Nerve-Gut Research Laboratory, Department of Gastroenterology, Hepatology and General Medicine, Royal Adelaide Hospital, Adelaide, South Australia 5000, Australia
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ABSTRACT |
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To investigate GABAB receptors along vagal afferent pathways, we recorded from vagal afferents, medullary neurons, and vagal efferents in ferrets. Baclofen (7-14 µmol/kg iv) reduced gastric tension receptor and nucleus tractus solitarii neuronal responses to gastric distension but not gastroduodenal mucosal receptor responses to cholecystokinin (CCK). GABAB antagonists CGP-35348 or CGP-62349 reversed effects of baclofen. Vagal efferents showed excitatory and inhibitory responses to distension and CCK. Baclofen (3 nmol icv or 7-14 µmol/kg iv) reduced both distension response types but reduced only inhibitory responses to CCK. CGP-35348 (100 nmol icv or 100 µmol/kg iv) reversed baclofen's effect on distension responses, but inhibitory responses to CCK remained attenuated. They were, however, reversed by CGP-62349 (0.4 nmol icv). In conclusion, GABAB receptors inhibit mechanosensitivity, not chemosensitivity, of vagal afferents peripherally. Mechanosensory input to brain stem neurons is also reduced centrally by GABAB receptors, but excitatory chemosensory input is unaffected. Inhibitory mechano- and chemosensory inputs to brain stem neurons (via inhibitory interneurons) are both reduced, but the pathway taken by chemosensory input involves GABAB receptors that are insensitive to CGP-35348.
-aminobutyric acid B receptors; ferret; mechanoreceptors; vagal
reflexes; cholecystokinin; nucleus tractus solitarii
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INTRODUCTION |
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GABAB receptor agonists, including baclofen, reduce triggering of transient lower esophageal sphincter (LES) relaxations and thereby inhibit gastroesophageal reflux in humans, ferrets, and dogs (12, 23, 24). Transient LES relaxations are mediated via a central vagal pathway (19, 26, 34), and their occurrence is increased after proximal gastric distension (22). Signaling from gastric vagal tension receptors to the central nervous system is therefore pivotal in the initiation of this motor pattern. Our evidence from an in vitro preparation indicates that baclofen inhibits the sensitivity of gastric vagal tension receptors to distension (28). These endings may therefore be the site of inhibition of transient LES relaxations. However, in vivo data are needed to corroborate these in vitro findings on afferent function. There is another population of gastroduodenal afferents, mucosal receptors, that respond to nutrient-related stimuli. It is not known how baclofen affects sensitivity of these endings to chemical stimuli such as cholecystokinin (CCK).
There is a dense distribution of GABAB receptors along central vagal pathways in the nucleus tractus solitarii (NTS) and dorsal vagal nucleus (25), which are equally plausible sites of the action of baclofen to reduce transient LES relaxations. Central effects of baclofen have been demonstrated on physiological vagal input to the NTS from other viscera (21, 32, 33, 35) and on electrically stimulated input from abdominal vagal afferents (36). These effects are invariably presynaptic, as demonstrated by experiments in brain stem slice preparations (16, 17). It is important to determine whether GABAB receptors have different influences on inputs to the brain stem from different populations of gastrointestinal vagal afferents and to establish whether inputs are affected after physiological activation. The four aims of this study were, first, to determine the effects of baclofen on responsiveness of gastric vagal tension receptors to physiological levels of distension; second, to determine its effects on a separate population of mucosal chemosensitive afferents; third, to evaluate the effects of baclofen on central processing of inputs from these populations of afferents; and fourth, to investigate specific aspects of the pharmacology of these effects by use of two GABAB receptor antagonists.
The influences of peripheral and central GABAB receptors were assessed by electrophysiological recording from single vagal neurons at three different sites along the vagal reflex pathway and by systemic and central administration of drugs. Gastric distension was used as a selective stimulus for smooth muscle tension receptors, and peripheral CCK was given to selectively activate gastroduodenal mucosal receptors (7). By virtue of these selective actions, we were able subsequently to determine functional GABAB receptor expression along the pathways arising from these two afferent populations. We recorded vagal afferent activity to determine the influences of peripheral GABAB receptors. The influence of GABAB receptors along the central vagal pathway was determined by recording the activity of neurons in the vagal sensory nucleus. Vagal motorneuron activity was also recorded from fibers projecting to the periphery. Our findings demonstrate multiple and selective actions of GABAB receptors in inhibition of vagal reflex pathways.
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MATERIALS AND METHODS |
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Experiments were conducted according to the guidelines of the Animal Ethics Committee of the Royal Adelaide Hospital and the Institute of Medical and Veterinary Sciences.
Surgery
Experiments were performed on 58 adult ferrets (Mustela putorius furo L.) weighing between 0.5 and 1.4 kg. Ferrets were initially anesthetized with urethane (1.25 g/kg ip) and given a lethal overdose of the anesthetic at the end of experiments. The left carotid artery was cannulated for blood pressure recordings, and the left jugular vein was cannulated for administration of intravenous drugs and further anesthetic as required to abolish the hindlimb pinch-withdrawal reflex. In 16 experiments, a small-diameter polyethylene catheter (OD 0.5 mm) was introduced into the atlantooccipital membrane and secured so that the tip lay in the fourth ventricle at the level of the obex for introduction of drugs centrally. After a midline laparotomy, a polyethylene cannula (OD 1.2 mm) was introduced via the aorta at the iliac bifurcation and positioned so that the tip lay at the celiac axis. It was used for close intra-arterial injections of CCK to stimulate mucosal receptors in the upper gastrointestinal tract (7). A saline-filled cannula was introduced via the pylorus in some experiments for cannulation of the whole stomach, or at the level of the incisura angularis in others for cannulation of the proximal stomach. The gut was closed off immediately distal to the cannula with a ligature. These cannulas were used for distension and intragastric pressure measurements. Only the proximal stomach preparation was used in experiments on afferents, whereas both preparations were used in experiments on efferents. Data on efferent responses from experiments with each method were combined, because GABAB receptor ligands were seen to have similar effects on efferent responses to whole and proximal gastric distension. The duodenum was cannulated in an oral direction proximal to the ligament of Treitz for bile drainage. Cannulas were exteriorized via the laparotomy, which was closed with towel clips.Nerve Recordings
Vagal afferent and efferent recordings. A paraffin pool was made in the neck by suturing skin and muscle to a steel ring. The right vagus nerve was supported by a small Perspex recording platform. Under a dissecting microscope (Olympus SZ60), the nerve sheath was split with a sharp blade over a length of ~5 mm. Fine filaments were dissected from the main nerve trunk and placed on a platinum hook recording electrode, with perineural connective tissue placed on an adjacent reference electrode. Recordings were made from 16 vagal afferent fibers by decentralizing a strand of nerve tissue from the main trunk, dissecting the peripheral cut end caudally over a distance of 2-3 mm, and placing it on the electrode. It is important to note that, when this technique was used, only centrally directed activity could be recorded. Recordings were made from 27 vagal preganglionic efferent fibers by following the reverse procedure, that is, by dissecting a deperipheralized strand rostrally and therefore recording caudally directed activity.
NTS recordings.
In addition to the general surgical procedures described above, a
bilateral thoracotomy was performed and animals were ventilated as
previously described (1). Animals were placed prone in a stereotaxic apparatus, and the brain stem was accessed by removing the
atlantooccipital membrane and dura mater. Glass microelectrodes filled
with 0.5 M sodium acetate and 2% Chicago blue with impedance >5 M
were advanced into the NTS in 5-µm steps with a stepper manipulator
at obex on either side by use of stereotaxic coordinates derived from
Boissonade et al. (13). Obex is defined for the purposes
of this study as the point at which the central canal of the spinal
cord opens dorsally and the floor of the fourth ventricle becomes
visible from a dorsal view. Thirty-eight units were recorded in 17 ferrets that showed responses to distension of the whole stomach (40 ml
saline), of which 28 were excitatory and 10 were inhibitory. These
pilot studies were performed to establish for the first time that
neurons responsive to gastric distension exist in the ferret NTS. In
five ferrets the effects of administration of baclofen (7 µmol/kg iv)
were assessed, and subsequently the effect of CGP-62349 (700 nmol/kg
iv) on two of these responses was observed.
Data processing. Electrical signals were amplified and filtered, and single units were discriminated from other units or noise by their particular shape, duration, and amplitude (JRak, Melbourne, Australia). In earlier studies, synchronized pulses corresponding to recognized spikes were fed into an Apple Macintosh IIci computer fitted with a NBMIO16 A-D card (National Instruments, Austin, TX). A pulse counter on the card was used to generate integrated output of action potential frequency. The integrated signal of spike frequency, together with measurements of intragastric and blood pressures, was displayed on screen, acquired, stored on hard disk, and analyzed off-line using Macintosh-based software (LabView, National Instruments). In later experiments, a micro 1401 interface (CED, Cambridge, UK) was used along with CED Spike 2 software for data acquisition with a Power Macintosh 7600/266 or G3. Spike 2 was also used to confirm accurate on-line discrimination of spikes by the JRak window discriminator. The filtered signal of electrical neural activity was stored on digital audio tape (Sony PCM2300).
Protocols
Gastric tension receptors were identified by their response to distension of the proximal stomach with a total of 15-25 ml of warm isotonic NaCl. The volume of saline used for distension was chosen according to the body weight of the ferret. Thus 15, 20, and 25 ml were used for animals of body weight ranges of 0.5-0.8, 0.8-1.1, and 1.1-1.4 kg, respectively. We have previously shown that discharge in vagal tension receptors is directly proportional to intraluminal pressure in the ferret (10), so distension volumes were chosen that led to similar increases in intraluminal pressure in all animals. Gastric distension caused a severalfold increase in the frequency of firing of a fiber. They were often identifiable also by their spontaneous patterns of discharge, which were clearly related to ongoing gastric contractile rhythms. After a recovery period ofMucosal receptors were identified by electrical stimulation (30 V, 0.5 ms, 1 Hz) delivered by exploration of the serosal surface of the
stomach with a hand-held electrode via the laparotomy. This yielded
action potentials on a 1:1 stimulus-response basis at a fixed latency.
Mucosal receptive fields were confirmed by stroking with the tip of the
intraluminal cannula (data not shown). Rapid, close intra-arterial
injections of CCK (100 pmol) were given into the celiac axis to
evaluate quantitatively the responsiveness of mucosal receptors
(7). This stimulus activated all mucosal receptors
identified. Mucosal and tension receptor responses were observed under
control conditions and 5 min after each drug treatment to allow
stabilization of resting discharge.
Efferent fiber responses and NTS neuronal responses were included for
analysis when they showed a 50% change in discharge during gastric
distension or after peripheral CCK administration and had no
cardiovascular or respiratory rhythms. Similar volumes were used for
proximal gastric distension as for afferent studies. Forty, fifty, and
sixty milliliters were used for distension of the whole stomach, which
gave rise to intraluminal pressures according to body weight comparable
to those volumes used for proximal gastric distension. Efferent and NTS
responses were observed under control conditions and
5 min after each
drug to allow stabilization of resting discharge. Drug treatments were
given peripherally (10 recordings of efferents, 5 recordings of NTS
neurons) or centrally (17 recordings of efferents).
Data Analysis
Basal discharge was assessed for 60 s upon commencing each experiment and after stabilization of discharge after administration of each drug. Resting discharge was calculated for 45 s before each stimulus. Tension receptor responses to gastric distension were measured as the change in mean discharge during distension. Responses showing phasic bursting patterns were assessed according to the peak frequency of the bursts relative to the interburst minimum frequency. Responses were quantified by expressing the minimum as a percentage of the peak, and they were categorized as follows: responses in which the minimum was >90% of the peak were classified as tonic, those in which it was <10% were classified as phasic, and those in between were classified as intermediate. Afferent responses to CCK are expressed as the total number of impulses evoked over the duration of the response until baseline discharge rate was reestablished.Efferent and NTS responses to distension were expressed as the change in mean discharge rate during the stimulus compared with the minute before. Because drug effects on responses were clearly independent of the direction of response to distension, inhibition and excitation were combined for statistical analysis and expressed as the change in discharge. Efferent responses to CCK are expressed as the duration of response. This was done because most inhibitory responses showed a 100% suppression of discharge both before and after drug treatment, and duration was found to be the most sensitive measure of changes in the profile of response. Data are expressed as means ± SE, with n the number of experiments. The Wilcoxon signed-rank test was used to assess changes in neuronal responses, because these data were not normally distributed. Likewise, a Kruskal-Wallis test was used with Dunn's post hoc test for experiments involving repeated measures.
Drugs
Baclofen, CGP-35348, and CGP-62349 were provided by AstraZeneca. CGP-62349 is a pure stereoisomer: 3-[(1R)-1-[[(2S)-2-hydroxy-3-[hydroxy[(4-methoxyphenyl)methyl]phosphinyl]propyl]amino]ethyl] benzoic acid. The compound henceforth referred to in this paper was in fact a diastereomeric mixture of this compound and 3-[(1S)-1-[[(2S)-2-hydroxy-3-[hydroxy[(4-methoxyphenyl)methyl]phosphinyl]propyl]amino]ethyl] benzoic acid. Urethane was obtained from Sigma-Aldrich (Sydney, Australia), and sulfated CCK-8 was from Auspep (Melbourne, Australia). CCK was initially dissolved in isotonic saline with 0.1% bovine serum albumin. All other drugs were dissolved in isotonic saline. ![]() |
RESULTS |
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Vagal Afferents
Vagal gastric tension receptor responses to proximal gastric
distension.
Our group has made previous detailed observations showing the
inhibitory effect of the GABAB receptor agonist baclofen on tension receptors in vitro (28). To corroborate these
findings in vivo, the responses of 12 gastric tension receptors to
proximal gastric distension (15-25 ml saline for 1 min) were
assessed. Distension was performed under control conditions, after
baclofen (7-14 µmol/kg iv), and again after the
GABAB receptor antagonist CGP-35348 (100 µmol/kg iv).
Under control conditions, distension led to excitation of afferent
discharge that was rapidly evoked and maintained throughout the
stimulus, as observed previously in the ferret (2, 7, 10).
Neural activity returned rapidly to predistension levels upon drainage.
Responses to distension often had periodic fluctuations that correlated
with the gastric pressure waves; these were classified according to the
dominant minimum-to-peak ratio as phasic (n = 4), tonic
(n = 3), or intermediate (n = 5)
responses (examples are shown in Fig. 1).
Gastric tension receptors were not excited by close intra-arterial
injections of CCK (100 pmol).
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Vagal mucosal receptor responses to peripheral CCK.
Baclofen was tested on four gastroduodenal vagal mucosal receptors.
Only four fibers were tested, because these fibers are difficult to
find in the ferret (see Ref. 7). The receptive fields of
these were located in the stomach (n = 3) and the
duodenum (n = 1). They responded to locally
administered CCK (100 pmol close ia) with an increase in
discharge exactly as previously reported (7). This
response was evoked within 10 s, and discharge returned slowly to basal levels over 5-10 min (see examples
in Fig. 2). Mucosal receptors did
not respond to distension of the regions containing their
receptive fields.
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Spontaneous discharge of vagal afferents. Vagal tension receptors showed a low-frequency irregular pattern of basal discharge (4.1 ± 1.0 impulses/s, n = 12) that bore no obvious relationship to respiratory or cardiovascular contractile rhythms in the absence of any intentional stimulus. This remained constant throughout the study before drug administration. Vagal mucosal receptors were silent before delivery of any stimulus, but in two units they developed an irregular low-frequency discharge during the experiment, as previously reported (7).
After administration of baclofen, the spontaneous discharge of vagal tension receptors was decreased significantly (4.1 ± 1.1 impulses/s under control conditions vs. 2.2 ± 0.6 impulses/s after baclofen, P < 0.01, n = 12). This effect of baclofen on spontaneous discharge was reversed by subsequent administration of the antagonist CGP-35348 (2.4 ± 0.7 impulses/s after baclofen vs. 3.8 ± 1.1 impulses/s after baclofen plus antagonist, P < 0.01, n = 10). No effect of baclofen was seen on the spontaneous discharge of the four mucosal receptors (0.5 ± 0.21 impulses/s control vs. 0.2 ± 0.04 impulses/s after baclofen, P = 0.22).NTS Neurons
Thirty-eight units were recorded in 17 ferrets that showed responses to distension of the whole stomach (40 ml saline). Twenty-eight responses were excitatory, and 10 were inhibitory. In five units the effects of administration of baclofen (7 µmol/kg iv) were assessed, and subsequently the effect of CGP-62349 (700 nmol/kg iv) on two of these responses was observed. Four of these units showed excitation, and one showed inhibition of discharge. All four excitatory responses were attenuated by baclofen by a mean of 59% (Fig. 3), whereas the inhibitory response was unchanged, although this unit showed 100% inhibition of firing before and after baclofen. In both cases in which CGP-62349 was given after baclofen, the response was reversed to become similar to control (Fig. 3).
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Spontaneous discharge of NTS units was reduced from 1.3 ± 0.5 to 0.2 ± 0.1 impulses/s (P < 0.001, n = 5) after baclofen. This increased to 0.4 ± 0.1 impulses/s after CGP-62349.
Vagal Efferents
Vagal efferent responses to gastric distension.
In 10 units, vagal efferent responses to gastric distension (40-60
ml) closely resembled those of gastric tension receptors described
above: they were rapidly evoked and maintained throughout the duration
of the distension until removal of the stimulus. In 8 units, inhibition
of discharge occurred, which was manifested as a mirror image of the
excitatory responses (Fig. 4), as
previously reported (9, 20).
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Vagal efferent responses to peripheral CCK.
Ten efferent units that responded to gastric distension also responded
to peripheral CCK (100 pmol close ia). Nine other units responded to
CCK only. Four units responded with excitation and 15 with inhibition
of discharge (see examples in Fig. 5).
Vagal efferent responses to CCK were similar to mucosal receptor
responses in terms of rapid onset and duration: they were <10 s in
latency and lasted 3-15 min before basal discharge levels were
reestablished.
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Spontaneous discharge of vagal efferents. Vagal efferents showed either no basal discharge or a low-frequency irregular pattern of discharge that bore no obvious relationship to respiratory, cardiovascular, or gastrointestinal contractile rhythms. This remained constant throughout the study before any drug administration. Administration of neither baclofen nor subsequent CGP-35348 significantly affected the basal discharge of the efferent fibers tested. Although group data showed no significant change, individual fibers often showed maintained increases or decreases in spontaneous discharge (Figs. 4 and 5). CNQX was without significant effect when group data on spontaneous efferent discharge were considered; however, again, individual experiments showed clear changes (e.g., Fig. 4).
Changes in Intragastric Pressure
Intragastric pressure was measured during gastric distension (whole and proximal stomach) under control conditions, after baclofen (7-14 µmol/kg iv or 3-6 nmol icv), and after CGP-35348 (100 µmol/kg iv or 100 nmol icv). The results from studies with systemic and central administration were combined because they were similar.The increase in intragastric pressure during gastric distension was significantly augmented after administration of baclofen (6.9 ± 0.5 mmHg under control conditions vs. 9.1 ± 0.8 mmHg after baclofen, P < 0.01, n = 23), indicating reduced gastric compliance. This was not significantly reversed after subsequent administration of CGP-35348 (8.5 ± 1.0 mmHg before vs. 7.6 ± .6 mmHg after CGP-35348, P > 0.05, n = 16). However, when data were compared for the effects of the GABAB receptor antagonist on intraluminal pressure in the proximal stomach only, there was a trend toward a significant reversal of the effect of baclofen (10.3 ± 1.8 vs. 7.0 ± 0.8, P = 0.06, n = 7).
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DISCUSSION |
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The results of this study indicate that GABAB receptors exert inhibitory effects along vagal pathways from the upper gastrointestinal tract to the brain stem. GABAB receptors inhibit mechanosensitivity of peripheral terminals of gastric tension receptors and, additionally, their central synaptic connections with brain stem neurons. The action of baclofen on these GABAB receptors was reversed by both selective GABAB receptor antagonists CGP-35348 and CGP-62349. The receptors are most likely presynaptic on afferent terminals, owing to the effects on both central inhibitory and excitatory pathways and our demonstration of the involvement of other (non-NMDA) receptors in synaptic transmission along this pathway. Vagal mucosal receptor sensitivity to CCK, on the other hand, was unaffected by baclofen. Central terminals of these afferents also appear to lack GABAB receptors, even though they utilize similar mechanisms of synaptic transmission to tension receptors (30). GABAB receptors are nonetheless manifested along the central pathway receiving inputs from mucosal afferents. These receptors are restricted to higher-order neurons because they selectively influence inhibitory pathways. The effect of baclofen on these receptors was insensitive to CGP-35348 but was antagonized by CGP-62349, providing evidence for pharmacological subtypes of GABAB receptors.
The responses of vagal gastric tension receptors to proximal gastric distension and the responses of vagal gastroduodenal mucosal receptors to CCK recorded in this study are directly comparable with those reported previously (2, 7, 10). Our results further demonstrate that baclofen reversibly inhibits responses of vagal tension receptors to gastric distension, confirming our in vitro observations (28). Baclofen concomitantly decreased gastric compliance, through a mechanism that was not investigated in this study. Decreased compliance was evidenced by increased intraluminal pressure during isovolumetric distension. A decrease in compliance normally leads to an increase in tension receptor responses in ferrets (10). Baclofen must therefore have inhibited mechanotransduction directly, in the face of an increased adequate stimulus. Baclofen also altered the response profile of some gastric tension receptors to distension, causing them to show significantly larger minimum-to-peak fluctuations. The predominant effect in these cases was to reduce the interburst minimum, which occurs when there is only a passive load on the ending. Our previous study, showing highly reproducible GABAB receptor inhibition of mechanosensitivity, was also performed using passive loads (28). The two studies together suggest that GABAB inhibition is manifest more during passive than active tension on the ending. Baclofen did not affect vagal mucosal receptor responses to CCK in this study, although only maximal doses of CCK were used, leaving the possibility for baclofen to exert subtle effects on these responses. Our previous in vitro data on baclofen and vagal mucosal receptors showed inhibition of esophageal mucosal receptor responses to stroking by baclofen (28). Although the two studies were performed under different conditions and receptive fields were investigated from different locations, the possibility arises that mucosal mechanosensitivity and chemosensitivity are affected differently by GABAB receptors.
The patterns of responses of vagal preganglionic efferent neurons to gastric distension were similar to those reported earlier, which are caused by activation of both vagal and nonvagal gastric mechanoreceptors (6, 20, 29). This is the first study to show effects of peripheral activation of mucosal receptors with CCK on vagal efferent discharge, although a vagal reflex triggered by this stimulus has been characterized previously (8), and convergence of inputs from mucosal and tension receptors onto vagal efferents has been demonstrated electrophysiologically by use of other stimuli for mucosal receptors (6, 9). The similarity of response profiles of the vagal afferents, NTS neurons, and efferent fibers to the same stimuli suggests that there is relatively little modulation of the signal along the central pathway. This signal is conserved despite the high degree of convergence from afferents of different modalities in different locations onto individual efferents that is evident from this and other studies (6, 9, 20, 29). The implication of strong connectivity between afferents and efferents correlates with anatomic and electrophysiological data on mono- and paucisynaptic connections (5, 31). In the case of inhibitory efferent responses to peripheral stimuli, an inhibitory interneuron must be interposed along the central pathway. This is most likely to be a GABAergic neuron from the results of intracellular recording and anatomic studies showing the predominance of GABA in inhibitory synaptic transmission in vagal nuclei (16-18).
This investigation included pilot studies to establish for the first time that neurons responsive to gastric distension exist in the ferret NTS, as would be predicted from studies in other species. Responses we observed in the ferret were comparable to those reported in the rat and the cat (3, 4, 27). Effects of baclofen on NTS responses to gastric distension were similar in magnitude and proportion affected to those on our vagal efferent responses described above. In both cases tested, these effects were reversible with the selective antagonist CGP-62349. Future studies will investigate GABAB receptors specifically in this nucleus in more detail by use of iontophoretic techniques.
We speculate that the GABAB receptors responsible for the effects of central baclofen on central neuronal responses to gastric distension are located presynaptically on the central terminations of gastric tension receptors that release glutamate. This is in keeping with demonstrations of presynaptic GABAB receptors in the brain stem of other species (16, 17). We suggest that GABAB receptors are presynaptic, because both inhibitory and excitatory efferent neuronal responses were affected alike by GABAB receptor ligands. This ties in with the demonstration of GABAB receptors on these afferents at their peripheral endings in this study and our previous study (28). In contrast, we suggest that GABAB receptors along the vagal pathway activated by mucosal afferents are probably restricted to second- or higher-order neurons, because only inhibitory efferent responses to CCK were affected by baclofen. The lack of central presynaptic receptors on mucosal afferents ties in with the lack of effect of baclofen on peripheral afferent responses. The GABAB receptors involved in modulating the response to CCK are insensitive to CGP-35348 at the doses used in this study, in contrast with the receptors modulating responses to gastric distension, upon which the effects of baclofen are fully reversible by CGP-35348, even on the same neuron. It is therefore logical to suggest that GABAB receptors with a peculiar pharmacology are involved in the central effects of baclofen on inhibitory efferent responses to peripheral CCK. Similar findings on pharmacology were made in studies of cortical synaptosomes, indicating that whereas GABAB heteroreceptors are sensitive to CGP-35348, GABAB autoreceptors are not (14, 15). The pharmacology of GABAB auto- and heteroreceptors is a complicated issue, in which different methods and the study of different brain regions produce conflicting results. Ours is the first evidence for heterogeneity of this kind in the response of a single neuron to two different afferent inputs in vivo. It is also worthy of note that the effect of baclofen on triggering of transient LES relaxations and reflux in conscious ferrets shows similar antagonist pharmacology to the pathway triggered by mucosal afferents in this study (12). A study of peripheral presynaptic actions of GABAB receptors on vagal motor outflow also showed this pattern of antagonist potency by use of broad ranges of dosage (11). Therefore, although potency of antagonists was not compared rigorously in the present study, our previous study indicates that the doses chosen are appropriate. The potential importance of pharmacological subtypes in therapeutic actions of GABAB receptor ligands is therefore emerging, but we do not yet have a definitive answer to the mechanism of GABAB receptor inhibition of transient LES relaxations. Although it was not the aim of this study to find such a mechanism, it is the subject of further investigation in our laboratory.
The effects of baclofen on efferent responses appeared to be an all-or-none phenomenon. Most vagal afferent, NTS, and efferent responses to gastric distension were abolished or potently reduced by baclofen. Efferent responses to distension were attenuated or abolished at the lowest central dose in most studies but were unchanged in other studies; these responses remained unchanged even when the central dose of baclofen was doubled and followed by peripheral administration. All-or-none effects of baclofen such as this were also found in previous investigations (28, 36). We interpret these findings to indicate that, although GABAB receptors are obviously important in modulating transmission from gastric tension receptors, they are restricted to a (major) subpopulation. The other subpopulation lacks GABAB receptors on both peripheral and central endings. An alternative explanation may be that drugs had insufficient access to the nuclei responsible in these experiments. However, cardiovascular changes were similar to those seen in experiments in which baclofen did not affect neuronal responses, and the systemic dose chosen is well recognized to have central effects accessed via the circulation (12).
In conclusion, we have found that GABAB receptors have selective influences on both peripheral and central vagal sensory transduction. Some of these receptors are likely to be those mediating therapeutic effects, which may underlie future treatment of gastroesophageal reflux disease.
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ACKNOWLEDGEMENTS |
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This work was supported by AstraZeneca. E. R. Partosoedarso was a recipient of the Royal Adelaide Dawes Postgraduate Research Scholarship.
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FOOTNOTES |
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Present address for E. R. Partosoedarso: Department of Pharmacology, Louisiana State University Health Science Center, 1901 Perdido St., New Orleans, LA 70112.
Address for reprint requests and other correspondence: L. A. Blackshaw, Nerve-Gut Research Laboratory, Level 1 Hanson Centre, Frome Road, Adelaide SA 5000, Australia (E-mail: ablacksh{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 7 August 2000; accepted in final form 8 November 2000.
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