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In this study we determined whether or not 5-hydroxytryptamine (5-HT) has an effect on the pacemaker activities of interstitial cells of Cajal (ICC) from the mouse small intestine. The actions of 5-HT on pacemaker activities were investigated using a whole-cell patch-clamp technique, intracellular Ca2+ ([Ca2+]i) analysis, and RT-PCR in ICC. Exogenously-treated 5-HT showed tonic inward currents on pacemaker currents in ICC under the voltage-clamp mode in a dose-dependent manner. Based on RT-PCR results, we found the existence of 5-HT2B, 3, 4, and 7 receptors in ICC. However, SDZ 205557 (a 5-HT4 receptor antagonist), SB 269970 (a 5-HT7 receptor antagonist), 3-tropanylindole - 3 - carboxylate methiodide (3-TCM; a 5-HT3 antagonist) blocked the 5-HT-induced action on pacemaker activity, but not SB 204741 (a 5-HT2B receptor antagonist). Based on [Ca2+]i analysis, we found that 5-HT increased the intensity of [Ca2+]i. The treatment of PD 98059 or JNK II inhibitor blocked the 5-HT-induced action on pacemaker activity of ICC, but not SB 203580. In summary, these results suggest that 5-HT can modulate pacemaker activity through 5-HT3, 4, and 7 receptors via [Ca2+]i mobilization and regulation of mitogen-activated protein kinases. [ABSTRACT FROM AUTHOR]

Hydrogen peroxide (H2O2) is involved in intestinal motility through changes of smooth muscle activity. However, there is no report as to the modulatory effects of H2O2 on interstitial cells of Cajal (ICC). We investigated the H2O2 effects and signal transductions to determine whether the intestinal motility can be modulated through ICC. We performed whole-cell patch clamp in cultured ICC from murine intestine and molecular analyses. H2O2 hyperpolarized the membrane and inhibited pacemaker currents. These effects were inhibited by glibenclamide, an inhibitor of ATP-sensitive K+ (KATP) channels. The free-radical scavenger catalase inhibited the H2O2-induced effects. MAFP and AACOCF3 (a cytosolic phospholipase A2 inhibitors) or SC-560 and NS-398 (a selective COX-1 and 2 inhibitor) or AH6809 (an EP2 receptor antagonist) inhibited the H2O2-induced effects. PD98059 (a mitogen activated/ERK-activating protein kinase inhibitor) inhibited the H2O2-induced effects, though SB-203580 (a p38 MAPK inhibitor) or a JNK inhibitor did not affect. H2O2-induced effects could not be inhibited by LY-294002 (an inhibitor of PI3-kinases), calphostin C (a protein kinase C inhibitor) or SQ-22536 (an adenylate cyclase inhibitor). Adenoviral infection analysis revealed H2O2 stimulated tyrosine kinase activity and AG 1478 (an antagonist of epidermal growth factor receptor tyrosine kinase) inhibited the H2O2-induced effects. These results suggest H2O2 can modulate ICC pacemaker activity and this occur by the activation of KATP channels through PGE2 production via receptor tyrosine kinase-dependent MAP kinase activation. [ABSTRACT FROM AUTHOR]

The interstitial cells of Cajal (ICC) are pacemaker cells in gastrointestinal tract and generate an electrical rhythm in gastrointestinal muscles. We investigated the possibility that PGE2 might affect the electrical properties of cultured ICC by activating ATPdependent K+ channels and, the EP receptor subtypes and the subunits of ATP-dependent K+ channels involved in these activities were identified. In addition, the regulation of intracellular Ca2+ ([Ca2+]i) mobilization may be involved the action of PGE2 on ICC. Treatments of ICC with PGE2 inhibited electrical pacemaker activities in the same manner as pinacidil, an ATPdependent K+ channel opener and PGE2 had only a dose-dependent effect. Using RT-PCR technique, we found that ATP-dependent K+ channels exist in ICC and that these are composed of Kir 6.2 and SUR 2B subunits. To characterize the specific membrane EP receptor subtypes in ICC, EP receptor agonists and RT-PCR were used: Butaprost (an EP2 receptor agonist) showed the actions on pacemaker currents in the same manner as PGE2. However sulprostone (a mixed EP1 and EP3 agonist) had no effects. In addition, RT-PCR results indicated the presence of the EP2 receptor in ICC. To investigate cAMP involvement in the effects of PGE2 on ICCs, SQ-22536 (an inhibitor of adenylate cyclase) and cAMP assays were used. SQ-22536 did not affect the effect of PGE2 on pacemaker currents, and PGE2 did not stimulate cAMP production. Also, we found PGE2 inhibited the spontaneous [Ca2+]i oscillations in cultured ICC. These observations indicate that PGE2 alters pacemaker currents by activating the ATP-dependent K+ channels comprised of Kir 6.2-SUR 2B in ICC and this action of PGE2 are through EP2 receptor subtype and also the activation of ATP-dependent K+ channels involves intracellular Ca2+ mobilization. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

We studied the modulation of pacemaker activities by bradykinin in cultured interstitial cells of Cajal (ICC) from murine small intestine with the whole-cell patch-clamp technique. Externally applied bradykinin produced membrane depolarization in the current-clamp mode and increased tonic inward pacemaker currents in the voltage-clamp mode.Pretreatment with bradykinin B1 antagonist did not block the bradykinin-induced effects on pacemaker currents. However, pretreatment with bradykinin B2 antagonist selectively blocked the bradykinin-induced effects. Also, only externally applied selective bradykinin B2 receptor agonist produced tonic inward pacemaker currents and ICC revealed a colocalization of the bradykinin B2 receptor and c-kit immunoreactivities, but bradykinin B1 receptors did not localize in ICC.External Na+-free solution abolished the generation of pacemaker currents and inhibited the bradykinin-induced tonic inward current. However, a Cl− channel blocker (DIDS) did not block the bradykinin-induced tonic inward current.The pretreatment with Ca2+-free solution and thapsigargin, a Ca2+-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker currents and suppressed the bradykinin-induced action.Chelerythrine and calphostin C, protein kinase C inhibitors or naproxen, an inhibitor of cyclooxygenase, did not block the bradykinin-induced effects on pacemaker currents.These results suggest that bradykinin modulates the pacemaker activities through bradykinin B2 receptor activation in ICC by external Ca2+ influx and internal Ca2+ release via protein kinase C- or cyclooxygenase-independent mechanism. Therefore, the ICC are targets for bradykinin and their interaction can affect intestinal motility.British Journal of Pharmacology (2006) 148, 918–926. doi:10.1038/sj.bjp.0706806; published online 19 June 2006 [ABSTRACT FROM AUTHOR]

Classical transient receptor potential channels (TRPCs) are thought to be candidates for the nonselective cation channels (NSCCs) involved in pacemaker activity and its neuromodulation in murine stomach smooth muscle. We aimed to determine the role of TRPC4 in the formation of NSCCs and in the generation of slow waves. At a holding potential of -60 mV, 50 μM carbachol (CCh) induced INSCC of amplitude [500.8 ± 161.8 pA (n = 8)] at -60 mV in mouse gastric smooth muscle cells. We investigated the effects of commercially available antibodies to TRPC4 on recombinant TRPC4 expressed in HEK cells and CCh-induced NSCCs in gastric smooth muscle cells. TRPC4 currents in HEK cells were reduced from 1525.6 ± 414.4 pA (n = 8) to 146.4 ± 83.3 pA (n = 10) by anti-TRPC4 antibody and INSCC amplitudes were reduced from 230.9 ± 36.3 pA (n = 15) to 49.8 ± 11.8 pA (n = 9). Furthermore, INSCC in the gastric smooth muscle cells of TRPC4 knockout mice was only 34.4 ± 10.4 pA (n = 8) at -60 mV. However, slow waves were still present in the knockout mice. Our data suggest that TRPC4 is an essential component of the NSCC activated by muscarinic stimulation in the murine stomach. [ABSTRACT FROM AUTHOR]

1: Interstitial cells of Cajal (ICCs) are pacemaker cells that activate the periodic spontaneous inward currents (pacemaker currents) responsible for the production of slow waves in gastrointestinal smooth muscle. The effects of noradrenaline on the pacemaker currents in cultured ICCs from murine small intestine were investigated by using whole-cell patch-clamp techniques at 30°C. 2: Under current clamping, ICCs had a mean resting membrane potential of -58±5?mV and produced electrical slow waves. Under voltage clamping, ICCs produced pacemaker currents with a mean amplitude of -410±57?pA and a mean frequency of 16±2?cycles?min-1. 3: Under voltage clamping, noradrenaline inhibited the amplitude and frequency of pacemaker currents and increased resting currents in the outward direction in a dose-dependent manner. These effects were reduced by intracellular GDPßS. 4: Noradrenaline-induced effects were blocked by propranolol (ß-adrenoceptor antagonist). However, neither prazosin (a1-adrenoceptor antagonist) nor yohimbine (a2-adrenoceptor antagonist) blocked the noradrenaline-induced effects. Phenylephrine (a1-adrenoceptor agonist) had no effect on the pacemaker currents, whereas isoprenaline (ß-adrenoceptor agonist) mimicked the effect of noradrenaline. Atenolol (ß1-adrenoceptor antagonist) blocked the noradrenaline-induced effects, but butoxamine (ß2-adrenoceptor antagonist) did not. In addition, BRL37344 (ß3-adrenoceptor agonist) had no effect on pacemaker currents. 5: 9-(Tetrahydro-2-furanyl)-9H-purine-6-amine (SQ-22536; adenylate cyclase inhibitor) and a myristoylated protein kinase A inhibitor did not inhibit the noradrenaline-induced effects and 8-bromo-cAMP had no effects on pacemaker currents. 8-Bromo-cGMP and SNAP inhibited pacemaker currents and these effects of SNAP were blocked by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; a guanylate cyclase inhibitor). However, ODQ did not block the noradrenaline-induced effects. 6: Neither tetraethylammonium (a voltage-dependent K+ channel blocker), apamin (a Ca2+-dependent K+ channel blocker) nor glibenclamide (an ATP-sensitive K+ channel blocker) blocked the noradrenaline-induced effects. 7: The results suggest that noradrenaline-induced stimulation of ß1-adrenoceptors in the ICCs inhibits pacemaker currents, and that this is mediated by the activation of G-protein. Neither adenylate cyclase, guanylate cyclase nor a K+ channel-dependent pathway are involved in this effect of noradrenaline.British Journal of Pharmacology (2004) 141, 670-677. doi:10.1038/sj.bjp.0705665 [ABSTRACT FROM AUTHOR]

Studies the regulation of ATP-sensitive K[sup +] (K[sub ATP]) currents in murine colonic myocytes with patch-clamp techniques. Measurement of the open probability of K[sub ATP] channels in cell-attached patches; Effects of PBDBu on the pinacidil-activated current in nondialyzed cells.

In this article, the author comments on a research conducted by W.W. Chan and H. Mashimo to investigate the effects of lubiprostone on intestinal smooth muscle contractions and pyloric sphincter tones. The author mentions that as per the study, lubiprostone has a role as a prokinetic agent in gastrointestinal (GI) tract motility but the underlying mechanisms of modulating contractions were not evaluated in the study.

Apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1) dysregulation has been identified in several human tumors and in patients with a variety of neurodegenerative diseases. However, the function of Ape1/Ref-1 is unclear. We show here that Ape1/Ref-1 increases the expression of glial cell-derived neurotropic factor (GDNF) receptor α1 (GFRα1), a key receptor for GDNF. Expression of Ape1/Ref-1 led to an increase in the GDNF responsiveness in human fibroblast. Ape1/Ref-1 induced GFRα1 transcription through enhanced binding of NF-κ complexes to the GFRα1 promoter. GFRα1 levels correlate proportionally with Ape1/Ref-1 in cancer cells. The knockdown of endogenous Ape1/Ref-1 in pancreatic cancer cells markedly suppressed GFRα1 expression and invasion in response to GNDF, while overexpression of GFRα1 restored invasion. In neuronal cells, the Ape1/Ref-1-mediated increase in GDNF responsiveness not only stimulated neurite outgrowth but also protected the cells from β-amyloid peptide and oxidative stress. Our results show that Ape1/Ref-1 is a novel physiological regulator of GDNF responsiveness, and they also suggest that Ape1/Ref-1-induced GFRα1 expression may play important roles in pancreatic cancer progression and neuronal cell survival. [ABSTRACT FROM AUTHOR]

The effects of calcitonin gene-related peptide (CGRP) on pacemaker currents in cultured interstitial cells of Cajal (ICC) from the mouse small intestine were investigated using the whole-cell patch clamp technique at 30∘C. Under voltage clamping at a holding potential of -70 mV, CGRP decreased the amplitude and frequency of pacemaker currents and activated outward resting currents. These effects were blocked by intracellular GDPβS, a G-protein inhibitor and glibenclamide, a specific ATP-sensitive K+ channels blocker. During current clamping, CGRP hyperpolarized the membrane and this effect was antagonized by glibenclamide. Pretreatment with SQ-22536 (an adenylate cyclase inhibitor) or naproxen (a cyclooxygenase inhibitor) did not block the CGRP-induced effects, whereas pretreatment with ODQ (a guanylate cyclase inhibitor) or LNAME (an inhibitor of nitric oxide synthase) did. In conclusion, CGRP inhibits pacemaker currents in ICC by generating nitric oxide via G-protein activation and so activating ATP-sensitive K+ channels. Nitric oxide- and guanylate cyclase- dependent pathways are involved in these effects. [ABSTRACT FROM AUTHOR]