Department of Veterinary Physiology, Free University of Berlin, 14163 Berlin, Germany
Submitted 13 September 2002 ; accepted in final form 19 February 2003
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ABSTRACT |
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sheep rumen; epithelial cells; intracellular magnesium; Mg2+-Cl cotransport; mag-fura-2
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.
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MATERIALS AND METHODS |
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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 612 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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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).
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RESULTS |
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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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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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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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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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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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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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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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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.
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DISCUSSION |
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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, 75145 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 3638% 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+.
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ACKNOWLEDGMENTS |
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This study was supported by Research Grant Schw 642 from the Deutsche Forschungsgemeinschaft.
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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.
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REFERENCES |
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