Hwangryunhaedok-tang induces the depolarization of pacemaker potentials through 5-HT_3 and 5-HT_4 receptors in cultured murine small intestine interstitial cells of Cajal

AIM To investigate the effects of a water extract of Hwangryunhaedok-tang(HHTE) on the pacemaker potentials of mouse interstitial cells of Cajal(ICCs).METHODS We dissociated ICCs from small intestines and cultured. ICCs were immunologically identified using an antic-kit antibody. We used the whole-cell patch-clamp configuration to record the pacemaker potentials generated by cultured ICCs under the current clamp mode(I = 0). All experiments were performed at 30 ℃-32 ℃RESULTS HHTE dose-dependently depolarized ICC pacemaker potentials. Pretreatment with a 5-HT_3 receptor anta-gonist(Y25130) or a 5-HT_4 receptor antagonist(RS39604) blocked HHTE-induced pacemaker potential depolarizations, whereas pretreatment with a 5-HT7 receptor antagonist(SB269970) did not. Intracellular GDPβS inhibited HHTE-induced pacemaker potential depolarization and pretreatment with a Ca~(2+)-free solution or thapsigargin abolished the pacemaker potentials. In the presence of a Ca~(2+)-free solution or thapsigargin, HHTE did not depolarize ICC pacemaker potentials. In addition, HHTE-induced pacemaker potential depolarization was unaffected by a PKC inhibitor(calphostin C) or a Rho kinase inhibitor(Y27632). Of the four ingredients of HHT, Coptidis Rhizoma and Gardeniae Fructus more effectively inhibited pacemaker potential depolarization.CONCLUSION These results suggest that HHTE dose-dependently depolarizes ICC pacemaker potentials through 5-HT_3 and 5-HT_4 receptors via external and internal Ca~(2+) regulation and via G protein-, PKC-and Rho kinase-independent pathways.     

Effects of Lizhong Tang on cultured mouse small intestine interstitial cells of Cajal

AIM:To investigate the effects of Lizhong Tang,an herbal product used in traditional Chinese medicine,on mouse small intestine interstitial cells of Cajal(ICCs).METHODS:Enzymatic digestions were used to dissociate ICCs from mouse small intestine tissues.The ICCs were morphologically distinct from other cell types in culture and were identified using phase contrast microscopy after verification with anti c-kit antibody.A whole-cell patch-clamp configuration was used to record potentials(current clamp) from cultured ICCs.All of the experiments were performed at 30-32 ℃.RESULTS:ICCs generated pacemaker potentials,and Lizhong Tang produced membrane depolarization in current-clamp mode.The application of flufenamic acid(a nonselective cation channel blocker) abolished the generation of pacemaker potentials by Lizhong Tang.Pretreatment with thapsigargin(a Ca 2+-ATPase inhibi-tor in the endoplasmic reticulum) also abolished the generation of pacemaker potentials by Lizhong Tang.However,pacemaker potentials were completely abolished in the presence of an external Ca 2+-free solution,and under this condition,Lizhong Tang induced membrane depolarizations.Furthermore,When GDPβ-S(1 mmol/L) was in the pipette solution,Lizhong Tang still induced membrane depolarizations.In addition,membrane depolarizations were not inhibited by chelerythrine or calphostin C,which are protein kinase C inhibitors,but were inhibited by U-73122,an active phospholipase C inhibitors.CONCLUSION:These results suggest that Lizhong Tang might affect gastrointestinal motility by modulating pacemaker activity in interstitial cells of Cajal.     

The mechanism and spread of pacemaker activity through myenteric interstitial cells of Cajal in human small intestine

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 Ca(2+) indicator fluo-4, and pacemaker activity was recorded via high-speed video imaging at 36.5 degrees C +/- 0.5 degrees C. RESULTS: Rhythmic, biphasic Ca(2+) 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 Ca(2+) 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 Ca(2+) transients in ICC-MY appeared to result from Ca(2+) influx through dihydropyridine-resistant Ca(2+) channels, whereas the plateau phase was attributed to Ca(2+) release from inositol 1,4,5-triphosphate receptor-operated Ca(2+) 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.     

Deficiency of interstitial cells of Cajal in the small intestine of patients with Crohn''s disease

OBJECTIVE: Interstitial cells of Cajal are critical for the generation of electrical slow waves that regulate the phasic contractile activity of the tunica muscularis of the GI tract. Under certain pathophysiological conditions loss of interstitial cells of Cajal may play a role in the generation of certain motility disorders. The aim of the present study was to determine if there is an abnormality in the density or distribution of interstitial cells of Cajal from patients with Crohn's disease. METHODS: Small intestines from control subjects and patients with Crohn's disease were examined using immunohistochemistry and antibodies against the Kit receptor, which is expressed in interstitial cells of Cajal within the tunica muscularis of the GI tract. The density and distribution of interstitial cells of Cajal were assessed in the longitudinal and circular muscle layers and in the myenteric and deep muscular plexus regions of Crohn's and control tissues. RESULTS: Tissues from Crohn's disease patients showed an almost complete abolition of interstitial cells of Cajal within the longitudinal and circular muscle layers and a significant reduction in numbers at the level of the myenteric and deep muscular plexuses. CONCLUSIONS: In tissues from Crohn's disease patients, the density of interstitial cells of Cajal was reduced throughout the tunica muscularis in comparison to control small intestines. The disturbance of intestinal motility that occurs in patients with Crohn's disease may be a consequence of the loss of or defects in specific populations of interstitial cells of Cajal within the tunica muscularis.     

Differential gene expression profile in the small intestines of mice lacking pacemaker interstitial cells of Cajal

Background  

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.     

High-conductance chloride channels generate pacemaker currents in interstitial cells of Cajal

BACKGROUND & AIMS: Interstitial cells of Cajal (ICCs) are responsible for slow, wave-driven, rhythmic, peristaltic motor patterns in the gastrointestinal tract. The aim was to identify and characterize the ion channels that generate the underlying pacemaker activity. METHODS: Single ion channel recordings were obtained from nonenzymatically isolated ICCs and studied by using the cell attached and inside-out configurations of the patch clamp technique. RESULTS: A high-conductance chloride channel was observed in ICCs that was spontaneously and rhythmically active at the same frequency as the rhythmic inward currents defining ICC pacemaker activity, 20-30 cycles/min at room temperature. Main conductance levels occurred between 122-144 pS and between 185-216 pS. Periodicity in the channel opening coincided with periodicity in membrane potential change, hence, at the single channel level, chloride channels were seen to be associated with the generation of rhythmic changes in membrane potential. CONCLUSIONS: ICCs harbor high-conductance chloride channels that participate in the generation of pacemaker activity and may become a target for pharmacologic treatment of gut motor disorders.     

Ultrastructure of interstitial cells of Cajal associated with deep muscular plexus of human small intestine.

Evidence showing that interstitial cells of Cajal have important regulatory functions in the gut musculature is accumulating. In the current study, the ultrastructure of the deep muscular plexus and associated interstial cells of Cajal in human small intestine were studied to provide a reference for identification and further physiological or pathological studies. The deep muscular plexus was sandwiched between a thin inner layer of smooth muscle (one to five cells thick) and the bulk of the circular muscle. Interstitial cells of Cajal in this region very much resembled smooth muscle cells (with a continuous basal lamina, caveolae, intermediate filaments, dense bodies, dense bands, and a well-developed subsurface smooth endoplasmic reticulum), but the arrangement of organelles was clearly different, and cisternae of granular endoplasmic reticulum were abundant. Interstitial cells of Cajal were distinguished from fibroblasts or macrophages in the region. They ramified in the inner zone of the outer division of circular muscle, penetrated the inner-most circular layer, and were also found at the submucosal border. They were in close, synapselike contact with nerve terminals of the deep muscular plexus, and only few gap junctions with other interstitial cells of Cajal or with the musculature were observed. Compared with interstitial cells of Cajal from other mammals, those associated with the deep muscular plexus in the human small intestine more closely resemble smooth muscle cells, and their organization appears more diffuse; however, the ultrastructure and organization of interstitial cells of Cajal is compatible with modulatory actions on the circular muscle also in humans.     

Differential gene expression in the murine gastric fundus lacking interstitial cells of Cajal

Background  

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/W ^V mutants provides a unique opportunity to study the molecular changes that are associated with the loss of these intercalating cells.     

Homing of the bone marrow-derived interstitial cells of Cajal is decreased in diabetic mouse intestine

Background: Interstitial cells of Cajal (ICCs), which express c‐Kit receptor tyrosine kinase (KIT), play an important role in gastrointestinal motility. Loss of ICCs likely contributes to diabetic gastrointestinal motility disorder, however, the mechanism of attrition remains unknown. Here, we test the hypothesis that the bone marrow‐derived progenitors are an important source of intestinal ICCs and that decreased homing of these progenitors in diabetes contributes to ICC diminution. Methods: Wild type mice were X‐ray irradiated, transplanted with bone marrow (BMT) from green fluorescence protein (GFP)‐transgenic (TG)‐mice and subsequently made diabetic by streptozotocin (STZ) injection. Intestinal homing of GFP‐positive bone marrow‐derived cells was examined 2 or 5 months after STZ treatment. Results: In the BMT‐mice, we found many GFP‐positive bone marrow‐derived cells (BMDCs) in most parts of the intestinal area, the number of BMDCs was significantly decreased in diabetic mice compared with nondiabetic controls. As a representative area, we further examined the myenteric plexus of the proximal small intestine, and found that the cell numbers of ICCs marked by c‐Kit‐positive immunoreactivity were decreased by more than 40% in diabetic versus nondiabetic mice. Furthermore, numbers of c‐Kit+/GFP+ and c‐Kit+/GFP‐ cells were similar in nondiabetic mice, and decreased by 45.8% and 42.0%, respectively, in diabetic mice. Conclusion: These results suggest that the decreased homing from the bone marrow is a major cause of ICC loss in the intestine in diabetes mellitus.