Characteristics of the Cholecystokinin-Induced Depolarization of Pacemaking Activity in Cultured Interstitial Cells of Cajal from Murine Small Intestine

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Background: Quercetin regulates gastrointestinal (GI) motor activity but the molecular mechanism involved has not been determined. The authors investigated the effects of quercetin, a flavonoid present in various foods, on the pacemaker activities of interstitial cells of Cajal (ICCs) in murine small intestine in vitro and on GI motility in vivo. Materials and Methods: Enzymatic digestion was used to dissociate ICCs from mouse small intestines. The whole-cell patch-clamp configuration was used to record pacemaker potentials in cultured ICCs in the absence or presence of quercetin and to record membrane currents of transient receptor potential melastatin (TRPM) 7 or transmembrane protein 16A (Tmem16A, anoctamin1 (ANO1)) overexpressed in human embryonic kidney (HEK) 293 cells. The in vivo effects of quercetin on GI motility were investigated by measuring the intestinal transit rates (ITRs) of Evans blue in normal mice. Results: Quercetin (100-200 μM) decreased the amplitudes and frequencies of pacemaker activity in a concentration-dependent manner in current clamp mode, but this action was blocked by naloxone (a pan-opioid receptor antagonist) and by GDPβS (a GTP-binding protein inhibitor). However, potassium channels were not involved in these inhibitory effects of quercetin. To study the quercetin signaling pathway, we examined the effects of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of guanylate cyclase, and of RP-8-CPT-cGMPS, an inhibitor of protein kinase G (PKG). These inhibitors blocked the inhibitory effects of quercetin on pacemaker activities. Also, L-NAME (100 μM), a non-selective NO synthase (NOS) inhibitor, blocked the effects of quercetin on pacemaker activity and quercetin stimulated cGMP production. Furthermore, quercetin inhibited both Ca²⁺-activated Cl^- channels (TMEM16A, ANO1) and TRPM7 channels. In vivo, quercetin (10-100 mg/kg, p.o.) decreased ITRs in normal mice in a dose-dependent manner. Conclusions: Quercetin inhibited ICC pacemaker activities by inhibiting TRPM7 and ANO1 via opioid receptor signaling pathways in cultured murine ICCs. The study shows quercetin attenuates GI tract motility, and suggests quercetin be considered the basis for the development of novel spasmolytic agents for the prevention or alleviation of GI motility dysfunctions.

Section: Introductionmentioning

confidence: 99%

Quercetin Inhibits Pacemaker Potentials via Nitric Oxide/cGMP-Dependent Activation and TRPM7/ANO1 Channels in Cultured Interstitial Cells of Cajal from Mouse Small Intestine

Gim¹,

Nam

Lee³

et al. 2015

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“…Smooth muscle cells respond to slow wave depolarization by activating L-type Ca 2+ channels [46]. In addition, smooth muscle response is regulated by neural inputs, and both excitatory and inhibitory enteric motor neurons are closely associated with ICC [14]. Thus, ICC play an important role in the determination and regulation of GI motility.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, the loss of ICC is implicated in several motility disorders, which suggests that ICC play an important role in the regulation of GI motility [13]. In addition, evidence indicates that endogenous agents, such as, neurotransmitters, hormones, and paracrine substances, modulate GI tract motility by influencing ICC [14]. It has been shown that the pacemaker activities of ICC in the murine small intestine are mainly due to periodic activations of nonselective cation channels (NSCCs) [15,16] or Cl - channels [17,18,19,20,21,22].…”

Section: Introductionmentioning

confidence: 99%

Characteristics of Gintonin-Mediated Membrane Depolarization of Pacemaker Activity in Cultured Interstitial Cells of Cajal

Kim

Nam

Kim

et al. 2014

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696

Background/Aims: Ginseng regulates gastrointestinal (GI) motor activity but the underlying components and molecular mechanisms are unknown. We investigated the effect of gintonin, a novel ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, on the pacemaker activity of the interstitial cells of Cajal (ICC) in murine small intestine and GI motility. Materials and Methods: Enzymatic digestion was used to dissociate ICC from mouse small intestines. The whole-cell patch-clamp configuration was used to record pacemaker potentials and currents from cultured ICC in the absence or presence of gintonin. In vivo effects of gintonin on gastrointestinal (GI) motility were investigated by measuring the intestinal transit rate (ITR) of Evans blue in normal and streptozotocin (STZ)-induced diabetic mice. Results: We investigated the effects of gintonin on pacemaker potentials and currents in cultured ICC from mouse small intestine. Gintonin caused membrane depolarization in current clamp mode but this action was blocked by Ki16425, an LPA1/3 receptor antagonist, and by the addition of GDPβS, a GTP-binding protein inhibitor, into the ICC. To study the gintonin signaling pathway, we examined the effects of U-73122, an active PLC inhibitor, and chelerythrine and calphostin, which inhibit PKC. All inhibitors blocked gintonin actions on pacemaker potentials, but not completely. Gintonin-mediated depolarization was lower in Ca²⁺-free than in Ca²⁺-containing external solutions and was blocked by thapsigargin. We found that, in ICC, gintonin also activated Ca²⁺-activated Cl^- channels (TMEM16A, ANO1), but not TRPM7 channels. In vivo, gintonin (10-100 mg/kg, p.o.) not only significantly increased the ITR in normal mice but also ameliorated STZ-induced diabetic GI motility retardation in a dose-dependent manner. Conclusions: Gintonin-mediated membrane depolarization of pacemaker activity and ANO1 activation are coupled to the stimulation of GI contractility through LPA1/3 receptor signaling pathways in cultured murine ICC. Gintonin might be a ingredient responsible for ginseng-mediated GI tract modulations, and could be a novel candidate for development as a prokinetic agent that may prevent or alleviate GI motility dysfunctions in human patients.

“…Accordingly, it has been proposed that TRPM7 and ANO1 is responsible for the pacemaker activity in ICCs and may be considered the potential targets for the pharmacological treatment of GI motility disorders. The absence of ICCs in GI tract causes abnormally slow electrical waves and reduces smooth muscle cell contractility and intestinal transit [25] and ICCs also mediate or transduce inputs from the enteric nervous system [28]. Therefore, ICCs play an important role in the determination and regulation of GI motility.…”

Section: Introductionmentioning

confidence: 99%

Menthol Modulates Pacemaker Potentials through TRPA1 Channels in Cultured Interstitial Cells of Cajal from Murine Small Intestine

Kim

Wie

So³

et al. 2016

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Background/Aims: ICCs are the pacemaker cells responsible for slow waves in gastrointestinal (GI) smooth muscle, and generate periodic pacemaker potentials in current-clamp mode. Methods: The effects of menthol on the pacemaker potentials of cultured interstitial cells of Cajal (ICCs) from mouse small intestine were studied using the whole cell patch clamp technique. Results: Menthol (1 - 10 μM) was found to induce membrane potential depolarization in a concentration-dependent manner. The effects of various TRP channel antagonists were examined to investigate the receptors involved. The addition of the TRPM8 antagonist, AMTB, did not block menthol-induced membrane potential depolarizations, but TRPA1 antagonists (A967079 or HC-030031) blocked the effects of menthol, as did intracellular GDP_βS. Furthermore, external and internal Ca²⁺ levels were found to depolarize menthol-induced membrane potentials, whereas external Na⁺ was not. Y-27632 (a Rho kinase inhibitor), SC-560 (a selective COX 1 inhibitor), NS-398 (a selective COX 2 inhibitor), ozagrel (a thromboxane A2 synthase inhibitor) and SQ-29548 (highly selective thromboxane receptor antagonist) were used to investigate the involvements of Rho-kinase, cyclooxygenase (COX), and the thromboxane pathway in menthol-induced membrane potential depolarizations, and all inhibitors were found to block the effect of menthol. Conclusions: These results suggest that menthol-induced membrane potential depolarizations occur in a G-protein-, Ca²⁺-, Rho-kinase-, COX-, and thromboxane A₂-dependent manner via TRPA1 receptor in cultured ICCs in murine small intestine. The study shows ICCs are targeted by menthol and that this interaction can affect intestinal motility.