Butyrate Permeation across the Isolated Ovine Reticulum Epithelium.

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.

Possible influence of free fatty acid receptors on pH regulation in the ruminal epithelium of sheep.

High amounts of short-chain fatty acids (SCFAs) occur in the ovine rumen and constitute the animal's main energy source. However, they lead to an acidification of the ruminal epithelium. Therefore, effective intracellular pH (pHi ) regulation by transport proteins like monocarboxylate transporter 1 (MCT1) and Na+ /H+ exchangers (NHEs) is pivotal to ruminants to avoid epithelial damage. SCFAs might function not only as nutrients but also as signalling molecules by activating free fatty acid receptors (FFARs) in the ruminal epithelium and thus influence pHi regulation. FFARs work as nutrient sensors, transducing their information by modulating cyclic adenosine monophosphate (cAMP) levels. We hypothesized that (FFAR-modulated) decreases in cAMP levels stimulate the activity of MCT1 and NHEs in the ruminal epithelium of sheep. We detected two FFARs (GPR109A and FFAR2) immunohistochemically in the ovine ruminal epithelium. Administration of 10 mM butyrate to Ussing chamber-mounted epithelia provoked a significant reduction in intraepithelial cAMP levels. However, application of the GPR109A agonist niacin did not affect cAMP levels. MCT1 activity was analysed by measuring transepithelial 14 C-acetate fluxes, which were not inhibited by forskolin-induced increased cAMP levels. The recovery of pHi after acidification was assessed as an indicator of NHE activity in primary cultured ruminal epithelial cells. Recovery was significantly reduced when cells with increased cAMP levels were subjected to the NHE inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (10 µM). Nonetheless, with augmented cAMP levels alone, NHE activity tended to decline. We hypothesize that modulation of cAMP levels by butyrate is accomplished by FFAR2 activation, regulating NHE activity for pHi homoeostasis at least in part.

Permeation of acetate across sheep ruminal epithelium is partly mediated by an anion channel.

The present study pursued the question if an anion channel could be partly responsible for the transport of acetate in the isolated ovine ruminal epithelium. Using the Ussing chamber technique, changes of short-circuit current (Isc) induced by mucosal or serosal addition of acetate was investigated. To further evaluate the Isc changes induced by acetate the epithelia were preincubated with ouabain or 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). In addition, unidirectional flux rates of 14C-acetate were measured at different transepithelial potential difference (Vt). At Vt=0mV, acetate addition to the mucosal side of epithelia incubated in ouabain/DIDS free solution resulted in an Isc decrease, whereas application to the serosal side induced an Isc increase. Absolute Isc changes were significantly larger after serosal than after mucosal acetate addition. Ouabain pre-incubation abolished these side-specific differences. Pre-incubation with DIDS on the mucosal side inhibited the current induced by subsequent mucosal acetate addition in a voltage dependent manner, whereas serosal DIDS pre-incubation had no effect on acetate-induced changes of Isc. Mucosal-to-serosal acetate flux was partly voltage-dependent. The serosal-to-mucosal flux of acetate was not influenced by Vt. The asymmetric changes of Isc after acetate application and the Vt dependence of the DIDS inhibition on the mucosal side indicate a partly electrogenic transcellular permeation of acetate which includes an apically located acetate conductance. It is suggested that this conductance may be important for epithelial cell homeostasis.

Glucose transport across lagomorph jejunum epithelium is modulated by AMP-activated protein kinase under hypoxia.

The gastrointestinal epithelium possesses adaptation mechanisms to cope with huge variations in blood flow and subsequently oxygenation. Since sufficient energy supply is crucial under hypoxic conditions, glucose uptake especially must be regulated by these adaptation mechanisms. Therefore, we investigated glucose transport under hypoxic conditions. Jejunal epithelia of rabbits were incubated in Ussing chambers under short-circuit current conditions. Hypoxia was simulated by gassing with 1% O2 instead of 100% O2. The activity of sodium-coupled glucose transporter-1 (SGLT-1) was assessed by measuring the increase of short circuit current ( Isc) after the addition of 2 mM glucose to the mucosal buffer solution. We observed decreased activity of SGLT-1 after hypoxia compared with control conditions. To investigate underlying mechanisms, epithelia were exposed to agonists and antagonists of AMP-activated protein kinase (AMPK) before assessment of SGLT-1-mediated transport and the pAMPK/AMPK protein ratio. Preincubation with the antagonist restored SGLT-1 activity under hypoxic conditions to the level of control conditions, indicating an involvement of AMPK in the downregulation of SGLT-1 activity under hypoxia, which was confirmed in Western blot analysis of pAMPK/AMPK. Transepithelial flux studies using radioactively labeled glucose, ortho-methyl-glucose, fructose, and mannitol revealed no changes after hypoxic incubation. Therefore, we could exclude a decreased transepithelial glucose transport rate and increased paracellular conductance under hypoxia. In conclusion, our study hints at a decreased activity of SGLT-1 under hypoxic conditions in an AMPK-dependent manner. However, transepithelial transport of glucose is maintained. Therefore, we suggest other transport mechanisms, especially glucose transporter 1 and/or 2 to substitute SGLT-1 under hypoxia. NEW & NOTEWORTHY To our knowledge, this is the first approach to simulate hypoxia and study its effects in the jejunal epithelium using the Ussing chamber technique. We were able show that AMPK plays a role in the downregulation of SGLT-1 and that there seems to be an upregulation of other glucose transport mechanisms in the apical membrane of lagomorph jejunum epithelium under hypoxia, securing the epithelial energy supply and thus integrity.

Epithelial capacity for apical uptake of short chain fatty acids is a key determinant for intraruminal pH and the susceptibility to subacute ruminal acidosis in sheep.

Subacute ruminal acidosis (SARA) is a common digestive disorder occurring in ruminants, with considerable variation in the severity of SARA observed among animals fed the same diet. Our aim in this study was to determine whether differences in the capacity of the ruminal epithelium for the apical uptake of acetate and butyrate (determined in Ussing chambers after slaughter) explains differences observed for the severity of a preceding episode of SARA in vivo. Adult sheep with an indwelling small ruminant ruminal pH measurement system (SRS) were randomly assigned to either a SARA induction treatment (oral drench containing 5 g glucose/kg body weight; n = 17) or a sham treatment (SHAM; n = 7; 12 mL water/kg body weight). Sheep receiving the glucose drench were further classified as nonresponders (NR; n = 7) or responders (RES; n = 7) according to their ruminal pH profile for the 3 h following the oral drench. Mean ruminal pH for the 3 h following the drench differed among groups (P < 0.001), with it being highest for SHAM (6.67 +/- 0.08), intermediate for NR (5.97 +/- 0.05), and lowest for RES (5.57 +/- 0.08) sheep. The apical uptake of acetate and butyrate did not differ between SHAM and RES sheep. However, NR sheep had greater in vitro apical uptake of acetate and butyrate and a higher plasma beta-hydroxybutyrate concentration than RES sheep, suggesting greater absorptive capacity for NR. Differences between NR and RES were attributed to greater bicarbonate-independent, nitrate-sensitive uptake of acetate (P = 0.007), a tendency for greater bicarbonate-dependent uptake of acetate (P = 0.071), and greater bicarbonate-independent uptake of butyrate (P = 0.022). These data indicate that differences in the rates and pathways for the uptake of acetate and butyrate explain a large proportion of the individual variation observed for the severity of SARA.