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
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ABSTRACT |
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
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INTRODUCTION |
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+.
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MATERIALS AND METHODS |
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).
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RESULTS |
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
( [Mg2+]i) in these media is
given. Values are means ± SE of 4 single experiments. * P < 0.05 vs. control.
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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 ( 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.
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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).
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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.
[Mg2+]i or magnitude of mean
[Na+]i change
( [Na+]i) after a time period of
100 s is given. Values are means ± SE (shown if larger than symbol
size) of 6 experiments.
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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.
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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.
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DISCUSSION |
[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 |
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.
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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