The intraruminal redox potential is stabilised by opposing influences during fermentation.

An optimal fermentation process in the forestomach is pivotal for the wellbeing and performance of ruminants. Complex carbohydrates are broken down into short-chain fatty acids (SCFA) which form the major energy source for the animal. A strong interrelationship of this process with intraruminal pH and redox potential (Eh) exists. These parameters can be measured with intraruminal sensors, but the interpretation of the measurements, especially of Eh, and their meaning for intraruminal homeostasis is not completely clear. In this study, factors influencing intraruminal Eh were elucidated. We hypothesised that intraruminal Eh is influenced by the fermentation process as such, but not by its end products SCFA. We measured Eh and pH in ruminal fluid from fasting cannulated sheep after the addition of 0.06 m Na-acetate, -propionate, -butyrate or glucose in vitro. Furthermore, we assessed the interrelation of pH and Eh. Basal Eh and pH values were -120 ± 41 mV and 7.0 ± 0.3, respectively, in native ruminal fluid in vitro. While the addition of SCFA did not induce any changes, glucose addition caused a significant decrease in both pH and Eh compared to the values before the addition (paired Student's t-test, p < 0.05). We attribute the decrease in Eh to an increased production of H2 in the process of generating SCFA, predominantly acetate. By titrating both native and particle-free ruminal fluid to more acidic and basic pH values (4.5-8.5), we found a non-linear inverse correlation of pH and Eh, counteracting the H2 -driven decrease of Eh during fermentation. Thus, the intraruminal Eh is influenced by pH and H2 output during SCFA formation. The opposed character of these factors stabilises the intraruminal homeostasis which might help maintain symbiotic microbiota in the rumen. Understanding, monitoring, and supporting this system will be an essential part of modern cattle production.

Ruminal epithelium: a checkpoint for cattle health.

The reticulorumen, as the main fermentation site of ruminants, delivers energy in the form of short-chain fatty acids (SCFA) for both the animal as well as the ruminal wall. By absorbing these SCFA, the ruminal epithelium plays a major role in the maintenance of intraruminal and intraepithelial acid-base homoeostasis as well as the balance of osmolarity. It takes up SCFA via several pathways which additionally lead to either a reduction of protons in the ruminal lumen or the secretion of bicarbonate, ultimately buffering the ruminal content effectively. Nutrition of the epithelium itself is achieved by catabolism of the SCFA, especially butyrate. Catabolism of SCFA also helps to maintain a concentration gradient across the epithelium to ensure efficient SCFA uptake and stability of the epithelial osmolarity. Furthermore, the ruminal epithelium forms a tight barrier against pathogens, endotoxins or biogenic amines, which may emerge from ruminal microorganisms and feed. Under physiological conditions, it reduces toxin uptake to a minimum. Moreover, the epithelium seems to have the ability to degrade biogenic amines like histamine. Nonetheless, in high performance production animals like dairy cattle, the reticulorumen is confronted with large amounts of rapidly fermentable carbohydrates. This may push the epithelium to its limits, even though it possesses a great capacity to adapt to varying feeding conditions. If the epithelial limit is exceeded, increasing amounts of SCFA lead to an acidotic imbalance that provokes epithelial damage and thereby elevates the entrance of pathogens and other potentially harmful substances into the animal's body. Hence, the ruminal epithelium lays the foundation for the animal's health, and in order to ensure longevity and high performance of ruminant farm animals, it should never be overburdened.

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.

The Fast Lane of Hypoxic Adaptation: Glucose Transport Is Modulated via A HIF-Hydroxylase-AMPK-Axis in Jejunum Epithelium.

The intestinal epithelium is able to adapt to varying blood flow and, thus, oxygen availability. Still, the adaptation fails under pathologic situations. A better understanding of the mechanisms underlying the epithelial adaptation to hypoxia could help to improve the therapeutic approach. We hypothesized that the short-term adaptation to hypoxia is mediated via AMP-activated protein kinase (AMPK) and that it is coupled to the long-term adaptation by a common regulation mechanism, the HIF-hydroxylase enzymes. Further, we hypothesized the transepithelial transport of glucose to be part of this short-term adaptation. We conducted Ussing chamber studies using isolated lagomorph jejunum epithelium and cell culture experiments with CaCo-2 cells. The epithelia and cells were incubated under 100% and 21% O2, respectively, with the panhydroxylase inhibitor dimethyloxalylglycine (DMOG) or under 1% O2. We showed an activation of AMPK under hypoxia and after incubation with DMOG by Western blot. This could be related to functional effects like an impairment of Na+-coupled glucose transport. Inhibitor studies revealed a recruitment of glucose transporter 1 under hypoxia, but not after incubation with DMOG. Summing up, we showed an influence of hydroxylase enzymes on AMPK activity and similarities between hypoxia and the effects of hydroxylase inhibition on functional changes.

Establishment and Characterization of an SV40 Large T Antigen-Transduced Porcine Colonic Epithelial Cell Line.

Continuous cell lines have become indispensable tools that have enabled investigations into cellular mechanisms by increasing experimental reproducibility and sample availability, and decreasing the use of experimental animals. To facilitate studies of epithelial barrier function of the porcine colon, we aimed to establish an epithelial cell line with an extended replicative capacity. Cells were isolated from the proximal colon of a 3-week-old piglet and transduced using a recombinant retroviral vector construct containing the simian virus 40 large T antigen (SV40 TAg). We established a clonal epithelial cell line, referred to as PoCo83-3, that stably expressed the SV40 TAg, verified at mRNA and protein levels. PoCo83-3 showed epithelial cell-specific features, such as cobblestone-like morphology, dome structure formation, the presence of apical microvilli, and the expression of keratin 18, E-cadherin and the tight junction-associated proteins zonula occludens-1, occludin, and claudin-1. To validate PoCo83-3 as an in vitro model in epithelial barrier research, proinflammatory cytokine-inducible alterations in barrier integrity were demonstrated by incubating the cells with TNF-α and IFN-γ for 48 h. These cytokine treatments promoted a decreased transepithelial electrical resistance. In summary, PoCo83-3 exhibited an extended life span and a differentiated phenotype while maintaining epithelial characteristics. Based on these results, we present this cell line as a valuable in vitro model for investigations of epithelial barrier function in the porcine colon.

Monocarboxylate transporters 1 and 4: expression and regulation by PPARα in ovine ruminal epithelial cells.

In the intact rumen epithelium, isoforms 1 and 4 of the monocarboxylate transporter (MCT1 and MCT4) are thought to play key roles in mediating transcellular and intracellular permeation of short-chain fatty acids and their metabolites and in maintaining intracellular pH. We examined whether both MCT1 and MCT4 are expressed at mRNA and protein levels in ovine ruminal epithelial cells (REC) maintained in primary culture and whether they are regulated by peroxisome proliferator-activated receptor-α (PPARα). Because both transporters have been characterized to function coupled to protons, the influence of PPARα on the recovery of intracellular pH after l-lactate exposure was evaluated by spectrofluorometry. MCT1 and MCT4 were detected using immunocytochemistry both at the cell margins and intracellularly in cultured REC. To test regulation by PPARα, cells were exposed to WY 14.643, a selective ligand of PPARα, for 48 h. The subsequent qPCR analysis resulted in a dose-dependent upregulation of MCT1 and PPARα target genes, whereas response of MCT4 was not uniform. Protein expression of MCT1 and MCT4 quantified by Western blot analysis was not altered by WY 14.643 treatment. l-Lactate-dependent proton export was blocked almost completely by pHMB, a specific inhibitor of MCT1 and MCT4. However, l-lactate-dependent, pHMB-inhibited proton export in WY 14.643-treated cells was not significantly altered compared with cells not treated with WY 14.643. These data suggest that PPARα is particularly regulating MCT1 but not MCT4 expression. Extent of lactate-coupled proton export indicates that MCT1 is already working on a high level even under unstimulated conditions.

Expression of the cobalamin transporters cubam and MRP1 in the canine ileum-Upregulation in chronic inflammatory enteropathy.

Chronic inflammatory enteropathy (CIE) in dogs, a spontaneous model of human inflammatory bowel disease (IBD), is associated with a high rate of cobalamin deficiency. The etiology of hypocobalaminemia in human IBD and canine CIE remains unknown, and compromised intestinal uptake of cobalamin resulting from ileal cobalamin receptor deficiency has been proposed as a possible cause. Here, we evaluated the intestinal expression of the cobalamin receptor subunits, amnionless (AMN) and cubilin (CUBN), and the basolateral efflux transporter multi-drug resistance protein 1 (MRP1) in 22 dogs with CIE in comparison to healthy dogs. Epithelial CUBN and AMN levels were quantified by confocal laser scanning microscopy using immunohistochemistry in endoscopic ileal biopsies from dogs with (i) CIE and normocobalaminemia, (ii) CIE and suboptimal serum cobalamin status, (iii) CIE and severe hypocobalaminemia, and (iv) healthy controls. CUBN and MRP1 expression was quantified by RT-qPCR. Receptor expression was evaluated for correlation with clinical patient data. Ileal mucosal protein levels of AMN and CUBN as well as mRNA levels of CUBN and MRP1 were significantly increased in dogs with CIE compared to healthy controls. Ileal cobalamin receptor expression was positively correlated with age, clinical disease activity index (CCECAI) score, and lacteal dilation in the ileum, inversely correlated with serum folate concentrations, but was not associated with serum cobalamin concentrations. Cobalamin receptor downregulation does not appear to be the primary cause of hypocobalaminemia in canine CIE. In dogs of older age with severe clinical signs and/or microscopic intestinal lesions, intestinal cobalamin receptor upregulation is proposed as a mechanism to compensate for CIE-associated hypocobalaminemia. These results support oral supplementation strategies in hypocobalaminemic CIE patients.

Cholinergic modulation of epithelial integrity in the proximal colon of pigs.

BACKGROUND: Within the gut, acetylcholine (ACh) is synthesised by enteric neurons, as well as by 'non-neuronal' epithelial cells. In studies of non-intestinal epithelia, ACh was involved in the generation of an intact epithelial barrier. In the present study, primary cultured porcine colonocytes were used to determine whether treatment with exogenous ACh or expression of endogenous epithelium-derived ACh may modulate epithelial tightness in the gastrointestinal tract. METHODS: Piglet colonocytes were cultured on filter membranes for 8 days. The tightness of the growing epithelial cell layer was evaluated by measuring transepithelial electrical resistance (TEER). To determine whether ACh modulates the tightness of the cell layer, cells were treated with cholinergic, muscarinic and/or nicotinic agonists and antagonists. Choline acetyltransferase (ChAT), cholinergic receptors and ACh were determined by immunohistochemistry, RT-PCR and HPLC, respectively. RESULTS: Application of the cholinergic agonist carbachol (10 µm) and the muscarinic agonist oxotremorine (10 µM) resulted in significantly higher TEER values compared to controls. The effect was completely inhibited by the muscarinic antagonist atropine. Application of atropine alone (without any agonist) led to significantly lower TEER values compared to controls. Synthesis of ACh by epithelial cells was proven by detection of muscarinic and nicotinic receptor mRNAs, immunohistochemical detection of ChAT and detection of ACh by HPLC. CONCLUSION: ACh is strongly involved in the regulation of epithelial tightness in the proximal colon of pigs via muscarinic pathways. Non-neuronal ACh seems to be of particular importance for epithelial cells forming a tight barrier.

Increased papillae growth and enhanced short-chain fatty acid absorption in the rumen of goats are associated with transient increases in cyclin D1 expression after ruminal butyrate infusion.

We tested the hypothesis that the proliferative effects of intraruminal butyrate infusions on the ruminal epithelium are linked to upregulation in cyclin D1 (CCND1), the cyclin-dependent kinase 4 (CDK4), and their possible association with enhanced absorption of short-chain fatty acids (SCFA). Goats (n=23) in 2 experiments (Exp.) were fed 200 g/d concentrate and hay ad libitum. In Exp. 1, goats received an intraruminal infusion of sodium butyrate at 0.3 (group B, n=8) or 0 (group C, n=7) g/kg of body weight (BW) per day before morning feeding for 28 d and were slaughtered 8 h after the butyrate infusion. In Exp. 2, goats (n=8) received butyrate infusion and feeding as in Exp. 1. On d 28, epithelial samples were biopsied from the antrium ruminis at 0, 3, and 7 h after the last butyrate infusion. In Exp. 1, the ruminal molar proportional concentration of butyrate increased in group B by about 110% after butyrate infusion and remained elevated for 1.5 h; thereafter, it gradually returned to the baseline (preinfusion) level. In group C, the molar proportional concentration of butyrate was unchanged over the time points. The length and width of papillae increased in B compared with C; this was associated with increased numbers of cells and cell layers in the epithelial strata and an increase in the surface area of 82%. The mRNA expression of CCND1 increased transiently at 3 h but returned to the preinfusion level at 7 h following butyrate infusion in Exp. 2. However, it did not differ between B and C in Exp. 1, in which the ruminal epithelium was sampled at 8 h after butyrate infusion. The mRNA expression of the monocarboxylate transporter MCT4, but not MCT1, was stably upregulated in B compared with C. The estimated absorption rate of total SCFA (%/h) increased in B compared with C. We conclude that transient increases in cyclin D1 transcription contribute to butyrate-induced papillae growth and subsequently to the increased absorption of SCFA in the ruminal epithelium of goats.

Intravenous infusions of glucose stimulate key lipogenic enzymes in adipose tissue of dairy cows in a dose-dependent manner.

The present study was investigated whether increasing amounts of glucose supply have a stimulatory effect on the mRNA abundance and activity of key lipogenic enzymes in adipose tissue of midlactation dairy cows. Twelve Holstein-Friesian dairy cows in midlactation were cannulated in the jugular vein and infused with either a 40% glucose solution (n=6) or saline (n=6). For glucose infusion cows, the infusion dose increased by 1.25%/d relative to the initial net energy for lactation (NEL) requirement until a maximum dose equating to a surplus of 30% NEL was reached on d 24. This maximum dose was maintained until d 28 and stopped thereafter (between d 29-32). Cows in the saline infusion group received an equivalent volume of 0.9% saline solution. Samples of subcutaneous adipose tissue were taken on d 0, 8, 16, 24, and 32 when surplus glucose reached 0, 10, 20, and 30% of the NEL requirement, respectively. The mRNA abundance of fatty acid synthase, cytoplasmic acetyl-coenzyme A synthetase, cytoplasmic glycerol 3-phosphate dehydrogenase-1, and glucose 6-phosphate dehydrogenase showed linear treatment × dose interactions with increasing mRNA abundance with increasing glucose dose. The increased mRNA abundance was paralleled by a linear treatment × dose interaction for fatty acid synthase and acetyl-coenzyme A synthetase enzymatic activities. The mRNA abundance of ATP-citrate lyase showed a tendency for linear treatment × dose interaction with increasing mRNA abundance with increasing glucose dose. The mRNA abundance of all tested enzymes, as well as the activities of fatty acid synthase and acetyl-coenzyme A synthetase, correlated with plasma glucose and serum insulin levels. In a multiple regression model, the predictive value of insulin was dominant over that of glucose. In conclusion, gradual increases in glucose supply upregulate key lipogenic enzymes in adipose tissue of midlactating dairy cows with linear dose dependency. Insulin appears to be critically involved in this regulation.

Establishment and characterization of porcine colonic epithelial cells grown in primary culture.

BACKGROUND: Primary cultures of epithelial cells are suitable models for studying epithelial function and, in particular, the regulation of epithelial tightness in vitro. The aim of our study was to develop a protocol for the isolation and culture of porcine colonic epithelial cells and to establish transepithelial electrical resistance (TEER) as a functional parameter for epithelial tightness. METHODS: Epithelial cells were obtained from the proximal colon of piglets by enzymatic dispase digestion. Cells were cultured on collagen-coated membrane supports for 21 days. The epithelial origin of the cells was shown by immunohistochemical detection of cytokeratin and zonula occludens protein 1 (ZO-1). Scanning electron microscopy, transmission electron microscopy and confocal microscopy were used for further morphological characterization. The integrity and tightness of the artificial epithelium were determined by measuring TEER. RESULTS: The cultured epithelial cells were immunoreactive for cytokeratin and ZO-1. They showed dense microvilli on their apical membranes and expression of Na(+)/K(+)-ATPase on their basolateral membranes. Adjacent cells were connected by tight junctions. We observed TEER to continuously increase up to 870 ± 38 Ω·cm(2) during the culture period. TEER correlated with the amount of epithelial cells expressing ZO-1. CONCLUSIONS: The properties of primary cultured epithelial cells resemble the structural properties of polarized colonic epithelium in vivo. Measurement of TEER seems to be suitable for studying epithelial tightness in vitro. We suggest that these primary epithelial cultures be used to investigate the regulation of the epithelial barrier function.

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

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.

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.

Cultured ruminal epithelial cells express a large-conductance channel permeable to chloride, bicarbonate, and acetate.

The absorption of short-chain fatty acids (SCFA) from the rumen requires efficient mechanisms for both apical uptake and basolateral extrusion. Previous studies suggest that the rumen expresses a basolateral chloride conductance that might be permeable to SCFA. In order to characterize this conductance in more detail, isolated cultured ruminal epithelial cells were studied with the patch-clamp technique, revealing a whole-cell conductance with p(Cl(-)) approximately p(NO(3) (-)) > p(HCO(3) (-)) > p(acetate(-)) > p(gluconate(-)). Currents could be blocked by diisothiocyanato-stilbene-2,2'-disulfonic acid (1 mmol l(-1) > 100 micromol l(-1)), 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (50 micromol l(-1)), niflumic acid (100 micromol l(-1)), and p-chloromercuribenzoate (1 mmol l(-1)). Single-channel conductance was 350 +/- 7 pS for chloride and 142 +/- 7 pS for acetate. Open probability could be fitted with a three-state gating model. We propose a role for this channel in mediating the permeation of chloride, bicarbonate, and acetate across the basolateral membrane of the ruminal epithelium.

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.

Intrinsic ruminal innervation in ruminants of different feeding types.

According to their feeding habits, ruminants can be classified as grazers, concentrate selectors and those of intermediate type. The different feeding types are reflected in distinct anatomical properties of the forestomachs. The present study was designed to investigate whether the intrinsic innervation patterns of the rumen (the main part of the forestomach) differ between intermediate types and grazers. Myenteric plexus preparations from the rumen of goats (intermediate type), fallow deer (intermediate type), cattle (grazer) and sheep (grazer) were analysed by immunohistochemical detection of the following antigens: Hu-protein (HuC/D), choline acetyltransferase (ChAT), nitric oxide synthase (NOS), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), substance P (SP), calbindin (CALB) and somatostatin (SOM). Myenteric ganglia of cattle contained 73 +/- 6 neurons per ganglion, whereas the ganglia of sheep were significantly smaller (45 +/- 18 neurons per ganglion). The ganglion density of the myenteric plexus was highest in fallow deer (15 +/- 3 ganglia per cm(2)) and lowest in cattle (6 +/- 1 ganglia per cm(2)). All myenteric neurons were either ChAT or NOS positive. The proportion of NOS-positive neurons was significantly lower in sheep (29.5 +/- 8.2% of all neurons) than in goats (44.2 +/- 9.8%). In all species, additional analysis of the different neuropeptides revealed the following subpopulations in descending order of percentile appearance: ChAT/SP > NOS/VIP/NPY > ChAT/- > NOS/NPY. Expression of CALB was detected in a minority of the ChAT-positive neurons in all species. Somatostatin immunoreactive somata were found only in preparations obtained from fallow deer and sheep. These data suggest that the rumen of grazers is under stronger cholinergic control than the rumen of species belonging to the intermediate type, although most subpopulations of neurons are present in all species. However, whether the strong mixing patterns of low quality roughage during digestion are enabled by the prominent excitatory input of the rumen of grazers requires elucidation in further studies.

Infection of the enteric nervous system by Borna disease virus (BDV) upregulates expression of Calbindin D-28k.

Borna disease virus (BDV) is a neurotropic agent infecting distinct neuronal subpopulations in the central nervous system of various mammalian species possibly including humans. Horses, a major natural host for BDV, show gastrointestinal dysfunctions besides characteristic neurological symptoms. Therefore, we hypothesized that enteric neurons may be targets of BDV replication. The presence of BDV-specific antigen in subpopulations of the ENS was investigated. Four-week-old Lewis rats were infected intracerebrally and sacrificed 4-14 weeks post infection (p.i.). BDV-immunoreactive neurons were found in submucous and myenteric neurons of the proximal colon. Fourteen weeks p.i., the proportion of BDV-positive neurons was 44+/-17 and 24+/-7% in the submucous and myenteric plexus, respectively. The majority of BDV-positive myenteric neurons showed immunoreactivity for choline acetyltransferase. Expression of Calbindin D-28k (CALB) was found in 96% of submucous and 67% of myenteric BDV-immunoreactive neurons. Additionally, the number of CALB-immunoreactive neurons was significantly higher in the myenteric plexus of infected rats compared to controls. These data indicate that BDV infects specific subpopulations of enteric neurons. Therefore, the ENS might serve as a site for BDV replication and as an immunoprivileged reservoir for BDV. In addition, upregulation of CALB in neurons of the myenteric plexus is probably induced during BDV-infection.

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.

High-performance liquid chromatographic method for the determination of histamine and 1-methylhistamine in biological buffers.

A method for the determination of histamine and its catabolite 1-methylhistamine (1-MH) was developed, using HPLC with fluorescence detection. Derivatization of both compounds occurred on-column with o-phthaldialdehyde dissolved in an alkaline borate buffer, followed by separation on a reversed phase C18 column. Histamine and 1-MH could be detected with comparable sensitivity (limit of quantification, 50 nM). The method was proven suitable to investigate catabolism of histamine by epithelia of pig colon. The method should be useful in research on histamine metabolism.