Mg2+ transport in sheep rumen
epithelium: evidence for an electrodiffusive uptake
mechanism
Monika
Schweigel1,
Ingo
Lang2, and
Holger
Martens1
1 Department of Veterinary
Physiology, Free University of Berlin, 14163 Berlin; and
2 Department of Zoophysiology and
Cell Biology, University of Potsdam, 14471 Potsdam, Germany
 |
ABSTRACT |
The potential difference (PD)-dependent
component of transcellular Mg2+
uptake in sheep rumen epithelium was studied. Unidirectional 28Mg2+
fluxes were measured at various transepithelial PD values, and the
unidirectional mucosal-to-serosal
28Mg2+
flux (JMgms) was
correlated with the PD across the apical membrane
(PDa) determined by mucosal
impalement with microelectrodes.
PDa was found to be
54 ± 5 mV, and JMgms was 65.9 ± 13.8 nmol · cm
2 · h
1
under short-circuit conditions. Hyperpolarization of the ruminal epithelium (blood-side positive) depolarized
PDa and, most noticeably, decreased JMgms. Further experiments were
performed with cultured ruminal epithelial cells (REC). With the aid of the fluorescence probe mag-fura 2, we measured the intracellular free
Mg2+ concentration
([Mg2+]i)
of isolated REC under basal conditions at various extracellular Mg2+ concentrations
([Mg2+]e)
and after alterations of the transmembrane voltage. Basal [Mg2+]i
was 0.54 ± 0.08 mM. REC suspended in media with
[Mg2+]e
between 0.5 and 7.5 mM showed an increase in
[Mg2+]i
that was dependent on
[Mg2+]e
and that exhibited a saturable component (Michaelis-Menten constant = 1.2 mM; maximum
[Mg2+]i = 1.26 mM). Membrane depolarization with high extracellular K+ (40, 80, or 140 mM
K+) and the
K+ channel blocker quinidine (50 and 100 µM ) resulted in a decrease in
[Mg2+]i.
On the other hand, hyperpolarization created by
K+ diffusion (intracellular
K+ concentration > extracellular
K+ concentration) in the presence
of valinomycin induced a 15% increase in
[Mg2+]i.
None of the manipulations had any effect on intracellular Ca2+ concentration and
intracellular pH. The results support the assumption that the membrane
potential acts as a principal driving force for
Mg2+ entry in REC and suggest that
the pathway for this electrodiffusive Mg2+ uptake across the luminal
membrane is a channel or a carrier.
sheep rumen; epithelial cells; magnesium transport; intracellular
magnesium; mag-fura 2
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INTRODUCTION |
THE RUMEN IS THE MOST important site of
Mg2+ absorption in the
gastrointestinal tract of sheep and maintains
Mg2+ homeostasis (35). Net
Mg2+ absorption across the rumen
epithelium occurs against an electrochemical gradient (3, 4,
22) and is mediated by an active transport mechanism (24).
It has long been known that an increase in
K+ intake and, consequently, in
ruminal K+ concentration
([K+]) decreases the
apparent digestibility of Mg2+ (9,
14, 30). This effect of K+ is
restricted to the forestomachs (35). The underlying mechanisms of this
impaired Mg2+ absorption from the
rumen have been studied intensively by in vivo and in vitro methods (4,
21-23), and there is now a growing body of evidence
that the active and transcellular component of net
Mg2+ uptake is significantly
decreased by high ruminal
[K+] (23).
Furthermore, the reduced net Mg2+
transport has been shown to be closely correlated with
electrophysiological changes of the rumen epithelium at high ruminal
[K+]. There is a
positive correlation between the
[K+] of the ruminal
fluid and the transepithelial potential difference (PDt) (8, 18, 32). This
K+-dependent increase of
PDt (blood-side positive) causes a
small and passive backflow of
Mg2+, probably via the
paracellular pathway from the blood side into the lumen (4, 21, 23).
Studies with microelectrodes have demonstrated that an increase in the
mucosal [K+] leads to
a reversible concentration-dependent depolarization of the potential
difference across the apical membrane of the rumen epithelium
(PDa); this depolarization is
accompanied by a decrease in the unidirectional mucosal-to-serosal
transcellular Mg2+ flux
(JMgms) (21). The
K+-induced changes of
Mg2+ fluxes, that is, a slight
increase in the serosal-to-mucosal Mg2+ flux
(JMgsm) and a marked reduction
in the JMgms, can be perfectly
simulated by corresponding alterations of
PDt by a voltage clamp (23).
These observations have led to a tentative model of luminal uptake of
Mg2+ in the rumen epithelium (21,
23), suggesting that Mg2+ enters
the apical membrane by a PD-dependent transport mechanism, or, because
K+ influences
PDa, by a
K+-sensitive transport mechanism
via a conductance or a carrier. The predominant driving force for this
Mg2+ uptake is probably
PDa and, to a small extent, the
chemical gradient between the luminal and cytosolic
Mg2+ concentrations
([Mg2+]).
Consequently, an ideal experiment for establishing this model of
Mg2+ transport should include the
simultaneous measurement of PDt, PDa, intracellular
[Mg2+]
([Mg2+]i),
and Mg2+ fluxes. Unfortunately, it
is not possible to measure Mg2+
transport and the other parameters directly and simultaneously with the
squamous, stratified, and keratinized rumen epithelium. However, an
indirect approach could give us the required information regarding
Mg2+ transport and
PDa. Any change of
PDt, which can be easily
manipulated in vitro in conventional Ussing chambers, induces
alterations of PDa and/or the
potential difference of the basolateral membrane (PDb). Hence, we have measured
unidirectional
28Mg2+
fluxes (JMgsm and
JMgms) at different
PDt values (blood-side positive)
and correlated the flux rates with
PDa as determined by mucosal
impalement with microelectrodes. To test the hypothesis of a
PD-dependent Mg2+ uptake in a more
direct way, we have performed experiments with cultured ruminal
epithelial cells (REC). With the aid of the fluorescence probe mag-fura
2, we have measured the free
[Mg2+]i
of isolated REC under basal conditions and after alterations of the
transmembrane voltage
(Em). The
obtained results clearly show PD-dependent changes of
Mg2+ transport across the sheep
rumen epithelium and of the
[Mg2+]i
and support the assumption of electrodiffusive
Mg2+ uptake across the luminal membrane.
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MATERIALS AND METHODS |
Materials.
Medium 199, trypsin, glutamine, antibiotics (gentamicin, nystatin, and
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, fura 2-AM,
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM, and
pluronic acid were from Molecular Probes (Eugene, OR). All other
chemicals were purchased from Sigma.
Epithelia.
Epithelia were taken from the ventral rumen sac of freshly slaughtered
sheep. Pieces of rumen wall were immediately immersed in the control
buffer solution (maintained in 38°C, gassed with 95%
O2-5%
CO2), and the epithelia were
stripped of the underlying muscle layers and the serosa.
Incubation.
Pieces of epithelia were mounted between the two halves of the
incubation chamber with an exposed area of 3.14 cm2 (for flux studies) or 0.79 cm2 (for microelectrode studies).
We minimized edge damage by using rings of silicone rubber on both
sides of the tissues. Flux chambers were connected to reservoirs
containing 16 ml buffer solution on each side. The solutions (standard
Ringer solution that contained in mM: 95 NaCl, 5 KCl, 25 NaHCO3, 1 Na2HPO4,
2 NaH2PO4,
10 glucose, 1 CaCl2, 2 MgCl2, 9 sodium-acetate, 9 sodium-propionate, 9 butyric acid, and 5 NaOH) were kept at 38°C
and were continuously stirred by the use of a gas lift system that
supplied 95% O2-5%
CO2. The microelectrode chamber
had a volume of 0.7 ml on the mucosal side and 0.4 ml on the serosal
side. Both chamber halves were perfused with solution from gassed
reservoirs, and the solutions were heated to 38°C immediately
before being added to the chamber.
Electrical measurements.
Chambers were connected to a computer-controlled voltage-clamp device
(AC Microclamp, Aachen, Germany) or to a voltage-clamp and
microelectrode device (Biomedical Instruments, Munich, Germany). PDt was measured via KCl (0.5 M)
agar bridges and calomel electrodes with reference to the mucosal
solution. Tissue conductance
(Gt) was
determined from the change in PDt
caused by bipolar current pulses of 100 µA of 100-ms duration. The
current was passed either through Ringer-agar bridges connected to
Ag-AgCl electrodes in 3 M KCl (flux chambers) or through rings of
Ag-AgCl electrodes (placed in each half of the microelectrode chamber).
In each setup, fluid resistance and junction potentials were measured
before mounting the epithelia and were corrected for during the experiments.
Microelectrodes.
Conventional microelectrodes were pulled from borosilicate glass (1.2 or 1.5 mm OD) and filled with 0.5 M KCl, yielding resistances of
15-30 M
. We impaled rumen epithelial cells across the apical membrane by means of a motorized micromanipulator with a Piezzo element
and measured PDa with reference to
the mucosal solution. PDa and
PDt were observed on an
oscilloscope and displayed on a chart recorder. Impalements were
accepted if 1) the change in PDa was abrupt while advancing
into the tissue, 2)
PDa remained stable for at least 1 min, and 3)
PDa returned to 0 ± 3 mV on withdrawal of the electrode.
Flux studies.
28MgCl2
(Department of Physics, Munich-Garching) was added to one side of the
epithelium to yield a specific activity of 2 kBq/µM in the buffer
solution. After an equilibrium period of 45 min, aliquots were sampled
at 30-min intervals and replaced by aliquots of unlabeled solution.
28Mg2+
was counted in a well-type crystal scintillation counter (Gammaszint 5300, Berthold). The counts were corrected for physical
decay. Unidirectional fluxes were calculated from the rate of tracer appearance on the other side of the tissue. Paired determinations of
fluxes were accepted if
Gt differed by
<25%.
Whole cell patch-clamp experiments.
To determine Em
of REC and to verify the effect of the manipulations that changed it,
we used standard electrophysiological methods. REC were routinely grown
as described under cell culture. Before the patch-clamp experiments,
cells were seeded onto uncoated glass coverslips for 1 day and then
transferred to a chamber mounted on the stage of an inverted microscope
(Axiovert, Zeiss) for recording. Resting membrane potential and cell
currents were recorded from single cells by using the conventional
patch-clamp technique in the whole cell mode under current clamp
(I = 0) or voltage clamp (V = 0) with an EPC-7 patch-clamp
amplifier (HEKA Elektronik). Computer-controlled voltage-clamp
protocols were used to generate current-voltage relationships (winTIDA
2.1, HEKA Elektronik). After low-pass filtering (0.8 kHz, four-pole
Bessel), data were sampled at 2 kHz and stored on hard disc. The
standard bath solution was the NaCl solution (in mM: 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES). When the
K+ content of the bath solution
was increased, an appropriate amount of NaCl was replaced by KCl.
Pipettes were filled with K+
solution (in mM: 110 potassium-gluconate, 25 KCl, 10 NaCl, 1 EGTA, 2 MgCl2, 2 ATP, and 10 HEPES) in all experiments.
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 (gentamicin 50 mg/l and kanamycin 100 mg/l) under an
atmosphere of humidified air-5%
CO2 at 38°C. Experiments were
performed between 6 and 12 days after seeding.
Measurement of cytoplasmic
Mg2+ and
Ca2+ by
spectrofluorometry.
Cells were loaded with either 5 µM mag-fura 2-AM or 10 µM fura 2-AM
for the determination of
[Mg2+]i
and intracellular Ca2+
concentration
([Ca2+]i),
respectively. Cells were subsequently washed twice in DPBS. The REC
were incubated for a further 30 min to allow for complete deesterfication and washed twice before measurement of fluorescence. Intracellular ion concentrations were determined by measuring the
fluorescence of the probe-loaded REC in the spectrofluorometer LS-50 B
(Perkin-Elmer) by using the fast filter accessory, which allowed the
measurement of fluorescence at 20-ms intervals with excitation for
mag-fura 2 and fura 2 at 340 and 380 nm and emission at 515 nm. All
measurements were made at 37°C in a 3-ml cuvette containing 2 ml
cell suspension under stirring. The
[Mg2+]i
and
[Ca2+]i
were calculated from the 340/380-nm ratio according to the formula of
Grynkiewicz et al. (16), with a dissociation constant of 1.5 mM and 224 nM, respectively, for the
mag-fura-2/Mg2+ and
fura-2/Ca2+ complexes. The minimum
(Rmin) and maximum
(Rmax) ratios were determined at
the end of each experiment by using digitonin.
Rmax for mag-fura 2 was obtained
by the addition of 25 mM MgCl2 in the absence of Ca2+ and
Rmin by addition of 50 mM EDTA, pH
7.2, to remove all Mg2+ from the
solution. Rmax for fura 2 was
obtained in solutions with 2 mM
Ca2+ and
Rmin by addition of 20 mM EGTA, pH
8.0.
Statistical analysis.
If not otherwise stated, data are presented as means ± SD.
Significance was determined by Student's
t-test or Tukey's ANOVA. Correlations
between variables were tested by calculating Pearson's product moment
correlation coefficients. P < 0.05 was considered significant. All statistical calculations were performed
using SigmaStat (Jandel Scientific).
 |
RESULTS |
PDt and unidirectional
28Mg2+
fluxes.
The unidirectional flux rates of
28Mg2+
are shown in Fig. 1. Increasing the
PDt (blood-side positive) induced
a decrease in the unidirectional
JMgms from 65.9 ± 13.8 nmol · cm
2 · h
1
under short-circuit conditions
(PDt = 0 mV) to 48.5 ± 8.4 (PDt = 15 mV), 40.6 ± 7.8 (PDt = 30 mV), and 31.5 ± 6.1 nmol · cm
2 · h
1
(PDt = 45 mV). The
28Mg2+
flux in the opposite direction
(JMgsm) was slightly enhanced
from 6.9 ± 1.2 nmol · cm
2 · h
1
(PDt = 0 mV) to 9.6 ± 2.2 (PDt = 15 mV), 14.1 ± 2.8 (PDt = 30 mV), and 17.5 ± 1.9 nmol · cm
2 · h
1
(PDt = 45 mV). These changes in
the unidirectional
28Mg2+
fluxes caused a significant decrease in net
28Mg2+
absorption (JMgnet) from 58.9 ± 12.8 nmol · cm
2 · h
1
to 38.9 ± 6.3, 26.5 ± 5.1, and 14.0 ± 4.5 nmol · cm
2 · h
1
at 0, 15, 30, or 45 mV, respectively.

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Fig. 1.
Unidirectional
28Mg2+
fluxes
(JMg2+)
at different transepithelial potential difference
(PDt) values (serosal-side
positive) determined in conventional Ussing chambers under
voltage-clamp conditions. Mucosal and serosal concentrations of
Mg2+ were identical (2 mM).
JMgms, mucosal-to-serosal
Mg2+ flux;
JMgsm, serosal-to-mucosal
Mg2+ flux. Values are means ± SD; n = 9-11.
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PDt and PDa.
Because simultaneous determinations of
28Mg2+
fluxes and PDa were not possible,
we tested the effect of applied changes in
PDt on
PDa in accompanying microelectrode
studies. During cell impalements, epithelia were voltage clamped to
various PDt values between 0 mV
and 80 mV, with the mucosal side as the reference. The applied PDt included the physiological
range of 20-60 mV (blood-side positive; Ref. 7). Under
short-circuit conditions (PDt = 0 mV), PDa was
54 ± 5.5 mV. Increasing PDt from 0 mV to 60 mV induced a reversible depolarization of
PDa to
10 ± 7 mV
(Fig. 2). The relationship between PDt and
PDa was linear:
PDa = 0.72 PDt
53.4 mV (r2 = 0.9; n = 13, P < 0.01).

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Fig. 2.
Potential difference of apical membrane
(PDa) as a function of
PDt (serosal-side positive)
Regression equation is given in PDt and
PDa. Values are means ± SD;
n = 15.
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Figure 3 shows
JMgms measured at defined
PDt values (0, 15, 30, and 45 mV;
serosal-side positive) as a function of
PDa. A depolarization of
PDa from
54 mV
(PDt = 0 mV) to
21 mV
(PDt = 45 mV) reduced
JMgms by 52%.

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Fig. 3.
JMgms measured at defined
PDt values (0-45 mV,
serosal-side positive; see Fig. 1) as a function of
PDa.
PDa was calculated by means of
regression equation (PDa = 0.72 PDt 53.4) determined from preceding microelectrode
studies (see Fig. 2). JMgms is
related to PDa in an exponential
fashion: JMgms = 18.7 + 6.08 · e( 0.038 · PDa).
Line is fitted to data by nonlinear regression analysis. There is a
strong negative correlation between
PDa and
JMgms; correlation coefficient = 0.99. Values are means ± SD; n = 9-11 for each equation.
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Free
[Mg2+]i.
The determination of
28Mg2+
fluxes of the whole epithelium clearly revealed that variations of
PDa (and
PDt) influenced transcellular Mg2+ transport. It was
hypothesized that depolarization of
PDa reduces Mg2+ uptake, which should cause
corresponding changes in
[Mg2+]i.
Because measurements of
[Mg2+]i
have not been made previously with isolated REC, the first series of
experiments was designed to study
[Mg2+]i
as a function of the extracellular
[Mg2+]
([Mg2+]e)
without manipulating the membrane potential. The basal cytosolic free
[Mg2+]i
of REC (cultured in the presence of
Ca2+ and
Mg2+ as described in
MATERIALS AND METHODS) was 0.54 ± 0.08 mM (n = 12), measured in
Ca2+-free and
Mg2+-free Hanks' balanced salt
solution. REC showed a rapid concentration-dependent increase in
[Mg2+]i
when
[Mg2+]e
was increased (Fig. 4). Figure
5 shows the effect of varying the bath
[Mg2+] on the
cytosolic [Mg2+] of
REC with a physiological Mg2+
content.
[Mg2+]i
of REC increased from 0.59 ± 0.13 mM
(n = 3) in media with 0.5 mM
Mg2+ to a maximum level of 1.26 ± 0.1 mM (n = 3) in solutions with a
[Mg2+]e of 7.5 mM. The changes in
[Mg2+]i
showed sigmoid saturation kinetics with a half-maximal saturation point
of 1.2 mM.

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Fig. 4.
Original tracings of measurements of intracellular
Mg2+ concentration
([Mg2+]i)
( ) and intracellular pH
(pHi)
( ) in ruminal epithelial cells (REC).
Extracellular [Mg2+]
([Mg2+]e)
was increased stepwise from 0 mM to a final
[Mg2+] of 1 mM; 0.5 mM
MgCl2 was added at time indicated
by arrows.
[Mg2+]i
and pHi were measured
simultaneously in mag-fura 2- and
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF)-loaded REC. BCECF signals were calibrated to
pHi values with 10 µM nigericin
in a high-K+ medium between pH 6.0 and 8.0. The pHi of REC was 7.1 ± 0.3 in time course of experiments.
Inset: original tracing of a
measurement of intracellular Ca2+
concentration in REC; there are no changes in intracellular
Ca2+ with increasing
[Mg2+]e.
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Fig. 5.
[Mg2+]i
as a function of
[Mg2+]e.
[Mg2+]e
was stepwise increased from 0 mM to 7.5 mM. Extracellular
Ca2+ concentration was 1 mM
throughout experiment; n = 3-6.
Line is drawn assuming sigmoidal kinetics (Hill Method) and is fitted
to data by nonlinear regression analysis. Parameters used are:
y0 = 0.54, a = 0.72, b = 2.84, c = 1.26, correlation coefficient = 0.997.
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Because we considered there to be a close connection between
Mg2+ uptake and the
Em of REC, we
measured
[Mg2+]i
in a further series of experiments after depolarization or hyperpolarization of
Em.
Effect of high extracellular
K+ and of quinidine on the
Em of REC.
The elevation of the extracellular
[K+]
([K+]e)
is a simple and often-applied method for depolarizing the
Em by reducing
K+ efflux via
K+ channels. Whole cell
patch-clamp experiments were carried out to verify the effects of
increased
[K+]e
on the Em of
isolated REC (Fig. 6). The REC resting
potential determined by conventional patch-clamp techniques was
24 ± 3 mV (n = 40 single
cells; maximum
70 mV, minimum
5 mV). An increase of
[K+]e
from 5 mM in control solutions to either 40 or 80 mM resulted in a
reversible depolarization of the REC
Em by ~15 and
32 mV, respectively (Fig. 6B). Like
high K+, the application of the
K+ channel blocker quinidine
depolarized the
Em of REC by
~30 mV.

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Fig. 6.
Effect of high extracellular K+
concentration
([K+]e)
on membrane potential
(Em) of REC. As
in fluorescence measurements, standard bath was NaCl solution. When
K+ content of bath solution was
increased, an appropriate amount of NaCl was replaced by KCl. Pipettes
were filled with K+ solution in
all experiments. A: original recording
showing depolarization after changing bath from a control solution with
5 mM K+ to a solution with 80 mM
K+.
B: summary of effects of high
[K+]e
on transmembrane voltage
(Em) of REC.
Values are means ± SD; n = 40, 7, and 3 for
[K+]e = 5, 40, and 80 mM, respectively.
Inset: whole cell currents from a
typical cell bathed in solutions with either 5, 40, or 80 mM
K+.
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Effect of high
[K+]e
and of quinidine on
[Mg2+]i
of REC.
The effects of increasing
[K+]e
on
[Mg2+]i
are summarized in Fig. 7. In all
experiments, high
[K+]e
led to a reduction of
[Mg2+]i.
As Fig. 7 shows, an increase in the
[K+]e
from 5 mM to 40, 80, or 140 mM decreased
[Mg2+]i
(P < 0.01) by 7 ± 2.7, 11 ± 2.6, and 42 ± 6.2%, respectively.

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Fig. 7.
Summary of effects of high
[K+]e
on
[Mg2+]i
of REC. Results are expressed as percent decrease from control
[Mg2+]i
([Mg2+]i
in media with a
[K+]e
of 5 mM = 100%).
[K+]e
was increased from 5 mM to 40, 80, or 140 mM. An increase in
[K+]e
decreased
[Mg2+]i
in all cases. Number of single experiments is shown in parentheses.
** P < 0.01 vs. control.
Inset: original recordings of 2 measurements showing effect of increasing bath
[K+] from 5 to 40 mM.
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Another series of experiments were performed with quinidine, a blocker
of K+ channels. The data are
summarized in Fig. 8. Application of
quinidine reduced
[Mg2+]i
significantly by ~17 ± 3.5% (50 µM quinidine) and 30 ± 3%
(100 µM quinidine) (P < 0.05).

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Fig. 8.
Effect of K+ channel blocker quinidine on free
[Mg2+]i of REC. Curves show results
of single experiments (n = 6), and
each point represents mean of repetitive measurements of
[Mg2+]i
under same conditions. Columns give mean values obtained without
quinidine (control) and with increasing quinidine concentrations in
bath.
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Effect of membrane hyperpolarization on
[Mg2+]i.
To test the effect of membrane hyperpolarization on
[Mg2+]i,
we enhanced the K+ diffusion by
application of 100 µM valinomycin, a
K+ ionophore, in the presence of
an outward K+ gradient
{intracellular
[K+]
([K+]i) > [K+]e}.
With 2 mM Mg2+ in the
extracellular solution, the resulting hyperpolarization of the REC
Em induced a
rapid 15% increase in
[Mg2+]i
(P < 0.01). Cells treated with
valinomycin and equal
[K+] on both membrane
sides
([K+]i = [K+]e = 140 mM) served as controls. Without a
K+ gradient, valinomycin
application resulted in a slow, not significant, elevation of
[Mg2+]i
by only 5% (Fig. 9).

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Fig. 9.
Effect of hyperpolarization of
Em generated by
outward K+ gradients
([K+]i = 140 mM > [K+]e = 5 mM) in presence of 100 µM valinomycin
(left).
Right: increase of
[Mg2+]i
after application of 100 µM valinomycin, but without a
K+ gradient
([K+]i = [K+]e = 140 mM) across cell membrane. Number of single experiments are shown
in parentheses.
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 |
DISCUSSION |
28Mg2+
fluxes.
A net absorption of
28Mg2+
is observed in the absence of transepithelial electrochemical
gradients, since JMgms is much
greater than JMgsm, which may
indicate that the passive and probably paracellular permeability of the rumen epithelium is low for Mg2+.
These data are in agreement with previously published results (21, 23).
In addition, our findings confirm the long-known negative correlation
between PDt and net
Mg2+ flux
(JMgnet) (4, 21-23, 35).
PDt/PDa and
JMgms.
PDt-dependent
Mg2+ absorption has been
previously reported in different parts of the kidney (1, 7, 33) and in
the gastrointestinal tract (19) but was mainly explained as
paracellular movement in these epithelia. In contrast, it has been
shown by Leonhard-Marek and Martens (21) that the majority of
PDt-dependent
JMgms in rumen epithelium is
electrogenic transport through the cell. In our study the reduction of
JMgms after an increase of
PDt is remarkably higher than the
increase in JMgsm, which
implies that predominantly the cellularly mediated
Mg2+ transport is affected.
This is supported by the microelectrode experiments showing that 72%
of a change in PDt is reflected in
a change of PDa (Fig. 2) and by
the close correlation between PDa
and JMgms. In considering that
the chemical gradient for Mg2+
(2-4 mM luminal and 1 mM intracellular) is small (12 mV) compared with PDa (
54 mV under
short-circuit conditions) and accounts for only 18% of the
electrochemical driving force
(
µMg) across the luminal
membrane, PDa is the major passive
driving force for luminal Mg2+
uptake into the epithelial cells.
A depolarization of PDa produced
by an increase of PDt was
accompanied by a decrease of
JMgms. These results are in
good agreement with data from a previous study (21) in which we
demonstrated that high mucosal
[K+] or blockage of an
apical K+ conductance depolarized
PDa and had the same diminishing
effect on JMgms. It was
hypothesized that depolarization of
PDa reduces
Mg2+ uptake into the REC, which
should cause corresponding changes in
[Mg2+]i.
Concentration dependence of
[Mg2+]i.
[Mg2+]i
of REC was determined for the first time in this study. Typically,
[Mg2+]i
is between 0.5 and 1.0 mM (5, 11, 13, 15, 17, 29, 34) and remains
constant, even when the cells are incubated in
Mg2+-rich media. In contrast, as
our results show, REC exhibits another behavior.
[Mg2+]i
rises from 0.59 ± 0.13 mM to 1.26 ± 0.1 mM as the
[Mg2+]e
is stepwise increased from 0.5 mM to 7.5 mM. It must be taken into
consideration that the experiments were performed with cell suspensions, where apical and serosal membranes are no longer separated
by tight junctions, and therefore the whole membrane was exposed to the
different [Mg2+]. In
the physiological situation only the apical membrane is exposed to high
[Mg2+] (up to 8 mM),
whereas the serosal or blood
[Mg2+] is lower and
relatively constant (0.8-1.2 mM). The rise in
[Mg2+]i
is obviously dependent on
[Mg2+]e
and exhibits a saturable component. Leonhard-Marek et al. (20) demonstrated at the tissue level that the process of
Mg2+ transport across the apical
membrane of the rumen epithelium involves a PD- or
K+-sensitive mechanism and a
second, parallel working PD-independent mechanism, which could result
from apical
Mg2+/H+
exchange or from cotransport of
Mg2+ with anions (20, 31). The
latter may become saturated at [Mg2+] in the rumen
liquid above 4 mM (4, 24). Even under basal conditions, these influx
pathways seem to be active and may therefore reflect the
Mg2+-absorbing ability of these cells.
Effect of Em
depolarization and hyperpolarization on
[Mg2+]i.
The results of the patch-clamp studies confirm that high
[K+]e
or application of quinidine, which has been shown to be an effective blocker of K+ channels in rumen
epithelium (2), led to a depolarization of the
Em of isolated
REC. These effects are consistent with the existence of a
K+ conductance in the cell
membrane of REC, as has been proposed from flux studies (2, 21). An
increase of the bath
[K+] or application of
quinidine also decreased
[Mg2+]i
in a dose-dependent manner, which implies an impairment of electrodiffusive Mg2+ uptake
mechanisms by a reduction of the transmembrane electrical gradient.
Quinidine has been demonstrated to inhibit both the Na+/H+
exchanger and the
Na+/Mg2+
exchanger (31), but the resulting effects (acidification of the cytosol
and reduction of Mg2+ efflux)
should increase, not decrease,
[Mg2+]i.
An enhancement of the Mg2+ efflux
by membrane depolarization is not in agreement with the results of flux
studies (23) and microelectrode experiments (21), in which
PDt is equally changed by a
K+ gradient or by a simple voltage
clamp. Identical PDt under both conditions lead to almost identical flux rates of
Mg2+ (23) and to a depolarization
of PDa (21). However,
PDb is hyperpolarized in
voltage-clamp experiments and slightly depolarized by high mucosal
[K+] (21). Similar
Mg2+ flux rates under both
conditions confirm the assumption of PD-dependent Mg2+ uptake across the luminal
membrane and excludes a significant effect of altered
PDb on
JMgms.
[Mg2+]i
increases with Em
hyperpolarization created by K+
diffusion
([K+]i > [K+]e)
in the presence of valinomycin. The voltage effects are consistent with
the hypothesis that electrodiffusive
Mg2+ transport driven by the
transmembrane electrical gradient is the predominant mode for
Mg2+ influx in REC. The kind of
uptake mechanism, that is, a channel or a carrier, cannot be defined
from these studies. Channels with Mg2+ permeability have been
demonstrated in toad rod outer segment (25) and the ciliate protozoa
Paramecium (26). Mg2+
uptake in cardiac myocytes (28), Madin-Darby canine kidney cells (27),
mouse distal convoluted tubule cells (6), and brush-border membrane
vesicles of trout kidneys (10) have also been observed to be influenced
by the transmembrane voltage, and the various authors assume that this
Mg2+ influx is mediated by an
ion channel. As in our studies, the applied methods, that is,
fluorescence spectroscopic measurement of
[Mg2+]i
by the aid of mag-fura 2 or
28Mg2+
uptake measurements, are not the methods of choice to establish unequivocally the presence of a channel. There is no doubt that further
studies are required to characterize the type of the
Mg2+ influx pathway in REC; this
was beyond the scope of the present study.
In conclusion, our results are in agreement with the well-known
antagonism between high ruminal
[K+], resulting in
electrophysiological changes in the rumen epithelium, and
Mg2+ transport. The previously
discussed suggestion of a correlation between
PDa and
Mg2+ transport is supported by the
data of the present study. Furthermore, the determination of
[Mg2+]i
has extended our knowledge of the total driving forces for Mg2+ uptake. The calculation of
µMg shows again that the
PDa is the major driving force for
the Mg2+ uptake. The chemical
gradient appears to be of minor importance under the conditions of the
present experiments. Our improved knowledge about the effects of
changes of the electrophysiological parameters on
Mg2+ transport should lead to a
better understanding of the pathogenesis of hypomagnesemia in
ruminants. Despite this improvement, no information is to date
available regarding the suggested transport protein (channel or
carrier) in the luminal membrane; this is also the case in other
Mg2+-transporting membranes and
should be the target of future research.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Jürgen Vormann for methodical assistance with
the first fluorescence measurements and for helpful discussions regarding measurement of intracellular ion concentrations by
fluorescence probes. We also thank Dr. Theresa Jones for linguistic corrections.
 |
FOOTNOTES |
This study was supported by Deutsche Forschungsgemeinschaft grants 642 (to M. Schweigel) and 699 (to H. Martens).
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 16 February 1999; accepted in final form 28 July 1999.
 |
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