Extracellular Magnesium Concentration Research Articles

Neurovirulent cytokines increase neuronal excitability in a model of coronavirus-induced neuroinflammation

BackgroundNeurological manifestations of severe coronavirus infections, including SARS-CoV-2, are wide-ranging and may persist following virus clearance. Detailed understanding of the underlying changes in brain function may facilitate the identification of therapeutic targets. We directly tested how neocortical function is impacted by the specific panel of cytokines that occur in coronavirus brain infection. Using the whole-cell patch-clamp technique, we determined how the five cytokines (TNFα, IL-1β, IL-6, IL-12p40 and IL-15 for 22–28-h) at concentrations matched to those elicited by MHV-A59 coronavirus brain infection, affected neuronal function in cultured primary mouse neocortical neurons.ResultsWe evaluated how acute cytokine exposure affected neuronal excitability (propensity to fire action potentials), membrane properties, and action potential characteristics, as well as sensitivity to changes in extracellular calcium and magnesium (divalent) concentration. Neurovirulent cytokines increased spontaneous excitability and response to low divalent concentration by depolarizing the resting membrane potential and hyperpolarizing the action potential threshold. Evoked excitability was also enhanced by neurovirulent cytokines at physiological divalent concentrations. At low divalent concentrations, the change in evoked excitability was attenuated. One hour after cytokine removal, spontaneous excitability and hyperpolarization of the action potential threshold normalized but membrane depolarization and attenuated divalent-dependent excitability persisted.ConclusionsCoronavirus-associated cytokine exposure increases spontaneous excitability in neocortical neurons, and some of the changes persist after cytokine removal.

Loss of CorA, the primary magnesium transporter of Salmonella, is alleviated by MgtA and PhoP-dependent compensatory mechanisms.

In many Gram-negative bacteria, the stress sigma factor of RNA polymerase, σS/RpoS, remodels global gene expression to reshape the physiology of stationary phase cells and ensure their survival under non-optimal growth conditions. In the foodborne pathogen Salmonella enterica serovar Typhimurium, σS is also required for biofilm formation and virulence. We have recently shown that a ΔrpoS mutation decreases the magnesium content and expression level of the housekeeping Mg2+-transporter CorA in stationary phase Salmonella. The other two Mg2+-transporters of Salmonella are encoded by the PhoP-activated mgtA and mgtB genes and are expressed under magnesium starvation. The σS control of corA prompted us to evaluate the impact of CorA in stationary phase Salmonella cells, by using global and analytical proteomic analyses and physiological assays. The ΔcorA mutation conferred a competitive disadvantage to exit from stationary phase, and slightly impaired motility, but had no effect on total and free cellular magnesium contents. In contrast to the wild-type strain, the ΔcorA mutant produced MgtA, but not MgtB, in the presence of high extracellular magnesium concentration. Under these conditions, MgtA production in the ΔcorA mutant did not require PhoP. Consistently, a ΔmgtA, but not a ΔphoP, mutation slightly reduced the magnesium content of the ΔcorA mutant. Synthetic phenotypes were observed when the ΔphoP and ΔcorA mutations were combined, including a strong reduction in growth and motility, independently of the extracellular magnesium concentration. The abundance of several proteins involved in flagella formation, chemotaxis and secretion was lowered by the ΔcorA and ΔphoP mutations in combination, but not alone. These findings unravel the importance of PhoP-dependent functions in the absence of CorA when magnesium is sufficient. Altogether, our data pinpoint a regulatory network, where the absence of CorA is sensed by the cell and compensated by MgtA and PhoP- dependent mechanisms.

High calcium concentrations reduce cellular excitability of mouse MNTB neurons

Calcium, a universal intracellular signaling molecule, plays essential roles in neural functions. Historically, in most in vitro brain slice electrophysiology studies, the extracellular calcium concentration ([Ca2+]e) in artificial cerebrospinal fluid is of a wide range and typically higher than the physiological value. At high [Ca2+]e, synaptic transmission is generally enhanced. However, the effects and the underlying mechanisms of calcium on intrinsic neuronal properties are diverse. Using whole-cell patch clamp in acute brainstem slices obtained from mice of either sex, we investigated the effects and the underlying mechanisms of high [Ca2+]e on intrinsic neuronal properties of neurons in the medial nucleus of the trapezoid body (MNTB), an auditory brainstem component in the sound localization circuitry. Compared to the physiological [Ca2+]e (1.2 mM), high [Ca2+]e at 1.8 and 2.4 mM significantly reduced the cellular excitability of MNTB neurons, resulting in decreased spike firing rate, depolarized spike threshold, and decreased the ability to follow high frequency inputs. High extracellular magnesium concentrations at 1.8 and 2.4 mM produced similar but less robust effects, due to surface charge screening. Upon high calcium application, voltage-gated sodium channel currents remained largely unchanged. Calcium-sensing receptors were detected in MNTB neurons, but blocking these receptors did not eliminate the effects of high calcium on spontaneous spiking. We attribute the lack of significant effects in these last two experiments to the moderate changes in calcium we tested. Our results call for the use of physiological [Ca2+]e in brain slice experiments.

High magnesium mitigates the vasoconstriction mediated by different types of calcium influx from monocrotaline-induced pulmonary hypertensive rats.

What is the central question of this study? What is the involvement of Mg2+ in mitigating the vasoconstriction in pulmonary arteries and smaller pulmonary arteries in the monocrotaline-induced pulmonary arterial hypertension (MCT-PAH) rat model? What are the main finding and its importance? Both store-operated Ca2+ entry- and receptor-operated Ca2+ entry-mediated vasoconstriction were enhanced in the MCT-PAH model. High magnesium inhibited vasoconstriction by directly antagonizing Ca2+ and increasing NO release, and this was more notable in smaller pulmonary arteries. Increased extracellular magnesium concentration has been shown to attenuate the endothelin-1-induced contractile response via the release of nitric oxide (NO) from the endothelium in proximal pulmonary arteries (PAs) of chronic hypoxic mice. Here, we further examined the involvement of Mg2+ in the inhibition of vasoconstriction in PAs and distal smaller pulmonary arteries (sPAs) in a monocrotaline-induced pulmonary arterial hypertension (MCT-PAH) rat model. The data showed that in control rats vasoconstriction in sPAs is more intense than that in PAs. In MCT-PAH rats, store-operated Ca2+ entry (SOCE)- and receptor-operated Ca2+ entry (ROCE)-mediated contraction were significantly strengthened. However, there was no upregulation of the vasoconstriction mediated by voltage-dependent calcium entry (VDCE). Furthermore, high magnesium greatly inhibited VDCE-mediated contraction in PAs rather than sPAs, which was the opposite of the ROCE-mediated contraction. Moreover, monocrotaline pretreatment partly eliminated the endothelium-dependent vasodilatation in PAs, which in sPAs, however, was still promoted by magnesium due to the increased NO release in pulmonary microvascular endothelial cells (PMVECs). In conclusion, the findings suggest that both SOCE- and ROCE-mediated vasoconstriction in the MCT-PAH model are enhanced, especially in sPAs. The inhibitory effect of high magnesium on vasoconstriction can be achieved partly by its direct role as a Ca2+ antagonist and partly by increasing NO release in PMVECs.

Effects of divalent cations on Schaffer collateral axon function.

Previously, we reported that distal Schaffer collaterals undergo biphasic changes in excitability during high-frequency stimulation (HFS), with an early hyper-excitability period followed by an excitability depression period. The extracellular divalent cations calcium and magnesium can regulate membrane excitability in neuronal tissue. Therefore, we hypothesized that altering the concentrations of extracellular calcium and magnesium would alter the biphasic excitability changes. We tested this hypothesis by recording distal Schaffer collateral fiber volleys in stratum radiatum of hippocampal area CA1 during 100Hz HFS in artificial cerebral spinal fluid (ACSF) containing normal and altered concentrations of extracellular divalent cations. Our normal ACSF contained 2.0mM calcium and 2.0mM magnesium. We examined four solutions with altered divalent cation concentrations: (1) high-calcium/low-magnesium (3.8mM/0.2mM), (2) low-calcium/high-magnesium (0.2mM/3.8mM), (3) high-calcium/normal-magnesium (3.8mM/2.0mM), or (4) normal-calcium/high-magnesium (2.0mM/10.0mM), and assessed the effects on Schaffer collateral responses. Increasing or decreasing extracellular calcium enhanced or reduced (respectively) the early hyper-excitable period whereas increasing extracellular magnesium reduced the later excitability depression. Because these results might be explained by altered calcium influx through voltage-gated calcium (CaV) channels, we tested CaV blockers (ω-agatoxin IVA, ω-conotoxin-GVIA, cadmium), but observed no effects on responses during HFS. Some of the effects of altered divalent cation concentration may be explained by altered membrane surface charge. Although this mechanism does not completely explain our findings, calcium influx through CaV channels is not required.

High Magnesium and Sirolimus on Rabbit Vascular Cells—An In Vitro Proof of Concept

Drug-eluting bioresorbable scaffolds represent the last frontier in the field of angioplasty and stenting to treat coronary artery disease, one of the leading causes of morbidity and mortality worldwide. In particular, sirolimus-eluting magnesium-based scaffolds were recently introduced in clinical practice. Magnesium alloys are biocompatible and dissolve in body fluids, thus determining high concentrations of magnesium in the local microenvironment. Since magnesium regulates cell growth, we asked whether high levels of magnesium might interfere with the antiproliferative action of sirolimus. We performed in vitro experiments on rabbit coronary artery endothelial and smooth muscle cells (rCAEC and rSMC, respectively). The cells were treated with sirolimus in the presence of different concentrations of extracellular magnesium. Sirolimus inhibits rCAEC proliferation only in physiological concentrations of magnesium, while high concentrations prevent this effect. On the contrary, high extracellular magnesium does not rescue rSMC growth arrest by sirolimus and accentuates the inhibitory effect of the drug on cell migration. Importantly, sirolimus and magnesium do not impair rSMC response to nitric oxide. If translated into a clinical setting, these results suggest that, in the presence of sirolimus, local increases of magnesium concentration maintain normal endothelial proliferative capacity and function without affecting rSMC growth inhibition and response to vasodilators.

Cation Flux and Electrolyte Composition of Frozen-Deglycerolized Blood

Cation flux and the extracellular and erythrocyte concentrations of sodium, potassium, calcium, magnesium, chloride, and acid soluble phosphorus were measured in fresh blood collected in various anticoagulant solutions, ACD blood stored at 4 C, and in frozen-deglycerolized blood stored at -80 to -120 C. Considerable variations in concentrations of extracellular and erythrocyte electrolytes were encountered due to certain characteristics of the anticoagulants employed and to the effects of storage, temperature, pH, and extracellular electrolyte concentration on erythrocyte electrolyte concentration. In contrast to ACD blood stored at 4 C, frozen-deglycerolized blood 1 day to 32 months old had slightly elevated erythrocyte concentrations of sodium and chloride and normal erythrocyte concentrations of potassium and phosphorus. Both ACD and frozen-deglycerolized blood had normal sodium and potassium fluxes. These data show that prolonged freezing and glycerolization of erythrocytes do not significantly impair the active transport processes responsible for maintenance of cellular electrolyte concentration.

Magnesium Ions Depolarize the Neuronal Membrane via Quantum Tunneling through the Closed Channels

Magnesium ions have many cellular actions including the suppression of the excitability of neurons; however, the depolarization effect of magnesium ions seems to be contradictory. Thus several hypotheses have aimed to explain this effect. In this study, a quantum mechanical approach is used to explain the depolarization action of magnesium. The model of quantum tunneling of magnesium ions through the closed sodium voltage-gated channels was adopted to calculate the quantum conductance of magnesium ions, and a modified version of Goldman–Hodgkin–Katz equation was used to determine whether this quantum conductance was significant in affecting the resting membrane potential of neurons. Accordingly, it was found that extracellular magnesium ions can exhibit a depolarization effect on membrane potential, and the degree of this depolarization depends on the tunneling probability, the channels’ selectivity to magnesium ions, the channels’ density in the neuronal membrane, and the extracellular magnesium concentration. In addition, extracellular magnesium ions achieve a quantum conductance much higher than intracellular ones because they have a higher kinetic energy. This study aims to identify the mechanism of the depolarization action of magnesium because this may help in offering better therapeutic solutions for fetal neuroprotection and in stabilizing the mood of bipolar patients.

Magnesium in schizophrenia.

Many psychopathological symptoms, including schizophrenia, can be associated with magnesium metabolism disturbances. In the literature, contradictory data exist regarding magnesium levels in patients with this disorder. However, this situation might be caused by determination of extracellular concentration of magnesium; although, this is mainly an intracellular ion. There are no data concerning determination of the ionized fraction of magnesium in patients with schizophrenia, while the ionized fraction represents 67% of the total pool of magnesium in healthy people. Also, the mechanism of magnesium action-the effect of magnesium ions on NMDA and GABA receptors-has not yet been fully investigated. There are preliminary studies aimed at increasing the effectiveness of schizophrenia pharmacotherapy via magnesium supplementation. Multidirectional activity of magnesium can significantly increase its therapeutic effect in psychiatry. This observation is confirmed by recent studies conducted by various research teams. However, further studies on the role of magnesium supplementation in patients with schizophrenia are needed.

Effect of altered extracellular magnesium concentration on the neuronal activity in different hippocampal regions of immature rats

Effect of altered extracellular magnesium concentration on the neuronal activity in different hippocampal regions of immature rats

Knockdown of SLC41A1 magnesium transporter promotes mineralization and attenuates magnesium inhibition during osteogenesis of mesenchymal stromal cells

BackgroundMagnesium is essential for numerous physiological functions. Magnesium exists mostly in bone and the amount is dynamically regulated by skeletal remodeling. Accelerating bone mass loss occurs when magnesium intake is insufficient; whereas high magnesium could lead to mineralization defects. However, the underlying magnesium regulatory mechanisms remain elusive. In the present study, we investigated the effects of high extracellular magnesium concentration on osteogenic differentiation of mesenchymal stromal/stem cells (MSCs) and the role of magnesium transporter SLC41A1 in the mineralization process.MethodsMurine MSCs derived from the bone marrow of BALB/c mouse or commercially purchased human MSCs were treated with osteogenic induction medium containing 5.8 mM magnesium chloride and the osteogenic differentiation efficiency was compared with that of MSCs in normal differentiation medium containing 0.8 mM magnesium chloride by cell morphology, gene expression profile of osteogenic markers, and Alizarin Red staining. Slc41a1 gene knockdown in MSCs was performed by siRNA transfection using Lipofectamine RNAiMAX, and the differentiation efficiency of siRNA-treated MSCs was also assessed.ResultsHigh concentration of extracellular magnesium ion inhibited mineralization during osteogenic differentiation of MSCs. Early osteogenic marker genes including osterix, alkaline phosphatase, and type I collagen were significantly downregulated in MSCs under high concentration of magnesium, whereas late marker genes such as osteopontin, osteocalcin, and bone morphogenetic protein 2 were upregulated with statistical significance compared with those in normal differentiation medium containing 0.8 mM magnesium. siRNA treatment targeting SLC41A1 magnesium transporter, a member of the solute carrier family with a predominant Mg2+ efflux system, accelerated the mineralization process and ameliorated the inhibition of mineralization caused by high concentration of magnesium. High concentration of magnesium significantly upregulated Dkk1 gene expression and the upregulation was attenuated after the Slc41a1 gene was knocked down. Immunofluorescent staining showed that Slc41a1 gene knockdown promoted the translocation of phosphorylated β-catenin into nuclei. In addition, secreted MGP protein was elevated after Slc41a1 was knocked down.ConclusionsHigh concentration of extracellular magnesium modulates gene expression of MSCs during osteogenic differentiation and inhibits the mineralization process. Additionally, we identified magnesium transporter SLC41A1 that regulates the interaction of magnesium and MSCs during osteogenic differentiation. Wnt signaling is suggested to be involved in SLC41A1-mediated regulation. Tissue-specific SLC41A1 could be a potential treatment for bone mass loss; in addition, caution should be taken regarding the role of magnesium in osteoporosis and the design of magnesium alloys for implantation.

136 - A Member of Solute Carrier Family SLC41 Regulates the Interaction of Magnesium and Mesenchymal Stromal Cells During Osteogenic Differentiation

As the second most abundant intracellular cation, magnesium exists mostly in bone and the amount of magnesium is dynamically regulated by skeletal remodeling. Previous studies showed that accelerating bone mass loss occurs in post-menopause animals when their magnesium intake is insufficient and increased magnesium intake alleviates the osteoporotic symptoms. However, high magnesium status has been reported to lead to mineralization defects possibly due to the magnesium substitution for calcium in hydroxyapatite structure. In the present study, we used murine and human mesenchymal stromal cells (MSCs) to investigate the effect of high extracellular magnesium concentration on osteogenic differentiation and the role of magnesium transporters in the mineralization process. We verified the differentiation efficiency by observing cell morphology, expression profiling of osteogenic marker genes, and performing alizarin red staining (Fig. 1) . The results showed that high concentration of extracellular magnesium ion inhibited mineralization during osteogenic differentiation of MSCs. Gene expressions of osterix, type I collagen alpha 1, and alkaline phosphatase were downregulated, as well as osteopontin and osteocalcin were upregulated in MSCs treated with high concentration of magnesium. siRNA treatment targeting the specific magnesium transporter, which is also a member of solute carrier family SLC41, accelerated the mineralization process and ameliorated the inhibition of mineralization caused by high concentration of magnesium. The sensitivity to extracellular magnesium was also diminished after the knockdown. Pathway analysis of differentially expressed genes indicated that Wnt signaling is involved in the regulation mediated by the magnesium transporter. The study suggests that caution should be taken when magnesium supplement used as a therapy for bone mass loss and provide insights into the design of magnesium alloys for implantation.

Elevation of Brain Magnesium Potentiates Neural Stem Cell Proliferation in the Hippocampus of Young and Aged Mice.

In the adult brain, neural stem cells (NSCs) can self-renew and generate all neural lineage types, and they persist in the sub-granular zone (SGZ) of the hippocampus and the sub-ventricular zone (SVZ) of the cortex. Here, we show that dietary-supplemented - magnesium-L-threonate (MgT), a novel magnesium compound designed to elevate brain magnesium regulates the NSC pool in the adult hippocampus. We found that administration of both short- and long-term regimens of MgT, increased the number of hippocampal NSCs. We demonstrated that in young mice, dietary supplementation with MgT significantly enhanced NSC proliferation in the SGZ. Importantly, in aged mice that underwent long-term (12-month) supplementation with MgT, MgT did not deplete the hippocampal NSC reservoir but rather curtailed the age-associated decline in NSC proliferation. We further established an association between extracellular magnesium concentrations and NSC self-renewal in vitro by demonstrating that elevated Mg(2+) concentrations can maintain or increase the number of cultured hippocampal NSCs. Our study also suggests that key signaling pathways for cell growth and proliferation may be candidate targets for Mg(2+) 's effects on NSC self-renewal. J. Cell. Physiol. 231: 1903-1912, 2016. © 2016 Wiley Periodicals, Inc.

Mechanisms of magnesium-induced vasodilation in cerebral penetrating arterioles

We investigated in cerebral penetrating arterioles the signaling mechanisms and dose-dependency of extracellular magnesium-induced vasodilation and also its vasodilatory effects in vessels preconstricted with agonists associated with delayed cerebral vasospasm following SAH. Male rat penetrating arterioles were cannulated. Their internal diameters were monitored. To investigate mechanisms of magnesium-induced vasodilation, inhibitors of endothelial function, potassium channels and endothelial impairment were tested. To simulate cerebral vasospasm we applied several spasmogenic agonists. Increased extracellular magnesium concentration produced concentration-dependent vasodilation, which was partially attenuated by non-specific calcium-sensitive potassium channel inhibitor tetraethylammonium, but not by other potassium channel inhibitors. Neither the nitric oxide synthase inhibitor L-NNA nor endothelial impairment induced by air embolism reduced the dilation. Although the magnesium-induced vasodilation was slightly attenuated by the spasmogen ET-1, neither application of PF2α nor TXA2 analog effect the vasodilation. Magnesium induced a concentration- and smooth muscle cell-dependent dilation in cerebral penetrating arterioles. Calcium-sensitive potassium channels of smooth muscle cells may play a key role in magnesium-induced vasodilation. Magnesium also dilated endothelium-impaired vessels as well as vessels preconstricted with spasmogenic agonists. These results provide a fundamental background for the clinical use of magnesium, especially in treatment against delayed cerebral ischemia or vasospasm following SAH.

Mechanisms of magnesium‐induced vasodilation in cerebral penetrating arterioles

Magnesium is a well-known neuroprotective and vasodilatory agent in experimental subarachnoid hemorrhage (SAH). We investigated the signaling mechanisms and dose-dependency of extracellular magnesium-induced vasodilation in cerebral penetrating arterioles and also its vasodilatory effects in vessels preconstricted with agonists associated with delayed cerebral vasospasm following SAH. Male rat penetrating arterioles were cannulated. Their internal diameters were monitored. To investigate mechanisms of magnesium-induced vasodilation, inhibitors of endothelial function, potassium channels and endothelial impairment were examined. To simulate cerebral vasospasm several spasmogenic agonists were applied. Increased extracellular magnesium concentration produced concentration-dependent vasodilation, which was partially attenuated by non-specific calcium-sensitive potassium channel inhibitor tetraethylammonium, but not by other potassium channnels inhibitors. Neither the nitric oxide synthase inhibitor L-NNA nor endothelial impairment induced by air embolism reduced the dilation. Although the magnesium-induced vasodilation was slightly attenuated by the spasmogen ET-1, neither application of PF2α nor TXA2 analog effect the vasodilation. Magnesium induced a concentration- and smooth muscle cell-dependent dilation in cerebral penetrating arterioles. Calcium-sensitive potassium channels of smooth muscle cells may play a key role in magnesium-induced vasodilation. These results provide a fundamental background for the clinical use of magnesium in SAH.

Extracellular Mg concentration and Ca blockers modulate the initial steps of the response of Th2 lymphocytes in co-culture with macrophages and dendritic cells

Magnesium is highly involved in the metabolic network such that even subtle disturbances in its homeostasis affect many cellular functions, including calcium homeostasis, signal transduction, energy metabolism, membrane stability and cell proliferation. Recently, magnesium level has been proposed to modulate the priming and activity of immune cells. We studied the behavior of antigen-presenting cells (APCs) and T lymphocytes after altering the magnesium/calcium balance. We used two different populations of primary APCs, i.e. bone marrow-derived dendritic cells and bone marrow-derived macrophages, while D10.G4.1 cells served as a model of responding Th2 cells. Our principal findings are the following: (i) the extracellular magnesium concentration had no significant impact on endocytosis by bone marrow-derived APCs, (ii) high concentrations of extracellular magnesium, with or without calcium antagonists, significantly decreased IL-4 and IL-10 secretion by Th2 cells in a co-culture system of APCs and Th2 lymphocytes, (iii) proliferation of Th2 cells in co-culture systems was significantly inhibited by calcium antagonists independently from extracellular magnesium concentrations. Our results suggest that alterations of magnesium and calcium homeostasis impact on some crucial steps of the immune response.

Effects of extracellular magnesium on the differentiation and function of human osteoclasts

Magnesium-based implants have been shown to influence the surrounding bone structure. In an attempt to partially reveal the cellular mechanisms involved in the remodelling of magnesium-based implants, the influence of increased extracellular magnesium content on human osteoclasts was studied. Peripheral blood mononuclear cells were driven towards an osteoclastogenesis pathway via stimulation with receptor activator of nuclear factor kappa-B ligand and macrophage colony-stimulating factor for 28days. Concomitantly, the cultures were exposed to variable magnesium concentrations (from either magnesium chloride or magnesium extracts). Osteoclast proliferation and differentiation were evaluated based on cell metabolic activity, total protein content, tartrate-resistant acid phosphatase activity, cathepsin K and calcitonin receptor immunocytochemistry, and cellular ability to form resorption pits. While magnesium chloride first enhanced and then opposed cell proliferation and differentiation in a concentration-dependent manner (peaking between 10 and 15mM magnesium chloride), magnesium extracts (with lower magnesium contents) appeared to decrease cell metabolic activity (≈50% decrease at day28) while increasing osteoclast activity at a lower concentration (twofold higher). Together, the results indicated that (i) variations in the in vitro extracellular magnesium concentration affect osteoclast metabolism and (ii) magnesium extracts should be used preferentially in vitro to more closely mimic the in vivo environment.

Mechanisms and Regulation of Renal Magnesium Transport

Magnesium's most important role is in the release of chemical energy. Although most magnesium is stored outside of the extracellular fluid compartment, the regulated value is blood magnesium concentration. Cellular magnesium and bone magnesium do not play a major role in the defense of blood magnesium concentration; the major role is played by the kidney, where the renal tubule matches the urinary magnesium excretion and the net entry of magnesium into the extracellular fluid. In the kidney, magnesium is reabsorbed in the proximal tubule, the thick ascending limb of the loop of Henle, and the distal convoluted tubule. Magnesium absorption is mainly paracellular in the proximal tubule and in the thick ascending limb of the loop of Henle, whereas it is transcellular in the distal convoluted tubule. Several hormones and extracellular magnesium itself alter the distal tubular handling of magnesium, but the hormone(s) regulating extracellular magnesium concentration remains unknown.

Extracellular magnesium and in vitro cell differentiation: different behaviour of different cells

The contribution of magnesium to cell differentiation is not clear. Some studies indicate that low extracellular magnesium promotes cell differentiation, while others reach opposite conclusions. We evaluated the effects of different concentrations of extracellular magnesium on the differentiation of three in vitro experimental models: human endothelial cells seeded onto Matrigel; phorbol ester-treated myeloid leukemia U937 cells; and 3T3-L1 pre-adipocytes exposed to a hormonal cocktail containing dexamethasone and insulin. The differentiation of endothelial cells and pre-adipocytes seems to be independent of extracellular Mg concentration. Conversely, magnesium deficiency retards, while high extracellular magnesium accelerates phorbol ester-induced U937 cell differentiation, probably by interfering with calcium homeostasis or with the activity of kinases. We conclude that the extracellular magnesium concentration affects the differentiation of various cell types.

Genetic switchboard for synthetic biology applications

A key next step in synthetic biology is to combine simple circuits into higher-order systems. In this work, we expanded our synthetic riboregulation platform into a genetic switchboard that independently controls the expression of multiple genes in parallel. First, we designed and characterized riboregulator variants to complete the foundation of the genetic switchboard; then we constructed the switchboard sensor, a testing platform that reported on quorum-signaling molecules, DNA damage, iron starvation, and extracellular magnesium concentration in single cells. As a demonstration of the biotechnological potential of our synthetic device, we built a metabolism switchboard that regulated four metabolic genes, pgi, zwf, edd, and gnd, to control carbon flow through three Escherichia coli glucose-utilization pathways: the Embden-Meyerhof, Entner-Doudoroff, and pentose phosphate pathways. We provide direct evidence for switchboard-mediated shunting of metabolic flux by measuring mRNA levels of the riboregulated genes, shifts in the activities of the relevant enzymes and pathways, and targeted changes to the E. coli metabolome. The design, testing, and implementation of the genetic switchboard illustrate the successful construction of a higher-order system that can be used for a broad range of practical applications in synthetic biology and biotechnology.