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Am J Physiol Gastrointest Liver Physiol 291: G1129-G1136, 2006. First published August 3, 2006; doi:10.1152/ajpgi.00518.2005
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NEUROREGULATION AND MOTILITY

Interstitial cells of Cajal and adaptive relaxation in the mouse stomach

Devika Dixit,1 Natalia Zarate,1 Louis W. C. Liu,1 Douglas R. Boreham,2 and Jan D. Huizinga1

1Intestinal Disease Research Program, Department of Medicine and 2Department of Medical Physics and Applied Radiation Sciences, McMaster University, Hamilton, Ontario, Canada

Submitted 3 November 2005 ; accepted in final form 26 May 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Interstitial cells of Cajal (ICC) are proposed to play a role in stretch activation of nerves and are under intense investigation for potential roles in enteric innervation. Most data to support such roles come from in vitro studies with muscle strips whereas data at the whole organ level are scarce. To obtain insight into the role of ICC in distention-induced motor patterns developing at the organ level, we studied distension-induced adaptive relaxation in the isolated whole stomach of wild-type and W/Wv mice. A method was developed to assess gastric adaptive relaxation that gave quantitative information on rates of pressure development and maximal adaptive relaxation. Pressure development was monitored throughout infusion of 1 ml of solution over a 10-min period. The final intraluminal pressure was sensitive to blockade of nitric oxide synthase, in wild-type and W/Wv mice to a similar extent, indicating NO-mediated relaxation in W/Wv mice. Adaptive relaxation occurred between 0.2 and 0.5 ml of solution infusion; this reflex was abolished by TTX, was not sensitive to blockade of nitric oxide synthase, but was abolished by apamin, suggesting that ATP and not nitric oxide is the neurotransmitter responsible for this intrinsic reflex. Despite the absence of intramuscular ICC (ICC-IM), normal gastric adaptive relaxation occurred in the W/Wv stomach. Because pressure development was significantly lower in W/Wv mice compared with wild type in all the conditions studied, including in the presence of TTX, ICC-IM may play a role in development of myogenic tone. In conclusion, a mouse model was developed to assess the intrinsic component of gastric accommodation. This showed that ICC-IM are not essential for activation of intrinsic sensory nerves nor ATP-driven adaptive relaxation nor NO-mediated relaxation in the present model. ICC-IM may be involved in regulation of (distention-induced) myogenic tone.

gastric motility; enteric nerves; enteric sensory nerves; inhibitory enteric neurotransmission; gastric accommodation

THE STOMACH GENERATES A VARIETY of motor patterns to perform its physiological functions. One of its major motor activities is the phenomenon of accommodation. Accommodation refers to an in vivo reflex that causes relaxation of the stomach musculature to avoid undue intraluminal pressure due to filling of the stomach that could lead to untimely gastric emptying and discomfort. This reflex involves extrinsic (vagal) and intrinsic enteric nerves, stimulated by distension. An important component of gastric accommodation in vivo is driven by the vagus through a vago-vagal reflex that includes activation of stretch receptors within the fundic musculature and involves gastric relaxation caused by the release of nitric oxide. Enteric (intrinsic) inhibitory nerves may function as the end structures of vagal inhibitory pathways but can also function independently of the central nervous system as shown by Paton and Vane (24) in the in vitro guinea pig stomach, and further characterized by Hennig et al. (15). Paton and Vane used the term "adaptive relaxation" for the intrinsic component of accommodation observed in the isolated stomach (24), and in this sense it will be used in the present study. Accommodation is usually discussed as a function of the fundus (fundic relaxation), but it is proven that the whole stomach is involved in this process (19), which makes the in vitro whole stomach preparation suitable to study adaptive relaxation.

Although the critical role of the enteric nerves in regulating these motor activities is unquestioned, the significance of interstitial cells of Cajal (ICC) in the various motor functions of the stomach is just beginning to be clarified. Little doubt exists about the role of ICC in generating gastric pacemaker activity that is critical for the peristaltic activity of the distal stomach (10, 28). More controversial are the possible roles of ICC in neural control of the various motor patterns. A role for intramuscular ICC (ICC-IM) in inhibitory innervation has been postulated on the basis of the observations that inhibitory innervation appears to be diminished in W/Wv mutant mice that lack intramuscular ICC (3, 29). On the other hand, when this hypothesis was tested in vivo in mice related to the innervation of the lower esophageal sphincter (LES), no clear evidence of abnormal reflex inhibition of the LES was observed (27).

ICC have also been proposed to play a role as stretch receptors involved in distension-induced activation of enteric nerves (25), and they have been found to be closely associated with intramuscular arrays, one of the two putative mechanoreceptors in gastrointestinal smooth muscle (13). Therefore, owing to the potential dual involvement of ICC-IM in afferent and efferent pathways, their role in the enteric reflex responsible for gastric adaptive relaxation upon distension could be of critical importance.

The objectives of the present study were to develop a method to quantify adaptive relaxation and then conduct comparative studies between wild-type and W/Wv mutant mice to evaluate the role of ICC in the process of adaptive relaxation.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Male wild-type and W/Wv mice (8–10 wk) were fasted for 24 h with free access to water. Mice were killed by cervical dislocation, and the stomachs were dissected by cutting the distal esophagus at 4 mm proximal to the gastroesophageal junction and at the duodenal end, 10 mm distal to the pylorus. Any gastric content that remained in the stomach was removed gently by infusion of Krebs solution. The composition of the Krebs solution was (in mM) 118 NaCl, 4.7 KCl, 1.0 NaH2PO4, 25 NaHCO3, 1.2 MgSO4, 11.0 glucose, 2.5 CaCl2; pH 7.4. A polyethylene tube (ID 0.38 mm, OD, 1.09 mm) was inserted through the lower esophageal sphincter into the stomach. The stomach was placed in the organ bath containing aerated (95% O2 and 5% CO2) Krebs solution at 37°C. A second polyethylene tube (ID 0.58 mm, OD 0.965 mm) attached to a pressure transducer connected to a Grass Polygraph machine (model 7D) with an analog and an electronic (computer) data-acquisition program (PolyVIEW 2.5) was inserted through the pyloric sphincter into the stomach. The other end of the tube that was inserted through the LES was attached to a peristaltic pump. This pump was used to infuse 1 ml of Krebs solution at a constant rate over 10 min. The stomach was allowed to equilibrate for 30 min before the first distension was conducted and between distensions if more than one was conducted in a particular set of experiments. After equilibration, the pump was turned on and intragastric pressure was recorded. After the 10-min distension, the stomach was allowed to remain at this volume for 5 min and pressure changes were recorded. Between distensions the stomachs were emptied by reversing the pump to minimize manual handling of the stomach. A 1-ml volume reflects the volume of an unfasted mouse stomach in both wild-type and W/Wv mice. A distension protocol over a 10-min time period but not a 1- or 3-min time period allowed development of adaptive relaxation as described in this paper in both wild-type and W/Wv mice (11). Distension curves were plotted using data (pressure and volume/time) taken every 15 s to assess pressure development and adaptive relaxation. Not more than two distensions were carried out per animal, to avoid changes due to diminishing viability; hence pharmacological studies were not done on the same animal. Distension experiments were repeated in the presence of a nitric oxide synthase inhibitor, NG-nitro-L-arginine (L-NNA; 2 x 10–4 M). Experiments were also conducted in the presence of tetrodotoxin (TTX; 2 x 10–7 M) and apamin (0.1 µM). Drugs were added to the Krebs solution in the organ bath 20–30 min before the distensions were carried out.

Animals were handled in accordance with McMaster University Animal Facility recommendations. The protocols used were approved by the McMaster University Animal Care Committee in accordance with guidelines from the Canadian Council on the Use of Laboratory Animals.

The term adaptive relaxation is used and not "accommodation" in the description of the experiments to clearly identify our study of the nonvagal relaxation of the stomach in response to distention; hence this study provides no information on vagally mediated accommodation.

Data are expressed as means ± SD. Comparisons within groups were made with the unpaired Student's t-test for two means and using one-way ANOVA with Bonferroni post hoc test for more than two means. P < 0.05 was considered to be significant. N, number of experiments; n, number of animals.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Adaptive relaxation. Stomachs were distended by infusion of a volume of 1 ml at a constant rate over a 10-min period. A typical distension curve showing the pressure vs. time relationship is shown in Fig. 1A. Three distinct phases in pressure development were evident. An initial phase was associated with an increase in pressure at a variable rate; a second phase showed a markedly reduced rate of pressure increase demonstrating adaptive relaxation; and the last phase showed an increase in the rate of pressure development. The second phase contained the point at which the lowest change in pressure over time occurred, the point of maximal adaptive relaxation. To quantify the pressure development and to determine the value of maximal adaptive relaxation, third order polynomial fitting was carried out. This allowed for an unbiased assessment of adaptive relaxation. To assess the rate of change in pressure along the distension curve, the first derivative was taken of the third order polynomial regression equation. This allowed us to determine the slopes, i.e., the rates of pressure change over volume, at all time points of the pressure-volume relationship (Fig. 1B). Such graphs showed a concave up shape; the pressure change gradually decreased until it reached a minimum. This point was termed the point of maximal adaptive relaxation because it shows the lowest pressure increase over volume infused (lowest rate of change). After this point, the rate of pressure development in the stomach began to increase again. To give a quantitative assessment the following parameters were assessed: the ({delta}P/{delta}V)initial, i.e., the initial slope at t = 0 min or 0 ml (as determined by the fitting equation, where P is pressure, V is volume, and {delta} is change), was determined to reflect the starting conditions of the experiment; the ({delta}P/{delta}V)ARmax, i.e., the slope of maximal adaptive relaxation, was determined, as well as ({delta}P/{delta}V)end, which reflected the final stage of the pressure development at t = 10 min (which is equivalent to volume = 1 ml).


Figure 1
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  		Fig. 1. Pressure development over time. A: wild-type mouse. One milliliter of Krebs solution was infused over 10 min in the isolated stomach, which was submerged in Krebs solution at 37°C. The intraluminal pressure was measured continuously. The figure depicts the relationship between intraluminal pressure and time. After 10 min the pump is turned off. A typical pressure-time curve of a wild-type mouse stomach shows 4 phases. The first 10 min depict pressure build-up over the distension protocol in which 1 ml of Krebs is being infused into the stomach at a constant rate. The first phase (first 2 min approximately) is the initial increase in pressure, followed by the second phase (from ~2 to ~6 min) of distension in which the change in pressure over time is greatly reduced, and is associated with the activation of inhibitory enteric nerves (see text). This is termed the adaptive relaxation phase. The third phase (from ~6 to 10 min) shows a return to a more marked increase in pressure over time. After 10 min the pump is turned off and pressure of the full stomach is measured for a further 5 min; this is the final fourth phase of a typical protocol. B: wild-type mouse, another example of an experiment following the same protocol as in A but now the pressure (P) is plotted against volume (V) over the first 10 min only ({lozenge}), with superimposed the fitted line [third order polynomial; y = 0.0004x3 – 0.025x2 + 0.716x + 1.1,965 (R2 = 0.9846)]. Also depicted is the first derivative of the fitted line ({blacklozenge}). The first derivative clearly shows that the pressure increase declines in magnitude in the first 5 min, up to the point where the {Delta}P/{Delta}V is smallest, the point of maximal adaptive relaxation. C: W/Wv mouse: pressure development vs. volume in the first 10 min ({square}), with superimposed the third order fitted line [y = 0.0025x3 – 0.0944x2 + 1.3697x – 1.5464 (R2 = 0.9653)]. Also depicted is the first derivative of the fitted line ({blacksquare}). Comparison of B and C suggests that adaptive relaxation is qualitatively similar in W/Wv and wild-type mice.

 
Because differences in size of the stomach may influence interpretation of the above data, wild-type and W/Wv mice used were littermates of equal weight between 17 and 22 g; in addition, the weight and volume of the stomachs were determined. Although in most paired experiments the weight of the stomach of W/Wv mice was higher, the difference was not large enough to make it statistically significant, suggesting a minor role if any in the differences described above. In 10 paired experiments, the stomach weight of wild-type mice was 0.30 ± 0.06 g and of W/Wv mice 0.36 ± 0.08 g (P = 0.08). The volume (water displacement of whole stomach) was 0.77 ± 0.12 and 0.83 ± 0.05 ml, respectively.

Analysis of adaptive relaxation in wild-type and W/Wv mice in basal conditions or in the presence of L-NNA. Maximal adaptive relaxation in wild-type mice occurred at 4.5 ± 1.3 min (0.45 ml). At this point the absolute pressure was 7.6 ± 1.2 cmH2O and the pressure development was at its lowest (12 cmH2O/ml), significantly different from both the initial slope (25 cmH2O/ml) and the end slope at the 10-min time point (36 cmH2O/ml) (P < 0.05; Table 1). To explore the role of NO in adaptive relaxation, a subsequent series of experiments was conducted in the same manner but in the presence of the nitric oxide synthase blocker L-NNA (2 x 10–4 M). All rates of pressure development were not significantly different from those obtained under control conditions, and, hence, blockade of NO synthesis did not influence the degree of adaptive relaxation (Table 1). In the presence of L-NNA, maximal adaptive relaxation occurred at 3.6 ± 0.8 min. At this point the absolute pressure was 5.6 ± 3.0 cmH2O and the pressure development was at its lowest (12 cmH2O/ml; Table 1); similar to controls, pressure development at the point of maximal adaptive relaxation was significantly different from the initial and final slope. Although NO did not significantly contribute to the adaptive relaxation, it did contribute to the overall impediment of distention-induced pressure development because the final pressure developed at 1 ml of distention was markedly increased in the presence of L-NNA compared with basal conditions (Table 2).


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  		Table 1. Rate of pressure development

 

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  		Table 2. Pressures at different time points and pharmacology

 
Examination of the pressure-volume relationship under control conditions in W/Wv mice identified marked adaptive relaxation (Fig. 1C). The presence of adaptive relaxation was confirmed by the fact that the first derivatives of the third order polynomial fits showed a concave up configuration and were similar to the findings observed in wild-type mouse stomach; ({delta}P/{delta}V)ARmax was significantly different from both ({delta}P/{delta}V)initial and ({delta}P/{delta}V)end (Table 1). The values obtained for the three slopes, ({delta}P/{delta}V)initial, ({delta}P/{delta}V)ARmax, as well as ({delta}P/{delta}V)end, were significantly smaller in W/Wv mice compared with wild-type mice (P < 0.05). This indicates that pressure development occurred at a slower rate in the W/Wv mice and that the rate of pressure change at the point of maximal adaptive relaxation was lower in W/Wv mice. The absolute value of the pressure at the point of maximal adaptive relaxation that occurred at time 5.1 ± 1.3 min was 4.3 ± 2.2 cmH2O, significantly lower in W/Wv mice than in the wild type, which had a value of 7.5 ± 3.5 cmH2O. Similarly, the final absolute pressure obtained after 10 min was 8.0 ± 1.4 cmH2O and was significantly lower than in wild-type animals (Table 2). To assess the role of NO in adaptive relaxation in W/Wv mice, experiments were conducted in the presence of L-NNA. The rates of pressure change were not different from those in the absence of L-NNA, suggesting that NO did not play a significant role in enteric adaptive relaxation in W/Wv mice (Table 1). Similar to the findings observed in wild-type mice, NO did contribute to the overall impediment of distension-induced pressure development because the final pressure developed at 10 min was markedly increased in the presence of L-NNA compared with the basal conditions (Table 2).

Pressure development in the presence of TTX or apamin. A typical pressure-volume relationship in the presence of TTX did not show an adaptive relaxation phase in both wild-type and W/Wv mice (Fig. 2). This was confirmed by the fact that the first derivatives of the third order polynomial curves did not show a concave up configuration but showed a concave down or a linear shape (Fig. 2A). Therefore, it was not feasible to analyze the data as done under control conditions or in the presence of L-NNA. The pressure development in the presence of TTX showed one of two patterns. The first was a near-linear development as shown in Fig. 2, A and B. The second, which was dominant in the W/Wv mice (Figs. 2C and 3C), showed a period of ~4 min in which there was little or no pressure development followed by a near-linear increase in pressure. Figure 2D shows that the linear development was typical of wild types whereas the second pattern with an inflection point at 0.4 ml (4 min) was typical of W/Wv animals. Hence, a third order polynomial fit did not represent the pressure development in the presence of TTX, confirming that the adaptive relaxation was evoked by enteric neural activity. On the basis of the observations of the second pattern, plots for the pressure development were divided in two parts before and after the 4-ml infusion and slopes corresponding to these two phases of pressure were determined (Table 3). Although the slopes of the two phases in the W/Wv mice were significantly different (P < 0.0002), this was not the case for wild-type mice, indicating a near-constant rate of increase of pressure development over the entire 10 min (1 ml) protocol in wild-type mice. In the presence of TTX, pressure development in the first 4 min was significantly less in the W/Wv compared with wild-type mice (P < 0.0002), which suggests a lower myogenic tone in the mutant mice. Pressure development may also be different if the stomach size was different. In wild-type mice, pressure development in the presence of apamin did not show adaptive relaxation according to the criteria developed for the experiments in the presence of TTX as described above. Because the pressure development was near linear in the presence of apamin (Fig. 3), the quantification of pressure development was expressed in a similar manner as with TTX (Table 3). The pressure development in the presence of apamin was much larger compared with control conditions and pressure development in the presence of TTX indicating that apamin blocked a significant inhibitory component, resulting in marked pressure development. At 10 min, in the presence of apamin, the absolute intraluminal pressure was ~40 cmH2O (Table 2), which was significantly higher compared with TTX and also significantly higher compared with control values and values in the presence of L-NNA. This indicates that it was apamin rather than L-NNA that inhibited the adaptive relaxation phase during pressure development in the absence of vagal influence. Pressure development in the presence of apamin could be influenced by a potential direct effect of apamin on smooth muscle cells by blocking potassium channels. In that case, initial pressure development in the presence of apamin in wild-type mice would likely be increased compared with control. This was not observed. First, common pressure-volume relationships as shown in Fig. 3A showed the opposite. Second, {delta}P/{delta}V over the first 4 min was 15.5 ± 6.1 cmH2O/ml (N + 10) in wild-type animals, whereas in the presence of apamin, this was 20.5 ± 15.7 (N + 7), not significantly different.


Figure 2
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  		Fig. 2. Pressure development in the presence of tetrodotoxin (TTX). To assess whether or not the adaptive relaxation phase as shown in Fig. 1 was truly a reflex mediated by intrinsic nerves, the same protocols were repeated in the presence of TTX. A: wild-type mouse: pressure development over time (volume) in the first 10 min ({square}), with superimposed the 3rd order fitted line [y = 0.6555x3 – 1.8211x2 + 7.2657x + 0.4511 (R2 = 0.9946)]. The pressure-volume relationship is almost linear, indicating that the adaptive relaxation phase as shown in Fig. 1 is not present. Also depicted is the first derivative of the fitted line ({blacksquare}), which shows that no "point of maximal adaptive relaxation" at around the 5-min time point (0.5 ml volume) can be identified. B and C: 2 experiments using the same protocol in different W/Wv mice showing different pressure developments in the presence of TTX. The pressure development shown in C was dominant. For discussion on fitting procedures, see RESULTS. D: average values of pressure development related to volume in the presence of TTX.

 

Figure 3
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  		Fig. 3. Comparison of typical pressure-volume relationships under various conditions in wild-type mice (A) and W/Wv mice (B). L-NNA, NG-nitro-L-arginine.

 

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  		Table 3. Rates of pressure change in the presence of TTX or apamin

 
In W/Wv mice, no significant adaptive relaxation was observed in the presence of apamin, similar to the situation in wild-type mice (Fig. 3B). In the presence of apamin, the rate of pressure development as well as the pressure at 10 min was much larger compared with pressure development in the presence of TTX (Table 3).

Pressure development at a constant volume of 1 ml. After infusion of 1 ml over 10 min, the volume was kept constant for 5 min to observe possible further adaptive relaxation. After 10 min, the pressure dropped ~20% and then stabilized within the 5-min period. The final pressure values are shown in Table 2. Because the drops in pressure values (%) were similar in the absence and presence of TTX, there was apparently no nerve-mediated relaxation; hence no second phase of adaptive relaxation occurred.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study investigated the intrinsic part of accommodation in the isolated whole stomach of the mouse: the nonvagally mediated adaptive relaxation (24). W/Wv mice showed marked adaptive relaxation, similar to wild-type mice, indicating that ICC-IM, which are absent in the corpus (26) and reduced to ~5% of their original numbers in the fundus (L. W. C. Liu, X.-Y. Wang, and J. D. Huizinga, unpublished observations), are not essential for this reflex, which is evoked by distention-induced activation of inhibitory enteric nerves. The adaptive relaxation was not affected by L-NNA but was abolished by TTX or apamin, indicating that apamin-sensitive neurotransmission was responsible for muscle relaxation. NO also contributed to retardation of pressure development in response to distension, although not in the process of adaptive relaxation. There was no difference between wild-type and W/Wv mice in this respect, suggesting that ICC-IM are not critical for NO to reach smooth muscle cells.

The present study demonstrates that an apamin-sensitive neurotransmitter, probably ATP, but not NO, is the primary component of the intrinsic (nonvagal) inhibitory reflex to distension in the murine stomach. This conclusion was made possible after development of a quantitative assessment of adaptive relaxation. The experimental protocol used, i.e., stomach distention induced by infusion of 1 ml over 10 min, resulted in adaptive relaxation occurring consistently around the 4-min mark followed by increasing pressure development. This allowed the fitting of the data using third order polynomial fitting, which established the inflection point where adaptive relaxation was maximal. This could be further demonstrated by calculating the first derivate of the third order polynomial. This resulted in quantitative information on the various stages of pressure development, which was then used to compare wild-type mice with W/Wv mutant mice. The fact that no further neurally mediated adaptive relaxation took place after the volume was kept constant at 1 ml suggests that adaptive relaxation was fully captured during the distention protocol. Hennig et al. demonstrated in the guinea pig stomach that pressure development induced by distention involved cholinergic excitation providing the tone and nitrergic and apamin-sensitive components providing adaptive relaxation (15). In the guinea pig, NO appeared to be the dominant neurotransmitter in the adaptive relaxation reflex. The pressure development in the guinea pig was described as having three phases, an initial phase involving the first 7 ml in which the pressure increase was relatively strong, a second phase in which pressure declined, and a third phase in which pressure increased again albeit at a slower rate compared with the initial phase. The {delta}P/{delta}V was calculated and termed resistivity by using linear regression over the first 7-ml infusion and over the total time of infusion. The slope involving total time of infusion was always lower than the slope covering only the first 7 ml because the former included the period of adaptive relaxation. The second phase that showed the pressure decline was not assessed with quantitative parameters. We were able to provide quantitative data on the phase of adaptive relaxation using third order polynomial fitting. This may be more difficult in the guinea pig because of the development of large phasic activities. Both the guinea pig and the mouse show significant variability between animals; hence quantification of data has to take into account that the physiological system has a wide range of normal behavior.

In vitro studies by Mashimo et al. (21) suggested two parallel and overlapping inhibitory pathways in the mouse fundus based on electrical stimulation of enteric nerves involving the neurotransmitters ATP and nitric oxide. ATP acting on P2 receptors opened apamin-sensitive potassium channels (1) to produce fast inhibitory junction potentials (IJPs) whereas NO mediated slow IJPs (21). Consistently, neurally induced relaxation of the mouse stomach was mediated in part by NO and in part blocked by apamin or P2Y receptor desensitization (23). In the rat stomach, similar results were obtained (18). Although Curro and coworkers (6) suggested that ATP might not play a crucial role as inhibitory neurotransmitter in the stomach, their model demonstrated only a small apamin-sensitive component, probably not strong enough to serve as a definite study on the role of ATP as neurotransmitter (17). In vivo, distention of the rat stomach evoked relaxation that was mediated by the vagus nerve associated with release of NO, a response that was abolished by vagotomy (30). Consistently, electrical stimulation of the vagus nerve in the nondistended isolated mouse stomach resulted in relaxation that was suppressed by nitric oxide synthase inhibitors (36). This same conclusion was reached using the guinea pig (9, 22). The preferential role of ATP in gastric adaptive relaxation, as observed in the present study, came therefore as a surprise. Interestingly, our conclusion that the nonvagal component of adaptive relaxation is not mediated by nitric oxide is consistent with observations in the denervated, vascularly perfused rat stomach in vitro showing that intra-arterial application of L-NNA methyl ester did not affect pressure increases evoked by gastric distention (30). The authors did not explore the role of ATP on gastric pressure development. According to the present study, it is likely that stretch activates inhibitory nerves in the mouse stomach inducing simultaneous release of both NO and ATP with ATP being the dominant neurotransmitter involved in adaptive relaxation. The dominant role of NO in vagally mediated relaxation suggests that NO is primarily of vagal origin or that intrinsic nitrergic neurons rely on the autonomic nervous system for their activation.

Nonneurally mediated distension-induced tone in the stomach of W/Wv mice is clearly different from wild-type mice, as measured in the presence of TTX. Furthermore, the intraluminal pressure throughout the distention protocol was always significantly lower in the W/Wv stomach compared with wild type. It appears therefore likely that ICC are involved in regulating myogenic tone. This is consistent with findings of a reduced LES tone in W/Wv mice (27). Similarly, the tone of the internal anal sphincter was shown to be significantly lower in W/Wv mice compared with wild type (12.8 vs. 9.5 mmHg) (31), although no significant difference was found in another study (21 vs. 15 mmHg) (8). The tone in the intestinal musculature of W/Wv mice appears to be reduced (14). Although eNOS has been suggested to generate NO for regulation of sphincter tone, eNOS is unlikely to be different in wild-type and W/Wv mice and may not contribute to the differences between wild-type and W/Wv mice (31).

The preserved adaptive relaxation reflex in W/Wv mice speaks for an intact afferent limb and makes a role for ICC in afferent pathways not apparent. Consistently, relaxation of the internal anal sphincter upon injection of 25 µl water in a latex balloon in the rectum to evoke a rectoanal inhibitory reflex was normal in W/Wv mice (31). However, when graded rectal distensions were evoked using 0.25–0.40 ml of air into a polyethylene balloon, the internal anal sphincter relaxation in W/Wv mice was significantly decreased (8). This might imply that ICC are only used in afferent pathways as stretch receptors or otherwise under specific conditions.

The close association between ICC and enteric nerves suggests that the functions of ICC whatever they might be can be modified by neural activity. Because ICC and smooth muscle function are closely linked, it follows that innervation of ICC will affect smooth muscle function. Linking ICC with innervation started with the original work of Cajal (4, 5) and was substantiated in subsequent morphological studies (2). Further ultrastructural studies, in which it was observed that enteric nerve varicosities make close or synapselike contact with ICC and not with smooth muscle cells, were consistent with physiological importance of ICC innervation (7, 32). Subsequent physiological studies were interpreted to show that ICC were necessary for smooth muscle innervation. Burns et al. (3) showed that the L-NNA-sensitive IJPs were absent in W/Wv stomach. This was further substantiated in the LES where NO-mediated IJPs and relaxations were found to be markedly reduced in W/Wv mice compared with wild type (35). In contrast, a study by Sivarao et al. (27) found that nitrergic relaxations of the LES in response to swallowing were normal in W/Wv mice in vivo, consistent with recent observations in the anal sphincter in vivo (8, 31). In light of these studies, it is likely that direct smooth muscle innervation is common. Close contact between nerves and smooth muscle cells do exist albeit at a relatively low density (12). Furthermore, there is no evidence that diffusion is a serious impediment to the relatively slow innervation of gut smooth muscle. The general statement that "transmitter released from enteric motor neurons binds primarily to receptors expressed by ICCs" (33) is therefore probably not correct as a general rule. However, it may apply to specific pathways of innervation such as vagal afferents (13, 16, 20) and vagal efferents involved in accommodation (30, 34). The process of adaptive relaxation, as measured in vitro, may occur in vivo as part of vagally mediated accommodation, or it may function as a separate phenomenon (30).

In summary, the present study demonstrates a method to quantify adaptive relaxation in the mouse stomach in vitro. There was no difference between adaptive relaxation in W/Wv mice compared with wild-type mice, and hence no evidence for a role of ICC in inhibitory innervation nor stretch-induced neural excitation could be deduced. Adaptive relaxation was primarily mediated by ATP. Distention-induced gastric intraluminal pressure was in part dependent on NO, in both wild-type and W/Wv mice.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Canadian Institutes of Health Research (CIHR) operating grants. N. Zarate was supported by a Canadian Association of Gastroenterology fellowship supported by Novartis-Bristol Myers Squibb. L. Liu was supported by a CIHR Clinical Scientist Award.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. D. Huizinga, McMaster Univ., Intestinal Disease Research Programme, Health Science Centre Rm. 3N5C, 1200 Main Street West, Hamilton, Ontario, L8N 3Z5, Canada (e-mail: huizinga{at}mcmaster.ca)

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
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  1. Banks BE, Brown C, Burgess GM, Burnstock G, Claret M, Cocks TM, and Jenkinson DH. Apamin blocks certain neurotransmitter-induced increases in potassium permeability. Nature 282: 415–417, 1979.[CrossRef][Medline]
  2. Boeke J. The sympathetic endformation, its synaptology, the interstitial cells, the periterminal network, and its bearing on the neurone theory discussion and critique. Acta Anat (Basel) 8: 18–61, 1949.[Web of Science][Medline]
  3. Burns AJ, Lomax AE, Torihashi S, Sanders KM, and Ward SM. Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach. Proc Natl Acad Sci USA 93: 12008–12013, 1996.[Abstract/Free Full Text]
  4. Cajal RS. Histologie du système nerveux de l'Homme et des Vertébrés. Grand sympathique. Paris: Maloine, 1911, p. 891–942.
  5. Cajal RS.Texture of the Nervous System of Man and the Vertebrates (vol. 3), edited by Pasik P and Pasik T. Vienna: Springer-Verlag, 2002.
  6. Curro D, De Marco T, and Preziosi P. Evidence for an apamin-sensitive, but not purinergic, component in the nonadrenergic noncholinergic relaxation of the rat gastric fundus. Br J Pharmacol 143: 785–793, 2004.[CrossRef][Web of Science][Medline]
  7. Daniel EE and Posey Daniel V. Neuromuscular structures in opossum esophagus: role of interstitial cells of Cajal. Am J Physiol Gastrointest Liver Physiol 246: G305–G315, 1984.[Abstract/Free Full Text]
  8. De Lorijn F, De Jonge WJ, Wedel T, Vanderwinden JM, Benninga MA, and Boeckxstaens GE. Interstitial cells of Cajal are involved in the afferent limb of the rectoanal inhibitory reflex. Gut 54: 1107–1113, 2005.[Abstract/Free Full Text]
  9. Desai KM, Sessa WC, and Vane JR. Involvement of nitric oxide in the reflex relaxation of the stomach to accommodate food or fluid. Nature 351: 477–479, 1991.[CrossRef][Medline]
  10. Dickens EJ, Edwards FR, and Hirst GD. Selective knockout of intramuscular interstitial cells reveals their role in the generation of slow waves in mouse stomach. J Physiol 531: 827–833, 2001.[Abstract/Free Full Text]
  11. Dixit D. Gastric Adaptive Relaxation in W/Wv Mutant and Irradiated Mice (PhD thesis). Hamilton, ON, Canada: McMaster University, 2005.
  12. Faussone-Pellegrini MS, Pantalone D, and Cortesini C. An ultrastructural study of the smooth muscle cells and nerve endings of the human stomach. J Submicrosc Cytol Pathol 21: 421–437, 1989.[Web of Science][Medline]
  13. Fox EA, Phillips RJ, Martinson FA, Baronowsky EA, and Powley TL. Vagal afferent innervation of smooth muscle in the stomach and duodenum of the mouse: morphology and topography. J Comp Neurol 428: 558–576, 2000.[CrossRef][Web of Science][Medline]
  14. Fujita A, Okishio Y, Fujinami K, Nakagawa M, Takeuchi T, Takewaki T, and Hata F. Role of the interstitial cells distributed in the myenteric plexus in neural reflexes in the mouse ileum. J Pharm Sci 96: 483–492, 2004.
  15. Hennig GW, Brookes SJ, and Costa M. Excitatory and inhibitory motor reflexes in the isolated guinea-pig stomach. J Physiol 501: 197–212, 1997.[Abstract/Free Full Text]
  16. Huizinga JD, Berezin I, Valdez DT, Xue S, and Diamant NE. Interstitial cells of Cajal as sensory receptors for vagal afferents in the cat esophagus (Abstract). Neurogastroenterol Motil 11: 266, 1999.
  17. Huizinga JD, Dixit D, and Zarate N. Intrinsic inhibitory innervation of the stomach (Letter to the Editor). Br J Pharmacol 146: 163–164, 2005.[CrossRef][Web of Science]
  18. Jenkinson KM and Reid JJ. The P(2)-purinoceptor antagonist suramin is a competitive antagonist at vasoactive intestinal peptide receptors in the rat gastric fundus. Br J Pharmacol 130: 1632–1638, 2000.[CrossRef][Web of Science]
  19. Lee KJ, Vos R, Janssens J, and Tack J. Differences in the sensorimotor response to distension between the proximal and distal stomach in humans. Gut 53: 938–943, 2004.[Abstract/Free Full Text]
  20. Liu LWC, Ye J, Wang L, Tougas G, Perdue MH, and Huizinga JD. Role of interstitial cells of Cajal as mechano-sensory receptors in the activation of gastric vagal afferent pathways (Abstract). Gastroenterology 122: A158, 2002.
  21. Mashimo H, He XD, Huang PL, Fishman MC, and Goyal RK. Neuronal constitutive nitric oxide synthase is involved in murine enteric inhibitory neurotransmission. J Clin Invest 98: 8–13, 1996.[Web of Science][Medline]
  22. Meulemans AL, Helsen LF, and Schuurkes JA. Role of NO in vagally mediated relaxations of guinea-pig stomach. Naunyn Schmiedebergs Arch Pharmacol 347: 225–230, 1993.[Web of Science][Medline]
  23. Mule F and Serio R. NANC inhibitory neurotransmission in mouse isolated stomach: involvement of nitric oxide, ATP and vasoactive intestinal polypeptide. Br J Pharmacol 140: 431–437, 2003.[CrossRef][Web of Science][Medline]
  24. Paton DM and Vane JR. Analysis of he responses of the isolated stomach to electrical stimulation and to drugs. J Physiol 165: 10–46, 1963.[Free Full Text]
  25. Rumessen JJ, Thuneberg L, and Mikkelsen HB. Nerve terminals and interstitial cell-types in plexus muscularis profundus (mouse small intestine). Scand J Gastroenterol Suppl 71: 145–146, 1982.[Medline]
  26. Seki K and Komuro T. Distribution of interstitial cells of Cajal and gap junction protein, Cx 43 in the stomach of wild-type and W/W (v) mutant mice. Anat Embryol (Berl) 206: 57–65, 2002.[CrossRef][Medline]
  27. Sivarao DV, Mashimo HL, Thatte HS, and Goyal RK. Lower esophageal sphincter is achalasic in nNOS–/– and hypotensive in W/Wv mutant mice. Gastroenterology 121: 34–42, 2001.[CrossRef][Web of Science][Medline]
  28. Suzuki H, Takano H, Yamamoto Y, Komuro T, Saito M, Kato K, and Mikoshiba K. Properties of gastric smooth muscles obtained from mice which lack inositol trisphosphate receptor. J Physiol 525: 105–111, 2000.[Abstract/Free Full Text]
  29. Suzuki H, Ward SM, Bayguinov YR, Edwards FR, and Hirst GD. Involvement of intramuscular interstitial cells in nitrergic inhibition in the mouse gastric antrum. J Physiol 546: 751–763, 2003.[Abstract/Free Full Text]
  30. Takahashi T and Owyang C. Characterization of vagal pathways mediating gastric accommodation reflex in rats. J Physiol 504: 479–488, 1997.[Abstract/Free Full Text]
  31. Terauchi A, Kobayashi D, and Mashimo H. Distinct roles of nitric oxide synthases and interstitial cells of Cajal in rectoanal relaxation. Am J Physiol Gastrointest Liver Physiol 289: G291–G299, 2005.[Abstract/Free Full Text]
  32. Thuneberg L. Interstitial cells of Cajal. In: Handbook of Physiology. The Gastrointestinal System. Bethesda, MD: Am. Physiol. Soc., sect. 6, vol. I, pt. 1, chapt. 10, 1989, p. 349–386.
  33. Ward SM. Interstitial cells of Cajal in enteric neurotransmission. Gut 47, Suppl 4: iv40–iv43, 2000.[Free Full Text]
  34. Ward SM, Beckett EA, Wang X, Baker F, Khoyi M, and Sanders KM. Interstitial cells of Cajal mediate cholinergic neurotransmission from enteric motor neurons. J Neurosci 20: 1393–1403, 2000.[Abstract/Free Full Text]
  35. Ward SM, Morris G, Reese L, Wang XY, and Sanders KM. Interstitial cells of Cajal mediate enteric inhibitory neurotransmission in the lower esophageal and pyloric sphincters. Gastroenterology 115: 314–329, 1998.[CrossRef][Web of Science][Medline]
  36. Yano S, Kiyota Y, Yamamoto M, and Watanabe K. Pharmacological features of non-adrenergic non-cholinergic (NANC) relaxation induced by electrical vagal stimulation in isolated mouse stomach. Jpn J Pharmacol 69: 9–15, 1995.[Medline]



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