Search

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

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. [ABSTRACT FROM AUTHOR]

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 CO2 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 CO2/HCO3- (10% and 25 mmol/l) 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 CO2 had no effect. Inhibition of Na+/H+ 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 CO2 on the function of existing urea transporters. [ABSTRACT FROM AUTHOR]

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 CO2 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 CO2/HCO3- (10% and 25 mmol/l) 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 CO2 had no effect. Inhibition of Na+/H+ 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 CO2 on the function of existing urea transporters. [ABSTRACT FROM AUTHOR]

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+ conductance in the apical membrane of ruminal epithelium. Using patch-clamped ruminal epithelial cells, we could observe a divalent sensitive, non- selective cation conductance (NSCC) with K+ permeability > Cs+ permeability > Na+ permeability. Conductance increased and rectification decreased when either Mg2+ or both Ca2+ and Mg2+ were removed from the internal or external solution or both. The conductance could be blocked by Ba2+, but not by tetraethylammonium (TEA). Subsequently, we studied this conductance measured as short-circuit current (Isc) in Ussing chambers. Forskolin, IBMX, and theophylline are known to block both Isc 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 Isc was followed by a subsequent increase. The cAMP-mediated increase in Ix was reduced by low serosal Na+ and serosal addition of imipramine or serosal amiloride and depended on the availability of mucosal magnesium. Luminal amiloride had no effect. Flux studies showed that low serosal Na+ reduced 28Mg fluxes from mucosal to serosal. The data suggest that cAMP stimulates basolateral Na+/Mg2+ 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. [ABSTRACT FROM AUTHOR]

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., (Copyright © 2014 the American Physiological Society.)