Tin protoporphyrin induces intestinal chloride secretion by inducing light oxidation processes

2007 ◽  
Vol 292 (5) ◽  
pp. C1906-C1914 ◽  
Author(s):  
Aliye Uc ◽  
Krzysztof J. Reszka ◽  
Garry R. Buettner ◽  
John B. Stokes
Keyword(s):  
Short Circuit ◽  
Specific Inhibitor ◽  
Chloride Secretion ◽  
Epithelial Cell Line ◽  
Intestinal Epithelial ◽  
Paramagnetic Resonance ◽  
Ussing Chambers ◽  
Oxidative Processes ◽  
Tin Protoporphyrin ◽  
Intestinal Chloride Secretion

Heme induces Cl− secretion in intestinal epithelial cells, most likely via carbon monoxide (CO) generation. The major source of endogenous CO comes from the degradation of heme via heme oxygenase (HO). We hypothesized that an inhibitor of HO activity, tin protoporphyrin (SnPP), may inhibit the stimulatory effect of heme on Cl− secretion. To test this hypothesis, we treated an intestinal epithelial cell line (Caco-2 cells) with SnPP. In contrast to our expectations, Caco-2 cells treated with SnPP had an increase in their short-circuit currents ( Isc) in Ussing chambers. This effect was observed only when the system was exposed to ambient light. SnPP-induced Isc was caused by Cl− secretion because it was inhibited in Cl−-free medium, with ouabain or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). The Cl− secretion was not via activation of the CFTR, because a specific inhibitor had no effect. Likewise, inhibitors of adenylate cyclase and guanylate cyclase had no effect on the enhanced Isc. SnPP-induced Isc was inhibited by the antioxidant vitamins, α-tocopherol and ascorbic acid. Electron paramagnetic resonance experiments confirmed that oxidative reactions were initiated with light in cells loaded with SnPP. These data suggest that SnPP-induced effects may not be entirely due to the inhibition of HO activity but rather to light-induced oxidative processes. These novel effects of SnPP-photosensitized oxidation may also lead to a new understanding of how intestinal Cl− secretion can be regulated by the redox environment of the cell.

scholarly journals Apical leptin induces chloride secretion by intestinal epithelial cells and in a rat model of acute chemotherapy-induced colitis

2010 ◽  
Vol 298 (5) ◽  
pp. G714-G721 ◽  
Author(s):  
Raschid M. Hoda ◽  
Michael Scharl ◽  
Stephen J. Keely ◽  
Declan F. McCole ◽  
Kim E. Barrett
Keyword(s):  
Epithelial Cells ◽  
Rat Model ◽  
Intestinal Inflammation ◽  
Intestinal Epithelial Cells ◽  
Short Circuit ◽  
Chloride Secretion ◽  
Secretory Diarrhea ◽  
Intestinal Epithelial ◽  
Sprague Dawley ◽  
Ussing Chambers

The purpose of this study was to investigate whether luminal leptin alters ion transport properties of the intestinal epithelium under acute inflammatory conditions. Monolayers of human intestinal T84 epithelial cells and a rat model of chemotherapy-induced enterocolitis were used. Cells were treated with leptin and mounted in Ussing chambers to measure basal and secretagogue-induced changes in transepithelial short-circuit current ( Isc). Furthermore, the role of MAPK and phosphatidylinositol 3-kinase (PI3K) signaling pathways in mediating responses to leptin was investigated. Acute colitis in Sprague-Dawley rats was induced by intraperitoneal injection of 40 mg/kg methotrexate. Leptin (100 ng/ml) induced a time-dependent increase in basal Isc in T84 intestinal epithelial cells ( P < 0.01). Moreover, pretreatment of T84 cells with leptin for up to 1 h significantly potentiated carbachol- and forskolin-induced increases in Isc. Pretreatment with an inhibitor of MAPK abolished the effect of leptin on basal, carbachol- and forskolin-induced chloride secretion ( P < 0.05). However, the PI3K inhibitor, wortmannin, only blunted the effect of leptin on forskolin-induced increases in Isc. Furthermore, leptin treatment evoked both ERK1/2 and Akt1 phosphorylation in T84 cells. In the rat model, luminal leptin induced significant increases in Isc across segments of proximal and, to a lesser extent, distal colon ( P < 0.05). We conclude that luminal leptin is likely an intestinal chloride secretagogue, particularly when present at elevated concentrations and/or in the setting of inflammation. Our findings may provide a mechanistic explanation, at least in part, for the clinical condition of secretory diarrhea both in hyperleptinemic obese patients and in patients with chemotherapy-induced intestinal inflammation.

Protease-activated receptor-2 stimulates intestinal epithelial chloride transport through activation of PLC and selective PKC isoforms

2009 ◽  
Vol 296 (6) ◽  
pp. G1258-G1266 ◽  
Author(s):  
Jacques Q. van der Merwe ◽  
France Moreau ◽  
Wallace K. MacNaughton
Keyword(s):  
Serine Proteases ◽  
Chloride Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Immunoblot Analysis ◽  
Chloride Secretion ◽  
Intestinal Epithelial ◽  
Ussing Chambers ◽  
Pkc Isoforms ◽  
Gf 109203X

Serine proteases play important physiological roles through their activity at G protein-coupled protease-activated receptors (PARs). We examined the roles that specific phospholipase (PL) C and protein kinase (PK) C (PKC) isoforms play in the regulation of PAR2-stimulated chloride secretion in intestinal epithelial cells. Confluent SCBN epithelial monolayers were grown on Snapwell supports and mounted in modified Ussing chambers. Short-circuit current ( Isc) responses to basolateral application of the selective PAR2 activating peptide, SLIGRL-NH2, were monitored as a measure of net electrogenic ion transport caused by PAR2 activation. SLIGRL-NH2 induced a transient Isc response that was significantly reduced by inhibitors of PLC (U73122), phosphoinositol-PLC (ET-18), phosphatidylcholine-PLC (D609), and phosphatidylinositol 3-kinase (PI3K; LY294002). Immunoblot analysis revealed the phosphorylation of both PLCβ and PLCγ following PAR2 activation. Pretreatment of the cells with inhibitors of PKC (GF 109203X), PKCα/βI (Gö6976), and PKCδ (rottlerin), but not PKCζ (selective pseudosubstrate inhibitor), also attenuated this response. Cellular fractionation and immunoblot analysis, as well as confocal immunocytochemistry, revealed increases of PKCβI, PKCδ, and PKCε, but not PKCα or PKCζ, in membrane fractions following PAR2 activation. Pretreatment of the cells with U73122, ET-18, or D609 inhibited PKC activation. Inhibition of PI3K activity only prevented PKCδ translocation. Immunoblots revealed that PAR2 activation induced phosphorylation of both cRaf and ERK1/2 via PKCδ. Inhibition of PKCβI and PI3K had only a partial effect on this response. We conclude that basolateral PAR2-induced chloride secretion involves activation of PKCβI and PKCδ via a PLC-dependent mechanism resulting in the stimulation of cRaf and ERK1/2 signaling.

scholarly journals Interferon-γ inhibits sirtuin 6 gene expression in intestinal epithelial cells through a microRNA-92b-dependent mechanism

2020 ◽  
Vol 318 (4) ◽  
pp. C732-C739
Author(s):  
Fangyi Liu ◽  
Xiao Wang ◽  
Hua Geng ◽  
Heng-Fu Bu ◽  
Peng Wang ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Protein Expression ◽  
Intestinal Epithelial Cells ◽  
Specific Inhibitor ◽  
In Silico Analysis ◽  
Interferon Γ ◽  
Luciferase Assay ◽  
Epithelial Cell Line ◽  
Dependent Manner ◽  
Intestinal Epithelial

Sirtuin 6 (Sirt6) is predominantly expressed in epithelial cells in intestinal crypts. It plays an important role in protecting intestinal epithelial cells against inflammatory injury. Previously, we found that colitis is associated with the downregulation of Sirt6 protein in the intestines. Here, we report that murine interferon-γ (Ifnγ) inhibits Sirt6 protein but not mRNA expression in young adult mouse colonocytes (YAMC, a mouse colonic epithelial cell line) in a dose- and time-dependent manner. Using microRNA array analysis, we showed that Ifnγ induces expression of miR-92b in YAMC cells. With in silico analysis, we found that the Sirt6 3′-untranslated region (UTR) contains a putative binding site for miR-92b. Luciferase assay showed that Ifnγ inhibited Sirt6 3′-UTR activity and this effect was mimicked by miR-92b via directly targeting the miR-92b seed site in the 3′-UTR of Sirt6 mRNA. Furthermore, Western blot demonstrated that miR-92b downregulated Sirt6 protein expression in YAMC cells. Blocking miR-92b with a specific inhibitor attenuated the inhibitory effect of Ifnγ on Sirt6 protein expression in the cells. Collectively, our data suggest that Ifnγ inhibits Sirt6 protein expression in intestinal epithelial cells via a miR-92b-mediated mechanism. miR-92b may be a novel therapeutic target for rescuing Sirt6 protein levels in intestinal epithelial cells, thereby protecting against intestinal mucosal injury caused by inflammation.

scholarly journals Nobiletin Stimulates Chloride Secretion in Human Bronchial Epithelia via a cAMP/PKA-Dependent Pathway

2015 ◽  
Vol 37 (1) ◽  
pp. 306-320 ◽  
Author(s):  
Yuan Hao ◽  
Cindy S.T. Cheung ◽  
Wallace C.Y. Yip ◽  
Wing-hung Ko
Keyword(s):  
Cystic Fibrosis ◽  
Adenylate Cyclase ◽  
Cell Line ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Epithelial Cell Line ◽  
Electrical Measurements ◽  
Cl Secretion ◽  
Bronchial Epithelia

Background/Aims: Nobiletin, a citrus flavonoid isolated from tangerines, alters ion transport functions in intestinal epithelia, and has antagonistic effects on eosinophilic airway inflammation of asthmatic rats. The present study examined the effects of nobiletin on basal short-circuit current (ISC) in a human bronchial epithelial cell line (16HBE14o-), and characterized the signal transduction pathways that allowed nobiletin to regulate electrolyte transport. Methods: The ISC measurement technique was used for transepithelial electrical measurements. Intracellular calcium ([Ca2+]i) and cAMP were also quantified. Results: Nobiletin stimulated a concentration-dependent increase in ISC, which was due to Cl- secretion. The increase in ISC was inhibited by a cystic fibrosis transmembrane conductance regulator inhibitor (CFTRinh-172), but not by 4,4'-diisothiocyano-stilbene-2,2'-disulphonic acid (DIDS), Chromanol 293B, clotrimazole, or TRAM-34. Nobiletin-stimulated ISC was also sensitive to a protein kinase A (PKA) inhibitor, H89, and an adenylate cyclase inhibitor, MDL-12330A. Nobiletin could not stimulate any increase in ISC in a cystic fibrosis (CF) cell line, CFBE41o-, which lacked a functional CFTR. Nobiletin stimulated a real-time increase in cAMP, but not [Ca2+]i. Conclusion: Nobiletin stimulated transepithelial Cl- secretion across human bronchial epithelia. The mechanisms involved activation of adenylate cyclase- and cAMP/PKA-dependent pathways, leading to activation of apical CFTR Cl- channels.

Substance P as a mediator of colonic secretory reflexes

1997 ◽  
Vol 272 (2) ◽  
pp. G238-G245 ◽  
Author(s):  
H. J. Cooke ◽  
M. Sidhu ◽  
P. Fox ◽  
Y. Z. Wang ◽  
E. M. Zimmermann
Keyword(s):  
Substance P ◽  
Messenger Rna ◽  
Short Circuit ◽  
Chronic Administration ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Chloride Secretion ◽  
Intestinal Epithelial ◽  
Axon Reflex ◽  
Reflex Pathways

The role of substance P in neural reflex pathways activated by stroking was investigated in muscle-stripped segments of distal colon from guinea pigs. Stroking the mucosal surface with a brush at 1 stroke/s evoked an increase in short-circuit current (Isc) indicative of chloride secretion. The response to mucosal stroking was maximally reduced by 69-75% by the antagonist GR-82334. The agonist [Sar9,Met(O2)11] substance P caused a bumetanide-sensitive increase in Isc when added to the mucosal or serosal bath. Ablation of extrinsic afferents with acute or chronic administration of capsaicin did not alter the mucosal stroking response. Reverse transcription-polymerase chain reaction and in situ hybridization revealed the presence of neurokinin1 (NK1) receptor messenger RNA in isolated colonocytes or crypt glands. Ligand binding of 125I-Bolton-Hunter-labeled substance P was inhibited by GR-82334. The 50% inhibitory concentration was 0.84 nM. The results demonstrate a role for substance P released from capsaicin-insensitive submucosal neurons and in mucosal stroking reflexes. The presence of NK1 receptors on isolated colonocytes suggests that appropriate elements are present for axon reflex activation of intestinal epithelial cells.

Prostaglandin-induced recovery of barrier function in porcine ileum is triggered by chloride secretion

1999 ◽  
Vol 276 (1) ◽  
pp. G28-G36 ◽  
Author(s):  
Anthony T. Blikslager ◽  
Malcolm C. Roberts ◽  
Robert A. Argenzio
Keyword(s):  
Short Circuit ◽  
Second Messengers ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Ringer Solution ◽  
Signal Recovery ◽  
Osmotic Gradient ◽  
Current Increase ◽  
Ussing Chambers

We have previously shown that PGI2 and PGE2 have a synergistic role in restoring electrical transepithelial resistance ( R) in ischemia-injured porcine ileum via the second messengers Ca2+ and cAMP. Because Ca2+ and cAMP stimulate Cl− secretion, we assessed the role of PG-induced Cl−secretion in recovery of R. Mucosa from porcine ileum subjected to ischemia for 45 min was mounted in Ussing chambers and bathed in indomethacin and Ringer solution. Addition of PGs stimulated a twofold increase in R, which was preceded by elevations in short-circuit current (increase of 25 μA/cm2). The PG-induced effect on R was partially inhibited with bumetanide, an inhibitor of Cl− secretion. The remaining elevations in R were similar in magnitude to those induced in ischemic tissues by amiloride, an inhibitor of Na+ absorption. Treatment with 10−4 M 8-bromo-cGMP or 300 mosM mucosal urea resulted in elevations in R similar to those attained with PG treatment. PGs signal recovery of Rvia induction of Cl−secretion and inhibition of Na+absorption, possibly by establishing a transmucosal osmotic gradient.

Protease-activated receptor-1 stimulates Ca2+-dependent Cl−secretion in human intestinal epithelial cells

2001 ◽  
Vol 281 (2) ◽  
pp. G323-G332 ◽  
Author(s):  
M. C. Buresi ◽  
E. Schleihauf ◽  
N. Vergnolle ◽  
A. Buret ◽  
J. L. Wallace ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Short Circuit ◽  
Intestinal Lumen ◽  
Epithelial Cell Line ◽  
Intracellular Camp ◽  
Intestinal Epithelial ◽  
Rt Pcr ◽  
Human Intestinal Epithelial Cells ◽  
Protease Activated Receptor 1

The thrombin receptor, protease-activated receptor-1 (PAR-1), has wide tissue distribution and is involved in many physiological functions. Because thrombin is in the intestinal lumen and mucosa during inflammation, we sought to determine PAR-1 expression and function in human intestinal epithelial cells. RT-PCR showed PAR-1 mRNA expression in SCBN cells, a nontransformed duodenal epithelial cell line. Confluent SCBN monolayers mounted in Ussing chambers responded to PAR-1 activation with a Cl−-dependent increase in short-circuit current. The secretory effect was blocked by BaCl2and the Ca2+-ATPase inhibitor thapsigargin, but not by the L-type Ca2+channel blocker verapamil or DIDS, the nonselective inhibitor of Ca2+-dependent Cl−transport. Responses to thrombin and PAR-1-activating peptides exhibited auto- and crossdesensitization. Fura 2-loaded SCBN cells had increased fluorescence after PAR-1 activation, indicating increased intracellular Ca2+. RT-PCR showed that SCBN cells expressed mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) and hypotonicity-activated Cl−channel-2 but not for the Ca2+-dependent Cl−channel-1. PAR-1 activation failed to increase intracellular cAMP, suggesting that the CFTR channel is not involved in the Cl−secretory response. Our data demonstrate that PAR-1 is expressed on human intestinal epithelial cells and regulates a novel Ca2+-dependent Cl−secretory pathway. This may be of clinical significance in inflammatory intestinal diseases with elevated thrombin levels.

Ion transport characteristics of the murine trachea and caecum

1992 ◽  
Vol 82 (6) ◽  
pp. 667-672 ◽  
Author(s):  
S. N. Smith ◽  
E. W. F. W. Alton ◽  
D. M. Geddes
Keyword(s):  
Cystic Fibrosis ◽  
Ion Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Open Circuit ◽  
Potential Difference ◽  
Cystic Fibrosis Gene ◽  
Tissue Type ◽  
Ussing Chambers

1. The basic defect in cystic fibrosis relates to abnormalities of ion transport in affected tissues, such as the respiratory and gastrointestinal tracts. The identification of the cystic fibrosis gene has enabled studies on the production of a cystic fibrosis transgenic mouse to be undertaken. Knowledge of normal ion transport will be necessary for the validation of any such animal model. We have therefore characterized selected responses of the murine trachea and caecum mounted in ‘mini’ Ussing chambers under open-circuit conditions. 2. Basal values for the trachea were: potential difference, 1.1 mV (sem 0.2; n=18); equivalent short-circuit current, 20.4 μA/cm2 (3.6); conductance, 18.2 mS/cm2 (1.7). Corresponding values for the caecum were: potential difference, 0.7 mV (0.1; n=18); equivalent short-circuit current, 11.0 μA/cm2 (1.6); conductance, 14.5 mS/cm2 (1.4). 3. Amiloride (10 μmol/l) produced a significant (P < 0.001) fall in potential difference of 43.0% (5.7) in the trachea, but had no significant effect in the caecum. 4. Subsequently, one of three protocols was used to assess the capacity of either tissue for chloride secretion. Addition of a combination of forskolin (1 μmol/l) and zardaverine (10 μmol/l) produced rises in the potential difference of 873% (509) in the trachea and 399% (202) in the caecum. Both A23187 (10 μmol/l) and phorbol dibutyrate (10 nmol/l) increased tracheal potential difference by 350% (182) and 147% (47), respectively. Neither had a significant effect in the caecum. 5. Subsequent addition of bumetanide caused a fall in the stimulated potential difference of between 39.8% and 71.7%, depending on secretagogue and tissue type. 6. When a homozygous transgenic cystic fibrosis mouse becomes available, these responses should allow such an animal to be distinguished from normal or heterozygous mice.

Hemin induces active chloride secretion in Caco-2 cells

2005 ◽  
Vol 289 (2) ◽  
pp. G202-G208 ◽  
Author(s):  
Aliye Uc ◽  
Russell F. Husted ◽  
Radhamma L. Giriyappa ◽  
Bradley E. Britigan ◽  
John B. Stokes
Keyword(s):  
Soluble Guanylate Cyclase ◽  
Short Circuit ◽  
Chloride Secretion ◽  
Electrolyte Transport ◽  
Intestinal Epithelial ◽  
Intestinal Fluid ◽  
Lung Epithelium ◽  
Electrolyte Homeostasis ◽  
Dependent Pathway ◽  
Short Circuit Currents

Enterocytes maintain fluid-electrolyte homeostasis by keeping a tight barrier and regulating ion channels. Carbon monoxide (CO), a product of heme degradation, modulates electrolyte transport in kidney and lung epithelium, but its role in regulating intestinal fluid-electrolyte homeostasis has not been studied. The major source of endogenous CO formation comes from the degradation of heme via heme oxygenase. We hypothesized that heme activates electrolyte transport in intestinal epithelial cells. Basolateral hemin treatment increased baseline Caco-2 cell short-circuit currents ( Isc) twofold (control = 1.96 ± 0.14 μA/cm2 vs. hemin = 4.07 ± 0.16 μA/cm2, P < 0.01); apical hemin had no effect. Hemin-induced Isc was caused by Cl− secretion because it was inhibited in Cl−-free medium, with ouabain, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), or DIDS. Apical electrogenic Na+ channel inhibitor benzamil had no effect on hemin-induced Isc. Hemin did not alter the ability of Caco-2 cells to respond maximally to forskolin, but a soluble guanylate cyclase inhibitor, [1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) inhibited the effects of hemin. A CO-releasing molecule, tricarbonyldichlororuthenium II, induced active Cl− secretion that was also inhibited with ODQ. We conclude that hemin induces active Cl− secretion in Caco-2 cells via a cGMP-dependent pathway. These effects are probably the consequence of CO formation. Heme and CO may be important regulators of intestinal fluid-electrolyte homeostasis.

scholarly journals Campylobacter concisus Impairs Sodium Absorption in Colonic Epithelium via ENaC Dysfunction and Claudin-8 Disruption

2020 ◽  
Vol 21 (2) ◽  
pp. 373 ◽  
Author(s):  
Praveen Kumar Nattramilarasu ◽  
Roland Bücker ◽  
Fábia Daniela Lobo de Sá ◽  
Anja Fromm ◽  
Oliver Nagel ◽  
...  
Keyword(s):  
Cell Model ◽  
Short Circuit ◽  
Intestinal Transport ◽  
Intestinal Lumen ◽  
Sodium Absorption ◽  
Epithelial Cell Line ◽  
Barrier Dysfunction ◽  
Campylobacter Concisus ◽  
Ussing Chambers ◽  
Ht 29

The epithelial sodium channel (ENaC) can increase the colonic absorptive capacity for salt and water. Campylobacter concisus is a common pathogenic epsilonproteobacterium, causing enteritis and diarrhea. It can induce barrier dysfunction in the intestine, but its influence on intestinal transport function is still unknown. Therefore, our study aimed to characterize C. concisus effects on ENaC using the HT-29/B6-GR/MR (epithelial cell line HT-29/B6 transfected with glucocorticoid and mineralocorticoid receptors) cell model and mouse colon. In Ussing chambers, C. concisus infection inhibited ENaC-dependent Na+ transport as indicated by a reduction in amiloride-sensitive short circuit current (−55%, n = 15, p < 0.001). This occurred via down-regulation of β- and γ-ENaC mRNA expression and ENaC ubiquitination due to extracellular signal-regulated kinase (ERK)1/2 activation, predicted by Ingenuity Pathway Analysis (IPA). In parallel, C. concisus reduced the expression of the sealing tight junction (TJ) protein claudin-8 and induced claudin-8 redistribution off the TJ domain of the enterocytes, which facilitates the back leakage of Na+ ions into the intestinal lumen. In conclusion, C. concisus caused ENaC dysfunction via interleukin-32-regulated ERK1/2, as well as claudin-8-dependent barrier dysfunction—both of which contribute to Na+ malabsorption and diarrhea.

Sign in / Sign up

Export Citation Format

Share Document