THEME
Physiology and Pathophysiology of the Interstitial Cell of Cajal: From Bench to Bedside
II. Gastric motility: lessons from mutant mice on slow waves and innervation

Jan D. Huizinga

McMaster University, Hamilton L8N 3Z5, Ontario, Canada


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
GENERAL INTRODUCTION TO ICC
ICC AND INNERVATION
ICC AND SLOW-WAVE GENERATION
THE ELECTROGASTROGRAM
ICC AND PACING
THE PYLORUS AND ICC...
REFERENCES

The stomach harbors a network of interstitial cells of Cajal (ICC) associated with Auerbach's plexus as well as intramuscular ICC within the muscle layers that make close apposition contact with nerve varicosities. ICC are critical for slow-wave generation, making ICC the pacemaker cells of the gut, allowing rhythmic peristaltic motor patterns in the mid- and distal stomach. ICC also play a role in neurotransmission, but its importance relative to direct muscle innervation is still under investigation. The role of ICC in many control functions of gastric motility in humans needs further examination. The pathophysiology of ICC in disease can be partially assessed by immunohistochemistry and electron microscopy on tissue samples. Electrogastrogram measurements may also play a role, but this technique needs further refinement. Communication between ICC and muscle may involve electrical coupling, metabolic coupling through gap junctions, or secretion of nitric oxide or carbon monoxide.

interstitial cells of Cajal; stomach; gastric emptying; innervation; distension


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
GENERAL INTRODUCTION TO ICC
ICC AND INNERVATION
ICC AND SLOW-WAVE GENERATION
THE ELECTROGASTROGRAM
ICC AND PACING
THE PYLORUS AND ICC...
REFERENCES

GASTRIC MOTILITY INVOLVES complex processes in which parts of the stomach relax to accept food from the esophagus, whereas other parts deliver chyme to the small bowel at a rate optimal for digestion and absorption. Before delivery to the small intestine, the stomach stores, mixes, grinds, and sorts food substances into liquid and solid components that are processed and cleared by different mechanisms. Both disease and surgery may cause disruption of any of these complex functions, leading to a variety of clinical disorders including delayed gastric emptying, postgastrectomy syndromes, and gastric atony. To understand the physiological basis for these alterations, a more detailed knowledge of the mechanisms underlying control of gastric motility is essential. Smooth muscle and enteric nerves together with interstitial cells of Cajal (ICC) form complex control systems. This review attempts to put recent work on ICC related to stomach motor physiology into context.


    GENERAL INTRODUCTION TO ICC
TOP
ABSTRACT
INTRODUCTION
GENERAL INTRODUCTION TO ICC
ICC AND INNERVATION
ICC AND SLOW-WAVE GENERATION
THE ELECTROGASTROGRAM
ICC AND PACING
THE PYLORUS AND ICC...
REFERENCES

There are two distinct classes of ICC in the stomach of humans and animal models (Fig. 1). The first type of ICC is spindle shaped and scattered throughout the circular and longitudinal muscle layers from the fundus into the distal antrum. These ICC are closely associated with both excitatory and inhibitory nerve fibers and identified as intramuscular ICC (ICC-IM). Functionally, they may serve as mediators interposing between enteric nerves and smooth muscle cells. In addition, they may serve to modify slow-wave activity. The second type of ICC is triangular or irregularly shaped with multiple processes forming highly branching networks in between the longitudinal and circular muscle layers of the corpus and antrum but not the fundus. The known function of these ICC is their role in pacemaker generation. These ICC are associated with Auerbach's plexus (ICC-AP). Because Auerbach's plexus is often called myenteric plexus, ICC-AP are also referred to as ICC-MY or IC-MY, although the term myenteric plexus really refers to all intrinsic neural structures within the musculature (34).


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Fig. 1.   Different classes of interstitial cells of Cajal (ICC) in the stomach muscle wall. A: multipolar ICC with triangular or stellate-shaped cell bodies comprise a network of ICC associated with the ganglia and nerves of Auerbach's plexus. This network is found in the corpus antrum region but not in the fundus. B: spindle-shaped ICC that are found throughout the circular and longitudinal muscle and are closely connected to enteric nerve varicosities. Pictures generated by Dr. X.-Y. Wang. Confocal microscopy of ICC in the mouse antrum (A) and fundus (B), stained using c-Kit antibodies.


    ICC AND INNERVATION
TOP
ABSTRACT
INTRODUCTION
GENERAL INTRODUCTION TO ICC
ICC AND INNERVATION
ICC AND SLOW-WAVE GENERATION
THE ELECTROGASTROGRAM
ICC AND PACING
THE PYLORUS AND ICC...
REFERENCES

The evidence that ICC-IM play a role in neurotransmission has been reviewed recently (40, 43). In the stomach, ICC-IM form close (~25 nm) contacts with enteric nerves that have nitric oxide (NO), acetylcholine, or substance P as their primary neurotransmitter (2, 41). W/Wv mutant mice have abnormal Kit protein that results in underdevelopment of certain classes of ICC, and these mice lack ICC-IM in the stomach and lower esophageal sphincter (LES). Ward and co-workers (2, 43) showed that the stomach of W/Wv mice has reduced NO-dependent postjunctional responses and suggested a critical role for ICC-IM in inhibitory innervation. However, Sivarao and co-workers (28) found normal LES relaxation in vivo in W/Wv mice, and they suggest that ICC-IM may not be essential for NO-mediated inhibitory innervation. The role of NO in the inhibitory innervation was demonstrated by lack of inhibition in neuronal NO synthase knockout mice (28). ICC-IM are also proposed to play a role in cholinergic excitatory responses in the fundus (41). Because smooth muscle cells are rich in muscarinic receptors and because neurotransmittor is released within the musculature on stimulation, direct cholinergic muscle innervation will occur as well. It is therefore to be investigated in which cholinergically mediated motor control functions ICC-IM play a complementary or exclusive role. All ICC are richly innervated with numerous close-apposition contacts. The conventional wisdom is that smooth muscle cells do not establish close contacts with nerves, the implication being that neurotransmitters have to travel a relatively large distance to reach smooth muscle receptors. Although many nerve varicosities do not appear to have close contact with muscle cells, close contacts do occur with membrane gaps as narrow as 20 nm without the basal lamina (11). Faussone-Pellegrini described such contacts in the human stomach, including protrusions from varicosities fitting into grooves in smooth muscle cells (Fig. 2) (10, 11). Close-apposition contacts between nerves and smooth muscle cells also occur in the mouse and rat stomach (M. S. Faussone-Pellegrini, personal communication). Nevertheless, the innervation of ICC is much more dense, with ICC often wedged between nerve and muscle. It is therefore likely that ICC-IM play an important complementary role in neurotransmission. It is also possible that ICC-IM are intermediaries for unique functional neural pathways yet to be characterized. It should not be forgotten in studies that use mutant mice with loss of ICC-IM that the mutation itself, or the loss of ICC specifically, may directly affect smooth muscle cells (see below).


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Fig. 2.   Close apposition contacts between ICC and muscle cells with nerves A: human stomach, corpus. A protrusion from a nerve varicosity fits perfectly into a groove formed by a smooth muscle cell. B: human stomach, lesser curvature. Two nerve endings, one close, the other near the same smooth muscle cell. C: human stomach within the circular muscle layer of the corpus. Nerve endings (*) are engulfed by ICC [stomach ICC (S-ICC)] making long close-apposition contact. Original figures (A and B) provided by Faussone-Pellegrini are simular to Figs. 28 and 29 from Ref. 11. C is adopted from Fig. 18 in Ref. 10.

A possible mechanism of communication between nerve and ICC and muscle is that neural activity initiates electrical and/or biochemical events in ICC that are transmitted to smooth muscle cells through gap junctions. However, not all ICC that are proposed to have a function in muscle innervation are coupled to smooth muscle cells by gap junctions; hence alternative mechanisms have to be considered (5). In addition, it is likely that products synthesized by ICC, such as NO and carbonmonoxide, will reach smooth muscle cells and influence their excitability (8, 27).


    ICC AND SLOW-WAVE GENERATION
TOP
ABSTRACT
INTRODUCTION
GENERAL INTRODUCTION TO ICC
ICC AND INNERVATION
ICC AND SLOW-WAVE GENERATION
THE ELECTROGASTROGRAM
ICC AND PACING
THE PYLORUS AND ICC...
REFERENCES

ICC-AP are essential for the generation of slow-wave activity. Bauer showed that slow-wave activity originated in the Auerbach's plexus region, where the ICC-AP are located (1). Simultaneous recording from ICC and smooth muscle cells in the guinea pig stomach showed that ICC generated "driving potentials" that were complemented by smooth muscle cells to produce complete slow waves (Fig. 3) (6). The frequency and propagation characteristics of the peristaltic contractions of the mid- and distal stomach are controlled by the slow waves. Excitation through distension and/or enteric neural activity generates smooth muscle action potentials that occur confined to the most depolarized phase of the slow waves. Because of this, the maximum frequency of the peristaltic contractions is the same as the frequency of the slow waves. Hence, whereas the enteric nerves are essential to evoke excitation, in the corpus-antrum it is not bolus-induced peristaltic reflexes that control propagation. The force of the contractions is determined by the degree of action potential generation on the plateau phase of the slow wave and changes in the plateau phase itself. This is determined by the level of excitation of the musculature, hence the level of distension and neural and hormonal input. Lammers and co-workers (17) showed the relationships between slow waves and action potentials. Figure 4 shows the action potentials occurring as "patches" associated with the slow-wave activity. The slow wave causes an area of depolarization, its extent determined by the length of the plateau phase. Within this area of depolarization, action potentials can be evoked that can propagate in several directions and for various lengths of time, depending on the degree of stimulation. The degree of stimulation determines how much of the slow wave is above threshold for the generation of action potentials and whether the slow-wave duration is increased. The overall contraction strength will be, in large part, determined by the extent of action potential generation. At the organ level, the propagation of the contraction wave will follow the aborally propagating slow wave.


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Fig. 3.   Spontaneous activity recorded from ICC within tissue reveals high-amplitude slow waves with very fast rate of rise. Spontaneous electrical activity was obtained from the guinea pig antrum in the presence of nifedipine. Slow-wave activity was recorded from cells recognized by injection of neurobiotin after recording of intracellular activity. Impalements of ICC gave large slow waves with a rate of rise that was much faster than that of slow waves from smooth muscle cells. Recordings from ICC (a) and smooth muscle cells (b) were recorded simultaneously. The slow wave appears first in the ICC. Reproduced from Fig. 3 in Dickens et al. (6).



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Fig. 4.   The pylorus is a region where slow-wave propagation is completely blocked. Propagation of slow waves and action potentials was recorded with a set of 240 electrodes on the pyloric region of the cat. This figure was reproduced and modified with permission from Fig. 3 in Lammers et al. (17).

W/Wv mutant mice provided the first real evidence that ICC-AP were critical for the generation of slow-wave activity (15, 32). The stomachs of W/Wv mice do not have ICC-IM but normal ICC-AP, and indeed slow-wave activity can be readily recorded (2). The normal fundus does not have ICC-AP, and no slow-wave activity is present. Early work in the 1970s from the laboratories of Szurszewski (7) and Tomita (24) described gastric slow-wave activity as being composed of two components. Apart from the regular basic slow wave, a secondary regenerative component was evident. On stimulation (by cholinergic agents for example), the duration and amplitude of the slow wave increased, and this secondary component was shown to be associated with contraction. In other words, in addition to the generation of action potentials, excitation causes changes in slow-wave activity that contribute to contractile activity. An increase in slow-wave duration may cause a decrease in frequency. Thus changes in slow-wave frequency can be a normal part of stomach functioning. Indeed, the slow-wave frequency decreases after a meal (37). Interestingly, Dickens et al. (5a) observed that the second component was missing in W/Wv mice and put forward the hypothesis that ICC-IM are involved in its generation. This was also suggested by Tomita and co-workers studying the guinea pig antrum (13). An alternative explanation could be that, without ICC-IM, intrinsic stimulation from enteric nerves through ICC-IM is diminished, which may lead to a poorly developed secondary component. Furthermore, lack of ICC may alter smooth muscle function because of loss of the hyperpolarizing influence of carbon monoxide production by ICC (22, 26, 44). Understanding of the precise genetic differences between mutant mice will eventually help to discriminate between these possibilities (31).

Mutant mice have also contributed to our knowledge about the intercellular mechanisms that trigger pacemaker ion channels that generate the ICC slow wave (Fig. 5). In inositol 1,4,5-trisphosphate (IP3) knockout mice, no slow-wave activity is recorded (30), providing further evidence for a role of IP3 in the activation of pacemaker currents (19, 42).


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Fig. 5.   Pacemaking in ICC may depend on inositol 1,4,5-trisphosphate (IP3)-stimulated calcium release. Spontaneous electrical activity recorded from circular smooth muscle of the gastric antrum of wild-type and IP3 knockout mice. Slow waves with action potentials are recorded from wild-type mice, but only bursts of action potentials are generated in the mutant mice, similar to results obtained in W/Wv mutant mice (20). Reproduced from Fig. 11 in Suzuki (29).


    THE ELECTROGASTROGRAM
TOP
ABSTRACT
INTRODUCTION
GENERAL INTRODUCTION TO ICC
ICC AND INNERVATION
ICC AND SLOW-WAVE GENERATION
THE ELECTROGASTROGRAM
ICC AND PACING
THE PYLORUS AND ICC...
REFERENCES

Does the electrogastrogram (EGG) reflect activity from ICC? Because the slow wave is initiated by ICC and because the EGG primarily records the slow-wave activity, ICC activity will be reflected in the EGG. However, the musculature and the enteric nervous system contribute to the overall appearance of the slow wave that is ultimately generated by the muscle cells and is picked up by EGG recording electrodes. Hence, abnormalities in the EGG do not necessarily reflect only abnormal ICC functioning. A change in slow-wave frequency that is established to occur after a meal (37) is likely a consequence of neural stimulation of the ICC and/or the musculature (see above). Apart from artifacts that can easily slip into the EGG analysis (38), the EGG as a measure of gastric motility is not well established. Although the slow wave itself (in addition to action potentials that are poorly, if at all, measured in the EGG) contributes to contractile activity, reliable correlations between such EGG changes and in vivo motor changes have not yet been established.


    ICC AND PACING
TOP
ABSTRACT
INTRODUCTION
GENERAL INTRODUCTION TO ICC
ICC AND INNERVATION
ICC AND SLOW-WAVE GENERATION
THE ELECTROGASTROGRAM
ICC AND PACING
THE PYLORUS AND ICC...
REFERENCES

Because ICC are pacemaker cells, can they be paced to alter motor activity? There is no question that electrical stimulation can increase slow-wave frequency or restore an abnormal slow-wave rhythm (Fig. 6). It has also been established that one can drive the frequency of the distal antral slow wave to frequencies higher then the intrinsic slow wave of the corpus and thereby force the direction of slow-wave propagation to occur from the pyloric region to the corpus. But can pacing restore delayed gastric emptying? Over the years, a lot of attention has been given to this possibility. First, it must be stated that in many cases in which pacing was considered for improvement of gastric emptying, no firm evidence existed that abnormal slow-wave activity was the primary cause of abnormal gastric emptying. Second, it was often assumed that an increase in slow-wave frequency would lead to an increase in rate of emptying. Two factors have to be in place for normal gastric emptying to occur with respect to the role of the distal stomach. A normal, aborally propagating slow wave has to occur, and this slow-wave activity has to be accompanied by pressure waves of sufficient amplitude (14). Thus the slow waves have to be bearing sufficient action potentials (spiking activity), which is usually provided by excitatory stimulation from the enteric nervous system. Hence, in cases in which abnormal emptying is primarily caused by insufficient excitation provided by the enteric nervous system, pacing the stomach to a higher frequency will not help. Even if the slow-wave rhythm is abnormal and the pacing would force it into a normal, aborally propagating rhythm, emptying might still not be improved because of a lack of forceful contractile activity (12). When an abnormal rhythm is the primary cause of delayed emptying, pacing may restore peristalsis (21). Electrical stimulation that causes pacing of slow waves may also initiate action potentials (Fig. 6), but this is not common. A beginning has been made with the electrical stimulation of excitatory nerves within the stomach to improve gastric emptying (23).


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Fig. 6.   Slow waves in the human stomach can be paced. Slow-wave activity was recorded with subcutaneously placed electrodes by Dr. W. Waterfall. The first recording is control slow-wave activity. The second recording is paced slow-wave activity at a higher frequency, with the pacing identified in the bottom trace. Note that the sharp peak preceding the paced slow waves is a stimulus artefact. Note that the electrical stimulation caused some slow waves to carry action potentials.


    THE PYLORUS AND ICC PATHOPHYSIOLOGY
TOP
ABSTRACT
INTRODUCTION
GENERAL INTRODUCTION TO ICC
ICC AND INNERVATION
ICC AND SLOW-WAVE GENERATION
THE ELECTROGASTROGRAM
ICC AND PACING
THE PYLORUS AND ICC...
REFERENCES

The normal pylorus has a rich complement of ICC-IM. These cells are markedly altered and reduced in number in pyloric stenosis as assessed by both immunohistochemistry and electron microscopy (18, 35). Abnormal motility in pyloric stenosis is likely a combination of lack of nitrergic nerves and ICC-IM, and both may develop to normal proportions over time. ICC-AP may serve as pacemaker cells orchestrating the isolated pyloric contractions but are unlikely to assist in coordination between gastric and duodenal activity. In a recent elegant study by Lammers and co-workers (17), the pylorus was shown as an area where slow-wave propagation is completely blocked (Fig. 4). This serves to allow the stomach and small intestine to generate independent propagating motor patterns involving ICC. Pyloric stenosis might be a case of delayed ICC development (36). Lack of ICC is also implicated in a study of idiopathic gastric perforation, in which three of seven neonates have complete absence of ICC and ICC were reduced in the other four (25). ICC-AP are probably functionally mature well before birth since strong slow wave rhythms were observed at 29 wk of gestation (3). Structurally, however, ICC may not be fully developed (4), which is a reminder that functional and structural maturity may not be perfectly correlated. Immunohistochemistry can show Kit-positive cells (excluding mast cells) that are likely to be ICC in the human gut musculature. The advantage of this technique is that it can give an overview of ICC distribution in sections using conventional immunohistochemistry and in whole mounts using confocal microscopy. Some quantification can also be attempted. Electron microscopy can identify ICC positively and can also provide details of structural abnormalities that may be undetected with immunohistochemistry (39). With the use of these techniques, the possible implication of ICC in the pathophysiology of gastric motor disorders will be elucidated in the coming years.


    ACKNOWLEDGEMENTS

The support of the Canadian Institutes of Health Research is gratefully acknowledged.


    FOOTNOTES

Abbreviations used for the various subtypes of ICC are those established by Thuneberg (9, 16, 33).

Address for reprint requests and other correspondence: J. Huizinga, McMaster Univ., 1200 Main St. West, Health Sciences Bldg. Rm 3N5C, Hamilton 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
TOP
ABSTRACT
INTRODUCTION
GENERAL INTRODUCTION TO ICC
ICC AND INNERVATION
ICC AND SLOW-WAVE GENERATION
THE ELECTROGASTROGRAM
ICC AND PACING
THE PYLORUS AND ICC...
REFERENCES

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Am J Physiol Gastrointest Liver Physiol 281(5):G1129-G1134
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