Effects of Histamine on Cultured Interstitial Cells of Cajal in Murine Small Intestine

Interstitial cells of Cajal (ICCs) are the pacemaker cells in the gastrointestinal tract, and histamine is known to regulate neuronal activity, control vascular tone, alter endothelial permeability, and modulate gastric acid secretion. However, the action mechanisms of histamine in mouse small intestinal ICCs have not been previously investigated, and thus, in the present study, we investigated the effects of histamine on mouse small intestinal ICCs, and sought to identify the receptors involved. Enzymatic digestions were used to dissociate ICCs from small intestines, and the whole-cell patch-clamp configuration was used to record potentials (in current clamp mode) from cultured ICCs. Histamine was found to depolarize resting membrane potentials concentration dependently, and whereas 2-PEA (a selective H1 receptor agonist) induced membrane depolarizations, Dimaprit (a selective H2-agonist), R-alpha-methylhistamine (R-alpha-MeHa; a selective H3-agonist), and 4-methylhistamine (4-MH; a selective H4-agonist) did not. Pretreatment with Ca(2+)-free solution or thapsigargin (a Ca(2+)-ATPase inhibitor in endoplasmic reticulum) abolished the generation of pacemaker potentials and suppressed histamine-induced membrane depolarization. Furthermore, treatments with U-73122 (a phospholipase C inhibitor) or 5-fluoro-2-indolyl des-chlorohalopemide (FIPI; a phospholipase D inhibitor) blocked histamine-induced membrane depolarizations in ICCs. On the other hand, KT5720 (a protein kinase A inhibitor) did not block histamine-induced membrane depolarization. These results suggest that histamine modulates pacemaker potentials through H1 receptor-mediated pathways via external Ca2+ influx and Ca2+ release from internal stores in a PLC and PLD dependent manner.

Comparison Analysis of Stress Radiography for the Evaluation of Posterior Knee Laxity / 中国运动医学杂志

Objective Stress radiography provides an objective tool to measure posterior knee instability.This study was conducted to evaluate the intraobserver and interobserver reliability of measurements using Telos device to quantify posterior knee instability,compared wim KT-1000 and PDT test for consistency analysis.Methods From October 2008 to June 2009,68 stress radiographs in 34 patients with posterior knee instability were taken using Telos device.The amount of posterior displacement on the radiographs was measured independently by 2 surgeons on 2 separate occasions.Changes in intraclass correlation coefficients(ICCs)were examined to assess the intraobserver and interobserver reliability of the measurement,and were compared with those from KT-1000 and PDT test for consistency analysis.Results Intraobserver ICC was 0.992,while interobserver ICC Was 0.991.There was no significant difierence between Telos and KT-1000 in pair-t test.The data from Telos device was consistent with KT-1000.The coincident ratio Of PDT test was 20% while the posterior displacement of the tibia calculated on stress radiography was 5-10 mm.The coincident ratio of the PDT was 71.4% while the posterior displacement of the tibia calculatcd from stress radiography was 10-15 mm.Conclusion Using Telos device for stress radiograph provides a reproducible method to quantify posterior knee instability,and the consistency between Telos divece and KT-1000 was reliable.The coincident ratio of the PDT test with stress radiography increased when the posterior displacement of the tibia from stress view became more severe.

The morphologic changes and significance of interstitial cells of Cajal-like cells in the bladder of diabetic guinea pig / 临床与实验病理学杂志

Purpose To clarify the morphologic changes and significance of interstitial cells of Cajal-like cells in the bladder of diabetic guinea pig.Methods The experimental guinea pigs were induced by a single intraperitoneal injection of streptozotocin(STZ).The diabetic guinea pig model, who were made succesful for 4 weeks, were detected through the urodynamic evaluation.The distribution of ICCs-like cells in the detrusor, which were taken from these guinea pigs,were quantitatively analyzed after frozen section and indirect immunofluorescence methods and observed by electron microscopy.Results In the diabetic group, the maximum detrusor pressure was signifently lower than controls (P＜0.01), maximum bladder capacity, compliance, residual volume and leak point pressures was significently higer than controls (P＜0.05 or P＜0.01);In detrusor, compared with control, the ICCs-like cells of diabetes group showed significently decreased in quantity. In diabetic bladder, the quantity of gap junctions between ICCs-like cells and cells around,such as smoth muscle cells,terminal nerves and other ICCs-like cells,decreased significently.And more cavities and less organoids occurred in cytoplasm of diabetic ICCs-like cells. Mitochondria became swollen or even disapeared, accompanied with swollen endocytoplasmic reticulum.Conclusion The decrease and injures of ICCs-like cells in bladder may be one of the mechanisms resulting in urodynamic changes of diabetic cystopathy.

pH-mediated Regulation of Pacemaker Activity in Cultured Interstitial Cells of Cajal

Interstitial cells of Cajal (ICCs) are pacemakers in gastrointestinal tracts, regulating rhythmicity by activating nonselective cation channels (NSCCs). In the present study, we investigated the general characteristics and pH-mediated regulation of pacemaker activity in cultured interstitial cells of Cajal. Under voltage clamp mode and at the holding potential of -60 mV, the I-V relationships and difference current showed that there was no reversal potential and voltage-independent inward current. Also, when the holding potentials were changed from +20 mV to -80 mV with intervals of 20 mV, there was little difference in inward current. In pacemaker activity, the resting membrane potential (RMP) was depolarized (In pH 5.5, 23+/-1.5 mV depolarized) and the amplitude was decreased by a decrease of the extracellular pH. However, in case of increase of extracellular pH, the RMP was slightly hyperpolarized and the amplitude was decreased a little. The melastatin type transient receptor potential (TRPM) channel 7 has been suggested to be required for intestinal pacemaking activity. TRPM7 produced large outward currents and small inward currents by voltage ramps, ranging from +100 to -100 mV from a holding potential of -60 mV. The inward current of TRPM7 was dramatically increased by a decrease in the extracellular pH. At pH 4.0, the average inward current amplitude measured at -100 mV was increased by about 7 fold, compared with the current amplitude at pH 7.4. Changes in the outward current (measured at +100 mV) were much smaller than those of the inward current. These results indicate that the resting membrane potential of pacemaking activity might be depolarized by external acidic pH through TRPM7 that is required for intestinal pacemaking activity.

The Role of Mitochondrial ATP-sensitive Potassium Channel on Intestinal Pacemaking Activity

Interstitial cells of Cajal (ICCs) are the pacemaker cells that generate slow waves in the gastrointestinal (GI) tract. In the present study, we investigated the effect of mitochondrial ATP-sensitive potassium (mitoKATP) channel on pacemaking activity in cultured ICCs from murine small intestine by using whole-cell patch clamp techniques. Under current clamp mode, at 10microM glibenclamide, there was no change in pacemaking activity of ICCs. At 30microM glibenclamide, an inhibitor of the ATP sensitive K+ channels, we could find two examples. If pacemaking activity of ICCs was irregulating, pacemaking activity of ICCs was changed into regulating and if in normal conditions, membrane potential amplitude was increased. At 50microM glibenclamide, the resting membrane potential was depolarized. At 3mM 5-HDA, an inhibitor of the mitoKATP channels, inhibited the pacemaking activity of ICCs. Both the amplitude and the frequency were decreased. At 5 mM 5-HDA, both the amplitude and the frequency were completely abolished. Diazoxide, an opener of the mitoKATP channels, was applied to examine its effect on pacemaking activity of ICCs. At 50microM concentration, the pacemaking activity of ICCs was inhibited. Both the amplitude and the frequency were decreased. At 1 mM concentration, both the amplitude and the frequency were completely abolished and the resting membrane potential was shaked. These results indicate that mitoKATP channel has an important role in pacemaking activity of ICCs.

PGE2 Regulates Pacemaker Currents through EP2-Receptor in Cultured Interstitial Cells of Cajal from Murine Small Intestine

The interstitial cells of Cajal (ICCs) are the pacemaker cells in gastrointestinal tract and generate electrical rhythmicity in gastrointestinal muscles. Therefore, ICC may be modulated by endogenous agents such as neurotransmitter, hormones, and prostaglandins (PGs). In the present study, we investigated the effects of prostaglandins, especially PGE2, on pacemaker currents in cultured ICCs from murine small intestine by using whole-cell patch clamp techniques. ICCs generated spontaneous slow waves under voltage-clamp conditions and showed a mean amplitude of -452+/-39 pA and frequency of 18+/-2 cycles/min (n=6). Treatments of the cells with PGE2 (1muM) decreased both the frequency and amplitude of the pacemaker currents and increased the resting currents in the outward direction. PGE2 had only inhibitory effects on pacemaker currents and this inhibitory effect was dose-dependent. For characterization of specific membrane EP receptor subtypes, involved in the effects of PGE2 on pacemaker currents in ICCs, EP receptor agonists were used: Butaprost (1muM), EP2 receptor agonist, reduced the spontaneous inward current frequency and amplitude in cultured ICCs (n=5). However sulprostone (1muM), a mixed EP1 and EP3 agonist, had no effects on the frequency, amplitude and resting currents of pacemaker currents (n=5). SQ-22536 (an inhibitor of adenylate cyclase; 100muM) and ODQ (an inhibitor of guanylate cyclase; 100muM) had no effects on PGE2 actions of pacemaker currents. These observations indicate that PGE2 alter directly the pacemaker currents in ICCs, and that the PGE2 receptor subtypes involved are the EP2 receptor, independent of cyclic AMP- and GMP-dependent pathway.