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Background & Aims Gastrointestinal (GI) motility is regulated by serotonin (5-hydroxytryptamine [5-HT]), which is primarily produced by enterochromaffin (EC) cells in the GI tract. However, the precise roles of EC cell-derived 5-HT in regulating gastric motility remain a major point of conjecture. Using a novel transgenic mouse line, we investigated the distribution of EC cells and the pathophysiologic roles of 5-HT deficiency in gastric motility in mice and humans. Methods We developed an inducible, EC cell–specific Tph1CreERT2/+ mouse, which was used to generate a reporter mouse line, Tph1-tdTom, and an EC cell–depleted line, Tph1-DTA. We examined EC cell distribution, morphology, and subpopulations in reporter mice. GI motility was measured in vivo and ex vivo in EC cell–depleted mice. Additionally, we evaluated 5-HT content in biopsy and plasma specimens from patients with idiopathic gastroparesis (IG). Results Tph1-tdTom mice showed EC cells that were heterogeneously distributed throughout the GI tract with the greatest abundance in the antrum and proximal colon. Two subpopulations of EC cells were identified in the gut: self-renewal cells located at the base of the crypt and mature cells observed in the villi. Tph1-DTA mice displayed delayed gastric emptying, total GI transit, and colonic transit. These gut motility alterations were reversed by exogenous provision of 5-HT. Patients with IG had a significant reduction of antral EC cell numbers and 5-HT content, which negatively correlated with gastric emptying rate. Conclusions The Tph1CreERT2/+ mouse provides a powerful tool to study the functional roles of EC cells in the GI tract. Our findings suggest a new pathophysiologic mechanism of 5-HT deficiency in IG.

Background & Aims In human and canine colon, both slow (slow waves, 2–8/min) and fast (myenteric potential oscillations [MPOs]; 16–20/min) electrical rhythms in the smooth muscle originate at the submucosal and myenteric borders, respectively. We used Ca 2+ imaging to investigate whether interstitial cells of Cajal (ICCs) at these borders generated distinct rhythms. Methods Segments of canine colon were pinned with submucosal or myenteric surface uppermost or cut in cross section. Tissues were loaded with a Ca 2+ indicator (fluo-4), and activity was monitored at 36.5 ± 0.5°C using an electron multiplying charge coupled device (EMCCD). Results Rhythmic, biphasic Ca 2+ transients (5–8/min), similar in waveform to electrical slow waves, propagated without decrement as a wave front (2–5 mm/s) through the ICC-SM network lying along the submucosal surface of the circular muscle (CM). In contrast, rhythmic intracellular Ca 2+ waves (∼16/min) and spontaneous reductions in Ca 2+ were observed in ICCs at the myenteric border (ICC-MY). Normally, intracellular Ca 2+ waves were unsynchronized between adjacent ICC-MY, although excitatory nerve activity synchronized activity. In addition, spontaneous reductions in Ca 2+ were observed that inhibited Ca 2+ waves. Nω-nitro-L-arginine (100 μmol/L; nitric oxide antagonist) blocked the reductions in Ca 2+ and increased the frequency (∼19/min) of intracellular Ca 2+ waves within ICC-MY. Conclusions ICC-SMs form a tightly coupled network that is able to generate and propagate slow waves. In contrast, Ca 2+ transients in ICC-MYs, which are normally not synchronized, have a similar duration and frequency as MPOs. Like MPOs, their activity is inhibited by nitrergic nerves and synchronized by excitatory nerves.

Background & Aims: Like the heart, intestinal smooth muscles exhibit electrical rhythmicity, which originates in pacemaker cells surrounding the myenteric plexus, called interstitial cells of Cajal (ICC-MY). In large mammals, ICC also line septa (ICC-SEP) between circular muscle (CM) bundles, suggesting they might be necessary for activating muscle bundles. It is important to determine their functional significance, because a loss of ICC in humans is associated with disordered motility. Our aims were therefore to determine the role of ICC-SEP in activating the thick CM in the human jejunum. Methods: The mucosa and submucosa were removed and muscle strips were cut and pinned in cross-section so that the ICC-MY and ICC-SEP networks and the CM could be readily visualized. The ICC networks and CM were loaded with the Ca 2+ indicator fluo-4, and pacemaker and muscle activity was recorded at 36.5 ± 0.5 ° C. Results: Ca 2+ imaging revealed that pacemaker activity in human ICC-MY can entrain ICC-SEP to excite CM bundles. Unlike the heart, pacemaker activity in ICC-MY varied in amplitude, propagation distance, and direction, leading to a sporadic activation of ICC-SEP. Conclusions: ICC-SEP form a crucial conduction pathway for spreading excitation deep into muscle bundles of the human jejunum, necessary for motor patterns underlying mixing. A loss of these cells could severely affect motor activity.

Background & Aims: Interstitial cells of Cajal (ICC) generate and propagate slow waves in the stomach. Gastric peristalsis depends on a proximal-to-distal gradient in slow wave frequency. We tested whether the gastric frequency gradient was an intrinsic property of ICC and whether dysrhythmias result from disruptions of ICC networks. Methods: We studied wild-type (WT) and W/W V mice, which have only myenteric (pacemaker) ICC in the stomach. ICC distributions were analyzed by Kit immunofluorescence. Pacemaking in tissues was studied by intracellular electrophysiologic recording and in cultured ICC by monitoring mitochondrial [Ca 2+ ] oscillations with rhod-2 fluorescence or membrane potential with DiBAC 4 (3) fluorescence. Results: Slow wave frequencies were constant throughout WT gastric muscle sheets containing corpus and antrum. Separating the antrum from the corpus caused a significant drop in antral slow wave frequency. ICC from WT antrums also displayed significantly slower pacemaker frequencies than corpus ICC, but the corpus pacemaker frequency dominated in cocultures of corpus and antrum ICC. Myenteric ICC networks were reduced in W/W V mice, particularly in the corpus. In W/W V mice, separating the antrum from the corpus failed to reduce antral slow wave frequency. Antral pacemaker frequency in ICC from W/W V stomachs was the same as in corpus ICC. Conclusions: The proximal-to-distal slow wave frequency gradient and entrainment of distal electrical activity by proximal pacemakers are fundamental properties of gastric ICC. Chronic depletion of ICC networks disrupts the proximal-to-distal frequency gradient, and emergence of ectopic pacemakers in the antrum may be caused by "reprogramming" of the ICC pacemaker apparatus. GASTROENTEROLOGY 2002;123:2028-2040

Background & Aims: Cyclooxygenase enzymes (COX) generate intermediates in the prostaglandin (PG) cascade. COX-1 is constitutively expressed in many cells, and COX-2 is typically thought to be an inducible isoform. Methods: We evaluated constitutive expression and function of COX-2 in murine gastric muscles. Results: Immunohistochemistry showed COX-2–like immunoreactivity (COX-2–LI) in myenteric neurons. Half the neurons with COX-2–LI expressed nitric oxide synthase (NOS). COX-2–LI was not observed in smooth muscle cells. Interstitial cells of Cajal within muscle layers (IC-IM) expressed COX-2–LI, suggesting a novel role for IC-IM. Molecular studies verified expression of COX-2 in gastric muscles. Quantitative polymerase chain reaction (PCR) showed equal expression of COX-1 and COX-2 in the antrum. COX-2 was more abundant in fundus. Indomethacin and GR253035X, a COX-2 inhibitor, increased antral phasic contractions and potentiated responses to ACh. Indomethacin, but not GR253035X, increased contractions and potentiated responses in tissues of COX-2 knockout mice. Indomethacin and GR253035X reduced tone in the fundus. Conclusions: COX-2 is constitutively expressed by IC-IM and neurons in the stomach and at levels similar to COX-1. Prostanoids produced by COX-2 regulate mechanical activities of fundus and antral muscles. GASTROENTEROLOGY 2002;122:1442-1454

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. GASTROENTEROLOGY 1999;117:140-148

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/W v ) 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/W v animals, NO-dependent inhibitory neurotransmission was reduced. Hyperpolarizations to sodium nitroprusside were also attenuated in W/W v 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. GASTROENTEROLOGY 1998;115:314-329

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. (Gastroenterology 1997 Jan;112(1):144-55)

Electrical rhythmicity in gastrointestinal muscles has been studied for a century, but the pacemakers driving this phenomenon have been elusive. Anatomic studies suggest that interstitial cells of Cajal (ICC) may be pacemakers and conductors of electrical activity. ICC may also mediate neurotransmission from enteric neurons. Functional evaluations of ICC include the following. (1) Electrophysiology experiments on dissected muscle strips show that slow waves originate from specific sites. These pacemaker areas are populated by networks of ICC that make gap junctions with smooth muscle cells. Removal of pacemaker regions interferes with slow wave generation and propagation. (2) Chemicals that label ICC histochemically can damage ICC and abolish rhythmicity. (3) isolated ICC are spontaneously active, and several voltage-dependent ion channels, including a low-threshold Ca2+ conductance, are expressed. (4) ICC are innervated by enteric neurons, and they respond to neurotransmitters. ICC may produce nitric oxide and amplify inhibitory neurotransmission. (5) Some classes of ICC fall to develop in animals with mutations in c-kit or stem cell factor, the ligand for c-Kit receptors. Without ICC, electrical slow waves are absent. Many questions remain about the function of ICC, but modern technologies should now facilitate rapid progress toward determining the role of these cells in normal physiology and pathological conditions.

Background & Aims Smooth muscle cells (SMCs) change phenotypes under various pathophysiological conditions. These changes are largely controlled by the serum response factor (SRF), a transcription factor that binds to CC (A/T) 6 GG (CArG) boxes in SM contractile genes. MicroRNAs (miRNA) regulate transitions among SMC phenotypes. The SMC miRNA transcriptome (SMC miRNAome) and its regulation by SRF have not been determined. Methods We performed massively parallel sequencing to identify gastrointestinal (GI) SMC miRNA transcriptomes in mice and humans. SMC miRNA transcriptomes were mapped to identify all CArG boxes, which were confirmed by SRF knockdown and microarrays. Quantitative polymerase chain reaction was used to identify SMC-phenotypic miRNAs in differentiated and proliferating SMCs. Bioinformatics and target validation analysis showed regulation of SMC phenotype by SRF-dependent, SMC-phenotype miRNAs. Results We cloned and identified GI miRNA transcriptomes using genome-wide analyses of mouse and human cells. The SM miRNAome consisted of hundreds of unique miRNAs that were highly conserved among both species. We mapped miRNAs CArG boxes and found that many had an SRF-dependent signature in the SM miRNAome. The SM miRNAs CArG boxes had several distinct features. We also identified approximately 100 SMC-phenotypic miRNAs that were induced in differentiated or proliferative SMC phenotypes. We showed that SRF-dependent, SMC-phenotypic miRNAs bind and regulate Srf and its cofactors, myocadin ( Myocd ) and member of ETS oncogene family Elk1 . Conclusions The GI SMC phenotypes are controlled by SRF-dependent, SMC-phenotypic miRNAs that regulate expression of SRF, MYOCD, and ELK1.

Background & Aims: It has been generally assumed that interstitial cells of Cajal (ICC) in the human gastrointestinal tract have similar functions to those in rodents, but no direct experimental evidence exists to date for this assumption. This is an important question because pathologists have noted decreased numbers of ICC in patients with a variety of motility disorders, and some have speculated that loss of ICC could be responsible for motor dysfunction. Our aims were to determine whether myenteric ICC (ICC-MY) in human jejunum are pacemaker cells and whether these cells actively propagate pacemaker activity. Methods: The mucosa and submucosa were removed, and strips of longitudinal muscle were peeled away to reveal the ICC-MY network. ICC networks were loaded with the Ca2+ indicator fluo-4, and pacemaker activity was recorded via high-speed video imaging at 36.5°C ± 0.5°C. Results: Rhythmic, biphasic Ca2+ transients (6.03 ± 0.33 cycles/min) occurred in Kit-positive ICC-MY. These consisted of a rapidly propagating upstroke phase that initiated a sustained plateau phase, which was associated with Ca2+ spikes in neighboring smooth muscle. Pacemaker activity was dependent on inositol 1,4,5-triphosphate receptor–operated stores and mitochondrial function. The upstroke phase of Ca2+ transients in ICC-MY appeared to result from Ca2+ influx through dihydropyridine-resistant Ca2+ channels, whereas the plateau phase was attributed to Ca2+ release from inositol 1,4,5-triphosphate receptor–operated Ca2+ stores. Conclusions: Each ICC-MY in human jejunum generates spontaneous pacemaker activity that actively propagates through the ICC network. Loss of these cells could severely disrupt the normal function of the human small intestine.

Background & Aims: Surgical manipulations of the gastrointestinal (GI) tract, including intestinal resection and anastomosis, lead to motility disorders including a decrease in phasic and segmental contractions. The aims of the present investigation were to determine mechanisms underlying the loss of motility in a murine model of intestinal resection and to follow-up the recovery of intestinal motility after surgical manipulation. Methods: Segments of ileum were removed from mice and the intestines were reconstructed. After surgery, the structure and activity of the ileal muscles, 0–5 cm oral and aboral to the site of resection, were examined at 5 and 24 hours with intracellular microelectrode recordings, isometric force measurements, Kit-like immunohistochemistry, and electron microscopy. Results: Five hours after surgery there was loss of electrical slow waves and phasic contractions in muscles near the site of resection. This defect decreased as a function of distance above and below the resection. Tissues in the affected region were poorly responsive to carbachol and transmural nerve stimulation. Kit-like immunohistochemistry revealed disruption in interstitial cell of Cajal (ICC) networks at the level of the myenteric and deep muscular plexuses. Lesions in ICCs decreased with distance from the site of resection. Slow waves and mechanical activity recovered at the site of anastomosis 24 hours after surgery and recovered more rapidly when tissues were incubated in the inducible nitric oxide synthase (iNOS) inhibitor, L-N 6 -(1-Iminoethyl) lysine hydrochloride (L-NIL). Conclusions: Loss of intestinal motility after surgery is associated with acute disruption of ICC networks, slow waves, and phasic contractions. This activity partially recovered within 24 hours after surgery.

Background & Aims: Development of interstitial cells of Cajal (ICC) requires signaling via Kit receptors. Kit is activated by stem cell factor (SCF), but the source of SCF in the bowel wall is unclear and controversy exists about whether enteric neurons express the SCF required for ICC development. Methods: Glial cell line–derived neurotrophic factor (GDNF) knockout mice, which lack enteric neurons throughout most of the gut, were used to determine whether neurons are necessary for ICC development. ICC distributions were determined with Kit immunofluorescence, and function of ICC was determined by intracellular electrical recording. Results: ICC were normally distributed throughout the gastrointestinal tracts of GDNF−/− mice. Intracellular recordings from aganglionic gastrointestinal muscles showed normal slow wave activity at birth in the stomach and small intestine. Slow waves developed normally in aganglionic segments of small bowel placed into organ culture at birth. Quantitative polymerase chain reaction showed similar expression of SCF in the muscles of animals with and without enteric neurons. Expression of SCF was demonstrated in isolated intestinal smooth muscle cells. Conclusions: These data suggest that enteric neurons are not required for the development of functional ICC. The circular smooth muscle layer, which develops before ICC, may be the source of SCF required for ICC development. GASTROENTEROLOGY 1999;117:584-594

Background & Aims: Enteric neurons in the murine intestine express stem cell factor (SCF), which may provide an important signal in the development of the interstitial cells of Cajal (ICC). Our aim was to identify the subpopulation(s) of myenteric neurons that express SCF. Methods: Myenteric plexus preparations from postnatal SCF-lacZ mice were processed for β-galactosidase histochemistry followed by immunohistochemistry. Results: Approximately 60% of the nitric oxide synthase–immunoreactive neurons, which projected to myenteric ganglia and to circular muscle, expressed SCF, and more than 80% of the calbindin-immunoreactive neurons, which projected exclusively to myenteric ganglia, expressed SCF. A small subpopulation of calretinin-immunoreactive neurons expressed SCF transiently. Many of the remainder of SCF-expressing neurons were choline acetyltransferase immunoreactive, but their projections are unknown. Conclusions: SCF-expressing neurons that project within the myenteric plexus may be an important source of SCF for the development of Kit-expressing ICC at this level. The only possible neuronal source of SCF for the ICC of the deep muscular plexus is a subpopulation of nitric oxide synthase–immunoreactive neurons. GASTROENTEROLOGY 1998;115:898-908

BACKGROUND & AIMS: Vasopressin, a circulating hormone and agent used to treat gastrointestinal bleeding, affects gastrointestinal motility. Both excitatory and inhibitory responses that may be caused by species differences or concentration-dependent effects of this hormone have been reported. This study examined the actions of vasopressin on the canine colon and studied the mechanism underlying the excitatory effects of vasopressin. METHODS: Intracellular microelectrodes and isometric force measurements were used to measure vasopressin responses in muscle strips. The patch-clamp technique was used to record arginine vasopressin activation of nonselective cation currents in isolated myocytes. RESULTS: Vasopressin (10(-12) to 10(-9) mol/L) increased electrical slow waves and enhanced phasic contractions. Atropine and tetrodotoxin did not alter the responses to vasopressin. In myocytes, vasopressin activated a nonselective cation conductance at concentrations that enhanced slow waves. Vasopressin (> 5 x 10(-8) mol/L) caused a tetrodotoxin- or NG-nitro-L-arginine methyl ester- insensitive inhibition of slow waves and contractile activity. A V1- receptor antagonist shifted the effects of vasopressin to higher concentrations. Immunohistochemistry showed expression of vasopressin- like immunoreactivity within the colonic wall, suggesting that local concentrations may exceed circulating levels. CONCLUSIONS: These data document the concentration dependence of the biphasic effects of vasopressin and provide a mechanism for the excitatory effects of vasopressin at physiological levels. (Gastroenterology 1997 Oct;113(4):1233-45)

Background: Nitric oxide and an apamin-sensitive transmitter may both contribute to neural inhibition in the human colon. The present study investigated the role of NO in regulating spontaneous rhythmic contractions and examined NO-dependent and independent components of neurally evoked hyperpolarization in the human colon. Methods: Mechanical and electrical activity were recorded from isolated circular muscle strips. Results: Rhythmic contractions were inhibited by nerve stimulation. This response was reduced by apamin, oxyhemoglobin, and l- N G -nitro arginine methyl ester (l-NAME). Electrical recording revealed two components of neurally evoked hyperpolarization: a fast hyperpolarization resulting from a single stimulus and a sustained hyperpolarization that developed with repetitive stimulation. Fast hyperpolarization was not affected by l-NAME or oxyhemoglobin but was significantly reduced by apamin. The sustained hyperpolarization was reduced by l-NAME or apamin. Exogenous NO and the P 2y receptor agonist 2-methylthio adenosine 5′-triphosphate (2-MATP) inhibited spontaneous contractions and produced hyperpolarization. Apamin reduced the effects of 2-MATP but not those of NO. Conclusions: The results support the concept that the inhibitory neurotransmission in the human colon involves two transmitters. A single stimulus results in an apamin-sensitive response. With multiple stimuli, a NO-dependent response develops and sums with the apamin-sensitive mechanism, producing sustained hyperpolarization and inhibition of contractions.