THEME
Physiology and Pathophysiology of the Interstitial Cell of Cajal: From Bench to Bedside
III. Interaction of interstitial cells of Cajal with neuromediators: an interim assessment

E. E. Daniel

Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
NEUROTRANSMITTER ACTIONS ON ICC...
NEUROTRANSMITTER ACTIONS ON ICC...
CLINICAL RELEVANCE
CONCLUSION
REFERENCES

Interstitial cells of Cajal (ICC) control gastrointestinal motility; some pace slow waves and others act in enteric neurotransmission. This review asks the question, does either class of ICC receive and respond to messages carried by neuromediators from these nerves? Relevant evidence includes the presence of receptors or responses to exogenous neuromediators and responses to endogenous neuromediators. Some pacemaking ICC networks have receptors for or respond to some exogenous neuromediators. None is known to respond to endogenous neuromediators. Intramuscular ICC have receptors for and respond to some neuromediators and are required in mice for responses to the exogenous and endogenous neuromediators nitric oxide and acetylcholine. The mechanisms underlying this requirement remain unclear. ICC pathologies exist, but their origins are unknown.

intestinal pacemaking; intestinal neurotransmission; nitric oxide; acetylcholine; substance P


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
NEUROTRANSMITTER ACTIONS ON ICC...
NEUROTRANSMITTER ACTIONS ON ICC...
CLINICAL RELEVANCE
CONCLUSION
REFERENCES

INTERSTITIAL CELLS OF CAJAL (ICC) provide myogenic control of gastrointestinal motility by initiating slow waves in distal stomach, intestine, and colon (3, 14, 25), and they are required for neurotransmission (23-26). ICC are in the myenteric plexus in distal stomach, intestine, and colon and the deep muscular plexus of intestine and submuscular plexus of colon as well as within the circular muscle layer (see, e.g., Refs. 14, 18). All are close to enteric nerves (see, e.g., Ref. 26). Major mediators controlling gastrointestinal motility are acetylcholine and substance P for excitation and nitric oxide (NO; possibly with CO) and vasoactive intestinal peptide (VIP; Refs. 14, 27) for inhibition. The relevant receptors are muscarinic for acetylcholine, neurokinin (especially NK1 receptor) for substance P, VIP1 and VIP2 for VIP, and cytosolic guanylate cyclase and other intracellular response systems for NO and CO (27). Here we ask the question, do ICC respond to chemical messages from enteric nerves?

Relevant evidence is that 1) ICC in various locations have receptors or response systems for neuromediators; 2) ICC respond to exogenous neuromediators; and 3) ICC respond under physiological conditions of neuromediator release. Evidence regarding responses of networks of pacemaking and intramuscular ICC to neuromediators is considered separately because these cells differ in developmental origin, structure, biochemical markers, and function (1, 5, 14, 18, 25, 26).


    NEUROTRANSMITTER ACTIONS ON ICC PACEMAKING NETWORKS
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ABSTRACT
INTRODUCTION
NEUROTRANSMITTER ACTIONS ON ICC...
NEUROTRANSMITTER ACTIONS ON ICC...
CLINICAL RELEVANCE
CONCLUSION
REFERENCES

In the canine stomach, slow waves initiated at a site along the greater curvature of the corpus spread circumferentially and distally to the pylorus. In the intestine and colon, slow waves spread circumferentially around the organ, but no individual slow waves travel over the length of the organ and the frequency of slow waves varies with their location. Other species studied appear similar. When uncoupled from more proximal ICC networks, distal ICC networks initiate slow wave activity but at lower frequencies temporally unrelated to proximal slow wave activities. Thus local effects on slow wave frequencies or coupling would have consequences for distal transit. Do extrinsic or intrinsic enteric nerves affect ICC and slow waves?

Vagotomy and sympathectomy do not abolish coordinated gastric slow waves and contractions. Sympathetic innervation acts indirectly in the gut by suppressing cholinergic activity. Adrenergic receptors are therefore likely to be on enteric nerves, not on ICC. Vagal stimulation has greater motor effects in the proximal gastrointestinal tract compared with the distal tract. Vagal activity slows slightly, whereas atropine or sympathetic activity increase, the frequency of gastric slow waves, and close intra-arterial injections of boluses of acetylcholine induce premature gastric slow waves. Thus vagal extrinsic nerves might modulate pacemaking of ICC in the gastric myenteric plexus.

In a recent study in guinea pig antrum, it was possible to penetrate ICC cells in situ with microelectrodes in addition to cells of the longitudinal and circular muscle layers (4). Vagal stimulation inhibited slow wave amplitude and contractile activity in muscle cells, and this was mediated by NO and another inhibitory mediator, possibly ATP. It did not affect electrical activity of pacemaking ("driving" cells or ICC) except for very small hyperpolarizations during the plateau of driving potentials. There was no effect on pacemaking frequency. The most likely explanations are that muscarinic receptors do not exist on these cells or that their activation does not affect pacemaking.

In mice, ICC are reported to have mRNA for receptors to muscarinic receptor types M2 and M3, neurokinin receptors NK1 and NK3, and inhibitory receptor VIP-1 when freshly isolated from the murine intestine myenteric plexus (5). The presence of mRNA for receptor proteins does not guarantee that the proteins are expressed, and evidence of expression of such receptors on myenteric plexus ICC is lacking. VIP nerves are very close to ICC in the submuscular plexus in canine colon, and these ICC may respond to VIP.

Neurotransmitters from enteric nerves might interact with pacemaking ICC networks during peristaltic reflexes, inhibiting slow wave amplitude distal to and enhancing it proximal to the site activation of the reflex. This would enhance proximally and decrease distally the probability of actions potentials on slow waves and contractions, promoting peristalsis. So far, no reports that this occurs are available.

Ca2+ waves were recorded in the longitudinal muscle of the guinea pig colon in vitro (17). Their properties were like those of slow waves generated by ICC as to frequency and anisotropic propagation (10 times faster axially compared with circumferentially). Also, multiple sites of initiation were found, and, as with slow waves, waves from one site propagated until extinguished by collision with waves from another pacemaker site or by reaching a region of refractoriness. Peristaltic activity induced a much higher density of pacemaker sites at proximal sites and obliterated them at distal sites. It is unclear whether this behavior at proximal sites reflects effects on slow waves or on action potentials during peristalsis. The behavior at distal sites might reflect abolition of action potentials rather than slow waves. Similar studies with simultaneous recording of electrical and Ca2+ waves would tell us whether and how pacemakers are modulated by enteric nerves.

Both neural and exogenous NO may affect pacemaking ICC. Slow wave frequency in the canine colon myenteric plexus region, isolated from the submuscular plexus, was decreased and configuration was changed by NO, acting on response systems involving cGMP (but not necessarily protein kinase G) (Ref. 9; K. M. Sanders, personal communication). ICC isolated from murine intestine respond to cyclic nucleotides (10), as do those of guinea pig intestine.

NO donors did not affect the frequency of spontaneous Ca2+ release in ICC isolated from the submuscular plexus of canine colon but increased their Ca2+ levels (13). Increased Ca2+ levels caused these ICC to produce a diffusible substance, apparently NO, which lowered Ca2+ levels in nearby smooth muscle. In slabs of canine intestine, neither exogenous NO, which markedly hyperpolarized muscle, nor VIP, which had less effect on membrane potential, affected slow wave frequency driven by ICC of the myenteric plexus. Obtaining evidence about neural modulation of slow wave amplitude or frequency from intracellular recordings in ICC during peristalsis would be difficult. Changes in slow wave amplitude during peristalsis would be easier to record from smooth muscle but might reflect actions on smooth muscle rather than on ICC.

Slow waves in the intestine appear to function to control excitability and to provide spatial and temporal coordination distally as well as locally in regions to which enteric nerves mediating the peristaltic reflex project. In mutant mice in which the myenteric ICC network and slow waves were missing, the peristaltic reflexes, proximal excitation, and distal inhibition to distension remained functional. However, aborad transit of intestinal content was severely impaired (3, 8). Thus slow waves and ICC pacemaker network collaboration was necessary for intestinal transit, but there was no evidence that they modulated one another.


    NEUROTRANSMITTER ACTIONS ON ICC OF DEEP MUSCULAR PLEXUS AND WITHIN CIRCULAR MUSCLE
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ABSTRACT
INTRODUCTION
NEUROTRANSMITTER ACTIONS ON ICC...
NEUROTRANSMITTER ACTIONS ON ICC...
CLINICAL RELEVANCE
CONCLUSION
REFERENCES

Nitric oxide synthase (NOS)- and VIP immunoreactivity (IR) are often colocalized in inhibitory nerves and are close to intramuscular ICC (14, 19, 24-26), as are cholinergic-IR (22, 25) and substance P-IR (6, 12, 15, 20-22) nerves. These ICC appear to be intercalated between nerve endings and smooth muscle on morphological grounds (26). Intramuscular ICC and those at the submuscular plexus of several species show clear evidence that they contain NK1 and NK3 or NK2 receptors on the basis of immunohistochemistry (6, 12, 15, 20-22). In guinea pig intestine, receptor occupation by substance P leads to internalization of these NK1 receptors, suggesting that they are capable of binding substance P and initiating response to it (11). No other evidence exists that these receptors function. Somatostatin receptors are also reported on these ICC (16).

Clear evidence exists that neuromediators from cholinergic and nitrergic nerves must interact with intramuscular ICC to initiate excitation or inhibition. The gastric fundus lacks an ICC network in the myenteric plexus, and slow waves are absent. Mice with a mutation in the gene c-kit lack ICC within circular muscle in the gastric fundus. However, both inhibitory nerve networks containing NOS and excitatory nerve networks containing acetylcholine are present in normal arrays. In normal mice, nerve stimulation can initiate inhibitory junction potentials and relaxation from NO release or excitatory junction potentials and contraction from acetylcholine release. Both responses are severely impaired in tissues from mutant mice. Moreover, exogenous NO, but not acetylcholine, had much-diminished effects on membrane potentials and contractile activity compared with tissue from wild-type mice (23, 26). Relaxation and hyperpolarization to stimulation of inhibitory nerves or release of NO were similarly impaired in the lower esophageal sphincter and pylorus of mutant mice lacking intramuscular ICC (24).

A simple explanation is that intramuscular ICC contain muscarinic receptors for acetylcholine as well as NO response systems. Evidence for the presence of muscarinic receptor mRNA in these ICC exists (5). Intramuscular ICC of several species have been shown to contain NOS-IR and may produce NO (2, 13). Some of them respond to NO donors by producing cGMP (13). However, smooth muscle cells also contain muscarinic receptors and response systems to NO. Therefore, the mediators should diffuse from nerves to the smooth muscle cells whether ICC are present or not. Instead, prolonged stimulation of enteric nerves in the presence of an anticholinesterase brought about only a small, slow excitatory response of the fundus in mutant mice lacking intramuscular ICC (23), but acetylcholine was able to depolarize or contract at least as well in mutant mice lacking ICC as in wild-type mice.

Morphological examination of the relations between nerve endings, ICC, and smooth muscle suggests that nerve endings are more frequently very close to intramuscular ICC and less frequently close to smooth muscle (26). Some intramuscular ICC have synapselike contacts with enteric nerves (26). Thus intramuscular ICC might be the preferred target for acetylcholine and NO because of their proximity. However, an explanation that the neurotransmitter does not usually reach the smooth muscle when released during normal activity of nerves cannot explain why an exogenous neurotransmitter, like an NO donor, has severely diminished effects in the absence of intramuscular ICC. Thus, although we have good evidence that intramuscular ICC are needed for neural signaling by the major neurotransmitters, the mechanisms underlying this need remain a mystery. For acetylcholine, only ICC may be easily accessible to nerve-released mediator. For NO, one possibility is that NO acts differently when released inside the cell after transformation, as is the case with sodium nitroprusside, compared with release outside the cell, as with nerve stimulation.


    CLINICAL RELEVANCE
TOP
ABSTRACT
INTRODUCTION
NEUROTRANSMITTER ACTIONS ON ICC...
NEUROTRANSMITTER ACTIONS ON ICC...
CLINICAL RELEVANCE
CONCLUSION
REFERENCES

There are no gastrointestinal diseases in which the absence or malfunction of pacemaking ICC networks has been shown to explain the pathophysiology fully. As recently reviewed in another themes article (7), there are number of diseases in which structural alterations in pacemaking ICC are found by some investigators. These include colon aganglionosis and chronic idiopathic intestinal pseudoobstruction, in which a reduction of pacemaking ICC may be found. However, so far there is no basis for deciding whether the changes in ICC networks are causes or consequences of the disease and no understanding of the mechanisms underlying changes in ICC structures. There are also several diseases with which absence or loss of intramuscular ICC and associated nerves is associated. These include achalasia, hypertrophic pyloric stenosis, and Hirshsprung disease. If human gastrointestinal ICC function as ICC do in mice, an absence of ICC would be sufficient to explain disease symptoms of loss of inhibitory nerve function. Frequently, both nerves and ICC are absent, and it seems likely that their development is intertwined. In developing canine intestinal tissues, the presence of nerve axons preceded the differentiation of intramuscular and deep muscular plexus ICC (1), suggesting that nerves come first. However, a temporal precedence does not establish a causal one.


    CONCLUSION
TOP
ABSTRACT
INTRODUCTION
NEUROTRANSMITTER ACTIONS ON ICC...
NEUROTRANSMITTER ACTIONS ON ICC...
CLINICAL RELEVANCE
CONCLUSION
REFERENCES

In contrast to ICC networks involved in pacing slow waves, there is compelling evidence that intramuscular ICC play crucial roles in neurotransmission of cholinergic excitation and nitrergic inhibition, at least in some tissues. The mechanisms whereby ICC fulfill these roles remain unclear. Studies in which recordings are made from ICC in situ such as those carried out in guinea pig stomach (4) are needed. Technical advances will probably be required to allow intracellular recording in situ from intramuscular ICC, such as ICC of the deep muscular plexus of intestine, while applying nerve stimulation. Our concepts of how gastrointestinal motor functions are controlled have changed dramatically since the discoveries of the roles of ICCs, but more change is likely in the future. The application of these concepts to the pathophysiology and therapy of gastrointestinal motor function has not yet occurred.


    ACKNOWLEDGEMENTS

I thank Dr. K. M. Sanders for advice.


    FOOTNOTES

Work from my laboratory was supported by the Medical Research Council of Canada.

Owing to space limitations, nearly all references to publications before 1998 have been eliminated. Readers who wish to see these references may obtain them in pdf or wpd format from the author by E-mail (edaniel{at}ualberta.ca).

Address for reprint requests and other correspondence: E. E. Daniel, Dept. of Pharmacology, Univ. of Alberta, 9-70 Medical Sciences Bldg., Edmonton, AB, Canada T6G 2H7 (E-mail: edaniel{at}ualberta.ca).


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
NEUROTRANSMITTER ACTIONS ON ICC...
NEUROTRANSMITTER ACTIONS ON ICC...
CLINICAL RELEVANCE
CONCLUSION
REFERENCES

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3.   Der-Silaphet Malysz, TJ, Hagel S, Arsenault LA, and Huizinga JD. Interstitial cells of Cajal direct normal propulsive contractile activity in the mouse small intestine. Gastroenterology 114: 724-736, 1998[ISI][Medline].

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5.   Epperson, A, Hatton WJ, Callaghan B, Doherty P, Walker RL, Sanders KM, Ward SM, and Horowitz B. Molecular markers expressed in cultured and freshly isolated interstitial cells of Cajal. Am J Physiol Cell Physiol 279: C529-C539, 2000[Abstract/Free Full Text].

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17.   Stevens, RJ, Publicover NG, and Smith TK. Induction and organization of Ca2+ waves by enteric neural reflexes. Nature 399: 62-66, 1999[ISI][Medline].

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20.   Vannucchi, MG, Corsani L, and Faussone-Pellegrini MS. Substance P immunoreactive nerves and interstitial cells of Cajal in the rat and guinea-pig ileum. A histochemical and quantitative study. Neurosci Lett 268: 49-52, 1999[ISI][Medline]. [Corrigenda. Neurosci Lett 272: Sept. 3, 1999; p. 72-73.]

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Am J Physiol Gastrointest Liver Physiol 281(6):G1329-G1332
0193-1857/01 $5.00 Copyright © 2001 the American Physiological Society




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