AJP - GI Journal of Neurophysiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 291: G246-G252, 2006. First published February 16, 2006; doi:10.1152/ajpgi.00419.2005
0193-1857/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/2/G246    most recent
00419.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Leonhard-Marek, S.
Right arrow Articles by Busche, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Leonhard-Marek, S.
Right arrow Articles by Busche, R.

MUCOSAL BIOLOGY

Effect of chloride on pH microclimate and electrogenic Na+ absorption across the rumen epithelium of goat and sheep

S. Leonhard-Marek,1 G. Breves,1 and R. Busche2

1Department of Physiology and 2Department of Biochemistry, School of Veterinary Medicine, Hannover, Germany

Submitted 6 September 2005 ; accepted in final form 10 February 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Active Na+ absorption across rumen epithelium comprises Na+/H+ exchange and a nonselective cation conductance (NSCC). Luminal chloride is able to stimulate Na+ absorption, which has been attributed to an interaction between Cl–/HCO3– and Na+/H+ exchangers. However, isolated rumen epithelial cells also express a Cl– conductance. We investigated whether Cl– has an additional effect on electrogenic Na+ absorption via NSCC. NSCC was estimated from short-circuit current (Isc) across epithelia of goat and sheep rumen in Ussing chambers. Epithelial surface pH (pHs) was measured with 5-N-hexadecanoyl-aminofluorescence. Membrane potentials were measured with microelelectrodes. Luminal, but not serosal, Cl– stimulated the Ca2+ and Mg2+ sensitive Isc. This effect was independent of the replacing anion (gluconate or acetate) and of the presence of bicarbonate. The mean pHs of rumen epithelium amounted to 7.47 ± 0.03 in a low-Cl– solution. It was increased by 0.21 pH units when luminal Cl– was increased from 10 to 68 mM. Increasing mucosal pH from 7.5 to 8.0 also increased the Ca2+ and Mg2+ sensitive Isc and transepithelial conductance and reduced the fractional resistance of the apical membrane. Luminal Cl– depolarized the apical membrane of rumen epithelium. 5-Nitro-2-(3-phenylpropylamino)-benzoate reduced the divalent cation sensitive Isc, but only in low-Cl– solutions. The results show that luminal Cl– can increase the microclimate pH via apical Cl–/HCO3– or Cl–/OH– exchangers. Electrogenic Na+ absorption via NSCC increases with pH, explaining part of the Cl– effects on Na+ absorption. The data further show that the Cl– conductance of rumen epithelium must be located at the basolateral membrane.

electrolyte transport; sodium absorption; forestomach; ruminants; microclimate

THE RUMEN, THE BIGGEST FORESTOMACH of ruminants, has a high significance for the absorption of sodium. Sodium intake with food is not very high (between 15 and 40 g/day for a cow, 2 g/day for a sheep), but during eating and especially during ruminating, substantial amounts of sodium enter the forestomachs with the saliva. This Na+ secretion by the salivary glands may reach more than 500 g/day in cows and 35 g/day in sheep (2, 14). Up to 50% of endogenous Na+ secretion is absorbed across the epithelium of the rumen (10), where two pathways contribute to active Na+ absorption: an electroneutral Na+/H+ exchange and an electrogenic Na+ conductance (25). Electrogenic Na+ uptake is especially important under conditions of sodium deficiency, when Na+ intake and Na+ concentration in the saliva are low (23). Ruminal electrogenic Na+ uptake ocurrs via a nonselective cation conductance (NSCC) which is regulated by extracellular Ca2+ and Mg2+ and by intracellular Mg2+ ions (19, 22).

Previous studies have shown that luminal chloride is able to stimulate Na+ absorption across the rumen wall in cattle and sheep (8, 24). This has been attributed to an interaction between Cl–/HCO3– and Na+/H+ exchange via the intracellular pH. Linear regression on Na+ and Cl– fluxes showed a high correlation coefficient between both transport rates and a coupling ratio of about 0.7 Cl–:1 Na+ in cattle (9) and 0.6 Cl–:1 Na+ in sheep (24). This coupling ratio suggests an indirect rather than a direct coupling mechanism. Alternatively, chloride absorption across the rumen, in addition to interacting with Na+/H+ exchange, might have an additional effect on Na+ conductance.

We wanted to know whether luminal chloride might have an effect on the electrogenic part of Na+ absorption via NSCC. For this purpose we measured the effect of chloride gradients on the Ca2+-sensitive Na+ current in Ussing chambers, on the apical membrane potential of rumen epithelial cells, and on the pH of the epithelial surface.

Our data suggest that the level of chloride on the luminal side can change the pH microclimate in the stratum corneum of the epithelial layer, which, in turn, increases the conductance for Na+ ions.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissues. The protocol of the animal treatment was approved and its conduct supervised by the respective animal protection officer of the institution. Adult male and female sheep and goats were killed by stunning with a commercial abattoir shooting apparatus and by bleeding from the carotids. Pieces of the ventral rumen wall were taken from slaughtered animals within 5 min after bleeding and immediately immersed in a buffer solution at 38°C, where the mucosa was stripped from the underlying muscle layers and the serosa.

Incubation and electrical measurements. Mucosal tissues were mounted between the two halves of incubation chambers with an exposed area of 1 or 3.14 cm2 (20). We minimized edge damage by placing rings of silicon rubber on both sides of the tissues. Ussing chambers were connected to reservoirs containing 15 ml buffer solution on each side. The solutions were kept at 38°C and were continuously stirred by the use of a gas lift system that supplied either 95% O2/5% CO2 or 100% O2. The chamber used for microelectrode studies (21) was perfused with solutions from gassed reservoirs driven by hydrostatic pressure. The chambers were connected to a computer-controlled voltage clamp device (AC Microclamp, K Mussler, Aachen, Germany) or to a voltage clamp and microelectrode device (Biomedical Instruments Munich, Germany). Transepithelial potential differences (Vt) were measured through buffer solution agar bridges and calomel electrodes with reference to the mucosal solution. Transepithelial conductances (Gt) were determined from the changes in Vt caused by bipolar current pulses of 100 µA/cm2 of 500-ms duration. The currents were passed through buffer solution agar bridges connected to Ag/AgCl electrodes in 3 M KCl (Ussing chambers) or through rings of Ag/AgCl electrodes placed in each half of the microelectrode chamber. In each setup, fluid resistances and junction potentials were measured before mounting the mucosal tissues and corrected for during the experiments. The experiments were performed under short-circuit conditions.

Microelectrodes. Conventional microelectrodes were pulled from borosilicate glass (outer diameter 1.2 or 1.5 mm) and filled with 0.5 M KCl, yielding resistances of 15–30 M{Omega}. We impaled rumen epithelial cells across the apical membrane using a motorized micromanipulator with piezo element and measured the apical membrane potential (Va) with reference to the mucosal solution. Impalements were accepted if 1) the change in Va was aprupt while advancing into the tissue, 2) Va remained stable for at least 1 min, and 3) Va returned to 0 ± 3 mV after withdrawing the electrode.

Measurements of surface pH. The surface pH (pHs) of the epithelia was measured according to the method of Genz et al. (12). 5-N-hexadecanoyl-aminofluorescein (HAF) is a pH-sensitive fluorescent dye that inserts in the outer leaflet of plasma membranes with the hexadecanoyl chain. Thereby, the fluorescent dye is fixed next to the surface of the epithelium allowing the continous measurement of pHs. For pH measurement, a piece of stripped ruminal epithelium was mounted in a microperfusion chamber (5) on the stage of a fluorescence microscope with the mucosal side directed to the objective. The mucosal side of the epithelium was superfused with HAF (15 µM in perfusion buffer) for 20 min to attach the dye to the epithelial surface (as shown in Fig. 1). Prior to the measurements, the epithelium was perfused for an additional 10 min with the experimental buffer. The fluorescence intensity of HAF (530 nm) was measured at two excitation wavelengths (436 and 485 nm) using an inverse microscope (Axiovert 35 M; Carl Zeiss, Oberkochen, Germany) equipped with a photomultiplier. The perfusion solutions could be changed independently at both sides of the tissues by using two microvalves (Hamilton, Bonaduz, Switzerland). The perfusion rate was 100 µl/min, driven by hydrostatic pressure. At the end of each experiment, a calibration with at least two calibration buffers of different pH was performed. The relationship between the ratio of the fluorescence signals and the pH has been shown to be linear between pH 7 and pH 8 (12). In the present study the fluorescence ratio changed between 1.64 (SD 0.16) at pH 7.0 and 3.01 (SD 0.33) at pH 8.0.


Figure 1
View larger version (25K):
[in this window]
[in a new window]
  		Fig. 1. Localization of 5-N-hexadecanoyl-aminofluorescein (HAF) in the stratum corneum of sheep rumen epithelium. Sections of HAF-labeled rumen epithelium. A: transmission image. B: fluorescence image. C: hematoxylin- and eosin-stained epithelium. A–C: scale bar = 100 µm.

 
Solutions. The standard solution contained (in mM) 140 Na+, 5.4 K+, 1.2 Ca2+, 1.2 Mg2+, 124 Cl–, 21 HCO3–, 2.4 HPO42–, 0.6 H2PO4–, and 10 glucose. In solutions with different chloride concentrations, Cl– was replaced by gluconate (or by acetate if specially indicated). Bicarbonate-free solutions were buffered with 8 mM HEPES. Ca2+-free solutions contained 0.5 mM EGTA. The solution used to transport the epithelia contained (in mM) 36 acetate, 15 propionate, and 9 butyrate in replacement for 60 mM Cl–. Bicarbonate solutions had a pH of 7.4 when gassed with 95% O2/5% CO2. The pH of the HEPES solutions was adjusted with Tris-OH. These solutions were gassed with 100% O2. Calibration buffers: for pH calibrations of HAF fluorescence, 10 µg/ml nigericin was added to the calibration buffers containing (in mM): 152 Cl–, 133.4 K+, 25 HEPES, 15 Na+, 1.8 Ca2+, 0.8 Mg2+, and 0.8 SO42–. The pH of these solutions was adjusted to defined pH in the range of 6.0 to 8.0 by different volumes of 1 N NaOH. In all buffers, osmolarity was adjusted with mannitol to 300 mosM.

Chemicals. 5-Nitro-2-(3-phenylpropylamino)-benzoate (NPPB) was dissolved in dimethyl sulfoxide (DMSO) and added in a volume of 2 µl DMSO per 10 ml buffer solution. This DMSO volume produced no electrophysiological effects in control tissues incubated in parallel. The pH-sensitive fluorescent dye HAF was purchased from Molecular Probes (Eugene, OR); NPPB and DMSO were from Sigma (Deisenhofen, Germany). All other chemicals were of analytical grade and were obtained from Merck (Darmstadt, Germany).

Statistics. Results are means ± SE; n designates the numbers of tissues or cells. Statistical significance was evaluated using analysis of variance or Student's t-test, paired or unpaired as appropriate.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of chloride on the Ca2+- and Mg2+-sensitive Na+ current. Isolated rumen epithelia from goat or sheep were incubated in standard buffer solution under short-circuit conditions. Changing to Ca2+- and Mg2+-free conditions on the luminal side increased short-circuit current (Isc), and addition of Ca2+ and Mg2+ ions to the luminal side decreased Isc, both as previously shown (19). Reducing the Cl– concentration on both sides of rumen epithelia had no effect on the baseline current but decreased the subsequent divalent cation-sensitive Isc (Fig. 2). This could be shown with epithelia from sheep and goat rumen.


Figure 2
View larger version (11K):
[in this window]
[in a new window]
  		Fig. 2. Influence of chloride on the short-circuit current (Isc) across goat rumen epithelium in the presence (gray bars) and in the absence (open bars) of Ca2+ and Mg2+ on the mucosal side. The given Cl– concentrations were present on both sides of the epithelia. Different solutions were applied consecutively to the same epithelia. Values are means ± SE; n = 5, *P < 0.05 vs. 124 mM Cl–.

 
We then changed repeatedly to Ca2+- and Mg2+-free conditions on the luminal side, while manipulating the Cl– concentration only on the luminal side, only on the serosal side, or on both sides of the epithelia. These experiments showed that luminal, but not serosal, Cl– accounted for the effects on the divalent cation-sensitive Isc in sheep and goat (Fig. 3).


Figure 3
View larger version (28K):
[in this window]
[in a new window]
  		Fig. 3. Mucosal Cl– stimulates the Isc across goat and sheep rumen epithelium in the absence of Ca2+ and Mg2+ on the mucosal side. Different solutions were applied consecutively to 3 neighboring epithelia from goat and 2 neighboring epithelia from sheep rumen. Values are means ± SE; n = 5 (goat) and n = 4 (sheep), *P < 0.05 vs. 124 mM Cl– (mucosal and serosal).

 
For these first experiments we had exchanged chloride for gluconate in the bathing solutions. Thus the results might have been due to a decrease in Cl– or an increase in gluconate concentration. To discriminate between both possibilities, we tested whether the replacing anion might be responsible for the effect attributed to chloride. Exchanging chloride for gluconate or acetate had the same immediate effect on Isc (Fig. 4). This Cl– effect was also shown in the absence of bicarbonate (HEPES-buffered solution, Fig. 4).


Figure 4
View larger version (30K):
[in this window]
[in a new window]
  		Fig. 4. Immediate effect of a reduced Cl– concentration on the Isc across goat rumen in the absence of Ca2+ and Mg2+ on the mucosal side. Cl– was replaced with acetate or gluconate and in the presence or absence of bicarbonate as indicated. The replacing anion did not influence this effect of Cl– on Isc. Values are means ± SE; number of experiments is given in parentheses.

 
A closer inspection of the time course of Isc measured at different Cl– concentrations showed two additional phenomena (Fig. 5). A drop in Cl– concentration was followed by an Isc decrease to the minimum value shown in Fig. 4. From this minimum value, Isc increased again when the epithelia were bathed in a low-Cl–, high-acetate solution (from 2.73 ± 0.25 to 3.65 ± 0.50 µeq·cm–2·h–1 within 10 min, n = 6, P < 0.05). In contrast, Isc remained on the minimum value when the epithelia were bathed in a low-Cl–, high-gluconate solution (2.10 ± 0.36 vs. 2.17 ± 0.34 µeq·cm–2·h–1 10 min later, n = 6). The exchange to a high-Cl– solution was followed by an overshoot in Isc (Fig. 5), which declined to a plateau value corresponding to the Isc under high-Cl– conditions shown in Fig. 4.


Figure 5
View larger version (9K):
[in this window]
[in a new window]
  		Fig. 5. Time course of Isc during changes in Cl– concentration on the luminal side of goat rumen epithelium as indicated. Values are means ± SE; n = 6.

 
Effects of chloride on apical membrane potential. Whole-cell patch-clamp experiments with isolated rumen epithelial cells have recently shown that these cells express a conductance for chloride (22). This Cl– conductance is not significantly altered by changes in external Ca2+ and Mg2+ ions (22) but might have an influence on the uptake of cations. To test whether Cl– conductances are also present in the intact tissue and whether luminal chloride might be able to alter the electric driving force for the uptake of cations, we impaled rumen epithelia with microelectrodes and measured the apical membrane potential at different mucosal Cl– concentrations. Serosal Cl– was kept constant at 120 mM.

Increasing the mucosal Cl– concentration from 0 to 120 mM induced a depolarization of the apical membrane in the presence and in the absence of divalent cations on the mucosal side (Fig. 6). In the presence of Ca2+ and Mg2+ ions, mucosal Cl– depolarized the Va from –55.9 ± 6.5 mV (0 mM Cl– mucosal) to –48.8 ± 6.0 mV (120 mM Cl– mucosal, n = 4, P < 0.01) without a significant effect on the fractional resistance of the apical membrane (fRa, 71 ± 2% and 69 ± 2%). In the absence of Ca2+ and Mg2+ ions, an increase in luminal Cl– depolarized Va from –44.8 ± 3.5 mV (0 mM Cl–) to –34.4 ± 3.2 (120 mM Cl–, n = 7, P < 0.001) and tended to reduce the fRa from 39 ± 5% to 34 ± 4% (P = 0.09). Membrane potentials were corrected for junction potentials according to Barry (3). Luminal Ca2+ and Mg2+ ions increased fRa (Fig. 6), as previously shown by Lang and Martens (18).


Figure 6
View larger version (53K):
[in this window]
[in a new window]
  		Fig. 6. Apical membrane potential of rumen epithelium (Va, bottom trace) and fractional resistance of the apical membrane (fRa, top trace) as influenced by luminal Cl– and luminal Ca2+ and Mg2+ concentrations (given in mM). Original trace from goat rumen. (Note that the trace for fRa appears with a technical time delay on the pen recorder. The addition of divalent cations causes an immediate hyperpolarization of Va and an immediate increase in fRa.)

 
Mucosal NPPB reduced Isc in low-chloride buffer. The arylaminobenzoate NPPB has been shown to block Cl– channels and Cl– transporters (6). When we applied 50 µM NPPB to the mucosal side of sheep rumen epithelium, in the absence of Ca2+ and Mg2+, NPPB tended to reduce Isc from 4.38 ± 0.69 to 3.95 ± 0.86 µeq·cm–2·h–1 (n = 4). In the standard buffer including 120 mM Cl–, this reduction, however, was not different from the time- and solvent-dependent control ({Delta}Isc = –0.43 ± 0.18 due to NPPB in DMSO vs. {Delta}Isc = –0.25 ± 0.06 µeq·cm–2·h–1 due to DMSO alone). In contrast, at low Cl– concentrations on the luminal side (10 mM Cl–) NPPB reduced Isc by 1.35 ± 0.37 µeq·cm–2·h–1, which differed significantly from the time- and solvent-dependent decrease of 0.13 ± 0.15 (n = 4). Experiments with goat rumen showed similar results. In the standard (high Cl–) buffer 50 µM NPPB reduced Isc by 0.42 ± 0.09 µeq·cm–2·h–1 (n = 5), whereas the time- and solvent (DMSO)-dependent decrease in Isc amounted only to 0.14 ± 0.04 µeq·cm–2·h–1 (n = 5). As in sheep, these experiments were performed under divalent cation-free conditions on the mucosal side. Because of the lower standard error, this NPPB effect was significantly different from the time-dependent decrease (P < 0.05). Again, the NPPB effect was significantly higher under Cl–-free conditions on the luminal side ({Delta}Isc = –1.73 ± 0.24 µeq·cm–2·h–1, n = 3, P < 0.001 vs. NPPB effect under high-Cl– conditions).

This NPPB action becoming obvious or enhanced at reduced Cl– concentrations suggests competition with the Cl– binding site of a transporter, rather than block of a channel.

Effects of chloride on pHs. Chloride absorption and bicarbonate secretion are partly dependent on each other in vivo; therefore, Cl–/HCO3– transporters have been suggested to be involved in ruminal Cl– absorption. Recently, the expression of different anion exchangers within the rumen papillae of sheep has been shown at the mRNA level (4). For the guinea pig colon it was shown that an apically located Cl–/HCO3– exchanger affects the pHs (12). We therefore tested whether chloride might exert an effect on the ruminal pH microclimate via these exchangers.

Incubating sheep ruminal epithelia with the pH-sensitive dye HAF for 20 min anchored the fatty acid tail of this dye molecule in the outermost layer of the epithelium (Fig. 1). When the epithelia were bathed in a low-chloride buffer (10 mM Cl–) on the mucosal side, the pHs amounted to 7.47 ± 0.03 (n = 10). This pHs was increased by 0.21 ± 0.01 (n = 10, P < 0.001) when the Cl– concentration was increased to 68 mM on the luminal side (Fig. 7).


Figure 7
View larger version (7K):
[in this window]
[in a new window]
  		Fig. 7. Influence of luminal Cl– concentration (in mM) on the surface pH (pHs) of sheep rumen epithelium. Representative trace measured with HAF.

 
A similar observation could be made in the absence of bicarbonate. In a HEPES-buffered solution the pHs amounted to 7.55 ± 0.03 under low-Cl– conditions on the mucosal side (25 mM Cl–). pHs increased by 0.12 ± 0.01 (n = 5, P < 0.001) upon change to a high-Cl– buffer on the luminal side (125 mM Cl–).

Effect of mucosal pH on Isc. To determine whether a small step in pHs might have an effect on the Na+ current via NSCC, we varied mucosal pH and measured basal and divalent cation-sensitive Isc and Gt across sheep rumen. This was done under SCFA- and bicarbonate-free conditions to minimize Na+ transport via Na+/H+ exchange.

In the presence of Ca2+ and Mg2+ on the mucosal side the increase in mucosal pH from 7.5 to 8.0 had no effect on Isc ({Delta}Isc = 0.09 ± 0.09 µeq·cm–2·h–1) and Gt ({Delta}Gt = 0.16 ± 0.18 mS/cm2, n = 9). When the same epithelia were incubated in the absence of divalent cations on the mucosal side, the increase in mucosal pH from 7.5 to 8.0 raised Isc by 0.19 ± 0.07 µeq·cm–2·h–1 (P < 0.05) and Gt by 0.39 ± 0.07 mS/cm2 (P < 0.001).

Effect of mucosal pH on apical membrane potential. If an increase in luminal pH is able to enhance the Na+ current through the divalent cation-sensitive pathway as suggested by the increases in Isc and Gt, then this should decrease the fRa and depolarize the Va. We therefore performed additional microelectrode experiments with sheep rumen under divalent cation-free conditions on the mucosal side and tested for the effect of a luminal pH increase from 7.5 to 8.0 on the Va and the fRa.

An increase in luminal pH depolarized the mean Va from –25.7 mV (at pH 7.5) to –24.5 mV (at pH 8.0) with a {Delta}Va of 1.2 ± 0.3 mV (n = 5, P < 0.01) and decreased the mean fRa from 49% to 45% with a {Delta}fRa of 4 ± 1% (n = 5, P < 0.05).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Physiological role of electrogenic Na+ transport in the rumen. In rumen epithelium the electrogenic Na+ transport mechanism works in parallel with a Na+/H+ exchange. At high concentrations Na+ is mainly transported via the electroneutral pathway, whereas at low-Na+ concentrations the electrogenic pathway predominates (23). High-energy diets have been shown to increase ruminal sodium absorption. This has been attributed to an increased transport via Na+/H+ exchange, since the stimulation could be blocked by a high dose of amiloride (1 mM). Na+/H+ exchange was also stimulated by the luminal presence of short-chain fatty acids (SCFA) and by a slight acidification of luminal pH (11). High-protein diets, on the other hand, can alkalinize the rumen contents. Under these conditions electrogenic Na+ absorption gains more importance, since a higher mucosal pH decreases the stimulatory effects of SCFA and CO2 on ruminal Na+/H+ exchange (11). A higher pH also reduces the concentration of free Ca2+ and Mg2+ ions in rumen fluid (13), which would at the same time relieve the blocking effect of these cations on the electrogenic Na+ transport. Sodium transport might be enhanced further if pH had a direct effect on the Na+ conductance. Such a direct stimulatory effect of mucosal pH on the Ca2+- sensitive current of monovalent cations has already been shown for amphibian epithelia (1, 17) and for cation currents through the epithelial Ca2+ channel ECaC (Ref. 29; now TRPV5). Our data suggest that pH can also stimulate the Ca2+- and Mg2+- sensitive Na+ current across rumen epithelium (see below).

Chloride effects via Cl– channels. In the current study chloride was able to stimulate the Ca2+- and Mg2+- sensitive Na+ current across the rumen, and this stimulation could be attributed to the presence of Cl– on the luminal side. Recent patch-clamp experiments with isolated rumen epithelial cells (REC) have shown that REC express a Cl– conductance (22). If this conductance were present in the intact tissue, and if it were localized in the apical membrane of epithelial cells, then an increase in luminal Cl– should hyperpolarize the transporting cells and thereby increase the driving force for the uptake of cations. We tested for this hypothesis with microelectrode experiments where we impaled rumen epithelium via the apical membrane. In contrast to the assumptions, an increase in luminal Cl– concentration did not hyperpolarize, but depolarized the rumen epithelium (Fig. 6). This observation strongly argues against an apical localization of Cl– channels in ruminal epithelium.

The Cl– conductances should thus be localized in the basolateral membranes of rumen epithelial cells and might contribute to the transepithelial transport of Cl–, as suggested for basolateral localized ClC-2 channels in the distal colon of guinea pigs (7). An increase in luminal Cl– concentration would then accumulate intracellular Cl– via electroneutral uptake across the apical membrane; this would be followed by electrogenic extrusion of Cl– across the basolateral membrane, thereby depolarizing the cell interior. A basolaterally localized Cl– conductance would thus be in line with the microelectrode experiments, whereas the Cl– effect on electrogenic Na+ transport must have another reason.

Chloride effects via anion exchangers. The current concept of chloride absorption across rumen epithelium involves an electroneutral Cl– uptake across the apical membrane via Cl–/HCO3– exchange, since removal of HCO3–/CO2 decreased the Cl– flux from the mucosal to the serosal side (8, 26). The hereby induced changes in HCO3– secretion might have an effect on local pH values. Studies with isolated REC (4) have shown that these cells need extracellular Cl– to recover from an alkaline load, suggesting apical and/or basolateral localized anion exchangers. We used the pH-sensitive dye HAF to investigate whether Cl– ions were able to change the pH microenvironment at the ruminal surface. HAF incorporates in the apical membrane of the outermost epithelial cells and inside epithelial cells that belong to the stratum corneum, the exterior cell layer of rumen epithelium (Fig. 1). Our data show that an increase in luminal Cl– concentration increases the pH of the epithelial surface. This would be in line with an apically localized Cl–/HCO3– exchange.

Investigating the effects of different combinations of SCFA on the pH microclimate was beyond the scope of the present study, but the regulatory increase in Isc in an acetate buffer (Fig. 5) suggests that an exchange of acetate for bicarbonate might as well have an effect on the pHs as it was described for butyrate at the basolateral pHs in the cecum by Kirschberger et al. (15) and therefore could also effect Isc. This would be in line with the concept of SCFA being partly absorbed via SCFA–/HCO3– exchange across rumen epithelium (16).

The present study shows additionally that an increase in luminal Cl– was also able to increase pHs in the absence of bicarbonate in the bathing solutions. This allows two explanations. First, cell metabolism might have provided sufficient HCO3– to run the exchanger even in the absence of extracellular HCO3–/CO2. We would, however, have expected a reduced exchange activity under these conditions since a switch from HCO3–/CO2-free to HCO3–/CO2-containing solutions is able to stimulate Cl– absorption (26). This argues against the first explanation. Second, bicarbonate might be replaced by OH– anions and allow for Cl– absorption via Cl–/OH– exchange. Different anion exchangers have been shown at the mRNA level in rumen epithelium and cultured ruminal epithelial cells (4), including AE2, DRA, and PAT1. These exchangers generally have a high affinity for HCO3– but can also transport OH– instead and function as a Cl–/OH– exchange (27, 28). This latter explanantion is supported by the observation that as for the pH effects, the presence of bicarbonate was also not necessary for the Cl– effects on Isc.

Effects of mucosal pH. The fluorescence experiments had shown that a variation in luminal Cl– was able to change the epithelial pHs by about 0.2 units to alkaline values. This is a pH range where electrogenic Na+ absorption gains importance for overall ruminal Na+ absorption. We therefore had to test whether a small change in luminal pH would be able to increase the Ca2+- and Mg2+- sensitive Isc. When divalent cations were present at the mucosal side, a variation in mucosal pH from 7.5 to 8.0 had no effect on Isc or Gt. This is in line with the observation that an increase in mucosal Cl– had no effect on Isc in the mucosal presence of divalent cations.

In the mucosal absence of divalent cations the same pH step from 7.5 to 8.0 increased Isc and Gt. These experiments were done with Na+ as the main cation in the buffer solution; so, the increase in Isc and Gt should reflect an increased Na+ absorption through the Ca2+-sensitive pathway at pH 8.0. Note that a change from low to high Cl– on the luminal side induced a small overshoot in the pHs as well as in the Isc recordings.

Microelectrode experiments under divalent cation-free conditions on the mucosal side showed that an increase in mucosal pH from 7.5 to 8.0 decreased the fRa and induced a small depolarization of the cells. Both observations are in line with a pH-dependent increase of the Ca2+-sensitive conductance and an enhanced Na+ diffusion via this pathway. The pH-induced changes in membrane potential seem very small; they are, however, in line with the changes in transepithelial potential that can be calculated from the Ussing chamber experiments. Furthermore, the increased Cl– effects on membrane potential and fRa seen in the absence of divalent cations are in the same range as the pH-dependent effects discussed here. These observations are in line with a Cl–-dependent change in pHs followed by an increased Na+ current through the divalent cation-sensitive pathway.

In conclusion, luminal chloride is able to increase the electrogenic (Ca2+ and Mg2+ sensitive) Na+ absorption across the rumen epithelium of sheep. Our data suggest that this increase is mediated via an increased activity of luminal Cl–/HCO3– or Cl–/OH– exchange, an increased pH in the microclimate of the epithelial surface, and a pH effect on the nonselective cation conductance responsible for Na+ absorption. The data further show that the Cl– conductance of rumen epithelium must be located at the basolateral membrane.


   ACKNOWLEDGMENTS
 
This project was supported by the Deutsche Forschungsgemeinschaft Le-824/2–1.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Leonhard-Marek, Dept. of Physiology, School of Veterinary Medicine, Bischofsholer Damm 15/102, D-30173 Hannover, Germany (e-mail: sabine.leonhard-marek{at}tiho-hannover.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Aelvoet I, Erlij D, and Van Driessche W. Activation and blockage of a calcium-sensitive cation-selective pathway in the apical membrane of toad urinary bladder. J Physiol 398: 555–574, 1988.[Abstract/Free Full Text]
  2. Bailey CB. Saliva secretion and its relation to feeding in cattle. 3. The rate of secretion of mixed saliva in the cow during eating, with an estimate of the magnitude of the total daily secretion of mixed saliva. Brit J Nutr 15: 443–451, 1961.[CrossRef][ISI][Medline]
  3. Barry PH. JPCalc, a software package for calculating liquid junction potential corrections in patch-clamp, intracellular, epithelial and bilayer measurements and for correcting junction potential measurements. J Neurosci Methods 51: 107–116, 1994.[CrossRef][ISI][Medline]
  4. Bilk S, Huhn K, Honscha KU, Pfannkuche H, and Gäbel G. Bicarbonate exporting transporters in the ovine ruminal epithelium. J Comp Physiol B 175: 365–374, 2005.[Medline]
  5. Busche R, Bartels J, Kirschberger S, and von Engelhardt W. Intracellular pH regulation of guinea-pig caecal and colonic enterocytes during and after loading with short-chain fatty acids and ammonia. Pflügers Arch 444: 785–794, 2002.[CrossRef][ISI][Medline]
  6. Cabantchik ZI and Greger R. Chemical probes for anion transporters of mammalian cell membranes. Am J Physiol Cell Physiol 262: C803–C827, 1992.[Abstract/Free Full Text]
  7. Catalan M, Cornejo I, Figueroa CD, Niemeyer MI, Sepulveda FV, and Cid LP. ClC-2 in guinea pig colon: mRNA, immunolabeling, and functional evidence for surface epithelium localization. Am J Physiol Gastrointest Liver Physiol 283: G1004–G1013, 2002.[Abstract/Free Full Text]
  8. Chien WJ and Stevens CE. Coupled active transport of Na and Cl across forestomach epithelium. Am J Physiol 223: 997–100, 1972.[Free Full Text]
  9. Diernæs L, Sehested J, Møller PD, and Skadhauge E. Sodium and chloride transport across the rumen epithelium of cattle in vitro: effect of short-chain fatty acids and amiloride. Exp Physiol 79: 755–762, 1994.[Abstract]
  10. Dobson A. Active transport through the epithelium of the reticulo-rumen sac. J Physiol 146: 235–251, 1959.[Free Full Text]
  11. Gäbel G, Vogler S, and Martens H. Short-chain fatty acids and CO2 as regulators of Na+ and Cl– absorption in isolated sheep rumen mucosa. J Comp Physiol B 161: 419–426, 1991.[Medline]
  12. Genz AK, v Engelhardt W, and Busche R. Maintenance and regulation of the pH microclimate at the luminal surface of the distal colon of guinea-pig. J Physiol 517: 507–519, 1999.[Abstract/Free Full Text]
  13. Johnson CL and Aubrey Jones DA. Effect of change of diet on the mineral composition of rumen fluid, on magnesium metabolism and on water balance in sheep. Brit J Nutr 61: 583–594, 1989.[Medline]
  14. Kay RNB. The rate of flow and composition of various salivary secretions in sheep and calves. J Physiol 150: 515–537, 1960.[Free Full Text]
  15. Kirschberger S, Busche R, and v Engelhardt W. Surface pH at the basolateral membrane of the caecal mucosa of guinea pig. J Membr Biol 169: 111–122, 1999.[CrossRef][ISI][Medline]
  16. Kramer T, Michelberger T, Gürtler H, and Gäbel G. Absorption of short-chain fatty acids across ruminal epithelium of sheep. J Comp Physiol B 166: 262–269, 1996.[CrossRef][Medline]
  17. Krattenmacher R, Voigt R, Heinz M, and Clauss W. Electrolyte transport through a cation-selective ion channel in large intestinal enterocytes of Xenopus laevis. J exp Biol 155: 275–290, 1991.[Abstract/Free Full Text]
  18. Lang I and Martens H. Na transport in sheep rumen is modulated by voltage-dependent cation conductance in apical membrane. Am J Physiol Gastrointest Liver Physiol 277: G609–G618, 1999.[Abstract/Free Full Text]
  19. Leonhard-Marek S. Divalent cations reduce the electrogenic transport of monovalent cations across rumen epithelium. J Comp Physiol B 172: 635–641, 2002.[CrossRef][Medline]
  20. Leonhard-Marek S, Gäbel G, and Martens H. Effects of short chain fatty acids and carbon dioxide on magnesium transport across sheep rumen epithelium. Exp Physiol 83: 155–164, 1998.[Abstract]
  21. Leonhard-Marek S and Martens H. Effects of potassium on magnesium transport across rumen epithelium. Am J Physiol Gastrointest Liver Physiol 271: G1034–G1038, 1996.[Abstract/Free Full Text]
  22. Leonhard-Marek S, Stumpff F, Brinkmann I, Breves G, and Martens. Basolateral Mg2+/Na+ exchange regulates apical non-selective cation channel in sheep rumen epithelium via cytosolic Mg2+. Am J Physiol Gastrointest Liver Physiol 288: G630–G645, 2005.[Abstract/Free Full Text]
  23. Martens H. Saturation kinetics of electrogenic and electroneutral Na transport of sheep rumen epithelium. Proc Soc Nutr Physiol 3: 87, 1994.
  24. Martens H and Blume I. Studies on the absorption of sodium and chloride from the rumen of sheep. Comp Biochem Physiol A 86: 653–656, 1987.[Medline]
  25. Martens H and Gäbel G. Transport of Na and Cl across the epithelium of ruminant forestomachs: Rumen and omasum. Comp Biochem Physiol A 90: 569–575, 1988.[Medline]
  26. Martens H, Gäbel G, and Strozyk B. Mechanism of electrically silent Na and Cl transport across the rumen epithelium of sheep. Exp Physiol 76: 103–114, 1991.[Abstract]
  27. Mount DB and Romero MF. The SLC26 gene family of multifunctional anion exchangers. Pflügers Arch 447: 710–721, 2004.[CrossRef][ISI][Medline]
  28. Romero MF, Fulton CM, and Boron WF. The SLC4 family of HCO3– transporters. Pflügers Arch 447: 495–509, 2004.[CrossRef][ISI][Medline]
  29. Vennekens R, Prenen J, Hoenderop JGJ, Bindels RJM, Droogmans G, and Nilius B. Modulation of the epithelial Ca2+ channel ECaC by extracellular pH. Pflügers Arch 442: 237–242, 2001.[CrossRef][ISI][Medline]



This article has been cited by other articles:


Home page
 J DAIRY SCIHome page
S. Leonhard-Marek, G. Becker, G. Breves, and B. Schroder
Chloride, Gluconate, Sulfate, and Short-Chain Fatty Acids Affect Calcium Flux Rates Across the Sheep Forestomach Epithelium
J Dairy Sci, March 1, 2007; 90(3): 1516 - 1526.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/2/G246    most recent
00419.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Leonhard-Marek, S.
Right arrow Articles by Busche, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Leonhard-Marek, S.
Right arrow Articles by Busche, R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.