Intracellular Free Magnesium Concentration Research Articles

Relationship between intracellular free magnesium concentration and the degree of insulin resistance in horses with equine metabolic syndrome

Relationship between intracellular free magnesium concentration and the degree of insulin resistance in horses with equine metabolic syndrome

Intracellular free magnesium concentration in healthy horses.

Equine metabolic syndrome (EMS) is a worldwide disease in horses that parallels human diabetes mellitus type 2. In both diseases, patients show an altered peripheral insulin sensitivity as a key feature. In humans, multiple studies have demonstrated the beneficial effect of magnesium supplementation on insulin sensitivity. However, serum magnesium levels vary and are therefore not a reliable indicator of the patients' magnesium status. Determining the intracellular free magnesium concentration appears to be a more sensitive diagnostic indicator. In this study, the free intracellular magnesium concentration was measured using mag-fura 2 spectrophotometry in blood lymphocytes in 12 healthy, non-obese horses at 9a.m., 12a.m. and 4p.m. to establish reference ranges according to a protocol designed for human blood lymphocytes. Additionally, the serum magnesium concentration was measured. In all horses, the total serum magnesium concentration was within the reference range. The mean free magnesium concentration in blood lymphocytes of all horses was 0.291±0.067mmol/L with no significant difference between the time points. The reference range for the free intracellular magnesium concentration in equine lymphocytes was set at 0.16-0.42mmol/L. The established values are slightly lower than those in healthy humans. The designed protocol for the measurement of the intracellular free magnesium concentration might be an excellent research tool to assess the cellular magnesium status and to reliably diagnose an altered magnesium homeostasis in EMS. Further studies shall elucidate possible alterations in cellular magnesium status in horses with EMS.

Mg2+‐Dependent Modulation of BKCa Channels by Genistein in Rat Arteriolar Smooth Muscle Cells

Genistein, a protein tyrosine kinase (PTK) inhibitor, regulates ion channel activities. However, the mechanism of action of genistein on large-conductance calcium-activated potassium (BK(Ca)) channels is unclear. This study aimed to investigate whether the mechanism of Mg(2+)-dependent modulation of BK(Ca) channel activity in vascular smooth muscle cells involved inhibition of phosphorylation by genistein or direct interaction between genistein and BK(Ca) channels. The whole-cell and inside-out patch-clamp techniques were used to measure BK(Ca) currents and the effects of genistein on BK(Ca) channel activities in rat mesenteric arteriolar smooth muscle cells. We found that the effects of genistein on BK(Ca) currents were Mg(2+)-dependent. Genistein (50 μM) inhibited BK(Ca) currents if the intracellular free magnesium concentration ([Mg(2+)]i) was 2 μM or 20 μM, but amplified BK(Ca) currents if [Mg(2+)]i was 200 μM or 2000 μM. The inhibitory effect of genistein on BK(Ca) currents was reversed by the protein tyrosine phosphatase inhibitor sodium orthovanadate (0.5 mM). Daidzein (50 μM), an inactive analogue of genistein, also amplified BK(Ca) currents, and its amplification was insensitive to orthovanadate. Another PTK inhibitor, tyrphostin 23 (50 μM), reduced the open probability of BK(Ca) channels. This inhibitory effect was weaker at 200 μM [Mg(2+)]i than at 2 μM [Mg(2+) ]i, and was countered by orthovanadate. Our results suggest that genistein amplifies BK(Ca) currents at a high [Mg(2+)]i, but inhibits BK(Ca) currents at a low [Mg(2+)]i. The mechanism of this biphasic effects involves PTK-independent amplification and [Mg(2+)]i -PTK-dependent inhibition.

Molecular determinants of magnesium-dependent synaptic plasticity at electrical synapses formed by connexin36.

Neuronal gap junction (GJ) channels composed of connexin36 (Cx36) play an important role in neuronal synchronization and network dynamics. Here we show that Cx36-containing electrical synapses between inhibitory neurons of the thalamic reticular nucleus are bi-directionally modulated by changes in intracellular free magnesium concentration ([Mg2+]i). Chimeragenesis demonstrates that the first extracellular loop of Cx36 contains a Mg2+-sensitive domain, and site-directed mutagenesis shows that the pore-lining residue D47 is critical in determining high Mg2+-sensitivity. Single channel analysis of Mg2+-sensitive chimeras and mutants reveals that [Mg2+]i controls the strength of electrical coupling mostly via gating mechanisms. In addition, asymmetric transjunctional [Mg2+]i induces strong instantaneous rectification, providing a novel mechanism for electrical rectification in homotypic Cx36 GJs. We suggest that Mg2+-dependent synaptic plasticity of Cx36-containing electrical synapses could underlie neuronal circuit reconfiguration via changes in brain energy metabolism that affects neuronal levels of intracellular ATP and [Mg2+]i.

Alcohol Intoxication May Exacerbate the Effects of Blunt Cranial Trauma Through Changes in Brain Free Magnesium Levels

Moderate to high levels of alcohol decrease brain intracellular free magnesium concentration, a factor known to be critical in brain injury. Phosphorus magnetic resonance spectroscopy was used to examine changes to brain free magnesium concentration after blunt cranial trauma in alcohol-intoxicated rats. Rats exposed acutely or chronically to alcohol sufficient to increase blood alcohol levels to between 150 and 350 mg/dL demonstrated a brain free magnesium level that was 20-50% less than in nonintoxicated animals (p < 0.01). After injury, brain free magnesium levels declined more rapidly and to a greater extent in alcohol-affected animals than in nonintoxicated control animals (p < 0.001). As both preinjury depletion of magnesium and degree of magnesium decline after brain injury have been associated with poor recovery, these findings suggest that moderate to severe alcohol intoxication may predispose the brain to a worse outcome by reducing brain free magnesium levels, both before and after injury.

Down-regulation of TRPM6-mediated magnesium influx by cyclosporin A

Transient receptor potential melastatin 6 (TRPM6) is distributed along the apical membrane of the renal tubular cells and is involved in the reabsorption of magnesium. In this study, we show that TRPM6 expression is suppressed by cyclosporin A (CsA) via a down-regulation of c-Fos expression. TRPM6 was expressed in NRK-52E, but not in Madin-Darby canine kidney cells. In contrast, its homolog, TRPM7, was equally expressed in both cells. In NRK-52E cells, CsA dose-dependently decreased TRPM6 expression without affecting TRPM7 expression. Magnesium load measurements revealed the rise in the intracellular free magnesium concentration ([Mg2+]i) to be inhibited by CsA. The transfection of TRPM6 siRNA decreased TRPM6 expression without affecting TRPM7 expression and inhibited the elevation of [Mg2+]i. CsA did not affect the intracellular distribution of nuclear factor of activated T cells (NFATc). Furthermore, TRPM6 expression was not changed by a NFATc inhibitor. Next, we examined the effect of CsA on the transcription factors c-Fos and c-Jun. CsA decreased c-Fos expression without affecting c-Jun expression. The transfection of c-Fos siRNA suppressed TRPM6 expression without affecting TRPM7 expression. We suggest that CsA decreases TRPM6 expression mediated by inhibition of c-Fos transcription, resulting in a decrease of renal Mg2+ reabsorption.

A method for measuring intracellular free magnesium concentration in platelets using flow cytometry.

Magnesium is the fourth most abundant cation in the body and is involved in over 302 enzymatic reactions. Basic science research has implicated magnesium deficiency as a cause of insulin resistance which is related to hypertension, diabetes, hyperlipidemia and increased cardiovascular risk. Research in magnesium deficiency states has been hindered because magnesium is an intracellular ion and difficult to measure. Our goal was to develop a reproducible assay to measure intracellular magnesium in platelets. Healthy volunteers agreed to have blood drawn for magnesium measurement. Platelet rich plasma was harvested from a venipuncture specimen and run through the flow cytometer. A standard titer curve using known increasing concentrations of magnesium chloride was created for each specimen, and then with the other half the specimen was run to measure the true intracellular free magnesium concentration. 15 adults agreed to volunteer for this experiment. All standard titer curves for all specimens had a correlation of > 0.99. The mean concentration of intracellular free magnesium was 450.05 microM with a range of 203.68 microM to 673.50 microM. Intracellular free magnesium can reliably and reproducibly be measured in platelets using Mag Green fluorescent dye and flow cytometry. This should advance our ability to study magnesium deficient states.

Activation of ferret erythrocyte Na+–K+–2Cl− cotransport by deoxygenation

Deoxygenation of ferret erythrocytes stimulates Na+-K+-2Cl- cotransport by 111% (s.d., 46) compared to controls in air. Half-maximal activation occurs at a PO2 of 24 mmHg (s.d., 2) indicating that physiological changes in oxygen tension can influence cotransport function. Approximately 25-35% of this stimulation can be attributed to the rise of intracellular free magnesium concentration that occurs on deoxygenation (from 0.82 (S.D., 0.07) to 1.40 mm (S.D., 0.17)). Most of the stimulation is probably caused by activation of a kinase which can be prevented or reversed by treating cells with the kinase inhibitors PP1 or staurosporine, or by reducing cell magnesium content to submicromolar levels. Stimulation by deoxygenation is comparable with that caused by calyculin A or sodium arsenite, compounds that cause a 2- to 3-fold increase in threonine phosphorylation of the cotransporter which can be detected with phospho-specific antibodies. However, the same approach failed to detect significant changes in threonine phosphorylation following deoxygenation. The results suggest that deoxygenation causes activation of a kinase that either phosphorylates the transporter, but probably not on threonine, or phosphorylates another protein that in turn influences cotransporter behaviour. They also indicate that more than one kinase and phosphatase are involved in cotransporter phosphorylation.

A Substance P Antagonist Increases Brain Intracellular Free Magnesium Concentration after Diffuse Traumatic Brain Injury in Rats

Objective: Magnesium (Mg) deficiency has been shown to increase substance P release and induce a pro-inflammatory response that can be attenuated with the administration of a substance P-antagonist. Neurogenic inflammation has also been implicated in traumatic brain injury (TBI), a condition where brain intracellular free magnesium (Mgf) decline is known to occur and has been correlated with functional outcome. We therefore examined whether a substance P antagonist restores brain intracellular free magnesium concentration following TBI.Methods: Male, adult Sprague-Dawley rats were injured using the Cernak impact acceleration model of diffuse TBI. At 30 min after injury, animals were administered either 0.25 mg/kg i.v. n-acetyl tryptophan or equal volume saline. Prior to and 4 h after induction of injury, phosphorus magnetic resonance spectra were acquired using a 7-tesla magnet interfaced with a Bruker console. Mgf was calculated from the chemical shift of the beta ATP. Before injury, Mgf was 0.51 ± 0.05 mM (SEM).Results: By 4 hr after injury, Mgf had significantly declined to 0.27 ± 0.02 mM in saline treated rats. In contrast, rats treated with n-acetyl tryptophan had a Mgf of 0.47 ± 0.06 mM at 4 h after injury, which was not significantly different from preinjury values. There were no significant differences in pH between the treatment groups.Conclusion: It seems that any beneficial effect of a substance P antagonist on functional outcome following TBI may be related to improvement in brain Mg homeostasis induced by the compound.

Magnesium influx enhanced by nitric oxide in hypertensive rat proximal tubule cells

An abnormal handling of renal magnesium has been suggested to cause salt-sensitive hypertension. The filtered magnesium is first reabsorbed in the proximal tubule. Amiloride has been shown to enhance renal magnesium conservation, but the regulatory mechanisms are unknown yet. High-salt (8% NaCl) diet decreased serum magnesium concentration, while increased urinary magnesium in Dahl salt-sensitive (DS) rat. Furthermore, the expression of nitric oxide synthase type 3 and nitric oxide (NO) content were decreased in high-salt loaded DS rat. In isolated proximal tubule cells, amiloride (0.1 mM) increased intracellular free magnesium concentration ([Mg 2+] i). However, the net [Mg 2+] i increase in the high-salt loaded DS rat was smaller than other groups. NOR1 (0.1 mM), a NO donor, restored the increase of [Mg 2+] i to the same level of other groups. On the contrary, l-NMMA (0.1 mM), an inhibitor of NO production, inhibited the increase of [Mg 2+] i in all groups. These results suggest that intracellular NO has an important role to up-regulate amiloride-elicited magnesium influx.

Subdural hematoma following traumatic brain injury causes a secondary decline in brain free magnesium concentration.

A number of studies have demonstrated that neurologic motor and cognitive deficits induced by traumatic brain injury (TBI) can be attenuated with administration of magnesium salts. However, many severe traumatic brain injuries have a significant hematoma that develops subsequent to the primary events, and it is unclear whether magnesium salts are effective in this situation. In the present study, an impact-acceleration rodent model of TBI was used to produce an injury that causes an extensive subdural hematoma in over 50% of injured animals. At 30 min after TBI, rats were randomly administered 250 micromoles/kg intravenous MgSO4 or equal volume saline before being monitored by magnetic resonance spectroscopy for 8 h to determine brain intracellular free magnesium concentration. Animals were then assessed for neurologic motor deficits over 1 week using a rotarod device, followed by postmortem examination for presence of subdural hematoma. Animals with subdural hematoma treated with MgSO4 showed no improvement in motor outcome when compared to nontreated controls. Animals with no visible subdural hematoma demonstrated a significant improvement (p < 0.05 by ANOVA) in rotarod scores with MgSO4 treatment. Brain free magnesium concentration in the magnesium treated/hematoma group demonstrated a biphasic decline made up of an immediate initial decline, recovery of brain magnesium levels with MgSO4 treatment, and then a significant second magnesium decline (p < 0.05). Such a secondary decline did not occur in the Mg treated/no hematoma animals. Our results suggest that development of a subdural hematoma following TBI results in a decline in brain magnesium, even after bolus administration of magnesium salts. Such effects of hematoma development will need to be considered in trials examining efficacy of magnesium salts as an intervention following TBI.

Intracellular Mg 2+ diffusion within isolated rat skeletal muscle fibers

Intracellular free magnesium concentration ([Mg 2+] i) was measured in enzymatically isolated rat skeletal muscle fibers using the fluorescent dye mag-indo-1. The change in [Mg 2+] i produced by a local intracellular microinjection of magnesium pidolate (magnesium pyrrolidone carboxylate) was measured at a given distance from the injection site. In one series of experiments this protocol was tested on isolated fibers that were completely embedded into silicone grease: under these conditions, the injection produced an increase in [Mg 2+] i that reached a steady level some time following the injection. The time-course of the [Mg 2+] i change could be well accounted for by a model of longitudinal diffusion. The mean apparent Mg 2+ diffusion coefficient ( D app) was 188±9 μm 2 s −1 ( n=16), approximately four times lower than the value measured in vitro. This reduction likely results from the effects of cytoplasmic viscosity and of Mg 2+ binding to low affinity static sites. Another series of measurements was performed on fibers that were either partially or completely free of silicone: under these conditions, the time course of the change in [Mg 2+] i was in many cases more complex than predicted by simple diffusion.

Physiological and pathophysiological role of magnesium in the cardiovascular system: implications in hypertension.

Attention is growing for a potential role of magnesium in the pathoetiology of cardiovascular disease. Magnesium modulates mechanical, electrical and structural functions of cardiac and vascular cells, and small changes in extracellular magnesium levels and/or intracellular free magnesium concentration may have significant effects on cardiac excitability and on vascular tone, contractility and reactivity. Thus, magnesium may be important in the physiological regulation of blood pressure whereas alterations in cellular magnesium metabolism could contribute to the pathogenesis of blood pressure elevation. Although most epidemiological and experimental studies support a pathological role for magnesium in the etiology and development of hypertension, data from clinical studies have been less convincing. Furthermore, the therapeutic value of magnesium in the management of essential hypertension is unclear. The present review discusses the molecular, biochemical, physiological and pharmacological roles of magnesium in the regulation of vascular function and blood pressure and introduces novel concepts relating to magnesium as a second messenger in intracellular signaling in cardiovascular cells. In addition, alterations in magnesium regulation in experimental and clinical hypertension and the potential antihypertensive therapeutic effects of magnesium are addressed.

Intracellular magnesium: transport and regulation in epithelial secretory cells.

The mechanisms of magnesium (Mg2+) transport, the regulation of intracellular Mg2+ concentrations and the relationship between Mg2+ and Ca2+ signaling during the stimulus-secretion coupling process in pancreatic acinar cells and other secretory epithelia are reviewed in this article. Our results demonstrate the existence of a Na+- and ATP-dependent transport system for Mg2+ extrusion from Mg2+-loaded cells. Moreover, employing such different techniques as spectrofluorimetry and atomic absorbance spectroscopy to measure intracellular free magnesium concentration [Mg2+]i from magfura-2-loaded acini and acinar cells and Mg2+ content in effluent samples from perfused pancreatic segments, respectively, confirm that secretagogues such as acetylcholine (ACh) and cholecystokinin-octapeptide (CCK-8) can evoke marked and significant extrusion of Mg2+ which is closely associated with the mobilization of intracellular calcium. These effects may be modulated by different mediators including cAMP, Protein Kinase C and nitric oxide/cGMP. This reduction in [Mg2+]i seems to be a prerequisite for optimal generation and maintenance of the calcium signal and subsequently, the secretion of enzymes, since an increase in extracellular Mg2+ concentration, [Mg2+]o and an increase in [Mg2+]i inhibit secretagogue-induced secretory responses, an effect exerted through a derangement of the calcium signaling events. In conclusion, the evidence presented in this review strongly supports an important modulatory role of magnesium in the control of secretory epithelial cells function.

Concentration of brain free magnesium following severe brain injury correlates with neurologic motor outcome

Recent studies have shown that brain intracellular free magnesium concentration significantly declines following mild to severe, focal and diffuse traumatic brain injury. However, little is known about how this decline or its attenuation by magnesium salts relates to neurologic outcome. This study uses phosphorus magnetic resonance spectroscopy and rotarod tests to characterise the relationship between brain free magnesium concentration and neurologic motor scores following severe, diffuse traumatic brain injury in rats. An intravenous bolus of MgSO 4 or MgCl 2 (100 μmoles/kg) at 30 min following brain injury significantly attenuated the postinjury brain free magnesium decline. This improved magnesium homeostasis was sustained for the entire postinjury monitoring period (1 week). There was an associated significant improvement in neurologic motor function in magnesium treated rats. Moreover, the brain free magnesium concentration over the one week period was linearly correlated with the neurologic motor function (r=0.70; P < 0.001) as assessed on a daily basis. We propose that brain free magnesium concentration may be used as a prognostic indicator of neurologic motor function after traumatic brain injury.

Lithium induced changes in intracellular free magnesium concentration in isolated rat ventricular myocytes.

We have examined the effect of exposing isolated rat ventricular myocytes to lithium while measuring cytosolic free magnesium ([Mg2+]i) and calcium ([Ca2+]i) levels with the fluorescent, ion sensitive probes mag-fura-2 and fura-2. There was a significant rise in [Mg2+]i after a 5 min exposure to a solution in which 50% of the sodium had been replaced by Li+, but not when the sodium had been replaced by bis-dimethylammonium (BDA). However, there were significant increases in [Ca2+]i when either Na+ substitute was used. The possibility that Li+, which enters the cells, interferes with the signal from mag-fura-2 was eliminated as Li+ concentrations up to 10 mM had no effect on the dye's fluorescence signal. A possible explanation for these findings is that Li+ displaces Mg2+ from intracellular binding sites. Having considered the binding constants for Mg2+ and Li+ to ATP, we conclude that Li+ can displace Mg2+ from Mg-ATP, thus causing a rise in [Mg2+]i. This work has implications for other studies where Li+ is used as a Na+ substitute.

Regulation of Intracellular Free Mg2+Concentration in Isolated Rat Hearts viaβ-adrenergic and Muscarinic Receptors

Regulation of the intracellular free magnesium concentration ([Mg2+]i) was investigated in isolated rat hearts, using31P-nuclear magnetic resonance (31P-NMR). [Mg2+]iwas found to be slowly and significantly decreased during prolonged application of isoproterenol (ISO) throughβ-adrenergic receptor stimulation, and restored by subsequent washouts. The ISO-induced decrease in [Mg2+]iwas antagonized by addition of a muscarinic receptor agonist, carbachol (CCh). In the presence of atropine, CCh did not exert this effect. A water-soluble forskolin derivative, NKH477, which directly activates adenylate cyclase, also caused a decrease in [Mg2+]i, which could be antagonized by CCh, but a greater concentration was required as compared to the ISO case. The manner of [Mg2+]iregulation mimicked those noted for the action potential duration and the Ca2+channel current, in which cAMP is known to act as a second messenger. Even in the presence of a Ca2+channel blocker, verapamil, [Mg2+]iwas reversibly decreased by ISO. Changes in the intracellular ATP concentration demonstrated any clear correlation with changes in [Mg2+]i. These results suggest that [Mg2+]ican be controlled by a balance of sympathetic and parasympathetic activities. cAMP may play a key role in the [Mg2+]iregulation viaβ-adrenergic and muscarinic receptors, although some other metabolic pathways also appear to be involved. Hormonally induced changes in [Mg2+]ihave possible clinical significance.

Measurements of Intracellular Mg 2+ Concentration in Mouse Skeletal Muscle Fibers with the Fluorescent Indicator Mag-Indo-1

Measurements of intracellular free magnesium concentration ([Mg 2+] i) were performed on enzymatically isolated skeletal muscle fibers from mice, using the fluorescent ratiometric indicator mag-indo-1. An original procedure was developed to calibrate the dye response within the fibers: fibers were first permeabilized with saponin in the presence of a given extracellular magnesium concentration and were then embedded in silicone grease. The dye was then pressure microinjected into the saponin-permeabilized silicone-embedded fibers, and fluorescence was measured. The results show that for all tested [Mg 2+], the value of the measured fluorescence ratio was higher than that found in aqueous solutions. Furthermore, the apparent binding curve that could be fit to the in vivo ratio data was shifted toward higher [Mg 2+] by a factor of ∼2. Using the in vivo calibration parameters, the mean resting [Mg 2+] i was found to be 1.53 ± 0.16 mM ( n = 7). In an attempt to gain insight into the myoplasmic magnesium buffering capacity, we measured, together with mag-indo-1 fluorescence, the current elicited by the application of carbamylcholine (CCh) to the endplate of isolated fibers, in the presence of a high extracellular magnesium concentration. The results show that, under these conditions, a change in [Mg 2+] i displaying a time course and amplitude qualitatively consistent with the CCh-induced inward current can be measured.

Free magnesium concentration in isolated rabbit hearts subjected to high dose isoproterenol infusion: a 31P NMR study

The hypothesis of magnesium deficiency in isoproterenol (ISO) induced myocardial injury has been investigated by 31P nuclear magnetic resonance spectroscopy. High energy phosphate concentrations, pHi, and intracellular free magnesium concentration ([Mg2+]i) were measured in isolated rabbit hearts perfused at constant flow and subjected to 10(-6)M isoproterenol during 30 min. Recent calibrations were used for [Mg2+]i measurements, and uncertainties on [Mg2+]i estimated values were calculated. During isoproterenol infusion, pHi, [PCr], and [ATP] decreased, while [P(i)] increased. When it was stopped, [PCr] completely repleted, whereas only a partial restoration was observed for pHi and [P(i)]. A rise of end-diastolic pressure and perfusion pressure expressed a contracture, concomitant with a lack of [ATP] recovery, which remained at 59 +/- 13% of the rest value. These results establish that 10(-6) M isoproterenol caused severe myocardial injury. [Mg2+]i increased from 0.70 mM at rest to 0.88 mM at the end of the isoproterenol period. Considering the estimated uncertainties on the [Mg2+]i values, this increase was not significant. After isoproterenol infusion, [Mg2+]i progressively decreased to reach 0.72 mM at 45 min recovery. It is concluded that isoproterenol myocardial toxicity may not be related to [Mg2+]i deficiency.

Magnesium sulphate improves neurologic outcome following severe closed head injury in rats

While recent evidence suggests that brain intracellular free magnesium concentration declines following severe diffuse traumatic brain injury, no studies have examined whether magnesium administration following such injury can improve subsequent neurologic outcome. The present study shows that MgSO 4 administered as a bolus at 30 min following severe closed head injury in rats significantly improves posttraumatic neurologic outcome as assessed by both rotarod and angleboard tests. Moreover, this improvement in outcome was evident with both intravenous and intramuscular drug administration. We conclude that parenteral administration of magnesium sulphate may be neuroprotective following severe closed head injury of a diffuse nature.