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.

Dexamethasone modulation of ion transport and fluid movement across airway epithelium

1993 ◽  
Vol 264 (4) ◽  
pp. L376-L381 ◽  
Author(s):  
M. Satoh ◽  
S. Shimura ◽  
H. Ishihara ◽  
K. Yamada ◽  
T. Masuda ◽  
...  
Keyword(s):  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Tracheal Epithelium ◽  
Fluid Movement ◽  
Airway Mucosa ◽  
Submucosal Glands ◽  
Superficial Epithelium ◽  
Dose Dependent

Electrolyte or fluid is secreted across airway mucosa by both superficial epithelium and submucosal glands. To understand the effect of glucocorticoid on fluid movement across airway mucosa, we examined the effects of dexamethasone (Dex) on bioelectric properties of canine and feline tracheal epithelium and on 22Na efflux from isolated feline tracheal submucosal glands. Potential difference (PD) and short-circuit current (SCC) across tracheal epithelium were measured using an Ussing chamber, and conductance (G) was calculated as the ratio SCC/PD. Isolated glands were loaded with 22Na, and the rate constant (RC) of Na2+ efflux was calculated by measuring the radioactivity of each effluent sample. After treatment with 10(-9) to 10(-5) M Dex for up to 6 h, the epithelium and isolated glands were stimulated with isoproterenol (ISP) and methacholine (MCh), respectively. Dex treatment did not alter significantly baseline values of PD, SCC, or RC. However, Dex treatment produced a dose-dependent attenuation of ISP-evoked epithelial PD and SCC and of MCh-evoked glandular RC. In canine epithelium, pretreatment with 10(-5) M Dex for 6 h reduced by 40% the ISP (10(-6) M)-evoked rise in PD and SCC, whereas G remained unchanged. After 10(-5) M Dex treatment for 6 h, MCh (10(-5) M)-evoked RC in isolated glands was significantly less than in control glands (MCh alone) by 23%. These findings suggest that glucocorticoid decreases the fluid secretion across the airway mucosa, especially when the mucosa are stimulated.

11-Dehydrocorticosterone, a glucocorticoid metabolite, inhibits aldosterone action in toad bladder

1991 ◽  
Vol 261 (5) ◽  
pp. F873-F879 ◽  
Author(s):  
A. S. Brem ◽  
K. L. Matheson ◽  
J. L. Barnes ◽  
D. J. Morris
Keyword(s):  
Sodium Transport ◽  
Cell Layer ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Hydroxysteroid Dehydrogenase ◽  
Toad Bladder ◽  
Compound A ◽  
Dose Dependent Fashion ◽  
Epithelial Cell Layer ◽  
Dose Dependent

The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) metabolizes glucocorticoid hormones and diminishes their ability to induce sodium transport. In these studies, we determined the location of this enzyme in toad bladder and assessed the biological role for its 11-dehydro end product. Employing a polyclonal antibody directed toward 11 beta-OHSD and immunofluorescence techniques, we located the enzyme in the epithelial cell layer of the toad bladder. Although corticosterone (10(-7) M) can partially suppress aldosterone (10(-7) M)-stimulated short-circuit current (SCC), a clear excess of corticosterone (10(-6) M) did not inhibit the aldosterone-induced induced (10(-8) M) rise in SCC (n = 6). The 11-dehydro product of corticosterone, 11-dehydrocorticosterone (compound A) added to the serosal bath suppressed aldosterone (10(-8) M) peak SCC (360 min) in a dose-dependent fashion reaching 46 +/- 5% of control values at 10(-5) M (n = 6; P less than 0.001). Compound A (10(-5) M) in the mucosal bath also was capable of partially inhibiting the peak aldosterone rise in SCC to 63 +/- 7% of control values with aldosterone at 10(-8) M (n = 6; P less than 0.01) and to 64 +/- 10% of control values with aldosterone at 10(-7) M (n = 9; P less than 0.01). Compound A alone at 10(-5) M did not have any effect on SCC. Isolated toad bladders were not able to transform compound A (at 10(-8) and 10(-5) M) back to corticosterone. Thus the 11-dehydro end product of 11 beta-OHSD (compound A) may play a biologic role by regulating a component of mineralocorticoid-induced sodium transport.

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.

ATP stimulates Ca2+ release from a rapidly exchanging pool in cultured rat epididymal cells

1993 ◽  
Vol 264 (6) ◽  
pp. C1388-C1394 ◽  
Author(s):  
A. Y. Leung ◽  
H. L. Tai ◽  
P. Y. Wong
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
The Other ◽  
Free Solution ◽  
Tetraacetic Acid ◽  
Cl Secretion ◽  
Transient Rise ◽  
Extracellular Compartment ◽  
Dose Dependent Fashion ◽  
Dose Dependent

A study was carried out to investigate an ATP-sensitive Ca2+ pool in rat epididymal cells and its role in transepithelial Cl- secretion. In normal buffered solution containing 2.5 mM free Ca2+, ATP triggered single calcium spikes in a dose-dependent fashion. In nominally Ca(2+)-free solution, the peaks of successive Ca2+ spikes diminished after repeated ATP stimulations. Addition of Sr2+ (2.5 mM) to Ca(2+)-free solution after ATP stimulation did not cause changes in fluorescence signals. However, in the presence of Sr2+, ATP gave rise to apparent repetitive Ca2+ spikes of similar magnitudes after repeated stimulations. Increasing the time of exposure in Ca(2+)-free solution containing 50 microM ethylene glycol-bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid rapidly decreased the intracellular Ca2+ concentration ([Ca2+]i) response to subsequent ATP stimulation. On the other hand, increasing the time of exposure in Sr(2+)-containing solution in Ca(2+)-depleted cells rapidly increased the apparent [Ca2+]i response to subsequent ATP stimulation. These observations suggested the existence of a Ca2+ pool that was rapidly exchanging with the extracellular compartment. Apical application of ATP elicited a transient rise in short-circuit current across the epididymal epithelium in a dose-dependent fashion, and the response was reduced by prior stimulation with thapsigargin. Ca2+ released from a rapidly exchanging ATP-sensitive store might stimulate Cl- secretion in the epididymis, thereby maintaining the electrolyte contents and fluidity of the epididymal microenvironment.

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.

scholarly journals Site-1 Protease-Derived Soluble (Pro)Renin Receptor Contributes to Angiotensin II–Induced Hypertension in Mice

Hypertension ◽  
2020 ◽  
Author(s):  
Ye Feng ◽  
Kexin Peng ◽  
Renfei Luo ◽  
Fei Wang ◽  
Tianxin Yang
Keyword(s):  
Blood Pressure ◽  
Angiotensin Ii ◽  
Collecting Duct ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Duct Cell ◽  
Ang Ii ◽  
Cortical Collecting Duct ◽  
Induced Hypertension

Activation of PRR ([pro]renin receptor) contributes to enhancement of intrarenal RAS and renal medullary α-ENaC and thus elevated blood pressure during Ang II (angiotensin II) infusion. The goal of the present study was to test whether such action of PRR was mediated by sPRR (soluble PRR), generated by S1P (site-1 protease), a newly identified PRR cleavage protease. F1 B6129SF1/J mice were infused for 6 days with control or Ang II at 300 ng/kg per day alone or in combination with S1P inhibitor PF-429242 (PF), and blood pressure was monitored by radiotelemetry. S1P inhibition significantly attenuated Ang II–induced hypertension accompanied with suppressed urinary and renal medullary renin levels and expression of renal medullary but not renal cortical α-ENaC expression. The effects of S1P inhibition were all reversed by supplement with histidine-tagged sPRR termed as sPRR-His. Ussing chamber technique was performed to determine amiloride-sensitive short-circuit current, an index of ENaC activity in confluent mouse cortical collecting duct cell line cells exposed for 24 hours to Ang II, Ang II + PF, or Ang II + PF + sPRR-His. Ang II–induced ENaC activity was blocked by PF, which was reversed by sPRR-His. Together, these results support that S1P-derived sPRR mediates Ang II–induced hypertension through enhancement of intrarenal renin level and activation of ENaC.

Ion transport by neonatal rabbit distal colon

1986 ◽  
Vol 250 (6) ◽  
pp. G754-G759 ◽  
Author(s):  
G. D. Potter ◽  
S. M. Burlingame
Keyword(s):  
Small Intestine ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Electrical Conductance ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Ion Fluxes ◽  
Adult Rabbit ◽  
Rabbit Colon ◽  
Edge Damage

The neonatal small intestine is characterized by electrical conductance and permeability to ions higher than in the corresponding adult intestine. To investigate whether this property of the neonate is limited to the small intestine, or extends to the colon, a modified Ussing chamber for determination of transmucosal potential difference (PD), short-circuit current (Isc), transepithelial conductance (Gt), and ion fluxes in the neonatal rabbit distal colon was constructed. After care to reduce edge damage, Gt for the neonatal colon was found to be 8.4 +/- 0.3 mS . cm2 and for adult colon in the same chamber, 7.4 +/- 0.5 (P greater than 0.05). Net Na and Cl fluxes under short-circuit conditions were similar to those obtained in adult colon. Unidirectional ion fluxes were also similar to those of the adult. Net Na flux (JNanet) was incompletely inhibited by 10(-4) M of amiloride. Response to replacement of Na, Cl, and HCO3-, respectively, in the bathing solutions was not different from that expected in adult rabbit colon. Thus differences between adult and neonatal rabbit colon were small, and the increased conductance and unidirectional ion fluxes characteristic of the neonatal small intestine were not evident in the neonatal rabbit distal colon.

Antisecretory effect of prostaglandin D2 in rat colon in vitro: action sites

1991 ◽  
Vol 260 (6) ◽  
pp. G904-G910 ◽  
Author(s):  
K. J. Goerg ◽  
C. Diener ◽  
M. Diener ◽  
W. Rummel
Keyword(s):  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Rat Colon ◽  
Prostaglandin D2 ◽  
Cyclic Monophosphate ◽  
Heat Stable ◽  
Antisecretory Effect ◽  
Flux Measurements

The effect of prostaglandin D2 (PGD2) on colonic ion transport was studied in the Ussing chamber. PGD2 (10(-6) M) decreased baseline short-circuit current (Isc) in two preparations of rat colon descendens, a mucosa-submucosa preparation with and a mucosa preparation without the submucosal plexus. In both preparations, PGD2 inhibited the neuronally mediated secretory responses to electric field stimulation, the sea anemone toxin ATX II, and different cholinergic agents. Unidirectional flux measurements revealed that PGD2 diminished the secretagogue-induced increase in the serosal-to-mucosal flux of Cl- and thereby inhibited net Cl- secretion. PGD2, however, had no effect on the adenosine 3',5'-cyclic monophosphate-mediated response to forskolin or vasoactive intestinal peptide or on guanosine 3',5'-cyclic monophosphate-mediated secretion induced by the heat-stable enterotoxin of Escherichia coli. The PGD2 also blocked the increase in Isc evoked by two neuronally acting inflammatory mediators, i.e., bradykinin and PGI2 in the mucosa-submucosa preparation, but had no effect on the response to PGE2. Consequently, PGD2 exerts an indirect antisecretory effect caused by an inhibition of enteric secretomotor neurons of both the submucosal and the mucosal plexus.

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