Agonists of protease-activated receptors 1 and 2 stimulate electrolyte secretion from mouse gallbladder

2007 ◽  
Vol 293 (1) ◽  
pp. G335-G346 ◽  
Author(s):  
Jacob G. Kirkland ◽  
Graeme S. Cottrell ◽  
Nigel W. Bunnett ◽  
Carlos U. Corvera
Keyword(s):  
Knockout Mice ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Gastrointestinal Diseases ◽  
Wild Type ◽  
Protease Activated Receptors ◽  
Intracellular Ca ◽  
Electrolyte Secretion ◽  
Homologous Desensitization

Cholecystitis is one of the most common gastrointestinal diseases. Inflammation induces the activation of proteases that can signal to cells by cleaving protease-activated receptors (PARs) to induce hemostasis, inflammation, pain, and repair. However, the distribution of PARs in the gallbladder is unknown, and their effects on gallbladder function have not been fully investigated. We localized immunoreactive PAR1 and PAR2 to the epithelium, muscle, and serosa of mouse gallbladder. mRNA transcripts corresponding to PAR1 and PAR2, but not PAR4, were detected by RT-PCR and sequencing. Addition of thrombin and a PAR1-selective activating peptide (TFLLRN-NH2) to the serosal surface of mouse gallbladder mounted in an Ussing chamber stimulated an increase in short-circuit current in wild-type but not PAR1 knockout mice. Similarly, serosally applied trypsin and PAR2 activating peptide (SLIGRL-NH2) increased short-circuit current in wild-type but not PAR2 knockout mice. Proteases and activating peptides strongly inhibited electrogenic responses to subsequent stimulation with the same agonist, indicating homologous desensitization. Removal of HCO3− ions from the serosal buffer reduced responses to thrombin and trypsin by >80%. Agonists of PAR1 and PAR2 increase intracellular Ca2+ concentration in isolated and cultured gallbladder epithelial cells. The COX-2 inhibitor meloxicam and an inhibitor of CFTR prevented the stimulatory effect of PAR1 but not PAR2. Thus PAR1 and PAR2 are expressed in the epithelium of the mouse gallbladder, and serosally applied proteases cause a HCO3− secretion. The effects of PAR1 but not PAR2 depend on generation of prostaglandins and activation of CFTR. These mechanisms may markedly influence fluid and electrolyte secretion of the inflamed gallbladder when multiple proteases are generated.

Extracellular UTP stimulates electrogenic bicarbonate secretion across CFTR knockout gallbladder epithelium

2000 ◽  
Vol 279 (1) ◽  
pp. G132-G138 ◽  
Author(s):  
Lane L. Clarke ◽  
Matthew C. Harline ◽  
Lara R. Gawenis ◽  
Nancy M. Walker ◽  
John T. Turner ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Receptor Activation ◽  
Biliary Disease ◽  
Anion Conductance ◽  
Intracellular Ca ◽  
Ph Stat ◽  
Chamber Studies

The loss of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial HCO3 − secretion contributes to the pathogenesis of pancreatic and biliary disease in cystic fibrosis (CF) patients. Recent studies have investigated P2Y2 nucleotide receptor agonists, e.g., UTP, as a means to bypass the CFTR defect by stimulating Ca2+-activated Cl− secretion. However, the value of this treatment in facilitating transepithelial HCO3 − secretion is unknown. Gallbladder mucosae from CFTR knockout mice were used to isolate the Ca2+-dependent anion conductance during activation of luminal P2Y2receptors. In Ussing chamber studies, UTP stimulated a transient peak in short-circuit current ( I sc) that declined to a stable plateau phase lasting 30–60 min. The plateau I sc after UTP was Cl− independent, HCO3 − dependent, insensitive to bumetanide, and blocked by luminal DIDS. In pH stat studies, luminal UTP increased both I sc and serosal-to-mucosal HCO3 − flux ( J s→m) during a 30-min period. Substitution of Cl− with gluconate in the luminal bath to inhibit Cl−/HCO3 −exchange did not prevent the increase in J s→mand I sc during UTP. In contrast, luminal DIDS completely inhibited UTP-stimulated increases in J s→m and I sc. We conclude that P2Y2 receptor activation results in a sustained (30–60 min) increase in electrogenic HCO3 − secretion that is mediated via an intracellular Ca2+-dependent anion conductance in CF gallbladder.

Cholinergic ion secretion in human colon requires coactivation by cAMP

1998 ◽  
Vol 275 (6) ◽  
pp. G1274-G1281 ◽  
Author(s):  
M. Mall ◽  
M. Bleich ◽  
M. Schürlein ◽  
J. Kühr ◽  
H. H. Seydewitz ◽  
...  
Keyword(s):  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Human Colon ◽  
Intracellular Camp ◽  
Camp Production ◽  
Ion Secretion ◽  
Negative Current ◽  
Intracellular Ca ◽  
Second Messenger Pathways

Cl−secretion in the colon can be activated by an increase of either intracellular Ca2+ or cAMP. In this study we examined a possible interdependence of the two second-messenger pathways in human colonic epithelium. When measured in a modified Ussing chamber, carbachol (CCH; 100 μmol/l, basolateral), via an increase in cytosolic Ca2+concentration ([Ca2+]i), activated a transient lumen-negative equivalent short-circuit current ( I sc) [change (Δ) in I sc = −79.4 ± 7.5 μA/cm2]. Previous studies indicated that intracellular Ca2+ directly acts on basolateral K+ channels, thus enhancing driving force for luminal Cl− exit. Increased intracellular cAMP (by basolateral addition of 100 μmol/l IBMX and 1 μmol/l forskolin) activated a sustained lumen-negative current (Δ I sc = −42.4 ± 7.2 μA/cm2) that was inhibited by basolateral trans-6-cyano-4-( N-ethylsulfonyl- N-methylamino)-3-hydroxy-2,2-dimethyl&2-chromane (10 μmol/l), a blocker of KvLQT1 channels. In the presence of elevated cAMP, the CCH-activated currents were augmented (Δ I sc = 167.7 ± 32.7 μA/cm2), suggesting cooperativity of the Ca2+- and cAMP-mediated responses. Inhibition of endogenous cAMP production by indomethacin (10 μmol/l) significantly reduced CCH-activated currents and even reversed the polarity in 70% of the experiments. The transient lumen-positive I sc was probably due to activation of apical K+channels because it was blocked by luminal Ba2+ (5 mmol/l) and tetraethylammonium (10 mmol/l). In the presence of indomethacin (10 μmol/l, basolateral), an increase of cAMP activated a sustained negative I sc. Under these conditions, CCH induced a large further increase in lumen-negative I sc(Δ I sc = −100.0 ± 21.0 μA/cm2). We conclude that CCH acting via [Ca2+]ican induce Cl− secretion only in the presence of cAMP, i.e., when luminal Cl− channels are already activated. The activation of a luminal and basolateral K+ conductance by CCH may be essential for transepithelial KCl secretion in human colon.

Colonic H+-K+-ATPase in K+conservation and electrogenic Na+ absorption during Na+ restriction

2001 ◽  
Vol 281 (6) ◽  
pp. G1369-G1377 ◽  
Author(s):  
Zachary Spicer ◽  
Lane L. Clarke ◽  
Lara R. Gawenis ◽  
Gary E. Shull
Keyword(s):  
Knockout Mice ◽  
Apical Membrane ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Wild Type ◽  
Restricted Diet ◽  
Serum Aldosterone

Upregulation of the colonic H+-K+- ATPase (cHKA) during hyperaldosteronism suggests that it functions in both K+conservation and electrogenic Na+ absorption in the colon when Na+-conserving mechanisms are activated. To test this hypothesis, wild-type ( cHKA +/+) and cHKA-deficient ( cHKA −/−) mice were fed Na+-replete and Na+-restricted diets and their responses were analyzed. In both genotypes, Na+ restriction led to reduced plasma Na+ and increased serum aldosterone, and mRNAs for the epithelial Na+ channel (ENaC) β- and γ-subunits, channel-inducing factor, and cHKA were increased in distal colon. Relative to wild-type controls, cHKA −/− mice on a Na+-replete diet had elevated fecal K+ excretion. Dietary Na+restriction led to increased K+ excretion in knockout but not in wild-type mice. The amiloride-sensitive, ENaC-mediated short-circuit current in distal colon was significantly reduced in knockout mice maintained on either the Na+-replete or Na+-restricted diet. These results demonstrate that cHKA plays an important role in K+ conservation during dietary Na+ restriction and suggest that cHKA-mediated K+ recycling across the apical membrane is required for maximum electrogenic Na+ absorption.

Role of luminal ATP in regulating electrogenic Na+ absorption in guinea pig distal colon

2002 ◽  
Vol 283 (2) ◽  
pp. G300-G308 ◽  
Author(s):  
Takeshi Yamamoto ◽  
Yuichi Suzuki
Keyword(s):  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Inhibitory Effect ◽  
Similar Degree ◽  
Maximum Inhibition ◽  
Luminal Side ◽  
Intracellular Ca

Extracellular ATP regulates a variety of functions in epithelial tissues by activating the membrane P2-receptor. The purpose of this study was to investigate the autocrine/paracrine regulation by luminal ATP of electrogenic amiloride-sensitive Na+ absorption in the distal colon from guinea pigs treated with aldosterone by measuring the amiloride-sensitive short-circuit current ( I sc) and 22Na+ flux in vitro with the Ussing chamber technique. ATP added to the luminal side inhibited the amiloride-sensitive I sc and22Na+ absorption to a similar degree. The concentration dependence of the inhibitory effect of ATP on amiloride-sensitive I sc had an IC50value of 20–30 μM, with the maximum inhibition being ∼50%. The effects of different nucleotides and of a nucleoside were also studied, the order of potency being ATP = UTP > ADP > adenosine. The effects of ATP were slightly, but significantly, reduced in the presence of suramin in the luminal solution. The inhibitory effect of luminal ATP was more potent in the absence of both Mg2+ and Ca2+ from the luminal solution. Pretreatment of the tissue with ionomycin or thapsigargin in the absence of serosal Ca2+ did not affect the percent inhibition of amiloride-sensitive I sc induced by ATP. Mechanical perturbation with a hypotonic luminal solution caused a reduction in amiloride-sensitive I sc, this effect being prevented by the presence of hexokinase, an ATP-scavenging enzyme. These results suggest that ATP released into the luminal side by hypotonic stimulation could exert an inhibitory effect on the electrogenic Na+ absorption. This effect was probably mediated by a P2Y2 receptor on the apical membrane of colonic epithelial cells, and a change in the intracellular Ca2+ concentration may not be necessary for this process.

scholarly journals Enterotoxigenicity of Mature 45-Kilodalton and Processed 35-Kilodalton Forms of Hemagglutinin Protease Purified from a Cholera Toxin Gene-Negative Vibrio cholerae Non-O1, Non-O139 Strain

2006 ◽  
Vol 74 (5) ◽  
pp. 2937-2946 ◽  
Author(s):  
A. Ghosh ◽  
D. R. Saha ◽  
K. M. Hoque ◽  
M. Asakuna ◽  
S. Yamasaki ◽  
...  
Keyword(s):  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Hela Cells ◽  
Histopathological Examination ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Toxin Gene ◽  
Dependent Manner ◽  
A Cell

ABSTRACT Cholera toxin gene-negative Vibrio cholerae non-O1, non-O139 strain PL-21 is the etiologic agent of cholera-like syndrome. Hemagglutinin protease (HAP) is one of the major secretory proteins of PL-21. The mature 45-kDa and processed 35-kDa forms of HAP were purified in the presence and absence of EDTA from culture supernatants of PL-21. Enterotoxigenicities of both forms of HAP were tested in rabbit ileal loop (RIL), Ussing chamber, and tissue culture assays. The 35-kDa HAP showed hemorrhagic fluid response in a dose-dependent manner in the RIL assay. Histopathological examination of 20 μg of purified protease-treated rabbit ileum showed the presence of erythrocytes and neutrophils in the upper part of the villous lamina propria. Treatment with 40 μg of protease resulted in gross damage of the villous epithelium with inflammation, hemorrhage, and necrosis. The 35-kDa form of HAP, when added to the lumenal surface of rat ileum loaded in an Ussing chamber, showed a decrease in the intestinal short-circuit current and a cell rounding effect on HeLa cells. The mature 45-kDa form of HAP showed an increase in intestinal short-circuit current in an Ussing chamber and a cell distending effect on HeLa cells. These results show that HAP may play a role in the pathogenesis of PL-21.

Basolateral Cl− uptake mechanisms in Xenopus laevis lung epithelium

2010 ◽  
Vol 299 (1) ◽  
pp. R92-R100 ◽  
Author(s):  
Jens Berger ◽  
Martin Hardt ◽  
Wolfgang G. Clauss ◽  
Martin Fronius
Keyword(s):  
Xenopus Laevis ◽  
Ion Transport ◽  
Anion Exchanger ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Transport Processes ◽  
Lung Epithelium ◽  
Active Ion Transport ◽  
Uptake Mechanisms

A thin liquid layer covers the lungs of air-breathing vertebrates. Active ion transport processes via the pulmonary epithelial cells regulate the maintenance of this layer. This study focuses on basolateral Cl− uptake mechanisms in native lungs of Xenopus laevis and the involvement of the Na+/K+/2 Cl− cotransporter (NKCC) and HCO3−/Cl− anion exchanger (AE), in particular. Western blot analysis and immunofluorescence staining revealed the expression of the NKCC protein in the Xenopus lung. Ussing chamber experiments demonstrated that the NKCC inhibitors (bumetanide and furosemide) were ineffective at blocking the cotransporter under basal conditions, as well as under pharmacologically stimulated Cl−-secreting conditions (forskolin and chlorzoxazone application). However, functional evidence for the NKCC was detected by generating a transepithelial Cl− gradient. Further, we were interested in the involvement of the HCO3−/Cl− anion exchanger to transepithelial ion transport processes. Basolateral application of DIDS, an inhibitor of the AE, resulted in a significantly decreased the short-circuit current (ISC). The effect of DIDS was diminished by acetazolamide and reduced by increased external HCO3− concentrations. Cl− secretion induced by forskolin was decreased by DIDS, but this effect was abolished in the presence of HCO3−. These experiments indicate that the AE at least partially contributes to Cl− secretion. Taken together, our data show that in Xenopus lung epithelia, the AE, rather than the NKCC, is involved in basolateral Cl− uptake, which contrasts with the common model for Cl− secretion in pulmonary epithelia.

scholarly journals Enterohemorrhagic E. coli (EHEC)—Secreted Serine Protease EspP Stimulates Electrogenic Ion Transport in Human Colonoid Monolayers

Toxins ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 351 ◽  
Author(s):  
C. Tse ◽  
Julie In ◽  
Jianyi Yin ◽  
Mark Donowitz ◽  
Michele Doucet ◽  
...  
Keyword(s):  
Ion Transport ◽  
Serine Protease ◽  
Shiga Toxin ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Adult Stem Cell ◽  
Short Circuit Current ◽  
Watery Diarrhea ◽  
E Coli ◽  
Electrogenic Ion Transport

One of the characteristic manifestations of Shiga-toxin-producing Escherichia coli (E. coli) infection in humans, including EHEC and Enteroaggregative E. coli O104:H4, is watery diarrhea. However, neither Shiga toxin nor numerous components of the type-3 secretion system have been found to independently elicit fluid secretion. We used the adult stem-cell-derived human colonoid monolayers (HCM) to test whether EHEC-secreted extracellular serine protease P (EspP), a member of the serine protease family broadly expressed by diarrheagenic E. coli can act as an enterotoxin. We applied the Ussing chamber/voltage clamp technique to determine whether EspP stimulates electrogenic ion transport indicated by a change in short-circuit current (Isc). EspP stimulates Isc in HCM. The EspP-stimulated Isc does not require protease activity, is not cystic fibrosis transmembrane conductance regulator (CFTR)-mediated, but is partially Ca2+-dependent. EspP neutralization with a specific antibody reduces its potency in stimulating Isc. Serine Protease A, secreted by Enteroaggregative E. coli, also stimulates Isc in HCM, but this current is CFTR-dependent. In conclusion, EspP stimulates colonic CFTR-independent active ion transport and may be involved in the pathophysiology of EHEC diarrhea. Serine protease toxins from E. coli pathogens appear to serve as enterotoxins, potentially significantly contributing to watery diarrhea.

Cellular pathways mediating tachykinin-evoked secretomotor responses in guinea pig ileum

1997 ◽  
Vol 273 (5) ◽  
pp. G1127-G1134 ◽  
Author(s):  
W. MacNaughton ◽  
B. Moore ◽  
S. Vanner
Keyword(s):  
Guinea Pig ◽  
Receptor Agonist ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Neurokinin A ◽  
Guinea Pig Ileum ◽  
Cellular Pathways ◽  
20 Nm

This study characterized tachykinin-evoked secretomotor responses in in vitro submucosal and mucosal-submucosal preparations of the guinea pig ileum using combined intracellular and Ussing chamber recording techniques. Superfusion of endogenous tachykinins substance P (SP), neurokinin A (NKA), and neurokinin B depolarized single submucosal neurons and evoked increased short-circuit current ( I sc) responses in Ussing chamber preparations. The NK1-receptor agonist [Sar9,Met(O2)11]SP [50% effective concentration (EC50) = 2 nM] depolarized all submucosal neurons examined. The NK3-receptor agonist senktide (EC50 = 20 nM) depolarized ∼50% of neurons examined, whereas the NK2-receptor agonist [Ala5,β-Ala8]NKA-(4—10) had no effect on membrane potential. [Sar9,Met(O2)11]SP and senktide evoked similar increases in I sc that were tetrodotoxin sensitive (91 and 100%, respectively) and were selectively blocked by the NK1antagonist CP-99,994 and the NK3antagonist SR-142801, respectively. Capsaicin-evoked increases in I sc were significantly inhibited (54%, P < 0.05) by CP-99,994 but not by SR-142801. Neither antagonist inhibited slow excitatory postsynaptic potentials. These findings suggest that tachykinin-evoked secretion in guinea pig ileum is mediated by NK1 and NK3 receptors on submucosal secretomotor neurons and that capsaicin-sensitive nerves release tachykinin(s) that activate the NK1 receptors.

Pharmacological modulation of ion transport across wild-type and ΔF508 CFTR-expressing human bronchial epithelia

2000 ◽  
Vol 279 (2) ◽  
pp. C461-C479 ◽  
Author(s):  
Daniel C. Devor ◽  
Robert J. Bridges ◽  
Joseph M. Pilewski
Keyword(s):  
Cystic Fibrosis ◽  
Ion Transport ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Bronchial Epithelium ◽  
Disulfonic Acid ◽  
Wild Type ◽  
Pharmacological Agents ◽  
Transport Defect

Forskolin, UTP, 1-ethyl-2-benzimidazolinone (1-EBIO), NS004, 8-methoxypsoralen (Methoxsalen; 8-MOP), and genistein were evaluated for their effects on ion transport across primary cultures of human bronchial epithelium (HBE) expressing wild-type (wt HBE) and ΔF508 (ΔF-HBE) cystic fibrosis transmembrane conductance regulator. In wt HBE, the baseline short-circuit current ( I sc) averaged 27.0 ± 0.6 μA/cm2 ( n = 350). Amiloride reduced this I sc by 13.5 ± 0.5 μA/cm2 ( n = 317). In ΔF-HBE, baseline I sc was 33.8 ± 1.2 μA/cm2 ( n = 200), and amiloride reduced this by 29.6 ± 1.5 μA/cm2 ( n = 116), demonstrating the characteristic hyperabsorption of Na+ associated with cystic fibrosis (CF). In wt HBE, subsequent to amiloride, forskolin induced a sustained, bumetanide-sensitive I sc(Δ I sc = 8.4 ± 0.8 μA/cm2; n = 119). Addition of acetazolamide, 5-( N-ethyl- N-isopropyl)-amiloride, and serosal 4,4′-dinitrostilben-2,2′-disulfonic acid further reduced I sc, suggesting forskolin also stimulates HCO3 − secretion. This was confirmed by ion substitution studies. The forskolin-induced I scwas inhibited by 293B, Ba2+, clofilium, and quinine, whereas charybdotoxin was without effect. In ΔF-HBE the forskolin I sc response was reduced to 1.2 ± 0.3 μA/cm2 ( n = 30). In wt HBE, mucosal UTP induced a transient increase in I sc (Δ I sc = 15.5 ± 1.1 μA/cm2; n = 44) followed by a sustained plateau, whereas in ΔF-HBE the increase in I sc was reduced to 5.8 ± 0.7 μA/cm2 ( n = 13). In wt HBE, 1-EBIO, NS004, 8-MOP, and genistein increased I sc by 11.6 ± 0.9 ( n = 20), 10.8 ± 1.7 ( n = 18), 10.0 ± 1.6 ( n = 5), and 7.9 ± 0.8 μA/cm2( n = 17), respectively. In ΔF-HBE, 1-EBIO, NS004, and 8-MOP failed to stimulate Cl− secretion. However, addition of NS004 subsequent to forskolin induced a sustained Cl−secretory response (2.1 ± 0.3 μA/cm2, n = 21). In ΔF-HBE, genistein alone stimulated Cl− secretion (2.5 ± 0.5 μA/cm2, n = 11). After incubation of ΔF-HBE at 26°C for 24 h, the responses to 1-EBIO, NS004, and genistein were all potentiated. 1-EBIO and genistein increased Na+ absorption across ΔF-HBE, whereas NS004 and 8-MOP had no effect. Finally, Ca2+-, but not cAMP-mediated agonists, stimulated K+ secretion across both wt HBE and ΔF-HBE in a glibenclamide-dependent fashion. Our results demonstrate that pharmacological agents directed at both basolateral K+ and apical Cl− conductances directly modulate Cl−secretion across HBE, indicating they may be useful in ameliorating the ion transport defect associated with CF.

Use of a modified Ussing chamber to monitor intestinal epithelial and smooth muscle functions

1991 ◽  
Vol 261 (1) ◽  
pp. G166-G170 ◽  
Author(s):  
Y. F. Li ◽  
N. W. Weisbrodt ◽  
Y. Harari ◽  
F. G. Moody
Keyword(s):  
Smooth Muscle ◽  
Strain Gauge ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Displacement Transducer ◽  
Organ Bath ◽  
Force Displacement ◽  
Dose Dependent ◽  
Muscle Functions

A technique that allows the simultaneous monitoring of epithelial and smooth muscle function was developed and used to study rat small intestine in vitro. A Ussing chamber was modified so that a strain gauge force transducer could be sewn to the serosal surface of an intestinal segment clamped in the chamber. The apparatus was used to monitor short-circuit current, potential difference, and resistance across the segment, and contractions of the longitudinal layer of the muscularis externa. Both spontaneous activity and responses to the application of carbachol were recorded. Carbachol applied to the serosal side induced dose-dependent increases in both short-circuit current and contractile force. The median effective doses of the two responses differed, with contractions being more sensitive to the drug. Carbachol applied to the mucosal side induced no changes in either epithelial or contractile activities. The ability of the serosal strain gauge transducer to monitor contractions faithfully was tested in an organ bath in which the gut segment was attached to an external force-displacement transducer. There was a close correlation between the dose-dependent increase in force in response to carbachol measured by the serosal transducer and that measured by the force-displacement transducer (r = 0.988). Thus our technique can be used to study simultaneously epithelial and smooth muscle functions of the intestine.

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