Functional Assessment of Intestinal Tight Junction Barrier and Ion Permeability in Native Tissue by Ussing Chamber Technique

10.3791/62468 ◽  
2021 ◽  
Author(s):  
Wendy Hempstock ◽  
Noriko Ishizuka ◽  
Hisaysohi Hayashi
Keyword(s):  
Tight Junction ◽  
Functional Assessment ◽  
Ussing Chamber ◽  
Ion Permeability ◽  
Native Tissue ◽  
Chamber Technique

Permeability of human jejunal segments to gonyautoxins measured by the Ussing chamber technique

Toxicon ◽  
2004 ◽  
Vol 44 (5) ◽  
pp. 521-528 ◽  
Author(s):  
Pamela Mardones ◽  
Darío Andrinolo ◽  
Attila Csendes ◽  
Néstor Lagos
Keyword(s):  
Ussing Chamber ◽  
Chamber Technique

Human zonulin, a potential modulator of intestinal tight junctions

2000 ◽  
Vol 113 (24) ◽  
pp. 4435-4440 ◽  
Author(s):  
W. Wang ◽  
S. Uzzau ◽  
S.E. Goldblum ◽  
A. Fasano
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Ussing Chamber ◽  
Intestinal Lumen ◽  
Epithelial Surface ◽  
Ussing Chambers ◽  
Zonula Occludens ◽  
Surface Receptor ◽  
Dynamic Structures ◽  
Zonula Occludens Toxin

Intercellular tight junctions are dynamic structures involved in vectorial transport of water and electrolytes across the intestinal epithelium. Zonula occludens toxin derived from Vibrio cholerae interacts with a specific intestinal epithelial surface receptor, with subsequent activation of a complex intracellular cascade of events that regulate tight junction permeability. We postulated that this toxin may mimic the effect of a functionally and immunologically related endogenous modulator of intestinal tight junctions. Affinity-purified anti-zonula occludens toxin antibodies and the Ussing chamber assay were used to screen for one or more mammalian zonula occludens toxin analogues in both fetal and adult human intestine. A novel protein, zonulin, was identified that induces tight junction disassembly in non-human primate intestinal epithelia mounted in Ussing chambers. Comparison of amino acids in the active zonula occludens toxin fragment and zonulin permitted the identification of the putative receptor binding domain within the N-terminal region of the two proteins. Zonulin likely plays a pivotal role in tight junction regulation during developmental, physiological, and pathological processes, including tissue morphogenesis, movement of fluid, macromolecules and leukocytes between the intestinal lumen and the interstitium, and inflammatory/autoimmune disorders.

Transport of selenium species in Caco-2 cells: Analytical approach employing the Ussing chamber technique and HPLC-ICP-MS

2013 ◽  
Vol 110 ◽  
pp. 8-14 ◽  
Author(s):  
Denis Pick ◽  
Christian Degen ◽  
Matthias Leiterer ◽  
Gerhard Jahreis ◽  
Jürgen W. Einax
Keyword(s):  
Analytical Approach ◽  
Ussing Chamber ◽  
Selenium Species ◽  
Icp Ms ◽  
Chamber Technique

Abstract MP10: Furin-Mediated Modification Is Required For Epithelial Sodium Channel-Activating Activity Of Soluble (Pro)Renin Receptor In Cultured Collecting Duct Cells

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
Huaqing Zheng ◽  
Changjiang zou ◽  
Tianxin Yang
Keyword(s):  
Cleavage Site ◽  
Collecting Duct ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Separate Experiment ◽  
Pcr Analysis ◽  
Furin Cleavage ◽  
Empty Vector ◽  
Furin Cleavage Site ◽  
Chamber Technique

(Pro)renin receptor (PRR) contains overlapping cleavage site for site-1 protease (S1P) and furin for generation of soluble PRR (sPRR). Although S1P-mediated cleavage mediates the release of sPRR, the functional implication of furin-mediated cleavage is unclear. Here we tested whether furin-mediated cleavage was required for the activity of sPRR in activating ENaC in cultured M1 cells. M1 cells were transfected with pcDNA3.4 containing full-length PRR with (Furin Mut) or without (WT) mutagenesis of furin cleavage site; empty vector (EM) served a control. As compared with EM, overexpression of WT and Furin Mut vectors induced a more than 16-fold comparable increase in the release of sPRR. Amiloride-sensitive short circuit current as assessed by Ussing chamber technique was elevated by overexpression of WT PRR which was reduced by 37% by Furin Mut (ENaC activity: 1.00 + 0.06 μA/cm 2 in EM, 1.67 + 0.05 μA/cm 2 in WT, and 1.04 + 0.07 μA/cm 2 in Furin Mut, p < 0.05). In parallel, the expression of α-ENaC but not β or γ subunit as assessed by immunoblotting and qRT-PCR analysis was elevated by WT PRR and this increase was blunted by Furin Mut. In a separate experiment, M1 cells were transfected with pcDNA3.4 containing cDNA for sPRR with S1P cleavage (AA 1-282) (sPRR-S1P) or with furin cleavage (AA 1-279) (sPRR-furin); empty vector was used as a control. Overexpression of cDNA for the two types of sPRR induced a significant and comparable increase in the release of sPRR. By Ussing chamber technique, ENaC activity was 1.00 + 0.09 μA/cm 2 in EM, 1.03 + 0.10 μA/cm 2 in sPRR-S1P, and 1.39 + 0.14 μA/cm 2 in sPRR-furin, p < 0.05. Lastly, 293 cells were pretreated for 1 h with furin inhibitor α1-antitrypsin Portland followed by transfection with empty vector, WT PRR, or Furin Mut vectors. sPRR in the condition medium was enriched by using protein centrifugal filter devices and applied to M1 cells for 10 min followed by measurement of ENaC activity. Pretreatment with furin inhibition attenuated ENaC-acting activity induced by overexpression of WT PRR. Overall, the three independent approaches consistently demonstrated that furin-mediated modification is required for the activity of sPRR to increase ENaC-mediated Na + transport in the CD cells.

Applicability of the Ussing Chamber Technique to Permeability Determinations in Functionally Distinct Regions of the Gastrointestinal Tract in the Rat

2003 ◽  
Vol 38 (7) ◽  
pp. 732-741 ◽  
Author(s):  
Bajka B. H. ◽  
Gillespie C. M. ◽  
Steeb C.-B. ◽  
Read L. C. ◽  
Howarth G. S.
Keyword(s):  
Gastrointestinal Tract ◽  
Ussing Chamber ◽  
Chamber Technique

Effect of ammonia on Na+ transport across isolated rumen epithelium of sheep is diet dependent

2003 ◽  
Vol 90 (4) ◽  
pp. 751-758 ◽  
Author(s):  
Khalid Abdoun ◽  
Katarina Wolf ◽  
Gisela Arndt ◽  
Holger Martens
Keyword(s):  
Cellular Uptake ◽  
Ussing Chamber ◽  
Ammonium Ion ◽  
Na Transport ◽  
Adaptation Mechanism ◽  
Rumen Epithelium ◽  
Ruminal Epithelium ◽  
Positive Effect ◽  
Chamber Technique

The cellular uptake of ammonia affects the intracellular pH (pHi) of polar and non-polar cells. A predominant uptake of NH3 and its intra-cellular protonation tend to alkalinise the cytoplasm, whereas a predominant uptake of NH4+ acidifies the cytoplasm by reversing this reaction. Hence, the well-known absorption of ammonia across the rumen epithelium probably causes a change in the pHi. The magnitude and direction of this change in pHi (acid or alkaline) depends on the relative transport rates of NH3 and NH4+. Consequently, the intra-cellular availability of protons will influence the activity of the Na+-H+ exchanger, which could affect transepithelial Na+ transport. The aim of the present study has been to test this possible interaction between ruminal ammonia concentrations and Na+ transport. The term ammonia is used to designate the sum of the protonated (NH4+) and unprotonated (NH3) forms. Isolated ruminal epithelium of sheep was investigated by using the Ussing-chamber technique in vitro. The present results indicate that ammonia inhibits Na+ transport across the rumen epithelium of hay-fed sheep, probably by binding intracellular protons and thus inhibiting Na+-H+ exchange. By contrast, ammonia stimulates Na+ transport in concentrate-fed and urea-fed sheep, which develop an adaptation mechanism in the form of an increased metabolism of ammonia in the rumen mucosa and/or an increased permeability of rumen epithelium to the charged ammonium ion (NH4+). Intracellular dissociation of NH4+ increases the availability of protons, which stimulate Na+ –H+ exchange. This positive effect of ruminal ammonia on Na+ absorption may significantly contribute to the regulation of osmotic pressure of the ruminal fluid, because intraruminal ammonia concentrations up to 40 mmol/l have been reported.

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