Anion-dependent Mg2+ influx and a role for a vacuolar H+-ATPase in sheep ruminal epithelial cells

Monika Schweigel and Holger Martens

Department of Veterinary Physiology, Free University of Berlin, 14163 Berlin, Germany

Submitted 13 September 2002 ; accepted in final form 19 February 2003


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The K+-insensitive component of Mg2+ influx in primary culture of ruminal epithelial cells (REC) was examined by means of fluorescence techniques. The effects of extracellular anions, ruminal fermentation products, and transport inhibitors on the intracellular free Mg2+ concentration ([Mg2+]i), Mg2+ uptake, and intracellular pH were determined. Under control conditions (HEPES-buffered high-NaCl medium), the [Mg2+]i of REC increased from 0.56 ± 0.14 to 0.76 ± 0.06 mM, corresponding to a Mg2+ uptake rate of 15 µM/min. Exposure to butyrate did not affect Mg2+ uptake, but it was stimulated (by 84 ± 19%) in the presence of . In contrast, Mg2+ uptake was strongly diminished if REC were suspended in -buffered high-KCl medium (22.3 ± 4 µM/min) rather than in HEPES-buffered KCl medium (37.5 ± 6 µM/min). After switching from high- to low-Cl solution, [Mg2+]i was reduced from 0.64 ± 0.09 to 0.32 ± 0.16 mM and the -stimulated Mg2+ uptake was completely inhibited. Bumetanide and furosemide blocked the rate of Mg2+ uptake by 64 and 40%, respectively. Specific blockers of vacuolar H+-ATPase reduced the [Mg2+]i (36%) and Mg2+ influx (38%) into REC. We interpret this data to mean that the K+-insensitive Mg2+ influx into REC is mediated by a cotransport of Mg2+ and Cl and is energized by an H+-ATPase. The stimulation of Mg2+ transport by ruminal fermentation products may result from a modulation of the H+-ATPase activity.

sheep rumen; epithelial cells; intracellular magnesium; Mg2+-Cl cotransport; mag-fura-2


IN RUMINATING ANIMALS, most of the required Mg2+ is absorbed from the forestomach by active, transcellular mechanisms. Uptake of Mg2+ into ruminal epithelial cells (REC) has been shown to be mediated by two parallel-working transport mechanisms. Part of the Mg2+ influx is K+ sensitive and may represent electrodiffusive Mg2+ uptake by an ion channel (35). The greater part (62%) is, however, K+ insensitive, and previous experiments carried out with isolated rumen epithelium and isolated ruminal epithelial cells (REC) in our laboratory have established that ruminal fermentation products [short-chain fatty acids (SCFA), CO2] stimulate transepithelial Mg2+ absorption (23) and Mg2+ influx into REC (37). Initially, it was assumed that the stimulation of Mg2+ transport was via Mg2+/H+ exchange in the apical membrane (23), by means of the proton load resulting from the absorption of SCFA in their protonized form and the intracellular hydration of CO2. More direct investigations with isolated REC have provided evidence for a symport of Mg2+ with anions and confirmed that SCFA and CO2 activate H+ efflux by the Na+/H+ exchanger (37). A positive relationship between intracellular H+ availability and transepithelial Mg2+ transport (23) can also be explained by the existence of an additional Na+-independent acid extrusion process (e.g., an H+ pump) that occurs in the cell membrane of REC and energizes Mg2+ uptake. To date, there is no information regarding the existence of such an active H+-extruding mechanism in REC. This therefore is explored in the present work. In addition, we have verified the influence of the predominant ruminal anions [, dissociated SCFA (SCFA), and Cl] on Mg2+ uptake.

To this purpose, we have performed experiments with isolated REC. With the aid of the fluorescent probes mag-fura 2 and BCECF, we have measured the intracellular free Mg2+ concentration ([Mg2+]i) and the intracellular pH (pHi) of REC under basal conditions and after changing the transmembrane chemical gradients for butyrate, , and Cl. To differentiate between K+-sensitive and -insensitive Mg2+ transport, some of the experiments have been carried out in high-K+/low-Na+ media. Additionally, transport inhibitors (loop diuretics, chlorothiazide, bafilomycin A1, and foliomycin) have been used to examine the possible role of an H+-ATPase in Mg2+-anion cotransport.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Materials. Medium 199, trypsin, glutamine, antibiotics (gentamycin, nystatin, kanamycin), and FCS were purchased from Sigma (St. Louis, MO). Dulbecco's phosphate-buffered saline (DPBS) and collagen were obtained from Biochrom (Berlin, Germany). Mag-fura-2 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. (9). Briefly, REC were isolated by fractional trypsination and grown in medium 199 containing 10% FCS, 1.36 mM glutamine, 20 mM HEPES, and antibiotics (gentamycin 50 mg/l, kanamycin 100 mg/l) in an atmosphere of humidified air-5% CO2 at 38°C. Experiments were performed 6–12 days after seeding.

Solutions. The control solutions were HEPES-buffered high-Na+, high-Cl solution (in mM: 145 NaCl, 5 KCl, 1 CaCl2, 2 MgCl2, 10 HEPES, 5 glucose, pH 7.4) and HEPES-buffered high-Na+/low-Cl solution (in mM: 110 Na-gluconate, 25 NaCl, 5 KCl, 1 CaCl2, 2 MgCl2, 10 HEPES, 5 glucose, pH 7.4). To inhibit the K+-sensitive part of Mg2+ uptake, cells were incubated in a HEPES-buffered high-K+ solution containing (in mM) 15 NaCl, 135 KCl, 1 CaCl2, 2 MgCl2, 10 HEPES, 5 glucose, pH 7.4. In butyrate-containing, HEPES-buffered high-NaCl solution, 20 mM NaCl was replaced by Na-butyrate. To investigate the effect of lowering the extracellular Cl concentration ([Cl]e) and the effect of on Mg2+ transport the composition of these solutions were changed. The composition of the modified experimental solutions is given in Table 1. All -containing solutions were preequilibrated with 95% air-5% CO2.


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Table 1. Composition of experimental solutions

 

Measurement of cytoplasmic Mg2+ and pH by spectrofluorometry. Cells were loaded with either 5 µM mag-fura-2 AM or 0.5 µM BCECF-AM for the determination of [Mg2+]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 were washed twice before measurement of fluorescence. Intracellular ion concentrations were determined by measuring the fluorescence of the probe-loaded REC in a spectrofluorometer (model LS-50 B; Perkin-Elmer), by using the fast-filter accessory, which allowed fluorescence to be measured at 20-ms intervals with excitation for mag-fura-2 at 340 and 380 nm and for BCECF at 440 and 480 nm and 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. To keep a constant time schedule, measurements were started consistently 5 min after transferring the cells from DPBS into the respective experimental solution. The measurements with -containing solutions were performed after the cell suspensions were preequilibrated with 95% air-5% CO2. During the experiments, the cuvette was tightly closed with a plastic cap to prevent CO2 leakage.

[Mg2+]i was calculated from the 340:380 nanometer ratio according to the formula of Grynkiewicz et al. (12) 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 the addition of 50 mM EDTA, pH 7.2, to remove all Mg2+ from the solution. BCECF signals were calibrated to ion concentrations by using the ionophore nigericin (10 µM) to equilibrate intra- and extracellular H+ concentration ([H+]). The procedure was repeated for various pH values between 6.0 and 8.0.

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


    RESULTS
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 MATERIALS AND METHODS
 RESULTS
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Some in vivo and in vitro studies showed an interrelationship between the intracellular supply of H+ and Mg2+ transport (7, 23). Therefore, we first studied the effect of various experimental manipulations on basal pHi and pHi-regulating mechanisms in REC.

Effect of butyrate, of , and of changing the extracellular [K+] and [Cl] on pHi of REC. Under control conditions (HEPES-buffered high-NaCl or high-KCl medium), baseline pHi was 6.83 ± 0.1 and 6.89 ± 0.03, respectively. Thus the resting pHi measured in the high-K+ medium was not significantly different from that measured in Na+ medium (Fig. 1). In contrast, pHi decreased to 6.68 ± 0.001 after switching from control solution (HEPES-buffered high-NaCl) to a HEPES-buffered high-Na+/low-Cl medium (Fig. 1). Incubation of REC in butyrate- and/or -containing solutions led to an intracellular acidification (Fig. 1). Thereupon, REC recovered to near control levels during the experimental period (Fig. 1). Neither increasing the extracellular K+ concentration ([K+]e) (from 5 to 135 mM) nor decreasing the [Cl]e (from 136/116 to 36 mM) affected the ability of REC to recover from the acid load. On average, the pHi recovered by 0.19 ± 0.05 pH units within 10 min, but slightly higher recovery rates of 0.24 ± 0.09 and 0.22 ± 0.06 units per 10 min were observed in Cl-reduced media with butyrate and/or , respectively.



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Fig. 1. Effects of butyrate and/or on intracellular pH (pHi) of ruminal epithelial cells (REC) incubated in high-NaCl, high-Na+/low-Cl, or high-KCl medium. Measurements were made after a 5-min preincubation in either HEPES-buffered solutions or media with butyrate (20 mM) and/or (5%/20 mM). Extracellular Cl concentration ([Cl]e) was 136 or 116 mM in high-Cl solutions and 36 mM in low-Cl solutions. External pH (pHe) was 7.4, and extracellular Mg2+ concentration ([Mg2+]e) was 2 mM in all solutions. Values are means ± SD of 4–6 single experiments. *P < 0.05 vs. control (HEPES-buffered high-NaCl or -KCl solutions).

 

Effect of butyrate and on [Mg2+]i and the Mg2+ uptake rate of REC incubated in high-NaCl or high-KCl media. Figure 2 shows a comparison of the effects of butyrate and/or on [Mg2+]i and Mg2+ influx in high-NaCl and -KCl media. The latter was used to eliminate the electrodiffusive K+-sensitive part of Mg2+ uptake (22, 35). Resting [Mg2+]i, determined in HEPES-buffered control solutions at the beginning of the experiments, was significantly lower in the high-NaCl medium (0.56 ± 0.14 mM) compared with that in high-KCl medium (0.88 ± 0.32 mM). In both media, an increase of [Mg2+]i was observed, which led to [Mg2+]i levels of 0.76 ± 0.06 mM (high-NaCl medium) and 1.26 ± 0.3 mM (high-KCl medium), respectively. As in our previous study (37), the Mg2+ influx was stimulated by the presence of butyrate and/or in high-NaCl solutions. After the 10-min measuring period, the [Mg2+]i was significantly higher (Fig. 2) in REC incubated in media with (0.92 ± 0.13 mM) or and butyrate (1.05 ± 0.4 mM) compared with that incubated in control medium (0.76 ± 0.06 mM). Additionally, Mg2+ uptake rates were stimulated by 84 ± 19 and 93 ± 40% in -buffered media without or with butyrate, respectively (Fig. 3). It is important to note that, in the presence of , neither the [Mg2+]i levels nor the Mg2+ uptake rates were significantly changed by the addition of butyrate. Furthermore, butyrate alone was not effective in increasing Mg2+ influx (12.8 ± 5 µM/min) compared with HEPES-buffered control medium (15 ± 1 µM/min).



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Fig. 2. Effects of butyrate and/or on intracellular Mg2+ concentration ([Mg2+]i) of REC incubated in high-NaCl or -KCl medium. Measurements were made after a 5-min preincubation in either control solution (HEPES-buffered high-NaCl or -KCl medium) or medium with butyrate (20 mM) and/or (5%/20 mM). Extracellular pH (pHe) was 7.4, and [Mg2+]e was 2 mM in all solutions. Values are means ± SE of 4 single experiments. *P < 0.05 vs. control.

 


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Fig. 3. Summary of the influence of extracellular Cl concentration ([Cl]e) and of butyrate on -stimulated Mg2+ uptake in REC. Results are expressed as %increase or decrease from uptake rate (15 ± 1 µM/min) in control medium (HEPES-buffered, high-NaCl medium). Bars represent means ± SE of 4 single experiments. *P < 0.05 vs. control.

 

The stimulating effect of on Mg2+ uptake was fully abolished if REC were incubated in a high-KCl medium, but this negative effect was compensated by supplementation of butyrate (Fig. 2 and 4). In the -buffered high-KCl medium, the rate of Mg2+ uptake was reduced to 22.3 ± 4 µM/min, which was significantly less than the rate observed in HEPES-buffered K+ medium (37.5 ± 6 µM/min) or in K+ medium with and butyrate (41.9 ± 12.4 µM/min) (Fig. 4).



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Fig. 4. Effect of butyrate and/or on the Mg2+ uptake rate of REC incubated in a high-KCl solution. Measurements were made after a 5-min preincubation in either HEPES-buffered control solution with a K+ concentration ([K+]) of 135 mM or in high-K+ medium with butyrate (20 mM) and/or (5%/20 mM). pHe was 7.4, and [Mg2+]e was 2 mM in all solutions. The 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.

 

Influence of a reduction of [Cl]e on [Mg2+]i and -stimulated Mg2+ uptake. Because, after SCFA and , Cl is the most abundant anion in the ruminal fluid, we then analyzed the role of the [Cl]e on [Mg2+]i and on the rate of Mg2+ uptake in REC. The cells were suspended in butyrate- and/or -containing high-Na+ solutions with a [Cl] of 116/136 or 36 mM, respectively.

As shown in Fig. 5, the [Mg2+]i of REC clearly depended on the extracellular Cl level. As [Cl]e was reduced, the initial [Mg2+]i fell from 0.64 ± 0.09 to 0.32 ± 0.16 mM (medium with ) and from 0.7 ± 0.26 to 0.37 ± 0.2 mM (medium with and butyrate), respectively. In all media, an increase of [Mg2+]i was observed, but the [Mg2+]i of REC incubated in the Cl-reduced media stayed well below that of cells in high-Cl media (Fig. 5). These effects of a [Cl]e reduction were independent of the presence of butyrate and/or in the experimental solutions.



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Fig. 5. [Mg2+]i of REC before and after a reduction of the [Cl]e. Cells were incubated in -buffered high-Na+ solution with or without butyrate and a [Cl]e of 116, 136, or 36 mM, respectively. Measurements were made after a 5-min preincubation in the respective medium. pHe was 7.4, and [Mg2+]e was 2 mM in all solutions. Values are means ± SE of 4 single experiments. Data with different lowercase letters in superscripts are significantly different with P < 0.05.

 

Furthermore, reduction of the [Cl]e diminished the rate of Mg2+ uptake. On exposure to medium with 36 mM Cl, the rate of Mg2+ uptake dropped from 28 ± 5 to 15 ± 5 µM/min (-containing Na+-solutions) and from 29.0 ± 10 to 20 ± 5 µM/min (- and butyrate-containing Na+ solutions). Figure 3 illustrates that the -dependent stimulation of Mg2+ uptake is completely abolished after the reduction of [Cl]e. Butyrate, on the other hand, can partly substitute for extracellular Cl under these experimental conditions (Fig. 3). As a result, the Mg2+ uptake rate was increased by 38 ± 18% compared with control values (in HEPES-buffered high-NaCl medium).

To show that the observed effects were induced by the decrease of [Cl]e, not by disturbing the content of other ions (, Na+) in the medium, we performed control experiments with gluconate as a substitute for Cl. The [Cl] of the solutions used in these experiments were reduced by replacing 100 mM (-containing solutions) or 80 mM (- and butyrate-containing solutions) of Cl by gluconate, leaving the concentration of all other ions unchanged. Under these conditions the same marked decrease of the [Mg2+]i was seen (results not shown). Furthermore, the Mg2+ uptake rate was reduced from 37 ± 5 to 19 ± 1 µM/min (high-Na+ solution with ) and from 31.5 ± 5 to 22 ± 4 µM/min (high-Na+ solution with and butyrate), respectively.

Effect of known inhibitors of cation-Cl cotransporters on [Mg2+]i and pHi. The loop-diuretics furosemide and bumetanide have been shown to inhibit anion-dependent electroneutral Mg2+ uptake in Yoshida ascites tumor cells (14). For this reason, we tested the effect of these inhibitors in a subsequent series of experiments. REC were suspended in high-K+ medium [to abolish the membrane potential (Em)] with and butyrate, and the [Mg2+]i was determined over a 10-min period. Compared with control conditions, we found a strong reduction of the [Mg2+]i after application of 100 µM furosemide or bumetanide, respectively. As shown in Fig. 6, control cells had an initial [Mg2+]i of 0.87 ± 0.12 mM, but this was only 0.55 ± 0.05 and 0.32 ± 0.09 mM in REC treated with 100 µM furosemide or bumetanide, respectively. Despite an increase of the [Mg2+]i in all experimental groups, the concentration determined at the end of the measuring period was significantly higher in control cells (1.07 ± 0.13 mM) compared with that in furosemide- or bumetanide-treated cells (furosemide, 0.76 ± 0.08; bumetanide, 0.43 ± 0.04).



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Fig. 6. Influence of loop diuretics and of chlorothiazide on [Mg2+]i of REC. Experiments were performed in high-KCl solution (extracellular [K+], 135 mM; [Cl]e, 116 mM) to eliminate the K+-sensitive part of Mg2+ influx. Measurements were made after a 5-min preincubation in either high-KCl medium with (5%/20 mM) and butyrate (20 mM) or the same medium with 100 µM each of furosemide (furo) bumetanide (bume), or chlorothiazide (CTZ); [Mg2+]e,2 mM; pHe 7.4. Values are means ± SE of 4 single experiments. **P < 0.01 vs. control.

 

Application of furosemide (100 µM) also influenced the pHi of REC. In inhibitor-treated cells, the initial decrease of pHi induced by CO2 and butyrate was more pronounced (6.45 ± 0.05) than in control cells (6.61 ± 0.08). The pHi recovery was retarded and amounted to 0.14 ± 0.03 compared with 0.19 ± 0.06 in control cells. In contrast, basal pHi and the ability of REC to recover from the acid load was not influenced by the presence of bumetanide in the extracellular solution.

The thiazide-type diuretic chlorothiazide was not effective in blocking Mg2+ uptake (Fig. 6) but increased basal pHi and stimulated the acid-induced pHi recovery (results not shown).

So far, the results confirm our previous conclusion (37) that the so-called K+-insensitive Mg2+ uptake mechanism is anion dependent and indicate a symport of Mg2+ with Cl. However, such a cotransport offers no direct explanation for the positive effects of SCFA and CO2 on Mg2+ transport. Our next hypothesis was that they activate a mechanism that increases the driving force for Mg2+-Cl cotransport and perhaps also for electrodiffusive Mg2+ influx. Because it seems from in vitro experiments with isolated epithelia that there is a coupling of H+ secretion and Mg2+ transport (23), we have tested the possibility that an H+-ATPase is involved in Mg2+ uptake.

Is there a role for an H+ pump in Mg2+ transport? To this purpose, we performed experiments with specific blockers of vacuolar H+-ATPases, namely bafilomycin A1 and foliomycin.

First, the effect of bafilomycin A1 (5 µM) on the free cytosolic [Mg2+] of REC incubated in a butyrate-containing, high-NaCl medium was examined. As shown in Fig. 7, bafilomycin A1 led to a reduction of the [Mg2+]i of REC. The [Mg2+]i, determined at the beginning of the measuring period (after a 5-min preincubation with or without inhibitor) was 0.76 ± 0.04 mM in control cells and 0.49 ± 0.08 mM in bafilomycin-A1-treated cells. After a further 10-min period, the [Mg2+]i of nontreated cells was increased to 0.83 ± 0.05 mM, compared with 0.59 ± 0.07 mM in REC exposed to the inhibitor.



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Fig. 7. Effect of the H+-ATPase inhibitor bafilomycin A1 on [Mg2+]i of REC. Measurements were made after a 5-min preincubation in HEPES-buffered high-NaCl medium with butyrate (20 mM) without or with 5 µM bafilomycin A1; pHe 7.4, [Mg2+]e, 2 mM. Values are means ± SE of 4 single experiments. *P < 0.05 vs. control.

 

Moreover, with the more specific inhibitor foliomycin (2 µM), we found a significant reduction of the rate of Mg2+ uptake (Fig. 8). REC incubated in the -containing high-NaCl solution took up Mg2+ at a rate of 47 ± 1 µM/min, but after application of foliomycin, the uptake rate was reduced to 30 ± 3 µM/min, which corresponded to a 36% decrease.



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Fig. 8. Inhibition of Mg2+ influx into REC by foliomycin. Measurements were made after a 5-min preincubation in -buffered high-NaCl medium without or with 2 µM foliomycin; [Mg2+]e, 2 mM; pHe 7.4. A: records from a typical experiment are shown. B: magnitude of the mean {Delta}[Mg2+]i for control cells and cells exposed to foliomycin; means ± SE of results from 3 single experiments are shown; **P < 0.01 vs. control.

 

With both bafilomycin A1 and foliomycin, the initial pHi (–0.05 ± 0.01; –0.074 ± 0.05) and the rate of pHi recovery (–0.04 ± 0.01; –0.07 ± 0.02) was reduced, compared with control values.


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
 REFERENCES
 
Modulation of pHi by , butyrate, [K+], and [Cl]. In a previous study (37), we have shown that there is no direct coupling between H+ efflux and Mg2+ influx. However, there is a possibility that the pHi may have indirect effects on Mg2+ transport. Therefore, we have tested the effects of various experimental manipulations on pHi. Changes from solutions containing HEPES to those containing and/or butyrate led to intracellular acidification, which can be explained partly by the permeation of the very lipid-soluble CO2 and/or the protonated form of the fatty acid across the cell membrane. Furthermore, with an extracellular pH (pHe) of 7.4 most of the butyrate (dissociation constant ~4.8) is present in dissociated form. Entry of SCFA into REC is mediated by an ion exchange for intracellular (21), thereby reducing the intracellular buffer capacity. Consequently, the pHi of REC decreases to a greater extent in butyratethan in -containing solutions. The CO2- and/ or butyrate-induced acidification rapidly activates cell-alkalinizing mechanisms, e.g., Na+/H+ exchanger and symport (27, 37) leading to pHi recovery. The rate of recovery of pHi seen in this study (0.17–0.24 pH units within 10 min) is consistent with our earlier data (37) and with results from the only other study dealing with pHi regulation in REC (27). Neither an increase of [K+]e (from 5 to 135 mM) nor a decrease of [Cl]e (from 116/136 to 36 mM) reduces the ability of REC to recover from an acid load. The pHi recovery rate is slightly higher in a medium with a reduced Cl content. Although the reason for this effect has not been investigated in detail, it can be attributed to an inhibition of the activity of the exchanger, which is present in the cell membrane of REC (24). Such an effect has been described repeatedly in other cell systems (15, 20) and is confirmed by the finding that pHi of REC is reduced in HEPES-buffered low-Cl medium.

Interrelationship between pHi acidification and [Mg2+]i. Some reports suggest a pHi-induced elevation of [Mg2+]i resulting from release of Mg2+ from intracellular compounds. In our study, proton loads evoked by switching from HEPES-buffered to - and/or butyrate-containing high-NaCl solutions led to marked pHi acidification but did not significantly alter the basal [Mg2+]i of REC. This corresponds well to the existence of REC showing spontaneously low pHi values of 6.4 ± 0.08, but basal [Mg2+]i levels (0.67 ± 0.09 mM) not different from those of normal REC (0.7 ± 0.05 mM; results not shown). Likewise, the elevation in [Mg2+]i observed during the time course of our experiments is not simply linked to a decrease in pHi. If it were, the effective pHi regulation back to near basal values should normalize [Mg2+]i. This was not the case, and, with the same or even stronger levels of acidification, the increase in [Mg2+]i is significantly reduced if REC were incubated in high-NaCl solutions with only butyrate, in Cl-reduced solutions, or in high-KCl medium with . These results suggest that REC, like other epithelial cells (30), regulate or stabilize their [Mg2+]i independently of the pHi or by means not directly related to pHi. Although the mechanisms have to be determined in detail, it seems that the Na+/Mg2+ exchanger, which we have shown to exist in the cell membrane of REC (37), contributes to this process.

Role of Na+/Mg2+ exchanger. On the other hand, it can be excluded that changes in the Na+/Mg2+ exchanger activity are responsible for the [Mg2+]i increase seen in this study. Most of our experiments have been performed in high-Na+ media (extracellular Na+ concentration, 75–145 mM) with an [Mg2+] of 2 mM. Under such conditions, the Na+/Mg2+ exchanger always operates in the forward mode, thereby mediating Mg2+ efflux and Na+ uptake (36). Therefore, its activity should result in an [Mg2+]i decrease rather than in the increase seen in our study. The latter effect would require an inhibition or reduced activity of the Na+/Mg2+ exchanger. This conclusion is supported by results from preliminary experiments with the nonspecific inhibitor amiloride. Application of amiloride in a low dosage of 100 µM led to the expected sharp increase in [Mg2+]i (results not shown). Furthermore, the higher [Mg2+]i starting levels measured in the high-K+/low-Na+ (135/15 mM) media are partly explicable by a reduced activity of the Na+/Mg2+ exchanger resulting from the lowering of the Na+ gradient across the cell membrane. It also explains the persistence of an "apparent" Mg2+ uptake after inhibiting K+-sensitive and -insensitive components of Mg2+ influx by incubation of REC in high-KCl media with either high , furosemide, or bumetanide.

Effect of butyrate and/or on [Mg2+]i and a comparison of Mg2+ uptake in high-NaCl and high-KCl medium. As in our previous study (37), the [Mg2+]i of REC incubated in a high-NaCl medium increases after exposure to butyrate and/or . This effect is in accordance with in vivo studies showing that the feeding of diets supplemented with easily fermentable carbohydrates (thereby increasing the intraruminal concentration of SCFA and ) leads to an elevation of the Mg2+ absorption (7, 11). In vitro experiments with isolated ruminal epithelia have confirmed these results and demonstrate that the so-called K+- or potential difference (PD)-insensitive component of Mg2+ uptake is stimulated specifically (23). After excluding a Mg2+/2H+ antiporter as the underlying mechanism, we have hypothesized the existence of a Mg2+-anion cotransport (37). The results obtained in the high-NaCl medium, viz., 1) that butyrate alone is not able to stimulate Mg2+ uptake and 2) that the rate of Mg2+ uptake is the same in -buffered media and -buffered media with additional butyrate, have led us to presume that the Mg2+ influx is coupled to uptake via a cotransport. Such a transport system has been shown by Günther et al. (14) in Yoshida ascites tumor cells. However, the existence of a cotransport is strongly opposed by the finding that the stimulating effect of is completely abolished after reduction of the [Cl]e. In the experiments performed by Günther et al. (14), even the total removal of Cl from the extracellular solution had no negative influence on the Mg2+ uptake as long as was present. Another result mitigating against a cotransport is the significant reduction of Mg2+ influx in the -buffered high-KCl medium.

Effect of [Cl]e. On the other hand, the [Cl]e directly influences the [Mg2+]i and the Mg2+ influx rate. Preincubation of REC in a Cl-reduced medium (36 mM) causes a decrease of the [Mg2+]i and a complete inhibition of the -stimulated Mg2+ uptake. We interpret this data as reflecting that the K+-insensitive Mg2+ influx in REC is mediated by a cotransport of Mg2+ with Cl. This idea is also supported by the sensitivity of [Mg2+]i and the Mg2+ uptake to the sulfamoyl-benzoic acid-type diuretics (loop diuretics), which are known inhibitors of cation-Cl cotransporters (26, 29). Bumetanide and furosemide (100 µM each) reduced the rate of Mg2+ influx by 64 and 40%, respectively, showing that bumetanide is the more potent blocker. This relative sensitivity to loop diuretics (bumetanide > furosemide) and the insensitivity to the thiazide-type agent chlorothiazide is typical for transport proteins belonging to the sodium-(potassium)-chloride cotransporter family (10, 32). It is important to note, however, that potassium is not universally required and that a number of studies have suggested the existence of bumetanide-sensitive, thiazide-insensitive Na+-Cl cotransporters (40). Anion-dependent transport systems for Mg2+ influx and Mg2+ efflux have been described previously (13, 19, 28). The existence of an Mg2+-Cl cotransporter in rumen epithelium is in accordance with results from controlled feeding trials with sheep showing a positive effect of Cl on Mg2+ absorption (34). Furthermore, in vitro experiments with isolated sheep rumen epithelia have revealed that the cellular component of Mg2+ absorption is strongly reduced by the removal of apical Cl. However, the effect of Cl withdrawal had been taken to reflect the coupling of an Mg2+/H+ exchanger and a exchanger (23). Because we have found no evidence for such a double ion exchange mechanism, we have tested an alternative hypothesis.

For some epithelia, it has been shown that a proton-motive force generated by electrogenic H+-ATPases in the apical membrane is utilized as a driving force for numerous transport processes (16, 18). Because the membrane potential has been shown to be the main driving force for Mg2+ uptake into REC (22, 35) and because of the positive relationship between intracellular H+ availability and transepithelial Mg2+ transport (23), we have tested whether a H+ pump is involved in Mg2+ uptake. For this purpose, we have used bafilomycin A1 and foliomycin, which are known to be potent inhibitors of vacuolar ATPases (V-ATPases) (3, 6). REC [Mg2+]i and Mg2+ influx are reduced by 36–38% after preincubation with bafilomycin A1 or foliomycin, respectively, supporting the idea that a vacuolar H+-ATPase energizes Mg2+ influx by generating an inside-negative membrane potential. The protons needed for this process are produced in cytoplasm from CO2 and water by the enzyme carbonic anhydrase. The presence of the latter in REC has been reported repeatedly (1, 2). For high concentrations of furosemide and related compounds, an interference with carbonic anhydrase has been shown (29). Thus some of the negative effects of furosemide on Mg2+ uptake may result from an inhibition of the enzyme. , which is also formed in this reaction, is secreted through the cell membrane in exchange for Cl and/or SCFA, but it is not clear whether Cl and SCFA compete for extracellular binding sites at a common anion-exchange mechanism or whether there are separate and exchangers (8, 21, 39). However, a reduction of the apical [Cl] or a Cl withdrawal from the luminal site of the epithelium leads to an elevation of the cellular SCFA transport (21, 39). This makes it likely that butyrate anions effect Mg2+ transport indirectly by stimulating secretion and that they therefore increase intracellular H+ availability to the H+ pump. In this way, butyrate can partly substitute for extracellular Cl in the -buffered solution. Because a great proportion (~95% under in vitro conditions) of absorbed butyrate is metabolized to CO2 in REC (38), the positive effect of SCFA also results from its delivering substrate to the carbonic anhydrase reaction. Oxidative metabolism of SCFA is an important energy source for the epithelial cells themselves (4), and the reversible disassembly of V-ATPase into its V0 and V1 subunits acts as a type of regulation of its activity in response to a drop in energy supply (25). The coupling of REC energy metabolism, H+-ATPase activity, and Mg2+ uptake provides an explanation of the positive effect of easily fermentable carbohydrates on Mg2+ absorption (7, 11). CO2 and SCFA also stimulate Mg2+ absorption from the large intestine (colon, cecum) of various species (5, 31, 33), but knowledge of the underlying mechanisms is limited. Holtug (17) has found evidence for SCFA-dependent active proton secretion in the colon of the hen. The implication of such a mechanism for Mg2+ transport in the large intestine of mammals remains to be shown.

In conclusion, our results are in agreement with the existence of an Mg2+-anion cotransport, most probably an Mg2+-Cl cotransport, in the cell membrane of REC. At this time, we cannot exclude that other ions () can substitute for Cl under certain conditions. Furthermore, it seems likely from our experiments with inhibitors of the vacuolar H+-ATPase (bafilomycin A1, foliomycin) that this Mg2+ uptake is energized by electrogenic proton pumps, which initially generate a transmembrane inside-negative voltage. The parallel influx of Mg2+ and Cl dissipates this voltage, which is an explanation for results showing the existence of a PD- and K+-dependent component and a so-called "PD- or K+-insensitive" component of ruminal Mg2+ transport (22, 35). The positive effect of ruminal fermentation products, such as SCFA/SCFA and CO2, on Mg2+ transport seems to be an indirect one and can be explained by their influence on the activity of a vacuolar H+-ATPase, thereby increasing the driving force for the uptake of Mg2+.


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

This study was supported by Research Grant Schw 642 from the Deutsche Forschungsgemeinschaft.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. Schweigel, Institute for Veterinary Physiology, Free University of Berlin, Oertzenweg 19b, 14163 Berlin, Germany (E-mail: shweigel{at}zedat.fu-berlin.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.


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