Intracellular Magnesium Levels Research Articles

MAGT1-mediated disturbance of Mg2+ homeostasis lead to exhausted of HBV-infected NK and CD8+ T cells

The magnesium transporter 1 (MAGT1) is a critical regulator of basal intracellular free magnesium ([Mg2+]i) levels. It has been shown that MAGT1 was involved in the disorder in Mg2+ homeostasis after Epstein-Barr virus (EBV) infection. Here, we identified the effects of MAGT1-mediated disturbance of Mg2+ homeostasis on chronic hepatitis B virus (HBV)-infected natural killer (NK) and CD8+ T cells. The expression of MAGT1 was gradually decreased with the increase of infected time in CD8+ T cells, but not with that in NK cells, of the patients. Decreased level of intracellular free Mg2+ ([Mg2+]i) leads to defective expression of programmed cell death 1 (PD-1) and the NK activating receptor (NKG2D) in NK and CD8+ T cells. Our data illustrate that [Mg2+]i plays a key role in control of HBV infection.

Increasing intracellular magnesium levels with the 31-amino acid MgtS protein

Synthesis of the 31-amino acid, inner membrane protein MgtS (formerly denoted YneM) is induced by very low Mg2+ in a PhoPQ-dependent manner in Escherichia coli Here we report that MgtS acts to increase intracellular Mg2+ levels and maintain cell integrity upon Mg2+ depletion. Upon development of a functional tagged derivative of MgtS, we found that MgtS interacts with MgtA to increase the levels of this P-type ATPase Mg2+ transporter under Mg2+-limiting conditions. Correspondingly, the effects of MgtS upon Mg2+ limitation are lost in a ∆mgtA mutant, and MgtA overexpression can suppress the ∆mgtS phenotype. MgtS stabilization of MgtA provides an additional layer of regulation of this tightly controlled Mg2+ transporter and adds to the list of small proteins that regulate inner membrane transporters.

Structural Basis of the Oncogenic Interaction of Phosphatase PRL-1 with the Magnesium Transporter CNNM2

Phosphatases of regenerating liver (PRLs), the most oncogenic of all protein-tyrosine phosphatases (PTPs), play a critical role in metastatic progression of cancers. Recent findings established a new paradigm by uncovering that their association with magnesium transporters of the cyclin M (CNNM) family causes a rise in intracellular magnesium levels that promote oncogenic transformation. Recently, however, essential roles for regulation of the circadian rhythm and reproduction of the CNNM family have been highlighted. Here, we describe the crystal structure of PRL-1 in complex with the Bateman module of CNNM2 (CNNM2BAT), which consists of two cystathionine β-synthase (CBS) domains (IPR000664) and represents an intracellular regulatory module of the transporter. The structure reveals a heterotetrameric association, consisting of a disc-like homodimer of CNNM2BAT bound to two independent PRL-1 molecules, each one located at opposite tips of the disc. The structure highlights the key role played by Asp-558 at the extended loop of the CBS2 motif of CNNM2 in maintaining the association between the two proteins and proves that the interaction between CNNM2 and PRL-1 occurs via the catalytic domain of the phosphatase. Our data shed new light on the structural basis underlying the interaction between PRL phosphatases and CNNM transporters and provides a hypothesis about the molecular mechanism by which PRL-1, upon binding to CNNM2, might increase the intracellular concentration of Mg2+ thereby contributing to tumor progression and metastasis. The availability of this structure sets the basis for the rational design of compounds modulating PRL-1 and CNNM2 activities.

Quadruplex-Flanking Stem Structures Modulate the Stability and Metal Ion Preferences of RNA Mimics of GFP.

The spinach family of RNA aptamers are RNA mimics of green fluorescent protein (GFP) that have previously been designed to address the challenges of imaging RNA inside living cells. However, relatively low levels of free intracellular magnesium limited the practical use of these aptamers. Recent cell-based selections identified the broccoli RNA aptamer, which requires less magnesium for fluorescence, but the basis for magnesium preference remained unclear. Here, we find that the broccoli RNA structure is very similar to that of baby spinach, a truncated version of the spinach aptamer. Differences in stability and metal ion preferences between these two aptamers, and among broccoli mutants, are primarily associated with the sequence and structure of predicted quadruplex-flanking stem structures. Mutation of purine-purine pairs in broccoli at the terminal stem-quadruplex transition caused reversion of broccoli to a higher magnesium dependence. Unique duplex-to-quadruplex transitions in GFP-mimic RNAs likely explain their sensitivity to magnesium for stability and fluorescence. Thus, optimizations designed to improve aptamers should take into consideration the role of metal ions in stabilizing the transitions and interactions between independently folding RNA structural motifs.

Inhibition of PRL-2·CNNM3 Protein Complex Formation Decreases Breast Cancer Proliferation and Tumor Growth

The oncogenic phosphatase of regenerating liver 2 (PRL-2) has been shown to regulate intracellular magnesium levels by forming a complex through an extended amino acid loop present in the Bateman module of the CNNM3 magnesium transporter. Here we identified highly conserved residues located on this amino acid loop critical for the binding with PRL-2. A single point mutation (D426A) of one of those critical amino acids was found to completely disrupt PRL-2·human Cyclin M 3 (CNNM3) complex formation. Whole-cell voltage clamping revealed that expression of CNNM3 influenced the surface current, whereas overexpression of the binding mutant had no effect, indicating that the binding of PRL-2 to CNNM3 is important for the activity of the complex. Interestingly, overexpression of the CNNM3 D426A-binding mutant in cancer cells decreased their ability to proliferate under magnesium-deprived situations and under anchorage-independent growth conditions, demonstrating a PRL-2·CNNM3 complex-dependent oncogenic advantage in a more stringent environment. We further confirmed the importance of this complex in vivo using an orthotopic xenograft breast cancer model. Finally, because molecular modeling showed that the Asp-426 side chain in CNNM3 buries into the catalytic cavity of PRL-2, we showed that a PRL inhibitor could abrogate complex formation, resulting in a decrease in proliferation of human breast cancer cells. In summary, we provide evidence that this fundamental regulatory aspect of PRL-2 in cancer cells could potentially lead to broadly applicable and innovative therapeutic avenues.

An Importance of Magnesium Deficiency in the Formation of Vegetative Disadaptation in Children and Adolescents with Digestive Tract Pathology

Aim. In the study of magnesium deficiency, a distinction is made between magnesium hypoelementosis and magnesium deficit. As a result, detection of the magnesium deficiency and its influence on vegetative disadaptation progression can help to correct damages in early stages and to decrease frequency of chronization of digestive tract disorders in children. Methods and results. 48 children at the ages from 7 to 17 years with complaints on sickliness, stomachache, dyspeptic symptoms were under the care. The first group contained of 28 children with organic digestive system disorders, the second – 20 children with functional digestive system disorders. In both groups serum and intracellular concentration of Mg, Holter monitoring of ECG were carried out. Conclusion. Among children and adolescents with digestive tract pathology, 4,3% of patients had magnesium deficit. Hypoelementosis was detected in 18,7% of cases, that demonstrates great prevalence of latent magnesium deficiency and the necessity of the intracellular content study. Indices of both intracellular (p≤0,05) and extracellular (p ≤0,001) magnesium in children from the 1st group were much lower than in children from the 2nd group. This demonstrates mutual influence of organic digestive tract pathology and magnesium deficit on the development and severity of pathologies. Direct and strong correlation line between intracellular magnesium level lowering and lowered indices of HRV time-line analysis was detected in the 1st group. Correlation absence in the 2nd group demonstrates the influence of magnesium deficiency on vegetative disadaptation development and provides change of digestive tract functional disorders to organic.

Mesendogen, a novel inhibitor of TRPM6, promotes mesoderm and definitive endoderm differentiation of human embryonic stem cells through alteration of magnesium homeostasis

The homo- and hetero-tetrameric channel complexes formed by transient receptor potential cation channel, subfamily M, member 6 (TRPM6) and 7 (TRPM7) (collectively referred to as TRPM6/TRPM7 channels in this study) are the major regulators of cellular magnesium uptake, yet the exact roles of TRPM6/TRPM7 channels and cellular magnesium homeostasis during development are poorly understood. Here, we report a novel small molecule Mesendogen (MEG) which robustly induces nearly homogeneous (≥85%) mesoderm and definitive endoderm (DE) differentiations of human embryonic stem cells (hESCs) in combination with growth factors. A kinome screen followed by loss-of-function experiments identified TRPM6 as the biological target of MEG. We demonstrated that MEG functions by inhibiting TRPM6/TRPM7 magnesium channel activity, as MEG reduced intracellular magnesium level, while TRPM6/TRPM7 channel modulation and magnesium-withdrawal phenocopied MEG at enhancing mesoderm and DE differentiations. This study discovers a robust chemical enhancer of hESC directed differentiation, and uncovers a novel regulatory role of cellular magnesium homeostasis during early embryonic cell fate specification.

Effects of transdermal magnesium chloride on quality of life for patients with fibromyalgia: a feasibility study

BackgroundFibromyalgia is a syndrome characterized by chronic pain, fatigue, depression, and sleep disturbances. Its primary cause is unclear. Several studies have reported decreased intracellular magnesium levels in patients with fibromyalgia and have found negative correlation between magnesium levels and fibromyalgia symptoms. ObjectiveTo gather preliminary data on whether transdermal magnesium can improve quality of life for women who have fibromyalgia. Design, setting, participants and interventionsThis is a patient questionnaires and survey in a fibromyalgia clinic at a tertiary medical center. Forty female patients with the diagnosis of fibromyalgia were enrolled. Each participant was provided a spray bottle containing a transdermal magnesium chloride solution and asked to apply 4 sprays per limb twice daily for 4 weeks. Participants were asked to complete the Revised Fibromyalgia Impact Questionnaire, SF-36v2 Health Survey, and a quality-of-life analog scale at baseline, week 2, and week 4. Main outcome measureQuestionnaire and survey scores, evaluated through intent-to-treat and per-protocol analyses. ResultsTwenty-four patients completed the study (mean [SD] age, 57.2 [7.6] years; white, 95%; mean body mass index, 31.3 kg/m2). With intention-to-treat analysis, Revised Fibromyalgia Impact Questionnaire subscale and total scores were significantly improved at week 2 and week 4 (total score, P = 0.001). Per-protocol analysis results were similar: all subscales of the Revised Fibromyalgia Impact Questionnaire were significantly improved at week 2 and week 4 (total score, P = 0.001). ConclusionThis pilot study suggests that transdermal magnesium chloride applied on upper and lower limbs may be beneficial to patients with fibromyalgia. Trial registrationClinicalTrials.gov. ldentifier NCT01968772.

Intracellular magnesium level determines cell viability in the MPP+ model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder resulting from mitochondrial dysfunction in dopaminergic neurons. Mitochondria are believed to be responsible for cellular Mg2+ homeostasis. Mg2+ is indispensable for maintaining ordinal cellular functions, hence perturbation of the cellular Mg2+ homeostasis may be responsible for the disorders of physiological functions and diseases including PD. However, the changes in intracellular Mg2+ concentration ([Mg2+]i) and the role of Mg2+ in PD have still been obscure. In this study, we investigated [Mg2+]i and its effect on neurodegeneration in the 1-methyl-4-phenylpyridinium (MPP+) model of PD in differentiated PC12 cells. Application of MPP+ induced an increase in [Mg2+]i immediately via two different pathways: Mg2+ release from mitochondria and Mg2+ influx across cell membrane, and the increased [Mg2+]i sustained for more than 16h after MPP+ application. Suppression of Mg2+ influx decreased the viability of the cells exposed to MPP+. The cell viability correlated highly with [Mg2+]i. In the PC12 cells with suppressed Mg2+ influx, ATP concentration decreased and the amount of reactive oxygen species (ROS) increased after an 8h exposure to MPP+. Our results indicate that the increase in [Mg2+]i inhibited cellular ROS generation and maintained ATP production, which resulted in the protection from MPP+ toxicity.

Cell viability evaluation of transdifferentiated endothelial-like cells by quantitative electron-probe X-ray microanalysis for tissue engineering.

Development of an efficient vascular substitute by tissue engineering is strongly dependent on endothelial cell viability. The aim of this study was to evaluate cell viability of transdifferentiated endothelial-like cells (Tr-ELC) by using for the first time electron probe X-ray microanalysis (EPXMA), not only to accurately analyze cell viability by quantifying the intracellular ionic concentrations, but also to establish their possible use in vascular tissue engineering protocols. Human umbilical cord Wharton's jelly stem cells (HWJSC) and endothelial cells from the human umbilical vein (HUVEC) were isolated and cultured. Transdifferentiation from HWJSC to the endothelial phenotype was induced. EPXMA was carried out to analyze HUVEC, HWJSC and Tr-ELC cells by using a scanning electron microscope equipped with an EDAX DX-4 microanalytical system and a solid-state backscattered electron detector. To determine total ion content, the peak-to-local-background (P/B) ratio method was used with reference to standards composed of dextran containing known amounts of inorganic salts. Our results revealed a high K/Na ratio in Tr-ELC (9.41), in association with the maintenance of the intracellular levels of chlorine, phosphorous and magnesium and an increase of calcium (p=0.031) and sulfur (p=0.022) as compared to HWJSC. Calcium levels were similar for HUVEC and Tr-ELC. These results ensure that transdifferentiated cells are highly viable and resemble the phenotypic and microanalytical profile of endothelial cells. Tr-ELC induced from HWJSC may fulfill the requirements for use in tissue engineering protocols applied to the vascular system at the viability and microanalytical levels.

Pharmacological Treatment may Impair Mineral Status in Blood

Hematological and biochemical blood parameters are rather stable in healthy subjects. However, there are many factors that can influence mineral status in blood, including diseases, diet, and drugs. It has been found that several medicines can alter both mineral concentrations and other mineral-status-related parameters in blood. In experimental and human studies, it has been shown that hypotension therapy disturbs zinc status in the body [1]. Some of our results suggest that indapamide and amlodipine treatment can decrease zinc concentration in serum and erythrocytes. Moreover, the decrease in zinc is accompanied by an increase in glucose serum levels [2]. In our experimental study, we observed that treatment with indapamide and amlodipine was associated with increased red blood cell counts and hematocrit values in the serum of spontaneously hypertensive rats [3]. In patients, high hematocrit could reflect the increase in red blood cell mass resulting from the reduction in plasma volume due to treatment with thiazide-type diuretics. Long-term therapy with captopril and enalapril may lead to zinc depletion in erythrocytes and monocytes. Preliminary data suggest that indapamide can cause a slight elevation in blood calcium levels. Several results indicate that loop diuretics, especially furosemide, reduce serum calcium and magnesium levels. It has also been observed that beta-blockers may induce hypocalcemia and hypomagnesemia [4]. Antiepileptic therapy with valproic acid may cause abnormalities in serum copper concentration. Some authors have found that therapy with valproic acid can diminish copper levels in erythrocytes and serum in epileptic patients [5,6]. Nonsteroidal anti-inflammatory drugs, when taken regularly, may cause bleeding from the gastrointestinal tract and iron loss with the blood. The use of aspirin and other medicines belonging to this group of drugs may therefore increase the risk of iron deficiency, which can be identified as a low hemoglobin and serum ferritin concentration [4,7]. In patients with digoxin therapy, a decrease in intracellular magnesium levels and a relatively low level of magnesium in serum were observed [8]. The long-term use of corticosteroids, cisplatin, cyclosporine is also associated with a depletion of magnesium in the serum and an increase in the risk of hypomagnesemia [4,9]. It is known that dietary restriction may impair mineral status; however, pharmacological treatment of obesity may also alter the concentration of minerals in the blood. In our clinical study, we found that 12 weeks of sibutramine therapy decreases zinc and increases magnesium concentrations in the serum, leading to mineral imbalances in obese women [10]. Oral contraceptives (OCs) are among the most commonly used drugs in developed countries. One of the side effects of this group of drugs is their influence on the mineral status in women. In several studies, it was observed that the use of oral contraceptives is associated with decreased zinc, magnesium, and selenium serum concentrations, and with increased copper levels in serum. It has also been found that oral contraceptives can increase ferritin and iron levels, TIBC, and mean corpuscular hemoglobin in serum, while decreasing red blood cells and hematocrit in whole blood in women. The results of human studies suggest that the effect of OCs on mineral status can vary with the form, dose, concomitant agents, and patient characteristics [4,11]. The mechanisms behind the observed interactions between drugs and minerals cannot always be explained. Some may be associated with changes in the excretion of minerals in the urine. Drugs may also influence the absorption and/or distribution of minerals in the body [4 ]. In conclusion, many drugs influence mineral status in the blood. Pharmacological treatment may require the monitoring of mineral concentrations in blood. Moreover, the interpretation of the results of morphological and biochemical analyses in blood requires a consideration of the drugs being used by the patient.

Structural studies of a novel phosphatase-transpoter interaction

The Phosphatase of Regenerating Liver (PRL) family, a subgroup of the protein tyrosine phosphatases, comprises three highly oncogenic members, PRL-1, -2 and -3, implicated in the progression of numerous cancer types. PRL-2 is overexpressed in breast cancer and was shown to promote mammary tumour growth in mice, but its full role in these oncogenic effects remains elusive. Recently, PRL-2 was found to interact with cyclin M3 (CNNM3), a magnesium transporter. To characterize this novel interaction, I crystallized PRL-2 in complex with the cytosolic cystathionine-β-synthase (CBS) domain of CNNM3. The binding surface consists of an elongated loop from CBS that makes contact close to the catalytic site of PRL-2. Site-directed CBS mutants confirmed the loop residues important for binding. PRL-2 and CBS bind particularly tightly, as determined by isothermal titration calorimetry, and PRL-2 enzyme assays revealed that CBS binding reduces the phosphatase's catalytic activity in vitro. The novel role of oncogenic PRL-2 as modulator of intracellular magnesium levels may represent the link between its overexpression and its effects on tumour growth. Small-molecule inhibitors of the PRL-2/CNNM3 complex formation are a potentially valuable tool for exploring the physiological function of this new interaction and may be used as future drug leads for the treatment of breast cancer.

The protein tyrosine phosphatase PRL-2 interacts with the magnesium transporter CNNM3 to promote oncogenesis

The three PRL (phosphatases of regenerating liver) protein tyrosine phosphatases (PRL-1, -2 and -3) have been identified as key contributors to metastasis in several human cancers, yet the molecular basis of their pro-oncogenic property is unclear. Among the subfamily of PRL phosphatases, overexpression of PRL-2 in breast cancer cells has been shown to promote tumor growth by a mechanism that remains to be uncovered. Here we show that PRL-2 regulates intracellular magnesium levels by forming a functional heterodimer with the magnesium transporter CNNM3. We further reveal that CNNM3 is not a phosphorylated substrate of PRL-2, and that the interaction occurs through a loop unique to the CBS pair domains of CNNM3 that exists only in organisms having PRL orthologs. Supporting the role of PRL-2 in cellular magnesium transport is the observation that PRL-2 knockdown results in a substantial decrease of cellular magnesium influx. Furthermore, in PRL-2 knockout mice, serum magnesium levels were significantly elevated as compared with control animals, indicating a pivotal role for PRL-2 in regulating cellular magnesium homeostasis. Although the expression levels of CNNM3 remained unchanged after magnesium depletion of various cancer cell lines, the interaction between endogenous PRL-2 and CNNM3 was markedly increased. Importantly, xenograft tumor assays with CNNM3 and a mutant form that does not associate with PRL-2 confirm that CNNM3 is itself pro-oncogenic, and that the PRL-2/CNNM3 association is important for conferring transforming activities. This finding is further confirmed from data in human breast cancer tissues showing that CNNM3 levels correlate positively with both PRL-2 expression and the tumor proliferative index. In summary, we demonstrate that oncogenic PRL-2 controls tumor growth by modulating intracellular magnesium levels through binding with the CNNM3 magnesium transporter.

Magnesium Sulfate Increases Intracellular Magnesium Reducing Inflammatory Cytokine Release in Neonates

Magnesium sulfate (MgSO4 ) exposure reduces the risk of cerebral palsy. As neonatal inflammatory cytokine levels strongly correlate with neurologic outcome, we hypothesize that MgSO4 decreases inflammatory cytokine production. We assessed the effect of MgSO4 on cellular magnesium levels, cytokine production, and release within THP-1 and cord blood mononuclear cells. MgSO4 exposure increased intracellular magnesium levels, reducing the frequency of THP-1 cells producing IL-1β, IL-8, and TNF-α following LPS stimulation. Significant reductions in the frequency of neonatal monocytes producing TNF-α (48%) and IL-6 (37%) were seen following LPS stimulation, and MgSO4 also significantly decreased the frequency of monocytes producing TNF-α (35%) under basal conditions. Decreased cytokine production was confirmed via ELISA, demonstrating a sustained effect and dose response. Magnesium also reduced cytokine production following stimulation with TLR ligands representing obstetrical infections (group B Streptococcus and Mycoplasma) and in preterm neonatal monocytes. MgSO4 increases intracellular magnesium, reducing inflammatory cytokine production and release, potentially elucidating the mechanism by which MgSO4 prevents cerebral palsy, eclampsia, and preterm birth.

Comparison of magnesium status using X-ray dispersion analysis following magnesium oxide and magnesium citrate treatment of healthy subjects

The magnesium content in food consumed in the Western world is steadily decreasing. Hypomagnesemia is associated with increased incidence of diabetes mellitus, metabolic syndrome, all-cause and coronary artery disease mortality. We investigated the impact of supplemental oral magnesium citrate versus magnesium oxide on intracellular magnesium levels ([Mg2+]i) and platelet function in healthy subjects with no apparent heart disease. In a randomized, prospective, double-blind, crossover study, 41 (20 women) healthy volunteers [mean age 53±8 (range 31-75) years] received either magnesium oxide monohydrate tablets (520 mg/day of elemental magnesium) or magnesium citrate tablets (295.8 mg/day of elemental magnesium) for one month (phase 1), followed by a four-week wash-out period, and then crossover treatment for one month (phase 2). [Mg2+]i was assessed from sublingual cells through x-ray dispersion (normal values 37.9±4.0 mEq/L), serum magnesium levels, platelet aggregation, and quality-of-life questionnaires were assessed before and after each phase. Oral magnesium oxide, rather than magnesium citrate, significantly increased [Mg2+]i (34.4±3 versus 36.3±2 mEq/L, p<0.001 and 34.7±2 versus 35.4±2 mEq/L, p=0.097; respectively), reduced total cholesterol (201±37 versus 186±27 mg/dL, p=0.016 and 187±28 versus 187±25 mg/dL, p=0.978; respectively) and low-density lipoprotein (LDL) cholesterol (128±22 versus 120±25 mg/dL, p=0.042 and 120±23 versus 121±22 mg/dL, p=0.622; respectively). Noteworthy is that both treatments significantly reduced epinephrine-induced platelet aggregation (78.9±16% versus 71.7±23%, p=0.013 and 81.3±15% versus 73.3±23%, p=0.036; respectively). Thus, oral magnesium oxide treatment significantly improved [Mg2+]i, total and LDL cholesterol compared with magnesium citrate, while both treatments similarly inhibited platelet aggregation in healthy subjects with no apparent heart disease.

The relationship between the antioxidant system and phycocyanin production in Spirulina maxima with respect to nitrate concentration

The relationship among antioxidant enzyme activities, such as iron-superoxide dismutase (FeSOD) and ascorbate-dependent peroxidase (AsA-POD), and phycocyanin, chlorophyll, total carotenoid, ascorbate contents, and lipid peroxidation (LPO) levels were investigated in Spirulina maxima SAG 84.79 in nitrate limited (10 mM), control (30 mM), and supplemented (50 mM) conditions. In the present study, extracellular nitrate and intracellular iron, magnesium, and copper levels were also investigated. The highest FeSOD and AsA-POD activities and chlorophyll content and the lowest LPO levels were found in control conditions on day 13: 21.9 ± 1.5, 5.7 ± 0.3 IU mg^{-1}, 16.2 ± 0.5 mg g^{-1}, and 0.322 ± 0.01 nmol malondialdehyde g^{-1} wet weight, respectively. The highest phycocyanin content was determined in nitrate supplemented conditions on day 13, at 190.68 ± 9.3 mg g^{-1}. The similar LPO levels observed in supplemented (50 mM nitrate) and control (30 mM) conditions showed that phycocyanin demonstrated effective protection of S. maxima membranes. The data revealed that its response against nitrate exposure was related to the antioxidant enzymes and compounds in the antioxidant defence system of S. maxima.

Magnesium used in bioabsorbable stents controls smooth muscle cell proliferation and stimulates endothelial cells in vitro

Magnesium-based bioabsorbable cardiovascular stents have been developed to overcome limitations of permanent metallic stents, such as late stent thrombosis. During stent degradation, endothelial and smooth muscle cells will be exposed to locally high magnesium concentrations with yet unknown physiological consequences. Here, we investigated the effects of elevated magnesium concentrations on human coronary artery endothelial and smooth muscle cell (HCAEC, HCASMC) growth and gene expression. In the course of 24 h after incubation with magnesium chloride solutions (1 or 10 mM) intracellular magnesium level in HCASMC raised from 0.55 ± 0.25 mM (1 mM) to 1.38 ± 0.95 mM (10 mM), while no increase was detected in HCAEC. Accordingly, a DNA microarray-based study identified 69 magnesium regulated transcripts in HCAEC, but 2172 magnesium regulated transcripts in HCASMC. Notably, a significant regulation of various growth factors and extracellular matrix components was observed. In contrast, viability and proliferation of HCAEC were increased at concentrations of up to 25 mM magnesium chloride, while in HCASMC viability and proliferation appeared to be unaffected. Taken together, our data indicate that magnesium halts smooth muscle cell proliferation and stimulates endothelial cell proliferation, which might translate into a beneficial effect in the setting of stent associated vascular injury.

Insights into Metalloregulation by M-box Riboswitch RNAs via Structural Analysis of Manganese-Bound Complexes

The M-box riboswitch couples intracellular magnesium levels to expression of bacterial metal transport genes. Structural analyses on other riboswitch RNA classes, which typically respond to a small organic metabolite, have revealed that ligand recognition occurs through a combination of base-stacking, electrostatic, and hydrogen-bonding interactions. In contrast, the M-box RNA triggers a change in gene expression upon association with an undefined population of metals, rather than responding to only a single ligand. Prior biophysical experimentation suggested that divalent ions associate with the M-box RNA to promote a compacted tertiary conformation, resulting in sequestration of a short sequence tract otherwise required for downstream gene expression. Electrostatic shielding from loosely associated metals is undoubtedly an important influence during this metal-mediated compaction pathway. However, it is also likely that a subset of divalent ions specifically occupies cation binding sites and promotes proper positioning of functional groups for tertiary structure stabilization. To better elucidate the role of these metal binding sites, we resolved a manganese-chelated M-box RNA complex to 1.86 Å by X-ray crystallography. These data support the presence of at least eight well-ordered cation binding pockets, including several sites that had been predicted by biochemical studies but were not observed in prior structural analysis. Overall, these data support the presence of three metal-binding cores within the M-box RNA that facilitate a network of long-range interactions within the metal-bound, compacted conformation.

Magnesium Modulates Amyloid-β Protein Precursor Trafficking and Processing

Alzheimer's disease (AD), the most common form of dementia, is characterized by the presence of excessive deposits of aggregated amyloid-beta (Abeta), which is derived from the amyloid-beta protein precursor (AbetaPP) following processing by beta- and gamma-secretase. Metal elements are implicated in the pathophysiology of AD. Magnesium affects many biochemical mechanisms vital for neuronal properties and synaptic plasticity, and magnesium levels were reported to be decreased in various tissues including brain of AD patients. However, the exact role of magnesium in the neurodegenerative process of AD remains elusive. In this study, we investigated the effects of physiological (0.8 mM, as normal control), low (0-0.4 mM), and high (1.2-4.0 mM) concentrations of extracellular magnesium ([Mg2+]o) on AbetaPP processing and Abeta secretion. Here we show the effects of varying [Mg2+]o on AbetaPP processing is time- and dose-dependent. After 24 h treatment, high [Mg2+]o increased C-terminal fragment-alpha (CTFalpha) levels and soluble alpha-secretase cleaved AbetaPP (sAbetaPPalpha) release via enhancing retention of AbetaPP on plasma membrane. In contrast, low [Mg2+]o enhanced CTFbeta accumulation and Abeta secretion, and reduced cell surface AbetaPP level. Varying [Mg2+]o did not alter protein contents of full length AbetaPP. However, decreased total intracellular magnesium level by magnesium deprivation over 24 hr impaired cell viability. Normal AbetaPP processing could be restored when magnesium was adjusted back to physiological concentration. These data demonstrate that AbetaPP processing can be modulated by magnesium and at high [Mg2+]o, AbetaPP processing favors the alpha-secretase cleavage pathway. Our findings suggest that supplementation of magnesium has a therapeutic potential for preventing AD.

The reproducibility of measurements of intramuscular magnesium concentrations and muscle oxidative capacity using 31P MRS.

31P magnetic resonance spectroscopy (31P MRS) has been used to measure intramuscular magnesium concentrations and muscle metabolism. Abnormal intramuscular magnesium has been reported in several patient populations with suspected metabolic disorders. The purpose of this study was to evaluate our ability to measure intramuscular magnesium and muscle metabolism in the quadriceps muscles of healthy subjects, and to test whether these measurements were influenced by prior exercise. Twelve normal, healthy male volunteers were tested in a 3 Tesla magnet on four separate days. Resting [Mg2+] was calculated from the heights and frequency shifts of the phosphate, phosphocreatine and ATP peaks. Phosphocreatine (PCr) recovery kinetics were measured after 30-39 second bouts of isometric exercise. Thirty minutes prior to the 3rd test session the subjects completed a 2 hour treadmill walk at 40-60% of heart rate reserve. Resting [Mg2+] averaged 0.388 mM and had an interclass correlation coefficient between days (ICC) of 0.352. The mean end exercise PCr was 47.6% and the mean end exercise pH was 6.97. PCr recovery averaged 39 seconds (p = 0.892) and had an ICC of 0.819. Prior long duration exercise did not produce significant alterations in either PCr recovery kinetics or intracellular magnesium levels (p = 0.440). In conclusion, the reproducibility of Resting [Mg2+] was less than that of PCr recovery measurements, and may reflect the sensitivity of these measurements to phasing errors. In addition, prior exercise is unlikely to alter measurements of resting metabolites or muscle metabolism suggesting that rigorous control of physical activity prior to metabolic testing is unnecessary.