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Am J Physiol Gastrointest Liver Physiol 294: G130-G138, 2008. First published November 1, 2007; doi:10.1152/ajpgi.00388.2007
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NEUROREGULATION AND MOTILITY

Differential effects of ASIC3 and TRPV1 deletion on gastroesophageal sensation in mice

Klaus Bielefeldt1 and Brian M. Davis1,2

Departments of 1Medicine and 2Neurobiology, University of Pittsburgh, Pittsburgh, Pennsylvania

Submitted 21 August 2007 ; accepted in final form 31 October 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Using a recently developed in vitro preparation of vagal afferent pathways, we examined the role of TRPV1 and ASIC3 on the mechano- and chemosensitive properties of gastroesophageal sensory neurons. Esophagus, stomach, and the intact vagus nerves up to the central terminations were carefully dissected from TRPV1 and ASIC3 knockout mice and wild-type controls. The organ preparation was placed in a superfusion chamber to obtain intracellular recordings from the soma of nodose neurons during luminal stimulation of esophagus and stomach. The proximal esophagus and distal stomach were separately intubated to allow perfusion and graded luminal distension. In wild-type mice, mechanosensitive neurons were activated by low distension pressures and encoded stimulus intensity over the entire range tested. Luminal acidification significantly transiently increased the resting frequency but did not alter responses to subsequent mechanical stimulation. ASIC3 and TRPV1 knockout significantly blunted responses to distension compared with wild-type controls, with deletion of TRPV1 having a more significant effect than ASIC3 deletion. Luminal acidification did not activate mechanosensory neurons in ASIC3 and TRPV1 knockout mice. Our data demonstrate a role of TRPV1 in chemo- and mechanosensation of gastroesophageal afferents. ASIC3 may contribute to acid sensation but plays a more subtle role in responses to distending stimuli. Considering the importance of acid in dyspeptic symptoms and gastroesophageal reflux, TRPV1 or ASIC3 may be an attractive target for treatment strategies in patients who do not respond to acid suppressive therapy.

visceral sensation; electrophysiology; mechanosensation; chemosensation

THE STOMACH CONTAINS a high proton concentration, which contributes to the normal digestive process and constitutes an important barrier to intestinal colonization with microorganisms. However, the high acidity is also a factor in the pathogenesis of common symptoms and diseases affecting the proximal gastrointestinal tract. Epidemiological studies suggest that up to 20% of adults experience heartburn or dyspeptic symptoms at least intermittently (1, 9, 17). Acid reflux into the esophagus and backdiffusion of acid into gastric mucosa due to impaired protective barrier function play a role in the development of these symptoms. Two distinct sensory pathways innervate the proximal gastrointestinal tract, with vagal afferents projecting to the brain stem via the nodose neuron and spinal afferents sending their processes via the dorsal root ganglia to the spinal cord (10). Although vagal pathways are thought to primarily regulate autonomic function, recent studies suggest that they may contribute to acid sensation and chemonociception (15, 26). We have recently shown that gastric sensory neurons respond to protons through activation of acid-sensitive ion channels (28). On the basis of functional and pharmacological properties of these currents, we concluded that gastric nodose neurons primarily express the proton-sensitive channel TRPV1 or a member of the family of acid-sensitive ion channels (ASIC), ASIC 3. This ASIC channel is characterized by rapid activation and desensitization. These studies were performed in cultured neurons and, thus, assessed responses to proton exposure of the soma rather than the terminals where sensory transduction takes place. Therefore, we decided to investigate whether these channels indeed contribute to acid sensation in esophagus and/or stomach. Using a newly developed in vitro preparation (3), we addressed the following hypotheses: 1) acid directly stimulates a subset of gastroesophageal afferents, 2) luminal acidification sensitizes gastroesophageal afferents to mechanical stimulation, and 3) TRPV1 and ASIC 3 are required for normal acid sensation.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals. All experiments were performed in male and female mice 4–8 wk of age, housed in a 12:12-h light-dark cycle with free access to water and food at the certified animal care facility of the University of Pittsburgh. Animal handling adhered to the Guide for the Care and Use of Laboratory Animals (National Research Council); all procedures were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. TRPV1 knockout mice and ASIC 3 knockout mice were obtained from Drs. M. Caterina (Johns Hopkins University) (5) and M. Price (University of Iowa) (24) and bred on a C57bl/6 background (The Jackson Laboratory, Bar Harbor, ME). C57bl/6 wild-type mice were used as controls. Homozygous deletion of TRPV1 or ASIC 3 was confirmed by PCR for each animal used in the studies (data not shown).

Preparation. Animals were euthanized with an overdose of pentobarbital and then transcardially perfused with carboxygenated, ice-cold artificial cerebrospinal fluid. During the initial perfusion and preparation, sodium chloride was replaced by an iso-osmolar concentration of sucrose (concentrations in mM: 253.9 sucrose, 1.9 KCl, 1.2 KH2PO4, 1.3 MgSO4, 2.4 CaCl2, 26.0 NaHCO3, 10.0 D-glucose). The duodenum, stomach, spleen, liver, and mediastinal structures including esophagus were rapidly mobilized up to the thoracic inlet. Neck, head, mediastinal organs, and proximal gastrointestinal tract were removed en bloc and placed in a dish constantly perfused with oxygenated chilled sucrose containing solution. All organs except for esophagus and stomach were dissected away. To minimize vagal damage, trachea, bronchial bifurcation, and right atrium were left intact. Right and left vagus nerves were identified and mobilized from the jugular foramen down to the thoracic inlet. The jugular foramina were opened and the nodose ganglia were freed from connective tissue and proximal projections cut close to their entry into the brain stem. Esophagus and larynx were cut from their attachment at the base of the skull and pharynx. The esophagus was intubated with small polyethylene tubing (PE 50) and perfused to empty the stomach. Once completed, the entire preparation was moved into the recording dish perfused with chilled and carboxygenated artificial cerebrospinal fluid (in mM: 127 NaCl, 1.9 KCl, 1.2 KH2PO4, 1.3 MgSO4, 2.4 CaCl2, 26.0 NaHCO3, 10.0 D-glucose). The temperature was slowly raised to 33°C. The esophagus was again intubated with PE tubing connected to a gravity-driven perfusion system for distension between 10 and 40 cmH2O. Similarly, PE tubing was inserted into the stomach through the pylorus for drainage. The tubing was connected to a pressure transducer to monitor intragastric pressure during distension. The position of proximal and distal tubes was secured through silk ligatures. The nodose ganglia were pinned with pins. A suction electrode was placed on the vagus (4–5 mm distal of the nodose ganglion) for electrical stimulation as described previously (3).

Electrophysiological recordings. Recordings were made from individual nodose neurons by using sharp electrodes (tip resistance > 100 M{Omega}) filled with 1 M potassium acetate. Intracellular penetrations were recognized by a sudden voltage change. Cells with axons projecting caudal to the suction electrode exhibited axon potentials in response to vagal stimulation. Cells were studied by using an Axoclamp 2B amplifier; data were digitized (CED Micro 1401; Cambridge Electronic Design, Cambridge, UK) with a sampling rate of 44 kHz and stored on a personal computer using Spike 2 software (Spike2 software Cambridge Electronic Design, Cambridge, UK). Cells generating an action potential in response to electrical stimulation of the vagus were included into the study if their resting membrane potential exceeded –35 mV and if they showed a distinct overshoot above 0 mV during the action potential. The stomach and esophagus were probed with a blunt glass rod (2 mm in diameter) to search for gastroesophageal afferents. If a cell responded, the area of the receptive field was determined by using the glass rod. Because the stomach was not opened up, only a rough determination could be performed. Therefore, we separated esophagus, cardia, forestomach, and glandular stomach as separate regions when assessing the localization of receptive fields. This was followed by stepwise distension with peak pressures of 10, 20, 30, and 40 cmH2O, using perfusion solution buffered to pH 7. Each distension trial lasted for 20 s and was followed by deflation of the stomach for 30 s. After the initial distension series, the stomach was continuously perfused with pH 7 solution for 3 min. Baseline activity before and after the 3 min perfusion was determined and averaged for a 10-s interval. This was followed by perfusion with an identical solution buffered to pH 4 with HCl, lasting 3 min. After baseline values were obtained prior to and at 3 min after luminal acidification, a second series of stepwise distension was performed.

Data analysis. For each cell, the resting membrane potential, conduction velocity, spike amplitude, action potential duration at 50% of peak amplitude, amplitude, and duration of the afterhyperpolarization were determined as described previously (3). For gastroesophageal afferents, we determined resting activity, responses to mechanical stimulation, and luminal acidification. To generate stimulus response functions, the mean action potential frequency during the 20-s stimulation period was plotted against the distension pressure. The time course of responses was analyzed by measuring spike frequency in 1-s bins. Because the kinetics of responses may differ between groups, we also compared the time-dependent changes during distension to different pressures. Changes in resting membrane potential during stimulation were expressed as a decrease in potential prior to the onset of an action potential before, and at the end of, the stimulation period. Because perfusion with control or acidic solution altered pressures, we could not reliably measure time courses of responses solely due to acid exposure. Therefore, only values before and 20 s after ending perfusion were obtained.

Statistical analysis. All data are given as means ± SE. Results were analyzed by the Mann-Whitney rank-sum test, a two-way ANOVA followed by Holm-Sidak methods for multiple group comparisons, or Fisher exact test where appropriate. A value of P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Basic properties of gastric nodose neurons. Recordings were obtained from a total of 514 neurons from 22 mice (wild-type: 11; ASIC3–/–: 5; TRPV1–/–: 6). When comparing basic properties of all nodose neurons, only the duration of afterhyperpolarization was significantly longer in ASIC3–/– mice compared with the other groups (Table 1). Consistent with our previous report (3), only 2 of the 514 nodose neurons had conductance velocities exceeding 1 m/s. Responses to gastroesophageal distension were obtained from 79 neurons (wild-type: 37; ASIC3–/–: 23; TRPV1–/–: 19) with receptive fields in the esophagus (13), cardia (8), forestomach (10), or glandular portion of the stomach (38). In the remaining 10 cells, the receptive field was in the stomach, but its location in the proximal or distal stomach could not be conclusively determined. All gastroesophageal neurons conducted in the C-fiber range without a difference between the groups (Table 2). As shown in Table 2, resting membrane potential, action potential amplitude and duration, and afterhyperpolarization amplitude and duration did not differ between the three groups of animals. Gastroesophageal sensory neurons exhibited little spontaneous activity with an average resting activity around 1 Hz (Table 2).


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  		Table 1. Basic properties of nodose neurons

 

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  		Table 2. Basic properties of gastroesophageal nodose neurons

 
Response to mechanical distension in wild-type mice. All afferents were selected based on their response to the mechanical search stimulus (blunt probing). A total of 37 cells responded to the search stimulus. A distinct receptive field could be identified in all but four cells (esophagus: 7; forestomach: 7; glandular stomach: 19). Gastroesophageal afferents encoded distending stimuli over the range of stimuli tested (Fig. 1). One of 19 cells tested over the entire range of stimulus intensities was quiescent at baseline and responded only to stimulus intensities of 20 cmH2O and higher. In an additional 12 cells, at least two different distension pressures were tested. Again, only one neuron required distension pressures of at least 20 cmH2O before responding. In gastric neurons, the onset of response was slow and generally took several seconds before reaching the peak (Fig. 2A). With distension pressures of 20 cmH2O or less, the majority of cells (n = 17/26) showed a gradual increase in spike frequency throughout the entire distension trial. With greater stimulus intensity (30 and 40 cmH2O), more cells showed accommodation. However, the peak frequency decreased by 50% or more in only 8 of 23 cells studied. This was associated with the development of an oscillatory pattern in two cells (Fig. 3). In three cells with receptive fields in the esophagus, distension triggered an instantaneous and rapidly adapting response that did not encode stimulus intensity over the range of pressures tested (Fig. 4). However, after a brief period of quiescence, these cells started to generate action potentials with greater distension pressures that continued to increase in frequency throughout the duration of the trial. In two cells, high response frequencies led to intermittent spike failure within the soma as shown by a distinct depolarization due to the invading action potential (Fig. 5). Responses to distension were associated with a progressive hyperpolarization at the end of the stimulation period as shown in Fig. 1C. The peak response frequency (r2 = 0.37), but not amplitude or duration of the afterhyperpolarization, was associated with the change in resting potential during prolonged stimulation.


Figure 1
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  		Fig. 1. Vagal sensory neurons respond to gastric distension over a wide range of stimulus intensities. Representative voltage tracings (A) show action potentials in response to distensions at 10 cm (a), 20 cm (b), 30 cm (c), and 40 cm water column (d). Calibration bars indicate 20 mV and 2 s, respectively. The stimulus response function for 19 cells is summarized in B. As can be seen in A, the increase in spike frequency is associated with a progressive hyperpolarization (C).

 

Figure 2
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  		Fig. 2. Time course of response to gastric distension. The spike frequency was calculated in 1-s bins and is plotted over the 20-s distension trial for the different peak pressures. A shows the results for different distension pressures in control animals (n = 19). The distension is indicated by the black bar. B–E: distension to different distension pressures (B, 10 cmH2O; C, 20 cmH2O; D, 30 cmH2O; E, 40 cmH2O) for wild-type mice (bullet; n = 19), ASIC3–/– mice ({square}; n = 8), and TRPV1–/– knockout mice ({circ}; n = 8). For details, please see text.

 

Figure 3
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  		Fig. 3. Voltage tracing of a gastric sensory neuron activated by distension. Gastric distension to 30 cmH2O (onset: arrow) triggered action potentials and progressive hyperpolarization with development of an oscillatory spike pattern (marked by the box).

 

Figure 4
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  		Fig. 4. Voltage tracing in A shows a rapid but short response to esophageal distension (30 cmH2O) followed by a second delayed increase in spike frequency. The receptive field was located in the midesophagus. No additional receptive fields were seen by blunt probing. Similar patterns were seen in 2 additional esophageal sensory neurons. The time course of responses for 3 different stimulus intensities is plotted in B. The black bar on top of the tracings indicates the stimulation period.

 

Figure 5
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  		Fig. 5. Failure of the invading spike during gastric distension. A gastric nodose neuron responds to distension with increasing spike frequency and hyperpolarization (top), eventually leading to intermittent spike failures of the invading action potential (bottom).

 
Response to luminal acidification. We compared baseline activity before and after a 3-min luminal perfusion at a rate of ~2 ml/min. Because luminal pressures varied during the period of flow, only values before and 30 s after the trial were determined. When control solution at pH 7 was used, baseline activity did not change significantly in four experiments (Fig. 6A). In contrast, there was a rise in baseline activity after luminal perfusion with pH 4 solution from 1.7 ± 0.2 to 2.9 ± 0.5 Hz with 5 of 10 cells showing an increase in baseline activity greater than the 95% confidence interval determined under control conditions. In seven cells, resting activity was determined 6 min after change to the higher proton concentration. The mean frequency increase rose from 1.7 ± 0.5 Hz at baseline to 3.1 ± 0.6 Hz after 3 min at pH 4 and fell back to 1.9 ± 0.3 Hz after an additional 3 min (P < 0.05; Fig. 6A). To determine a potential effect of luminal acidification on mechanosensation, we performed a second series of distension. As shown in Fig. 7A, there was no significant effect on the response to distension.


Figure 6
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  		Fig. 6. Baseline spike frequency before and after perfusion with control or acidic solution (pH 4) in wild-type mice (A; n = 11). B and C: baseline spike frequency and effects of pH at 3 min in ASIC3 (n = 8) and TRPV1 knockout mice (n = 5), respectively. *P < 0.05 compared with baseline.

 

Figure 7
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  		Fig. 7. Effects of luminal acidification on gastroesophageal mechanosensitivity. Stimulus response function before and after perfusion with pH 4 solution was determined by stepwise distension. In 19 wild-type mice (A), luminal acidification did not alter mechanosensitivity (P = 0.27). ASIC3 knockout mice (B) had similar response to gastroesophageal distension as controls (dotted line) before and after acidification (n = 10; P = 0.94 compared with control). In contrast, TRPV1 knockout mice (C; n = 11) showed significantly decreased mechanosensitivity compared with wild-type controls (dotted line; P < 0.01). However, luminal acidification did not alter the response.

 
Effect of ASIC3 deletion on gastroesophageal sensory function. Basic properties of nodose neurons from ASIC3 knockout mice projecting to esophagus or stomach did not differ from wild-type controls (Table 2). Receptive fields could be identified in 18 of 23 neurons (esophagus: 7; forestomach: 3; glandular stomach: 8). In eight cells stepwise distension was performed over the entire range of stimulus intensities. An additional two cells were tested with at least two different distension pressures. On the basis of a stimulus response function, responses to gastric distension also did not differ from wild-type controls (Fig. 7B). Because response kinetics may differ between groups, we performed a more detailed analysis of time-dependent changes in spike frequency at the different distension pressures. At low pressures, responses were significantly lower in ASIC3–/– mice compared with controls (t = 5.1 and t = 5.6 for 10 and 20 cmH2O, respectively; P < 0.01) and did not differ from TRPV1–/– mice (t = 0.96 and t = 0.92 for 10 and 20 cmH2O, respectively). At 30 cmH2O, responses in ASIC3–/– mice differed from controls (t = 2.0; P < 0.05) and TRPV1–/– mice (t = 3.1; P < 0.01). In contrast, distension at 40 cmH2O triggered similar increases for control and ASIC3–/– (t = 1.3; P = 0.19; Fig. 2, B–E). As described above, we noted several distinct response patterns that differed in adaptation. Although Fig. 2, B–E suggests a slower onset of response, we qualitatively observed rapidly adapting cells in ASIC3–/– mice. Consistent with this observation, there was no significant interaction between group and time course in the statistical analysis (P = 0.99 for 10 cmH2O; P = 0.98 for 20 cmH2O; P = 0.82 for 30 cmH2O; P = 0.96 for 40 cmH2O). As already indicated by comparable responses to high distension pressures, the peak spike frequency did not differ between ASIC3 knockout mice and controls (10.3 ± 2.1 vs. 10.3 ± 1.6 Hz for ASIC3–/– and wild type, respectively). Two of eight neurons tested showed a response to luminal acidification without any difference in mean spike frequency before and after switching to pH 4 solution (Fig. 6B). Repeated distension in the presence of luminal acid also did not alter the stimulus response function (Fig. 7B).

Effect of TRPV1 deletion on gastroesophageal sensory function. Similar to ASIC3 knockout mice, we did not observe differences in resting activity when comparing TRPV1 knockout mice with wild-type controls (Table 2). Distinct receptive fields could be localized in all 19 cells responsive to the search stimulus (esophagus: 6; forestomach: 2; glandular stomach: 11). Compared with wild-type mice, responses to distension were significantly blunted (Fig. 7C). A more detailed analysis of response kinetics shows significant differences at all distension pressures compared with controls (t = 5.2 at 10 cmH2O; t = 7.0 at 20 cmH2O; t = 5.7 at 30 cmH2O; t = 5.6 at 40 cmH2O; P < 0.01). Consistent with these results, four cells required distending pressures of at least 20 cm before responses were noted. None of the five neurons tested showed a response to luminal acidification (Fig. 6C). Similarly, there was no significant difference between distension responses obtained at pH 7 and pH 4 (Fig. 7C).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have recently described a novel technique to study gastroesophageal afferents in vitro (3). The main goal of this series of experiments was to examine acid sensation in the proximal gastrointestinal tract. Considering the clinical importance of esophageal acid sensation, we decided to modify our approach, because opening the small mouse esophagus to expose the luminal surface would likely result in damage of small vagal branches. Whereas the fluid perfusion and distension more closely resembles the typical physiological events of filling and emptying, it does not allow direct comparisons with most in vitro studies of gastroesophageal vagal afferents, which used punctate stimuli, mucosal brushing with small filaments, and/or defined stretch (16, 20, 22, 29–31). In addition to these differences in stimulation paradigms, we used longer distension trials to assure complete distension.

The basic properties of nodose neurons did not differ from the previously reported results (3). All cells studied had conductance velocities in the C fiber range, consistent with anatomic studies of the subdiaphragmatic vagus in the rat showing mostly thin, unmyelinated axons (23). When comparing nodose neurons of the three different experimental groups, we noted a slightly but significantly longer duration of the afterhyperpolarization in cells recorded in ASIC3 knockout animals compared with the two other groups. We did not systematically examine responses to defined suprathreshold stimulation in all neurons. In the smaller and thus potentially underpowered sample of gastroesophageal neurons, we did not discern such a difference. In addition, the average peak frequency during gastric distension did not differ between ASIC3 knockout mice and wild-type controls. The underlying mechanism for the increase in ASIC3 afterhyperpolarization duration remains unclear and will require additional studies using more detailed electrophysiological characterization.

Vagal afferents formed a relatively homogeneous group with all fibers responding to low-intensity stimulation. Fluid distension typically triggered a gradual increase in response with limited adaptation during the 20-s stimulation, which is similar to results obtained with teased-fiber technique in rats (19). The delay in responses differs from findings obtained during mechanical stimulation of opened preparation through stretch or punctate stimulation and largely reflects the slow filling of the esophagus and stomach through a relatively small catheter. Essentially all cells encoded stimuli over the entire range of intensities tested. In three neurons, we observed a rapid and saturating transient response reminiscent of results described for the rapidly adapting "mucosal" afferents (22) or "nonnociceptive" A fibers (29) described in the guinea esophagus. In contrast to mucosal afferents, all mechanosensitive neurons included in our study responded to stretch. Moreover, the stimulation revealed a second phase of activation that differentially encoded increasing stimulus intensities. Although the different methodologies and/or experimental animals do not allow direct comparison, our results raise several interesting questions. First, do low-threshold mechanosensors contribute to sensation and/or perception of high-intensity stimulation? Second, is the differential response due to activation of different mechanosensory channels? Third, do epithelial signals or other paracrine influences contribute to the apparent dichotomous response patterns? Fourth, are anatomical equivalents with peripheral sprouting and different specialized endings responsible for the biphasic response? An alternative explanation may focus on the nature of the stimulus. The stimulus was continuously applied but may initially lead to distension (circular stretch) and later lengthening of the esophagus (longitudinal stretch). Nonetheless, this explanation still requires coding of two distinct phenomena by likely distinct sensory structures. The relatively small number of these neurons (only three in our series) will hinder our ability to answer these questions experimentally.

Interestingly, we noted action potential failures in two neurons during stimulation. Although a spike invaded the soma (our recording site) it did not pass 0 mV. Such action potential failures have previously been observed during electrical stimulation of rabbit and cat nodose neurons (11). Although the true physiological relevance is not known, computer modeling suggests that the low amplitude of the invading spike may not suffice to assure central propagation of the spikes (4). These modeling results may not necessarily apply to pseudo-unipolar mammalian sensory neurons. However, our findings raise the question whether the primary afferent neuron may function as a dynamic filter, potentially limiting sensory transmission. Parallel recordings from a peripheral site (i.e., the soma) and central terminations are required to definitively answer this point.

Molecular and electrophysiological studies have clearly demonstrated that vagal sensory neurons projecting to the proximal gastrointestinal tract express proton-gated ion channels (7, 21, 28, 32). We thus tested whether luminal acidification directly activated gastroesophageal sensory neurons. Five of 11 mechanosensitive neurons examined also responded to luminal acidification. The increase in activity was small and returned to baseline within 6 min. These results are consistent with recordings from the rat esophagus where about one-third of the mechanosensitive afferents respond to acid (16). In contrast, Page et al. (22) only detected one proton-sensitive fiber when studying 11 gastroesophageal afferents in mice. Although we do not know the reason for the different results, it is likely that a shorter exposure time in the latter series contributed to the apparent discrepancies. Moreover, we applied acidic solution through luminal perfusion rather than instilling it into a cylinder placed over the receptive field. The continuing flow may affect the unstirred superficial water layer and further increase proton diffusion into the mucosa. Very few nerve endings project into the epithelial layer of the esophagus (2). Thus acid has to diffuse through the protective epithelial layer, before activating nerve terminals and potentially triggering action potential generation. Shorter exposure time will limit the diffusion and proton concentration in the vicinity of the sensory terminal, resulting in lower response rates. With the longer exposure time, diffusing hydrogen ions may reach areas that are normally not exposed to high proton concentration due to protective mechanisms, primarily submucosal blood flow. Although the organ was superfused by buffered solution, the lack of submucosal blood flow in vitro may lead to activation of afferents that play no physiological role in acid sensation. Yet these afferents could contribute to sensation of acid in disease states, such as esophagitis or peptic ulcer disease, when the mucosal integrity is disrupted.

We have previously reported sensitization of mechanosensitive fibers after administration of acid into the rat stomach (13). Using a lower proton concentration and shorter exposure time to limit the likelihood of injury, we did not observe any interaction between the sequential chemical and mechanical stimulation.

In the rat, we have identified TRPV1 and ASIC3 as the predominant proton-sensitive ion channel in gastric sensory neurons based on electrophysiological and pharmacological properties (28). We, therefore, investigated the effect of gene knockout of these ion channels on the sensory properties of gastroesophageal sensory neurons. Consistent with a potential role in acid sensation, ASIC3 and TRPV1 knockout mice did not respond to luminal acidification, providing further support for their role in acid sensation.

ASIC3 is a member of the large family of ion channels (Degenerins) that was initially identified in mechanosensitive neurons of Caenorhabditis elegans (14). This certainly led to speculation about potential contribution of these channels in mechanosensation. Only nonselective blockers are currently available, preventing the identification of a potential contribution of different ASIC subunits to mechanosensation with pharmacological tools. Using ASIC3 knockout mice, we also examined responses to mechanical stimulation and noted a blunted response at low stimulation intensities compared with wild-type controls. These results at least partially confirm previously published data, showing a decreased response of gastric afferents to circumferential stretch in the same knockout strain (21). However, at higher stimulus intensities, we did not observe differences between ASIC3–/– and control animals, which differs from the previously reported results. This apparent discrepancy is likely due to a relatively subtle effect of ASIC3 on gastroesophageal mechanosensation. Only one of two functionally distinct fiber types ("muscular" afferents) showed altered properties in ASIC3 knockout mice; in addition, gastric emptying, which is modulated by vagovagal reflexes, was unchanged (21). Other reports also leave a confusing picture. In isolated neurons, deletion of ASIC3 did not alter current responses to mechanical stimulation (8). In the colon, ASIC3 deletion blunted mechanosensation. However, amiloride, a nonselective ASIC blocker, did not mimic the effects of ASIC3 deletion on colonic afferents in vitro (12). ASIC3 knockout mice exhibited decreased responses in a model of muscle hyperalgesia (27). The interpretation of these results is complicated by the fact that ASIC channels are comprised of multiple subunits in a hetero-oligomeric complex. To circumvent this problem, Mogil et al. (18) generated a mouse with dominant negative ASIC3 transgene, which exhibits increased rather than decreased cutaneous sensitivity. Thus the effects of ASIC3 gene deletion are likely indirect, requiring additional studies to identify the underlying mechanism.

Consistent with previously published reports on jejunal (25) and colonic (12) mechanosensation, we noted a significant decrease in mechanosensitivity of gastroesophageal afferent neurons in TRPV1 neurons. Some members of the TRP family, but not TRPV1, are gated by mechanical stimuli (6). Thus the effects on mechanosensation, and perhaps also the blunted acid sensation, are due to indirect effects on excitability. We did not directly examine the cellular excitability of all neurons in TRPV1 knockout animals. Whereas the threshold for activation was significantly higher in the gastroesophageal afferents from TRPV1 knockout animals compared with the other groups, the peak frequency (14.7 ± 2.7 Hz) was not different from controls. Prior studies have demonstrated TRPV1 expression in only a subset of vagal sensory neurons (3, 32). In about one-quarter of the cells tested, we did not observe responses to the lowest stimulus intensity, which always triggered an increase in spike frequency in controls. Individual stimulus response functions overlapped between all groups. Thus it is possible that the significant difference between groups is indeed due to the significant effect on the subgroup of neurons that normally express TRPV1, although we cannot exclude nonspecific effects of TRPV1 deletion. As already discussed above, the findings cannot be explained by the known properties of TRPV1 but suggest that TRPV1 indirectly modulates sensory function.

In conclusion, our data clearly confirm a role of TRPV1 in chemo- and mechanosensation of gastroesophageal afferents. ASIC3 may contribute to acid sensation and plays a minor role in responses to distending stimuli. Considering the importance of acid in dyspeptic symptoms and gastroesophageal reflux, TRPV1 and ASIC3 may be an attractive target for treatment strategies in patients who do not respond to acid suppressive therapy.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Bielefeldt, Univ. of Pittsburgh, Division of Gastroenterology, 200 Lothrop St., Pittsburgh, PA 15213 (e-mail: bielefeldtk{at}dom.pitt.edu)

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.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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