculated from individual experiments illus-
trated in A, a Schild plot was created generat-
ing a regression slope equaling 1.06 ? 0.12 and
n ? 5 animals (see Table 1).
Comparisons of experimental functional pKbvalues with binding affinity pKivalues for selective ?AR antagonists for mouse ?2AR subtypes
pKbvalues represent the negative logarithm10of the Kband are expressed as the mean. Schild regression slopes are expressed as the mean slope ? S.E. and were determined
in three to five separate experiments using brain slices from WT mice. Reported pKivalues are from binding affinity studies using recombinant mouse ?2AR clones expressed
in COS-7 cells. pKbvalue was calculated using a single 10 ?M concentration of JP-1302.
1.06 ? 0.12
1.01 ? 0.07
0.89 ? 0.08
0.97 ? 0.06
aLink et al. (1992).
bChruscinski et al. (1992).
Goldenstein et al.
values of 5.92 (Chruscinski et al., 1992) and 6.96 (Link et al.,
1992) did not. If the ?2BAR made a significant contribution,
the slope of the Schild plot should have been less than 1.
These results further confirm that this response is primarily
mediated by an ?2AAR.
Effects of Pertussis Toxin on EPI-Mediated Inhibi-
tion of CA3 Epileptiform Activity. Pertussis toxin (PTX)
blocks the receptor-mediated activation of Gi/oproteins. We
used PTX to assess which G protein types are involved in the
inhibitory effects of EPI. Extracellular field potential record-
ings of epileptiform burst frequency were used to generate
concentration-response curves using increasing amounts of
EPI in untreated control slices or in slices treated with 5
?g/ml PTX for 7 to 8 h. As illustrated in Fig. 7, the mean
concentration-response curve for nontreated control slices
was fit best by a unity-slope sigmoidal model with a calcu-
lated EC50value of 12 ? 3.9 nM and a maximum effect of
74 ? 6.1% (n ? 13 slices). Conversely, for PTX-treated slices
from the same mice, the mean concentration-response curve
showed minimal inhibition (?25%) (n ? 12 slices). These
results indicate that inhibition of mouse hippocampal CA3
epileptiform activity in response to EPI is mediated by either
Gior Goproteins and not Gsor Gqproteins.
EPI-Mediated Inhibition of CA3 Epileptiform Activity
in Slices from G?o
response and which type of inhibitory G protein may be in-
volved, we used mice with a knock-in G? subunit mutation
(G184S) that renders G?oand G?i2proteins incapable of bind-
ing to the RGS protein. This results in the loss of RGS-mediated
inhibition of the G?oand G?i2protein and enhances G?-specific
effects in tissues with responses under RGS control. An in-
crease in response with one of these RGS-insensitive G proteins
would implicate that G protein as contributing to the response
G?o?/GS, or G?i2?/GS slices were pretreated with the GABA
blocker picrotoxin, ?AR blocker timolol, and NE transporter
reuptake inhibitor desipramine. Extracellular field potential
using increasing amounts of EPI. Inhibition of frequency burst
discharges was significantly more potent in brain slices from
G?omice, with an EC50of 2.5 ? 0.9 nM (n ? 23 slices) versus
litter mate control mice (EC50? 19 ? 5 nM, n ? 21 slices) (Fig.
G184SHeterozygous (G?o?/GS) and
G184SHeterozygous (G?i2?/GS) Knock-in Mice. To de-
(EC50? 19 ? 5 nM, n ? 32 slices) compared with the WT
controls (EC50? 23 ? 7 nM, n ? 22 slices) (Fig. 8B). These
results indicate the EPI-mediated inhibition of mouse hip-
pocampal CA3 epileptiform activity involves a G?omechanism
under strong negative regulation by RGS proteins.
The role of catecholamines in seizures and epilepsy is com-
plicated, but it is clear that endogenous EPI and NE can
protect against many types of seizures (Weinshenker and
Szot, 2002). Agonists at all three types of AR (?, ?1, and ?2)
can be antiepileptic, but the most consistent findings show
that ?2AR agonists are generally anticonvulsant, and selec-
tive ?2AR antagonists are proconvulsant (Weinshenker and
Szot, 2002). Consequently, we focused the current study on
the hippocampus, which plays an important role in the com-
mon clinical condition of temporal lobe seizures, to begin to
dissect mechanisms underlying the antiepileptic actions of
?2AR agonists. We used both pharmacological and mouse
genetic models to define the receptor and G protein involved
in the EPI-mediated antiepileptiform activity in the hip-
pocampus. We have confirmed the role of the ?2AAR in inhi-
bition of hippocampal CA3 epileptiform activity in mice, as
shown previously by a pharmacological approach in rats (Ju-
rgens et al., 2007). We built upon these findings by demon-
strating that this involves a PTX-sensitive Gi/o-type G pro-
mutant knock-in mice, we show that endogenous RGS pro-
tein action on G?ostrongly suppresses this signal, implicat-
ing G?oas a mediator of the response. In contrast, G?i2
seems not to be involved. These findings enhance our under-
standing of the mechanism underlying ?2AAR-mediated in-
hibition of hippocampal epileptiform activity by NE and sug-
gest a novel approach to antiepileptic drug therapies.
The ?2AAR is the predominant ?2AR in the CNS, and it has
been implicated as the primary anticonvulsant ?2AR in rat
hippocampus in vitro (Jurgens et al., 2007) and in mouse in
vivo (Janumpalli et al., 1998). A previous study using dopa-
mine ?-hydroxylase, ?2AAR, and ?2CAR-KO mice showed
that the proconvulsant effects of ?2AR agonists were medi-
ated by the ?2AAR autoreceptor, which decreases NE release,
whereas the anticonvulsant effects of ?2AR agonists were
Fig. 4. Correlation between the functional affinity estimates (pKb) to the equilibrium dissociation constants (pKi) for various selective ?2AR
antagonists. Using the pKband pKivalues from Table 1, correlation analyses were performed for the ?2AAR (A), the ?2BAR (B), and the ?2CAR (C).
G?o/RGS Regulation of ?2AAdrenergic Receptor Inhibition
mediated by ?2AARs on target neurons (Szot et al., 2004). In
the present study, we confirm the results of these findings in
mouse using both pharmacological (antagonist pKb) and ge-
netic (?2AAR- and ?2CAR-KO) approaches. Despite expres-
sion of the ?2CAR in hippocampus, it does not seem to con-
tribute at all to the antiepileptiform activity of EPI and NE
(Fig. 5). Likewise, the ?2BAR does not seem to play a role
(Fig. 6). Neither were ?1ARs involved in this particular re-
sponse (Fig. 3 and Table 1). This level of receptor subtype-
specificity does not, however, provide any significant thera-
peutic advance on its own, because the ?2AAR is also the
major receptor involved in the antihypertensive therapeutic
effect of ?2AR agonists and in their major sedative side effect
as well (MacMillan et al., 1998). Thus, we pursued subse-
quent steps in the downstream signaling.
The ?2ARs are known to couple primarily to Gi/ofamily G
proteins with subsequent actions on several effector systems,
including inhibition of adenylyl cyclase, inhibition of voltage-
gated calcium channels, and activation of G protein-coupled
inwardly rectifying K?currents (Offermanns, 2003). The Gi/o
protein family is also strongly regulated by the 20-plus mem-
ber RGS protein family (Neubig and Siderovski, 2002), which
has been implicated as a potential drug target (Zhong and
Neubig, 2001; Roman et al., 2007). We first confirmed that
the ?2AAR response in hippocampus was PTX-sensitive, in-
dicating a role for the Gi/ofamily. The small residual effect
after PTX treatment (?1/3 of the control response) could be
due to incomplete modification of the Gi/oproteins during the
7- to 8-h pretreatment period, because many studies use an
overnight (?12 h) treatment with PTX. Alternatively, a non–
PTX-sensitive protein like Gzcould play a small role.
To further examine which Gi/osubtype(s) can mediate EPI’s
effect, mice with knock-in mutant RGS-insensitive G?oor G?i2
were used. The knock-in mice differ from WT only in the pres-
G? subunit and the subsequent GTPase acceleration (Fu et al.,
2004; Huang et al., 2006). Consequently, this mutation results
in prolonged and enhanced activation of the modified G protein,
which increases signal transduction by both the ? and ?? sub-
units derived from that G protein. The heterozygous G?oRGS-
insensitive [G?o(?/G184S)] knock-in animals showed a 7-fold
leftward shift of the EPI dose-response curve (2.5 versus 19
nM), whereas there was no significant difference in potency
between the heterozygous G?i2RGS-insensitive mouse (19 nM)
and its control (23 nM). The pronounced effect even in the
heterozygous mouse is not surprising. RGS proteins can accel-
erate G protein deactivation nearly 1000-fold (Mukhopadhyay
and Ross, 1999; Lan et al., 2000), dramatically suppressing G
regulatory effect, so it produces a gain-of-function phenotype in
Fig. 5. Effects of EPI on hippocampal CA3 epileptiform activity in brain
slices from ?2AAR- and ?2CAR-KO mice. Extracellular field potential
recordings of epileptiform burst frequency were used to generate concen-
tration-response curves using increasing amounts of EPI in the presence
of 100 ?M picrotoxin, 30 ?M timolol, and 0.5 ?M desipramine. Concen-
tration-response curves for EPI were plotted as a percentage of decrease
(reduction) in epileptiform burst frequency. For the ?2AAR-KO mice, the
mean concentration-response curve for 41 slices from 12 animals was fit
best by a linear regression line. In contrast, the mean concentration-
response curve for 39 brain slices from 15 ?2CAR-KO mice was fit best by
a nonvariable sigmoidal model with a calculated EC50value of 37 ? 12
nM, which was not significantly different from the potency of 31 ? 8.1 nM
calculated for EPI in slices from WT mice (see Fig. 2). The efficacy of EPI
at reducing the frequency of epileptiform bursts in slices from ?2CAR-KO
mice was 64 ? 3.9%, which was significantly different from the 8.7 ?
3.3% inhibition for EPI in slices from ?2AAR-KO mice.
Fig. 6. Schild regression analysis using the selective ?2AAR ligand oxymetazoline in slices from ?2CAR-KO mice. A, consecutive EPI concentration-
response curves demonstrate a concentration-dependent effect of the selective ?2AAR ligand, oxymetazoline, on the EPI-mediated inhibition of
hippocampal CA3 epileptiform activity in brain slices from ?2CAR-KO mice. Pretreatment with 100 nM (E), 300 nM (f), and 1000 nM (?)
concentrations of this antagonist produced consecutive parallel rightward shifts of the EPI curve that were significantly different from control (F)
(EC50? 205 ? 48, 738 ? 267, and 2317 ? 980 nM, respectively, versus 33 ? 9 nM for control). B, using dose ratios calculated from individual
experiments illustrated in A, a Schild plot was created generating a regression slope equaling 1.16 ? 0.12 and an x-intercept correlating to a pKbvalue
of 7.50, n ? 7 animals. This pKbvalue matched the binding affinity of oxymetazoline (pKi? 7.49) for the mouse ?2AAR, but not the mouse ?2BAR (pKi?
5.92) (Chruscinski et al., 1992) or mouse ?2CAR (pKi? 6.96) (Link et al., 1992).
Goldenstein et al.
which even half of the G protein removed from this suppression
could produce a marked increase in signaling. Previous studies
with the G?i2
cant effects in heterozygous mice (Huang et al., 2006). Thus,
these results show that RGS proteins play a key role in regu-
lating the ?2AAR-mediated hippocampal CA3 antiepileptiform
effect and suggest that the G?osubtype of Gi/oproteins is
involved in the signaling mechanism, whereas G?i2seems not
G184Sknock-in mutants have also shown signifi-
pertussis toxin-sensitive G proteins such as G?i1or G?i3, but
the evidence clearly indicates that G?odoes play a role.
Several important questions remain. Although the G?o
RGS-insensitive mouse shows that an RGS protein is in-
volved in this system, it does not reveal which of the 20-plus
RGS proteins (Hollinger and Hepler, 2002; Neubig and Sid-
erovski, 2002) are important. Given that nearly 15 different
RGS proteins can function as a GTPase-activating protein for
G?o, it may be difficult to establish which one(s) are involved.
Furthermore, it is possible that more than one RGS protein
may work in a redundant manner in this system. That said,
the RGS7 family of RGS proteins (RGS6, 7, 9, and 11) rep-
resent intriguing candidates because they are relatively se-
lective for G?oin vitro (Lan et al., 2000). A second question is
whether the same enhancement of ?2AAR agonist anticon-
vulsant actions will be seen in vivo. Studies are currently
underway to assess this question.
The present study suggests two strategies that may pro-
vide improved therapeutics for adrenergic agonist anticon-
vulsants. First, an ?2AAR agonist that can selectively acti-
vate G?oversus G?i2or other Gifamily members could lead
to improved potency and/or reduced side effects. It would also
be important for such a compound to preferentially activate
the ?2AARs on target neurons over ?2AAR autoreceptors that
would decrease NE release. This could be achieved by a
“functionally selective” (Urban et al., 2007) ?2AAR agonist.
Second, RGS proteins have been implicated as potential ther-
apeutic targets. Several peptide (Jin et al., 2004; Young et al.,
2004; Roof et al., 2006) and nonpeptide (Roman et al., 2007)
RGS inhibitors have been described. To date, none are active
in vivo for pharmacological studies, but the identification of
the involved RGS protein and the creation of an inhibitor
that could target it could either produce anticonvulsant ef-
fects through endogenous NE or potentially reduce side ef-
Fig. 7. PTX reduces the EPI-mediated inhibition of hippocampal CA3
epileptiform bursts. Extracellular field potential recordings of epilepti-
form burst frequency were used to generate concentration-response
curves using increasing amounts of EPI in untreated control slices (F) or
slices treated (E) with 5 ?g/ml PTX for 7 to 8 h. Concentration-response
curves for EPI were plotted as a percentage of decrease (reduction) in
epileptiform burst frequency. The mean concentration-response curve for
nontreated control slices was fit best by a nonvariable sigmoidal model
with a calculated EC50value of 12 ? 3.9 nM and an efficacy of 74 ? 6.1%
(n ? 13 slices from 6 animals). In contrast, for PTX-treated slices from
these same mice, the mean concentration-response curve was fit best by
a linear regression line and had an efficacy of 24 ? 13% (n ? 12 slices
from 6 animals).
Fig. 8. EPI-mediated inhibition of hippocampal CA3 epileptiform bursts is significantly enhanced in brain slices from G?o?/GS mice but not G?i2?/GS
mice. Extracellular field potential recordings were used to generate concentration-response curves using increasing amounts of EPI (F) in the presence
of 30 ?M timolol and 0.5 ?M desipramine. Concentration-response curves for EPI were plotted as a percent reduction in epileptiform burst frequency.
Each individual experiment best fit to a nonvariable sigmoidal curve. A, there was a significant difference in the potencies (EC50values) calculated
for EPI in brain slices from G?o?/GS mice (?) (2.5 ? 0.9 nM, n ? 23 slices from 6 animals) versus litter mate control mice (F) (19 ? 5 nM, n ? 21
slices from 6 animals). B, in contrast, the EPI-mediated inhibition of epileptiform activity was unchanged in brain slices from G?i2?/GS mice (?) (19 ?
5 nM, n ? 32 slices from 6 animals) compared with WT control mice (F) (23 ? 7 nM, n ? 22 slices from 8 animals). There was no significant difference
in the efficacy of EPI among these four groups (G?o?/GS, 74 ? 3.8%; G?olitter mate control, 74 ? 4.4%; G?i2?/GS, 73 ? 2.8%; G?i2WT control, 75 ?
G?o/RGS Regulation of ?2AAdrenergic Receptor Inhibition
fects on the treatment with ?2AAR-selective agonists in pa-
tients with epilepsy.
In summary, we have defined the receptor (?2AAR), a G
protein (G?o), and a regulatory mechanism (RGS proteins)
that are important for the antiepileptiform actions of NE and
EPI in the hippocampus, a key site of seizure activity in
many patients. These advances provide a theoretical ratio-
nale for future, novel therapeutic approaches.
We thank Sarah J. Boese, Chris W. D. Jurgens, Brandi A. Kaster,
Jasmine J. O’Brien, and Danielle D. Schlosser for help with the
experiments, Karen L. Cisek for assistance in editing the manu-
script, and Dr. James E. Porter for advice about data acquisition and
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Address correspondence to: Dr. Van A. Doze, Department of Pharmacology,
Physiology and Therapeutics, School of Medicine and Health Sciences, Uni-
versity of North Dakota, 501 North Columbia Road, Stop 9037, Grand Forks,
ND 58202-9037. E-mail: firstname.lastname@example.org
Goldenstein et al.