scholarly journals Butyrate Permeation across the Isolated Ovine Reticulum Epithelium

Animals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 2198
Author(s):  
Reiko Rackwitz ◽  
Franziska Dengler ◽  
Gotthold Gäbel
Keyword(s):  
Fatty Acids ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Short Chain Fatty Acids ◽  
Monocarboxylate Transporters ◽  
Buffer Solution ◽  
Mean Values ◽  
Ussing Chambers ◽  
Rumen Epithelium ◽  
The Mean

We hypothesized that, due to the high pH of this compartment, the reticulum epithelium displays particular features in the transport of short-chain fatty acids (SCFA). Ovine reticulum epithelium was incubated in Ussing chambers using a bicarbonate-free buffer solution containing butyrate (20 mmol L−1). p-hydroxymercuribenzoic acid (pHMB), 5-(N-Ethyl-N-isopropyl)amiloride (EIPA), or ouabain were added to the buffer solution as inhibitors of monocarboxylate transporters, sodium-proton-exchangers, or the Na+/K+-ATPase, respectively. The short-circuit current (Isc) and transepithelial conductance (Gt) were monitored continuously while the flux rates of 14C-labelled butyrate were measured in the mucosal-to-serosal (Jmsbut) or serosal-to-mucosal direction (Jsmbut). Under control conditions, the mean values of Isc and Gt amounted to 2.54 ± 0.46 µEq cm−2 h−1 and 6.02 ± 3.3 mS cm−2, respectively. Jmsbut was 2.1 ± 1.01 µmol cm−2 h−1 on average and about twice as high as Jsmbut. Incubation with ouabain reduced Jmsbut, while Jsmbut was not affected. The serosal addition of EIPA did not affect Jmsbut but reduced Jsmbut by about 10%. The addition of pHMB to the mucosal or serosal solution reduced Jmsbut but had no effect on Jsmbut. Mucosally applied pHMB provoked a transient increase in the Isc. The serosal pHMB sharply reduced Isc. Our results demonstrate that butyrate can be effectively transported across the reticulum epithelium. The mechanisms involved in this absorption differ from those known from the rumen epithelium.

scholarly journals Butyrate Protects Porcine Colon Epithelium from Hypoxia-Induced Damage on a Functional Level

Nutrients ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 305
Author(s):  
Franziska Dengler ◽  
Anika Kraetzig ◽  
Gotthold Gäbel
Keyword(s):  
Fatty Acids ◽  
Intracellular Signaling ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Short Chain Fatty Acids ◽  
Protective Mechanism ◽  
Bacterial Fermentation ◽  
Ussing Chambers ◽  
Pathological Conditions ◽  
Simulated Hypoxia

The large intestinal epithelium is confronted with the necessity to adapt quickly to varying levels of oxygenation. In contrast to other tissues, it meets this requirement successfully and remains unharmed during (limited) hypoxic periods. The large intestine is also the site of bacterial fermentation producing short-chain fatty acids (SCFA). Amongst these SCFA, butyrate has been reported to ameliorate many pathological conditions. Thus, we hypothesized that butyrate protects the colonocytes from hypoxic damage. We used isolated porcine colon epithelium mounted in Ussing chambers, incubated it with or without butyrate and simulated hypoxia by changing the gassing regime to test this hypothesis. We found an increase in transepithelial conductance and a decrease in short-circuit current across the epithelia when simulating hypoxia for more than 30 min. Incubation with 50 mM butyrate significantly ameliorated these changes to the epithelial integrity. In order to characterize the protective mechanism, we compared the effects of butyrate to those of iso-butyrate and propionate. These two SCFAs exerted similar effects to butyrate. Therefore, we propose that the protective effect of butyrate on colon epithelium under hypoxia is not (only) based on its nutritive function, but rather on the intracellular signaling effects of SCFA.

Short-chain fatty acids inhibit cAMP-mediated chloride secretion in rat colon

1996 ◽  
Vol 271 (6) ◽  
pp. C1853-C1860 ◽  
Author(s):  
P. C. Dagher ◽  
R. W. Egnor ◽  
A. Taglietta-Kohlbrecher ◽  
A. N. Charney
Keyword(s):  
Fatty Acids ◽  
Short Circuit ◽  
Short Chain Fatty Acids ◽  
Short Chain ◽  
Rat Colon ◽  
Sprague Dawley ◽  
T84 Cells ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Chain Fatty Acids

Butyrate stimulates salt absorption in mammalian colon. We examined whether butyrate also affects Cl- secretion. Mucosal segments of distal colon of male Sprague-Dawley rats and T84 cells were studied in Ussing chambers. In control colon, 1 mM dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) increased short-circuit current (Isc) and serosal-to-mucosal Cl- flux (JsmCl) by 3.2 +/- 0.8 and 2.9 +/- 0.8 mueq.cm-2.h-1, respectively. Mucosal or serosal 25 mM butyrate prevented DBcAMP-induced increases in Isc and JsmCl. Four and eight millimolar butyrate caused half-maximal inhibition of the increases in JsmCl and Isc, respectively. Butyrate also inhibited basal JsmCl (by 2.0 +/- 0.4 mueq.cm-2.h-1) but not carbachol-mediated Cl- secretion. The relative inhibitory potency at 25 mM of other short-chain fatty acids (SCFA) paralleled their degree of cellular metabolism: butyrate > acetate = propionate > isobutyrate. At 25 mM, all SCFA reduced mucosal intracellular pH (pHi) transiently by 0.1 pH unit. In intact T84 cells, 50 mM butyrate inhibited the DBcAMP-induced rise in Isc by 55%. In T84 cells with nystatin-permeabilized basolateral membranes, butyrate inhibited the increase in Isc by 82%. We conclude that butyrate inhibits basal and cAMP-mediated Cl- secretion by a mechanism independent of pHi, possibly located at the apical membrane.

Effect of chloride on pH microclimate and electrogenic Na+ absorption across the rumen epithelium of goat and sheep

2006 ◽  
Vol 291 (2) ◽  
pp. G246-G252 ◽  
Author(s):  
S. Leonhard-Marek ◽  
G. Breves ◽  
R. Busche
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Epithelial Surface ◽  
Ussing Chambers ◽  
Rumen Epithelium ◽  
Microclimate Ph ◽  
Cation Conductance ◽  
Sheep Rumen

Active Na+ absorption across rumen epithelium comprises Na+/H+ exchange and a nonselective cation conductance (NSCC). Luminal chloride is able to stimulate Na+ absorption, which has been attributed to an interaction between Cl−/HCO3− and Na+/H+ exchangers. However, isolated rumen epithelial cells also express a Cl− conductance. We investigated whether Cl− has an additional effect on electrogenic Na+ absorption via NSCC. NSCC was estimated from short-circuit current ( Isc) across epithelia of goat and sheep rumen in Ussing chambers. Epithelial surface pH (pHs) was measured with 5- N-hexadecanoyl-aminofluorescence. Membrane potentials were measured with microelelectrodes. Luminal, but not serosal, Cl− stimulated the Ca2+ and Mg2+ sensitive Isc. This effect was independent of the replacing anion (gluconate or acetate) and of the presence of bicarbonate. The mean pHs of rumen epithelium amounted to 7.47 ± 0.03 in a low-Cl− solution. It was increased by 0.21 pH units when luminal Cl− was increased from 10 to 68 mM. Increasing mucosal pH from 7.5 to 8.0 also increased the Ca2+ and Mg2+ sensitive Isc and transepithelial conductance and reduced the fractional resistance of the apical membrane. Luminal Cl− depolarized the apical membrane of rumen epithelium. 5-Nitro-2-(3-phenylpropylamino)-benzoate reduced the divalent cation sensitive Isc, but only in low-Cl− solutions. The results show that luminal Cl− can increase the microclimate pH via apical Cl−/HCO3− or Cl−/OH− exchangers. Electrogenic Na+ absorption via NSCC increases with pH, explaining part of the Cl− effects on Na+ absorption. The data further show that the Cl− conductance of rumen epithelium must be located at the basolateral membrane.

Functional and molecular biological evidence of SGLT-1 in the ruminal epithelium of sheep

2000 ◽  
Vol 279 (1) ◽  
pp. G20-G27 ◽  
Author(s):  
Jörg R. Aschenbach ◽  
Heike Wehning ◽  
Martina Kurze ◽  
Elisabeth Schaberg ◽  
Hermann Nieper ◽  
...  
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Short Chain Fatty Acids ◽  
Glucose Absorption ◽  
Minor Importance ◽  
Mrna Levels ◽  
Ussing Chambers ◽  
Ruminal Epithelium ◽  
Cloned Cdna ◽  
Mucosal Side

Because of the effective catabolism ofd-glucose to short-chain fatty acids by intraruminal microorganisms, the absorption of d-glucose from the rumen was thought to be of minor importance. However, clinical studies suggested that significant quantities of d-glucose are transported from the ruminal contents to the blood. We therefore tested the ruminal epithelium of sheep for the presence of Na+-glucose cotransporter 1 (SGLT-1) on both the functional and mRNA levels. In the absence of an electrochemical gradient, 3- O-methylglucose (3-OMG) was net absorbed across isolated ruminal epithelia mounted in Ussing chambers. The net transport of 3-OMG followed Michaelis-Menten kinetics and was sensitive to phlorizin or decreasing Na+concentrations. The mucosal addition of 10 mM d-glucose induced an immediate, phlorizin-sensitive increase in short-circuit current ( Isc). Isccould also be increased by serosal addition of d-glucose or d-mannose, but electrogenic uptake of d-glucose or 3-OMG added on the mucosal side was still detectable after serosal stimulation of Isc. RT-PCR using primers specific for the ovine intestinal SGLT-1 with subsequent TA cloning and sequencing revealed 100% identity between the cloned cDNA and mRNA fragment 187–621 of ovine intestinal SGLT-1. In conclusion, the ruminal epithelium has a high-affinity SGLT-1, which indicates that it maintains the capacity for d-glucose absorption.

Protein kinase C potentiates cAMP-stimulated mouse duodenal mucosal bicarbonate secretion in vitro

2004 ◽  
Vol 286 (5) ◽  
pp. G814-G821 ◽  
Author(s):  
Bi-Guang Tuo ◽  
Jimmy Y. C. Chow ◽  
Kim E. Barrett ◽  
Jon I. Isenberg
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Duodenal Mucosa ◽  
Bicarbonate Secretion ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ussing Chambers ◽  
Duodenal Bicarbonate Secretion

PKC has been shown to regulate epithelial Cl- secretion in a variety of models. However, the role of PKC in duodenal mucosal bicarbonate secretion is less clear. We aimed to investigate the role of PKC in regulation of duodenal mucosal bicarbonate secretion. Bicarbonate secretion by murine duodenal mucosa was examined in vitro in Ussing chambers using a pH-stat technique. PKC isoform expression and activity were assessed by Western blotting and in vitro kinase assays, respectively. PMA (an activator of PKC) alone had no effect on duodenal bicarbonate secretion or short-circuit current ( Isc). When PMA and dibutyryl-cAMP (db-cAMP) were added simultaneously, PMA failed to alter db-cAMP-stimulated duodenal bicarbonate secretion or Isc ( P > 0.05). However, a 1-h preincubation with PMA potentiated db-cAMP-stimulated duodenal bicarbonate secretion and Isc in a concentration-dependent manner (from 10-8 to 10-5M) ( P < 0.05). PMA preincubation had no effects on carbachol- or heat-stable toxin-stimulated bicarbonate secretion. Western blot analysis revealed that PKCα, -γ, -ϵ, -θ, -μ, and -ι/λ were expressed in murine duodenal mucosa. Ro 31–8220 (an inhibitor active against PKCϵ, -α, -β, and -γ), but not Gö 6983 (an inhibitor active against PKCα, -γ, -β, and -δ), reversed the potentiating effect of PMA on db-cAMP-stimulated bicarbonate secretion. PMA also time- and concentration-dependently increased the activity of PKCϵ, an effect that was prevented by Ro 31–8220 but not Gö 6983. These results demonstrate that activation of PKC potentiates cAMP-stimulated duodenal bicarbonate secretion, whereas it does not modify basal secretion. The effect of PKC on cAMP-stimulated bicarbonate secretion is mediated by the PKCϵ isoform.

Effect of pH on chloride absorption in the flounder intestine

1988 ◽  
Vol 255 (2) ◽  
pp. G247-G252 ◽  
Author(s):  
A. N. Charney ◽  
J. I. Scheide ◽  
P. M. Ingrassia ◽  
J. A. Zadunaisky
Keyword(s):  
Small Intestine ◽  
Short Circuit ◽  
Ph Effect ◽  
Short Circuit Current ◽  
Bathing Solution ◽  
Solution Ph ◽  
Transport Pathway ◽  
Ussing Chambers ◽  
Chloride Absorption ◽  
In Series

Chloride absorption in the small intestine of the winter flounder, Pseudopleuronectes americanus, is reported to be sensitive to ambient pH. We studied this sensitivity in isolated stripped intestinal mucosa mounted in modified Ussing chambers. Unidirectional 36Cl fluxes (JClm----s, JCls----m) were measured under short-circuited conditions in bathing solutions containing various combinations of HCO3- (0-20 mM), partial pressure of CO2 (0-36 mmHg), and pH (6.77-7.85). We found that JClm----s, net 36Cl flux (JClnet), and short-circuit current (Isc) increased and JCls----m decreased predominately in response to increases in bathing solution pH. There was a linear relationship between pH and both JClnet (r = 0.92, P less than 0.01) and Isc (r = 0.96, P less than 0.005) between pH 6.77 and 7.74. The pH effect was completely reversible, did not require either CO2 or HCO3-, and was not affected by the presence of mucosal barium at 1 mM. Mucosal bumetanide (0.1 mM) completely inhibited the pH effect. These data suggest that the process by which Cl- is absorbed in the flounder intestine is sensitive to pH. The data do not indicate whether pH affects Na+-K+-2Cl- cotransport or a Cl- transport pathway in series with this process. The direction of Cl- absorption in response to pH contrasts with inverse relation of pH and Cl- absorption in mammalian small intestine.

Active electrolyte transport in mammalian buccal mucosa

1988 ◽  
Vol 255 (3) ◽  
pp. G286-G291 ◽  
Author(s):  
R. C. Orlando ◽  
N. A. Tobey ◽  
V. J. Schreiner ◽  
R. D. Readling
Keyword(s):  
Buccal Mucosa ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Electrical Potential ◽  
Short Circuit Current ◽  
Electrolyte Transport ◽  
Electrical Potential Difference ◽  
Ussing Chambers ◽  
Net Fluxes

The transmural electrical potential difference (PD) was measured in vivo across the buccal mucosa of humans and experimental animals. Mean PD was -31 +/- 2 mV in humans, -34 +/- 2 mV in dogs, -39 +/- 2 mV in rabbits, and -18 +/- 1 mV in hamsters. The mechanisms responsible for this PD were explored in Ussing chambers using dog buccal mucosa. After equilibration, mean PD was -16 +/- 2 mV, short-circuit current (Isc) was 15 +/- 1 microA/cm2, and resistance was 1,090 +/- 100 omega.cm2, the latter indicating an electrically "tight" tissue. Fluxes of [14C]mannitol, a marker of paracellular permeability, varied directly with tissue conductance. The net fluxes of 22Na and 36Cl were +0.21 +/- 0.05 and -0.04 +/- 0.02 mueq/h.cm2, respectively, but only the Na+ flux differed significantly from zero. Isc was reduced by luminal amiloride, serosal ouabain, or by reducing luminal Na+ below 20 mM. This indicated that the Isc was determined primarily by active Na+ absorption and that Na+ traverses the apical membrane at least partly through amiloride-sensitive channels and exits across the basolateral membrane through Na+-K+-ATPase activity. We conclude that buccal mucosa is capable of active electrolyte transport and that this capacity contributes to generation of the buccal PD in vivo.

Mechanisms of sodium and chloride transport across equine tracheal epithelium

1990 ◽  
Vol 259 (6) ◽  
pp. L459-L467 ◽  
Author(s):  
G. J. Tessier ◽  
T. R. Traynor ◽  
M. S. Kannan ◽  
S. M. O3'Grady
Keyword(s):  
Chloride Transport ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Tracheal Epithelium ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Cotransport System ◽  
Ethanesulfonic Acid

Equine tracheal epithelium, stripped of serosal muscle, mounted in Ussing chambers, and bathed in plasmalike Ringer solution generates a serosa-positive transepithelial potential of 10–22 mV and a short-circuit current (Isc) of 70–200 microA/cm2. Mucosal amiloride (10 microM) causes a 40–60% decrease in Isc and inhibits the net transepithelial Na flux by 95%. Substitution of Cl with gluconate resulted in a 30% decrease in basal Isc. Bicarbonate substitution with 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid decreased the Isc by 21%. The Cl-dependent Isc was inhibited by serosal addition of 1 mM amiloride. Bicarbonate replacement or serosal amiloride (1 mM) inhibits the net Cl flux by 72 and 69%, respectively. Bicarbonate replacement significantly reduces the effects of serosal amiloride (1 mM) on Isc, indicating its effect is HCO3 dependent. Addition of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP; 100 microM) causes a 40% increase in Isc. This effect is inhibited by subsequent addition of 10 microM serosal bumetanide. Bumetanide (10 microM) reduces net Cl secretion following stimulation with 8-BrcAMP (100 microM). Serosal addition of BaCl2 (1 mM) causes a reduction in Isc equal to that following Cl replacement in the presence or absence of 100 microM cAMP. These results suggest that 1) Na absorption depends on amiloride-inhibitable Na channels in the apical membrane, 2) Cl influx across the basolateral membrane occurs by both a Na-H/Cl-HCO3 parallel exchange mechanism under basal conditions and by a bumetanide-sensitive Na-(K?)-Cl cotransport system under cAMP-stimulated conditions, and 3) basal and cAMP-stimulated Cl secretion depends on Ba-sensitive K channels in the basolateral membrane.

Alterations in capsaicin-evoked electrolyte transport during the evolution of guinea pig TNBS ileitis

2002 ◽  
Vol 282 (6) ◽  
pp. G972-G980 ◽  
Author(s):  
Paula Miceli ◽  
Gerald P. Morris ◽  
Wallace K. MacNaughton ◽  
Stephen Vanner
Keyword(s):  
Substance P ◽  
Guinea Pig ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Electrolyte Transport ◽  
Secretory Function ◽  
Trinitrobenzenesulfonic Acid ◽  
Ussing Chambers ◽  
Circulating Cytokines

The efferent secretomotor activity of capsaicin-sensitive nerves was monitored during the evolution of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced ileitis in the guinea pig by recording changes in short-circuit current (Δ I sc) in response to capsaicin, substance P (SP), and carbachol. Submucosal-mucosal preparations mounted in standard Ussing chambers were studied at time 0, at 8 h, and 1, 3, 5, 7, 14, and 30 days following the intraluminal instillation of TNBS or saline. Maximal Δ I scresponses to capsaicin were dramatically attenuated (54%) by 24 h. By day 7, SP- and TTX-insensitive carbachol-stimulated Δ I sc were also significantly reduced. Similar attenuation in capsaicin and carbachol responses was observed in jejunal tissue 20 cm proximal to the inflamed site at day 7. These studies demonstrate that efferent secretomotor function of capsaicin-sensitive nerves is maintained early in TNBS ileitis but significantly reduced by 24 h. By day 7, defects in enterocyte secretory function at inflamed and noninflamed sites also occurred, an effect that may be mediated by circulating cytokines.

ENaC- and CFTR-dependent ion and fluid transport in mammary epithelia

2001 ◽  
Vol 281 (2) ◽  
pp. C633-C648 ◽  
Author(s):  
Sasha Blaug ◽  
Kevin Hybiske ◽  
Jonathan Cohn ◽  
Gary L. Firestone ◽  
Terry E. Machen ◽  
...  
Keyword(s):  
Tight Junctions ◽  
Fluid Transport ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Equivalent Circuit Analysis ◽  
Mean Values ◽  
Apical Side

Mammary epithelial 31EG4 cells (MEC) were grown as monolayers on filters to analyze the apical membrane mechanisms that help mediate ion and fluid transport across the epithelium. RT-PCR showed the presence of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial Na+ channel (ENaC) message, and immunomicroscopy showed apical membrane staining for both proteins. CFTR was also localized to the apical membrane of native human mammary duct epithelium. In control conditions, mean values of transepithelial potential (apical-side negative) and resistance ( R T) are −5.9 mV and 829 Ω · cm2, respectively. The apical membrane potential ( V A) is −40.7 mV, and the mean ratio of apical to basolateral membrane resistance ( R A/ R B) is 2.8. Apical amiloride hyperpolarized V A by 19.7 mV and tripled R A/ R B. A cAMP-elevating cocktail depolarized V A by 17.6 mV, decreased R A/ R B by 60%, increased short-circuit current by 6 μA/cm2, decreased R T by 155 Ω · cm2, and largely eliminated responses to amiloride. Whole cell patch-clamp measurements demonstrated amiloride-inhibited Na+ currents [linear current-voltage ( I-V) relation] and forskolin-stimulated Cl−currents (linear I-V relation). A capacitance probe method showed that in the control state, MEC monolayers either absorbed or secreted fluid (2–4 μl · cm−2 · h−1). Fluid secretion was stimulated either by activating CFTR (cAMP) or blocking ENaC (amiloride). These data plus equivalent circuit analysis showed that 1) fluid absorption across MEC is mediated by Na+ transport via apical membrane ENaC, and fluid secretion is mediated, in part, by Cl− transport via apical CFTR; 2) in both cases, appropriate counterions move through tight junctions to maintain electroneutrality; and 3) interactions among CFTR, ENaC, and tight junctions allow MEC to either absorb or secrete fluid and, in situ, may help control luminal [Na+] and [Cl−].

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