scholarly journals Activation of TRPA1 by luminal stimuli induces EP4-mediated anion secretion in human and rat colon

2012 ◽  
Vol 302 (7) ◽  
pp. G690-G701 ◽  
Author(s):  
Izumi Kaji ◽  
Yukiko Yasuoka ◽  
Shin-ichiro Karaki ◽  
Atsukazu Kuwahara
Keyword(s):  
Host Defense ◽  
Transient Receptor Potential ◽  
Defense Mechanism ◽  
Ussing Chamber ◽  
Pcr Analysis ◽  
Rat Colon ◽  
Receptor Subtype ◽  
Neural Pathways ◽  
Dependent Manner ◽  
Anion Secretion

In gastrointestinal (GI) physiology, anion and fluid secretion is an important function for host defense and is induced by changes in the luminal environment. The transient receptor potential A1 (TRPA1) channel is considered to be a chemosensor in several sensory tissues. Although the function of TRPA1 has been studied in GI motility, its contribution to the transepithelial ion transport system has rarely been discussed. In the present study, we investigated the secretory effect of the potential TRPA1 agonist allyl isothiocyanate (AITC) in rat and human colon using an Ussing chamber. The mucosal application of AITC (10−6-10−3 M) induced Cl− and HCO3− secretion in a concentration-dependent manner, whereas the serosal application induced a significantly weaker effect. AITC-evoked anion secretion was attenuated by tissue pretreatment with piroxicam and prostaglandin (PG) E2; however, this secretion was not affected by TTX, atropine, or extracellular Ca2+ depletion. These experiments indicate that TRPA1 activation induces anion secretion through PG synthesis, independent of neural pathways in the colon. Further analysis also indicates that AITC-evoked anion secretion is mediated mainly by the EP4 receptor subtype. The magnitude of the secretory response exhibited segmental heterogeneity in rat colon. Real-time PCR analysis showed the segmental difference was corresponding to the differential expression of EP4 receptor and cyclooxygenase-1 and -2. In addition, RT-PCR, in situ hybridization, and immunohistochemical studies showed TRPA1 expression in the colonic epithelia. Therefore, we conclude that the activation of TRPA1 in colonic epithelial cells is likely involved in the host defense mechanism through rapid anion secretion.

Effects of luminal thymol on epithelial transport in human and rat colon

2011 ◽  
Vol 300 (6) ◽  
pp. G1132-G1143 ◽  
Author(s):  
Izumi Kaji ◽  
Shin-ichiro Karaki ◽  
Atsukazu Kuwahara
Keyword(s):  
Ion Transport ◽  
Transient Receptor Potential ◽  
Epithelial Transport ◽  
Ussing Chamber ◽  
Rat Colon ◽  
Small Intestinal Mucosa ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Anion Secretion ◽  
Serotonin Secretion

Gut lumen is continually exposed to a great variety of agents, including noxious compounds. Chemical receptors that detect the luminal environment are thought to play an important role as sensors and to modulate gastrointestinal functions. Recently, it has been reported that odorant receptors (ORs) are expressed in the small intestinal mucosa and that odorants stimulate serotonin secretion. However, ion transport in the responses to odorants has rarely been discussed, particularly in relation to the large intestine. In the present study, we examined the effects of the OR ligand thymol on ion transport in human and rat colonic epithelia using an Ussing chamber. In the mucosal-submucosal preparations, the mucosal addition of thymol evoked anion secretion concentration dependently. In addition, dextran (4 kDa) permeability was enhanced by the mucosal treatment with thymol. The response to thymol was not affected by tetrodotoxin (TTX) or piroxicam treatments in human or rat colon. Thymol-evoked electrogenic anion secretion was abolished under Ca2+-free conditions or mucosal treatment with transient receptor potential (TRP) A1 blocker (HC-030031). Pretreatment of thymol did not affect electrical field stimulation-evoked anion secretion but significantly attenuated short-chain fatty acid-evoked secretion in a concentration-dependent manner. OR1G1 and TRPA1 expression was investigated in isolated colonic mucosa by RT-PCR. The present results provide evidence that the OR ligand thymol modulates epithelial permeability and electrogenic anion secretion in human and rat colon. The anion secretion by luminal thymol is most likely mediated by direct activation of TRPA1 channel. We suggest that the sensing and responding to odorants in the colon also plays a role in maintaining intestinal homeostasis.

Evidence for age-associated reduction in acetylcholine release and smooth muscle response in the rat colon

1994 ◽  
Vol 267 (4) ◽  
pp. G515-G522 ◽  
Author(s):  
D. Roberts ◽  
D. Gelperin ◽  
J. W. Wiley
Keyword(s):  
Calcium Channels ◽  
Acetylcholine Release ◽  
Calcium Influx ◽  
Muscle Tension ◽  
Calcium Ionophore ◽  
Rat Colon ◽  
Neural Pathways ◽  
Dependent Manner ◽  
Ach Release ◽  
Age Dependent

The effect of aging was examined on cholinergically mediated contractions and acetylcholine (ACh) release in isolated colonic segments from Fischer (F344 x BN) F1 rats, 4-8 mo (postpubertal) and 22-28 mo (senescent) of age. This species demonstrates age-dependent slowing of colonic transit. Muscle tension response to electrical stimulation of cholinergic neural pathways and application of ACh was significantly decreased in preparations from senescent compared with postpubertal animals. We focused on the hypothesis that aging was associated with reduced ACh release that resulted from decreased calcium influx through membrane calcium channels. Aging did not affect either the synthesis of [3H]ACh from [3H]choline or the percentage of 3H released in the form of [3H]ACh. However, elevated KCl-evoked release of [3H]ACh was significantly reduced in tissue from senescent compared with postpubertal animals. Treatment with the calcium ionophore ionomycin increased [3H]ACh release in tissue from senescent animals to near postpubertal levels. However, increasing extracellular calcium concentration ([Ca2+]o) from 1.2 to 5 mM did not increase the amount of transmitter release in tissue from senescent animals to the levels observed with 1.2 mM [Ca2+]o in postpubertal tissue. The neuronal calcium channel antagonist omega-conotoxin GVIA inhibited acetylcholine release in a concentration-dependent manner with half-maximal inhibitory values of 1.8 and 8.2 nM for senescent and postpubertal preparations, respectively. In summary, age-dependent reduction in ACh release was observed in the rat colon myenteric plexus that may, in part, be associated with decreased calcium influx via membrane calcium channels.

scholarly journals Involvement of Sphingosine 1-Phosphate in Palmitate-Induced Non-Alcoholic Fatty Liver Disease

2016 ◽  
Vol 40 (6) ◽  
pp. 1637-1645 ◽  
Author(s):  
Frdoos Al Fadel ◽  
Susann Fayyaz ◽  
Lukasz Japtok ◽  
Burkhard Kleuser
Keyword(s):  
Liver Fibrosis ◽  
Smooth Muscle Actin ◽  
Sphingolipid Metabolism ◽  
Pcr Analysis ◽  
Receptor Subtype ◽  
Dependent Manner ◽  
Alcoholic Fatty Liver ◽  
S1p Receptors ◽  
Sphingosine 1 Phosphate ◽  
Bioactive Secondary Metabolite

Background/Aims: Ectopic lipid accumulation in hepatocytes has been identified as a risk factor for the progression of liver fibrosis and is strongly associated with obesity. In particular, the saturated fatty acid palmitate is involved in initiation of liver fibrosis via formation of secondary metabolites by hepatocytes that in turn activate hepatic stellate cells (HSCs) in a paracrine manner. Methods: α-smooth muscle actin-expression (α-SMA) as a marker of liver fibrosis was investigated via western blot analysis and immunofluorescence microscopy in HSCs (LX-2). Sphingolipid metabolism and the generation of the bioactive secondary metabolite sphingosine 1-phosphate (S1P) in response to palmitate were analyzed by LC-MS/MS in hepatocytes (HepG2). To identify the molecular mechanism involved in the progression of liver fibrosis real-time PCR analysis and pharmacological modulation of S1P receptors were performed. Results: Palmitate oversupply increased intra- and extracellular S1P-concentrations in hepatocytes. Conditioned medium from HepG2 cells initiated fibrosis by enhancing α-SMA-expression in LX-2 in a S1P-dependent manner. In accordance, fibrotic response in the presence of S1P was also observed in HSCs. Pharmacological inhibition of S1P receptors demonstrated that S1P3 is the crucial receptor subtype involved in this process. Conclusion: S1P is synthesized in hepatocytes in response to palmitate and released into the extracellular environment leading to an activation of HSCs via the S1P3 receptor.

scholarly journals Involvement of TRP channels in the CO2 chemosensitivity of locus coeruleus neurons

2011 ◽  
Vol 105 (6) ◽  
pp. 2791-2801 ◽  
Author(s):  
Ningren Cui ◽  
Xiaoli Zhang ◽  
Jyothirmayee S. Tadepalli ◽  
Lei Yu ◽  
Hongyu Gai ◽  
...  
Keyword(s):  
Locus Coeruleus ◽  
Transient Receptor Potential ◽  
Trp Channels ◽  
Brain Slices ◽  
Receptor Potential ◽  
Input Resistance ◽  
Pcr Analysis ◽  
Dependent Manner ◽  
Intracellular Acidosis ◽  
Cationic Channels

Catecholaminergic neurons in the locus coeruleus (LC) play a role in the ventilatory response to hypercapnia. Here, we show evidence for the involvement of transient receptor potential (TRP) channels. We found that the input resistance was reduced during an exposure to 8% CO2 in ∼35% LC neurons in mouse brain slices, accompanied by depolarization and higher firing activity. The neuronal responses suggest the opening of Na+ or nonselective cationic channels instead of the closure of K+ channels. As a major group of cationic channels, the TRP channels are expressed in the brain, some of which are activated by acidic pH. We therefore screened all representative TRP channels using the quantitative real-time PCR analysis. High levels of mRNA expression of TRPC5, TRPM2, and TRPM7 were found in the LC tissue. Of them, the TRPC5 transcript was the most abundant. The TRPC5 channel was activated by extracellular acidification when expressed in human embryonic kidney (HEK) cells. The TRPC5 currents started to be activated at pH 7.4 with pKa 6.9. The TRPC5 currents were also activated by isohydric hypercapnic and intracellular acidosis in a Ca2+-dependent manner. Consistently, the LC neurons were stimulated by both extra- and intracellular acidosis. The stimulatory effect of hypercapnia on LC neurons was eliminated by selective TRPC inhibitor SKF-96365 with and without the blockade of synaptic transmission. Single-cell PCR analysis indicated that TRPC5 mRNAs existed in the LC neurons. Thus these results strongly suggest that the TRP channels are likely to play a role in the CO2 chemosensitivity of LC neurons, especially TRPC5.

scholarly journals Cholinergic-induced anion secretion in murine jejunal enteroids involves synergy between muscarinic and nicotinic pathways

2020 ◽  
Vol 319 (2) ◽  
pp. C321-C330
Author(s):  
Kelli Johnson ◽  
Jianyi Yin ◽  
Julie G. In ◽  
Subhash Kulkarni ◽  
Pankaj Pasricha ◽  
...  
Keyword(s):  
Nicotinic Receptors ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Intestinal Transport ◽  
Short Circuit Current ◽  
Muscarinic Agonist ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Anion Secretion ◽  
Qrt Pcr

Acetylcholine induces robust electrogenic anion secretion in mammalian intestine and it has long been hypothesized that it mediates the epithelial response through the M3 and, to a lesser extent, the M1 muscarinic receptors in the mouse. However, nicotinic receptors have recently been identified in intestinal enterocytes by quantitative real-time (qRT)-PCR/RNAseq, although any direct influence on intestinal transport has not been identified. We tested the hypothesis that cholinergic-induced anion secretion in the intestine is a result of both muscarinic and nicotinic pathways that are intrinsic to the intestinal epithelia. We developed a method to generate mouse jejunal enteroid monolayers which were used to measure active electrogenic anion secretion by the Ussing chamber/voltage-clamp technique. Here, we show that the cholinergic agonist carbachol (CCh) and the muscarinic agonist bethanechol (BCh) stimulate short-lived, concentration-dependent anion secretion in the epithelial cell-only enteroid monolayers. The muscarinic antagonist atropine completely inhibited CCh- and BCh-induced secretion, while the nicotinic antagonist hexamethonium reduced the CCh response by ~45%. While nicotine alone did not alter anion secretion, it increased the BCh-induced increase in short-circuit current in a concentration-dependent manner; this synergy was prevented by pretreatment with hexamethonium. In addition to being sensitive to hexamethonium, monolayers express both classes of cholinergic receptor by qRT-PCR, including 13 of 16 nicotinic receptor subunits. Our findings indicate that an interaction between muscarinic and nicotinic agonists synergistically stimulates anion secretion in mouse jejunal epithelial cells and identify a role for epithelial nicotinic receptors in anion secretion.

Secretory effects of a luminal bitter tastant and expressions of bitter taste receptors, T2Rs, in the human and rat large intestine

2009 ◽  
Vol 296 (5) ◽  
pp. G971-G981 ◽  
Author(s):  
Izumi Kaji ◽  
Shin-ichiro Karaki ◽  
Yasuyuki Fukami ◽  
Masaki Terasaki ◽  
Atsukazu Kuwahara
Keyword(s):  
Large Intestine ◽  
Bitter Taste ◽  
Physiological Function ◽  
Ussing Chamber ◽  
Taste Receptor ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Gi Tract ◽  
Concentration Dependent Manner ◽  
Anion Secretion

Taste transduction molecules, such as Gαgust, and taste receptor families for bitter [taste receptor type 2 (T2R)], sweet, and umami, have previously been identified in taste buds and the gastrointestinal (GI) tract; however, their physiological functions in GI tissues are still unclear. Here, we investigated the physiological function and expression of T2R in human and rat large intestine using various physiological and molecular biological techniques. To study the physiological function of T2R, the effect of a bitter compound, 6- n-propyl-2-thiouracil (6-PTU), on transepithelial ion transport was investigated using the Ussing chamber technique. In mucosal-submucosal preparations, mucosal 6-PTU evoked Cl−and HCO3−secretions in a concentration-dependent manner. In rat middle colon, levels of 6-PTU-evoked anion secretion were higher than in distal colon, but there was no such difference in human large intestine. The response to 6-PTU was greatly reduced by piroxicam, but not by tetrodotoxin. Additionally, prostaglandin E2concentration-dependently potentiated the response to 6-PTU. Transcripts of multiple T2Rs (putative 6-PTU receptors) were detected in both human and rat colonic mucosa by RT-PCR. In conclusion, these results suggest that the T2R ligand, 6-PTU, evokes anion secretion, and such response is regulated by prostaglandins. This luminal bitter sensing mechanism may be important for host defense in the GI tract.

H. pyloriacutely inhibits gastric secretion by activating CGRP sensory neurons coupled to stimulation of somatostatin and inhibition of histamine secretion

2013 ◽  
Vol 304 (8) ◽  
pp. G715-G722 ◽  
Author(s):  
Muhammad Zaki ◽  
Philip E. Coudron ◽  
Robert W. McCuen ◽  
Leslie Harrington ◽  
Shijian Chu ◽  
...  
Keyword(s):  
Acid Secretion ◽  
Sensory Neurons ◽  
Cell Function ◽  
Ussing Chamber ◽  
Acute Administration ◽  
Neural Pathways ◽  
Dependent Manner ◽  
Histamine Secretion ◽  
H Pylori ◽  
Stimulation Of

Acute Helicobacter pylori infection produces hypochlorhydria. The decrease in acid facilitates survival of the bacterium and its colonization of the stomach. The present study was designed to identify the pathways in oxyntic mucosa by which acute H. pylori infection inhibits acid secretion. In rat fundic sheets in an Ussing chamber, perfusion of the luminal surface with H. pylori in spent broth (103–108cfu/ml) or spent broth alone (1:105to 1:100final dilution) caused a concentration-dependent increase in somatostatin (SST; maximal: 200 ± 20 and 194 ± 9% above basal; P < 0.001) and decrease in histamine secretion (maximal: 45 ± 5 and 48 ± 2% below basal; P < 0.001); the latter was abolished by SST antibody, implying that changes in histamine secretion reflected changes in SST secretion. Both responses were abolished by the axonal blocker tetrodotoxin (TTX), the sensory neurotoxin capsaicin, or the CGRP antagonist CGRP8-37, implying that the reciprocal changes in SST and histamine secretion were due to release of CGRP from sensory neurons. In isolated rabbit oxyntic glands, H. pylori inhibited basal and histamine-stimulated acid secretion in a concentration-dependent manner; the responses were not affected by TTX or SST antibody, implying that H. pylori can directly inhibit parietal cell function. In conclusion, acute administration of H. pylori is capable of inhibiting acid secretion directly as well as indirectly by activating intramural CGRP sensory neurons coupled to stimulation of SST and inhibition of histamine secretion. Activation of neural pathways provides one explanation as to how initial patchy colonization of the superficial gastric mucosa by H. pylori can acutely inhibit acid secretion.

scholarly journals Pyrrolizidine Alkaloids Induce Cell Death in Human HepaRG Cells in a Structure-Dependent Manner

2020 ◽  
Vol 22 (1) ◽  
pp. 202
Author(s):  
Josephin Glück ◽  
Julia Waizenegger ◽  
Albert Braeuning ◽  
Stefanie Hessel-Pras
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Pyrrolizidine Alkaloids ◽  
Defense Mechanism ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Dependent Manner ◽  
Human Hepatoma Cell

Pyrrolizidine alkaloids (PAs) are a group of secondary metabolites produced in various plant species as a defense mechanism against herbivores. PAs consist of a necine base, which is esterified with one or two necine acids. Humans are exposed to PAs by consumption of contaminated food. PA intoxication in humans causes acute and chronic hepatotoxicity. It is considered that enzymatic PA toxification in hepatocytes is structure-dependent. In this study, we aimed to elucidate the induction of PA-induced cell death associated with apoptosis activation. Therefore, 22 structurally different PAs were analyzed concerning the disturbance of cell viability in the metabolically competent human hepatoma cell line HepaRG. The chosen PAs represent the main necine base structures and the different esterification types. Open-chained and cyclic heliotridine- and retronecine-type diesters induced strong cytotoxic effects, while treatment of HepaRG with monoesters did not affect cell viability. For more detailed investigation of apoptosis induction, comprising caspase activation and gene expression analysis, 14 PA representatives were selected. The proapoptotic effects were in line with the potency observed in cell viability studies. In vitro data point towards a strong structure–activity relationship whose effectiveness needs to be investigated in vivo and can then be the basis for a structure-associated risk assessment.

scholarly journals Targeting a Lipid Desaturation Enzyme, SCD1, Selectively Eliminates Colon Cancer Stem Cells through the Suppression of Wnt and NOTCH Signaling

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 106
Author(s):  
Yeongji Yu ◽  
Hyejin Kim ◽  
SeokGyeong Choi ◽  
JinSuh Yu ◽  
Joo Yeon Lee ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Notch Signaling ◽  
Cultured Cells ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Dependent Manner ◽  
Lipid Desaturation ◽  
Novel Target

The elimination of the cancer stem cell (CSC) population may be required to achieve better outcomes of cancer therapy. We evaluated stearoyl-CoA desaturase 1 (SCD1) as a novel target for CSC-selective elimination in colon cancer. CSCs expressed more SCD1 than bulk cultured cells (BCCs), and blocking SCD1 expression or function revealed an essential role for SCD1 in the survival of CSCs, but not BCCs. The CSC potential selectively decreased after treatment with the SCD1 inhibitor in vitro and in vivo. The CSC-selective suppression was mediated through the induction of apoptosis. The mechanism leading to selective CSC death was investigated by performing a quantitative RT-PCR analysis of 14 CSC-specific signaling and marker genes after 24 and 48 h of treatment with two concentrations of an inhibitor. The decrease in the expression of Notch1 and AXIN2 preceded changes in the expression of all other genes, at 24 h of treatment in a dose-dependent manner, followed by the downregulation of most Wnt- and NOTCH-signaling genes. Collectively, we showed that not only Wnt but also NOTCH signaling is a primary target of suppression by SCD1 inhibition in CSCs, suggesting the possibility of targeting SCD1 against colon cancer in clinical settings.

scholarly journals Potentiation of TRPC5 by Protons

2007 ◽  
Vol 282 (46) ◽  
pp. 33868-33878 ◽  
Author(s):  
Marcus Semtner ◽  
Michael Schaefer ◽  
Olaf Pinkenburg ◽  
Tim D. Plant
Keyword(s):  
Phospholipase C ◽  
Transient Receptor Potential ◽  
Single Channel ◽  
Receptor Potential ◽  
High Sensitivity ◽  
Transient Receptor Potential Channel ◽  
Extracellular Ph ◽  
Dependent Manner ◽  
Open Probability ◽  
Transient Receptor

Mammalian members of the classical transient receptor potential channel subfamily (TRPC) are Ca2+-permeable cation channels involved in receptor-mediated increases in intracellular Ca2+. TRPC4 and TRPC5 form a group within the TRPC subfamily and are activated in a phospholipase C-dependent manner by an unidentified messenger. Unlike most other Ca2+-permeable channels, TRPC4 and -5 are potentiated by micromolar concentrations of La3+ and Gd3+. This effect results from an action of the cations at two glutamate residues accessible from the extracellular solution. Here, we show that TRPC4 and -5 respond to changes in extracellular pH. Lowering the pH increased both G protein-activated and spontaneous TRPC5 currents. Both effects were already observed with small reductions in pH (from 7.4 to 7.0) and increased up to pH 6.5. TRPC4 was also potentiated by decreases in pH, whereas TRPC6 was only inhibited, with a pIC50 of 5.7. Mutation of the glutamate residues responsible for lanthanoid sensitivity of TRPC5 (E543Q and E595Q) modified the potentiation of TRPC5 by acid. Further evidence for a similarity in the actions of lanthanoids and H+ on TRPC5 is the reduction in single channel conductance and dramatic increase in channel open probability in the presence of either H+ or Gd3+ that leads to larger integral currents. In conclusion, the high sensitivity of TRPC5 to H+ indicates that, in addition to regulation by phospholipase C and other factors, the channel may act as a sensor of pH that links decreases in extracellular pH to Ca2+ entry and depolarization.

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