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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega . 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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.

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.

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.

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) (<UNL>1</UNL>) and intracellular pH (pHi) (<UNL>2</UNL>) 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.

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+.

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.

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.

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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 (Delta 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 Delta 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastroint Liver Physiol 277(5):G976-G982
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