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Abdoun K, Stumpff F, Rabbani I, Martens H. Modulation of urea transport across sheep rumen epithelium in vitro by SCFA and [CO.sub.2]. Am J Physiol Gastrointest Liver Physiol 298: G190-G202, 2010. First published November 19, 2009; doi:10.1152/ajpgi.00216.2009.--Urea transport across the gastrointestinal tract involves transporters of the urea transporter-B group, the regulation of which is poorly understood. The classical stimulatory effect of [CO.sub.2] and the effect of short-chain fatty acids (SCFA) on the ruminal recycling of urea were investigated by using Ussing chamber and microelectrode techniques with isolated ruminal epithelium of sheep. The flux of urea was found to be phloretin sensitive and passive. At a luminal pH of 6.4, but not at 7.4, the addition of SCFA (40 mmol/l) or [CO.sub.2]/[HCO.sub.3.sup.-] (10% and 25 mmol/1) led to a fourfold increase in urea flux. The stepwise reduction of luminal pH in the presence of SCFA from 7.4 to 5.4 led to a bell-shaped modification of urea transport, with a maximum at pH 6.2. Lowering the pH in the absence of SCFA or [CO.sub.2] had no effect. Inhibition of [Na.sup.+]/[H.sup.+] exchange increased urea flux at pH 7.4, with a decrease being seen at pH 6.4. In experiments with double-barreled, pH-sensitive microelectrodes, we confirmed the presence of an apical pH microclimate and demonstrated the acidifying effects of SCFA on the underlying epithelium. We confirm that the permeability of the ruminal epithelium to urea involves a phloretin-sensitive pathway. We present clear evidence for the regulation of urea transport by strategies that alter intracellular pH, with permeability being highest after a moderate decrease. The well-known postprandial stimulation of urea transport to the rumen in vivo may involve acute pH-dependent effects of intraruminal SCFA and [CO.sub.2] on the function of existing urea transporters. pHi; urea transporter-B; short-chain fatty acids; microclimate; volatile fatty acid doi: 10.1152/ajpgi.00216.2009

The present study investigated the significance of apical transport proteins for ruminal acetate absorption and their interaction with different anions. In anion competition experiments in the washed reticulorumen, chloride disappearance rate (initial concentration, 28 mM) was inhibited by the presence of a short-chain fatty acid mixture (15 or 30 mM of each acetate, propionate, and butyrate). Disappearance rates of acetate and propionate, but not butyrate (initial concentration, 25 mM each) were diminished by 40 or 80 mM chloride. In isolated ovine ruminal epithelia mounted in Ussing chambers, an increase in chloride concentration from 4.5 to 90 mM led to a decrease of apical acetate uptake at a concentration of 0.5 mM. Mucosal nitrate inhibited acetate uptake most potently whereas sulfate had no effect. Decreasing mucosal pH from 7.4 to 6.1 approximately doubled uptake of acetate both at 0.5 and 10 mM, but this doubling was almost abolished when HC[O.sup.-.sub.3] was absent. The stimulated uptake at mucosal pH 6.1 consisted of a bicarbonate-dependent, nitrate-inhibitable part ([K.sub.m] = 54 mM) and a bicarbonate-independent component ([K.sub.m] = 12 mM) that was also sensitive to nitrate inhibition. Maximal uptake was three times larger for bicarbonate-dependent vs. bicarbonate-independent uptake. Mucosal addition of 200 [micro]M DIDS, 400 [micro]M p-chloromercuribenzene sulfonic acid, 800 [micro]M p-hydroxymercuribenzoic acid, or 100 [micro]M phloretin had no effects on acetate uptake although the latter two inhibited L-lactate uptake. Our data conclusively show a dominant involvement of proteins in apical acetate uptake. Previously described pH effects on acetate absorption originate mainly from modulation of acetate/bicarbonate exchange. Additionally, there is bicarbonate-independent uptake of acetate anions that is protein coupled but not via monocarboxylate cotransporter. anion exchanger; bicarbonate transport; forestomach; monocarboxylate cotransporter; short-chain fatty acids

Ammonia is an abundant fermentation product in the forestomachs of ruminants and the intestine of other species. Uptake as N[H.sub.3] or N[H.sup.+.sub.4] should modulate cytosolic pH and sodium-proton exchange via [Na.sup.+]/[H.sup.+] exchanger (NHE). Transport rates of [Na.sup.+], N[H.sup.+.sub.4], and N[H.sub.3] across the isolated rumen epithelium were studied at various luminal ammonia concentrations and pH values using the Ussing chamber method. The patch-clamp technique was used to identify an uptake route for N[H.sup.+.sub.4]. The data show that luminal ammonia inhibits electroneutral Na transport at pH 7.4 and abolishes it at 30 mM (P < 0.05). In contrast, at pH 6.4, ammonia stimulates Na transport (P < 0.05). Flux data reveal that at pH 6.4, ~70% of ammonia is absorbed in the form of N[H.sup.+.sub.4], whereas at pH 7.4, uptake of N[H.sub.3] exceeds that of NH[sup.+.sub.4] by a factor of approximately four. The patch-clamp data show a quinidine-sensitive permeability for NH[sup.+.sub.4] and [K.sup.+] but not [Na.sup.+]. Conductance was 135 [+ or -] 12 pS in symmetrical N[H.sub.4]Cl solution (130 mM). Permeability was modulated by the concentration of permeant ions, with [P.sub.K] > [P.sub.NH4] at high and [P.sub.NH4] > [P.sub.K] at lower external concentrations. Joint application of both ions led to anomalous mole fraction effects. In conclusion, the luminal pH determines the predominant form of ammonia absorption from the rumen and the effect of ammonia on electroneutral Na transport. Protons that enter the cytosol through potassium channels in the form of NH[sup.+.sub.4] stimulate and nonionic diffusion of N[H.sub.3] blocks NHE, thus contributing to sodium transport and regulation of pH. rumen; ammonia; sodium transport; sodium-hydrogen exchanger; potassium channel

High potassium diets lead to an inverse regulation of sodium and magnesium absorption in ruminants, suggesting some form of cross talk. Previous Ussing chamber experiments have demonstrated a divalent sensitive [Na.sup.+] conductance in the apical membrane of ruminal epithelium. Using patch-clamped ruminal epithelial cells, we could observe a divalent sensitive, nonselective cation conductance (NSCC) with [K.sup.+] permeability > [Cs.sup.+] permeability > [Na.sup.+] permeability. Conductance increased and rectification decreased when either [Mg.sup.2+] or both [Ca.sup.2+] and [Mg.sup.2+] were removed from the internal or external solution or both. The conductance could be blocked by [Ba.sup.2+], but not by tetraethylammonium (TEA). Subsequently, we studied this conductance measured as short-circuit current ([I.sub.sc]) in Ussing chambers. Forskolin, IBMX, and theophylline are known to block both [I.sub.sc] and Na transport across ruminal epithelium in the presence of divalent cations. When the NSCC was stimulated by removing mucosal calcium, an initial decrease in [I.sub.sc] was followed by a subsequent increase. The cAMP-mediated increase in [I.sub.sc] was reduced by low serosal [Na.sup.+] and serosal addition of imipramine or serosal amilofide and depended on the availability of mucosal magnesium. Luminal amiloride had no effect. Flux studies showed that low serosal [Na.sup.+] reduced [sup.28]Mg fluxes from mucosal to serosal. The data suggest that cAMP stimulates basolateral [Na.sup.+]/[Mg.sup.2+] exchange, reducing cytosolic Mg. This increases sodium uptake through a magnesium-sensitive NSCC in the apical membrane. Likewise, the reduction in magnesium uptake that follows ingestion of high potassium fodder may facilitate sodium absorption, as observed in studies of ruminal osmoregulation. Possibly, grass tetany (hypomagnesemia) is a side effect of this useful mechanism. magnesium transport; nonselective cation channel; sodium absorption; rumen

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). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Magnesium (Mg) is an essential mineral without a specific hormonal regulation system. Mg-homeostasis and blood Mg-concentration depends on a continuous and sufficient absorption from the rumen. Ruminal absorption is essential for Mg-homeostasis. The apparent Mg-digestibility exhibits large variations and is influenced by the K content of the diet, the Na-status and the fermentation products short chain fatty acids (SCFA), CO2, NH4+ and pH. A meta-analysis of Mg-digestibility in dairy cows has shown that only the K-content reduces Mg-digestibility under practical feeding conditions of dairy cows. [ABSTRACT FROM AUTHOR]

A 15-year-old donkey gelding was referred to the Equine Clinic of the Freie Universität Berlin because of acute onset of anorexia, bradycardia and multiple syncopes. The clinical examination revealed an irregular heartbeat (11-18 bpm) and syncopes. A continuous ECG revealed absence of P-waves and an irregular ventricular escape rhythm. An echocardiography revealed a pericardial effusion with reduction of left and right ventricular function. Laboratory abnormalities revealed increased troponin I, alpha hydroxybutyrate dehydrogenase and symmetric as well as asymmetric dimethylarginine. Increased concentrations of the cardiac glycoside digitoxin was evident in serum. Clinical findings were consistent with myocarditis with pericardial effusion and atrial standstill. Because of increasing severity of symptoms and grave prognosis the donkey was humanely euthanized. Histopathological examination of the cardiac atria including the sinoatrial node revealed severe, subacute, diffuse, suppurative myocarditis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 NO that are associated with global warming. In ruminants, channel-mediated uptake of NH 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 NH that could be blocked in a voltage-dependent manner by divalent cations. In the native epithelium, NH depolarized the apical potential, acidified the cytosol and induced a rise in short-circuit current ( I) 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 in Na or NH containing solutions in a dose-dependent manner and modulated transepithelial Ca 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 NH, Na, and Ca across the rumen involves TRP channels, with TRPV3 and TRPA1 emerging as prime candidate genes. TRP channels may also contribute to the transport of NH across other epithelia. [ABSTRACT FROM AUTHOR]