Nerve-Gut Research Laboratory, Level 1 Hanson Centre, Adelaide SA 5000, Australia
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ABSTRACT |
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GABAB-receptor (GABABR) agonists reduce
transient lower esophageal sphincter relaxation (TLESR) and reflux
episodes through an action on vagal pathways. In this study, we
determined whether GABABR are expressed on vagal afferent
neurones and whether they modulate distension-evoked discharge of vagal
afferents in the isolated stomach. Vagal mehanoreceptor activity was
recorded following distensions of the isolated ferret proximal stomach
before and after perfusion with the GABABR-selective
agonists baclofen and 3-aminopropylphosphinic acid (3-APPiA).
Retrograde labeling and immunohistochemistry were used to identify
GABABR located on vagal afferent neurones in the nodose
ganglia. Vagal afferent fibers responded to isovolumetric gastric
distension with an increase in discharge. The
GABAB-receptor agonists baclofen (5 × 105 M) and 3-APPiA (10
6 to
10
5 M) but not muscimol (GABAA-selective
agonist: 1.3 × 10
5 M) significantly decreased
afferent distension-response curves. The effect of baclofen (5 × 10
5 M) was reversed by the GABAB-receptor
antagonist CGP 62349 (10
5 M). Over 93% of retrogradely
labeled gastric vagal afferents in the nodose ganglia expressed
immunoreactivity for the GABABR. GABABR
expressed on vagal afferent fibers directly inhibit gastric mechanosensory activity. This is likely a contributing mechanism to the
efficacy of GABAB-receptor agonists in reducing TLESR and reflux episodes in vivo.
gastric mechanoreceptors; vagus nerve
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INTRODUCTION |
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THE PROXIMAL STOMACH PLAYS an important physiological role in the venting of air from the stomach (i.e., belching). Additionally, the movement of acidic gastric contents into the distal esophagus during periods of gastroesophageal reflux is allowed to occur through transient lower esophageal sphincter relaxation (TLESR) generated in response to signaling from the proximal stomach. Distension-evoked TLESR occurs via vagovagal pathways (23), allowing the formation of a common cavity and facilitating retrograde movement of gastric contents. The evidence that the proximal stomach is pivotal in the generation of TLESR arises from gastric compartmentalization studies (11) and the efficacy of fundoplication in reducing distension-evoked TLESR (29).
The GABAB-receptor agonist baclofen has been shown to reduce the incidence of TLESR in dogs (19) and to reduce both TLESR rates and reflux episodes in ferrets (8) and humans (20). Thus GABAB-receptor agonists may have therapeutic potential in the treatment of gastroesophageal reflux disease. Within the context of gastric vagovagal reflexes, the mechanisms and sites of action of baclofen are uncertain. GABABR are distributed centrally in brain stem nuclei involved in gastrointestinal regulation, such as the nucleus of the solitary tract (NTS) and dorsal motor nucleus of the vagus (DMV) (22), including DMV neurons projecting to the ferret lower esophageal sphincter (LES) (24). Central application of baclofen has been shown to inhibit NTS neurons receiving gastric vagal inputs (34) in addition to inhibiting vagal efferent responses to gastric distension (26). In the periphery, GABABR inhibit vagal motor pathways to the LES (7, 30) but do not alter the relaxation profile of the LES during TLESR (19, 20), suggesting minimal physiological significance of this coupling to motor function. With regard to sensory innervation of the upper gastrointestinal tract, baclofen selectively inhibits vagal esophageal mechanoreceptor responses to stretch in vitro (25) and gastric tension receptor responses to filling in vivo (26). This suggests that part of the effects of baclofen may be mediated through an inhibition of sensory signaling via the vagus nerve. However, studies are yet to investigate the influence of GABABR on the function of gastric vagal mechanoreceptors in a controlled environment and in response to physiological stimuli. Given their importance in mediating distension-evoked gastric reflexes, we aimed to characterize vagal afferent fiber responses to proximal gastric distension and investigate the effects of both GABAA and GABABR on vagal mechanosensory activity in an intact isolated preparation of the ferret stomach. To determine the anatomical localization of GABABR on vagal afferent neurones innervating the proximal stomach, combined retrograde labeling and immunohistochemical studies were performed.
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MATERIALS AND METHODS |
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Animal preparation. Experiments were performed on adult ferrets of either sex weighing 500-800 g. All studies were performed in accordance with the guidelines of the Animal Ethics Committee of the Royal Adelaide Hospital. Animals had free access to water and a standard diet but were fasted from food overnight before experimentation. Esophagogastric tissue was obtained from ferrets following exsanguination under general anesthesia (pentobarbital sodium, 60 mg/kg ip).
Isolated gastric preparation.
The esophagus and stomach were removed and placed in ice-cold Krebs
solution bubbled with carbogen (95% O2, 5%
CO2). The area encompassing the pylorus and antrum was
removed, and a large-bore cannula was inserted and secured with cotton
suture for air infusion and monitoring of intragastric pressure. The
vagus nerves were dissected away from their course down the esophagus
to a level ~1 cm from the esophagogastric junction. The proximal
esophagus was resected, and the remainder was tied off at the proximal
margin with cotton suture. The vagus nerves were carefully cleared of sheathing, and each nerve was then tied with a length of cotton suture.
The preparation was placed on its side and secured in a custom-designed
acrylic 30-ml organ bath (Danz Instrument Service, Adelaide, South
Australia). A thin layer of parafilm was placed on top of the stomach
to prevent drying of the uppermost portion. The preparation was
serosally perfused in carbogenated Krebs solution at 34°C of the
following composition (in mM): 118 NaCl, 25 NaHCO3, 4.6 KCl, 1.2 MgSO4, 1.3 NaH2PO4, 11 glucose, and 2.5 CaCl2. A relatively low organ bath
temperature was used, because this has previously been shown to inhibit
signs of deterioration in the isolated rat stomach (33).
Pretreatment with nifedipine (106 M) was used to inhibit
smooth muscle activity so that afferent responses did not occur as a
result of localized muscle contraction. At the end of experimentation,
the stomach was inspected for signs of edema, then opened
longitudinally, and inspected for signs of mucosal sloughing. None of
the preparations used were found to have such evidence of
deterioration. In our hands, experiments were performed for up to
8 h without macroscopic evidence of deterioration.
Nerve fiber recordings, data acquisition, and analysis. The left or right vagus nerve was drawn through a small hole in the main organ bath chamber via the cotton tie and into an isolated recording chamber filled with paraffin oil. Under a dissecting microscope, a longitudinal incision was made in the nerve sheath, exposing free fibers. With the use of fine forceps, nerve fibers were teased onto platinum recording electrodes for measurement of isolated nerve activity. The reference electrode was connected to a thin section of nerve connective tissue.
The amplified signals of neural activity and intragastric pressure were acquired to computer hard disk following digitization through a micro1401 interface (CED, Cambridge, UK). On-line acquisition and off-line single-unit discrimination were performed using Spike 2 software (version 3.16, CED). Gastric pressure was recorded using a Sorenson Transpac transducer (Abbott Laboratories, Chicago, IL) before being amplified (Polygraf, Synectics Medical, Stockholm, Sweden) and digitized through the micro1401 interface. Changes in unit discharge frequency and intragastric pressure were measured as the difference in mean values recorded during the drug distension period and the equivalent period immediately before distension.Protocol: distension and drug treatments. Distension volumes were administered via the pyloric catheter and were scaled to the body weight of the individual ferret, with volumes of 2, 5, and 10 ml used for the maximal (800 g) ferret body weights. Distension-response curves were constructed following noncumulative inflation of the isolated stomach (2-10 ml) for 20 s, with a 3-min period between individual distensions. Units were identified as distension-sensitive mechanoreceptors via a combination of criteria including the presence of spontaneous discharge, increase in discharge in response to distension (5 ml air), and unit discharge in response to blunt serosal probing (blunt metal rod, 2-mm OD). Gentle probing of the gastric serosa was also used to localize afferent receptive fields.
Of the 16 preparations studied, each was tested with a single pharmacological intervention only [baclofen ± CGP 62349 (n = 7), 3-aminopropylphosphinic acid (3-APPiA) at mutiple concentrations (n = 5), or muscimol ± bicucculine (n = 4)] and served as its own control. Distensions were performed in triplicate for control preparations and following drug pretreatments. Distensions were also performed in the presence of nifedipine; thus all experimental in vitro gastric compliance measurements occurred in the presence of the L-type calcium-channel blockade. All drug perfusions were allowed to equilibrate with the organ bath for 10 min before unit responses to gastric distension were tested.Retrograde tracing and immunohistochemistry. Four male ferrets, weighing between 0.9 and 1.2 kg and fasted overnight preceding surgery, were anesthetized with isoflurane (2.5% in O2), and the proximal stomach was exposed by a midline laparotomy. A total of 50 µl of 0.5% cholera toxin B subunit conjugated to FITC (CTB-FITC; List Biological, Campbell, CA) was injected subserosally into the proximal stomach in 5-µl aliquots via a Hamilton microsyringe. Injections were made ~1-1.5 cm from the esophagogastric junction on both the anterior and posterior sides of the stomach. Gauze was then used to dry any leakage from the injection sites, the laparotomy was closed, and Teramycin was administered (200 mg/ml, 0.1 ml sc). All animals recovered well from surgery and were routinely monitored over 2 days. Survival time was 4 days, based on tracing kinetics reported previously (15), after which ferrets were anesthetized with urethane (1.25 g/kg ip) and perfused transcardially with 0.1 M PBS at 4°C (pH 7.4, 500 ml) and 10% formaldehyde at 4°C (1 liter). Left and right nodose ganglia and cerebellum were removed, postfixed, and cryoprotected in 30% sucrose at 4°C for 24-48 h. Frozen transverse sections at 20 µm were cut serially through the rostrocaudal extent of the nodose ganglia, and coronal sections taken from an ipsilateral quadrant of the cerebellum were placed onto silane-coated slides (silane 2%; 3-aminopropyl-triethoxy-silane; Sigma-Aldrich, New South Wales, Australia) in preparation for immunohistochemistry.
GABABR immunoreactivity (GABABRI) was detected using a guinea pig anti-rat GABABRI polyclonal primary IgG (Calbiochem-Novabiochem, Victoria, Australia) and a goat anti-guinea pig secondary IgG conjugated to Alexa Fluor 546 (Molecular Probes, Eugene, OR). The pan-GABABRI primary IgG was raised against a COOH-terminal sequence of GABABRI. Sections were dried at room temperature (10 min) and rinsed in PBS + 0.2% Triton X-100 (PBS-T; Sigma-Aldrich, pH 7.4) to facilitate antibody penetration. Sections were then treated with 10% normal goat serum (NGS) in PBS-T to saturate nonspecific binding sites. After incubation overnight with the GABABRI primary IgG (1:1,400 in 10% NGS/PBS-T), unbound antibody was removed with PBS-T, sections were incubated with the secondary goat anti-guinea pig IgG (1:200 in PBS-T), and it was washed again. Sections were drained and mounted with ProLong Antifade (Molecu the blocking solution served as negative controls.Visualization and quantification. Epifluorescent imaging of GABABRI was performed in the cerebellar sections to confirm the specificity and optimal conditions for the GABABRI primary IgG in the ferret, based on evidence for GABABRI localization in the rat cerebellum (28). High-power images were obtained on an Olympus BX-51 microscope equipped with an excitation filter for Alexa Fluor 546 (Olympus Imaging Unit, Biochemistry Dept., Univ. of Adelaide) and acquired to an Olympus DP11 digital camera. Images were imported unmodified into Microsoft PhotoEditor or Adobe PhotoShop software and adjusted for brightness and contrast.
Epifluorescent imaging of CTB-FITC and GABABRI in nodose ganglion sections was performed as for cerebellum, using specific excitation filters for FITC or Alexa Fluor 546, respectively. The comparative use of wide (BP460-490, BA510)- and narrow-band (BP460-490, BA510-550) FITC filters enabled us to discount sources of autofluorescence in nodose sections. Proximal stomach-innervating neurones were counted in six to eight whole ganglion profiles representative of the rostrocaudal extent of each ganglion. Labeled neurones were counted only for "central profile counts," indicative of completely labeled cells, whereas cell fragments were ignored (1). Total neuronal counts were also calculated in these representative sections, using differential interference contrast light microscopy to provide a basis for calculation of percent single- and dual-labeled neurones. Multiple high-power images from each representative section were acquired separately using FITC or Alexa Fluor 546 emission filters to the DP11 digital camera, and neurones were counted using Microsoft PhotoEditor software. Adobe PhotoShop software was then used to prepare separate images of CTB-FITC (green) and Alexa Fluor 546 (red)-labeled neurones to identify GABABRI-positive neurones innervating the proximal stomach.Statistical analysis. Data were expressed as means ± SE of a number of animals (n). Statistical analysis was performed using repeated-measures one- or two-way ANOVA, where appropriate, with group means compared using Bonferroni's multiple-comparison test (Prism 3.02, GraphPad, San Diego, CA). A P value of <0.05 was considered significant.
Drugs.
(±)-Baclofen was obtained from Research Biochemicals International
(Natick, MA). The concentrations of (±)-baclofen and 3-APPiA were
chosen on the basis of concentrations used in previous studies (7, 10, 25). Dr. A. Lehmann (AstraZeneca R&D, Molndal, Sweden) kindly provided the 3-APPiA and CGP 62349. Muscimol
hydrobromide and bicucculine methobromide were obtained from
Sigma-Aldrich (Sydney, Australia). All drugs were dissolved in saline.
(±)-Baclofen stock solution (102 M) was dissolved in
saline and HCl (10
4 M).
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RESULTS |
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Characteristics of vagal gastric mechanoreceptors.
A total of 16 proximal gastric distension-sensitive vagal afferent
fibers was studied. Units often displayed a regular discharge without
distension (Table 1: mean firing
frequency of all units = 1.23 ± 0.3 Hz). Spontaneous
frequency of discharge was generally greater for units located on or
near the LES. Vagal mechanoreceptors responded to air insufflation of
the proximal stomach with a volume-dependent increase in afferent
discharge that was linear over the range of 2-10 ml (Fig.
1). After removal of the distension
stimulus, a brief period of reduced firing was observed, followed by
resumption of spontaneous activity (Fig. 1). During gastric distension,
intragastric pressure increases were volume dependent and reached a
plateau at ~25 mmHg at the higher levels of distension (Fig. 1).
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GABABR-mediated effects on vagal mechanosensory fibers
and gastric compliance.
Superfusion of baclofen (5 × 105 M) in place of
regular Krebs solution elicited a 30% reduction in basal discharge
frequencies (Table 1, n = 7). Baclofen also
decreased vagal afferent fiber discharge in response to gastric
distension over the full range of volumes and maximally at 10 ml (Figs.
2B and 3, *P < 0.05 vs. control, n = 7). On addition of the
GABABR antagonist CGP 62349 (10
5 M), both
basal and distension-evoked discharge frequencies were significantly
reversed to levels similar to those observed in control preparations
(Table 1). Baclofen had no significant effect on the intragastric
pressure response to gastric distension (Table 2, n = 7). After
pretreatment with CGP 62349 (10
5 M), intragastric
pressure increases in this instance were significantly reduced vs.
control (Figs. 2B and 3, *P < 0.05, n = 7).
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GABAAR-mediated effects on vagal mechanosensory fibers
and gastric compliance.
The GABAAR agonist muscimol (1.3 × 105
M) significantly increased basal afferent discharge by 31% (Table 1,
P < 0.05 vs. control, n = 4). This was
not reversed by the GABAAR antagonist bicucculine, instead
resulting in a further increase in discharge frequency (10
5 M; P < 0.001 vs. control,
n = 4). Muscimol (1.3 × 10
5 M) had
no significant effect on the vagal afferent response to gastric
distension (Fig. 5, n = 4). Similarly, vagal afferent discharge in response to gastric
distension was also unaffected in the presence of both muscimol and
bicucculine (1 × 10
5 M; Fig. 5, n = 4). There were no significant differences in gastric pressure during
distension after pretreatment with either muscimol (1.3 × 10
5 M) or muscimol (1.3 × 10
5 M) and
bicucculine (1 × 10
5 M; Table 2, n = 4).
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Retrograde labeling and immunohistochemical localization of the
GABABR.
Injection of CTB-FITC into the proximal gastric serosa resulted in
dense but discrete cytoplasmic labeling of cell bodies within the
nodose ganglia (Fig. 6, C and
D). Cell counts revealed that 13.0 ± 2.9% of labeled
left nodose ganglion neurones and 9.4 ± 1.8% of labeled right
nodose ganglion neurones innervated the proximal stomach.
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DISCUSSION |
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In the present study, we have shown that GABABRs selectively inhibit both spontaneous activity and distension-evoked increases in gastric vagal afferent discharge. These findings support recent evidence demonstrating an inhibitory effect of baclofen on gastric mechanosensitive vagal afferent fibers in vivo (26) and stretch-sensitive esophageal vagal fibers in the ferret in vitro (25). In addition, we have demonstrated a similar effect of the potent GABABR agonist 3-APPiA on distension-evoked discharge and shown that GABABR may be localized anatomically on vagal afferent fibers innervating the stomach.
The effects of baclofen were reversed with the GABABR antagonist CGP 62349, indicating that the response was due specifically to the action of baclofen on GABABR. The GABABR agonist 3-APPiA, which is a 10- to 20-fold more potent agonist than baclofen (13), elicited a marked and steep concentration-dependent reduction in distension-evoked activity. Given the relative lack of effect of other transmitter pathways on gastric mechanosensitivity (16, 33) and the lack of effect of baclofen on chemosensitive pathways from the ferret stomach (26), GABABR agonists represent a powerful pharmacological tool for inhibition of esophageal and gastric vagal mechanosensory activity.
Baclofen and 3-APPiA each had varying effects on the spontaneous basal activity of gastric mechanoreceptors. There was a trend toward an increased basal activity following 3-APPiA treatment. This may be related to a part-nonselective action of 3-APPiA, which has been reported to possess activity at GABAA receptors (32). The nonreversible increase in basal activity elicited by the GABAAR agonist muscimol supports a possible action of 3-APPiA at GABAARs.
No action on distension-evoked activity of gastric mechanoreceptors was observed when muscimol was used at an established effective concentration (4). Whereas GABAAR have been shown to activate nodose ganglion cells (4), they also inhibit stimulated vagal afferent neurons in the rat (34). Our data would suggest that GABAAR are relatively unimportant as targets on vagal pathways from the proximal stomach compared with GABABR.
It is unknown whether endogenous GABA can act to modulate physiological levels of gastric mechanoreceptor activity; certainly, GABA is found throughout many parts of the gastrointestinal tract. An endogenous source of GABA acting on vagal afferents may arise from GABA-immunoreactive nerve fibers found in the myenteric ganglia of the stomach (14), a region where the putative vagal sensory terminals, termed intraganglionic laminar endings, also occur (27).
Effects of GABABR agonists on intragastric pressure were minimal in contrast to their effects on mechanoreceptor responses to distension. This is primarily because gastric compliance was recorded under conditions of L-type calcium-channel blockade with nifedipine; therefore, any effects of GABA-receptor agonists on gastric wall tension would be limited to non-L-type calcium channels, such as nonselective cation channel conductance (17) or T-type calcium-channel activation (18). Additionally, compliance changes would be limited to the control of intragastric pressure via local reflex accommodatory mechanisms (9), which are negligible in the isolated stomach (31). This will differ considerably from the situation that occurs in vivo, where vagovagal reflexes allow for greater and more complex control of compliance (3). This complexity is demonstrated by baclofen's capacity to reduce compliance in response to gastric distension in the ferret in vivo, an effect likely to be mediated centrally and manifest via the overriding inhibition of tonic vagal inhibitory motor outflow to the stomach (2). It is not clear why baclofen and CGP 62349 together elicited a significant decrease in compliance vs. control, but nonspecific actions on gastric motility have been ascribed to CGP 62349 at micromolar concentrations (8).
Immunohistochemistry for the GABABR (GABABRI-LI) combined with retrograde neuronal tracing from the proximal stomach (CTB-FITC) demonstrated the presence of GABAB-LI on vagal afferent neurones innervating this region. Cell counts of somata within the nodose ganglia sections labeling positive to CTB-FITC show that 9-13% of the total population of vagal afferents innervate the area of the proximal stomach injected with neuronal tracer. These fibers may be a mixture of mechanoreceptors and afferent fibers possessing other modalities such as mucosal receptors. In this study, gastric mechanoreceptive fields were identified electrophysiologically in the regions injected with neuronal tracer, suggesting that labeled neurones are representative of recorded neurones. Whereas labeling of fibers en passage cannot be excluded, it is unlikely that afferent fibers of passage from distal regions of the stomach would be labeled due to the extensive arborization of gastric divisions of dorsal and ventral vagal trunks proximal to the injection sites (21). In addition, the CTB-based label has been shown to display negligible labeling after both intraperitoneal injection and perivagal administration to the ferret cervical vagus (15).
GABABRI-LI was colocalized in nodose ganglia somata also containing CTB-FITC, demonstrating that vagal afferent neurones innervating the proximal stomach express GABABR. We used cerebellar cortex to determine the optimal conditions for GABABRI IgG binding, because there is a distinct pattern of labeling observed in this region (5, 12, 22, 28). Whereas the GABABRI primary IgG we used is not specific for the 1a or 1b splice-receptor isoforms in the rat, the strong labeling we observed in Purkinje cells and lack of labeling in the granule cell layer suggest that the antibody preferentially binds the 1b isoform in the ferret, assuming it is similar to the rat (28). GABABRI-LI was expressed in >90% of somata in the nodose ganglia, suggesting GABABR may serve regulatory functions in many vagal afferent neurones innervating visceral organs. Evidence that baclofen modifies vagal cardiovascular and respiratory reflexes via presynaptic inhibition of vagal afferent terminations in the dorsal medulla (6) is supported by our observation of extensive labeling in the nodose ganglia.
In conclusion, GABABR expressed on gastric vagal afferent neurones directly and reversibly inhibit gastric vagal mechanoreceptor responses to distension, a mechanism that may contribute to the efficacy of baclofen in the treatment of gastroesophageal reflux disease.
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ACKNOWLEDGEMENTS |
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Many thanks to B. Dixon from the Olympus Imaging Unit, Biochemistry Dept., Univ. of Adelaide, for fluorescence imaging facilities. AstraZeneca is gratefully acknowledged for financial support of this project.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. D. Smid, Nerve-Gut Research Laboratory, Level 1 Hanson Centre, Frome Rd, Adelaide SA 5000, Australia (E-mail: ssmid{at}mail.rah.sa.gov.au).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 July 2001; accepted in final form 5 September 2001.
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