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.

Results of recent in vitro experiments suggest that essential oils (EO) may not only influence ruminal fermentation but also modulate the absorption of cations like Na+, Ca2+ and NH4 + across ruminal epithelia of cattle and sheep through direct interaction with epithelial transport proteins, such as those of the transient receptor potential family. The aim of the current study was to examine this hypothesis by testing the effect of a blend of essential oils (BEO) on cation status and feed efficiency in lactating dairy cows. In the experiment, 72 dairy cows in mid-to-end lactation were divided into two groups of 36 animals each and fed the same mixed ration with or without addition of BEO in a 2×2 cross-over design. Feed intake, milk yield and composition, plasma and urine samples were monitored. Feeding BEO elevated milk yield, milk fat and protein yield as well as feed efficiency, whereas urea levels in plasma and milk decreased. In addition, plasma calcium levels increased significantly upon BEO supplementation, supporting the hypothesis that enhanced cation absorption might contribute to the beneficial effects of these EO.

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.

It has long been established that the absorption of short-chain fatty acids (SCFA) across epithelia stimulates sodium proton exchange. The apically released protons are not available as countercations for the basolateral efflux of SCFA anions and a suitable transport model is lacking. Patch clamp and microelectrode techniques were used to characterize an anion conductance expressed by cultured cells of the sheep and bovine rumen and the sheep omasum and to localize the conductance in the intact tissue. Cells were filled with a Na-gluconate solution and superfused with sodium salts of acetate, propionate, butyrate, or lactate. Reversal potential rose and whole cell current at +100 mV decreased with the size of the anion. Anion-induced currents could be blocked by diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS), NPPB (200 μmol l(-1)), or pCMB (1 mmol l(-1)). In patches of bovine ruminal cells, single channels were observed with a conductance for chloride (327 ± 11 pS), acetate (115 ± 8 pS), propionate (102 ± 10 pS), butyrate (81 ± 2 pS), and gluconate (44 ± 3 pS). Channels expressed by sheep rumen and omasum were similar. Microelectrode experiments suggest basolateral localization. In conclusion, forestomach epithelia express basolateral maxi-anion channels with a permeability sequence of chloride > acetate > propionate > butyrate. SCFA absorption may resemble functionally coupled transport of NaCl, with the Na(+)/K(+)-ATPase driving the basolateral efflux of the anion through a channel. Since protons are apically extruded, the model accurately predicts that influx of buffers with saliva is essential for the pH homeostasis of the ruminant forestomach.

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.

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.

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.

No abstract