Sean M. Ward

Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine.

1. Interstitial cells of Cajal (ICs) have been proposed as pacemakers in the gastrointestinal tract. We studied the characteristics and distribution of ICs and electrical activity of small intestinal muscles from mice with mutations at the dominant‐white spotting/c‐kit (W) locus because the tyrosine kinase function of c‐kit may be important in the development of the IC network. 2. W/WV mutants (days 3‐30 postpartum) had few ICs in the myenteric plexus region compared with wild type (+/+) siblings. The few ICs present were associated with neural elements and lay between myenteric ganglia and the longitudinal muscle layer. 3. Electrical recordings from intestinal muscle strips showed that electrical slow waves were always present in muscles of +/+ siblings, but were absent in W/WV mice. 4. Muscles from W/WV mice responded to stimulation of intrinsic nerves. Neural responses, attributed to the release of acetylcholine, nitric oxide and other unidentified transmitters, were recorded. 5. These findings are consistent with the hypothesis that ICs are a critical element in the generation of electrical rhythmicity in intestinal muscles. The data also show that neural regulation of gastrointestinal muscles can develop independently of the IC network. 6. W locus mutants provide a powerful new model for studies of the physiological role of ICs and the significance of electrical rhythmicity to normal gastrointestinal motility.

C-kit-dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract

In vivo injection of a neutralizing, monoclonal antibody (ACK2) to the receptor tyrosine kinase (c-kit) disrupts the normal motility patterns of the mouse small intestine. Immunohistochemical studies showed that cells expressing c-kit-like immunoreactivity (c-kit-LI) decreased in numbers in response to ACK2, but the identity of these cells is unknown. We investigated the identity and development of the cells that express c-kit-LI in the mouse small intestine and colon. Cells in the region of the myenteric plexus and deep muscular plexus of the small intestine and in the subserosa, in the myenteric plexus region, within the circular and longitudinal muscle layers, and along the submucosal surface of the circular muscle in the colon were labeled with ACK2. The distribution of cells that express c-kit-LI was the same as that of interstitial cells (ICs). In whole-mount preparations cells with c-kit-LI were interconnected, forming a netword similar to the network formed by cells that stained with methylene blue, which has been used as a marker for ICs in the mouse gastrointestinal tract. Immunocytochemistry verified that ICs were labeled with ACK2. Multiple injections of animals with ACK2 between days 0 and 8 post partum (pp) caused a dramatic reduction in the number of ICs compared to control animals. From an ultrastructural point of view, the proliferation and development appeared to be suppressed in some classes of ICs, while others displayed an altered course of development. Functional studies showed that the decrease in ICs was accompanied by a loss of electrical rhythmicity in the small intestine and reduced neural responses in the small bowel and colon. Morphological experiments showed that c-kit-positive cells are ICs, and physiological evidence reinforced the concept that ICs are involved in generation of rhythmicity and translation of neural inputs in gastrointestinal smooth muscles. Controlling the development of ICs provides a powerful new tool for the investigation of the physiological role of these cells.

Pacemaking in interstitial cells of Cajal depends upon calcium handling by endoplasmic reticulum and mitochondria

1 Pacemaker cells, known as interstitial cells of Cajal (ICC), generate electrical rhythmicity in the gastrointestinal tract. Pacemaker currents in ICC result from the activation of a voltage‐independent, non‐selective cation conductance, but the timing mechanism responsible for periodic activation of the pacemaker current is unknown. 2 Previous studies suggest that pacemaking in ICC is dependent upon metabolic activity 1y1yand1 Ca2+ release from intracellular stores. We tested the hypothesis that mitochondrial Ca2+ handling may underlie the dependence of gastrointestinal pacemaking on oxidative metabolism. 3 Pacemaker currents occurred spontaneously in cultured ICC and were associated with mitochondrial Ca2+ transients. 4 Inhibition of the electrochemical gradient across the inner mitochondrial membrane blocked Ca2+ uptake and pacemaker currents in cultured ICC and blocked slow wave activity in intact gastrointestinal muscles from mouse, dog and guinea‐pig. 5 Pacemaker currents and rhythmic mitochondrial Ca2+ uptake in ICC were also blocked by inhibitors of IP3‐dependent release of Ca2+ from the endoplasmic reticulum and by inhibitors of endoplasmic reticulum Ca2+ reuptake. 6 Our data suggest that integrated Ca2+ handling by endoplasmic reticulum and mitochondria is a prerequisite of electrical pacemaking in the gastrointestinal tract.

Interstitial Cells of Cajal Mediate Enteric Inhibitory Neurotransmission in the Lower Esophageal and Pyloric Sphincters

BACKGROUND & AIMS Previous studies have suggested that a specific class of interstitial cells of Cajal (ICC) act as mediators in nitrergic inhibitory neurotransmission. The aim of this investigation was to examine the role of intramuscular ICC (IC-IM) in neurotransmission in the murine lower esophageal (LES) and pyloric sphincters (PS). METHODS Immunohistochemistry and electrophysiology were used to study the distribution and role of IC-IM. RESULTS The LES and PS contain spindle-shaped IC-IM, which form close relationships with nitric oxide synthase-containing nerve fibers. The PS contains ICC within the myenteric plexus and c-Kit immunopositive cells along the submucosal surface of the circular muscle. IC-IM were absent in the LES and PS of c-kit (W/Wv) mutant mice. Using these mutants, we tested whether IC-IM mediate neural inputs in the LES and PS. Although the distribution of inhibitory nerves was normal in W/Wv animals, NO-dependent inhibitory neurotransmission was reduced. Hyperpolarizations to sodium nitroprusside were also attenuated in W/Wv animals. CONCLUSIONS The data suggest that IC-IM play an important role in NO-dependent neurotransmission in the LES and PS. IC-IM may be the effectors that transduce NO signals into hyperpolarizing responses. Loss of IC-IM may interfere with relaxations and normal motility in these sphincters.

Spontaneous electrical rhythmicity in cultured interstitial cells of Cajal from the murine small intestine

1 Interstitial cells of Cajal (ICC) are pacemaker cells in the small bowel, and therefore this cell type must express the mechanism responsible for slow wave activity. Isolated ICC were cultured for 1–3 days from the murine small intestine and identified with c‐Kit‐like immunoreactivity (c‐Kit‐LI). 2 Electrical recordings were obtained from cultured ICC with the whole‐cell patch clamp technique. ICC were rhythmically active, producing regular slow wave depolarizations with waveforms and properties similar to slow waves in intact tissues. 3 Spontaneous activity of c‐Kit‐LI cells was inhibited by reduced extracellular Na+, gadolinium, and reduced extracellular Ca2+. The activity was not affected by nisoldipine. Voltage clamp studies showed rhythmic inward currents that were probably responsible for the slow wave activity. The current‐voltage relationship showed that the spontaneous currents reversed at about +17 mV. These observations are consistent with the involvement of a non‐selective cation current in the generation of slow waves, but do not rule out contributions from other conductances or transporters. 4 A Ba2+‐sensitive inwardly rectifying K+ current in c‐Kit‐LI cells that may be involved in slow wave repolarization and maintenance of a negative potential between slow waves was also found. Similar pharmacology was observed in studies of intact murine intestinal muscles. 5 Cultured ICC may be a useful model for studying the properties and pharmacology of some of the ionic conductances involved in spontaneous rhythmicity in the gastrointestinal tract.

Blockade of Kit signaling induces transdifferentiation of interstitial cells of Cajal to a smooth muscle phenotype

BACKGROUND & AIMS Interstitial cells of Cajal (ICC) serve as pacemaker cells and mediators of neurotransmission from the enteric nervous system to gastrointestinal muscles. ICC develop from mesenchymal cells that express c-Kit, and signaling via Kit receptors is necessary for normal development of ICC. We studied the fate of functionally developed ICC after blockade of Kit receptors to determine whether ICC undergo cell death or whether the phenotype of the cells is modified. The fate of undeveloped ICC was also investigated. METHODS Neutralizing, anti-Kit monoclonal antibody (ACK2) was administered to mice for 8 days after birth. ICC in the small intestine were examined by immunohistochemistry and electron microscopy. Occurrence of apoptosis was also assayed. RESULTS When Kit receptors were blocked, ICC nearly disappeared from the small intestine. Apoptosis was not detected in regions where ICC are normally distributed. Remaining Kit-immunopositive cells in the pacemaker region of the small intestine developed ultrastructural features similar to smooth muscle cells, including prominent filament bundles and expression of the muscle-specific intermediate filament protein, desmin, and smooth muscle myosin. ICC of the deep muscular plexus normally develop after birth in the mouse. Precursors of these cells remained in an undifferentiated state when Kit was blocked. CONCLUSIONS These data, along with previous studies showing that ICC in the pacemaker region of the small intestine and longitudinal muscle cells develop from the same Kit-immunopositive precursor cells, suggest inherent plasticity between the ICC and smooth muscle cells that is regulated by Kit-dependent cell signaling.

Expression of anoctamin 1/TMEM16A by interstitial cells of Cajal is fundamental for slow wave activity in gastrointestinal muscles

Interstitial cells of Cajal (ICC) generate pacemaker activity (slow waves) in gastrointestinal (GI) smooth muscles, but the mechanism(s) of pacemaker activity are controversial. Several conductances, such as Ca2+‐activated Cl− channels (CaCC) and non‐selective cation channels (NSCC) have been suggested to be involved in slow wave depolarization. We investigated the expression and function of a new class of CaCC, anoctamin 1 (ANO1), encoded by Tmem16a, which was discovered to be highly expressed in ICC in a microarray screen. GI muscles express splice variants of the Tmem16a transcript in addition to other paralogues of the Tmem16a family. ANO1 protein is expressed abundantly and specifically in ICC in all regions of the murine, non‐human primate (Macaca fascicularis) and human GI tracts. CaCC blocking drugs, niflumic acid and 4,4′‐diisothiocyano‐2,2′‐stillbene‐disulfonic acid (DIDS) reduced the frequency and blocked slow waves in murine, primate, human small intestine and stomach in a concentration‐dependent manner. Unitary potentials, small stochastic membrane depolarizations thought to underlie slow waves, were insensitive to CaCC blockers. Slow waves failed to develop by birth in mice homozygous for a null allele of Tmem16a (Tmem16atm1Bdh/tm1Bdh) and did not develop subsequent to birth in organ culture, as in wildtype and heterozygous muscles. Loss of function of ANO1 did not inhibit the development of ICC networks that appeared structurally normal as indicated by Kit antibodies. These data demonstrate the fundamental role of ANO1 in the generation of slow waves in GI ICC.

Distribution of the vanilloid receptor (VR1) in the gastrointestinal tract

The gastrointestinal (GI) tract responds to a variety of stimuli through local and centrally mediated pathways. Changes in the intestinal microenvironment are sensed by vagal, spinal, and intrinsic primary afferent fibers. Sensory nerve endings located close to the lumen of the GI tract respond to pH, chemical composition of lumenal contents, or distortion of the mucosa. Afferents within the muscle layers are thought to be tension sensitive, whereas those located within the myenteric plexus are also thought to respond to changes in chemical composition and humoral substances. Subpopulations of these afferent fibers are activated by capsaicin. However, the exact location of these nerves is currently not known. The vanilloid receptor (VR1) is a nonselective cation channel that is activated by capsaicin, acid, and temperature. Antibodies to VR1 make it possible to determine the location of these afferents, their morphology, and their relationships with enteric nerves and other cell types in the GI tract. VR1‐like immunoreactivity was observed on nerves within myenteric ganglia and interganglionic fiber tracts throughout the GI tract. VR1 nerves were also observed within the muscle layers and had an irregular profile, with varicose‐like swellings along their lengths. Blood vessels within the GI wall had VR1‐immunoreactive nerve fibers associated with them. VR1‐like nerves and other immunopositive cells were also observed within the mucosa. In summary, VR1‐like immunoreactivity was found in several locations within the GI tract and may provide sensory integration of chemical, physical, or inflammatory stimuli. VR1‐like fibers appear to be predominantly spinal in origin, but a few vagal VR1‐like fibers exist in the stomach. J. Comp. Neurol. 465:121–135, 2003.

Development of c-Kit-positive cells and the onset of electrical rhythmicity in murine small intestine

BACKGROUND & AIMS Little is known about the development of interstitial cells (ICs), yet these cells are important in electrical rhythmicity and neurotransmission in the gastrointestinal tract. This study characterized the development of ICs and the onset of electrical rhythmicity in the murine intestine. METHODS Antibodies against c-Kit (e.g., the receptor for stem cell factor) were used to label ICs of the small intestines of embryos and neonatal mice. Labels for enteric neuroblasts and smooth muscle cells were used to study neighboring cells. Development was examined also with electron microscopy and electrophysiological techniques. RESULTS c-Kit-like immunoreactivity (c-Kit-LI) was detected in gastrointestinal tissues at embryonic day 12.5. Labeled cells were distributed along the outer perimeter of the intestine and had morphological features of neither smooth muscle cells nor ICs. Cells with c-Kit-LI were nonneural and seemed to be common precursors for longitudinal muscle cells and ICs of the myenteric plexus region (IC-MY). Longitudinal muscle cells lost c-Kit by E18, whereas IC-MY continued c-Kit expression into adulthood. Electrical rhythmicity developed after IC-MY, and longitudinal muscle cells became separate entities. ICs in the deep muscular plexus region developed after birth. CONCLUSIONS ICs have a nonneural origin. Common precursors yield IC-MY and longitudinal muscle cells. Development of IC-MY correlates with the initiation of electrical rhythmicity.

Interstitial cells of Cajal in the guinea-pig gastrointestinal tract as revealed by c-Kit immunohistochemistry

Abstract.Interstitial cells of Cajal (ICC) of various morphologies have been described in the gastrointestinal (GI) tracts of mammals. Different classes of ICC are likely to have different functional roles. ICC of the mouse GI tract have been shown to express c-kit, a proto-oncogene that codes for a receptor tyrosine kinase. We have studied the distribution of ICC within the guinea pig GI tract using antibodies to c-Kit protein and immunohistochemical techniques. c-Kit-like immunoreactivity revealed at least 6 types of ICC: (1) intramuscular ICC (IC-IM1) that lie within the muscle layers of the esophagus, stomach, and cecum, (2) ICC within the myenteric plexus region (IC-MY1) in the corpus, antrum, small intestine, and colon,(3) ICC that populate the deep muscular plexus of the small intestine (IC-DMP), (4) ICC at the submucosal surface of the circular muscle layer in the colon (IC-SM), (5) stellate ICC that are closely associated with the myenteric plexus (IC-MY2) and orientated toward the longitudinal muscle layer in the colon, and (6) branching intramuscular ICC (IC-IM2) in the proximal colon within the circular and longitudinal muscle layers. c-Kit immunohistochemistry appears to be an excellent and selective technique for labeling ICC of the guinea-pig GI tract. Labeling of these cells at the light-microscopic level provides an opportunity for characterizing the distribution, density, organization, and relationship between ICC and other cell types.