Mechanisms of Mg2+ transport in cultured ruminal epithelial cells

Monika Schweigel1, Jürgen Vormann2, and Holger Martens1

1 Department of Veterinary Physiology, Free University of Berlin, 14163 Berlin; and 2 Department of Molecular Biology and Biochemistry, Free University of Berlin, 14195 Berlin, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Net Mg2+ absorption from the rumen is mainly mediated by a transcellular pathway, with the greater part (62%) being electrically silent. To investigate this component of Mg2+ transport, experiments were performed with isolated ruminal epithelial cells (REC). Using the fluorescent indicators mag-fura 2, sodium-binding benzofuran isophthalate, and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, we measured the intracellular free Mg2+ concentration ([Mg2+]i), the intracellular Na+ concentration ([Na+]i), and the intracellular pH (pHi) of REC under basal conditions, after stimulation with butyrate and HCO-3, and after changing the transmembrane chemical gradients for Mg2+, H+, and Na+. REC had a mean resting pHi of 6.83 ± 0.1, [Mg2+]i was 0.56 ± 0.14 mM, and [Na+]i was 18.95 ± 3.9 mM. Exposure to both HCO-3 and HCO-3/butyrate led to a stimulation of Mg2+ influx that amounted to 27.7 ± 5 and 29 ± 10.6 µM/min, respectively, compared with 15 ± 1 µM/min in control solution. The increase of [Mg2+]i was dependent on extracellular Mg2+ concentration ([Mg2+]e). Regulation of pHi has been demonstrated to be Na+ dependent and is performed, for the most part, by a Na+/H+ exchanger. The recovery of pHi was fully blocked in nominally Na+-free media, even if [Mg2+]e was stepwise increased from 0 to 7.5 mM. However, an increase of [Mg2+]i was observed after reversing the transmembrane Na+ gradient. This rise in [Mg2+]i was pH independent, K+ insensitive, dependent on [Mg2+]e, imipramine and quinidine sensitive, and accompanied by a decrease of [Na+]i. The results are consistent with the existence of a Na+/Mg2+ exchanger in the cell membrane of REC. The coupling between butyrate, CO2/HCO-3, and Mg2+ transport may be mediated by another mechanism, perhaps by cotransport of Mg2+ and HCO-3.

sheep rumen; epithelial cells; magnesium transport; intracellular magnesium; sodium/magnesium antiport; mag-fura 2


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS NOW WELL ESTABLISHED that the forestomachs are the main and most important sites of Mg2+ absorption in ruminants (34). Many in vivo and in vitro experiments have been performed on Mg2+ transport and the mechanisms of impaired Mg2+ absorption by high ruminal K+ concentrations (3, 22, 24). The obtained data have revealed that net Mg2+ absorption from the rumen occurs against an electrochemical gradient (3, 24) and is mainly mediated by an active transcellular pathway (26). The mechanisms of this cellular Mg2+ transport, that is, luminal uptake and basolateral extrusion, are not well understood. So far, investigations at the tissue level suggest that two different transport mechanisms exist for the apical uptake of Mg2+. Part of the unidirectional mucosal-to-serosal Mg2+ flux (J Mgms) is potential difference (PD) dependent and K+ sensitive and may represent passive Mg2+ uptake by a channel (22, 31). Another somewhat greater part (62% of J Mgms) is electrically silent, dependent on luminal short chain fatty acids (SCFA), CO2/HCO-3, and Cl-, and may in part result from apical Mg2+/H+ exchange (23). The existence of a Mg2+/H+ exchange as a second PD-independent Mg2+ uptake mechanism has been derived indirectly from several observations. Feeding high levels of easily fermentable carbohydrates to ruminants increases Mg2+ availability (10, 13). Such a diet leads to an alteration of microbial activity and composition in the rumen contents. Among other parameters, the concentrations of SCFA and CO2/HCO-3 are increased; these are the major end products of the microbial digestion of carbohydrates in the ruminal lumen and stimulate Mg2+ net absorption in vivo (10, 27) and in vitro (23). In vitro experiments with isolated sheep rumen epithelium have revealed that this increase in net Mg2+ absorption is entirely attributable to a stimulation of J Mgms, which is specifically reduced by removal of SCFA, CO2/HCO-3, and Cl- (23). Because the stimulating effect of SCFA on J Mgms depends on their lipid solubility (acetate < propionate < butyrate) and because the carbonic anhydrase inhibitor ethoxyzolamide reduces J Mgms in SCFA-free buffer, it has been suggested that the PD-independent stimulation of ruminal Mg2+ transport depends on permeant anions that supply substrates for Mg2+/H+ and Cl-/HCO-3 exchange mechanisms in the apical membrane of the ruminal epithelium (23). SCFA and CO2/HCO-3 are the predominant anions in the ruminal fluid and are readily absorbed by the stratified epithelium of the rumen (7, 28). Therefore, it has been hypothesized that the supply of H+ for this exchange comes in part from the intracellular dissociation of SCFA that are absorbed in their nonionized form by diffusion and from the intracellular hydration of CO2, produced in the lumen by microbial fermentation and in the mucosa by SCFA catabolism (23). In such a model, the effect of Cl- withdrawal could be explained by a reduction of the Cl-/HCO-3 exchange activity that is present in the ruminal epithelium (25).

Only a few data are available regarding the basolateral Mg2+ efflux from the epithelial cells. The significant uphill electrical gradient (basolateral PD = 50-70 mV) for the basolateral exit of Mg2+ would suggest the participation of an energy-dependent mechanism. Because inhibition of the Na+-K+-ATPase by ouabain reduces the net movement of Mg2+ by 90% (26), and because of the correlation between net Mg2+ and net Na+ efflux from the rumen (3), it is assumed that Mg2+ efflux takes place via nonelectrogenic Na+/Mg2+ exchange, by utilization of the electrochemical gradient for Na+ (generated by Na+-K+-ATPase).

The existence of the proposed transport proteins for apical Mg2+/H+ and basolateral Na+/Mg2+ exchange has not as yet been shown directly. Therefore, the aim of the present study has been to obtain preliminary information about the PD-independent component of Mg2+ transport and possible transport proteins involved at the cellular level. To this purpose, we have performed experiments with isolated ruminal epithelial cells (REC). With the aid of the fluorescent probes mag-fura 2, sodium-binding benzofuran isophthalate (SBFI), and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), we have measured the free intracellular Mg2+ concentration ([Mg2+]i), the intracellular Na+ concentration ([Na+]i), and the intracellular pH (pHi) of REC under basal conditions, after stimulation with butyrate and HCO-3, and after changing the transmembrane chemical gradients for Mg2+, H+, and Na+.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Medium 199, trypsin, glutamine, antibiotics (gentamycin, nystatin, and kanamycin), and FCS were purchased from Sigma (St. Louis, MO). Dulbecco's PBS (DPBS) and collagen were obtained from Biochrom (Berlin, Germany). Mag-fura 2-AM, SBFI-AM, BCECF-AM, and pluronic acid were from Molecular Probes (Eugene, OR). All other chemicals were purchased from Sigma.

Cell culture. Primary cultures of REC were prepared as described by Galfi et al. (12). Briefly, REC were isolated by fractional trypsination and grown 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-5% CO2 at 38°C. Experiments were performed between 6 and 12 days after seeding.

Solutions. The control solution was the NaCl solution (in mM: 145 NaCl, 5 KCl, 1 CaCl2, 2 MgCl2, 10 HEPES, and 5 glucose, pH 7.4). In HCO-3-buffered solutions 20 mM NaCl was replaced by NaHCO3, and in butyrate-containing solutions a further 20 mM NaCl was replaced by sodium butyrate. All HCO-3-containing solutions were preequilibrated with 5% CO2 and 95% air. In Na+-free solutions, NaCl was replaced by N-methyl-D-glucamine (NMDG)-Cl. The high-K+ solution contained (in mM) 15 NaCl, 135 KCl, 1 CaCl2, 2 MgCl2, 10 HEPES, and 5 glucose, pH 7.4. When the Mg2+ content of any solution was increased, an appropriate amount of NaCl, KCl, or NMDG-Cl was replaced to maintain osmolarity.

Measurement of cytoplasmic Mg2+, Na+, and pH by spectrofluorometry. Cells were loaded with 5 µM mag-fura 2-AM, 10 µM SBFI-AM, or 0.5 µM BCECF-AM for the determination of [Mg2+]i, [Na+]i, and pHi, respectively. Cells were subsequently washed twice in DPBS. REC were incubated for a further 30 min to allow for complete deesterification and washed twice before measurement of fluorescence. Intracellular ion concentrations were determined by measuring the fluorescence of the probe-loaded REC in the LS-50 B spectrofluorometer (Perkin-Elmer), by using the fast filter accessory, which allowed fluorescence to be measured at 20-ms intervals, with excitation for mag-fura 2 and SBFI at 340 and 380 nm and for BCECF at 440 and 480 nm, and with emission at 515 nm. All measurements were made at 37°C in a 3-ml cuvette containing 2 ml cell suspension (10% cytocrit) under stirring. The measurements with HCO-3-containing solutions were done after the cell suspensions were preequilibrated with 5% CO2 and 95% air. During the experiments the cuvette was tightly closed with a plastic cap to prevent CO2 leakage.

[Mg2+]i was calculated from the 340/380-nm ratio according to the formula of Grynkiewicz et al. (14) by using a dissociation constant of 1.5 mM for the mag-fura-2/Mg2+ complex. The minimum (Rmin) and maximum (Rmax) ratios were determined at the end of each experiment by using digitonin. Rmax was found by the addition of 25 mM MgCl2 in the absence of Ca2+, and Rmin was obtained by addition of 50 mM EDTA, pH 7.2, to remove all Mg2+ from the solution.

BCECF and SBFI signals were calibrated to ion concentrations by using the ionophores nigericin (10 µM) and gramicidin (10 µM) to equilibrate intra- and extracellular concentrations of H+ and Na+. The procedure was repeated for various pH values between 6.0 and 8.0 and for [Na+] between 0 and 160 mM.

Statistical analysis. If not otherwise stated, data are presented as means ± SE. Significance was determined by Student's t-test or Tukey's ANOVA as appropriate. Correlations between variables were tested by calculating Pearson's product moment correlation coefficients. P < 0.05 was considered significant. All statistical calculations were performed with SigmaStat (Jandel Scientific).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of butyrate and HCO-3 on pHi and [Mg2+]i. SCFA and CO2/HCO-3 stimulate Mg2+ net absorption across rumen epithelium in vivo and in vitro. Therefore, our initial goal was to define the acute cellular response to SCFA and/or HCO-3 exposure in terms of both [Mg2+]i and pHi. Mag-fura 2-loaded and BCECF-loaded REC in suspension were equilibrated in control solution without butyrate and HCO-3, in Na+ medium with 20 mM butyrate and 20 mM HCO-3, or in Na+ medium with only 20 mM HCO-3, and [Mg2+]i and pHi were measured over a 10-min period. Extracellular pH (pHe) was 7.4, and extracellular [Mg2+] ([Mg2+]e) was 2 mM in all solutions. Cells in control solution had a mean resting pHi of 6.83 ± 0.1. On exposure to medium with 20 mM HCO-3 or with 20 mM HCO-3 and 20 mM butyrate, pHi dropped to 6.67 ± 0.2 and 6.58 ± 0.13, respectively, and then recovered after 10 min to 6.87 ± 0.2 and 6.76 ± 0.12 (Fig. 1). The resting [Mg2+]i, determined at the beginning of the experiments, was not significantly different in experiments performed with HCO-3 and HCO-3/butyrate media (0.64 ± 0.09 mM and 0.7 ± 0.26 mM) compared with that in HCO-3- and butyrate-free control solution (0.56 ± 0.14 mM), but [Mg2+]i starting levels tended to be higher with butyrate and/or HCO-3 in the bath solution. In all media, an increase of [Mg2+]i was observed. The increase of [Mg2+]i was higher (Fig. 2) in media with HCO-3 (27.7 ± 5.0 µM/min) or HCO-3 and butyrate (29.0 ± 10.6 µM/min) compared with control medium (15.0 ± 1.0 µM/min).


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Fig. 1.   Effect of HCO-3 and butyrate on intracellular pH (pHi) of ruminal epithelial cells (REC). Measurements were made after a 5-min preincubation in either control solution (Na+ medium without butyrate or HCO-3) or medium with 20 mM butyrate and/or HCO-3. Values are means ± SE of 4 single experiments. * P < 0.05 for pHi starting level of REC in HCO-3/butyrate medium vs. control.



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Fig. 2.   Effect of HCO-3 and butyrate on intracellular Mg2+ concentration ([Mg2+]i) of REC. Measurements were made after a 5-min preincubation in either control solution (Na+ medium without butyrate or HCO-3) or medium with 20 mM butyrate and/or HCO-3. Magnitude of mean [Mg2+]i change (Delta [Mg2+]i) in these media is given. Values are means ± SE of 4 single experiments. * P < 0.05 vs. control.

These results, that is, the alkalization of pHi and increase in [Mg2+]i, are in agreement with the hypothesis of a Mg2+/H+ exchange mechanism. However, other mechanisms could also contribute to these effects.

Experimental evidence of the Na+/H+ antiporter. It has been observed previously that SCFA stimulate the transepithelial Na+ absorption from the ruminal fluid and that this effect is sensitive to high doses (1 mM) of the diuretic amiloride (11, 25). It is thought that acidification caused by nonionic diffusion of SCFA activates Na+/H+ exchange in the luminal membrane of rumen epithelium. To demonstrate the presence and activity of Na+/H+ exchange, we have measured pHi under several conditions known to inhibit this antiporter: 1) after addition of amiloride or ethylisopropylamiloride, which are recognized inhibitors of the antiporter (20); 2) after substituting extracellular Na+ with NMDG, thus reversing the transmembrane Na+ gradient, which is the driving force for Na+/H+ exchange; and 3) after addition of 10-4 M 8-bromo-cAMP, which decreases the activity of the antiporter through cAMP-dependent protein kinase (5, 35). The results of these experiments are presented in Fig. 3A. We have also shown by control experiments that pHi recovery after butyrate exposure is dependent on extracellular Na+ concentration ([Na+]e) (Fig. 3B). Therefore, it is likely that part of the effects of butyrate and/or HCO-3 exposure on the pHi of REC (initial decrease and recovery to near control level) are attributable to Na+/H+ exchange and that this activity can mask or overlap Mg2+/H+ exchange.


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Fig. 3.   A: effect of various experimental conditions known to inhibit Na+/H+ antiport on pHi in REC. Magnitude of mean pHi change (Delta pHi) is given for each condition. In all cases, pHi fell significantly (* P < 0.05). Bars represent means ± SE, and number of single experiments is shown in parentheses. EIPA, ethylisopropylamiloride. B: original tracing of a measurement of pHi in REC. There is no pHi recovery in cells exposed to butyrate if Na+/H+ exchanger is completely blocked by removal of extracellular Na+ [replaced by N-methyl-D-glucamine (NMDG)-Cl]. [Na+], Na+ concentration. Trace is representative of 6 independent experiments.

Effects of Na+ withdrawal on [Mg2+]i, [Na+]i, and pHi. To avoid Na+/H+ exchange and other pH-regulating mechanisms, further experiments were performed with Na+- and HCO-3-free media. Figure 4 shows the intracellular H+ concentration ([H+]i) and [Na+]i of REC before and after removal of extracellular Na+. As [Na+]e was reduced to zero, pHi fell from 6.86 ± 0.32 to 6.4 ± 0.14 and [Na+]i fell from 18.95 ± 3.9 to 10.3 ± 4.7 mM. Under these conditions, the Na+/H+ exchanger is not capable of extruding protons, and, as a result, [H+]i increases about three times (Fig. 4). If there is a Mg2+/2H+ exchanger in the cell membrane of REC, increasing [Mg2+]e should decrease [H+]i and increase [Mg2+]i under these experimental conditions. The results are shown in Fig. 5. As expected, [Mg2+]i increased from 0.87 ± 0.19 to 1.15 ± 0.45 mM when [Mg2+]e was stepwise increased from 0 to 2.5, 5.0, and 7.5 mM; on the contrary, pHi shifted toward more acidic pH values and fell from 6.42 ± 0.2 to 6.24 ± 0.2 during exposure to high [Mg2+]e. Recovery of pHi did not occur before Na+ was added to the bath.


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Fig. 4.   Intracellular concentrations of H+ and Na+ ([H+]i and [Na+]i) of REC before and after omission of Na+ from extracellular solution. NaCl was isosmotically replaced by NMDG-Cl. Number of single experiments is shown in parentheses. ** P < 0.01 vs. control (solution with a [Na+] of 145 mM).



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Fig. 5.   Cytoplasmic pH and Mg2+ responses to addition of extracellular Mg2+ in REC exposed to Na+-free NMDG medium. Under these experimental conditions, H+ gradient was outwardly directed (pHi << extracellular pH), and we simultaneously generated an inwardly directed Mg2+ gradient {extracellular [Mg2+] ([Mg2+]e) >> [Mg2+]i} by increasing the [Mg2+] in the medium from 0 to 2.5, 5.0, and 7.5 mM in a stepwise manner. A: records from a typical experiment are shown. B: means ± SE of results from 6 different experiments are shown; pHi and [Mg2+]i are shown plotted against time. There was an inverse relationship between [Mg2+]i and pHi in all experiments (r = -0.98).

Interrelationship between [Na+]i and [Mg2+]i. As shown above, the [Mg2+]i of REC increased continuously when the cells were exposed to Na+-free media and [Mg2+]e was stepwise increased from 0 mM to 2.5, 5.0, and 7.5 mM. At the same time, [Na+]i fell from 10.2 ± 4.8 to 8.4 ± 4.4 mM. An example for this interrelationship is given by the original recordings presented in Fig. 6. After readdition of Na+ to the extracellular solution, [Na+]i increased and the [Mg2+]i increase was blocked completely (Fig. 6). Figure 7 shows in summary that the increase in [Mg2+]i and the decrease in [Na+]i are substantially reduced if the [Na+]e is increased. In Na+-free media, [Mg2+]i increased by 114 ± 31, 189 ± 47, and 248 ± 64 µM, and [Na+]i decreased by 1.3 ± 0.3, 1.6 ± 0.2, and 1.9 ± 0.3 mM at [Mg2+]e of 2.5, 5.0, and 7.5 mM, respectively. In media containing 145 mM Na+, a rise in [Mg2+]i and a fall in [Na+]i was not detectable until the external Mg2+ concentration was 7.5 mM. Compared with Na+-free media, there were only slight changes in [Mg2+]i and [Na+]i; [Mg2+]i rose by 33 ± 24 and 87 ± 9 µM and [Na+]i fell by 0.2 and 0.9 mM in Na+ media with 5 and 7.5 mM Mg2+, respectively.


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Fig. 6.   Effect of variation in extracellular [Na+] ([Na+]e) and [Mg2+]e on [Mg2+]i and [Na+]i in REC. Representative original recordings for [Mg2+]i (top) and [Na+]i (bottom) are shown. After reversing Na+ gradient by exposing cells to Na+-free NMDG solution, [Mg2+]e was increased in a stepwise manner from 0 to 2.5, 5.0, and 7.5 mM. Subsequently, Na+ was readded to bath solution. Tracings are representative of 6 separate experiments.



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Fig. 7.   Changes of [Mg2+]i and [Na+]i of REC as a function of [Mg2+]e and [Na+]e. Cytosolic Mg2+ and Na+ were measured in Na+-free NMDG medium or in control solution with a [Na+] of 145 mM. [Mg2+]e was increased stepwise from 0 mM to 2.5, 5.0, and 7.5 mM. Delta [Mg2+]i or magnitude of mean [Na+]i change (Delta [Na+]i) after a time period of 100 s is given. Values are means ± SE (shown if larger than symbol size) of 6 experiments.

To characterize this Mg2+ uptake, [Mg2+]i was measured in REC suspended in nominally Na+-free medium with various Mg2+ concentrations (0, 2.5, 5.0, and 7.5 mM) for a 10-min period. As shown in Fig. 8, the rate of Mg2+ influx was dependent on [Mg2+]e and, compared with control values (in nominally Mg2+-free medium), [Mg2+]i increased by 47 ± 18, 91 ± 24, and 67 ± 13% with 2.5, 5.0, and 7.5 mM Mg2+ in the bath, respectively. A maximal increase of [Mg2+]i was seen with 5 mM Mg2+ in the medium. Application of imipramine or quinidine reduced this increase significantly (P < 0.05). Figure 9 shows that reversing the transmembrane Na+ gradient in the presence of 5 mM Mg2+ in the extracellular solution increased [Mg2+]i by 49.1 ± 8.1 µM/min. This elevation was reduced to 34.6 ± 9.0 and 24.6 ± 5.5 µM/min by the application of 100 µM or 500 µM imipramine and to 37.6 ± 5.2 µM/min by the application of 100 µM quinidine. The pHi did not change in the time course of the experiment.


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Fig. 8.   Increase of [Mg2+]i in relation to [Mg2+]e. Cytosolic Mg2+ was measured in Na+-free NMDG medium with various [Mg2+] [0 (control), 2.5, 5.0, and 7.5 mM]. Number of single experiments is shown in parentheses. * P < 0.05 vs. control (nominally Mg2+- and Na+-free medium).



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Fig. 9.   Influence of imipramine and quinidine on the [Mg2+]i change in Na+-free solution with an [Mg2+]e of 5 mM. Number of single experiments is shown in parentheses. ** P < 0.01 vs. control.

Influence of K+-rich medium on [Mg2+]i and pHi. Because it is known that the membrane potential (Em) acts as a driving force for Mg2+ uptake, and since removal of Na+ may have hyperpolarized the Em of REC, Em was short circuited by increasing the external K+ concentration in the next series of experiments.

REC were suspended in control solution (145 mM Na+ and 5 mM K+) or in high-K+ medium (15 mM Na+ and 135 mM K+), and [Mg2+]i and pHi were measured over a 10-min period. Cells in high-K+ medium had a mean resting pHi of 6.81 ± 0.03, which was not significantly different from the resting pHi in Na+ medium (6.83 ± 0.1), and there was no change in pHi over the 10-min period. In contrast, [Mg2+]i increased if cells were incubated in K+ medium or in Na+ medium. As shown in Fig. 10, on exposure of the cells to high-K+ medium, [Mg2+]i rose by 37.5 ± 8.0 µM/min, an increase that was twice that in cells in Na+ medium (19.0 ± 12.0 µM/min).


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Fig. 10.   [Mg2+]i response to an increase in extracellular K+ concentration ([K+]e). REC were exposed to either control solution (145 mM Na+ and 5 mM K+) or high-K+ medium (15 mM Na+ and 135 mM K+). Values are means ± SE of 4 single experiments. * P < 0.05 vs. control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

[Mg2+]i under control conditions. The basal [Mg2+]i of REC (0.56 ± 0.14 mM) is within the range of 0.5-1.0 mM that has been reported for other cell types (6, 30, 32).

Modulation of [Mg2+]i and pHi by HCO-3 and butyrate. The finding that butyrate and HCO-3 increase [Mg2+]i of REC compared with the control value is in agreement with results from in vivo and in vitro studies at the tissue level (23, 27). The in vivo results have been explained partly on the basis of the stimulating effects of SCFA and CO2 on the blood flow in the rumen wall (27, 33), but such an effect can be excluded under in vitro conditions. This is also true for changes in pHe, which is known to decrease after supplementation of readily fermentable carbohydrates, thereby increasing Mg2+ solubility. Exposure of REC to butyrate- and/or HCO-3-containing solutions leads to an initial decrease of pHi accompanied by an increase of [Mg2+]i that is independent of the presence of extracellular Mg2+ and attributable to the mobilization of Mg2+ from intracellular buffering systems. The pHi-dependent intracellular redistribution of Mg2+ is responsible for the higher [Mg2+]i starting levels of REC exposed to butyrate and/or HCO-3. The magnitude of the initial changes in pHi and hence the evoked increase in [Mg2+]i are reduced when the pH-buffering capacity of the cytosol is increased, as in HCO-3-buffered solutions without butyrate. If mobilization from intracellular stores is the only source of the observed elevation of [Mg2+]i, then it would be expected that [Mg2+]i decreases to control levels on pHi recovery. This is not the case, and, in addition, it has been shown that 1) with the same level of acidification (by removal of extracellular Na+), the increase in [Mg2+]i is significantly higher in Mg2+-containing solutions compared with Mg2+-free media, 2) exposure of cells to Na+ media with 2 mM Mg2+ and HCO-3 alone or with both butyrate and HCO-3 increases [Mg2+]i to the same extent (Fig. 2), although the initial pHi decrease is more pronounced in media with butyrate, and 3) significant differences in the [Mg2+]i of REC exposed to HCO-3 and butyrate compared with those suspended in control solution has been found only in media containing Mg2+. Therefore, the modulation of [Mg2+]i by butyrate and HCO-3 is probably associated with the movement of Mg2+ across the cell membrane. This raises the question as to what constitutes the transport mechanism.

Role of Na+/H+ exchange. It has long been known that feeding diets supplemented with easily fermentable carbohydrates and thus increasing the intraruminal concentration of SCFA and CO2/HCO-3 leads to a stimulation of fluid and electrolyte absorption (10, 13, 27). As in a variety of other epithelia (1, 29), it is well established for the sheep rumen that SCFA (and HCO-3) stimulate Na+ transport via Na+/H+ exchange in the apical membrane (11, 25). Considering the limited cellular supply of H+, recirculation of H+ is essential for such a system, and SCFA and CO2 may serve this function. The existence of a Na+/H+ exchange mechanism in the cell membrane of ruminal epithelial cells has been confirmed by the results of the present study. The finding that ethylisopropylamiloride, amiloride, and cAMP (Fig. 3A) affect basal pHi suggests that a Na+/H+ exchanger is also active in "resting" REC at physiological pHi (6.83 ± 0.1). The Na+ potential (ENa+) is larger than the H+ potential (EH+), i.e., +54 mV compared with -35 mV, and sufficient to drive the Na+/H+ exchange, even under basal conditions. As expected, exposure of REC to HCO-3 and HCO-3/ butyrate media leads to an initial decrease of pHi, resulting from the uptake of nonionized butyrate and/or CO2. After entry into the cells, the protonated form of butyrate readily dissociates because of the low acidic dissociation constant (pKa) value (~4.8) of the SCFA, thereby delivering H+ to the cell interior. The intracellular hydration of CO2 supplies not only H+ but also HCO-3, and therefore pHi decreases to a lesser extent after HCO-3 exposure. However, in both cases, there is a substantial elevation of [H+]i coupled with an increased driving force for H+ secretion (and Na+ uptake), that is, an ENa+- EH+ of +98.4 mV (HCO-3 medium) and +104.0 mV (HCO-3/butyrate medium) compared with +88.6 mV under basal conditions. The acidification activates Na+/H+ exchange, and it is therefore not surprising that pHi recovers to near control levels during the experimental period.

Is there an Mg2+/2H+ exchange in REC? Because Mg2+ absorption from the ruminal fluid is also stimulated by SCFA and CO2/HCO-3, an attractive hypothesis is that such a model may also apply to electroneutral Mg2+ uptake and that a Mg2+/H+ exchange mechanism is present in the apical membrane of ruminal epithelium. To exclude influences of the Na+/H+ exchanger, experiments were performed in Na+-free NMDG media. Under these conditions, the Na+/H+ exchanger is not capable of extruding protons (ENa+- EH+ = -135 mV) and could even cause proton influx. In comparison, the driving force for an electroneutral Mg2+/2H+ exchange (EMg2+- 2EH+) is +159, +180, and +194 mV for an [Mg2+]e of 2.5, 5.0, or 7.5 mM, respectively. Therefore, a Mg2+/2H+ exchanger, if present in the cell membrane of REC, should work as a H+ -exporting and Mg2+-importing mechanism and hence result in pHi recovery accompanied by an increase of [Mg2+]i. However, the results show that cells take up Mg2+ but pHi remains acidic (Fig. 5). The chemical PDs for Mg2+ and H+ that must be considered the only driving forces for electroneutral ion exchange are optimal under our experimental conditions (2.5-7.5 mM [Mg2+]e >> 0.99-1.15 mM [Mg2+]i; 6.4-6.2 pHi << 7.4 pHe). In the physiological situation the chemical gradient for Mg2+ (2-4 mM luminal and about 1 mM intracellular) is small, and with normal, low pH values (5.5-6.5) in the ruminal fluid the H+ gradient is inwardly, not outwardly, directed. Together, these data and considerations indicate that Mg2+ uptake is not directly coupled to an efflux of H+ via the proposed Mg2+/H+ exchanger. Furthermore, under our experimental conditions, we do not have any evidence for the existence of a Na+-independent H+ extrusion. But it cannot be precluded from the presented results that CO2/HCO-3 or SCFA (butyrate) stimulate an additional acid extrusion process across the luminal membrane (e.g., an H+ pump or H+ conductance) that energizes Mg2+ uptake, for example by hyperpolarizing the Em. Another possible explanation for the increase of [Mg2+]i after exposure to butyrate and HCO-3 is a cotransport of Mg2+ with anions (e.g., Cl-, SCFA-, and HCO-3). Because [Mg2+]i increases to the same extent with HCO-3 alone and with HCO-3 and butyrate in the media, and because it is known that SCFA increase the production and secretion of HCO-3 in vivo (9), a Mg2+-2HCO-3 cotransporter seems to be a possible candidate for electroneutral Mg2+ uptake in REC. A furosemide- and bumetanide-sensitive electroneutral Mg2+-HCO-3 cotransport has been shown by Günther et al. (17) in Yoshida ascites tumor cells. Further experiments are necessary to test this hypothesis in REC.

Na+-dependent Mg2+ uptake. REC showed a pHi- and K+-insensitive increase of [Mg2+]i that seems to be dependent on the Na+ gradient across the cell membrane. In cells in nominally Na+-free NMDG media, we have demonstrated an elevation of [Mg2+]i accompanied by a decrease in [Na+]i. The magnitude of the observed rise in [Mg2+]i is dependent on the [Mg2+]e, reaching maximal values with 5 mM Mg2+ in the medium. Addition of Na+ to the bathing solution stops this Mg2+ uptake, and, in media containing 145 mM Na+, it is difficult to measure any significant change in [Mg2+]i unless [Mg2+]e is 7.5 mM. The interactions between external Na+ and [Mg2+]i described here are consistent with the existence of a Na+/Mg2+ antiporter in REC, a conclusion that is supported by the imipramine and quinidine sensitivity of the Mg2+ uptake in Na+-free media. A Na+/Mg2+ antiport has been proposed as the regulator of [Mg2+]i in a variety of cells and is the best-characterized Mg2+-transport protein (2, 15). At physiological intra- and extracellular [Na+] (145 mM [Na+]e >> 20 mM [Na+]i), a 2Na+/Mg2+ antiport will obviously mediate Mg2+ efflux; this has been confirmed by preliminary experiments with Mg2+-loaded (by A-23187) REC, where we have demonstrated a Mg2+ efflux in Mg2+-free high-Na+ media (results not shown). Since JMgms decreases on reduction of the serosal Na+ concentration (21), the exchanger is probably located mainly at the basolateral side of the epithelium. Exposing REC to Na+-free media, thus reversing the transmembrane Na+ gradient (0.005 mM [Na+]e << 9 mM [Na+]i), changes the driving force (2ENa+- EMg2+) for an electroneutral 2Na+/Mg2+ antiport to -430 mV, compared with +83 mV under control conditions. This indicates that the transporter should operate in the reverse mode, thereby increasing the Mg2+ influx and Na+ efflux. As expected, [Mg2+]i rises (from 0.87 to 1.15 mM) and [Na+]i falls (from 8.9 to 8.36 mM) in the Na+-free period. Because of the presence of other transport mechanisms, all of which are active in intact cells, it is impossible to measure the precise stoichiometry of the Na+/Mg2+ exchanger in these experiments. However, the exchanger is probably PD independent and therefore electroneutral because it also works in high-K+ media.

In addition to the PD-independent Mg2+ uptake (23), a second, parallel working PD-dependent mechanism exists for ruminal Mg2+ transport (22, 31). The main driving force for Mg2+ uptake by the latter mechanism is the Em. However, in media with 135 mM K+ and only 15 mM Na+, Em is short circuited; therefore, only the chemical gradient can drive Mg2+ uptake. Nevertheless, our experimental results show that Mg2+ uptake in high-K+ medium is twice (38 ± 8 µM/min) that in media with 145 mM Na+ and 5 mM K+ (19 ± 12 µM/min). These results are consistent with the idea that, even in high-K+/low-Na+ media, the Na+/Mg2+ antiport has been switched to the reverse mode, thereby mediating Mg2+ influx. Mg2+ uptake via a reverse-operating Na+/Mg2+ antiport has been observed in rat and ferret erythrocytes (8, 16), in neurons and neutrophil glial cells (18, 19), and in liver cells (4). Probably in REC, as in rat and ferret erythrocytes, the Na+ gradient is an additional driving force for the Na+/Mg2+ exchanger, determining the direction of transport. To date, a physiological implication of Mg2+ uptake via a Na+/Mg2+ antiport is uncertain, and the in vivo function of the transporter may be the production of net Mg2+ efflux.

In conclusion, our results have confirmed, at the cellular level, that Mg2+ influx is stimulated by butyrate and HCO-3, but we have no experimental evidence for the existence of a Mg2+/H+ antiport in the cell membrane of REC. The coupling between SCFA, CO2/HCO-3, and Mg2+ transport seems to be mediated by another mechanism, perhaps by a cotransport of Mg2+ and anions such as HCO-3. We have demonstrated the existence of a Na+/Mg2+ exchange mechanism in the plasma membrane of REC. Under physiological conditions, this transport pathway would be expected to use the inwardly directed Na+ gradient for Mg2+ efflux, but we have also shown a Mg2+ influx mediated by reversal of the Na+/Mg2+ exchanger. Thus the Na+ gradient seems to be an important factor for Mg2+ absorption across the ruminal epithelium.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the valuable assistance of Almut Böttcher. We also thank Dr. Theresa Jones for linguistic corrections.


    FOOTNOTES

This study was supported by a research grant from the Deutsche Forschungsgemeinschaft (Schw 642).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. Schweigel, Institut f. Vet.-Physiologie, FU-Berlin, Oertzenweg 19b, 14163 Berlin, Germany (E-mail: shweigel{at}vetmed.fu-berlin.de).

Received 9 August 1999; accepted in final form 18 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Argenzio, R. A., N. Miller, and W. v. Engelhardt. Effect of volatile fatty acids on water and ion absorption from the goat colon. Am. J. Physiol. 229: 997-1002, 1975[ISI][Medline].

2.   Blatter, L. A. Intracellular free magnesium in frog skeletal muscle studied with a new type of magnesium-selective microelectrode: interactions between magnesium and sodium in the regulation of [Mg]i. Pflügers Arch. 416: 238-246, 1990[ISI][Medline].

3.   Care, A. D., R. C. Brown, A. R. Farrar, and D. W. Pickard. Magnesium absorption from the digestive tract of sheep. Q. J. Exp. Physiol. 69: 577-587, 1984[ISI][Medline].

4.   Cefaratti, C., A. Romani, and A. Scarpa. Characterization of two Mg2+ transporters in sealed plasma membrane vesicles from rat liver. Am. J. Physiol. Cell Physiol. 275: C995-C1008, 1998[Abstract/Free Full Text].

5.   Clarke, J. D., E. J. Cragoe, and L. E. Limbird. alpha 2-Adrenergic receptors regulate Na+-H+ exchange via a cAMP-dependent mechanism. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 259: F977-F985, 1990[Abstract/Free Full Text].

6.   Dai, L.-J., and G. A. Quamme. Intracellular [Mg2+] and magnesium depletion in isolated renal thick ascending limb cells. J. Clin. Invest. 88: 1255-1264, 1991[ISI][Medline].

7.   Doreau, M., E. Ferchal, and Y. Beckers. Effects of level of intake and of available volatile fatty acids on the absorptive capacity of sheep rumen. Small Ruminant Res. 25: 99-105, 1997[ISI].

8.   Flatman, P. W., and L. M. Smith. Sodium-dependent magnesium uptake by ferret red cells. J. Physiol. (Lond.) 443: 217-230, 1991[Abstract].

9.   Gäbel, G., M. Bestmann, and H. Martens. Bikarbonattransport im Pansen von Schafen: Einfluß der Diät und von kurzkettigen Fettsäuren und Chlorid. J. Anim. Physiol. Anim. Nutr. 62: 20-21, 1989.

10.   Gäbel, G., H. Martens, M. Sündermann, and P. Galfi. The effect of diet, intraruminal pH and osmolarity on sodium, chloride and magnesium absorption from the temporarily isolated and washed reticulo-rumen of sheep. Q. J. Exp. Physiol. 72: 501-511, 1987[ISI][Medline].

11.   Gäbel, G., S. Vogler, and H. Martens. Short-chain fatty acids and CO2 as regulators of Na+ and Cl- absorption in isolated sheep rumen mucosa. J. Comp. Physiol. B Biochem. Syst. Environ. Physiol. 161: 419-426, 1991[ISI][Medline].

12.   Galfi, P., S. Neogrady, and F. Kutas. Culture of ruminal epithelial cells from bovine ruminal mucosa. Vet. Res. Comm. 4: 295-300, 1980[ISI].

13.   Giduck, S. A., and J. P. Fontenot. Utilization of magnesium and other macrominerals in sheep supplemented with different readily-fermentable carbohydrates. J. Anim. Sci. 65: 1667-1673, 1987[ISI][Medline].

14.   Grynkiewicz, G., M. Poenie, and T. Tsien. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260: 3440-3450, 1985[Abstract].

15.   Günther, T., and J. Vormann. Mg2+ efflux is accomplished by an amiloride-sensitive Na+/Mg2+ antiport. Biochem. Biophys. Res. Commun. 130: 540-545, 1985[ISI][Medline].

16.   Günther, T., and J. Vormann. Reversibility of Na+/Mg2+ antiport in rat erythrocytes. Biochim. Biophys. Acta 1234: 105-110, 1995[ISI][Medline].

17.   Günther, T., J. Vormann, and R. Averdunk. Characterization of furosemide-sensitive Mg2+ influx in Yoshida ascites tumor cells. FEBS Lett. 197: 297-300, 1986[ISI][Medline].

18.   Günzel, D., and W.-R. Schlue. Sodium-magnesium antiport in Retzius neurons of the leech Hirudo medicinalis. J. Physiol. (Lond.) 491: 595-608, 1996[Abstract].

19.   Hintz, K., D. Günzel, and W.-R. Schlue. Na+-dependent regulation of the free Mg2+ concentration in neuropile glial cells and P neurones of the leech Hirudo medicinalis. Pflügers Arch. 437: 354-362, 1999[ISI][Medline].

20.   Kleyman, T. R., and E. J. Cragoe. Amiloride and its analogs as tools in the study of ion transport. J. Membr. Biol. 105: 1-21, 1988[ISI][Medline].

21.   Leonhard-Marek, S., and H. Martens. Influences of Na on Mg transport across sheep rumen epithelium. Proc. Soc. Nutr. Physiol. 3: 88, 1994.

22.   Leonhard-Marek, S., and H. Martens. Effects of potassium on magnesium transport across rumen epithelium. Am. J. Physiol. Gastrointest. Liver Physiol. 271: G1034-G1038, 1996[Abstract/Free Full Text].

23.   Leonhard-Marek, S., H. Martens, and G. Gäbel. Effects of short chain fatty acids and carbon dioxide on magnesium transport across sheep rumen epithelium. Exp. Physiol. 83: 155-164, 1998[Abstract].

24.   Martens, H., and I. Blume. Effect of intraruminal sodium and potassium concentrations and of the transmural potential difference on magnesium absorption from the temporarely isolated rumen of sheep. Q. J. Exp. Physiol. 71: 409-415, 1986[ISI][Medline].

25.   Martens, H., G. Gäbel, and H. Strozyk. Mechanism of electrically silent Na and Cl transport across the rumen epithelium of sheep. Exp. Physiol. 76: 103-113, 1991[Abstract].

26.   Martens, H., and J. Harmeyer. Magnesium transport by isolated rumen epithelium of sheep. Res. Vet. Sci. 24: 161-168, 1978[ISI][Medline].

27.   Martens, H., G. Heggemann, and K. Regier. Studies on the effect of K, Na, NH+4, VFA and CO2 on the net absorption of magnesium from the temporarily isolated rumen of heifers. J. Vet. Med. Ser. A 35: 73-80, 1988[ISI].

28.   Masson, M. J., and A. T. Phillipson. The absorption of acetate, propionate and butyrate from the rumen of sheep. J. Physiol. (Lond.) 113: 189-206, 1951[ISI].

29.   Petersen, K.-U., J. R. Wood, G. Schulze, and K. Heintze. Stimulation of gallbladder fluid and electrolyte absorption by butyrate. J. Membr. Biol. 62: 183-193, 1981[ISI][Medline].

30.   Raju, B., E. Murphy, L. A. Levy, R. D. Hall, and R. E. London. A fluorescent indicator for measuring cytosolic free magnesium. Am. J. Physiol. Cell Physiol. 256: C540-C548, 1989[Abstract/Free Full Text].

31.   Schweigel, M., I. Lang, and H. Martens. Mg2+ transport in sheep rumen epithelium: evidence for an electrodiffusive uptake mechanism. Am. J. Physiol. Gastrointest. Liver Physiol. 277: G976-G982, 1999[Abstract/Free Full Text].

32.   Silverman, H. S., F. Di Lisa, R. C. Hui, H. Miyata, S. J. Sollott, R. G. Hansford, E. G. Lakatta, and M. D. Stern. Regulation of intracellular free Mg2+ and contraction in single adult mammalian cardiac myocytes. Am. J. Physiol. Cell Physiol. 266: C222-C233, 1994[Abstract/Free Full Text].

33.   Thorlacius, S. O. Effect of steam-volatile fatty acids and carbon dioxide on blood content of rumen papillae of the cow. Am. J. Vet. Res. 33: 427-430, 1972[ISI][Medline].

34.   Tomas, F. M., and B. J. Potter. The effect and site of action of potassium upon magnesium absorption in sheep. Aust. J. Agric. Res. 27: 873-880, 1976[ISI].

35.   Yun, C. H., S. Oh, M. Zizak, D. Steplock, S. Tsao, C.-M. Tse, E. J. Weinman, and M. Donowitz. cAMP-mediated inhibition of the epithelial brush border Na+/H+ exchanger, NH3, requires an associated regulatory protein. Proc. Natl. Acad. Sci. USA 94: 3010-3015, 1997[Abstract/Free Full Text].


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