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.

Ion transport across cat and ferret tracheal epithelia

1986 ◽  
Vol 61 (3) ◽  
pp. 1065-1070 ◽  
Author(s):  
R. J. Corrales ◽  
D. L. Coleman ◽  
D. B. Jacoby ◽  
G. D. Leikauf ◽  
H. L. Hahn ◽  
...  
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Free Medium ◽  
Mucin Secretion ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Mucosal Side

Sheets of trachea from ferret and cat were mounted in Ussing chambers and continuously short circuited. Under resting conditions, in both the cat and ferret there was little or no Cl secretion, and Na absorption accounted for most of the short-circuit current (Isc). Ouabain (10(-4) M, serosal bath) reduced Isc to zero in 30–60 min. This decline was matched by a decrease in net Na absorption. Amiloride (10(-4) M, luminal bath) caused a significant decrease in Isc and conductance (G) in both species. Bumetanide (10(-4) M, serosal bath) had negligible effects on Isc and G. In both species, isoproterenol increased Isc by stimulating Cl secretion. Methacholine induced equal amounts of Na and Cl secretion, with little change in Isc. In the cat, prostaglandins E2 and F2 alpha and bradykinin increased Isc, responses which were abolished in Cl-free medium. In open-circuited cat tissues, Na flux from the serosal to mucosal side was measured simultaneously with the secretion of nondialyzable 35S. Prostaglandins E1, E2, and F2 alpha, histamine, bradykinin, methacholine and isoproterenol all increased both Na and 35S-mucin secretion.

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 Effects of Combined or Along VFA, pH, Lipopolysaccharide and Histamine on the Rumen Epithelial Permeability of Dairy Goats In Vitro

2021 ◽  
Vol 26 (01) ◽  
pp. 79-86
Author(s):  
YY Sun
Keyword(s):  
Short Circuit ◽  
Barrier Dysfunction ◽  
Dairy Goats ◽  
Ussing Chambers ◽  
Ruminal Ph ◽  
Buffer Solutions ◽  
Ruminal Epithelium ◽  
Mucosal Side ◽  
Epithelial Barrier Dysfunction

This study investigated whether concurrent presence of lipopolysaccharide (LPS) and histamine (HIS) have the potential to increase permeability of the ruminal epithelium at physiological pH and acidotic ruminal pH. Nine 2.5-year-old female lactating Saanen dairy goats (42.79 ± 5.61 kg of BW; Mean ± SD) were used as a ruminant model. ruminal epithelium of goats were collected and mounted in Ussing chambers on their mucosal side in different gradient buffer solutions (pH 7.4, 5.5 and 5.2) containing LPS (0, 30 and 60 KEU·mL-1) or HIS (0, 0.5 and 10 ng·mL-1). The rumen epithelial electrophysiological indexes, such as short-circuit (Isc), tissue conductance (Gt) and the permeability of marker molecules of different sizes (horseradish peroxidase, HRP and fluorescein 5(6)-isothiocyanate, FITC) from the mucosal to the serosal side, were measured. Both Isc and Gt were increased, accompanied by enhanced flux of FITC, with a decrease of mucosal pH (P < 0.05). The addition of LPS at mucosal pH 5.2 significantly increased Isc, Gt and FITC flux rates and decreased potential difference (PD) (P < 0.05). Additionally, the concurrent presence of LPS and HIS at both physiological and acidotic ruminal pH also significantly increased the permeability of ruminal epithelium asevidenced by increasing Isc, Gt and FITC flux rates and decreasing PD. In summary, our results have shown that concurrent presence of LPS 60 KEU‧mL-1 and HIS 10 ng‧mL-1 at mucosal pH 5.5 can increase the permeability of ruminal epithelium. The combination of low pH and both high LPS and HIS may increase vulnerability to aggravated rumen epithelial barrier dysfunction. © 2021 Friends Science Publishers

Opiate activity and transepithelial passage of intact beta-casomorphins in rabbit ileum

1987 ◽  
Vol 253 (6) ◽  
pp. G737-G744 ◽  
Author(s):  
D. Tome ◽  
A. M. Dumontier ◽  
M. Hautefeuille ◽  
J. F. Desjeux
Keyword(s):  
Short Circuit ◽  
Opiate Receptors ◽  
Short Circuit Current ◽  
Enzymatic Digestion ◽  
Rabbit Ileum ◽  
Ussing Chambers ◽  
Luminal Side ◽  
Mucosal Side ◽  
Natural Peptides ◽  
Hydrolysis Of

The functional significance of the presence of opioid peptides in enzymatic digestion of food proteins remains uncertain. The effect of natural beta-casomorphins (beta-CMs), beta-CM-5 and beta-CM-4-NH2 (morphiceptine), and the analogue beta-[D-Ala2,4,Tyr5]CM-5-NH2 were studied in isolated rabbit ileum mounted in Ussing chambers. All three peptides caused a naloxone-reversible reduction in short-circuit current (Isc) after addition at a concentration of 10(-4) M to the serosal side of the tissue. After addition to the mucosal side, only the analogue beta-[D-Ala2,4,Tyr5]CM-5-NH2 reduced Isc. Natural beta-CMs were degraded by the intestinal mucosa, and no intact transepithelial passage was detected for these peptides, whereas beta-[D-Ala2,4,Tyr5]CM-5-NH2 was demonstrated to cross the epithelium intact when added at a concentration of 10(-3) M in the mucosal reservoir (mucosal-to-serosal flux = 3.5 +/- 1.2 nmol.h-1.cm-2). These results show that both natural beta-CMs and the protected analogue have an opiate activity on intestinal electrolyte transport. Their action from the luminal side of the intestine seems to depend on the transfer of the intact peptides from the luminal to the blood side of the tissue where opiate receptors are located. This action is prevented by luminal hydrolysis of the natural peptides.

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.

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.

Suppression of gastric acid secretion by furosemide in isolated gastric mucosa of guinea pig

1980 ◽  
Vol 239 (6) ◽  
pp. G532-G535 ◽  
Author(s):  
A. Ayalon ◽  
A. Corcia ◽  
G. Klemperer ◽  
S. R. Caplan
Keyword(s):  
Guinea Pig ◽  
Acid Secretion ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Electrical Conductance ◽  
Short Circuit Current ◽  
Cl Transport ◽  
Consequent Reduction ◽  
Net Flux ◽  
Mucosal Side

The effect of furosemide on acid secretion and Cl- transport was studied in isolated fundic mucosa of the guinea pig. Furosemide (10(-3) M), applied to the serosal side produced an immediate effect on the short-circuit current (Isc), lowering it by 47 +/- 2%. Potential difference decreased by 29 +/- 3%, electrical conductance by 18 +/- 4%, acid secretion by 38 +/- 1%, and net flux of Cl- from serosal-to-mucosal side by 37%. Application of the drug to the mucosal side produced similar effects on acid secretion and on the electrical parameters. It is suggested that furosemide blocks the entrance of Cl-, by the Na+--Cl- cotransport mechanism, through the basolateral membrane of the secreting cell. The consequent reduction in electrogenic Cl- transport would cause Isc and acid secretion to decrease. A reduction of Cl- conductance of the apical membrane, upon mucosal application of the drug, would cause similar effects on acid secretion and Cl- transport.

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.

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