Kenton M. Sanders

Recent studies have demonstrated that intramuscular interstitial cells of Cajal (ICC) are preferential targets for neurotransmission in the stomach. Terminals of enteric motor neurones also form tight, synaptic‐like contacts with ICC in the small intestine and colon, but little is known about the role of these cells in neurotransmission. ICC at the deep muscular plexus (ICC‐DMP) of the small intestine express neurokinin 1 receptors (NK1R) and internalize these receptors in response to exogenous substance P. We used NK1R internalization as an assay of functional innervation of ICC‐DMP in the murine small intestine. Under basal conditions NK1R‐like immunoreactivity (NK1R‐LI) was mainly observed in ICC‐DMP (519 cells counted, 100% were positive) and myenteric neurones. ICC‐DMP were closely apposed to substance P‐containing nerve fibres. Of 338 ICC‐DMP examined, 65% were closely associated with at least one substance P‐positive nerve fibre, 32% were associated with at least two, 2% were associated with more than two nerve fibres and 1% with none. After electrical field stimulation (EFS, 10 Hz; 1 min) NK1R‐LI was internalized in more than 80% of ICC‐DMP, as compared to 10% of cells before EFS. Internalization of NK1R was not observed in myenteric ICC or smooth muscle cells in response to nerve stimulation. Internalization of NK1R‐LI was blocked by the specific NK1 receptor antagonist WIN 62577 (1 μm) and by tetrodotoxin (0.3 μm), suggesting that internalization resulted from stimulation of receptors with neurally released neurokinins. These data suggest that ICC‐DMP are primary targets for neurokinins released from enteric motor neurones in the intestine.

Considerable work has led many to conclude that interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. These cells form electrically coupled networks within the pacemaker regions of the GI tract, and ICC are electrically coupled to smooth muscle cells. ICC express unique ion channels that periodically produce inward (pacemaker) currents. Recent work has suggested that the inward current is produced by a calcium (Ca²⁺)‐regulated, nonselective cation conductance. Channels responsible for this conductance oscillate in open probability in response to the periodic drop in intracellular Ca²⁺ concentration during the slow wave cycle. Pacemaker activity generates slow waves that are propagated actively through ICC networks. Depolarization coordinates the pacemaker activity through the ICC network by activating a dihydropyridine‐resistant Ca²⁺ conductance. Entry of small amounts of Ca²⁺ into ICC entrains spontaneous pacemaker activity and produces cell‐to‐cell propagation of slow waves. This review discusses the mechanisms and conductances involved in generation and propagation of electrical slow waves in ICC.

We previously identified eight known and novel genes differentially expressed in the small intestines of wild type and W/W ^V mice, which have greatly reduced populations of the interstitial cells of Cajal, that are responsible for the generation of electrical slow waves, by using a differential gene display method. By using the same method we isolated additional candidate genes that were specifically down- or up-regulated in W/W ^V mice. Novel transcripts were designated as DDWMEST. We isolated seven candidates that were specifically down- or up-regulated in W/W ^V mice. Two novel transcripts, DDWMEST 1 and -91 were increased in both fed and fasted W/W ^V mice. Expression of another five genes was suppressed in W/W ^V mice: ARG2 (Arginase II), ONZIN (encoding leukemia inhibitory factor regulated protein), and three novel transcripts: DDWMEST62, -84, and -100. Together with the previous report, we identified fifteen differentially expressed genes in total in the small intestines of W/W ^V mice. Eight of these genes were reduced in the jejunums of W/W ^V mice compared to age matched wild type mice, whereas the other seven genes showed an increase in expression. Differential expression was the same in fasted and fed animals, suggesting that the differences were independent of the dietetic state of the animal. Several known and novel genes are differentially expressed in the small intestines of W/W ^V mice. Differential gene comparison might contribute to our understanding of motility disorders associated with the loss of the interstitial cells of Cajal.

The muscle layers of murine gastric fundus have no interstitial cells of Cajal at the level of the myenteric plexus and only possess intramuscular interstitial cells and this tissue does not generate electric slow waves. The absence of intramuscular interstitial cells in W/WV mutants provides a unique opportunity to study the molecular changes that are associated with the loss of these intercalating cells. The gene expression profile of the gastric fundus of wild type and W/WV mice was assayed by murine microarray analysis displaying a total of 8734 elements. Queried genes from the microarray analysis were confirmed by semi-quantitative reverse transcription-polymerase chain reaction. Twenty-one genes were differentially expressed in wild type and W/WV mice. Eleven transcripts had 2.0-2.5 fold higher mRNA expression in W/WV gastric fundus when compared to wild type tissues. Ten transcripts had 2.1-3.9 fold lower expression in W/WV mutants in comparison with wild type animals. None of these genes have ever been implicated in any bowel motility function. These data provides evidence that several important genes have significantly changed in the murine fundus of W/WV mutants that lack intramuscular interstitial cells of Cajal and have reduced enteric motor neurotransmission.

Neurokinins contribute to the neural regulation of gastrointestinal (GI) smooth muscles. We studied responses of murine colonic smooth muscle cells to substance P (SP) and NK₁ and NK₂ agonists using confocal microscopy and the patch clamp technique. Colonic myocytes generated localized Ca²⁺ transients that were coupled to spontaneous transient outward currents (STOCs). SP (10⁻¹⁰ M) increased Ca²⁺ transients and STOCs. Higher concentrations of SP (10⁻⁶ M) increased basal Ca²⁺ and inhibited Ca²⁺ transients and STOCs. Effects of SP were due to increased Ca²⁺ entry via L‐type Ca²⁺ channels, and were mediated by protein kinase C (PKC). Nifedipine (10⁻⁶ M) and the PKC inhibitor, GF 109203X (10⁻⁶ M) reduced L‐type Ca²⁺ current and blocked the effects of SP. SP responses depended upon parallel stimulation of NK₁ and NK₂ receptors. NK₁ agonist ([Sar⁹,Met(O₂)¹¹]‐substance P; SSP) and NK₂ agonists (neurokinin A (NKA) or GR‐64349) did not mimic the effects of SP alone, but NK₁ and NK₂ agonists were effective when added in combination (10⁻¹⁰–10⁻⁶ M). Consistent with this, either an NK₁‐specific antagonist (GR‐82334; 10⁻⁷ M) or an NK₂‐specific antagonist (MEN 10,627; 10⁻⁷ M) blocked responses to SP (10⁻⁶ M). Ryanodine (10⁻⁵ M) blocked the increase in Ca²⁺ transients and STOCs in response to SP (10⁻¹⁰ M). Our findings show that low concentrations of SP, via PKC‐dependent enhancement of L‐type Ca²⁺ current and recruitment of ryanodine receptors, stimulate Ca²⁺ transients. At higher concentrations of SP (10⁻⁶ M), basal Ca²⁺ increases and spontaneous Ca²⁺ transients and STOCs are inhibited. British Journal of Pharmacology (2003) 138, 1233–1243. doi:10.1038/sj.bjp.0705139

Peristaltic contractions in the stomach are regulated by the spread of electrical slow waves from the corpus to the pylorus. Gastric slow waves are generated and propagated by the interstitial cells of Cajal (ICC). All regions distal to the dominant pacemaker area in the corpus are capable of generating slow waves, but orderly gastric peristalsis depends upon a frequency gradient in which the corpus pacemaker frequency exceeds the antral frequency. Cholinergic, muscarinic stimulation enhances pacemaker frequency. We investigated this phenomenon using intact murine gastric muscles and cultured ICC. Acetylcholine (ACh) increased the frequency of slow waves in antrum and corpus muscles. The increase was significantly greater in the antrum. ACh and carbachol (CCh) increased the pacemaker currents in cultured ICC. At high doses of CCh, transient pacemaker currents fused into sustained inward currents that persisted for the duration of stimulation. The effects of CCh were blocked by low doses of the M₃ receptor antagonist 1‐dimethyl‐4‐diphenylacetoxypiperidinium. Frequency enhancement by CCh was not affected by forskolin, but the phospholipase C inhibitor U‐73122 inhibited both the increase in frequency and the development of tonic inward currents. 2‐Aminoethyldiphenyl borate also blocked the chronotropic responses to CCh. Inhibitors of protein kinase C did not block responses to CCh. These studies show that mice are an excellent model for studying mechanisms that regulate gastric slow‐wave frequency. CCh, apparently via production of inositol 1,4,5‐trisphosphate, accelerates the frequency of pacemaker activity. High concentrations of CCh may block the entrainment of pacemaker currents, resulting in a tonic inward current.

Electrical slow waves in gastrointestinal (GI) muscles are generated by pacemaker cells, known as interstitial cells of Cajal (ICC). The pacemaker conductance is regulated by periodic release of Ca²⁺ from inositol 1,4,5‐trisphosphate (IP₃) receptor‐operated stores, but little is known about how slow waves are actively propagated. We investigated voltage‐dependent Ca²⁺ currents in cultured ICC from the murine colon and small intestine. ICC, identified by kit immunohistochemistry, were spontaneously active under current clamp and generated transient inward (pacemaker) currents under voltage clamp. Depolarization activated inward currents due to entry of Ca²⁺. Nicardipine (1 μM) blocked only half of the voltage‐dependent inward current. After nicardipine, there was a shift in the potential at which peak current was obtained (‐15 mV), and negative shifts in the voltage dependence of activation and inactivation of the remaining voltage‐dependent inward current. The current that was resistant to dihydropyridine (I_VDDR) displayed kinetics, ion selectivity and pharmacology that differed from dihydropyridine‐sensitive Ca²⁺ currents. I_VDDR was increased by elevating extracellular Ca²⁺ from 2 to 10 mm, and this caused a +30 mV shift in reversal potential. I_VDDR was blocked by Ni²⁺ (100 μM) or mebefradil (1 μM) but was not affected by blockers of N‐, P‐ or Q‐type Ca²⁺ channels. Equimolar replacement of Ca²⁺ with Ba²⁺ reduced I_VDDR without effects on inactivation kinetics. BayK8644 had significantly less effect on I_VDDR than on I_VDIC. In summary, two components of inward Ca²⁺ current were resolved in ICC of murine small intestine and colon. Since slow waves persist in the presence of dihydropyridines, the dyhydropyridine‐resistant component of inward current may contribute to slow wave propagation.

Several human motility disorders have been shown to be associated with loss or defects in interstitial cells of Cajal (ICC) networks. Because tissue samples for these studies were taken from patients with well-advanced motility problems, it is difficult to determine whether the loss of ICC is a cause or a consequence of the disease process. To establish the cause-and-effect relationship of ICC loss in motility disorders, it may be feasible to use animal models in which ICC are lost as motility dysfunction develops. Several models with defects in ICC networks have been developed, and these include animals with defects in the Kit signaling pathway (e.g., white-spotting mutants that have defects in Kit receptors; steel mutants that have mutations in stem cell factor, the ligand for Kit; and animals that are chronically treated with reagents that block Kit or downstream signaling proteins). ICC do not die when Kit signaling is blocked, rather, they redifferentiate into a smooth musclelike phenotype. Diabetic animals (NOD/LtJ mice), animals with chronic bowel obstruction, and inflammatory bowel models also have defects in ICC networks that have been associated with motility disorders. By studying these models with molecular and genomic techniques it may be possible to determine the signals that cause loss of ICC and find ways of restoring ICC to dysfunctional tissues. This article discusses recent progress in the utilization of animal models to study the consequences of losing ICC on the development of motility disorders.

PGE₂ has been linked to the production of gastric arrhythmias such as tachygastria. The interstitial cells of Cajal (ICC) generate electrical rhythmicity in gastrointestinal muscles, and may therefore be a target for PGE₂ in gastric muscles. We cultured ICC from the murine gastric antrum, verified that cells were Kit immunoreactive, and measured spontaneous slow waves. These events were caused by spontaneous inward (pacemaker) currents that were not blocked by nifedipine. Forskolin and 8-bromoadenosine 3′:5′-cyclic monophosphate (8-Br-cAMP) reduced the frequency of pacemaker currents in ICC and of slow waves in intact antral muscles. The effects of forskolin and 8-Br-cAMP were not blocked by inhibitors of protein kinase A, suggesting that cAMP has direct effects on pacemaker activity. PGE₂ mimicked the effects of forskolin and 8-Br-cAMP on ICC, but increased slow-wave frequency in intact muscles. Therefore, the chronotropic effects of specific prostaglandin EP receptor agonists were examined. Butaprost and ONO-AE1-329, EP₂ and EP₄ receptor agonists, mimicked the effects of forskolin and 8-Br-cAMP on ICC and intact muscles. Sulprostone (EP₃>EP₁ agonist), GR63799, and ONO-AE-248 (EP₃ agonists) enhanced the frequencies of pacemaker currents in ICC and slow waves in intact muscles. The effects of sulprostone were not blocked by SC-19220, an EP₁ receptor antagonist. These observations suggest that the positive chronotropic effects of PGE₂ in intact muscles are mediated by EP₃ receptor stimulation. The effects of PGE₂ in intact muscles may be dependent upon the relative expression of EP receptors and/or proximity of receptors to sources of PGE₂.