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Recent studies of diabetic animal models suggest an important role of ICC in the pathogenesis of gastropathy. The aim of this study was to characterize the ultrastructural features of ICC and record the electrical properties in the stomach of patients with type 2 DM. Gastric specimens were obtained from 13 diabetic patients and 6 control subjects with gastric cancer that underwent gastrectomy. All specimens were taken from disease-free areas. The samples were processed for both electron microscopic and electrophysiologic examination. The characteristic ultrastructural changes of the ICC were observed in both the nucleus and cytoplasm in patients with type 2 DM. Wrinkling of the nuclear envelope and changes in the cytoplasm such as dilatation of the endoplasmic reticulum, an increase of autophagic vacuoles, were more frequently observed in the diabetic patients. Apoptosis characterized by nuclear karyorrhexis or pyknosis was observed only in the diabetic patients. Slow waves were recorded in the circular muscle of stomach. In diabetic patients, the mean resting membrane potential was higher and amplitude was lower than controls. These changes of electrical activities of slow waves were accompanied with ultrastructural changes of ICC, particularly the characteristic nuclear changes. In human diabetic patients, the characteristic ultrastructural changes of ICC such as preapoptosis, accompanied with electrical dysrhythmia of slow waves, were observed. These results show several evidence converging to support that degeneration of the ICC may be associated with the pathogenesis of diabetic gastropathy.

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.

Interstitial cells of Cajal (ICC) are a fundamental component of the pacemaker apparatus of the gastrointestinal (GI) tract. ICC generate pacemaker currents that are the basis for slow wave activity in GI muscles. ICC form a network of cells connected by gap junctions that run around and along the phasic regions of the GI tract. ICC possess specialized conductances that allow them to generate pacemaker activity and serve as the pathway for active propagation of slow waves. Pacemaker currents are attributed to a Ca(2+)-inhibited, voltage-independent, non-selective cation conductance that has similar properties to the conductance elicited by expression of transient receptor potential-C4. Propagation occurs through a voltage-dependent mechanism, and data suggest that the factor coupling pacemaker units in ICC is Ca(2+) entry. ICC express a dihydropyridine-resistant, voltage-dependent Ca(2+) conductance that is important in slow wave propagation. Work is underway to determine the molecular identities of these conductances.

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

In order to learn about the endogenous Ca 2+ -buffering in the cytoplasm of chick dorsal root ganglion (DRG) neurons and the distance separating the ryanodine receptor Ca 2+ release channels (RyRs) from the plasma membrane, we monitored the amplitude and time course of Ca 2+ -activated Cl – currents (l Cl(Ca) ) in protocols that manipulated Ca 2+ -buffering. l Cl(Ca) was activated by Ca 2+ influx via voltage-gated Ca 2+ channels or by Ca 2+ release via RyRs activated by 10 mM caffeine. l Cl(Ca) was measured in neurons at 20°C and 35°C using the amphotericin perforated patch technique that preserves endogenous Ca 2+ -buffering, or at 20°C in neurons dialyzed with pipette solutions designed to replace the endogenous Ca 2+ buffers. The amplitude of l Cl(Ca) activated by Ca 2+ influx or Ca 2+ at 20°C was similar in the amphotericin neurons and neurons dialyzed with an ‘unbuffered’ pipette solution containing 10 mM citrate and 3 mM ATP as the only Ca 2+ binding molecules. Thus, endogenous mobile Ca 2+ buffers are relatively unimportant in chick DRG neurons. Warming the neurons from 20°C to 35°C increased the amplitude and the rate of deactivation of l Cl(Ca) consistent with an increased rate of Ca 2+ buffering by fixed endogenous Ca 2+ -buffers. Dialysis with 2 mM EGTA/0.1 μM free Ca 2+ reduced the amplitude and increased the rate of deactivation of l Cl(Ca) activated by Ca 2+ influx and abolished l Cl(Ca) activated by Ca 2+ release. Dialysis with 2 mM BAPTA/0.1 μM free Ca 2+ abolished l Cl(Ca) activated by Ca 2+ influx or release. Dialysis with 42 mM HEEDTA/0.5 μM free Ca 2+ caused the persistent activation of l Cl(Ca) . Calculations using a Ca 2+ -diffusion model suggest that the voltage-gated Ca 2+ channels and the Ca 2+ -activated Cl – channels are separated by 50–400 nm and that the RyRs are more than 600 nm from the plasma membrane.

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)

Neurons that synthesize nitric oxide from arginine produce stoichiometric amounts of citrulline. We investigated whether nitric oxide-releasing enteric neurons have the capacity to recycle citrulline to arginine and thereby sustain nitrergic neurotransmission. Argininosuccinate synthetase-like immunoreactivity and argininosuccinate lyase-like immunoreactivity, enzymes capable of citrulline to arginine conversion, were both localized in discrete populations of myenteric and submucosal neurons in the canine proximal colon. Argininosuccinate synthetase-like immunoreactivity and argininosuccinate lyase-like immunoreactivity were also found in varicose nerve fibers within the circular and longitudinal muscle layers. Argininosuccinate synthetase-like immunoreactivity and argininosuccinate lyase-like immunoreactivity co-localized with neuronal β-nicotinamide adenine dinucleotide phosphate diaphorase staining, a marker for nitric oxide synthase. The functional significance of argininosuccinate synthetase-like immunoreactivity and argininosuccinate lyase-like immunoreactivity was shown by testing the effects of exogenous citrulline on responses to enteric inhibitory nerve stimulation, which were assessed by measuring contractions, inhibitory junction potentials and electrical slow waves. As shown previously, arginine analogues ( l -nitroarginine methyl ester or l -nitroarginine; 100 μM) inhibited nitric oxide-dependent responses, and excess l -arginine restored inhibitory responses. Citrulline alone (0.1–2 mM) had no effect on nitrergic transmission under control conditions, but in the presence of l -nitroarginine methyl ester or l -nitroarginine, citrulline (0.1–2 mM) restored nitrergic transmission in a concentration-dependent manner. Other neutral amino acids ( l -serine, l -leucine) did not mimic the effects of citrulline. Taken together, these data suggest that enteric nitrergic neurons have the enzymatic apparatus and functional capability of recycling citrulline to arginine.

A subpopulation of interstitial cells (ICs) are interposed between nerve terminals and smooth muscle cells in the gastrointestinal tract and may participate in neuromuscular transmission. These cells appear to be targets for NO released from enteric inhibitory nerves and respond to exogenous NO with: (i) an elevation in cGMP levels; (ii) an increase in intracellular Ca2+; (iii) and release of a diffusible substance that has tentatively been identified as NO. For the latter to be possible, ICs must express a constitutive isoform of NOS. This study characterized the expression of NOS-like immunoreactivity (NOS-LI) in ICs of the canine colon using 3 antibodies raised against the 2 known constitutive forms of NOS (i.e., neural (nNOS) and endothelial (eNOS) isoforms). Antibodies raised against cNOS and an antibody raised against rat cerebellar nNOS labeled ICs along the submucosal surface of the circular muscle layer (IC-SM), along the surface of septa that separate the circular muscle into fiber bundles (IC-SM), and in the myenteric region between the circular and longitudinal muscle layers (IC-MY). Another antibody raised against rat cerebellar nNOS failed to label ICs. Cultured IC-SM also expressed NOS-LI, suggesting that this feature of the IC phenotype survives culture conditions. Arteriolar endothelial cells in the canine colon were labeled with the same 2 antibodies that labeled ICs, suggesting there are significant structural similarities between NO synthases in ICs and endothelial cells. The data suggest that IC-SM and IC-MY express a constitutive form of NOS. Synthesis of NO by ICs may influence electrical rhythmicity and may serve to amplify and even propagate enteric inhibitory neurotransmission.

There is growing evidence that nitric oxide serves as a neurotransmitter released from enteric inhibitory nerves in the gastrointestinal tract. The distribution of nitric oxide synthase suggests that nitric oxide may also be a neurotransmitter within enteric ganglia. Since many actions of nitric oxide are mediated by stimulation of soluble guanylate cyclase and a subsequent increase in 3',5'-cyclic guanosine monophosphate (cGMP) concentration, targets for nitric oxide in the canine proximal colon were investigated by immunohistochemical localization of cGMP. In the presence of phosphodiesterase inhibitors (M&B 22948, 100 microM and 3-isobutyl-1-methyl-xanthine, 1 mM), exogenous nitric oxide and electrical field stimulation caused an accumulation of cGMP-like immunoreactivity in several cell-types including colonic smooth muscle cells. cGMP-like immunoreactivity was also observed in a subpopulation of neurons in both myenteric and submucosal ganglia. Sequential labeling with the NADPH diaphorase technique showed that 94% of neurons that responded to exogenous nitric oxide with an increase in cGMP-like immunoreactivity were NADPH diaphorase negative. None of the myenteric neurons that responded to electrical field stimulation with an increase in cGMP-like immunoreactivity were NADPH diaphorase positive, and only one submucosal neuron with cGMP-like immunoreactivity was also NADPH diaphorase positive. The electrical field-stimulated increase in cGMP-like immunoreactivity was blocked by nitroarginine (100 microM). An increase in cGMP-like immunoreactivity also occurred in interstitial cells located at the submucosal surface of the circular muscle layer. These cells are interposed between nerve varicosities and smooth muscle cells and may partially mediate neuromuscular transmission. Sodium nitroprusside and nitric oxide also caused an accumulation of cGMP-like immunoreactivity in smooth muscle cells of intramural arterioles and venules. The results of this study further support the role of nitric oxide as a neurotransmitter in colonic muscles, and provide support for the hypothesis that interstitial cells are functionally innervated by enteric inhibitory neurons. The data also suggest that nitric oxide may serve as a neurotransmitter in enteric ganglia.

test) with aging. To determine whether these pro-inflammatory cytokines affected ENSC survival, TUNEL assays were performed on ENSCs cultured in conditioned media from young (CM-young) and old (CM-old) LMMP. CM-old caused a over a 2-fold increase in apoptosis compared to CM-young, an effect completely reversed by anti-IL6 neutralizing antibody (11.1±0.6% for CM-old, 4.9±1.2% for CM-young, 4.9±1.4% for CM-old+anti-IL6, p

Introduction: Motor functions of the stomach have traditionally been regarded to differ by stomach region. However, to date there have been few studies on the gastric contractility of the human stomach. Objectives: The aim of the present study was therefore to examine the response to acetylcholine and electrical field stimulation (EFS) of the human fundus, corpus and antrum. Methods: Gastric muscles were obtained from 71 patients (44 males, 27 females with an average age of 60.7 yrs; 34 85 yrs) undergoing gastric cancer surgery. Muscle strips (n = 71) were isolated and attached to a fixed mount and to an isometric strain gauge. The muscle strips were studied In Vitro for their contractile responses to acetylcholine (n=25) and electrical field stimulation (EFS, n=46). N-nitro-L-arginine (L-NA), atropine and tetrodotoxin (TTX) were added to assess the nitro oxide-mediated changes and cholinergic neural pathway involved in EFS-induced contractions. Results: Spontaneous contractile activity was observed in muscle strips of stomach regions. In the antrum, acetylcholine induced increase in contraction frequency and peak contraction in dose dependent pattern, but no significant change was noticed in muscle tone. In corpus and fundus, acetylcholine induced dose-dependent increase in peak contraction and muscle tone, but there was no significant change in frequency. The response to EFS also differed by stomach region. EFS produced no significant change in contractile activity in 22 of 46 muscle strips. When EFS evoked frequency-dependent contraction or relaxation in muscle strips, contraction was primarily observed in the muscle strips from the antrum, and relaxation was primarily observed in the muscle strips of the corpus and fundus. The addition of LNA (100 uM) to the muscle bath converted the EFS-induced relaxation to contractions and increased the EFS-induced contractions. EFS-induced contractions were abolished by atropine (1uM) or TTX (1uM). Conclusions: Our study demonstrated that the response to acetylcholine and electrical field stimulation on human gastric fundus and corpus is different with that of human gastric antrum.