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.

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.

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.

scholarly journals Regulation of electrolyte transport with IL-1β in rabbit distal colon

1995 ◽  
Vol 4 (1) ◽  
pp. 61-66 ◽  
Author(s):  
F. R. Homaidan ◽  
H. Desai ◽  
L. Zhao ◽  
G. Broutman ◽  
R. Burakoff
Keyword(s):  
Inflammatory Bowel Disease ◽  
Protein Synthesis ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Chloride Secretion ◽  
Electrolyte Transport ◽  
Interleukin 1Β ◽  
Prostaglandin E ◽  
Inflammatory Bowel

Interletrkin-1β levels are elevated in inflammatory bowel disease. In this study the mechanism by which interleukin-1β affects electrolyte transport in the rabbit distal colon, was investigated. Interleukin-1β caused a delayed increase in short-circuit current (Isc) which was attributed to protein synthesis since the effect was inhibited by cycloheximide. The interleukin-1β induced increase in Iscwas not affected by amiloride treatment but was completely inhibited by bumetanide or in chloride-free buffer and by indomethacin. Prostaglandin E2levels increased in tissue treated with interleukin-1β, but this increase was reversed by cycloheximide. These data suggest that interleukin-1β causes its effect via a yet to be identified second messenger, by increasing chloride secretion through a prostaglandin E2mediated mechanism.

Regulation of anion secretion by nitric oxide in human airway epithelial cells

2001 ◽  
Vol 281 (2) ◽  
pp. L450-L457 ◽  
Author(s):  
Marek Duszyk
Keyword(s):  
Nitric Oxide ◽  
Soluble Guanylate Cyclase ◽  
Short Circuit ◽  
Biological Significance ◽  
Short Circuit Current ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
No Donor ◽  
Anion Secretion ◽  
Dependent Pathway

Nitric oxide (NO) is continuously produced and released in human airways, but the biological significance of this process is unknown. In this study, we have used Calu-3 cells to investigate the effects of NO on transepithelial anion secretion. An inhibitor of NO synthase, NG-nitro-l-arginine methyl ester, reduced short- circuit current ( Isc), whereas an NO donor, S-nitrosoglutathione (GSNO), increased Isc, with an EC50∼1.2 μM. The NO-activated current was inhibited by diphenylamine-2-carboxylate, clotrimazole, and charybdotoxin. Selective permeabilization of cell membranes indicated that NO activated both apical anion channels and basolateral potassium channels. An inhibitor of soluble guanylate cyclase, 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one, prevented activation of Iscby NO but not by 8-bromo-cGMP, suggesting that NO acts via a cGMP-dependent pathway. Sequential treatment of cells with forskolin and GSNO or 1-ethyl-2-benzimidazolinone and GSNO showed additive effects of these chemicals on Isc. Interestingly, GSNO elevated intracellular Ca2+concentration ([Ca2+]i) but had no effect on Iscactivated by thapsigargin. These results show that NO activates transepithelial anion secretion via a cGMP-dependent pathway that involves cross talk between NO and [Ca2+]i.

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.

Role of calcium in the regulation of colonic secretion in the rat

1983 ◽  
Vol 244 (5) ◽  
pp. G552-G560 ◽  
Author(s):  
T. W. Zimmerman ◽  
J. W. Dobbins ◽  
H. J. Binder
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Extracellular Calcium ◽  
Electrolyte Transport ◽  
Rat Colon ◽  
Sodium Absorption ◽  
Chloride Absorption

In vitro experiments were performed in rat colon to define the role of calcium in the regulation of electrolyte transport. Neither basal net sodium absorption (JNanet) nor JClnet was affected by varying serosal calcium from 0 to 3.0 mM, but both were decreased by 4.8 mM calcium. Removal of serosal calcium completely inhibited the effect of bethanechol, a muscarinic cholinergic agonist, which inhibits neutral sodium-chloride absorption in 1.2 mM calcium. In contrast, theophylline significantly decreased JNanet and JClnet both in the presence and absence of calcium, but the effects of theophylline were significantly less in calcium-free media. In 3.0 mM calcium bethanechol inhibited JCLnet significantly greater than JNanet and in 4.8 mM calcium bethanechol decreased JClnet equivalent to the increase in short-circuit current without significantly altering JNanet. We conclude that 1) high [Ca2+] inhibits net sodium and net chloride absorption; 2) the alteration of electrolyte transport by bethanechol is dependent on extracellular calcium, and the alteration of electrolyte transport by theophylline is not dependent on extracellular calcium but may be dependent on intracellular calcium; and 3) in addition to inhibition of neutral NaCl absorption, bethanechol stimulates chloride secretion.

Neurohumoral control of esophageal epithelial electrolyte transport

1980 ◽  
Vol 239 (1) ◽  
pp. G5-G11 ◽  
Author(s):  
D. D. Boyd ◽  
C. N. Carney ◽  
D. W. Powell
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Electrolyte Transport ◽  
Sodium Absorption ◽  
Metabolic Substrates ◽  
Submucosal Glands ◽  
Neurohumoral Control

The neurohumoral control of epithelial esophageal electrolyte transport was investigated by studying the effect of various hormones and neuroeffector agents on the potential difference (PD) in vivo or on the electrical parameters of electrolyte transport in vitro. The rabbit esophagus, which has no submucosal esophageal glands, demonstrated no effect of pentagastrin, cholecystokinin octapeptide, or synthetic secretin in vivo, and no effect of these hormones or of vasopressin, aldosterone, carbachol, epinephrine, or cAMP in vitro. The rabbit esophagus did respond to metabolic substrates (glucose) in vitro by increasing sodium absorption. In contrast, the opossum esophagus, which contains extensive submucosal glands, had a lower electrical resistance, PD, short-circuit current, and sodium absorption with higher chloride secretion. This esophagus responded to carbachol and epinephrine by sodium and chloride secretion. We believe that only the submucosal glands of the esophagus are under significant neurohumoral control while the sodium transporting function of the stratified squamous epithelium of this organ is important in maintaining its barrier function.

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.

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