Brain Research 992 (2003) 69 – 75 Characterization of the GABAA receptor in the brain of the adult male bullfrog, Rana catesbeiana David M. Hollis*, Sunny K. Boyd Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, USA Accepted 21 August 2003 Little is known about the properties of GABA receptors in the amphibian brain. The GABAA receptor is widespread in the mammalian brain, and can be specifically labeled with the receptor agonist [3H]muscimol. The binding of [3H]muscimol to membrane preparations from the brainof the bullfrog, Rana catesbeiana, was investigated in kinetic, saturation, and inhibition experiments to determine whether this speciespossessed a GABAA-like receptor. Binding of 20 nM [3H]muscimol to membranes was specific and could be displaced by 1 mM GABA.
Association binding curves showed that steady state occurred rapidly, within 2 min, and dissociation occurred within 5 min. The receptor wassaturable with a single, high-affinity binding site (KD = 19.2 nM; Bmax = 1.8 pmol/mg protein). Binding of [3H]muscimol was inhibited in a dose-dependent fashion by muscimol, GABA, bicuculline methiodide, and bicuculline (in order of potency). Baclofen (at doses from 10! 9 to 10! 3M) failed to displace [3H]muscimol. The binding characteristics and ligand specificity of [3H]muscimol binding sites in the bullfrog brainsupport the hypothesis that this amphibian possesses a GABAA-like receptor protein similar to the GABAA receptor characterized in mammals.
D 2003 Elsevier B.V. All rights reserved.
Theme: Neurotransmitters, modulators, transporters, and receptorsTopic: GABA receptors Keywords: Amphibian; Bicuculline; GABA; Muscimol 42,45,59,63]. There are nonetheless significant differencesin receptor structure between birds and mammals The amino acid neurotransmitter GABA interacts with Both GABAA and GABAB receptor subtypes may be three different families of receptors in mammalian tissues: the present in reptiles, but there are currently very few studies GABAA, GABAB and GABAC receptor families In the Turtle brain [3H]flunitrazepam binding and func- brain, the GABAA receptor is the most broadly distributed of tional studies support the presence of GABA receptors with these subtypes and is responsible for many diverse and binding characteristics similar to rat receptors important actions in the central nervous system GABA and GABA analog binding suggests the presence 50,51,56,57,64]. Evidence from representative species in of both high affinity GABAA receptor and GABAA/benzo- most vertebrate classes suggests that major elements of the diazepine receptor subtypes in an elasmobranch and teleosts GABAergic system have been conserved. For example, the The presence of similar GABA avian brain also possesses receptors from the GABAA and receptor families, with similar pharmacology, is thus sup- GABAB families Binding of the mammalian ported in ectothermic vertebrate species studied so far. On GABAA receptor agonist, [3H]muscimol, occurs in most of the other hand, very little is known of the actual structure and the same regions in the quail brain as in the rat brain function of GABA receptors in these classes.
GABA binding to the GABAA receptor plays important roles In amphibians, there is pharmacological and/or immuno- in audition and learning and memory in birds cytochemical evidence for members of all three GABAreceptor families in brain or retinal tissue. GABAA-,GABAB- and GABAC-receptor specific synthetic analogs * Corresponding author. Department of Zoology, Oregon State alter the activity of cells in the retina and olfactory bulb of University, Corvallis, OR 97331, USA. Tel.: +1-541-737-5348; fax: +1- frogs and salamanders A GABA E-mail address: [email protected] (D.M. Hollis).
antibody labels cells in the frog retina GABAA- 0006-8993/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2003.08.030 D.M. Hollis, S.K. Boyd / Brain Research 992 (2003) 69–75 specific analog treatment alters neuroendocrine control of a- citrate, pH = 7.4). Two bullfrog brains were used per exper- melanocyte stimulating hormone, neurosteroid biosynthesis, iment. The homogenate was then centrifuged at 20,000 " g auditory integration, and sexual behavior in amphibians for 20 min. The resultant supernatant was decanted and the However, none of the GABA receptor pellet resuspended in fresh buffer. This wash procedure was types has been well characterized in an amphibian. Further- repeated an additional five times. After the final wash and more, there is some evidence that GABA exerts unique removal of supernatant, the pellet was placed at ! 80 jC.
effects in amphibians. For example, GABA receptor-medi- After a minimum of 18 h, the pellet was resuspended in 50 ated presynaptic inhibition in frog spinal cord preparations volumes of Tris – citrate and washed two more times to yield is not the same as that in rats These differences a mixed membrane preparation with endogenous GABA may be due to receptors with unique characteristics.
removed. The mixed membrane preparation was divided We used [3H]muscimol, a high-affinity GABAA agonist into aliquots and the appropriate amount of [3H]muscimol in in mammals, to characterize putative GABAA-like receptors buffer was added to aliquots of 750 Al incubation volume in the adult bullfrog, Rana catesbeiana. Early work in with final protein concentrations of 0.2 – 1 mg/ml. All bullfrogs showed binding sites for [3H]GABA in brain determinations within a single experiment were made in and spinal cord This ligand, however, can bind to triplicate, thus each 750 Al aliquot allowed for three repli- all three receptor subtypes and even to transporters. The cates of 250 Al. Membrane protein concentration was presence of GABAA-type receptors, in particular, in frog determined by the method of Bradford with bovine brain is supported by three studies. First, patch-clamp serum albumin as standard.
studies on neurons from the optic tectum of frog (Ranapipiens) tadpoles have shown that bicuculline sensitive 2.2. Binding experiments GABA receptors exert a profound effect on visualresponses Second, an antibody against mammalian Most experiments were performed with [3H]muscimol GABAA receptor h2/3 subunits labels the brain of R.
concentrations at 20nM (based on the methods of Tavolaro et pipiens Third, the autoradiographic distribution of al. specific activity = 20.0 or 28.5 Ci/mmol; NENk, [3H]muscimol and [3H]flunitrazepam has been described Boston, MA, USA). For association experiments, each set of in the frog, Rana esculenta However, the character- triplicates was terminated (see below) after progressively istics of [3H]muscimol binding were not described, so it is longer [3H]muscimol incubation periods. Because of the not known whether the kinetics, affinity, concentration, or extremely rapid association of [3H]muscimol, each 250 ligand specificity of frog [3H]muscimol binding sites are Al replicate was assayed separately. For the dissociation similar to those sites in mammals. We used membrane assay, the receptor preparation was incubated with 20nM preparations to characterize [3H]muscimol binding sites in [3H]muscimol for 20 min to reach equilibrium. After 20 min, the bullfrog brain.
three 250-Al aliquots were used to determine mean totalbinding. Unlabeled GABA (1 mM) was then added and thereaction terminated at subsequent times. The saturation 2. Materials and methods binding isotherm for [3H]muscimol was determined follow-ing the methods of Basile Total binding was determined 2.1. Tissue and membrane receptor preparation by incubating membranes with different concentrations of[3H]muscimol for 90min before reactions were terminated Adult male bullfrogs (R. catesbeiana) were purchased (see below). Tissue samples from the liver, spleen, testis, and from C. Sullivan Company (Nashville, TN). Bullfrogs were retina of bullfrogs were assayed for [3H]muscimol specific housed in the lab on a 12L/12D controlled photoperiod at 17 binding at 20 nM using identical procedures as saturation jC in large tanks (50 " 21 " 21 cm) with flow-through binding. To determine ligand specificity, 20 nM [3H]musci- water and maintained on a diet of goldfish. Bullfrogs were mol was added to receptor preparations with increasing cryoanaesthetized, rapidly decapitated and the brains re- concentrations of different putative GABA receptor agonists moved (including brainstem, but not spinal cord) and and antagonists and incubated for 20 min. Compounds weighed immediately before membrane preparation. All experiments were performed in accordance with the NIH Cat. no. M1523), GABA (Cat no. A5835), bicuculline Guide for the Care and Use of Laboratory Animals and had (Cat. no. B9130), bicuculline methiodide (Cat. no. B6889), been approved by the University of Notre Dame IACUC.
All chemicals and incubations were kept at 4 jC or on ice noic acid; Cat. no. B5399). All compounds were purchased unless otherwise noted.
from Sigma, St. Louis, MO, USA. Specific binding of each Receptor preparation was based on the methods of Basile compound was normalized to percent of the control, which , with modification. Brains were homogenized using a had an equal volume of buffer added. Nonspecific binding of PolytronR (Brinkmann Instruments, Westbury, NY) for 12 s [3H]muscimol was determined in the presence of 1 mM at approximately 1/4 full speed in 50 volumes (g brain tissue unlabeled GABA. Non-specific binding was subtracted from wet weight to ml of buffer) of ice-cold buffer (50mM Tris – the total binding to determine specific binding.
D.M. Hollis, S.K. Boyd / Brain Research 992 (2003) 69–75 2.3. Reaction termination ear regression, ANOVA and post hoc tests. Also, F-testscompared fits for one- and two-site binding models for All binding experiments were terminated by rapid vacu- Scatchard analysis, plus one- and two-site competition um filtration. Aliquots of 250 Al of treated mixed membrane models. Linearizing inhibition data was performed to detect preparations were placed on Whatman GF/C glass micro- the possibility of multiple classes of binding sites fibre filters presoaked for 15 min with 0.03% polyethyleni-mine in deionized, distilled H2O. Membranes on filters werethen washed twice with 3 ml rinses of 50 mM ice-cold Tris – citrate. Filters were placed in vials with 10 ml of scintillationfluid (ScintiSafek 30%; Fisher Scientific, Pittsburgh, PA), Bullfrog brain membranes possessed specific binding shaken overnight, and counted on a liquid scintillation sites for [3H]muscimol. Specific binding of [3H]muscimol was approximately 50% of the total binding. This binding Representative results (from at least three experiments of was tissue- and ligand-specific, saturable, and could be the same type) are shown. Data were analyzed using displaced by 1 mM GABA.
GRAPHPAD PRISM (v. 3.0; Graph Pad, San Diego, CA, The kinetics of [3H]muscimol binding in the bullfrog brain USA), which performed transformations, linear and nonlin- showed both rapid association and dissociation The Fig. 1. Kinetics of 20 nM [3H]muscimol binding in bullfrog brain membrane homogenates (mean F S.E.M. of triplicates from a single representativeexperiment). (A) Association of [3H]muscimol at 4 jC. (Inset) Pseudo first-order association plot (r2 = 0.91). (B) Dissociation of [3H]muscimol at 4 jC with theaddition of 1mM GABA following 20 min association. (Inset) Semi-logarithmic plot of dissociation (r2 = 0.88).
D.M. Hollis, S.K. Boyd / Brain Research 992 (2003) 69–75 Fig. 2. Saturation binding isotherm for [3H]muscimol in bullfrog brain homogenates. Points are means F S.E.M. from triplicate determinations (some pointshad very small S.E.M. determinations, between 1 and 23 fmol/mg protein, and error bars are thus obscured by the symbols). (Inset) Scatchard replot ofsaturation data (r2 = 0.684).
association rate of specifically bound [3H]muscimol was the Nonlinear regression and Scatchard analysis indi- more rapid of the two, as steady state was reached in just over cated a single, high affinity binding site with a KD of 2 min at 4 jC The association rate constant (k+ 1) 19.2 F 1.9 nM and a Bmax = 1.8 pmol/mg protein ( was 0.22 nM/min and the observed association was linear with log transformation (inset). The dissociation of Mammalian GABAA receptor agonists and antagonists specifically bound [3H]muscimol was nearly as fast as its displaced [3H]muscimol from bullfrog membranes in a association, with less than 10% of specific binding remaining concentration-dependent manner Unlabeled musci- after 5 min The dissociation rate constant (k! 1) mol was the most potent inhibitor of [3H]muscimol specific was 0.15 nM/min (inset).
binding with an EC50 of 0.23 AM. GABA was the next most The binding of [3H]muscimol to bullfrog brain mem- potent competitor, with an EC50 of 1.6 AM, followed by branes was saturable with increased ligand concentrations bicuculline methiodide (EC50 = 13.2 AM), and bicuculline . Specifically bound [3H]muscimol increased with (EC50 = 32.4 AM). Baclofen, a specific mammalian GABAB increasing concentrations of the radioligand and saturated at a receptor agonist, failed to influence [3H]muscimol binding concentration of approximately 40 nM of free radioligand at concentrations of between 10 pmol and 10 AM.
Fig. 3. Inhibition of [3H]muscimol binding by different chemicals. Points are mean F S.E.M. of triplicates.
D.M. Hollis, S.K. Boyd / Brain Research 992 (2003) 69–75 The distribution of [3H]muscimol binding sites was salmon brain membranes Although the satura- tissue-specific. Apart from the brain, specific binding was tion binding isotherm for bullfrogs definitively showed only detected in membrane preparations from the retina and one class of [3H]muscimol binding site, there were two testes. No specific binding was observed in membrane indirect indications that a second (lower affinity) binding preparations from the liver and spleen.
site may exist. First, the EC50 of muscimol in inhibitionexperiments was about an order of magnitude higher thanexpected, when compared to the affinity of [3H]muscimol from saturation experiments. A likely explanation is thepresence of a low affinity site not detected by saturation The binding of [3H]muscimol to bullfrog brain mem- binding. Second, inhibition plots from bicuculline and branes was tissue- and ligand-specific, time-dependent, of bicuculline methiodide inhibition experiments in bullfrog high affinity and of limited capacity. This supports the brain suggested the presence of two classes of binding sites hypothesis that the amphibian brain possesses a GABAA- In the mammalian brain, the low affinity binding site type receptor. This is the first report of kinetics, saturation of the GABAA receptor preferentially binds [3H]bicuculline binding, and ligand specificity for any GABA analog in methochloride Thus, the use of a different radiolabeled amphibian brain.
analog (such as [3H]bicuculline, or [3H]bicuculline metho- The association rate of 20 nM [3H]muscimol in bullfrog chloride) might identify more GABAA receptor subtypes in membrane preparations at 4 jC was extremely rapid, reach- the bullfrog brain.
ing steady state in just over 2 min. This is very similar to The density of [3H]muscimol binding sites (Bmax = 1.8 [3H]muscimol association seen in bovine brain preparations pmol/mg protein) in bullfrog brain membranes was similar at 25 jC Whether temperature affects [3H]muscimol to bovine cerebral cortex (3.5 pmol/mg protein), and codfish binding is still questionable as past work has yielded brain (2.73 pmol/mg protein) Furthermore, the contrasting results Dissociation of [3H]muscimol in number of [3H]muscimol binding sites was similar to the the presence of unlabeled GABA was also quite rapid. This number of binding sites observed with [3H]GABA (1.6 short displacement time ( < 5 min) is also found in the pmol/mg protein) in the same species [3H]GABA mammalian brain, in the presence of excess unlabeled can theoretically bind to all three classes of GABA receptors GABA or muscimol while [3H]muscimol should bind only to the GABAA class.
The affinity of the bullfrog [3H]muscimol binding site The similarity in the number of sites detected with these two was similar to that of other GABAA receptors. Binding of ligands suggests that the GABAA class constitutes the vast [3H]muscimol in bullfrog brain preparations revealed a majority of GABA receptors in bullfrog brain. The concen- single, high affinity binding site with a KD of 19.2 nM, tration of [3H]muscimol binding sites in R. esculenta was which is in the range considered to indicate a high affinity also estimated with in vitro quantitative autoradiography site This KD is consistent with muscimol's greater The range of concentrations across brain areas was affinity for the GABAA receptor than GABA, as seen in within an order of magnitude of our estimate from bullfrog mammals The binding affinity of [3H]GABA in whole brain homogenates. In contrast, there are markedly bullfrog brain (58 nM) and spinal cord (33 nM) membrane lower numbers of [3H]GABA binding sites observed in preparations is lower The high affinity binding of brain membranes of salmon (16.6 – 41.4 fmol/mg protein) [3H]muscimol in bullfrogs is similar to the affinity in rat (13 and 42 nM), bovine (Bos taurus; 10 nM), and codfish The GABAA receptor agonists and antagonists inhibited (Gadus morrhua; 13.5 nM) brain Thus, the [3H]muscimol specific binding in a concentration-dependent high affinity binding site of the GABAA receptor appears manner. The rank order of potency was muscimol>GABA> conserved in vertebrate evolution.
bicuculline methiodide>bicuculline. GABA is also a more Binding of [3H]muscimol to one versus two classes of potent inhibitor than bicuculline when [3H]GABA is the binding sites varies with species and assay conditions. Our labeled ligand This rank order is similar to that seen in procedure reveals high and low affinity [3H]muscimol rat, bovine, and codfish brain membranes, with muscimol binding sites in rat membrane preparations Visual being the most potent competitor followed by GABA and examination of the Scatchard replot (inset) suggests bicuculline methiodide and/or bicuculline The that bullfrogs might also have two binding sites. However, EC50 of bicuculline methiodide in bullfrog brain (13.2 the presence of only a single, high affinity binding site was AM) was very similar to that of the codfish (15.6 AM) determined using computer assisted nonlinear regression However, both muscimol and GABA had higher EC50s analysis Only one class of binding site was also found than observed in other vertebrates. Finally, in bullfrogs, the in bullfrog brain when [3H]GABA was used as the ligand mammalian GABAB receptor agonist baclofen did not One high-affinity binding site for [3H]muscimol is inhibit [3H]muscimol binding. This supports the hypothesis present in codfish as well as rat under some assay that [3H]muscimol binding in bullfrog brain is to a GABAA- conditions Two classes of [3H]muscimol or [3H]GABA like receptor, rather than a GABAB-like receptor The binding sites are more typically found in rat, bovine and pharmacological properties of the GABAA-like receptor in D.M. Hollis, S.K. Boyd / Brain Research 992 (2003) 69–75 the bullfrog brain are thus similar to receptors in other [3H]Ro 15-1788 binding sites to brain membrane of the saltwater vertebrates. Species differences in EC Mugil cephalus, Comp. Biochem. Physiol C Toxic. Pharmacol. 128 50s may indicate that (2001) 291 – 297.
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