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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]

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 NH4Cl 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. [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]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [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]

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. [ABSTRACT FROM PUBLISHER]

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]

Abstract: Many organic anions bind free Ca2+, the total concentration of which must be adjusted in experimental solutions. Because published values for the apparent dissociation constant (K app) describing the Ca2+ affinity of short chain fatty acids (SCFAs) and gluconate are highly variable, Ca2+ electrodes coupled to either a 3M KCl or a Na+ selective electrode were used to redetermine K app. 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 K app 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 K app either after correction for these LJP changes or via direct reference to a Na+ electrode showed that SCFAs do not bind Ca2+ and that the K app for the binding of Ca2+ to gluconate at pH 7.4, ionic strength 0.15M, and 23°C was 52.7mM. [Copyright &y& Elsevier]

Abstract: The ionized concentration of calcium in physiological buffers ([Ca2+]) is normally calculated using either tabulated constants or software programs. To investigate the accuracy of such calculations, the [Ca2+] in EGTA [ethylene glycol-bis(β-aminoethylether)-N,N,N|,N|-tetraacetic acid], BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N|,N|-tetraacetic acid], HEDTA [N-(2-hydroxyethyl)-ethylenediamine-N,N|,N|-triacetic acid], and NTA [N,N-bis(carboxymethyl)glycine] buffers was estimated using the ligand optimization method, and these measured values were compared with calculated values. All measurements overlapped in the pCa range of 3.51 (NTA) to 8.12 (EGTA). In all four buffer solutions, there was no correlation between measured and calculated values; the calculated values differed among themselves by factors varying from 1.3 (NTA) to 6.9 (EGTA). Independent measurements of EGTA purity and the apparent dissociation constants for HEDTA and NTA were not significantly different from the values estimated by the ligand optimization method, further substantiating the method. Using two calibration solutions of pCa 2.0 and 3.01 and seven buffers in the pCa range of 4.0–7.5, calibration of a Ca2+ electrode over the pCa range of 2.0–7.5 became a routine procedure. It is proposed that such Ca2+ calibration/buffer solutions be internationally defined and made commercially available to allow the precise measurement of [Ca2+] in biology. [Copyright &y& Elsevier]

The forestomachs of cows and sheep have historically served as important models for the study of epithelial transport. Thus, the ruminal epithelium was among the first tissues in which absorption of chloride against an electrochemical gradient was observed, requiring a tight paracellular barrier to prevent back-leakage. However, little is known about ruminal barrier function, despite the considerable implications for ruminant health. The tight junction proteins of the omasum have never been investigated, and no cell culture model exists. We present a new method for the isolation of cells from forestomach epithelia. Protein expression of cells and source tissues of sheep were studied using western blot, PCR and confocal laser scanning microscopy. Cultured cells were characterized by transepithelial resistance (TER) measurements and patch clamping. Cells developed TER values of 729±134 Ω cm2 (rumen) and 1522±126 O cm2 (omasum). Both primary cells and source epithelia of rumen and omasum expressed cytokeratin, occludin and claudins 1, 4 and 7 (but not claudins 2, 3, 5, 8 and 10), consistent with the observed paracellular sealing properties. Staining for claudin-1 reached the stratum basale. The full mRNA coding sequence of claudins 1, 4 and 7 (sheep) was obtained. Patch-clamp analyses of isolated cells proved expression of an anion conductance with a permeability sequence of gluconate

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]

PURPOSE. Flufenamic acid relaxes trabecular meshwork,a smooth muscle-like tissue involved in the regulation of ocular outflow inthe eye. In this study, we attempted to determine if ionic channels are involvedin this response. METHODS. Cultured human (HTM) and bovine (BTM) trabecular meshwork cellswere investigated using the patch-clamp technique. RESULTS. In trabecular meshwork, flufenamic acid (10[sup -5] M) reversibly stimulated outward current to 406 ± 71% of initial outwardcurrent level in BTM (n = 10) and 294 ± 75% of initial current levelin HTM (n = 12) in all cells investigated; no significant differences emerged.The response was dosage-dependent. Replacement of potassium in all solutionseliminated the response to flufenamic acid (n = 4, BTM). Blocking K[sub ATP] channels with glibenclamide (10[sup -5] M, n = 6) and small-conductancecalcium-activated potassium channels with apamin (10[sup -6] M,n = 5) had no effect. A direct effect on calcium channels could also not bedetected. Blockage of the large-conductance calcium-activated potassium channel(maxi-K) by iberiotoxin (10[sup -7] M) suppressed 87 ± 9%(n = 6; HTM) and 91 ± 10% (n = 6; BTM) of the response. Depleting thecells of calcium did not significantly alter the response to flufenamic acid. CONCLUSIONS. Flufenamic acid stimulates maxi-K channels in trabecular meshworkof both human and bovine origin. This should lead to hyperpolarization, closureof L-type channels and lowered cytosolic calcium levels, possibly explainingthe relaxation observed in response to this substance. [ABSTRACT FROM AUTHOR]