Splice-variant 1 of the ancient domain protein 2 (ACDP2) complements the magnesium-deficient growth phenotype of Salmonella enterica sv. typhimurium strain MM281.

Evidence arguing for the existence of genes encoding for proteins directly involved in the transport of Mg2+ through the cytoplasmic membrane have accumulated over the last few years. Gene ACDP2 (ancient conserved domain protein 2; old name CNNM2, cyclin M2) is one such gene. ACDP2 is a distant homologue of the bacterial gene corC, which is known to be involved in cobalt resistance. We have previously demonstrated that the over-expression of the human Mg2+ carrier SLC41A1 partly complements the Mg2+-dependent growth deficiency of Salmonella strain MM281 (triple disruptant in genes: mgtA, mgtB and corA) cultivated in media containing growth non-permissive [Mg2+]e. We have used the same approach to examine whether over-expressed human ACDP2 has a similar efficacy to complement growth deficiency of the MM281 strain in media containing growth non-permissive [Mg2+]e. Two splicing variants of the ACDP2 gene have been tested. Here, we show that over-expressed isomorph 1 is efficient in restoring growth of the MM281 strain in media containing growth non-permissive [Mg2+]e, whereas isomorph 2 is not. Therefore, we conclude that ACDP2sp.v.1 is a functional Mg2+-transporting entity per se. Our conclusion is supported by the measurable Mg2+ influx seen in MM281 bacteria over-expressing ACDP2sp.v.1 but not in MM281 bacteria over-expressing ACDP2sp.v.2 or in cells transformed with the empty vector.

The vacuolar-type H-ATPase in ovine rumen epithelium is regulated by metabolic signals.

In this study, the effect of metabolic inhibition (MI) by glucose substitution with 2-deoxyglucose (2-DOG) and/or application of antimycin A on ovine rumen epithelial cells (REC) vacuolar-type H(+)-ATPase (vH(+)-ATPase) activity was investigated. Using fluorescent spectroscopy, basal pH(i) of REC was measured to be 7.3 +/- 0.1 in HCO(3) (-)-free, glucose-containing NaCl medium. MI induced a strong pH(i) reduction (-0.44 +/- 0.04 pH units) with a more pronounced effect of 2-DOG compared to antimycin A (-0.30 +/- 0.03 versus -0.21 +/- 0.03 pH units). Treatment with foliomycin, a specific vH(+)-ATPase inhibitor, decreased REC pH(i) by 0.21 +/- 0.05 pH units. After MI induction, this effect was nearly abolished (-0.03 +/- 0.02 pH units). In addition, membrane-associated localization of vH(+)-ATPase B subunit disappeared. Metabolic control of vH(+)-ATPase involving regulation of its assembly state by elements of the glycolytic pathway could provide a means to adapt REC ATP consumption according to energy availability.

Modulation of TRPM6 and Na(+)/Mg(2+) exchange in mammary epithelial cells in response to variations of magnesium availability.

Mammary epithelial cells (HC11) chronically adapted to grow in a low-magnesium (0.05 mM vs. 0.5 mM) or in a high-magnesium (40 mM) medium were used to investigate on the mechanisms of cell magnesium transport under conditions of non-physiological magnesium availability. Magnesium influx was higher in low-magnesium cells compared to control or high-magnesium cells, whereas magnesium efflux was higher in high-magnesium cells compared to control and low-magnesium cells. Magnesium efflux was partially inhibited by imipramine, inhibitor of the Na(+)/Mg(2+) exchange. Using a monoclonal antibody detecting a approximately 70 kDa protein associated with Na(+)/Mg(2+) exchange activity, we found that the expression levels of this protein were proportional to magnesium efflux capacity, that is, high-magnesium cells > control cells > low-magnesium cells. As for magnesium influx, this was abolished by Co(III)hexaammine, inhibitor of magnesium channels. Surprisingly, we found that cells grown in low magnesium upregulated mRNA for the magnesium channel TRPM6, but not for other channels like TRPM7 or MagT1. TRPM6 mRNA was also rapidly upregulated or downregulated in HC11 cells deprived of magnesium or in low-magnesium cells re-added with magnesium, respectively. TRPM6 protein levels, as assessed by Western blot and immunofluorescence, underwent similar changes under comparable conditions. We propose that mammary epithelial cells adapt to decreased magnesium availability by upregulating magnesium influx via TRPM6, and counteract increased magnesium availability by increasing magnesium efflux primarily via Na(+)/Mg(2+) exchange. These results show, for the first time, that TRPM6 contributes to regulating magnesium influx in mammary epithelial cells, similar to what is known for intestine and kidney.

Rumen epithelial cells adapt magnesium transport to high and low extracellular magnesium conditions.

A protein of approximately 70-kDa was identified as a candidate Na+/Mg2+ exchanger in rumen epithelial cells (REC). Melastatin-related Transient Receptor Potential 7 (TRPM7) and Magnesium Transporter 1 (MagT1) transcripts and, from them, encoded proteins were also detected. The regulation of these Mg transport pathways by extracellular [Mg] changes was the main focus of this study. Therefore, a 24-h pre-incubation of ovine REC in control (1.2 mM), low (0.12 mM)-Mg, and high (5 mM)-Mg medium was performed. Na+/Mg2+ exchangers, TRPM7 and MagT1 abundance and activity were investigated by Western blot analysis, flow cytometry, immunocytochemistry and fluorescence spectroscopic measurements of [Mg2+]i changes. Inhibitors were employed to differentiate Na+/Mg2+ exchanger-mediated (imipramine) and channel-mediated (cobalt(III)hexaammine, nitrendipine) Mg transport. Basal [Mg2+]i (0.40 +/- 0.02 mM) was not influenced by pre-incubation in low- or high-Mg medium. However, compared with control REC (4.1 +/- 0.7 microM/min), such cells showed reduced (2.8 +/- 0.6 microM/min) or elevated (6.4 +/- 0.9 microM/min) Mg extrusion rates that correlated with a decreased (25%) and increased (38%) expression of the putative Na+/Mg2+ exchanger protein, respectively. Low- and high-Mg pre-incubated REC were both characterized by an increased (30-40%) influx capacity. In low-Mg REC, the latter resulted mainly from a strong activation of the TRPM7-related transport component. The data thus clearly demonstrate the intrinsic regulation of REC transmembrane Mg transport.

Expression and functional activity of the Na/Mg exchanger, TRPM7 and MagT1 are changed to regulate Mg homeostasis and transport in rumen epithelial cells.

The present study was performed to show the molecular identity of functionally characterized Mg transport pathways in rumen epithelial cells (REC) and to investigate the effects of extracellular [Mg] changes on their expression and activity. By using RT- PCR, Western blot, flow cytometry and immunocytochemistry, TRPM7, MagT1 and a Na+/Mg2+ exchanger were found in REC. Compared with control conditions ([Mg]e = 1.2 mM), a decreased or increased MagT1 (-30%; 20%) and Na+/Mg2+ exchanger (-25%; 40%) protein abundance was observed after a 24-h incubation of REC in low (0.12 mM)- and high (5 mM)-Mg medium, respectively. To determine the Mg transport capacity, [Mg2+]i changes were measured by use of mag-fura 2. The basal [Mg2]i (0.43 +/- 0.03 mM) was not influenced by the [Mg] of the pre-incubation medium. However, compared to control cells, REC incubated in low- or high-Mg medium showed significantly reduced (59%) and elevated (57%) Mg extrusion rates, respectively. In addition, they were characterized by an increased influx capacity (30-40%). In low-Mg cells the latter results mainly from a strong TRPM7 related transport component whereas in high-Mg cells the imipramine-sensitive, the Na+/Mg2+ exchanger-mediated transport component causes this effect. In conclusion, TRPM7, MagT1 and a Na+/Mg2+ exchanger are shown to be the main Mg transport proteins in REC and their expression and functional activity is influenced by the cellular Mg status. The latter responses permit adaptation of epithelial Mg absorption and enable REC to maintain a physiological [Mg2+]i which is a prerequisite for various cell functions.

SLC41A1 is a novel mammalian Mg2+ carrier.

The molecular biology of mammalian magnesium transporters and their interrelations in cellular magnesium homeostasis are largely unknown. Recently, the mouse SLC41A1 protein was suggested to be a candidate magnesium transporter with channel-like properties when overexpressed in Xenopus laevis oocytes. Here, we demonstrate that human SLC41A1 overexpressed in HEK293 cells forms protein complexes and locates to the plasma membrane without, however, giving rise to any detectable magnesium currents during whole cell patch clamp experiments. Nevertheless, in a strain of Salmonella enterica exhibiting disruption of all three distinct magnesium transport systems (CorA, MgtA, and MgtB), overexpression of human SLC41A1 functionally substitutes these transporters and restores the growth of the mutant bacteria at magnesium concentrations otherwise non-permissive for growth. Thus, we have identified human SLC41A1 as being a bona fide magnesium transporter. Most importantly, overexpressed SLC41A1 provide HEK293 cells with an increased magnesium efflux capacity. With outwardly directed Mg(2+) gradients, a SLC41A1-dependent reduction of the free intracellular magnesium concentration accompanied by a significant net decrease of the total cellular magnesium concentration could be observed in such cells. SLC41A1 activity is temperature-sensitive but not sensitive to the only known magnesium channel blocker, cobalt(III) hexaammine. Taken together, these data functionally identify SLC41A1 as a mammalian carrier mediating magnesium efflux.

Molecular identification, immunolocalization, and functional activity of a vacuolar-type H(+)-ATPase in bovine rumen epithelium.

In this study, we have studied the expression, localization, and functionality of vacuolar-type H(+)-ATPase (vH(+)-ATPase) and Na(+)/K(+)-ATPase in the bovine rumen epithelium. Compared with the intracellular pH (pH(i)) of control rumen epithelial cells (REC; 7.06 +/- 0.07), application of inhibitors selective for vH(+)-ATPase (foliomycin) and Na(+)/K(+)-ATPase (ouabain) reduced pH(i) by 0.10 +/- 0.03 and 0.18 +/- 0.03 pH-units, respectively, thereby verifying the existence of both functional proteins. Results from qRT-PCR and immunoblotting clearly confirm the expression of vH(+)-ATPase B subunit in REC. However, the amount of Na(+)/K(+)-ATPase mRNA and protein is tenfold and 11-fold of those of vH(+)-ATPase subunit B, respectively, reflecting a lower overall abundance of the latter in REC. Na(+)/K(+)-ATPase immunostaining has revealed the protein in the plasma membrane of all REC from the stratum basale to stratum granulosum, with the highest abundance in basal cells. In contrast, the vH(+)-ATPase B subunit has been detected in groups of cells only, mainly localized in the stratum spinosum and stratum granulosum of the epithelium. Furthermore, vH(+)-ATPase has been detected in the cell membrane and in intracellular pools. Thus, functional vacuolar-type H(+) pumps are expressed in REC and probably play a role in the adaptation of epithelial transport processes.

Methane production in cattle calculated by the nutrient composition of the diet.

In this study data originating from complete metabolic trials with cattle of both sexes, fed 337 rations at feed intake levels between one to three times maintenance energy requirement were used to regress the total CH4 emission to the level of DM intake and to the nutrient composition, respectively. A major component of the measured CH4 emission cannot be explained by DM intake but is rather due to differences in dietary nutrient composition. The amount of digestible nutrients especially of the carbohydrate fraction (starch, sugar, N-free residuals) are reliable to estimate CH4 release with high precision (r2 = 0.885). Its production rate increased to 1.9-fold higher levels (range 1.8-2.1) per g of N-free residuals compared to that induced per g of protein, starch or sugar, respectively. Furthermore, diets rich in fat reduced CH4 formation in the rumen. The regression equations fit a wide range of diets and diet compositions, and more importantly, they are applicable to various types of production systems.

A vH+-ATPase is present in cultured sheep ruminal epithelial cells.

In this study, the existence and functional activity of a vacuolar-type H(+)-ATPase (vH(+)-ATPase) was explored in primary cultures of sheep ruminal epithelial cells (REC). The mRNA transcripts of the E and B subunits of vH(+)-ATPase were detectable in RNA from REC samples by RT-PCR. Immunoblotting of REC protein extractions with antibodies directed against the B subunit of yeast vH(+)-ATPase revealed a protein band of the expected size (60 kDa). Using the fluorescent indicator BCECF and selective inhibitors (foliomycin, HOE 694, S3226), the contribution of vH(+)-ATPase and Na(+)/H(+) exchanger (NHE) subtype 1 and 3 activity to the regulation of intracellular pH (pH(i)) was determined in nominally HCO(3)(-)-free, HEPES-buffered NaCl medium containing 20 mM of the short-chain fatty acid butyrate as well as after reduction of the extracellular Cl(-) concentration ([Cl(-)](e)) from 136 to 36 mM. The initial pH(i) of REC was 7.4 +/- 0.1 in nominally HCO(3)(-)-free, HEPES-buffered NaCl medium and 7.0 +/- 0.1 after acid loading with butyrate. Selective inhibition of the vH(+)-ATPase with foliomycin decreased pH(i) by 0.19 +/- 0.03 pH units. On the basis of the observed decreases in pH(i) resulting from inhibition of vH(+)-ATPase as well as of subtypes 1 and 3 of NHE, vH(+)-ATPase activity appears to account for approximately 30% of H(+) extrusion, whereas the activities of NHE subtypes 3 and 1 account for 20 and 50% of H(+) extrusion, respectively. Lowering of [Cl(-)](e) induced a pH(i) decrease (-0.51 +/- 0.03 pH units) and impaired pH(i) recovery from butyrate-induced acid load. Moreover, reduction of [Cl(-)](e) abolished the inhibitory effect of foliomycin and markedly reduced the HOE 694- and S3226-sensitive components of pH(i), indicating a role of Cl(-) in the function of these H(+) extrusion mechanisms. We conclude that a vH(+)-ATPase is expressed in ovine REC and plays a considerable role in the pH(i) regulation of these cells.

Characterization of the Na+-dependent Mg2+ transport in sheep ruminal epithelial cells.

This study examines the routes by which Mg2+ leaves cultured ovine ruminal epithelial cells (REC). Mg2+-loaded (6 mM) REC were incubated in completely Mg2+-free solutions with varying Na+ concentrations, and the Mg2+ extrusion rate was calculated from the increase of the Mg2+ concentration in the incubation medium determined with the aid of the fluorescent probe mag-fura 2 (Na+ salt). In other experiments, REC were also studied for the intracellular free Mg2+ concentration ([Mg2+]i; using mag-fura 2), the intracellular Na+ concentration (using Na+-binding benzofuran isophthalate), the intracellular cAMP concentration ([cAMP]i; using an enzyme-linked immunoassay), and Na+/Mg2+ exchanger existence [using a monoclonal antibody (mAb) raised against the porcine red blood cell Na+/Mg2+ exchanger]. Mg2+-loaded REC show a Mg2+ efflux that was strictly dependent on extracellular Na+. The Mg2+ extrusion rate increased from 0.018+/-0.009 in a Na+-free medium to 0.73+/-0.3 mM.l cells-1.min-1 in a 145 mM Na+ medium and relates to extracellular Na+ concentration ([Na+]e) according to a typical saturation kinetic (Km value for [Na+]e=24 mM; maximal velocity=11 mM.l cells-1.min-1). Mg2+ efflux was reduced by imipramine (48%) and increased after application of dibutyryl-cAMP (55%) or PGE2 (17%). These effects are completely abolished in Na+-free media. Furthermore, an elevation of [cAMP]i led to an [Mg2+]i decrease that amounted to 375+/-105 microM. The anti-Na+/Mg2+ exchanger mAb inhibits Mg2+ extrusion; moreover, it detects a specific 70-kDa immunoreactive band in protein lysates of ovine REC. The data clearly demonstrate that a Na+/Mg2+ exchanger is existent in the cell membrane of REC. The transport protein is the main pathway (97%) for Mg2+ extrusion and can be assumed to play a considerable role in the process of Mg2+ absorption as well as the maintenance of the cellular Mg2+ homeodynamics.

Luminal hyperosmolarity decreases Na transport and impairs barrier function of sheep rumen epithelium.

The effects of luminal hyperosmolarity on Na and Cl transport were studied in rumen epithelium of sheep. An increase of luminal osmotic pressure with mannitol (350 and 450 mosm/l) caused a significant increase of tissue conductance, G (T), which is linearly correlated with flux rates of (51)Cr-EDTA and indicates an increase of passive permeability. Studies with microelectrodes revealed, that an increase of the osmotic pressure caused a significant increase of the conductance of the shunt pathway from 1.23 +/- 0.10 (control) to 1.92 +/- 0.14 mS cm(-2) (450 mosm/l) without a change of fractional resistance. Hyperosmolarity significantly increased J (sm) and reduced J (net) Na. The effect of hyperosmolarity on J (ms) Na is explained by two independent and opposed effects: increase of passive permeability and inhibition of the Na(+)/H(+) exchanger. Hypertonic buffer solution induced a decrease of the intracellular pH (pH(i)) of isolated ruminal cells, which is consistent with an inhibition of Na(+)/H(+) exchange, probably isoform NHE-3, because NHE-3-mRNA was detectable in rumen epithelium. These data are in contrast to previous reports and reveal a disturbed Na transport and an impaired barrier function of the rumen epithelium, which predisposes translocation of rumen endotoxins and penetration of bacteria.

Fluorescence measurements of free [Mg2+] by use of mag-fura 2 in Salmonella enterica.

The Mg2+ fluorescent dye mag-fura 2, entrapped in cells or organelles, has frequently been used for dual excitation ratio-metric determinations of free ionic Mg2+ concentrations in eukaryotic, mostly mammalian cells. Here we report its successful application to measure free Mg2+ concentrations ([Mg2+]i) in Salmonella enterica cells. When kept in nominally Mg2+ free buffer (resting conditions), the [Mg2+]i of wild-type cells has been determined to be 0.9 mM. An increase in the external Mg2+ concentration ([Mg2+]e) resulted in a rapid increase of [Mg2+]i, saturating within a few seconds at about 1.5 mM with [Mg2+]e of 20 mM. In contrast, cells lacking the Mg2+ transport proteins CorA, MgtA, MgtB failed to show this rapid increase. Instead, their [Mg2+]i increased steadily over extended periods of time and saturated at concentrations below those of wild-type cells. Mg2+ uptake rates increased more than 15-fold when corA was overexpressed in these mutant cells. Uptake of Mg2+ into corA expressing cells was strongly stimulated by nigericin, which increased the membrane potential DeltaPsi at the expense of DeltapH, and drastically reduced by valinomycin, which decreased the membrane potential DeltaPsi. These results reveal mag-fura 2 as a useful indicator to measure steady-state [Mg2+]i values in resting bacterial cells and to determine Mg2+ uptake rates. They confirm the role of CorA as the major Mg2+ transport protein and reveal the membrane potential as driving force for Mg2+ uptake into S. enterica cells.

Mrs2p is an essential component of the major electrophoretic Mg2+ influx system in mitochondria.

Steady-state concentrations of mitochondrial Mg(2+) previously have been shown to vary with the expression of Mrs2p, a component of the inner mitochondrial membrane with two transmembrane domains. While its structural and functional similarity to the bacterial Mg(2+) transport protein CorA suggested a role for Mrs2p in Mg(2+) influx into the organelle, other functions in cation homeostasis could not be excluded. Making use of the fluorescent dye mag-fura 2 to measure free Mg(2+) concentrations continuously, we describe here a high capacity, rapid Mg(2+) influx system in isolated yeast mitochondria, driven by the mitochondrial membrane potential Deltapsi and inhibited by cobalt(III)hexaammine. Overexpression of Mrs2p increases influx rates 5-fold, while the deletion of the MRS2 gene abolishes this high capacity Mg(2+) influx. Mg(2+) efflux from isolated mitochondria, observed with low Deltapsi only, also requires the presence of Mrs2p. Cross-linking experiments revealed the presence of Mrs2p-containing complexes in the mitochondrial membrane, probably constituting Mrs2p homo- oligomers. Taken together, these findings characterize Mrs2p as the first molecularly identified metal ion channel protein in the inner mitochondrial membrane.

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

The K+-insensitive component of Mg2+ influx in primary culture of ruminal epithelial cells (REC) was examined by means of fluorescence techniques. The effects of extracellular anions, ruminal fermentation products, and transport inhibitors on the intracellular free Mg2+ concentration ([Mg2+]i), Mg2+ uptake, and intracellular pH were determined. Under control conditions (HEPES-buffered high-NaCl medium), the [Mg2+]i of REC increased from 0.56 +/- 0.14 to 0.76 +/- 0.06 mM, corresponding to a Mg2+ uptake rate of 15 microM/min. Exposure to butyrate did not affect Mg2+ uptake, but it was stimulated (by 84 +/- 19%) in the presence of CO2/HCO(-)3. In contrast, Mg2+ uptake was strongly diminished if REC were suspended in HCO(-)3-buffered high-KCl medium (22.3 +/- 4 microM/min) rather than in HEPES-buffered KCl medium (37.5 +/- 6 microM/min). After switching from high- to low-Cl- solution, [Mg2+]i was reduced from 0.64 +/- 0.09 to 0.32 +/- 0.16 mM and the CO2/HCO(-)3-stimulated Mg2+ uptake was completely inhibited. Bumetanide and furosemide blocked the rate of Mg2+ uptake by 64 and 40%, respectively. Specific blockers of vacuolar H+-ATPase reduced the [Mg2+]i (36%) and Mg2+ influx (38%) into REC. We interpret this data to mean that the K+-insensitive Mg2+ influx into REC is mediated by a cotransport of Mg2+ and Cl- and is energized by an H+-ATPase. The stimulation of Mg2+ transport by ruminal fermentation products may result from a modulation of the H+-ATPase activity.