Concentrations Of Extracellular Mg2 Research Articles

The Effects of Sirolimus and Magnesium on Primary Human Coronary Endothelial Cells: An In Vitro Study.

Drug eluting magnesium (Mg) bioresorbable scaffolds represent a novel paradigm in percutaneous coronary intervention because Mg-based alloys are biocompatible, have adequate mechanical properties and can be resorbed without adverse events. Importantly, Mg is fundamental in many biological processes, mitigates the inflammatory response and is beneficial for the endothelium. Sirolimus is widely used as an antiproliferative agent in drug eluting stents to inhibit the proliferation of smooth muscle cells, thus reducing the occurrence of stent restenosis. Little is known about the potential interplay between sirolimus and Mg in cultured human coronary artery endothelial cells (hCAEC). Therefore, the cells were treated with sirolimus in the presence of different concentrations of extracellular Mg. Cell viability, migration, barrier function, adhesivity and nitric oxide synthesis were assessed. Sirolimus impairs the viability of subconfluent, but not of confluent cells independently from the concentration of Mg in the culture medium. In confluent cells, sirolimus inhibits migration, while it cooperates with Mg in exerting an anti-inflammatory action that might have a role in preventing restenosis and thrombosis.

Staphylococcus-Induced Bacteriospermia In Vitro: Consequences on the Bovine Spermatozoa Quality, Extracellular Calcium and Magnesium Content.

Bacterial contamination of bovine ejaculates intended for artificial insemination may be reflected in a significant economic loss due to unsuccessful fertilization as well as health issues of the recipients. The Staphylococcus genus represents a large part of bacteriocenosis of bovine ejaculates. Therefore, this study aims to get a closer look on the effects of Staphylococcus-induced bacteriospermia under in vitro conditions on bovine sperm quality. Prior to inducing bacteriospermia, spermatozoa were separated from each ejaculate using Percoll® Plus gradient medium in order to limit the effects only to the selected bacterial species. Seven Staphylococcus species previously isolated from bovine semen were used for our experiments at a turbidity of 0.5 McFarland (equivalent to 1.5 × 108 colony-forming units per mL). The contaminated semen samples were incubated at 37 °C and at times of 0, 2, and 4 h, motility, mitochondrial membrane potential, reactive oxygen species (ROS) generation, sperm DNA fragmentation, and magnesium (Mg) and calcium (Ca) extracellular concentration were analyzed and compared with the control group (uncontaminated). The results showed no significant changes at the initial measurement. However, significant adverse effects were observed after 2 h and 4 h of incubation. Most notably, the presence of S. aureus, S. warneri, S. kloosii, and S. cohnii caused a significantly increased ROS production, leading to sperm DNA fragmentation, changes in the mitochondrial membrane potential, and a decreased sperm motility. Furthermore, the presence of Staphylococcus species led to lower extracellular concentrations of Mg and Ca. In conclusion, the overgrowth of Staphylococcus bacteria in bovine semen may contribute to oxidative stress resulting in sperm DNA fragmentation, altered mitochondrial membrane potential, and diminished sperm motility.

TRPM7 and MagT1 regulate the proliferation of osteoblast-like SaOS-2 cells through different mechanisms.

A correct magnesium (Mg2+) intake is essential for bone health. In particular, Mg2+ deficiency inhibits the proliferation of osteoblast-like SaOS-2 cells by increasing nitric oxide (NO) production through the upregulation of inducible NO synthase. At the moment, little is known about the expression and the role of TRPM7, a channel/enzyme involved in Mg2+ uptake, and MagT1, a Mg2+ selective transporter, in SaOS-2 cells. Here, we demonstrate that TRPM7 is not modulated by different extracellular concentrations of Mg2+ and its silencing exacerbates growth inhibition exerted by low Mg2+ through the activation of inducible NO synthase and consequent accumulation of NO. Moreover, MagT1 is upregulated in SaOS-2 cultured in high Mg2+ and its silencing inhibits the growth of SaOS-2 cultured in media containing physiological or high Mg2+, without any modulation of NO production. We propose that TRPM7 and MagT1 are both involved in regulating SaOS-2 proliferation through different mechanisms.

Magnesium Is a Key Regulator of the Balance between Osteoclast and Osteoblast Differentiation in the Presence of Vitamin D3

Magnesium (Mg) is crucial for bone health. Low concentrations of Mg inhibit the activity of osteoblasts while promoting that of osteoclasts, with the final result of inducing osteopenia. Conversely, little is known about the effects of high concentrations of extracellular Mg on osteoclasts and osteoblasts. Since the differentiation and activation of these cells is coordinated by vitamin D3 (VD3), we investigated the effects of high extracellular Mg, as well as its impact on VD3 activity, in these cells. U937 cells were induced to osteoclastic differentiation by VD3 in the presence of supra-physiological concentrations (>1 mM) of extracellular Mg. The effect of high Mg concentrations was also studied in human bone-marrow-derived mesenchymal stem cells (bMSCs) induced to differentiate into osteoblasts by VD3. We demonstrate that high extra-cellular Mg levels potentiate VD3-induced osteoclastic differentiation, while decreasing osteoblastogenesis. We hypothesize that Mg might reprogram VD3 activity on bone remodeling, causing an unbalanced activation of osteoclasts and osteoblasts.

Extracellular magnesium and calcium blockers modulate macrophage activity

Magnesium (Mg) possesses anti-inflammatory properties, partly because it antagonizes calcium (Ca) and inhibits L-type Ca channels. Our aim was to determine the effects of different concentrations of extracellular Mg, with or without Ca-channel blockers, in macrophages. A macrophage-like cell line J774.E was cultured in different concentrations of extracellular Mg and exposed to i) the phorbol ester PMA to induce the production of reactive oxygen species ii) lipopolysaccharide to induce the production of pro-inflammatory cytokines, or iii) ovalbumin to study endocytosis. The Ca antagonists verapamil and/or TMB-8 were used to interfere with Ca homeostasis. Different concentrations of extracellular Mg did not impact on endocytosis, while Ca antagonists markedly decreased it. Low extracellular Mg exacerbated, whereas Ca antagonists inhibited, PMA-induced production of free radicals. Ca blockers prevented lipopolysaccharide-induced transcription and release of IL-1β, IL-6 and TNF-α, while extracellular Mg had only a marginal effect. Ca channel inhibitors markedly reduced the activity of J774.E cells, thus underscoring the critical role of Ca in the non-specific immune response, a role which was, in some instances, also modulated by extracellular Mg.

Extracellular magnesium and calcium reduce myotonia in isolated ClC‐1 chloride channel‐inhibited human muscle

Experimental myotonia induced in rat muscle by ClC-1 chloride channel-inhibited has been shown to be related inversely to extracellular concentrations of Mg(2+) and Ca(2+) ([Mg(2+) ]o and [Ca(2+) ]o) within physiological ranges. Because this implicates a role for [Mg(2+)]o and [Ca(2+)]o in the variability of symptoms among myotonia congenita patients, we searched for similar effects of [Mg(2+)]o and [Ca(2+)]o on myotonia in human muscle. Bundles of muscle fibers were isolated from abdominal rectus in patients undergoing abdominal surgery. Myotonia was induced by ClC-1 inhibition using 9-anthracene carboxylic acid (9-AC) and was assessed from integrals of force induced by 5-Hz stimulation for 2 seconds. Myotonia disappeared gradually when [Mg(2+)]o or [Ca(2+)]o were elevated throughout their physiological ranges. These effects of [Mg(2+)]o and [Ca(2+)]o were additive and interchangeable. These findings suggest that variations in symptoms in myotonia congenita patients may arise from physiological variations in serum Mg(2+) and Ca(2+).

Nitric oxide mediates low magnesium inhibition of osteoblast-like cell proliferation

An adequate intake of magnesium (Mg) is important for bone cell activity and contributes to the prevention of osteoporosis. Because (a) Mg is mitogenic for osteoblasts and (b) reduction of osteoblast proliferation is detected in osteoporosis, we investigated the influence of different concentrations of extracellular Mg on osteoblast-like SaOS-2 cell behavior. We found that low Mg inhibited SaOS-2 cell proliferation by increasing the release of nitric oxide through the up-regulation of inducible nitric oxide synthase (iNOS). Indeed, both pharmacological inhibition with the iNOS inhibitor l-N6-(iminoethyl)-lysine-HCl and genetic silencing of iNOS by small interfering RNA restored the normal proliferation rate of the cells.Because a moderate induction of nitric oxide is sufficient to potentiate bone resorption and a relative deficiency in osteoblast proliferation can result in their inadequate activity, we conclude that maintaining Mg homeostasis is relevant to ensure osteoblast function and, therefore, to prevent osteoporosis.

Rotenone reduces Mg 2+-dependent block of NMDA currents in substantia nigra dopamine neurons

Rotenone is a pesticide that has been successfully used to produce a rodent model of Parkinson's disease. We reported previously that rotenone potently augmented N-methyl- d-aspartate (NMDA)-evoked currents in rat dopamine neurons via a tyrosine kinase-dependent mechanism. In this study, we investigated the effect of rotenone on the current–voltage relationship of NMDA-induced currents in substantia nigra zona compacta neurons recorded with whole-cell patch pipettes in slices of rat brain. In a physiologic concentration of extracellular Mg 2+ (1.2 mM), a 30 min perfusion with rotenone (100 nM) produced a marked increase in current evoked by bath application of NMDA (30 μM), especially when measured at relatively hyperpolarized currents. In the presence of rotenone, NMDA currents lost the characteristic region of negative-slope conductance that is normally produced by voltage-dependent block by Mg 2+. The voltage-dependent effect of rotenone on NMDA currents was mimicked by a low extracellular concentration of Mg 2+ (0.2 mM) and was antagonized by a high level of Mg 2+ (6.0 mM). Moreover, we report that the tyrosine kinase inhibitor genistein blocked the ability of rotenone to augment NMDA receptor currents. These results suggest that rotenone potentiates NMDA currents by a tyrosine kinase-dependent process that attenuates voltage-dependent Mg 2+ block of NMDA-gated channels. These results support the hypothesis that an excitotoxic mechanism might participate in rotenone-induced toxicity of midbrain dopamine neurons.

Divalent cations modulate the integrin‐mediated malignant phenotype in pancreatic cancer cells

We have previously demonstrated that pathophysiological shifts in the concentrations of extracellular Mg(2+) and Ca(2+) activate the alpha(2)beta(1) integrin-mediated malignant phenotype on type I collagen in pancreatic cancer cells, as evidenced by increased adhesion, migration and proliferation. In the present study, we examined the integrin and divalent cation specificity of pancreatic cancer cell interactions with other physiologically relevant extracellular matrix proteins, including fibronectin, type IV collagen, laminin and vitronectin. Our results indicate that, like alpha(2)beta(1) integrin-mediated interactions with type I collagen, beta(1) integrin-mediated adhesion to fibronectin, type IV collagen and laminin are promoted by Mg(2+) but not by Ca(2+). On vitronectin, cells attach via alpha(v)beta(5) and beta(1) integrins, and in the presence of either divalent cation. We also demonstrate that, like type I collagen, pancreatic cancer cell migration and proliferation on fibronectin, laminin and type IV collagen is maximal when Mg(2+) is present at concentrations that promote optimal adhesion and Ca(2+) is present at concentrations less than Mg(2+). On vitronectin, Panc-1 cell migration is maximal with decreased Mg(2+) and increased Ca(2+), but the reverse is true for BxPC-3 cells. Both cell lines exhibited maximal proliferation with increased Mg(2+) and decreased Ca(2+), however. Together with evidence indicating that the in vivo local tumor microenvironment contains increased Mg(2+) and decreased Ca(2+), our studies demonstrate that such divalent cation shifts could activate the integrin-mediated malignant phenotype in pancreatic cancer.

Activation of the α2β1 integrin-mediated malignant phenotype on type I collagen in pancreatic cancer cells by shifts in the concentrations of extracellular Mg2+ and Ca2+

The authors have previously demonstrated that alpha(2)beta(1) integrin-mediated pancreatic cancer cell adhesion to Type I collagen is Mg(2+)-dependent, inhibited by Ca(2+), and that this integrin, purified from cell lysates using Type I-collagen-sepharose in Mg(2+), can be eluted with Ca(2+). In the present study, the authors examined the divalent cation-dependency of alpha(2)beta(1) integrin-mediated pancreatic cancer cell adhesion, migration and proliferation on Type I collagen, an extracellular matrix protein shown to be highly up-regulated, and to promote the malignant phenotype in vitro and in vivo. The results indicate that cells attach to Type I collagen maximally when Mg(2+) is greater than 1 mM, and that addition of increasing concentrations of Ca(2+) reduces this adhesion. These effects are reversible, in that previous cell attachment in Mg(2+) can be reversed by adding Ca(2+), and vice versa. They also demonstrate that pancreatic cancer cells migrate and proliferate on Type I collagen in Mg(2+) alone, but maximally when Mg(2+) is present at concentrations that promote maximal cell adhesion and Ca(2+) is present at concentrations less than Mg(2+). Cell adhesion and proliferation assays, as well as affinity chromatography on Type I collagen using anti-integrin function-blocking monoclonal antibodies indicate that the effects of these divalent cation shifts are mediated specifically by the alpha(2)beta(1) integrin. As pancreatic juice contains over 1,200-fold more Mg(2+) than Ca(2+) and solid tumors are characterized by increased magnesium load, these data indicate that such pathophysiological divalent cation shifts could be involved in the activation of the alpha(2)beta(1) integrin-mediated malignant phenotype on Type I collagen in the pancreatic cancer.

Calcium-dependent subthreshold oscillations determine bursting activity induced by N-methyl-D-aspartate in rat subthalamic neurons in vitro.

We used whole-cell patch recordings in current clamp to investigate the ionic dependence of burst firing induced by N-methyl-d-aspartate (NMDA) in neurons of the subthalamic nucleus (STN) in slices of rat brain. NMDA (20 microm) converted single-spike firing to burst firing in 87% of STN neurons tested. NMDA-induced bursting was blocked by AP5 (50 microm), and was not mimicked by the non-NMDA receptor agonist AMPA (0.6 microm). Tetrodotoxin (1 microm) converted bursts to oscillations of membrane potential, which were most robust when oscillations ranged between -50 and -70 mV. The NMDA bursts were blocked by an elevated extracellular concentration of Mg(2+), but superfusate containing no added Mg(2+) either reduced or increased burst firing, depending upon the amount of intracellular current injection. Block of K(+) conductances by apamin and tetraethylammonium prolonged burst duration, but iberiotoxin had no effect. NMDA-induced burst firing and membrane oscillations were completely blocked by superfusate containing no added Ca(2+), and they were significantly reduced when patch pipettes contained BAPTA. Selective antagonists for T-type (mibefradil, 10 microm), L-type (nifedipine, 3 microm), and N-type (omega-conotoxin GVIA, 1 micro m) Ca(2+) channels had no effect on NMDA burst firing. Superfusate containing a low concentration of Na(+) (20 mm) completely abolished NMDA-induced burst firing. Flufenamic acid (10 microm), which blocks current mediated by Ca(2+)-activated nonselective cation channels (I(CAN)), reversibly abolished NMDA-depended bursting. These results are consistent with the hypothesis that NMDA-induced burst firing in STN neurons requires activation of either an I(CAN) or a Na(+)-Ca(2+) exchanger.

Glycosylation influences voltage-dependent gating of cardiac and skeletal muscle sodium channels.

The role of glycosylation on voltage-dependent channel gating for the cloned human cardiac sodium channel (hH1a) and the adult rat skeletal muscle isoform (microl) was investigated in HEK293 cells transiently transfected with either hH1a or microl cDNA. The contribution of sugar residues to channel gating was examined in transfected cells pretreated with various glycosidase and enzyme inhibitors to deglycosylate channel proteins. Pretreating transfected cells with enzyme inhibitors castanospermine and swainsonine, or exo-glycosidase neuroaminidase caused 7 to 9 mV depolarizing shifts of V(1/2) for steady-state activation of hH1a, while deglycosylation with corresponding drugs elicited about the same amount of depolarizing shifts (8 to 9 mV) of V(1/2) for steady-state activation of microl. Elevated concentrations of extracellular Mg(2+) significantly masked the castanospermine-elicited depolarizing shifts of V(1/2) for steady-state activation in both transfected hH1a and microl. For steady-state activation, deglycosylation induced depolarizing shifts of V(1/2) for hH1a (10.6 to 12 mV), but hyperpolarizing shifts for microl (3.6 to 4.4 mV). Pretreatment with neuraminidase had no significant effects on single-channel conductance, the mean open time, and the open probability. These data suggest that glycosylation differentially regulates Na channel function in heart and skeletal muscle myocytes.

Individual Subunits Contribute Independently to Slow Gating of Bovine EAG Potassium Channels

The bovine ether à go-go gene encodes a delayed rectifier potassium channel. In contrast to other delayed rectifiers, its activation kinetics is largely determined by the holding potential and the concentration of extracellular Mg2+, giving rise to slowly activating currents with a characteristic sigmoidal rising phase. Replacement of a single amino acid in the extracellular linker between transmembrane segments S3 and S4 (L322H) strongly reduced the prepulse dependence and accelerated activation by 1 order of magnitude. In addition, compared with the wild type, the half-activation voltage of this mutant was shifted by more than 30 mV to more negative potentials. We used dimeric and tetrameric constructs of the bovine eag1 gene to analyze channels with defined stoichiometry of mutated and wild-type subunits within the tetrameric channel complexes. With increasing numbers of mutated subunits, the channel activation was progressively accelerated, and the sigmoidicity of the current traces was reduced. Based on a quantitative analysis, we show that the slow gating, typical for EAG channels, is mediated by independent conformational transitions of individual subunits, which gain their voltage dependence from the S4 segment. At a given voltage, external Mg2+ increases the probability of a channel subunit to be in the slowly activating conformation, whereas mutation L322H strongly reduces this probability.

In vivo microtubule dynamics during experimentally induced conversions between tubulin assembly states in Allogromia laticollaris.

A distinctive property of foraminiferan tubulin is that, in addition to microtubules (MTs), it exists in an alternate assembly state, helical filaments. Here, we have examined in vivo MT dynamics during experimentally induced conversions between these two assembly states in the reticulopods of the marine foraminiferan Allogromia laticollaris. Exposure to high extracellular concentrations of Mg2+ (165 mM) resulted in a complete conversion of MTs into helical filaments. However, Mg2+ treatment also induced a retrograde movement of organelles and cytoplasm, and it was necessary to inhibit this response in order to assess the effects of assembly state changes on individual MTs. This was accomplished by simultaneous treatment with high extracellular Mg2+ and 2,4-dinitrophenol (DNP). The resulting loss in MTs was detected by video enhanced DIC (VEC-DIC) microscopy as either an endwise MT shortening (at an average rate of 474 microns/min) or transformation into one or more irregularly shaped fibrils, which we termed residual fibrils. Correlative immunofluorescence and video microscopy showed residual fibrils to be composed of helical filaments. Removal of extracellular Mg2+/DNP initiated a reversal in assembly state, from helical filaments into MTs, which was completed within 5 min. VEC-DIC microscopy showed that MTs reformed by an endwise lengthening at an average rate of 216 microns/min. These results suggest that conversion between alternate tubulin assembly states provides a more rapid means to build and dismantle MTs than conventional subunit-driven pathways.

NMR studies of the effect of Mg 2 on post-ischemic recovery of ATP and intracellular sodium in perfused kidney

Ischemia is often implicated as a cause of acute renal failure. We have investigated the effect of various concentrations of extracellular Mg 2+ on the post-ischemic recovery of ATP and low intracellular Na + in the isolated perfused rat kidney using 31P and triple-quantum filtered (TQ) 23Na-NMR spectroscopy. Following a 1 h period of stopped flow ischemia, the kidneys exposed to 0.3 MM Mg 2+ throughout the experiment exhibited a significantly ( p < 0.05) decreased post-ischemic fractional recovery of ATP (56 ± 7%) as well as a significantly ( p < 0.05) increased accumulation of P i (250 ± 30%) as compared to kidneys exposed to 1.2 MM Mg 2+ throughout (88 ± 5% recovery of [ATP] and 158 ± 8% accumulation of [P i]). Kidneys exposed to 0.3 MM Mg 2+ during the pre-ischemic and ischemic periods but to 1.2 mM Mg 2+ during reperfusion also showed better recovery of ATP (83 ± 6%) and lower accumulation of P i (143 ± 8%) compared to kidneys exposed to low Mg 2+ (0.3 mM) throughout the experiment. Measurements of the 23Na TQ signal following ischemia-reperfusion revealed that kidneys exposed to 1.2 mM Mg 2+ exhibited significantly improved maintenance of low intracellular Na + as compared to those exposed to 0.3 MM Mg 2+ ([Na +] i = 107 ± 7% in 1.2 mM Mg 2+ vs. 152 ± 3% in 0.3 mM Mg 2+. No significant difference was found in the pre-ischemic basal intracellular free Ca 2+ level (as measured by 19F-NMR in combination with 5 FBAPTA) between kidneys perfused with 1.2 mM and 0.3 MM Mg 2+, and comparable depletion of ATP occurred during ischemia under both experimental conditions. These data indicate that increased extracellular Mg 2+ has a protective effect against post-ischemic damage, probably related to its role in resynthesis of ATP during post-ischemic reperfusion. Our results would imply a greater vulnerability of the kidney to ischemic damage in hypomagnesemic clinical conditions such as alcoholism and diabetes.

Changes in the Concentrations of Extracellular Mg++ and Ca++ Down-Regulate E-Cadherin and Up-Regulate α2β1 Integrin Function, Activating Keratinocyte Migration on Type I Collagen

We have demonstrated recently that shifts in the concentrations of extracellular Mg++ and Ca++ occur during cutaneous injury in vivo. These shifts correlate well with the timing of migration of various cell types involved in wound healing, including keratinocytes. In the present study, we examined the potential of such cation shifts to activate the keratinocyte migratory phenotype. In modified Boyden chamber migration assays, alpha 2 beta 1 integrin-mediated migration of human keratinocytes (HaCaT) on type I collagen was supported by Mg++ but not by Ca++ alone. Migration could be increased up to twofold, however, by using both cations in combination, as long as the Mg++ concentration was in the optimal range for migration in Mg++ only (1-3 mM) and Ca++ was present at concentrations of approximately 0.1-1 mM. Further examination of this divalent-cation-induced migratory keratinocyte phenotype demonstrated that, as Mg++ is elevated and Ca++ is reduced, mature E-cadherin and cell-cell contacts are reduced and the alpha 2 beta 1 integrin is redistributed from cell-cell contacts to the periphery. These in vitro observations corroborate what occurs in vivo at the keratinocyte migrating front during wound healing. Together these data suggest that changes in the concentrations of extracellular Mg++ and Ca++ can regulate the competitive interplay between Ca(++)-dependent E-cadherin-mediated and Mg(++)-dependent alpha 2 beta 1-integrin-mediated adhesion, promoting the development of an activated keratinocyte phenotype.

Effect of magnesium ions on rabbit detrusor contractility and intracellular free calcium.

Magnesium (Mg2+) is one of the most abundant ions in the body. In the human body, Mg2+ plays important roles including cofactors in many crucial enzyme systems, especially those involving energy transfer, storage and utilization. Alteration of the concentration of Mg2+ may cause neuromuscular hyperactivity, psychiatric disturbances, calcium/potassium abnormalities, and overactivity of cardiac muscle. Most information on the effect of Mg2+ on muscle contraction has been obtained from studies on cardiac, skeletal, and vascular muscle; much less is known about the effect of Mg2+ in other smooth muscle systems. In the current study, we investigated the effect of Mg2+ on the contraction and intracellular free calcium of rabbit urinary bladder detrusor muscle in response to carbachol and transmural field stimulation (FS). The results can be summarized as follows: (1) Reduction of the concentration of magnesium [Mg2+] from normal Tyrode's solution enhanced the spontaneous basal activity, whereas addition of Mg2+ gradually abolished this spontaneous activity. (2) Muscle contraction induced by FS or carbachol was enhanced in Mg(2+)-free Tyrode's solution. Addition of Mg2+ inhibited the response to both forms of stimulation in a dose-dependent manner. (3) Inhibitory effects of Mg2+ were potentiated when the Ca2+ concentration in the Tyrode's solution was reduced to 0.6 mM, whereas increasing the extracellular concentration of Ca2+ (5.4 mM) reduced the inhibitory effects of Mg2+. (4) Using FURA-2 to monitor intracellular free calcium simultaneous with contractile tension, we demonstrated that the alterations in the contractile responses observed at different concentrations of extracellular Mg2+ correlated with similar changes in intracellular free calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional regulation of the human integrin VLA-1 (CD49a/CD29) by divalent cations and stimulatory β1 antibodies

We have investigated the regulation by divalent cations Mg 2+, Ca 2+ and Mn 2+ of the functional activity of the human integrin VLA-1 expressed on neuroblastoma NB100 cells. VLA-1-mediated adhesion of NB100 cells to ligand collagen type I was supported by either mM concentrations of extracellular Mg 2+ or μM levels of Mn 2+. In contrast, Ca 2+ alone did not induce activation of VLA-1 but exerted a potent inhibitory effect on the Mg 2+-supported cell adhesion. We have also demonstrated that VLA-1 can be directly activated by the stimulatory monoclonal antibody TS2/16 specific for the integrin β1 subunit, resulting in effective adhesion of NB100 cells to type I collagen. This study has been possible by using a novel blocking VLA-α1 specific monoclonal antibody, 5E8D9.

Regulation of alpha 2 beta 1-mediated fibroblast migration on type I collagen by shifts in the concentrations of extracellular Mg2+ and Ca2+.

Extracellular Ca2+ can reverse the Mg(2+)-dependent, alpha 2 beta 1-mediated adhesion of WI38 human fibroblasts to type I collagen substrates. Affinity chromatography data also demonstrate that Ca2+ can specifically elute the fibroblast alpha 2 beta 1 integrin bound to type I collagen-Sepharose in Mg2+. In modified Boyden chamber migration assays, Mg2+ alone supports the alpha 2 beta 1-mediated migration of fibroblasts on type I collagen substrates, while Ca2+ does not. However, a twofold enhancement in migration was observed when combinations of the two cations were used, with optimal migration observed when the Mg2+/Ca2+ ratio was higher than one. Inhibitory mAbs directed against various integrin subunits demonstrate that these observed cation effects appear to be mediated primarily by alpha 2 beta 1. These data, together with reports that under certain physiological conditions significant fluctuations in the concentrations of extracellular Ca2+ and Mg2+ can take place in vivo, suggest that the ratio between these two cations is involved in the up- and downregulation of integrin function, and thus, may influence cell migratory behavior.

MgtA and MgtB: prokaryotic P-type ATPases that mediate Mg2+ influx.

The gram-negative bacterium Salmonella typhimurium possesses three distinct Mg2+ transport systems, encoded by the corA, mgtA, and mgtB loci. The CorA transport system is the constitutive Mg2+ influx system. It can also mediate Mg2+ efflux at very high extracellular Mg2+ concentrations. In contrast, the MgtA and MgtB Mg2+ transport systems are normally expressed only at low extracellular Mg2+ concentrations. A strain of S. typhimurium was constructed by mutagenesis which lacks Mg2+ transport and requires 100 mM Mg2+ for growth. Using this strain, both the MgtA and MgtB transport systems were cloned by complementation of the strains inability to grow without Mg2+ supplementation. After sequencing and further genetic analysis, the MgtB system appears to be an operon composed of the mgtC and mgtB genes (5' to 3'). The downstream mgtB gene encodes the 102 kDa MgtB protein which by sequence analysis is clearly a P-type ATPase. Interestingly, while MgtB has relatively poor homology to other known prokaryotic P-type ATPases, it is highly homologous to mammalian reticular Ca(2+)-ATPases. MgtC is a 22.5 kDa hydrophobic membrane protein that lacks homology to any known protein. Transposon insertions in this gene abolish uptake by the MgtB transport system. We hypothesize that MgtC is a subunit of the MgtB ATPase involved either in proper insertion of MgtB into the membrane or possibly in binding of extracellular Mg2+ for delivery to the ATPase subunit. The sequence of the MgtA gene has recently been completed, and it too is a P-type ATPase more similar to eukaryotic than prokaryotic P-type ATPases. Expression of both MgtA and MgtB are highly regulated by the concentration of extracellular Mg2+. Transcription of mgtB can be increased about 1000 fold by lowering Mg2+ from 1 mM to 1 microM. Likewise, when mgtB is expressed from a multicopy plasmid, a similar decrease in extracellular Mg2+ greatly increases transport. Under growth conditions of limiting Mg2+, MgtB becomes the dominant Mg2+ influx system in S. typhimurium. Even so, since MgtB (and MgtA) mediate only influx of Mg2+, it is unclear why the cell requires energy from ATP to mediate Mg2+ entry into the cell down a large electrochemical gradient. Further studies of the structure-function and energetics of these novel Mg2+ influx P-type ATPases should yield insights into the function of P-type ATPases in general as well as information about the regulation of cellular Mg2+ fluxes.