Effects of butyrate- on ruminal Ca2+ transport: evidence for the involvement of apically expressed TRPV3 and TRPV4 channels.

The ruminal epithelium absorbs large quantities of NH4+ and Ca2+. A role for TRPV3 has emerged, but data on TRPV4 are lacking. Furthermore, short-chain fatty acids (SCFA) stimulate ruminal Ca2+ and NH4+ uptake in vivo and in vitro, but the pathway is unclear. Sequencing of the bovine homologue (bTRPV4) revealed 96.79% homology to human TRPV4. Two commercial antibodies were tested using HEK-293 cells overexpressing bTRPV4, which in ruminal protein detected a weak band at the expected ~ 100 kDa and several bands ≤ 60 kDa. Immunofluorescence imaging revealed staining of the apical membrane of the stratum granulosum for bTRPV3 and bTRPV4, with cytosolic staining in other layers of the ruminal epithelium. A similar expression pattern was observed in a multilayered ruminal cell culture which developed resistances of > 700 Ω · cm2 with expression of zonula occludens-1 and claudin-4. In Ussing chambers, 2-APB and the TRPV4 agonist GSK1016790A stimulated the short-circuit current across native bovine ruminal epithelia. In whole-cell patch-clamp recordings on HEK-293 cells, bTRPV4 was shown to be permeable to NH4+, K+, and Na+ and highly sensitive to GSK1016790A, while effects of butyrate- were insignificant. Conversely, bTRPV3 was strongly stimulated by 2-APB and by butyrate- (pH 6.4 > pH 7.4), but not by GSK1016790A. Fluorescence calcium imaging experiments suggest that butyrate- stimulates both bTRPV3 and bTRPV4. While expression of bTRPV4 appears to be weaker, both channels are candidates for the ruminal transport of NH4+ and Ca2+. Stimulation by SCFA may involve cytosolic acidification (bTRPV3) and cell swelling (bTRPV4).

Beyond Ca2+ signalling: the role of TRPV3 in the transport of NH4.

Mutations of TRPV3 lead to severe dermal hyperkeratosis in Olmsted syndrome, but whether the mutants are trafficked to the cell membrane or not is controversial. Even less is known about TRPV3 function in intestinal epithelia, although research on ruminants and pigs suggests an involvement in the uptake of NH4+. It was the purpose of this study to measure the permeability of the human homologue (hTRPV3) to NH4+, to localize hTRPV3 in human skin equivalents, and to investigate trafficking of the Olmsted mutant G573S. Immunoblotting and immunostaining verified the successful expression of hTRPV3 in HEK-293 cells and Xenopus oocytes with trafficking to the cell membrane. Human skin equivalents showed distinct staining of the apical membrane of the top layer of keratinocytes with cytosolic staining in the middle layers. Experiments with pH-sensitive microelectrodes on Xenopus oocytes demonstrated that acidification by NH4+ was significantly greater when hTRPV3 was expressed. Single-channel measurements showed larger conductances in overexpressing Xenopus oocytes than in controls. In whole-cell experiments on HEK-293 cells, both enantiomers of menthol stimulated influx of NH4+ in hTRPV3 expressing cells, but not in controls. Expression of the mutant G573S greatly reduced cell viability with partial rescue via ruthenium red. Immunofluorescence confirmed cytosolic expression, with membrane staining observed in a very small number of cells. We suggest that expression of TRPV3 by epithelia may have implications not just for Ca2+ signalling, but also for nitrogen metabolism. Models suggesting how influx of NH4+ via TRPV3 might stimulate skin cornification or intestinal NH4+ transport are discussed.

Evaluation of Different Blood Parameters From Endurance Horses Competing at 160 km.

The purpose of this study was to assess a change in different blood parameters before and after a 160 km endurance race and to evaluate differences in cardiac biomarkers between horses that completed the race and horses that did not. The study population consisted of 52 healthy endurance horses. Horses participating in the study were assigned to three groups: horses that successfully completed the race ("finishers"), horses that failed to qualify at the veterinary check for primarily metabolic reasons ("metabolic") and horses that failed to qualify at the veterinary check for primarily gait related reasons ("gait related"). The latter two groups were combined to form a final group of "non-finishers" that were excluded for either "gait related" or "metabolic" disorders. Venous blood samples were taken before and after the endurance race. Serum and EDTA-plasma were analyzed for cardiac troponin I (cTNI), heart fatty acid binding protein (HFABP), alpha-hydroxybutyrate dehydrogenase (α-HBDH), atrial natriuretic peptide (ANP), lactate dehydrogenase (LDH), symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA). Lactate dehydrogenase (P = .001), SDMA (P= .001) and ADMA (P= .002) increased significantly after the endurance race in the finisher group. A significant increase in cTNI and α-HBDH concentration after the endurance race compared to the values before the endurance race was detected in the finisher (P= .001, P= .001) and gait related group (P= .002, P= .007). The longer the distance completed, the more these five blood parameters increased. No differences between the groups could be found and none of the measured blood parameters showed significant differences among groups before or after racing.

The TRPA1 Agonist Cinnamaldehyde Induces the Secretion of HCO3- by the Porcine Colon.

A therapeutic potential of the TRPA1 channel agonist cinnamaldehyde for use in inflammatory bowel disease is emerging, but the mechanisms are unclear. Semi-quantitative qPCR of various parts of the porcine gastrointestinal tract showed that mRNA for TRPA1 was highest in the colonic mucosa. In Ussing chambers, 1 mmol·L-1 cinnamaldehyde induced increases in short circuit current (ΔIsc) and conductance (ΔGt) across the colon that were higher than those across the jejunum or after 1 mmol·L-1 thymol. Lidocaine, amiloride or bumetanide did not change the response. The application of 1 mmol·L-1 quinidine or the bilateral replacement of 120 Na+, 120 Cl- or 25 HCO3- reduced ΔGt, while the removal of Ca2+ enhanced ΔGt with ΔIsc numerically higher. ΔIsc decreased after 0.5 NPPB, 0.01 indometacin and the bilateral replacement of 120 Na+ or 25 HCO3-. The removal of 120 Cl- had no effect. Cinnamaldehyde also activates TRPV3, but comparative measurements involving patch clamp experiments on overexpressing cells demonstrated that much higher concentrations are required. We suggest that cinnamaldehyde stimulates the secretion of HCO3- via apical CFTR and basolateral Na+-HCO3- cotransport, preventing acidosis and damage to the epithelium and the colonic microbiome. Signaling may involve the opening of TRPA1, depolarization of the epithelium and a rise in PGE2 following a lower uptake of prostaglandins via OATP2A1.

TRPV3 and TRPV4 as candidate proteins for intestinal ammonium absorption.

AIM: Absorption of ammonia from the gut has consequences that range from encephalitis in hepatic disease to global climate change induced by nitrogenous excretions from livestock. Since patch clamp data show that certain members of the transient receptor potential (TRP) family are permeable to NH4+ , participation in ammonium efflux was investigated. METHODS: Digesta, mucosa and muscular samples from stomach, duodenum, jejunum, ileum, caecum and colon of pigs were analysed via colourimetry, qPCR, Western blot, immunohistochemistry and Ussing chambers. RESULTS: qPCR data show high duodenal expression of TRPV6. TRPM6 was highest in jejunum and colon, with expression of TRPM7 ubiquitous. TRPM8 and TRPV1 were below detection. TRPV2 was highest in the jejunum but almost non-detectable in the colon. TRPV4 was ubiquitously expressed by mucosal and muscular layers. TRPV3 mRNA was only found in the mucosa of the caecum and colon, organs in which NH4+ was highest (>7 mmol·L-1 ). Immunohistochemically, an apical expression of TRPV3 and TRPV4 could be detected in all tissues, with effects of 2-APB and GSK106790A supporting functional expression. In symmetrical NaCl Ringer, removal of mucosal Ca2+ and Mg2+ increased colonic short circuit current (Isc ) and conductance (Gt ) by 0.18 ± 0.06 µeq·cm-2 ·h-1 and 4.70 ± 0.85 mS·cm-2 (P < .05, N/n = 4/17). Application of mucosal NH4 Cl led to dose-dependent and divalent-sensitive increases in Gt and Isc , with effects highest in the caecum and colon. CONCLUSION: We propose that TRP channels contribute to the intestinal transport of ammonium, with TRPV3 and TRPV4 promising candidate proteins. Pharmacological regulation may be possible.

Design and Testing of Efficient Mucus-Penetrating Nanogels-Pitfalls of Preclinical Testing and Lessons Learned.

Mucosal surfaces pose a challenging environment for efficient drug delivery. Various delivery strategies such as nanoparticles have been employed so far; yet, still yielding limited success. To address the need of efficient transmucosal drug delivery, this report presents the synthesis of novel disulfide-containing dendritic polyglycerol (dPG)-based nanogels and their preclinical testing. A bifunctional disulfide-containing linker is coupled to dPG to act as a macromolecular crosslinker for poly-N-isopropylacrylamide (PNIPAM) and poly-N-isopropylmethacrylamide (PNIPMAM) in a precipitation polymerization process. A systematic analysis of the polymerization reveals the importance of a careful polymer choice to yield mucus-degradable nanogels with diameters between 100 and 200 nm, low polydispersity, and intact disulfide linkers. Absorption studies in porcine intestinal tissue and human bronchial epithelial models demonstrate that disulfide-containing nanogels are highly efficient in overcoming mucosal barriers. The nanogels efficiently degrade and deliver the anti-inflammatory biomacromolecule etanercept into epithelial tissues yielding local anti-inflammatory effects. Over the course of this work, several problems are encountered due to a limited availability of valid test systems for mucosal drug-delivery systems. Hence, this study also emphasizes how critical a combined and multifaceted approach is for the preclinical testing of mucosal drug-delivery systems, discusses potential pitfalls, and provides suggestions for solutions.

The TRPV3 channel of the bovine rumen: localization and functional characterization of a protein relevant for ruminal ammonia transport.

Large quantities of ammonia (NH3 or NH4+) are absorbed from the gut, associated with encephalitis in hepatic disease, poor protein efficiency in livestock, and emissions of nitrogenous climate gasses. Identifying the transport mechanisms appears urgent. Recent functional and mRNA data suggest that absorption of ammonia from the forestomach of cattle may involve TRPV3 channels. The purpose of the present study was to sequence the bovine homologue of TRPV3 (bTRPV3), localize the protein in ruminal tissue, and confirm transport of NH4+. After sequencing, bTRPV3 was overexpressed in HEK-293 cells and Xenopus oocytes. An antibody was selected via epitope screening and used to detect the protein in immunoblots of overexpressing cells and bovine rumen, revealing a signal of the predicted ~ 90 kDa. In rumen only, an additional ~ 60 kDa band appeared, which may represent a previously described bTRPV3 splice variant of equal length. Immunohistochemistry revealed staining from the ruminal stratum basale to stratum granulosum. Measurements with pH-sensitive microelectrodes showed that NH4+ acidifies Xenopus oocytes, with overexpression of bTRPV3 enhancing permeability to NH4+. Single-channel measurements revealed that Xenopus oocytes endogenously expressed small cation channels in addition to fourfold-larger channels only observed after expression of bTRPV3. Both endogenous and bTRPV3 channels conducted NH4+, Na+, and K+. We conclude that bTRPV3 is expressed by the ruminal epithelium on the protein level. In conjunction with data from previous studies, a role in the transport of Na+, Ca2+, and NH4+ emerges. Consequences for calcium homeostasis, ruminal pH, and nitrogen efficiency in cattle are discussed.

Assessment of magnesium intake according to requirement in dairy cows.

To date, no specific hormonal regulation system has been identified for homoeostatic control of the essential mineral Mg. In cattle, the maintenance of physiological plasma Mg concentration depends on gastrointestinal absorption, primarily from the rumen, which serves as a pool for covering the requirement. Whereas a possible surplus (absorption greater than requirement) is rapidly excreted by the kidneys, a shortage (absorption lower than requirement) cannot be compensated for by mobilization from the large Mg pool in bones or soft tissue, so that the maintenance of the necessary physiological Mg concentration in plasma relies on continuous and sufficient absorption. Our knowledge concerning the site and mechanisms of Mg absorption has improved during the last few decades, and meta-analyses of the absorption of Mg in dairy cows have shown that the K content has a pronounced negative effect on Mg digestibility. The current recommendations of Mg intake propose a constant percentage of Mg and emphasize the depressive effect of high potassium (K) intake on Mg absorption. The current knowledge about the antagonism between K intake and Mg absorption allows a more flexible solution which includes the K content of the diet. An assessment of Mg intake is proposed that incorporates the improved knowledge of Mg absorption, metabolism and requirement. Within this framework, an equation is derived that allows a prediction of the amount of Mg required to compensate for dietary K content, the goal being to avoid both possible undernutrition or an unnecessary surplus of dietary Mg.

A comparative study of ammonia transport across ruminal epithelia from Bos indicus crossbreds versus Bos taurus.

Absorption of ammonia from the rumen of cattle decreases nitrogen availability for fermentational protein synthesis, leading to increased competition of cattle with humans for protein and enhancing the release of toxic nitrogenous compounds into the environment. Given that differences in feeding and breeding might induce differences in ruminal ammonia transport, we compared electrophysiological, histological, and molecular biological characteristics of ruminal epithelia of Bos indicus crossbreds (Sahiwal-Mix, SWM) with those of Bos taurus (Holstein-Friesian, HF). As in HF, the stratified cornified epithelium of SWM expressed claudin 1 and 4. Measurements of ammonia flux (HF) and serosal pH (both breeds) suggested that at a mucosal pH of 6.4, net transport primarily occurred as NH4 + . As shown previously for HF, NH4 + induced a concentration-dependent rise in short circuit current (Isc ) in SWM that could be further stimulated by the TRP channel agonist menthol. Relative mRNA expression levels for TRPV3, TRPV4, TRPM6, and TRPM7 were significantly lower in SWM than in HF, with TRPA1 expression near the limit of detection. We conclude that uptake of ammonia from the rumen of both breeds occurs electrogenically as NH4 + with functional and molecular biological evidence pointing towards involvement of TRPV3 and TRPV4.

The bovine TRPV3 as a pathway for the uptake of Na+, Ca2+, and NH4+

Absorption of ammonia from the gastrointestinal tract results in problems that range from hepatic encephalopathy in humans to poor nitrogen efficiency of cattle with consequences for the global climate. Previous studies on epithelia and cells from the native ruminal epithelium suggest functional involvement of the bovine homologue of TRPV3 (bTRPV3) in ruminal NH4+ transport. Since the conductance of TRP channels to NH4+ has never been studied, bTRPV3 was overexpressed in HEK-293 cells and investigated using the patch-clamp technique and intracellular calcium imaging. Control cells contained the empty construct. Divalent cations blocked the conductance for monovalent cations in both cell types, with effects higher in cells expressing bTRPV3. In bTRPV3 cells, but not in controls, menthol, thymol, carvacrol, or 2-APB stimulated whole cell currents mediated by Na+, Cs+, NH4+, and K+, with a rise in intracellular Ca2+ observed in response to menthol. While only 25% of control patches showed single-channel events (with a conductance of 40.8 ± 11.9 pS for NH4+ and 25.0 ± 5.8 pS for Na+), 90% of bTRPV3 patches showed much larger conductances of 127.8 ± 4.2 pS for Na+, 240.1 ± 3.6 pS for NH4+, 34.0 ± 1.7 pS for Ca2+, and ~ 36 pS for NMDG+. Open probability, but not conductance, rose with time after patch excision. In conjunction with previous research, we suggest that bTRPV3 channels may play a role in the transport of Na+, K+, Ca2+ and NH4+ across the rumen with possible repercussions for understanding the function of TRPV3 in other epithelia.

Magnesium homeostasis in cattle: absorption and excretion.

Magnesium (Mg2+) is an essential mineral without known specific regulatory mechanisms. In ruminants, plasma Mg2+ concentration depends primarily on the balance between Mg2+ absorption and Mg2+ excretion. The primary site of Mg2+ absorption is the rumen, where Mg2+ is apically absorbed by both potential-dependent and potential-independent uptake mechanisms, reflecting involvement of ion channels and electroneutral transporters, respectively. Transport is energised in a secondary active manner by a basolateral Na+/Mg2+ exchanger. Ruminal transport of Mg2+ is significantly influenced by a variety of factors such as high K+ concentration, sudden increases of ammonia, pH, and the concentration of SCFA. Impaired Mg2+ absorption in the rumen is not compensated for by increased transport in the small or large intestine. While renal excretion can be adjusted to compensate precisely for any surplus in Mg2+ uptake, a shortage in dietary Mg2+ cannot be compensated for either via skeletal mobilisation of Mg2+ or via up-regulation of ruminal absorption. In such situations, hypomagnesaemia will lead to decrease of a Mg2+ in the cerebrospinal fluid and clinical manifestations of tetany. Improved knowledge concerning the factors governing Mg2+ homeostasis will allow reliable recommendations for an adequate Mg2+ intake and for the avoidance of possible disturbances. Future research should clarify the molecular identity of the suggested Mg2+ transport proteins and the regulatory mechanisms controlling renal Mg excretion as parameters influencing Mg2+ homeostasis.

A look at the smelly side of physiology: transport of short chain fatty acids.

Fermentative organs such as the caecum, the colon, and the rumen have evolved to produce and absorb energy rich short chain fatty acids (SCFA) from otherwise indigestible substrates. Classical models postulate diffusional uptake of the undissociated acid (HSCFA). However, in net terms, a major part of SCFA absorption occurs with uptake of Na+ and resembles classical, coupled electroneutral NaCl transport. Considerable evidence suggests that the anion transporting proteins expressed by epithelia of fermentative organs are poorly selective and that their main function may be to transport acetate-, propionate-, butyrate- and HCO3- as the physiologically relevant anions. Apical uptake of SCFA thus involves non-saturable diffusion of the undissociated acid (HSCFA), SCFA-/HCO3- exchange via DRA (SLC26A3) and/or SCFA--H+ symport (MCT1, SLC16A1). All mechanisms lead to cytosolic acidification with stimulation of Na+/H+ exchange via NHE (SLC9A2/3). Basolaterally, Na+ leaves via the Na+/K+-ATPase with recirculation of K+. Na+ efflux drives the transport of SCFA- anions through volume-regulated anion channels, such as maxi-anion channels (possibly SLCO2A1), LRRC8, anoctamins, or uncoupled exchangers. When luminal buffering is inadequate, basolateral efflux will increasingly involve SCFA-/ HCO3- exchange (AE1/2, SCL4A1/2), or efflux of SCFA- with H+ (MCT1/4, SLC16A1/3). Furthermore, protons can be basolaterally removed by NHE1 (SCL9A1) or NBCe1 (SLC4A4). The purpose of these transport proteins is to maximize the amount of SCFA transported from the tightly buffered ingesta while minimizing acid transport through the epithelium. As known from the rumen for many decades, a disturbance of these processes is likely to cause severe colonic disease.

Evidence for the functional involvement of members of the TRP channel family in the uptake of Na(+) and NH4 (+) by the ruminal epithelium.

Large quantities of protein are degraded in the fermentative parts of the gut to ammonia, which is absorbed, detoxified to urea, and excreted, leading to formation of nitrogenous compounds such as N2O that are associated with global warming. In ruminants, channel-mediated uptake of NH4 (+) from the rumen predominates. The molecular identity of these channels remains to be clarified. Ruminal cells and epithelia from cows and sheep were investigated using patch clamp, Ussing chamber, microelectrode techniques, and qPCR. In patch clamp experiments, bovine ruminal epithelial cells expressed a conductance for NH4 (+) that could be blocked in a voltage-dependent manner by divalent cations. In the native epithelium, NH4 (+) depolarized the apical potential, acidified the cytosol and induced a rise in short-circuit current (I sc) that persisted after the removal of Na(+), was blocked by verapamil, enhanced by the removal of divalent cations, and was sensitive to certain transient receptor potential (TRP) channel modulators. Menthol or thymol stimulated the I sc in Na(+) or NH4 (+) containing solutions in a dose-dependent manner and modulated transepithelial Ca(2+) fluxes. On the level of messenger RNA (mRNA), ovine and bovine ruminal epithelium expressed TRPA1, TRPV3, TRPV4, TRPM6, and TRPM7, with any expression of TRPV6 marginal. No bands were detected for TRPV1, TRPV5, or TRPM8. Functional and molecular biological data suggest that the transport of NH4 (+), Na(+), and Ca(2+) across the rumen involves TRP channels, with TRPV3 and TRPA1 emerging as prime candidate genes. TRP channels may also contribute to the transport of NH4 (+) across other epithelia.

Impact of Increasing Dietary Calcium Levels on Calcium Excretion and Vitamin D Metabolites in the Blood of Healthy Adult Cats.

BACKGROUND: Dietary calcium (Ca) concentrations might affect regulatory pathways within the Ca and vitamin D metabolism and consequently excretory mechanisms. Considering large variations in Ca concentrations of feline diets, the physiological impact on Ca homeostasis has not been evaluated to date. In the present study, diets with increasing concentrations of dicalcium phosphate were offered to ten healthy adult cats (Ca/phosphorus (P): 6.23/6.02, 7.77/7.56, 15.0/12.7, 19.0/17.3, 22.2/19.9, 24.3/21.6 g/kg dry matter). Each feeding period was divided into a 10-day adaptation and an 8-day sampling period in order to collect urine and faeces. On the last day of each feeding period, blood samples were taken. RESULTS: Urinary Ca concentrations remained unaffected, but faecal Ca concentrations increased (P < 0.001) with increasing dietary Ca levels. No effect on whole and intact parathyroid hormone levels, fibroblast growth factor 23 and calcitriol concentrations in the blood of the cats were observed. However, the calcitriol precursors 25(OH)D2 and 25(OH)D3, which are considered the most useful indicators for the vitamin D status, decreased with higher dietary Ca levels (P = 0.013 and P = 0.033). Increasing dietary levels of dicalcium phosphate revealed an acidifying effect on urinary fasting pH (6.02) and postprandial pH (6.01) (P < 0.001), possibly mediated by an increase of urinary phosphorus (P) concentrations (P < 0.001). CONCLUSIONS: In conclusion, calcitriol precursors were linearly affected by increasing dietary Ca concentrations. The increase in faecal Ca excretion indicates that Ca homeostasis of cats is mainly regulated in the intestine and not by the kidneys. Long-term studies should investigate the physiological relevance of the acidifying effect observed when feeding diets high in Ca and P.

Determination of Henry's constant, the dissociation constant, and the buffer capacity of the bicarbonate system in ruminal fluid.

Despite the clinical importance of ruminal acidosis, ruminal buffering continues to be poorly understood. In particular, the constants for the dissociation of H2CO3 and the solubility of CO2 (Henry's constant) have never been stringently determined for ruminal fluid. The pH was measured in parallel directly in the rumen and the reticulum in vivo, and in samples obtained via aspiration from 10 fistulated cows on hay- or concentrate-based diets. The equilibrium constants of the bicarbonate system were measured at 38°C both using the Astrup technique and a newly developed method with titration at 2 levels of partial pressure of CO2 (pCO2; 4.75 and 94.98 kPa), yielding mean values of 0.234 ± 0.005 mmol ∙ L(-1) ∙ kPa(-1) and 6.11 ± 0.02 for Henry's constant and the dissociation constant, respectively (n/n = 31/10). Both reticular pH and the pH of samples measured after removal were more alkalic than those measured in vivo in the rumen (by ΔpH = 0.87 ± 0.04 and 0.26 ± 0.04). The amount of acid or base required to shift the pH of ruminal samples to 6.4 or 5.8 (base excess) differed between the 2 feeding groups. Experimental results are compared with the mathematical predictions of an open 2-buffer Henderson-Hasselbalch equilibrium model. Because pCO2 has pronounced effects on ruminal pH and can decrease rapidly in samples removed from the rumen, introduction of a generally accepted protocol for determining the acid-base status of ruminal fluid with standard levels of pCO2 and measurement of base excess in addition to pH should be considered.

Down-regulation of monocarboxylate transporter 1 (MCT1) gene expression in the colon of piglets is linked to bacterial protein fermentation and pro-inflammatory cytokine-mediated signalling.

The present study investigated the influence of bacterial metabolites on monocarboxylate transporter 1 (MCT1) expression in pigs using in vivo, ex vivo and in vitro approaches. Piglets (n 24) were fed high-protein (26 %) or low-protein (18 %) diets with or without fermentable carbohydrates. Colonic digesta samples were analysed for a broad range of bacterial metabolites. The expression of MCT1, TNF-α, interferon γ (IFN-γ) and IL-8 was determined in colonic tissue. The expression of MCT1 was lower and of TNF-α and IL-8 was higher with high-protein diets (P< 0·05). MCT1 expression was positively correlated with l-lactate, whereas negatively correlated with NH3 and putrescine (P< 0·05). The expression of IL-8 and TNF-α was negatively correlated with l-lactate and positively correlated with NH3 and putrescine, whereas the expression of IFN-γ was positively correlated with histamine and 4-ethylphenol (P< 0·05). Subsequently, porcine colonic tissue and Caco-2 cells were incubated with Na-butyrate, NH4Cl or TNF-α as selected bacterial metabolites or mediators of inflammation. Colonic MCT1 expression was higher after incubation with Na-butyrate (P< 0·05) and lower after incubation with NH4Cl or TNF-α (P< 0·05). Incubation of Caco-2 cells with increasing concentrations of these metabolites confirmed the up-regulation of MCT1 expression by Na-butyrate (linear, P< 0·05) and down-regulation by TNF-α and NH4Cl (linear, P< 0·05). The high-protein diet decreased the expression of MCT1 in the colon of pigs, which appears to be linked to NH3- and TNF-α-mediated signalling.

Modulation of sheep ruminal urea transport by ammonia and pH.

Ruminal fermentation products such as short-chain fatty acids (SCFA) and CO2 acutely stimulate urea transport across the ruminal epithelium in vivo, whereas ammonia has inhibitory effects. Uptake and signaling pathways remain obscure. The ruminal expression of SLC14a1 (UT-B) was studied using polymerase chain reaction (PCR). The functional short-term effects of ammonia on cytosolic pH (pHi) and ruminal urea transport across native epithelia were investigated using pH-sensitive microelectrodes and via flux measurements in Ussing chambers. Two variants (UT-B1 and UT-B2) could be fully sequenced from ovine ruminal cDNA. Functionally, transport was passive and modulated by luminal pH in the presence of SCFA and CO2, rising in response to luminal acidification to a peak value at pH 5.8 and dropping with further acidification, resulting in a bell-shaped curve. Presence of ammonia reduced the amplitude, but not the shape of the relationship between urea flux and pH, so that urea flux remained maximal at pH 5.8. Effects of ammonia were concentration dependent, with saturation at 5 mmol/l. Clamping the transepithelial potential altered the inhibitory potential of ammonia on urea flux. Ammonia depolarized the apical membrane and acidified pHi, suggesting that, at physiological pH (< 7), uptake of NH4 (+) into the cytosol may be a key signaling event regulating ruminal urea transport. We conclude that transport of urea across the ruminal epithelium involves proteins subject to rapid modulation by manipulations that alter pHi and the cytosolic concentration of NH4 (+). Implications for epithelial and ruminal homeostasis are discussed.

Measuring Ca(2+) binding to short chain fatty acids and gluconate with a Ca(2+) electrode: role of the reference electrode.

Many organic anions bind free Ca(2+), the total concentration of which must be adjusted in experimental solutions. Because published values for the apparent dissociation constant (Kapp) describing the Ca(2+) affinity of short chain fatty acids (SCFAs) and gluconate are highly variable, Ca(2+) electrodes coupled to either a 3M KCl or a Na(+) selective electrode were used to redetermine Kapp. All solutions contained 130mM Na(+), whereas the concentration of the studied anion was varied from 15 to 120mM, replacing Cl(-) that was decreased concomitantly to maintain osmolarity. This induces changes in the liquid junction potential (LJP) at the 3M KCl reference electrode, leading to a systematic underestimation of Kapp if left uncorrected. Because the Na(+) concentration in all solutions was constant, a Na(+) electrode was used to directly measure the changes in the LJP at the 3 M KCl reference, which were under 5mV but twice those predicted by the Henderson equation. Determination of Kapp either after correction for these LJP changes or via direct reference to a Na(+) electrode showed that SCFAs do not bind Ca(2+) and that the Kapp for the binding of Ca(2+) to gluconate at pH 7.4, ionic strength 0.15M, and 23°C was 52.7mM.