Decline in intracellular free Mg2+ is associated with irreversible tissue injury after brain trauma

Much of the tissue damage resulting from trauma to the central nervous system appears to result from secondary, delayed biochemical changes that follow primary mechanical injury. However, the early biochemical events remain to be elucidated. In the present studies, we have used phosphorus (31P) magnetic resonance spectroscopy (MRS) to examine in vivo, the temporal changes in brain intracellular free Mg2+ concentration following fluid percussion head injury in rats. We report that injury caused a profound and rapid decrease in intracellular free Mg2+ which was significantly correlated with the severity of injury. At high levels of injury, the decrease in intracellular free Mg2+ concentration was associated with a decrease in total Mg2+ concentration as determined by atomic absorption spectrophotometry. Prophylactic treatment with MgSO4 prevented the post-traumatic decrease in intracellular free Mg2+ and resulted in a significant improvement in acute neurological outcome. Because magnesium is essential for a number of critical enzyme reactions, including those of glycolysis, oxidative and substrate level phosphorylation, protein synthesis, and phospholipid synthesis, changes in free Mg2+ after brain trauma may represent a critical early factor leading to irreversible tissue damage.     

Multinuclear NMR studies of intracellular cations in perfused hypertensive rat kidney.

We have investigated hypertension-associated alterations in intracellular cations in the kidney by measuring intracellular pH, free Mg2+, free Ca2+, and Na+ concentrations in perfused normotensive and hypertensive rat (8-14 weeks old) kidneys using 31P, 19F, and double quantum-filtered (DQ) 23Na NMR. The effects of both anoxia and ischemia on the 23Na DQ signal confirmed its ability to detect changes in intracellular Na+. However, there was a sizable contribution of the extracellular Na+ to the 23Na DQ signal of the kidney. The intracellular free Ca2+ concentration, measured using 19F NMR and 5,5'difluoro-1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid, also increased dramatically during ischemia; the increase could be partly reversed by reperfusion. No significant differences were found between normotensive and hypertensive kidneys in the ATP level, intracellular pH, intracellular free Mg2+, and the 23Na DQ signal or in the extent of the extracellular contribution to the 23Na DQ signal. Oxygen consumption rates were also similar for the normotensive (5.02 +/- 0.46 mumol of O2/min/g) and hypertensive (5.47 +/- 0.42 mumol O2/min/g) rat kidneys. The absence of a significant difference in intracellular pH, Na+ concentration, and oxygen consumption between normotensive and hypertensive rat kidneys suggests that an alteration in the luminal Na+/H+ antiport activity in hypertension is unlikely. However, a highly significant increase (64%, p less than 0.01) in free Ca2+ concentration was found in perfused kidneys from hypertensive rats (557 +/- 48 nM, blood pressure = 199 +/- 5 mmHg, n = 6) compared with normotensive rats (339 +/- 21 nM, blood pressure = 134 +/- 6, n = 4) indicating altered renal calcium homeostasis in essential hypertension. An increase in intracellular free Ca2+ concentration without an accompanying change in the intracellular Na+ suggests, among many possibilities, that the Ca2+/Mg(2+)-ATPase may be inhibited in the hypertensive renal tissue.     

Competition between Li+ and Mg2+ in neuroblastoma SH-SY5Y cells: a fluorescence and 31P NMR study.

Because Mg2+ and Li+ ions have similar chemical properties, we have hypothesized that Li+/Mg2+ competition for Mg2+ binding sites is the molecular basis for the therapeutic action of lithium in manic-depressive illness. By fluorescence spectroscopy with furaptra-loaded cells, the free intracellular Mg2+ concentration within the intact neuroblastoma cells was found to increase from 0. 39 +/- 0.04 mM to 0.60 +/- 0.04 mM during a 40-min Li+ incubation in which the total intracellular Li+ concentration increased from 0 to 5.5 mM. Our fluorescence microscopy observations of Li+-free and Li+-loaded cells also indicate an increase in free Mg2+ concentration upon Li+ incubation. By 31P NMR, the free intracellular Mg2+ concentrations for Li+-free cells was 0.35 +/- 0. 03 mM and 0.80 +/- 0.04 mM for Li+-loaded cells (final total intracellular Li+ concentration of 16 mM). If a Li+/Mg2+ competition mechanism is present in neuroblastoma cells, an increase in the total intracellular Li+ concentration is expected to result in an increase in the free intracellular Mg2+ concentration, because Li+ displaces Mg2+ from its binding sites within the nerve cell. The fluorescence spectroscopy, fluorescence microscopy, and 31P NMR spectroscopy studies presented here have shown this to be the case.     

Magnesium sulfate attenuates increased blood-brain barrier permeability during insulin-induced hypoglycemia in rats

Magnesium probably protects brain tissue against the effects of cerebral ischemia, brain injury and stroke through its actions as a calcium antagonist and inhibitor of excitatory amino acids. The effects of magnesium sulfate on cerebrovascular permeability to a dye, Evans blue, were studied during insulin-induced hypoglycemia with hypothermia in rats. Hypoglycemia was induced by an intramuscular injection of insulin. After giving insulin, each animal received MgSO4 (270 mg/kg) ip, followed by a 27 mg/kg dose every 20 min for 2.5 h. Plasma glucose and Mg2+ levels of animals were measured. Magnesium concentrations increased in the serum following MgSO4 administration (6.05+/-0.57 vs. 2.58+/-0.14 mg/dL in the Mg2+ group, and 7.14+/-0.42 vs. 2.78+/-0.06 mg/dL in the insulin + Mg2+ group, P < 0.01). Plasma glucose levels decreased following hypoglycemia (4+/-0.66 vs. 118+/-2.23 mg/dL in the insulin group, and 7+/-1.59 vs. 118+/-4.84 mg/dL in the insulin + Mg2+ group, P < 0.01). Blood-brain barrier permeability to Evans blue considerably increased in hypoglycemic rats (P < 0.01). In contrast, blood-brain barrier permeability to Evans blue was significantly reduced in treatment of hypoglycemic rats with MgSO4 (P < 0.01). These results indicate that Mg2+ greatly reduced the passage of exogenous vascular tracer bound to albumin into the brain during hypoglycemia with hypothermia. Mg2+ could have protective effects on blood-brain barrier permeability against insulin-induced hypoglycemia.     

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

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

Binding of S-adenosylhomocysteine to hydroxyindole O-methyltransferase

Mg2+-selective microelectrodes have been used to measure the intracellular free Mg2+ concentration in frog skeletal muscle fibers. Glass capillaries with a tip diameter of less than 0.4 micron were backfilled with the Mg2+ sensor, ETH 1117. In the absence of interfering ions, they gave Nernstian responses between 1 and 10 mM free Mg2+. In the presence of an ionic environment resembling the myoplasm, the microelectrode response was sub Nernstian (18-24 mV) but still useful. The electrodes were calibrated before and after muscle-fiber impalements . In quiescent fibers from sartorius muscle (Rana pipiens), with resting membrane potentials not less than -82 mV, the intracellular free Mg2+ concentration was 3.8 +/- 0.41 (S.E.) mM (n = 58) at 22 degrees C. No significant change in the intracellular free Mg2+ was observed following extensive (approx. 6 h) incubation in Mg2+-free media. Increasing the external concentration of magnesium from 4 to 20 mM (approx. 15 min) produced a slow and small enhancement (1.8 mM) of [Mg2+]i, which was fully reverted when the divalent cation was removed from the bathing solution. No change in ionic magnesium resting concentration was observed when the muscle fibers were treated either with caffeine 3 mM or with Na+-free solutions. In depolarized muscle fibers (-23 +/- 2.7 mV) treated with 100 mM K+, the myoplasmic [Mg2+] was 3.7 +/- 0.45 (S.E.) mM, n = 6, immediately after the spontaneous relaxation of the contracture. Similar determinations in muscle fibers during stimulation at low frequency (5 Hz), and after fatigue development, showed no changes in the concentration of free cytosolic Mg2+. These results point out that [Mg2+]i is not modified under these three different experimental conditions.     

Secretagogue-induced mobilization of an intracellular Mg2+ pool in rat sublingual mucous acini.

The regulation of the intracellular free Mg2+ concentration ([Mg2+]i) was monitored in rat sublingual mucous acini using dual wavelength microfluorometry of the Mg(2+)-sensitive dye mag-fura-2. Acini attached to coverslips and superfused continuously with a Mg(2+)-containing medium (0.8 mM) have a steady-state [Mg2+]i of 0.35 +/- 0.01 mM. Adjusting the extracellular Mg2+ concentration to 0 and 10 mM or removing extracellular Na+ did not alter the resting [Mg2+]i. Stimulation with the Ca(2+)-mobilizing, muscarinic agonist, carbachol, induced a sustained increase in [Mg2+]i (approximately 50%; t1/2 < 20 s; Kd approximately 1.5 microM), the magnitude and the duration of which were unchanged in Mg(2+)-depleted medium indicating that the rise in [Mg2+]i was generated by Mg2+ release from an intracellular Mg2+ pool. Forskolin, which increases the intracellular cAMP content, produced a small, transient increase in the [Mg2+]i (< 10%). Muscarinic stimulation in a Ca(2+)-free medium blunted the initial increase in [Mg2+]i by approximately 50%, whereas the sustained increase in [Mg2+]i was lost. When the muscarinic-induced increase in [Ca2+]i was blocked by 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate, an inhibitor of the agonist-sensitive intracellular Ca2+ release pathway, both the initial and the sustained phases of the increase in [Mg2+]i were virtually eliminated. Thapsigargin and 2,5-di-(terbutyl)-1,4-benzohydroquinone, which increase [Ca2+]i by inhibiting microsomal Ca(2+)-ATPase, caused a dramatic increase in [Mg2+]i. Stimulation in a Na(+)-free medium or in the presence of bumetanide, an inhibitor of Na+/K+/2Cl- cotransport, blunted the agonist-induced rise in [Mg2+]i (approximately 50%), whereas ouabain, a Na+,K(+)-ATPase inhibitor, had no significant effect. FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), a mitochondrial uncoupler, mobilized an intracellular Mg2+ pool as well. The carbachol-induced increase in [Mg2+]i was markedly inhibited by FCCP (approximately 80%), suggesting that the same pool(s) of Mg2+ were primarily involved. The above results provide strong evidence that Ca(2+)-mobilizing agonists increase cytoplasmic free [Mg2+] by releasing an intracellular pool of Mg2+ that is associated with a rise in the [Na+]i.     

Estrogen-induced changes in high-energy phosphate metabolism in rat uterus: 31P NMR studies

Changes in the concentrations of high-energy phosphate metabolites were measured by 31P NMR spectroscopy of surviving rat uteri from 0-48 h following estrogen administration. Concentrations (millimoles per kilogram wet weight) of these metabolites in the untreated immature uterus, measured at 4 degrees C, were found to be the following: creatine phosphate (CP), 2.1 +/- 0.2; nucleoside triphosphates, mainly adenosine 5'-triphosphate (ATP), 4.6 +/- 0.4; phospho monoesters, primarily sugar phosphates (SP), 5.4 +/- 0.7; and inorganic phosphate (Pi), 0.8 +/- 0.4. Adenosine 5'-diphosphate (ADP) concentration was estimated to be approximately 40 mumol/kg wet weight from the assumed equilibrium of the creatine kinase reaction. The concentration of CP, and to lesser extent ATP and SP, declined within the first 1.5-3 h after injection of 17 beta-estradiol, returned to control values between 6 and 12 h, and then increased, reaching maximal concentrations at 24 h. From the fractions of the total soluble ATP in free and Mg2+-bound forms, [free Mg2+] in the untreated uterus was estimated to be 0.2-0.4 mmol/kg wet weight. An increase in [free Mg2+] in the uterus was detected 1.5 h after estrogen injection. A subsequent parallel increase in the ratio of ATP to CP concentrations suggests that estrogen can also affect the apparent creatine kinase equilibrium by modulating [free Mg2+].     

Phosphatic metabolites, intracellular pH and free [Mg2+] in single, intact porcine carotid artery segments studied by 31P-NMR

Superfused porcine carotid artery segments (approximately 7 cm lengths) were analyzed by 31P-NMR spectroscopic methods to characterize the 31P spectrum of arterial smooth muscle and to determine the influence of passive stretch (intraluminal pressurization, 95-100 mmHg) on cellular phosphatic metabolite levels, intracellular pH and free magnesium concentration ([Mg2+free]i). Equilibrated, single, intact arteries were studied under steady-state, constant flow conditions at 37 degrees C. Phosphoethanolamine, phosphocholine, inorganic phosphate (Pi), phosphocreatine (PCr) and nucleoside triphosphates (NTP), primarily ATP, were the principle metabolites detected in the 31P-NMR spectrum of intact arterial smooth muscle. The concentration of these metabolites and intracellular pH, as determined from the referenced chemical shift of Pi, were unaffected by pressurization. The PCr:Pi ratios determined for nonpressurized (flaccid) and pressurized arteries were 1.2 +/- 0.1 and 1.3 +/- 0.3, respectively. Intracellular pH averaged 7.02 +/- 0.02 (mean +/- 1 S.D.) for flaccid arteries vs. 7.03 +/- 0.05 for pressurized arteries. The upfield chemical shift of the beta-ATP peak, which has been described in other types of smooth muscle, was also observed in these experiments. Interestingly, pressurization significantly shifted the resonance position of this peak, which was interpreted to represent a change in [Mg2+free]i. The average [Mg2+free]i of flaccid artery preparations was computed to be 0.54 +/- 0.03 x 10(-3) M, as compared to 0.99 +/- 0.10 x 10(-3) M for pressurized arteries. This change in [Mg2+free]i was evident within the first hour following pressurization and persisted thereafter. These findings suggest that altering the resting length of vascular smooth muscle produces a change in [Mg2+free]i. This shift in free Mg2+ levels may act as a metabolic signal triggering a change in vascular smooth muscle metabolism, an effect which has been reported to occur in smooth muscle in response to stretch.     

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

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

Activation of Na+/Mg2+ antiport in thymocytes by cAMP.

Mg2+ efflux from Mg(2+)-loaded rat thymocytes was stimulated by 0.1 mM dibutyryl cAMP (db cAMP). The activation of Mg2+ efflux by db cAMP was more expressed at lower Mg(2+)-loading. cAMP induced only a very small increase in the concentration of intracellular free Mg2+ which cannot explain the activation of Na+/Mg2+ antiport. From these results it was concluded that cAMP increases the affinity of the Na+/Mg2+ antiporter for intracellular Mg2+, probably by phosphorylation.     

31P-NMR studies of metabolite compartmentation in Fasciola hepatica

Fasciola hepatica, the common liver fluke, is an anaerobic parasitic worm. Possible compartmentation of metabolites between different cell types, metabolic compartments, and free and macromolecule-bound species was investigated using 31P-NMR. A spectrum of the intact worm shows unusual metabolic features, among which are large amounts of glycerolphosphorylcholine, phospholipids mobile on the NMR time-scale, and free cytosolic ADP. Spectra from cells as different as those in oral sucker tissue and eggs showed similar features. Acidosis after serotonin administration was associated with parallel changes in chemical shifts of intracellular Pi and glucose 6-phosphate, suggesting that they are in the same metabolic compartment. Although 13.4 +/- 1.1 mumol/g wet wt. (n = 3) Mg2+ is present in fluke tissue, a considerable fraction is sequestered out of the cytosol. The intracellular free [Mg2+] was independently estimated from the chemical shifts of ATP and ADP as 1.6 +/- 0.5 mM and 2.9 +/- 0.7 mM, respectively. Quantitation of observable phosphate-containing metabolites in whole tissue and in perchlorate extracts demonstrated that 60% of the total ADP and 50% of the total Pi are 'NMR-invisible' in the intact fluke and therefore probably bound to macromolecules in the cells. The apparent ATP/ADP X Pi free concentration ratio is much lower in this anaerobic tissue than in mammalian oxidative tissues.     

Apparent intracellular Mg2+ buffering in neurons of the leech Hirudo medicinalis

The apparent intracellular Mg2+ buffering, or muffling (sum of processes that damp changes in the free intracellular Mg2+ concentration, [Mg2+](i), e.g., buffering, extrusion, and sequestration), was investigated in Retzius neurons of the leech Hirudo medicinalis by iontophoretic injection of H+, OH-, or Mg2+. Simultaneously, changes in intracellular pH and the intracellular Mg2+, Na+, or K+ concentration were recorded with triple-barreled ion-selective microelectrodes. Cell volume changes were monitored measuring the tetramethylammonium (TMA) concentration in TMA-loaded neurons. Control measurements were carried out in electrolyte droplets (diameter 100-200 microm) placed on a silver wire under paraffin oil. Droplets with or without ATP, the presumed major intracellular Mg2+ buffer, were used to quantify the pH dependence of Mg2+ buffering and to determine the transport index of Mg2+ during iontophoretic injection. The observed pH dependence of [Mg2+](i) corresponded to what would be expected from Mg2+ buffering through ATP. The quantity of Mg2+ muffling, however, was considerably larger than what would be expected if ATP were the sole Mg2+ buffer. From the decrease in Mg2+ muffling in the nominal absence of extracellular Na+ it was estimated that almost 50% of the ATP-independent muffling is due to the action of Na+/Mg2+ antiport.     

Axoplasmic free magnesium levels and magnesium extrusion from squid giant axons

The free magnesium concentration in the axoplasm of the giant axon of the squid, Loligo pealei, was estimated by exploting the known sensitivity of the sodium pump to intracellular Mg2+ levels. The Mg- citrate buffer which, when injected into the axon, resulted in no change in sodium efflux was in equilibrium with a Mg2+ level of about 3- -4 mM. Optimal [Mg2+] for the sodium pump is somewhat higher. Total magnesium content of axoplasm was 6.7 mmol/kg, and that of hemolymph was 44 mM. The rate coefficient for 28Mg efflux was about 2 X 10(-3) min-u for a 500-mum axon at 22-25degreesC, with a very high temperature coefficient (Q10=4-5). This efflux is inhibited 95% by injection of apyrase and 75% by removal of external sodium, and seems unaffected by membrane potential or potassium ions. Increased intracellular ADP levels do not affect Mg efflux nor its requirement for Na+/o, but extracellularl magnesium ions do. Activation of 28Mg efflux by Na+/o follows hyperbolic kinetics, with Mg2+/o reducing the affinity of the system for Na+/o. Lanthanum and D600 reversibly inhibit Mg efflux. In the absence of both Na+ and Mg2+, but not in their presence, removal of Ca2+ from the seawater vastly increased 28Mg efflux; this efflux was also strongly inhibited by lanthanum. A small (10(-14) mol cm-2) extra Mg efflux accompanies the conduction of an action potential.     

Effect of anoxia on intracellular ATP, Na+i, Ca2+i, Mg2+i, and cytotoxicity in rat hepatocytes.

The effects of anoxia were studied in freshly isolated rat hepatocytes maintained in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer (KHB). Cytosolic free calcium (Ca2+i) was measured with aequorin, intracellular sodium (Na+i) with SBFI, intracellular pH (pHi) with BCECF, lactic dehydrogenase (LDH) by the increase in NADH absorbance during lactate oxidation to pyruvate, ATP by 31P NMR spectroscopy in real time, and intracellular free Mg2+ (Mg2+i) from the chemical shift of beta-ATP relative to alpha-ATP in the NMR spectra. Anoxia was induced by perfusing the cells with KHB saturated with 95% N2, 5% CO2. After 1 h of anoxia, beta-ATP fell 66%, and 85% after 2 h, while the Pi/ATP ratio increased 10-fold from 2.75 to 28.3. Under control conditions, the resting cytosolic free calcium was 127 +/- 6 nM. Anoxia increased Ca2+i in two distinct phases: a first rise occurred within 15 min and reached a mean value of 389 +/- 35 nM (p less than 0.001). A second peak reached a maximum value of 1.45 +/- 0.12 microM (p less than 0.001) after 1 h. During the first hour of anoxia, Na+i increased from 15.9 +/- 2.4 mM to 32.2 +/- 1.2 mM (p less than 0.001), Mg2+i doubled from 0.51 +/- 0.05 to 1.12 +/- 0.01 mM (p less than 0.001), and pHi decreased from 7.41 +/- 0.03 to 7.06 +/- 0.1 (p less than 0.001). LDH release doubled during the first hour and increased 6-fold during the second hour of anoxia. Upon reoxygenation, ATP, Ca2+i, Mg2+i, Na+i, and LDH returned near the control levels within 45 min. To determine whether the increased LDH release was related to the rise in Ca2+i, and whether the increased Ca2+i was caused by Ca2+ influx, the cells were perfused with Ca(2+)-free KHB (+ 0.1 mM EGTA) during the anoxic period. After 2 h of anoxia in Ca(2+)-free medium, beta-ATP again fell 90%, but Ca2+i, after the first initial peak, fell below control levels, and LDH release increased only 2.7-fold. During reoxygenation, Ca2+i, ATP, Na+i, and LDH returned near the control levels within 45 min. These results suggest that the rise in Ca2+i induced by anoxia is caused by an influx of Ca2+ from the extracellular fluid, and that LDH release and cell injury may be related to the resulting rise in Ca2+i.     

Effects of intracellular and extracellular concentrations of Ca2+, K+, and Cl- on the Na+-dependent Mg2+ efflux in rat ventricular myocytes

Intracellular Mg2+ concentration ([Mg2+]i) was measured in rat ventricular myocytes with the fluorescent indicator furaptra (25 degrees C). After the myocytes were loaded with Mg2+, the initial rate of decrease in [Mg2+]i (initial Delta[Mg2+]i/Deltat) was estimated upon introduction of extracellular Na+, as an index of the rate of Na+-dependent Mg2+ efflux. The initial Delta[Mg2+]i/Deltat values with 140 mM [Na+]o were essentially unchanged by the addition of extracellular Ca2+ up to 1 mM (107.3+/-8.7% of the control value measured at 0 mM [Ca2+]o in the presence of 0.1 mM EGTA, n=5). Intracellular loading of a Ca2+ chelator, either BAPTA or dimethyl BAPTA, by incubation with its acetoxymethyl ester form (5 microM for 3.5 h) did not significantly change the initial Delta[Mg2+]i/Deltat: 115.2+/-7.5% (seven BAPTA-loaded cells) and 109.5+/-10.9% (four dimethyl BAPTA loaded cells) of the control values measured in the absence of an intracellular chelator. Extracellular and/or intracellular concentrations of K+ and Cl- were modified under constant [Na+]o (70 mM), [Ca2+]o (0 mM with 0.1 mM EGTA), and membrane potential (-13 mV with the amphotericin-B-perforated patch-clamp technique). None of the following conditions significantly changed the initial Delta[Mg2+]i/Deltat: 1), changes in [K+]o between 0 mM and 75 mM (65.6+/-5.0% (n=11) and 79.0+/-6.0% (n=8), respectively, of the control values measured at 140 mM [Na+]o without any modification of extracellular and intracellular K+ and Cl-); 2), intracellular perfusion with K+-free (Cs+-substituted) solution from the patch pipette in combination with removal of extracellular K+ (77.7+/-8.2%, n=8); and 3), extracellular and intracellular perfusion with K+-free and Cl--free solutions (71.6+/-5.1%, n=5). These results suggest that Mg2+ is transported in exchange with Na+, but not with Ca2+, K+, or Cl-, in cardiac myocytes.     

Alterations in Ionized and Total Blood Magnesium After Experimental Traumatic Brain Injury

Experimental evidence suggests that magnesium plays a role in the pathophysiological sequelae of brain injury. The present study examined the variation of blood ionized and total magnesium, as well as potassium, sodium, and ionized calcium, after experimental fluid percussion brain injury in rats. Blood ionized magnesium concentration significantly declined from 0.45 +/- 0.02 to 0.32 +/- 0.02 mM by 30 min postinjury and stayed depressed for the 24-h study period in vehicle-treated rats. Blood total magnesium concentration was 0.59 +/- 0.01 mM and remained stable over time in brain-injured vehicle-treated animals. When magnesium chloride (125 micromol/rat) was administered 1 h postinjury, ionized magnesium levels were restored by 2 h postinjury and remained at normal values up to 24 h following brain trauma. Magnesium treatment also significantly reduced posttraumatic neuromotor impairments 1 and 2 weeks after the insult, but failed to attenuate spatial learning deficits. A significant positive and linear correlation could be established between ionized magnesium levels measured 24 h postinjury and neuromotor outcome at 1 and 2 weeks. We conclude that acute ionized magnesium measurement may be a predictor of long-term neurobehavioral outcome following head injury and that delayed administration of magnesium chloride can restore blood magnesium concentration and attenuate neurological motor deficits in brain-injured rats.     

Basal intracellular free Mg2+ concentration in smooth muscle cells of guinea pig tenia cecum: intracellular calibration of the fluorescent indicator furaptra

Longitudinal muscle strips dissected from tenia cecum of guinea pig were loaded with the Mg2+ indicator, furaptra, and the relation between the fluorescent ratio signal (R) and cytoplasmic free Mg2+ concentration ([Mg2+]i) was studied in smooth muscle cells at 25 degrees C. After the application of ionophores (4-bromo-A23187, monensin, and nigericin), a small immediate offset of R (deltaRjump) was followed by a slow change in R (deltaRslow), which reached a steady level within 2-5 h. The deltaRjump was independent of Mg2+ concentration in solution ([Mg2+]o), and was thought to be unrelated to the change in [Mg2+]i. The direction of the deltaRslow depended on [Mg2+]o with a reversal at approximately 1 mM [Mg2+]o. The intracellular calibration curve was constructed from the steady levels of deltaRslow, and the dissociation constant was 5.4 mM. With the intracellular calibration curve and correction for the deltaRjump, basal [Mg2+], was estimated to be 0.98 +/- 0.05 mM (mean +/- SE, n = 12). When the same calibration was applied to A7r5 cells and rat ventricular myocytes, estimates of basal [Mg2+]i of these cells were 0.74 +/- 0.02 mM (n = 33) and 1.13 +/- 0.06 mM (n = 9), respectively. These results suggest that the basal [Mg2+] level is approximately 1 mM at least in some types of smooth muscle cells, as generally found in striated muscles.     

Ca2+, Mg2+ and (adenosine diphosphate ribose)n in cellular response to irradiation

A model of cellular response to irradiation involving adenosine diphosphate ribosylation (ADP-ribosylation) is proposed. Its main assumptions are (a) control of accessibility of sites for ADP-ribosylation in chromatin by free Ca2+/Mg2+ ratio; and (b) regulation of the Ca2+/Mg2+ ratio by factors affecting intracellular free Ca2+ concentration; the regulation would be mediated by mitochondria. The model seeks to explain the mechanism of action of radiomodifiers such as caffeine, local anaesthetics, polyamines and 2,4-dinitrophenol.     

19F-NMR study of the effect of lead on intracellular free calcium in human platelets

Lead has been shown to affect calcium homeostasis. However, there is no prior evidence to indicate an effect of low concentrations of lead in the environment (approximately 1 microM) on the intracellular free Ca2+ concentration in any human tissue. We have investigated the effect of lead on the intracellular free Ca2+ concentration of human blood platelets using 19F-NMR and a fluorinated intracellular Ca2+ indicator. We report a basal intracellular free Ca2+ value of 172 +/- 8 nM. Treatment with 1, 5, 10 and 25 microM Pb2+ resulted in average increases in intracellular free Ca2+ of 39%, 91%, 135% and 172%, respectively. The percent increase in intracellular free Ca2+ was linearly and positively correlated with the log of Pb2+ concentration. Using atomic absorption spectroscopy, a significant increase in total calcium of approx. 10 nmol/mg protein was found in 25 microM Pb2+ treated platelets. This indicates that influx of external Ca2+ contributes to the observed increase in free Ca2+. The results provide an explanation for the previously reported effects of lead on platelet function, and suggest a mechanism for low level lead-induced hypertension.