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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2008 Jan 21;153(6):1195–1202. doi: 10.1038/sj.bjp.0707681

The role of GABAA receptors in the control of transient lower oesophageal sphincter relaxations in the dog

H Beaumont 1,*, A-C Jönsson-Rylander 2, K Carlsson 2, S Pierrou 2, M Ahlefelt 2, L Brändén 2, J Jensen 2, G E Boeckxstaens 1, A Lehmann 2
PMCID: PMC2275437  PMID: 18204479

Abstract

Background and purpose:

Transient lower oesophageal sphincter relaxations (TLESRs) are triggered by activation of mechanosensitive gastric vagal afferents and are the major cause of gastroesophageal reflux and therefore an important target for therapeutic intervention in gastroesophageal reflux disease (GERD). Activation of the metabotropic GABAB receptor has shown to inhibit TLESRs. The aim of the present study was to assess the role of the ionotropic GABAA receptor in the regulation of TLESRs.

Experimental approach:

TLESRs were quantified using Dentsleeve manometry in dogs, and GABAA agonists were given i.v. prior to gastric distension. Immunohistochemistry and RT-PCR were used to localize GABAA receptors in the dog nodose ganglion, the source of vagal afferents which initiate TLESRs.

Key results:

The prototypical GABAA agonist muscimol produced a dose-dependent inhibition of TLESRs ranging from 19 to 56%. The two other GABAA agonists evaluated, isoguvacine and 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP), as well as the GABAA positive allosteric modulator diazepam, had no major effects on TLESRs. Evaluation of higher doses was limited by emesis (THIP and isoguvacine) or restlessness/sedation (diazepam). Of the predominant GABAA receptor subunits (α, β and γ components), α and β but not γ were detected in the dog nodose ganglion by RT-PCR, while immunohistochemistry in addition demonstrated nerve fibres expressing the γ subunit.

Conclusions and implications:

The present observations demonstrate that GABAA receptors exert an inhibitory control of TLESRs. These results warrant further studies using GABAA isoform-selective agonists to define the identity of receptors involved.

Keywords: gastroesophageal reflux disease, GABAA, muscimol, TLESR, dogs

Introduction

Gastroesophageal reflux disease is one of the most common disorders of the gastrointestinal tract and is characterized by symptoms of heartburn, regurgitation and retrosternal pain (McDougall et al., 1998; Valle et al., 1999). Transient lower oesophageal sphincter relaxation (TLESR) is the major mechanism underlying gastroesophageal reflux, both in healthy volunteers and in gastroesophageal reflux disease (GERD) patients (Holloway, 2000). Pharmacological inhibition of TLESRs is therefore a potential target for the treatment of GERD. TLESRs are triggered by gastric distension, leading to a vagally mediated reflex pathway involving mechanosensitive gastric vagal afferents, integrative brainstem centres and vagal efferents to the lower oesophageal sphincter (LES) (Mittal et al., 1995).

γ-Aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the central nervous system, acting through either ionotropic GABAA and GABAC or metabotropic GABAB receptors. The GABAA/C receptors are ligand-gated ion channels, which induce rapid synaptic inhibition upon activation. This contrasts to the GABAB receptor, which couples to G-proteins and produces longer lasting inhibitory signals (Berthele et al., 2001). Inhibitory GABAB receptors are present on gastric mechanoreceptors (Smid et al., 2001) and on vagal afferent terminals in the dorsal medulla, and have shown to inhibit transmitter release in vagal nuclei (Brooks et al., 1992). Previous studies showed that the GABAB-receptor agonist baclofen significantly reduces the rate of TLESRs in dogs, ferrets and humans (Blackshaw et al., 1999; Lehmann et al., 1999; Lidums et al., 2000; Zhang et al., 2002). Baclofen also reduces the number of reflux episodes and reflux symptoms in GERD patients (Ciccaglione and Marzio, 2003; Koek et al., 2003; Vela et al., 2003). However, the side-effect profile of baclofen makes it less attractive for clinical use. It is unknown if ionotropic GABAA receptors play a role similar to that of GABAB receptors.

γ-Aminobutyric acid-A receptors are also widely expressed in the central and peripheral nervous systems, and mediate fast postsynaptic inhibition. The GABAA receptor complex is a pentameric assembly of subunits forming a chloride channel and, depending on subunit configuration, benzodiazepine, barbiturate and neuroactive steroid sites (Mehta and Ticku, 1999). The various subunits of the GABAA receptors are α1–α6, β1–β3, γ1–γ3 and δ (Macdonald and Olsen, 1994). Each GABAA receptor consists of a combination of various subunits. Coexpression of α-, β- and γ-subunits is required for the formation of a fully functional GABAA receptor (Saha et al., 2001). GABA and other directly acting GABAA-receptor agonists bind specifically to a recognition site located between an α and a β subunit, whereas the benzodiazepine ligands bind to an allosteric site located between an α- and a γ-subunit (Ebert et al., 1994; Krogsgaard-Larsen et al., 2004). However, for the formation of the GABAA receptor, colocalization of these three types of subunits is not an absolute requirement. It is known that mice lacking the γ2-subunit were able to express functional GABAA receptors, but were lacking the benzodiazepine-binding site (Gunther et al., 1995). Other studies on recombinant GABAA receptors also showed that the combination of the α1- and β2-subunits produces functional GABAA receptors, but the γ2-subunit is required to express benzodiazepine binding (Pritchett et al., 1989; Pregenzer et al., 1993). Positive GABAA modulators, like benzodiazepines, facilitate GABA-mediated Cl− flux and have sedative, anxiolytic and anticonvulsant effects.

γ-Aminobutyric acid-A agonism has been found to excite (Zagorodnyuk et al., 2002), inhibit (Yuan et al., 1998) or have no effect (Smid et al., 2001) on vagal afferents, the key initiators of TLESRs. Whether these contradictory results are due to species or methodological differences is unknown, but they warrant studies on the effects of peripherally restricted agonists on TLESRs. On the other hand, GABAA receptors mediate inhibition in the dorsal vagal complex, the central relay station translating afferent signalling into efferent firing producing TLESRs. It can therefore be speculated that central GABAA agonism may modify the peripheral actions of GABAA agonists.

These hypotheses were tested by assessing the potential for peripheral agonistic effects by determining GABAA receptor subunit expression in the dog nodose ganglion (NG), the origin of vagal afferents. Furthermore, to characterize peripheral and central involvement of GABAA receptors in the regulation of TLESRs in dog, the effects of two centrally acting GABAA agonists, muscimol and THIP (4,5,6,7-tetrahydroisoxazolo [5,4-c] pyridine-3-ol), and the non-selective, peripherally acting agonist isoguvacine, as well as the positive GABAA modulator diazepam were studied. The selection of a benzodiazepine and THIP was based on the finding that the former selectively enhances the function of synaptic α1β2γ2S GABAA receptors, whereas the latter preferentially activates extrasynaptic α4β3δ GABAA receptors (Krogsgaard-Larsen et al., 2004).

Materials and methods

Animals

All animal procedures were approved by the Ethical Committee for Animal Experiments of the Göteborg region. Adult male and female Labrador retrievers were used in the experiments. Cervical oesophagostomies were made and the dogs were accustomed to rest in a Pavlov stand after recovery from surgery. Before each experiment, the dogs were fasted overnight, with free access to water. A washout of at least 3 days was allowed between experiments.

Protocol for in vivo experiments

The dogs were intubated with a water-perfused Dentsleeve multilumen catheter to record gastric, LES and oesophageal pressures. An antimony pH electrode was placed 3 cm above the lower oesophageal sphincter to measure acid reflux episodes, and a water-perfused catheter was placed in the hypopharynx to measure swallows.

Transient lower oesophageal sphincter relaxations were stimulated by infusion into the stomach through the central lumen of the assembly of a acidified liquid nutrient (30 ml kg−1), followed by air insufflation (100 ml min−1) to maintain gastric pressure between 9 and 11 mm Hg.

The number of TLESRs was measured during a 45-min period starting from the infusion of the liquid. TLESRs were defined by a rapid decrease of LES pressure (>1 mm Hg s−1) to a value <2 mm Hg above gastric pressure and a duration of >1 s, without any pharyngeal signal, <2 s before onset (Lehmann et al., 1999).

Muscimol, isoguvacine and diazepam were administered as intravenous boluses (muscimol and isoguvacine 0.5 ml kg−1; diazepam 1 mg kg−1, containing 5 mg ml−1) 10 min before start of the experiment. THIP was administered according to an infusion protocol of 1 ml kg−1 infused over 30 min. The infusion started 15 min before the infusion of liquid into the stomach. Except for TLESRs and acid-reflux episodes, basal LES pressure, duration of TLESR, latency time from start to first TLESR and swallowing rate were determined. Basal LES pressure was defined as the average pressure between the sleeve and the intragastric pressure during the 45-min period. All swallow- and TLESR-related LES pressure changes were excluded.

Acid exposure was expressed as the percentage of the 45-min period during which oesophageal pH was <4. Reflux episodes were defined as a drop in pH >1 unit within 5 s. The average pH in the 15 s following nadir pH should be <4.

Immunohistochemistry

Primary antibodies

Various antibodies used against different GABAA receptor subunits were as follows: mouse monoclonal anti-GABAA-α (Biosite AB, Täby, Sweden), mouse monoclonal anti-GABAA-α1 (Chemicon International Inc. and Boehringer, Ingelheim, Germany), goat polyclonal anti-GABAA-α2 and anti-GABAA-α3 (Santa Cruz Biotechnology Inc.), mouse monoclonal anti-GABAA-β (Chemicon International Inc., Temecula, California, USA) and rabbit polyclonal anti-GABAA-γ2 (Chemicon International Inc.).

Immunolabelling procedure

Fresh dog brain tissue and dog nodose ganglia were harvested and fixed in formaldehyde (formalin 10%) for at least 12 h. The tissue was dehydrated in increasing concentrations of alcohol and xylene, and then embedded in paraffin and sectioned at 4 μm using a microtome (Leica Microsystems, Wetzlar, Germany). Tissue sections were deparaffinized and rehydrated through xylene, graded ethanol series, distilled water and phosphate-buffered saline (PBS). Different pretreatments tried as antigen retriever were as follows: no pretreatment, trypsin, high-pressure boiling with Diva or Borg solution in a Decloaker (Biocare Medical, CA, USA) or microwave boiling with a citrate buffer solution (0.01 M, pH 6). Sections were then incubated for 5 min in 3% hydrogen peroxidase (Merck, Darmstadt, Germany) in distilled water, washed twice in PBS and preincubated with 10% normal donkey serum in PBS for 20 min at room temperature, and then incubated with primary antibodies to one of the GABAA receptor subunits. The antibodies were diluted in ChemMate antibody diluent (Dako Cytomation, Glostrup, Denmark) 1:25 (α and γ) and 1:75 (β), and incubations were performed overnight at room temperature. The sections were washed three time in PBS and then incubated in biotinylated donkey anti-rabbit, anti-mouse or anti-goat IgG (Jackson Immunoresearch, West Grove, PA, USA; dilution 1:500) for 1 h at room temperature. After washing the sections three times in PBS, the avidin–biotin complex (ABC) solution (Vectastain Elite Standard kit; Vector Laboratories, Burlingame, CA, USA) was applied for 45 min at room temperature. Following rinsing in PBS, immunoreactivity was visualized using 3-amino-9-ethylcarbazole for 10–20 min at room temperature. Finally, sections were counterstained with Mayer's haematoxylin and coverslipped using a water-based mounting media (Quick Mount, Daido Sangyo, Japan).

Negative controls included sections that were incubated in the presence of negative control immunoglobulin fraction from non-immunized rabbits or goats in the same dilution as the primary antibodies, or as antigen–antibody preabsorption experiments with the native antigen preincubated at 4 °C for 24 h with the diluted antibody solution. Dog cerebellum was used as positive control.

Images were captured with the image programme Picsara (Euromed Networks, Stockholm, Sweden) from the light microscope, using a Sony 3CCD video camera. The images were imported into Adobe Photoshop or Microsoft Photo Editor for minor adjustments of brightness, contrast and sharpness.

Reverse transcription-PCR

Nodose ganglion and cerebellum (positive control) were taken from the same dog. RNA was prepared (TRIzol reagent; Invitrogen, Carlsbad, California, USA) and reverse transcribed into cDNA using oligo(dT) and random primers (iScript cDNA Synthesis kit; BioRad, Hercules, California, USA). The cDNA was amplified by reverse transcription-PCR (RT-PCR) (Taqman Universal PCR Master Mix; Applied Biosystems, Foster City, California, USA) using primer pairs and probes for GABAA receptor subunits α1, β2, β3 and γ1 (Eurogentec SA, Seraing, Belgium). All primer sequences are listed in Table 1. Real-time PCR was performed using a 7500Real Time PCR System (Applied Biosystems). Product specificity of the PCR products was confirmed by agarose gel electrophoresis.

Table 1.

Primers used in PCR experiments

Subunit Primer sequence Primer length
α1f 5′-AATTTGGCCAGGGGTGAC-3′ 18
α1r 5′-GAAAGCTATTCTTGACAGTCGGTC-3′ 24
α1 probe 5′-TGGCTTAGCCACGATTGCTAAAAGTGC-3′ 27
β2f 5′-GATGTGGAGAGTCCGGAAAAAG-3′ 22
β2r 5′-CTTTCAGGAGTCTATCCACTGTCTCTT-3′ 27
β2 probe 5′-TGTGCGCAGAGTGTCAATGACCCTAGTAA-3′ 29
β3f 5′-CAAAGAATGACCGTTCCAAG-3′ 20
β3r 5′-TGAGTTGTCAAAGGGTCGTG-3′ 20
β3 probe 5′-CATGAGCATCCACCCGATTGC-3′ 21
γ1f 5′-CAACAAACTTCGCCCAGATA-3′ 20
γ1r 5′-TTTAAACGACTGTCAAACCAGG-3′ 22
γ1 probe 5′-TGGGATCAACTGGTCCAATGCTG-3′ 23
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f=forward; PCR=polymerase chain reaction; r=reverse.

Data analysis

Each dog served as its own control. All variables were calculated on the basis of the mean of five preceding control experiments. Data are presented as mean±s.e.mean. Statistical analysis was performed using paired Student's t-tests. A P-value <0.05 was considered statistically significant.

Drugs

All compounds were from Tocris, Bristol, UK, and were dissolved in physiological saline (0.9% NaCl), except for diazepam, which was from Dumex-Alpharma, Copenhagen, Denmark, and was delivered as a solution, which was directly used at a dose of 3.5 μmol kg−1 (1 mg kg−1). The doses of muscimol, which were tested, were 8.2 μmol kg−1 (1 mg kg−1), 0.82 μmol kg−1 (0.1 mg kg−1), 0.27 μmol kg−1 (0.033 mg kg−1) and 0.082 μmol kg−1 (0.01 mg kg−1). A dose of 8.2 μmol kg−1 (1.3 mg kg−1) and 5.7 μmol kg−1 (1 mg kg−1) was used for isoguvacine and THIP, respectively.

Results

In vivo experiments

TLESRs

The GABAA agonist muscimol produced a dose-dependent inhibition of TLESRs over the range of doses used (Figure 1; n=6 for all doses). Surprisingly, at the lowest and highest dose of muscimol no side effects occurred, whereas at the intermediate doses emesis occurred just after drug administration. No sedative effects were seen after muscimol. The two other GABAA agonists evaluated, isoguvacine (8.2 μmol kg−1) and THIP (5.7 μmol kg−1), as well as the GABAA-positive allosteric modulator, diazepam (3.5 μmol kg−1), had no major effect on TLESRs at the doses tested (isoguvacine, 0±12% change; THIP, 15±9% inhibition; diazepam, 29±12% inhibition; Figure 2), and were limited by emesis (isoguvacine and THIP) or restlessness and sedation (diazepam).

Figure 1.

Figure 1

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Dose-dependent effect of muscimol on the occurrence of TLESRs. The effect is expressed in mean values (a) and as percent inhibition of control (b). The number of TLESRs was significantly reduced at all doses except the lowest dose of muscimol. P-values are shown above each set of experiments (paired Student's t-test; n=6 for each dose). TLESR, transient lower oesophageal sphincter relaxation.

Figure 2.

Figure 2

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Isoguvacine (a), THIP (b) and diazepam (c) had no significant effect on the number of TLESRs. THIP, 4,5,6,7-tetrahydroisoxazolo [5,4-c] pyridine-3-ol; TLESR, transient lower oesophageal sphincter relaxation.

Basal LES pressure

Basal LES pressure was variably affected by muscimol (Figure 3), reaching statistical significance only after one intermediate dose (0.82 μmol kg−1; P<0.04). The other compounds showed no significant effect on basal LES pressure.

Figure 3.

Figure 3

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Effect of muscimol on basal LES pressure (LESp). The effects were small and highly variable; only at 0.82 μmol kg−1 was there a significant increase in LESp (P<0.04; paired Student's t-test; n=6 for each dose). LES, lower oesophageal sphincter.

Reflux and other parameters

Reflux episodes were significantly reduced by 25±8.8% after 0.27 μmol kg−1 muscimol (controls 3.2±0.37; muscimol 0.27 μmol kg−1 2.4±0.4; P<0.03). The other doses of muscimol, isoguvacine, THIP and diazepam showed no significant effect on reflux episodes (data not shown). Swallowing rate and latency time to the first TLESR were not significantly affected by any of the tested compounds (data not shown).

Immunohistochemistry

We were unable to detect any immunoreactivity, in either NG or cerebellum, with the GABAA-α and -α1-specific antibodies. Immunoreactivity of the GABAA-α2 and -α3 subunits was observed scattered throughout the dog NG, where it was localized in the cell bodies (Figure 4). The immunoreactivity in the cerebellum (positive control tissue) of GABAA-α2 and -α3 was found in the granular cell layer and in the fibres of the white matter. The β-subunit was intensely found in the granular cell layer of the cerebellum, but not in the NG. GABAA-γ2 immunoreactivity in the NG was confined to fine varicose fibres surrounding the somata. This staining was not seen using the IgG-negative controls (Figure 5). In the cerebellum, weak γ2 immunoreactivity was found in the Purkinje cells and some staining was seen in the endothelial cells of the brain and NG.

Figure 4.

Figure 4

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(a) Photomicrograph showing the dog NG, with cell bodies staining positively for GABAA-receptor α3-subunits. (b) Preabsorption control experiments for GABAA α3-subunits. Scale bar=50 μm. GABAA, γ-aminobutyric acid-A; NG, nodose ganglion.

Figure 5.

Figure 5

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(a) Photomicrograph showing the dog NG, with fibres staining positively for GABAA-receptor γ2-subunits (arrows). (b) IgG control experiment for GABAA γ2-subunits. Scale bar=20 μm. GABAA, γ-aminobutyric acid-A; NG, nodose ganglion.

Reverse transcription-PCR

γ-Aminobutyric acid-A subunits α1, β2 and β3, but not γ1, were detected in the dog NG by PCR analysis. mRNA expression of these GABAA subunits in the dog cerebellum was used as positive control (Figure 6).

Figure 6.

Figure 6

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Results of RT-PCR, with the different GABAA receptor subunits expressed in dog NG (a) and cerebellum as positive control (b). There is abundant expression of β3-subunits, but lack of γ1-subunit expression in the dog NG. GABAA, γ-aminobutyric acid-A; NG, nodose ganglion; RT-PCR, reverse transcription-PCR.

Discussion

In the present study, we investigated the effects of GABAA agonists on the occurrence of TLESRs and the expression of GABAA subunit receptors in the NG of the dog. This study showed a significant and dose-dependent inhibition, up to 56%, of TLESRs by the centrally acting GABAA agonist muscimol in the dog. This suggests that the use of GABAA-receptor agonists may be a novel strategy in the treatment of GERD. However, muscimol was less effective in reducing TLESRs than the GABAB-receptor agonist baclofen, which previously showed an up to 90% reduction of postprandial TLESRs in dogs (Lehmann et al., 2002). The maximal effect of baclofen has been reproduced in several independent studies in our laboratory (Lehmann et al., unpublished observations), and so it was not deemed necessary to include a separate baclofen dose group in the current study. If the difference between muscimol and baclofen in terms of efficacy can be generalized to their respective targets, it appears that GABAB-receptor agonists would offer a more attractive option than GABAA-receptor agonists in the quest for novel drugs for treatment of GERD.

Muscimol has been reported to inhibit TLESRs in ferrets, but only at a dose (10 μmol kg−1), which induced sedation (Blackshaw et al., 1999). In the current experiments, reduction of TLESRs could not be secondary to sedation since this side effect was not seen at the doses used. In contrast to the effect of muscimol, the less potent GABAA agonist THIP (Kemp et al., 1986) and the peripherally restricted GABAA agonist isoguvacine (Krogsgaard-Larsen et al., 1981) showed no effect on the occurrence of TLESRs. The emetogenic effects of both THIP and isoguvacine limited further studies with higher doses. THIP has been administered in baboons at a dose ranging from 0.25 to 8.0 mg kg−1, intravenously (Meldrum and Horton, 1980). However, in our study, emesis occurred at doses higher than 1 mg kg−1, even in the absence of intubation and administration of a high gastric load. THIP has potent anxiolytic and analgesic properties in man, but is known to have approximately 8–10 times lower affinity for GABAA receptors compared with muscimol (Meldrum and Horton, 1980; Huckle, 2004; Vyazovskiy et al., 2005). Isoguvacine has poor penetration into the CNS (Rode et al., 2005) and acts as a peripherally restricted GABAA agonist (Krogsgaard-Larsen et al., 1981). However, the hypothesis that a peripherally restricted agonist would augment or reduce the number of TLESRs by activating or inhibiting vagal afferents could not be confirmed in our study. This may suggest that a central but not peripheral site of action of GABAA agonists plays a critical role in the control of TLESRs. Although there was a tendency towards inhibition of TLESRs, the positive GABAA modulator diazepam had no statistically significant effect. In analogy with mice devoid of the γ2-subunit, the lack of γ2-subunit expression in vagal afferents in our study may render them insensitive to benzodiazepines (Gunther et al., 1995; Mehta and Ticku, 1999), and therefore an effect of diazepam on the rate of TLESRs through binding to vagal afferent terminals might be not anticipated.

In addition, general sedation is thought to reduce the number of TLESRs (Dent et al., 1980; Cox et al., 1988). In our study, however, sedation occurred with diazepam, but no significant reduction in TLESRs was seen. It should be noted that the onset of sedation in the dogs followed a period of restless behaviour, and any effect of sedation on TLESRs could be masked. However, TLESRs were seen throughout the total study time after diazepam, and they peaked during the first 20 min as in control experiments. It would be important to assess whether there is a GABAA tone controlling TLESR by using receptor antagonists. However, GABAA-receptor antagonists can trigger epileptic seizures and ethical considerations prevent such experiments in dogs.

Muscimol has no effect on basal LES pressure in ferrets (Blackshaw et al., 1999), but it slightly increased LES pressure in dogs. This effect is likely to be mediated in the hindbrain, since muscimol has been shown to elevate basal LES pressure in cats upon hindbrain microinjection (Washabau et al., 1995). In the management of GERD, stimulation of LES pressure is considered to have a beneficial effect in patients who display long periods of low or no LES pressure, which possibly promote reflux. There were discrepancies between the effect of muscimol on TLESRs and acid reflux episodes, since the two highest doses significantly attenuated TLESRs but not reflux. However, this difference is probably more apparent than real, as the number of reflux episodes was quite low and changes were therefore difficult to identify. Also, the pHmetric method used failed to detect superimposed reflux,that is, reflux occurring shortly after a preceding reflux episode, which already has acidified the distal oesophagus.

The distribution of various GABAA subunits in the NG has to our knowledge not been described before in any species, including the dog. Interestingly, GABA itself has been detected immunocytochemically in the feline NG (Stoyanova, 2004). Its function there, if any, is obscure. Immunohistochemical studies on GABAA subunits in peripheral ganglia have been performed in a few cases. For instance, in situ hybridization showed expression of α2, β2 and γ2 in dorsal root ganglia of rats (Persohn et al., 1991) and all GABAA receptors subunits were detected in rat spinal ganglia by immunohistochemistry (Yamamoto et al., 2002). Although the present study focused on another peripheral ganglion, the NG, published data support the presence of GABAA-receptor subunits in peripheral ganglion cells. We showed immunoreactivity with the α2- and α3-subunits in the cell bodies of the dog NG, and with γ2 in pericellular fibres. In line with this, additional RT-PCR showed α1, β2 and β3, but no γ1-subunit expression in the dog NG. We were unable to detect any immunoreaction with the α- and α1-specific antibodies in our paraffin-embedded material. This is surprising as the predominant GABAA-receptor subunit combination throughout the brain is composed of α1β2/3γ2 (Fritschy and Mohler, 1995; Mohler et al., 1995; Nusser et al., 1995). The fact that most other studies have been performed on frozen material could explain this difference in observed immunoreactivity. Presence of the α1-subunit was nevertheless indicated by RT-PCR, and we can therefore speculate that several α-subunits are present in the NG along with β-subunits, especially β-3, according to the RT-PCR experiments. However, our immunohistochemical studies did not demonstrate convincing β2 staining in the NG. A possible explanation for this could be the fact that the antibodies used were not directed against dog GABAA. An interesting finding is the immunohistochemical localization of the γ2-subunit in pericellular nerve fibres rather than in the ganglion cells of the NG. Previous studies have demonstrated the presence of such pericellular fibres in the NG of the monkey, rabbit and pigeon (Katz and Karten, 1980; Ling et al., 1992). Also GABAB receptor-1a immunoreactivity has been found on fibres surrounding the nerve cell bodies in guinea pig NG (Zagorodnyuk et al., 2002).

In conclusion, the present study revealed the expression of GABAA receptor subunits in the dog NG. In addition, we showed the involvement of GABAA receptors in the control of TLESRs. The GABAA-receptor agonist muscimol reduced the rate of TLESRs by 19–56% depending on dose. The potential of GABAA agonists as inhibitors of TLESRs, and therefore as future anti-reflux agents, depends on their therapeutic margin. Whereas the currently available GABAA-stimulating drugs registered for other indications carry a side-effect profile not compatible with their use in GERD, emerging GABAA subunit-selective compounds (Basile et al., 2004) may be useful in this context. Therefore, further studies are warranted in which the effects of GABAA subtype-selective compounds on TLESRs are assessed.

Acknowledgments

This study was supported by AstraZeneca, Mölndal, Sweden and was part of a collaboration between AstraZeneca and the Department of Gastroenterology of the Academic Medical Centre, Amsterdam, The Netherlands.

Abbreviations

GERD

gastroesophageal reflux disease

LES

lower oesophageal sphincter

PBS

phosphate-buffered saline

THIP

4,5,6,7-tetrahydroisoxazolo [5,4-c] pyridine-3-ol

TLESR

transient lower oesophageal sphincter relaxation

Conflict of interest

ACJR, KC, SP, MA, LB, JJ and AL are all full-time employees of AstraZeneca. GEB has participated as an investigator in clinical studies sponsored by AstraZeneca.

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