1Department of Physiology, School of Veterinary Medicine, Hannover, Germany; and 2Department of Veterinary Physiology, Free University of Berlin, Berlin, Germany
Submitted 25 June 2004 ; accepted in final form 12 November 2004
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ABSTRACT |
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magnesium transport; nonselective cation channel; sodium absorption; rumen
Despite the economic importance of ruminants, the complex functions of rumen epithelium are only partly understood. The rumen, the biggest forestomach, has to ensure an optimal environment for fermentation. Stratified, squamous epithelium such as that found lining the rumen is not usually considered an absorptive type of epithelium. However, numerous and richly vascularized papillae ensure absorption of many nutrients across the ruminal wall. Thus up to 50% of the sodium entering the forestomachs is transported across this epithelium (2). The forestomachs are the main site of Mg2+ uptake in ruminants (24, 35), which has to be considerable to compensate for secretion of this element in milk.
Absorption rates of electrolytes across the rumen vary with the dietary state of the animal, suggesting a precise regulation of ion transport processes (6, 8). In general, absorption of electrolytes has to take place in a manner that does not endanger vital functions. In particular, osmolarity has to be regulated to prevent fluid loss from the plasma in the ruminal cavity, and excessive absorption of potassium has to be avoided to prevent hyperkalemia. When dietary intake of potassium is high, as when high-potassium fertilizer is used, the rumen epithelium responds by adjusting osmolarity of the ruminal content through an increase in the rate of sodium absorption (14, 30, 32). Simultaneously, magnesium absorption decreases, which can lead to fatal hypomagnesemias in ruminants (grass tetany) if high potassium intake persists. In this study, we attempted to find out if these two phenomena are in any way linked to each other.
Active Na+ absorption across the rumen epithelium involves active extrusion by an Na+-K+-ATPase on the basolateral side, whereas uptake on the apical side utilizes both Na+/H+ exchange and an electrogenic Na+ conductance that can be measured as short-circuit current (Isc) in Ussing chamber experiments. This conductance is resistant to low doses of amiloride (22) and can be stimulated by depolarization of the apical membrane. The conductance can be measured in the presence of physiological concentrations of divalent cations, where Na+ current is equivalent in magnitude to the sodium flux measured when electroneutral sodium transport is blocked (22). It is enhanced by the elimination of free Ca2+ and Mg2+ from the mucosal side, and conducts K+ in addition to Na+ (14, 15, 17, 26).
Mg2+ absorption across the rumen epithelium involves basolateral Mg2+/Na+ exchange (19, 29), whereas various pathways for apical uptake have been suggested. Voltage-independent Mg2+ uptake mechanisms may include Mg2+/2H+ exchange (stimulated by fermentation products, such as short-chain fatty acids and CO2) (16, 18) and/or Mg2+-anion cotransport (28). In addition, a further electrodiffusive, voltage-dependent pathway exists. High potassium intake depolarizes the apical membrane, which reduces both magnesium absorption rates and cytosolic magnesium levels (20, 29).
Two different methods were used to investigate if the increase in Na+ absorption that follows an elevation of luminal potassium is linked to changes in the level of intracellular Mg2+ in rumen epithelium.
In patch-clamp experiments, cytosolic levels of magnesium were altered directly in isolated cultured ruminal epithelial cells via infusion of different solutions in the cell. In Ussing chamber experiments, the interaction of sodium with 28Mg flux rates was studied, using native preparations of ruminal epithelium. In these experiments, changes in cAMP were used to alter cytosolic magnesium levels pharmacologically, with impact both on magnesium and sodium transport. cAMP has been shown to stimulate Mg2+/Na+ exchange in several tissues, including the ruminal epithelium, thereby decreasing intracellular Mg2+ (3, 10, 23, 25).
The patch-clamp data show that rumen epithelial cells (REC) express a nonselective cation channel that opens when magnesium alone or both calcium and magnesium are removed from either the extracellular or the intracellular side, or both. Channel rectification is influenced by the presence of divalents, suggesting a voltage-dependent blocking mechanism. In Ussing chamber experiments, we were able to demonstrate that basolateral Mg2+ extrusion is Na+ dependent and can be stimulated by cAMP, thus leading to lower levels of cytosolic Mg2+ and an increase in Na+ current through the tissue. Thus magnesium may be an important second messenger in the regulation of sodium transport across the ruminal wall. We suggest that, physiologically, stimulation of sodium absorption by decreased levels of cytosolic magnesium may be an important pathway for osmoregulation after ingestion of high potassium fodder, with clinical manifestations of hypomagnesaemia occurring as a side effect if high potassium intake persists.
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METHODS |
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Pieces of the ventral rumen wall were taken from slaughtered adult sheep within 5 min after bleeding and immediately immersed in buffer solution at 38°C, where the mucosa was stripped from the underlying muscle layers and the serosa. The mucosa was then used to prepare either cell cultures for patch-clamp experiments or used directly for Ussing chamber measurements.
Cell Culture
Primary cultures of REC were kindly prepared by Schweigel and Martens (28). Briefly, REC were isolated by fractional trypsination and grown on glass coverslips in medium 199 containing 10% FCS, 1.36 mM glutamine, 20 mM HEPES, and antibiotics (50 mg/l gentamycin and 100 mg/l kanamycin) in an atmosphere of humidified air with 5% CO2 at 38°C. The number of cornified cells in the culture increased rapidly after seeding. Cover slips were removed from the culture dishes for experiments 68 days after seeding or, alternately, 15 days after reseeding from primary culture. Immediately before the experiments, cells were exposed to a trypsin/EDTA solution (0.005/002%; Biochrom) for 30 s to 1 min to facilitate seal formation. Only cells without signs of cornification were used for experiments. Cells remaining in the culture dishes all cornified if left undisturbed for 2 to 3 wk.
Patch-Clamp Experiments
All patch-clamp experiments were performed essentially as in a previous study (31) using an EPC 9 patch-clamp amplifier (HEKA Elektronic, Lambrecht, Germany) and TIDA for Windows Software (HEKA Elektronic). Positive ions flowing into the pipette correspond to a negative current and are depicted in Figs. 17 as going downward. Capacitance and access resistance were corrected using TIDA software.
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Solutions for the Patch-Clamp Experiments
Basically, four different pipette solutions and seven extracellular solutions were used (Table 1). In some bath solutions, gluconate was used to replace chloride in solutions that were otherwise not altered in composition. In some pipette solutions, CsCl or Cs-methanesulfonate was used to replace choline chloride with otherwise identical components. All bath solutions were titrated to pH 7.4; all pipette solutions were titrated to pH 7.2, using Tris-OH. Osmolarity was adjusted by adding appropriate amounts of the dominant salt. In the pipette solutions, calcium was buffered to the physiological range below 300 nM using EGTA of 97% purity. Because EGTA binding with magnesium is weak, ionized concentration of magnesium should roughly equal the total amount (13, 21).
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Current values for analysis were obtained after a certain stabilization had occurred, immediately before the application of the next solution. Absolute current values from different cells varied greatly because of differing cell sizes. To compare the data, two methods were employed. In the first approach, current was expressed in percent of current level measured at the beginning of the experiment at 100 mV pipette potential in "NaCl + Ca2+ + Mg2+" solution, thus obtaining a certain correction for both cell size and leakage but making it impossible to compare cells with different intracellular (pipette) solutions. In another approach, current was normalized to the capacity as measured by Tida for Windows software. This approach yielded absolute current density values for different pipette solutions that could then be compared.
Reversal potentials (Erev) were estimated by linear regression between the current values just above and just below the zero level for each cell and corrected for liquid junction potential.
Rectification scores were obtained by determining the value of the slope of the current-voltage relationship between 120 and 100 mV pipette potential and dividing it by that between 0 and 20 mV. {In practice, Rect = [I(120 mV) I(100 mV)]/[I(0 mV) I(20 mV)], where I(E) is the current at a potential of "E"}.
Electrical Measurements in Ussing Chambers
Mucosal tissues were mounted between the two halves of incubation chambers with an exposed area of 1 or 3.14 cm2. Edge damage was minimized by placing rings of silicon rubber on both sides of the tissues. Incubation 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 chambers were connected to a computer-controlled voltage-clamp device (AC Microclamp, Aachen, Germany). Transepithelial potential differences (Vt) were measured through buffer solution agar bridges and calomel electrodes with reference to the mucosal solution. Tissue 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. 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.
Flux Studies
Epithelia were incubated in Ussing chambers under short-circuit conditions (see above). For the measurement of unidirectional Mg2+ flux rates, 28MgCl2 (Dept. of Physics, Garching, Germany) was added to one side of the epithelium to yield a specific activity of 2 kBq/µmol in the buffer solution. After an equilibration period of 45 min, aliquots were taken at 30-min intervals from the unlabeled side and replaced by aliquots of unlabeled solution. 28Mg was counted in a crystal scintillation counter (Gammaszint 5300; Berthold), and countings were corrected for physical decay. Unidirectional fluxes were calculated from the rate of tracer appearance at the unlabeled side of the tissue. Paired determinations of fluxes were accepted if Gt differed by <25%. Unless otherwise stated, different experimental conditions were tested with adjacent pieces of tissue from the same animal.
Solutions for Ussing Chamber and Flux Measurements
The standard solution contained (in mmol/l): 140 Na+, 5.4 K+, 1.2 Ca2+, 1.2 Mg2+, 124 Cl, 21 HCO3, 2.4 HPO, 0.6 H2PO
, and 10 glucose. In the short-chain fatty acid buffer used to prepare and transport the tissues, 60 mmol/l Cl was replaced by 36 acetate, 15 propionate, and 9 butyrate. In the low Na buffer, 119 mmol/l Na+ was replaced by N-methyl-D-glucamine (NMDG+). Ca2+-free solutions contained 0.5 mmol/l EGTA. The pH of the solutions was 7.4 when gassed with 95% O2-5% CO2. Indomethacin (105 mol/l) was added to all solutions to prevent a stimulation of the cAMP pathway by the production of prostaglandins within the rumen tissue.
The low Na+ solutions used for the Mg2+ flux studies contained (in mmol/l): 5, 10, or 30 Na+, 25, 20, or 0 NMDG+, 5 K+, 1 Ca2+, 2 Mg2+, 48 choline+, 85 Cl, 1 HPO, 2 H2PO
, 13 acetic acid, 13 propionic acid, 13 butyric acid, and 10 glucose. The pH of these solutions was adjusted to 7.4 with Tris-OH, and they were gassed with 100% O2.
Osmolarity was adjusted to 300 mosmol/l with mannitol.
Chemicals
Forskolin and amiloride were dissolved in DMSO and added in a maximal volume of 10 µl DMSO/10 ml buffer solution. This DMSO volume produced no electrophysiological effects in control tissues incubated in parallel. IBMX and imipramine were dissolved in aqueous stock solutions. Theophylline and indomethacin were directly included in the buffer solutions. Forskolin, amiloride, DMSO, IBMX, imipramine, medium 199, trypsin, glutamine, antibiotics (gentamycin, nystatin, and kanamycin) and FCS were purchased from Sigma (St. Louis, MO). Dulbecco's PBS was obtained from Biochrom (Berlin, Germany). All other chemicals were of analytical grade and were obtained from Merck (Darmstadt, Germany) or from Sigma.
Statistics
Results are given as means ± SE; n designates the numbers of tissues or cells. Statistical significance was evaluated using ANOVA or Student's t-test, paired or unpaired as appropriate. Differences were assumed significant when P values were lower than 0.05 and highly significant when P values were lower than 0.01.
As a cross-check to assess the validity of the calculations of significance, the group of cells measured with "K-gluconate + Ca2+ + Mg2+" in the patch pipette was split into two subgroups of 7 cells each with cells measured in months 1 and 2 in the first group and cells from the months 3 and 4 in the second group. The current densities of these two groups were then compared with each other using Student's t-test. No significant differences (P < 0.05) emerged between cells exposed to identical solutions but measured in different months. In terms of reproducibility of the various parameters, P values for current densities lay around P = 0.1, those for rectification scores around P = 0.5, whereas Erev were practically identical with P values approaching P = 1.
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RESULTS |
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Effect of external Ca2+ and Mg2+ on whole cell currents. To mimic physiological conditions, the cells were filled with a high-potassium, low-chloride solution containing calcium buffered with EGTA (K-gluconate + Ca2+ + Mg2+). The cells were superfused with a NaCl buffer solution (NaCl + Ca2+ + Mg2+). A pipette potential of 100 mV induced an outward current of 27 ± 5 pA/pF (n = 17), which was set to 100% (see METHODS). In the same cells, a pipette potential of 120 mV induced an inward current of 14 ± 4 pA/pF, corresponding to 51 ± 4% of initial outward current level.
After current had stabilized, calcium and magnesium were removed from the external solution. This induced a great increase in inward current, which changed from 51 ± 4 to 116 ± 17% (P = 0.0003, n = 17) and from 14 ± 4 to 24 ± 8 pA/pF (n = 14, P = 0.04), whereas changes in outward current at a pipette potential of 100 mV did not reach significance (29 ± 10 pA/pF, P = 0.8; Fig. 1, A and C). Erev increased (Table 2).
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It appears that REC express a nonselective cation channel that is inwardly rectifying under the conditions described above and opens when external divalents are removed.
Substitution of chloride by gluconate in the outside medium. When NaCl was replaced by sodium gluconate in the divalent-containing bath solution, outward current sank significantly to 14 ± 4 pA/pF (n = 5, P = 0.01) or from 100% (NaCl) to 40 ± 4% (P = 0.0006; sodium gluconate), whereas inward current did not change significantly (P = 0.07; Fig. 1, B and C). Erev rose, reflecting the decrease in chloride influx (Table 2).
Superfusion of cells with divalent free potassium gluconate solution resulted in an outward current level of 208 ± 116% or 59 ± 36 pA/pF, marginally down from 259 ± 128 (KCl 0 Ca2+ 0 Mg2+; P = 0.05, n = 5). Inward current remained the same as in divalent free KCl solution (733 ± 230% or 171 ± 77 pA/pF). Erev rose.
By subtracting the current levels of individual cells in sodium gluconate solution from those in NaCl solution, it is possible to obtain difference currents that correspond to the chloride conductance of the cells. This maneuver resulted in values of 5 ± 2 pA/pF (120 mV) and 17 ± 9 pA/pF (100 mV). When the same calculation was carried out for the divalent free KCl and potassium gluconate solutions, chloride conductance was 6 ± 2 pA/pF (120 mV) and 22 ± 7 pA/pF (100 mV), which corresponds remarkably well to the levels obtained for the divalent-containing NaCl solution (P = 0.9 and 0.7).
We conclude that REC express a chloride conductance that does not seem to be significantly altered by changes in external calcium and magnesium. In divalent-containing solutions, this conductance contributes significantly to the Erev, whereas in divalent-free solutions, cation conductances are more relevant.
Effect of internal Ca+ and Mg2+ on whole cell currents. To assess the influence of divalent cations from the intracellular side, we removed Ca2+ and Mg2+ from the pipette solution in a second series of experiments (potassium gluconate 0 Ca2+ 0 Mg2+). When divalent cations were removed from the extracellular NaCl buffer under these conditions, not just inward but also outward current level increased significantly (Fig. 3). At 100 mV, current level increased from 100% to 162 ± 26% (n = 10, P = 0.04), corresponding to 36 ± 10 and 59 ± 16 pA/pF (n = 8, P = 0.04). Inward current at 120 mV increased from 56 ± 7 to 202 ± 39% of initial outward current (P = 0.003) or, in relation to capacitance, from 21 ± 6 to 57 ± 16 pA/pF (n = 8, P = 0.03).
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Thus, in the absence of divalent cations from the intracellular solution, the removal of divalents from the extracellular side induced higher inward (P = 0.02) and higher outward (P = 0.001) currents than in the presence of internal Ca2+ and Mg2+ (Fig. 2). The divalent-sensitive current was higher both at hyperpolarized levels (P = 0.03) and at depolarized levels (P = 0.01). It appears that both influx of sodium and efflux of potassium through the divalent-sensitive pathway is enhanced by the removal of divalents from the internal solution. Inward rectification was reduced by removal of internal divalents.
The Erev tended to be higher than those obtained with divalent-containing pipette solutions (Table 2), but the difference was not significant. This may reflect the fact that a greater efflux of potassium compensates for a greater influx of sodium.
Currents in cells filled with choline chloride. In the experiments presented so far, both chloride and potassium conductances contribute significantly to the currents. To eliminate the contributions of potassium to the whole cell current, the cells were filled with a (unphysiological) solution in which choline and chloride were the predominant ions (choline chloride +Ca2+ + Mg2+). Under these conditions, the theoretical Erev are 1 mV for chloride and 0 mV for potassium.
Removing external divalents from an NaCl buffer increased inward current from 13 ± 2 to 26 ± 4 pA/pF (P = 0.00001, n = 21; or, in percent, from 79 ± 5 to 219 ± 34%, P = 0.0003), whereas outward current changed less dramatically from 16 ± 3 pA/pF (100%) to 19 ± 4 pA/pF (114 ± 7%; P = 0.02). Values recovered to 8 ± 2 (82 ± 11%) and 12 ± 3 (84 ± 4%) pA/pF upon readdition of external divalents (Fig. 4A and Fig 5, AC).
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Calculation of the divalent-sensitive current yielded mean inward and outward current values of 12 ± 1 and 3 ± 1 pA/pF, in remarkably good agreement with those obtained with divalent-containing potassium gluconate solution in the cells (P = 0.8 and P = 0.6, respectively; Fig. 2). The Erev of the divalent-sensitive current of the choline chloride-filled cells (26 ± 4 mV) was significantly higher than that of cells filled with potassium gluconate solution (P = 0.01). Rectification score was higher than that observed in potassium gluconate pipette solution (8 ± 2) because of the reduction in potassium efflux.
This current could be blocked by adding 5 mM BaCl2 to the external solution, resulting in inward currents that were significantly lower than those observed in calcium- and magnesium-free solution (18 ± 5 pA/pF or 101 ± 13%, n = 5, P = 0.003), whereas outward current remained the same (divalent-containing solution: P = 0.9; divalent-free solution: P = 0.3). Erev fell to 3 ± 2 mV, approaching the chloride equilibrium potential of 1 mV. TEA-Cl (5 mM) did not have a significant effect on the divalent-sensitive current (n = 4; Fig. 4B).
Again, an even greater effect could be observed when divalents were removed with potassium as the major cation in the external solution, with inward current rising from 14 ± 2 to 24 ± 3 pA/pF (n = 4, P = 0.04; or from 119 ± 13 to 358 ± 77%; Fig. 4A and Fig. 5, DF). Outward current level did not change significantly. Erev rose to higher levels than those in NaCl buffer (Table 2). The divalent-sensitive potassium current, calculated as described above for NaCl solution, yielded a Erev of 38 ± 11 mV, approaching the Erev for potassium.
Neither inward nor outward current nor Erev changed when choline chloride was used to substitute for NaCl in the external solution (P = 0.3, P = 0.6, and P = 0.9, n = 8). Removal of divalent cations from this solution did not change inward or outward current significantly (P = 0.1 and P = 0.4, respectively; Fig. 5, GI).
In summary, we conclude that a change in internal monovalent cations (from K to choline) and internal anions (from gluconate to chloride) does not significantly alter the properties of the nonselective cation channel in ruminal epithelial cells.
Currents in cells filled with cesium chloride. In a further series of experiments, cells were filled with a pipette solution in which cesium replaced choline as the major ion in a solution otherwise identical to the one above. Erev was negative and significantly lower than in the choline chloride-filled cells, reflecting a significant amount of Cs efflux, exceeding influx of Na (Table 2).
Elevation of potassium in the external solution increased inward current from 44 ± 6 to 86 ± 29 pA/pF (or from 86 ± 6 to 153 ± 22% of initial outward current, P < 0.000001, n = 10), whereas outward current (55 ± 8 pA) remained stable (P = 0.5; Fig. 6, AC). Erev increased significantly to a positive value, reflecting that conductance for potassium exceeds that of Cs. The increase in Erev (when changing from NaCl to KCl solution extracellular) was comparable to that observed in cells filled with choline chloride solution (Table 2), indicating that internal Cs does not interfere with potassium influx into the cell.
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Removal of divalent cations in KCl solution induced an increase in inward current to 210 ± 29% (n = 4), exceeding the value in normal KCl solution (p = 0.01; Fig. 6, B and C). Outward current did not differ from the value measured in divalent-free NaCl solution (P = 0.2). Erev was higher than in the NaCl-containing solutions but significantly lower than that measured when cells were filled with choline chloride solution.
As before, we calculated a divalent-sensitive current from these values with inward divalent-sensitive current in NaCl solution remarkably close to that observed for the choline chloride-filled cells (12 ± 3 pA/pF, n = 4, P = 0.7). Differences in divalent-sensitive outward current level between the cells filled with choline or CsCl did not reach significance at +100 mV (7.3 ± 3 pA/pF, P = 0.4). However, around 0 mV, divalent-sensitive current in the CsCl-filled cells was significantly higher (P = 0.001) than in the choline chloride group, indicating efflux of Cs+. Erev of the divalent-sensitive current was 0.7 ± 0.8 mV for the NaCl solutions and 5.7 ± 0.8 mV for the KCl solutions (P = 0.003).
A further series of experiments was performed using cesium methanesulfonate instead of CsCl. As to be expected, Erev was significantly lower than in cells filled with either CsCl or choline chloride. Currents were outwardly rectifying (Fig. 6E), reflecting the reduction in the efflux of chloride at negative potential levels in the cesium methanesulfonate-filled cells. Inward current was stimulated by KCl solution, reaching a significance level upon removal of divalent cations (P = 0.04; Fig. 6D). Erev remained close to the original (negative) value throughout this maneuver, demonstrating stability of the seal while suggesting that, as before, removal of divalents stimulates both influx of K and efflux of Cs.
In summary, the data support our assumption of a nonselective cation channel in ruminal epithelial cells. The data suggest that there is some efflux of Cs+ from the cells under control conditions that increases when external divalent cations are removed.
Effect of removing intracellular Mg2+ from cells filled with choline chloride solution. In a final series of experiments, we omitted magnesium from the internal solution, in the continued presence of calcium (choline chloride + Ca2+ 0 Mg2+).
Under these conditions, the removal of divalent cations from the extracellular NaCl buffer raised both mean outward and mean inward currents significantly (Fig. 7). Mean current at 100 mV rose from 100 to 176 ± 32% and from 30 ± 5 to 42 ± 6 pA/pF (P = 0.03, n = 13). Current at 120 mV rose from 88 ± 7 to 215 ± 44% and from 26 ± 4 to 49 ± 7 pA/pF (P = 0.003). These values are significantly higher than those observed for the pipette solution that contained magnesium (P = 0.007 for both), so that removal of cytosolic magnesium enhances inward current both in the presence and in the absence of external calcium and magnesium.
When the divalent-sensitive current was calculated from these values, we obtained an inward current density of 25 ± 6 pA/pF and an outward current density of 11 ± 4 pA/pF. These values were significantly higher than those obtained in the presence of intracellular magnesium (P = 0.01 and P = 0.03). Rectification score decreased to 2.9 ± 0.8, lower than that obtained for Mg2+-containing choline chloride pipette solution (8 ± 2).
When removal of divalents was performed in solutions that did not contain EGTA (nominally calcium-free NaCl buffer), a smaller but also highly significant increase in inward current could be observed (P = 0.008), whereas outward current remained unaltered (P = 0.3, n = 5). Conversely, inward current did not change significantly when divalents were removed either in nominally free or EGTA-buffered choline chloride solution (P = 0.8, n = 3 and P = 0.9, n = 4).
Erev did not change significantly when divalents were removed from the choline chloride buffer solution. However, when these Erev were compared with those observed in NaCl buffer, they were found to be significantly lower (Table 2), reflecting a decrease in the influx of sodium when choline was used to substitute this ion. This is in contrast to cells filled with magnesium-containing pipette solution, where no changes in Erev could be detected when external sodium was replaced with choline (P = 0.9, n = 8).
Erev in divalent-free KCl solution was significantly higher than that both in divalent-containing and divalent-free NaCl buffer (Table 2). As in the case of sodium, the omission of magnesium from the choline chloride pipette solution resulted in a higher potassium influx than in cells that contained magnesium (P = 0.02).
Addition of extracellular magnesium alone by switching the bath solution from "NaCl 0 Ca2+ 0 Mg2+" to "NaCl 0 Ca2+ + Mg2+" (in the presence of 0.5 mmol/l EGTA) reduced both inward and outward current significantly from 42 ± 8 to 38 ± 8 pA/pF (n = 4, P = 0.03) at 100 mV and from 50 ± 11 to 40 ± 13 pA/pF (n = 4, P = 0.01) at 120 mV, almost completely to the original level in NaCl buffer (NaCl + Ca2+ + Mg2+). Erev remained unaltered.
In summary, these experiments show that influx of sodium and potassium in ruminal epithelial cells is blocked by external calcium, magnesium, and barium and enhanced by the removal of cytosolic magnesium alone or by removal of both cytosolic calcium and magnesium.
Ussing Chamber Experiments
Effect of serosal Na+ concentration on Mg2+ fluxes across rumen epithelium. In the first Ussing chamber experiments, we studied the influence of serosal Na+ on ruminal Mg2+ absorption. Reduction of the serosal Na+ concentration from 30 to 10 and 5 mmol/l while maintaining mucosal Na+ constant at 30 mmol/l led to a significant reduction in mucosal-to-serosal Mg2+ fluxes (JMgms). Because serosal-to-mucosal Mg2+ fluxes (JMgsm) were not significantly affected, the decreased serosal Na+ concentration resulted in a decreased net absorption of Mg2+ (Table 3). Mucosal Na+ was set to 30 mmol/l to prevent a significant increase in serosal Na+ during the experiments.
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Effects of forskolin, IBMX, or theophylline on Isc. Previous studies (5, 38) have shown that elevation of cAMP suppresses sodium transport through ruminal epithelium, possibly by inhibition of electroneutral sodium transport via the apical sodium proton exchanger. On the other hand, cAMP has been shown to stimulate Na+/Mg2+ exchange in various tissues, including the ruminal epithelium (3, 10, 23, 25), decreasing the level of cytosolic magnesium. According to the patch-clamp data in this study, this should stimulate electrogenic sodium transport via nonselective cation conductance (NSCC), and thus Isc.
To decrease the relative contribution of electroneutral sodium exchange to the total transport of sodium across the apical membrane, mucosal Ca2+ was removed, stimulating the divalent-sensitive conductance. Next, cAMP level was elevated pharmacologically to stimulate basolateral Mg2+ extrusion via the Na+/Mg2+ exchanger. Under these conditions, the combined addition of forskolin and IBMX (10 µmol/l each) induced a biphasic response in Isc. A short decrease (1 Isc) was followed by a sustained increase in current (
2 Isc) and transepithelial conductance (Gt; Fig. 8 and Table 4). Neighbor epithelia, incubated in the presence of mucosal Ca2+, only showed a decrease in Isc (Fig. 8). Because the mucosal addition of drugs was more effective (Table 4), this application side was chosen for further experiments. When manipulating intracellular cAMP by the mucosal addition of different drugs, the combined addition of forskolin and IBMX (10 µmol/l each) had a higher effect on Isc (
2 Isc = +0.89 ± 0.09 µeq·cm2·h1) than forskolin (10 µmol/l,
2 Isc = +0.62 ± 0.09), IBMX (10 µmol/l,
2 Isc = +0.34 ± 0.07), or theophylline (10 mmol/l,
2 Isc = +0.42 ± 0.01) alone (P < 0.05, n = 35). Decreasing mucosal Na+ concentration from 140 to 21 mmol/l by replacement with NMDG+ abolished the forskolin-induced increase in Isc (Fig. 9).
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However, preincubation of the epithelia with 1 mmol/l amiloride and thereby block of apical Na+/H+ exchange had no effect on the subsequent Isc increase induced by forskolin and IBMX (2 Isc = +0.91 ± 0.07 µeq·cm2·h1 in the presence of amiloride and +0.89 ± 0.09 µeq·cm2·h1 in its absence; n = 6 and 3).
cAMP effect depended on the mucosal presence of Mg2+. The divalent cation-sensitive Isc can be stimulated to even higher levels by the removal of both mucosal Ca2+ and Mg2+. However, consecutive additions of forskolin in the mucosal presence of Mg2+ or Ca2+ showed that the Isc increase resulting from forskolin addition could only be seen in the mucosal presence of Mg2+ (Table 5).
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In the absence of both Ca2+ and Mg2+ from the luminal side, forskolin induced the usual decrease in Isc from 3.35 ± 0.35 to 2.40 ± 0.26 µeq·cm2·h1 (n = 8, P < 0.01) with no significant alteration in current after this first decline. Transepithelial conductance was reduced from 4.75 ± 0.23 to 4.26 ± 0.24 mS/cm2 (P < 0.05) in this experiment.
It appears that, in the absence of mucosal Mg2+, cytosolic Mg2+ levels are so low before the elevation of cAMP that no effect can be observed. Thus the observation that cAMP increased Isc (and Gt) in the presence of Mg2+, but not in its absence, is in line with the assumption that cAMP-mediated decreases in cytosolic magnesium open the nonselective cation channel in ruminal epithelial cells, resulting in an increase in Isc.
Forskolin had no measurable effect on the kinetics of the Mg2+-induced Isc decrease.
Next, we tried the effect of varying mucosal Mg2+ concentration. When preincubating one epithelium of each pair with forskolin (10 µmol/l), the subsequent mucosal addition of 1 mmol/l MgCl2 reduced the Isc from 3.63 ± 0.23 to 1.67 ± 0.19 µeq·cm2·h1 in the absence of forskolin and from 2.54 ± 0.31 to 1.29 ± 0.23 µeq·cm2·h1 in the presence of forskolin (n = 7). The data suggest that forskolin reduces the blocking effect of mucosal Mg2+ from Isc = 1.96 ± 0.12 to
Isc = 1.26 ± 0.10 µeq·cm2·h1 (P < 0.05). However, the Isc values before Mg2+ addition also differed significantly (P < 0.05). After the kinetic of the Mg2+-induced decrease in Isc, the half-maximal decrease of Isc was achieved 18 s after Mg2+ addition, irrespective of a preincubation with forskolin (Fig. 10).
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DISCUSSION |
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When the potassium content in the diet of ruminants is elevated by use of artificial fertilizing techniques in farming, absorption of sodium across the rumen is elevated to ensure that the sum of the concentrations of these two ions remains at a constant level of 140 ± 10 mmol/l (14, 32, 37), with sodium varying physiologically between 21 and 145 mmol/l. This prevents loss of water in the ruminal cavity while limiting the amount of potassium absorbed and thus avoiding hyperkalemia. Simultaneously, the rate of magnesium uptake across the rumen is reduced, which can lead to fatal states of hypomagnesaemia if high potassium intake persists over a longer period of time (24).
In this study, we attempted to find out if these two phenomena are in any way linked to each other.
Ruminal electrogenic Na+ transport varies both with the apical membrane potential and with the luminal concentration of divalent cations. Removal of Ca2+ and Mg2+ from the mucosal side of rumen epithelium leads to parallel increases in Isc and Gt carried by Na+, Rb+, or K+, a depolarization of the apical membrane potential of REC, and a decrease of the fractional apical resistance (14, 17). In an Na+ buffer, this divalent cation-sensitive increase in Isc is accompanied by an increase in Na+ flux from the mucosal to the serosal side in the same order of magnitude, whereas the paracellular pathway does not seem to be affected (26). A prolonged Mg2+ deprivation from the mucosal side enhances the Ca2+-sensitive Isc across goat rumen, which suggests that not only extra- but also intracellular Mg2+ contribute to the blocking effect on Isc (17).
Thus it seems possible that electrogenic sodium transport across ruminal epithelium is regulated by cytosolic magnesium. It is known that high ruminal potassium depolarizes the apical membrane, decreases the uptake of magnesium via an electrodiffusive mechanism (20, 29), and thereby reduces cytosolic magnesium. We suggest that this opens an apical nonselective cation channel that mediates the well-known increase in sodium uptake across the ruminal epithelium (30) that follows the ingestion of high-potassium fodder. Stimulation of cellular magnesium efflux via Na+/Mg2+ exchange can lead to similar effects (Fig. 12).
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Using the whole cell configuration of the patch-clamp technique, we were able to demonstrate the existence of a nonselective cation channel in REC that is regulated not only by external but also by internal magnesium. Removal of magnesium alone or of calcium and magnesium in combination from the external side of the membrane resulted in a significant increase in the influx of sodium and potassium, but not of choline in the cell, with concomitant rises in Erev. Likewise, permeability for sodium and potassium could be stimulated by the removal of magnesium alone or both calcium and magnesium from the cytosolic side of the membrane. The conductance could be blocked by 5 mM barium on the external side, in parallel to previous transepithelial studies (17), but not by 5 mM TEA-Cl. Filling the cells with CsCl did not interfere with influx of K+ in the cell either in the absence or presence of divalents. Erev indicate a basic conductance for Cs that can be stimulated by the removal of divalent cations from the external medium.
With the use of the Goldman-Hodgkin-Katz equation {Erev,K Erev,Na = ln[(PK[K]o)/(PNa[Na]o)]; see Eq. 14.17 in Ref.12}, it is possible to determine relative permeability ratios PK/PNa of the membrane in divalent-free medium from Erev. This yields values of PK/PNa = 2.1 ± 0.5 (for potassium gluconate + Ca2+ + Mg2+ pipette solution), PK/PNa = 1.4 ± 0.2 (for "choline chloride + Ca2+ + Mg2+" pipette solution), PK/PNa = 1.3 ± 0.1 (for "CsCl + Ca2+ + Mg2+" pipette solution), and PK/PNa = 1.2 ± 0.2 (for "choline chloride + Ca2+ 0 Mg2+" pipette solution). It should be noted that these values contain contributions of potassium conductances that are insensitive to divalent cations. In the cells filled with CsCl, Erev changed from negative values in NaCl solution to positive values in KCl solution, indicating a permeability sequence of K+ > Cs+ > Na+ (Eisenmann III or IV).
The Goldman-Hodgkin-Katz theory can also be used to calculate theoretical values for the rectification score resulting from the concentration gradient of Na+ and K+ across the membrane (see Eq. 14.14 in Ref. 12). Diffusion of these two ions through a plain pore yields a current of
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Using the approximate values for PK/PNa as determined above, values for i(E) can be determinined for various potential levels (E). The rectification score (Rect) can then be determined as described in METHODS:
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With the use of this equation, the rectification score for potassium gluconate pipette solution (in NaCl buffer) is 0.69, and that for the choline chloride pipette solution (in NaCl buffer) 1.88. Thus the experimentally determined rectification scores were all significantly higher than these theoretical values, reflecting the fact that the rectification of the divalent-sensitive currents in these experiments cannot be explained by the Goldman-Hodgkin-Katz theory alone.
Rectification decreased markedly when either magnesium alone or calcium and magnesium were removed from the internal solution, suggesting a voltage-dependent block along the lines of the classical inward rectifier model (12).
The mechanism by which Mg2+ block inwardly rectifying potassium channels has been studied extensively. These channels are blocked by Mg2+ that obstruct the internal mouth of the channel pore. If external potassium is elevated, the channel opens. The explanation given for this is that K+ ions entering the cell seem to literally "kick" the blocking magnesium out of the pore. The same effect can be achieved if the cell is hyperpolarized: the cytosol is now negative, the outside of the cell positive, and the potential along the (conducting) channel pore repells positive ions from the inner mouth of the channel into the cell. Thus inwardly rectifying potassium channels open when the cell is hyperpolarized, due to the fact that the internal block by magnesium is relieved. The same effect can be achieved when internal magnesium levels drop.
In the channel discussed here, we suggest that depolarization of the cytosol relieves the block from the external side by preventing positive divalents from entering (and blocking) the pore from the outside, whereas hyperpolarization relieves the blocking effect of divalents that enter the pore from the internal side. It should be noted that strong inward rectification resulting from internal magnesium has been reported for the transient receptor potential vanilloid type 6 channel (36).
In addition to the nonselective cation current, we were able to show that ruminal epithelial cells also express a chloride conductance that does not appear to be involved in the stimulation of inward current by the removal of divalents.
As shown in micropuncture experiments, changes in mucosal sodium concentration have only small effects on the apical membrane potential at resting potential (14, 19). A significant increase in sodium conductance emerges only when the cells are depolarized (thus reducing cytosolic magnesium levels), or when divalents were removed from the external solution (14). In patch-clamp experiments, where cytosolic magnesium levels are clamped to a defined level, we were able to show that depolarization alone has no effect, whereas removal of cytosolic magnesium significantly enhances sodium conductance at all potential levels. Thus it appears that the electrogenic sodium conductance of the ruminal cell is not opened directly by depolarization alone but via a drop in cytosolic magnesium that is correlated with this depolarization (20, 27).
In summary, our data indicate that lowering the level of cytosolic magnesium enhances sodium uptake through the nonselective cation channel in ruminal epithelial cells.
Basolateral Mg2+ Extrusion via Mg2+/Na+ Exchange
Previous studies suggest cross talk between nonselective cation channels and Na+/Ca2+ exchange (31). In this study, we were able to confirm the existance of Na+/Mg2+ exchange in rumen epithelium (23, 29), demonstrate that is is located on the basolateral side of the tissue, and provide evidence for transepithelial transport of magnesium via this exchanger. The data in this study speak for an interaction with magnesium-sensitive apical nonselective cation channels (14, 17).
Net Mg2+ absorption was significantly reduced when serosal Na+ concentration was lowered (Table 3). This suggests basolateral localization of Na+/Mg2+ exchange in rumen epithelium, as recently demonstrated in rat hepatocytes (25). Imipramine-sensitive interaction between Mg2+ and Na+ has recently been shown in isolated cultured REC, with a Km for Na+ of 24 mmol/l (23, 29).
Regulation of Ruminal Mg2+/Na+ Exchange and Transepithelial Mg2+ Absorption Through cAMP
The cAMP effects on ruminal Isc were biphasic. An initial decrease in Isc could be seen in the presence and absence of mucosal cations and has already been reported in previous studies (5, 7, 38). It can be explained with an inhibitory impact of cAMP on the electroneutral Na+/H+ exchanger in the apical membrane and a reduced electrogenic extrusion via Na+-K+-ATPase (5). A secondary increase in Isc could only be seen in the mucosal absence of Ca2+, i.e., when the relative contribution of the divalent cation-sensitive pathway to total sodium transport was enhanced (Fig. 8). Replacing Na+ with NMDG+ abolished the effect of forskolin on Isc (Fig. 9). The increase in Isc by forskolin is consistent with a reduction of magnesium block from the cytosolic side.
Removal of Mg2+ from the mucosal side (Table 5) obliterated the secondary increase in sodium conductance. It appears that, in the absence of mucosal Mg2+, cytosolic Mg2+ levels are so low before the elevation of cAMP that no effect can be observed. Likewise, the presence of serosal Na+ was necessary for the secondary increase, demonstrating that the assumption of a direct effect of cAMP on the apical nonselective cation channel is not sufficient to explain the data in this study. However, an additional direct stimulation of this channel by cAMP cannot and should not be excluded.
cAMP has been shown to increase Mg2+ extrusion in various cell types, including REC, probably via an activation of Mg2+/Na+ exchange (10, 23, 25). The activity of such an exchange system can be reduced by the application of imipramine and amiloride or by a decrease in extracellular Na+ concentration (4, 9, 11, 23). All three blocking conditions, applied to the serosal side of rumen epithelium, reduced the forskolin-mediated increase in Isc and therefore confirm the regulation of basolateral Mg2+/Na+ exchange through cAMP (Fig. 11). Depending on the rate-limiting step in transepithelial Mg2+ transport, a stimulation of Mg2+ extrusion might increase overall transepithelial Mg2+ absorption. The Mg2+ flux studies with theophylline indicate that this may be the case.
We conclude that rumen epithelium expresses a nonselective cation channel with PK > PCs > PNa, which is blocked by Mg2+ alone, or Ca2+ and Mg2+ in combination on the extracellular and intracellular side, as well as by Ba2+.
Mg2+ absorption across rumen epithelium involves an Na+ dependent extrusion mechanism at the basolateral side. cAMP activates basolateral Mg2+/Na+ exchange, which results in a reduction of the Mg2+ block from the cytosolic side of the nonselective cation channel and an increase in current.
We suggest that the decrease in cytosolic magnesium that follows the ingestion of high-potassium fodder by ruminants stimulates electrogenic sodium absorption through the nonselective cation channel. In the natural habitat of ruminants, where fodder is usually low in potassium, this mechanism appears useful in regulating ruminal osmolarity while averting the (immediate) danger of an uncontrolled increase in the uptake of potassium. Potentially fatal consequences from the reduced magnesium uptake should only emerge after prolonged exposure to a high-potassium diet, as when artificial fertilizing techniques are used.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
* S. Leonhard-Marek and F. Stumpff contributed equally to this work.
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