Magnesium Efflux Research Articles

Characterization of the calcium paradox in the isolated perfused frog heart: enzymatic, ionic, contractile and electrophysiological studies

The effect of perfusion temperature and duration of calcium deprivation on the occurrence of the calcium paradox was studied in the isolated frog heart. Loss of electrical and mechanical activity, ion fluxes, creatine kinase and protein release were used to define cell damage. Perfusion was performed at 22, 27, 32, and 37 degrees C, and calcium deprivation lasted 10, 20, 30, or 40 min. At 22 degrees C and 27 degrees C even a prolonged calcium-free perfusion failed to induce a calcium paradox. After 30 min of calcium-free perfusion at 37 degrees C ventricular activity ceased and a major contraction occurred followed by an increase in resting tension. During the 15-min re-perfusion period the release of creatine kinase was 158.24 +/- 2.49 IU.g dry wt-1, and the total amount of protein lost was 70.37 +/- 0.73 mg.g dry wt-1, while lower perfusion temperatures resulted in a decreased loss of protein and creatine kinase. Ion fluxes in the perfusion effluent indicate that during re-perfusion a massive calcium influx accompanied by a potassium and a magnesium efflux, and an apparent sodium efflux, occur at a perfusion temperature of 37 degrees C after 30 min of calcium deprivation. The results suggest that the basic principles and damaging effects of calcium overloading are common to both mammalian and frog hearts.

An ATP-dependent Na +/Mg 2+ countertransport is the only mechanism for Mg extrusion in squid axons

The components of magnesium efflux in squid axons have been studied under internal dialysis and voltage clamp conditions. The present report rules out the existence of an ATP-dependent, Na 0- and Mg 0-independent Mg 2+ efflux (ATP-dependent Mg 2+ pump) leaving the Mg 2+Na + exchange system as the only mechanism for Mg 2+ extrusion. The main features of the Mg 2+ efflux are: (1) The efflux is completely dependent on ATP. (2) The efflux can be activated either by external Na + (forward Mg 2+Na + exchange) or external Mg 2+ (Mg 2+Mg 2+ exchange). (3) The mobility of the Mg 2+ exchanger in the Na 0 +-loaded form is greater than that in the Mg 2+-loaded one. (4) In variance with the Na +Ca 2+ exchange mechanism, Mg 2+Mg 2+ exchange is not activated by external monovalent cations. (5) ATPγS replaces ATP in activating Mg 2+Na + exchange suggesting that a phosphorylation/dephosphorylation process regulates this transport mechanism.

Saturation kinetics of magnesium efflux across the rumen wall in heifers.

1. Absorption of magnesium from the temporarily-isolated, emptied and washed rumen of four 10-12-month-old heifers was measured at five different Mg concentrations (2.5, 5.0, 7.5, 12.5 and 20 mmol/l). 2. Net absorption of Mg was observed at all concentrations. Saturation occurred at a concentration of 12.5 mmol Mg/l. 3. Using an Eadie-Hofstee plot, the Michaelis-Menten constant (Km; 11.43 mmol/l) and maximal efflux (Vmax; 120.3 mumol/min) were estimated. 4. The phenomenon of saturation supports the assumption that Mg transport across the rumen wall of heifers is an active process.

Properties of Ca2+- or Mg2+-dependent ATPase in rat heart sarcolemma.

Rat heart sarcolemma was shown to hydrolyze ATP in the presence of Ca2+ or Mg2+; Ka values for Ca2+ and Mg2+ were in the range of 0.58-0.67 and 0.72-0.83 mM, whereas Vmax values were 33-38 and 21-28 mumol Pi/mg per hr, respectively. Both Ca2+ ATPase and Mg2+ ATPase showed low- and high-affinity sites for ATP; the Km value for the low-affinity sites for both enzyme activities was 300-325 microM, whereas Km values for high-affinity sites were 75-85 and 100-108 microM, respectively. The pattern of nucleotide hydrolysis in the presence of Ca2+ was found to be different from that with Mg2+. Although both high concentrations of ADP and Pi inhibited the enzyme activities, Mg2+ ATPase was more sensitive to ADP and less sensitive to Pi in comparison to Ca2+ ATPase. Storage of sarcolemma at about 0 degrees C showed a greater increase in ATP hydrolysis with Ca2+ than with Mg2+. The inhibitory effect of Mg2+ on Ca2+ ATPase, unlike that of Ni2+, Co2+, and Cu2+, was more than that on Mg2+ ATPase. Treatment of membranes with sodium dodecylsulfate or deoxycholate produced a greater reduction in Mg2+ ATPase than in Ca2+ ATPase. These results further support the view that Ca2+ ATPase and Mg2+ ATPase may be two separate enzymes in heart sarcolemma. It is suggested that Ca2+-dependent ATPase may be involved in opening calcium channels for the entry of calcium, whereas Mg2+ ATPase may serve as a Mg2+ pump mechanism for the efflux of magnesium from the cardiac cell.

Stimulation of sugar transport in rat soleus muscle by prolonged cooling at 0 degrees C.

The uptake of D-xylose by isolated rat soleus muscle (measured at 37 degrees C) was stimulated by prolonged cooling at 0 degrees C. The effect of cooling reached a maximum value after 3 h and was reversed on rewarming; reversal was temperature-dependent. Cooling stimulated xylose uptake sub-maximally compared with the effect of insulin (100 U/l). Xylose uptake in cooled muscle was further stimulated by insulin, but not by anoxia. The effect of cooling and its reversal were still demonstrable in the presence of ouabain (1 mmol/l), or when unidirectional efflux of calcium and magnesium from the muscle was induced by EDTA (5 mmol/l). The ionophore, A23187 (2.5 mg/l), depressed the effect of cooling in the presence of EDTA but not in the presence of EGTA. It is concluded that cooling disrupts and intracellular magnesium-p]ump and that muscle sugar transport is consequentially stimulated through an increase in cytoplasmic magnesium.

Efflux of magnesium and potassium ions from liver mitochondria induced by inorganic phosphate and by diamide.

Addition to rat liver mitochondria of 2 mM inorganic phosphate or 0.15 mM diamide, a thiol-oxidizing agent, induced an efflux of endogenous Mg2+ linear with time and dependent on coupled respiration. No net Ca2+ release occurred under these conditions, while a concomitant release of K+ was observed. Mg2+ efflux mediated either by Pi or low concentration of diamide was completely prevented by EGTA, Ruthenium red, and NEM. These reagents also inhibited the increased rate of state 4 respiration induced both by Pi and diamide. At higher concentrations (0.4 mM), diamide induced an efflux of Mg2+ which was associated also with a release of endogenous Ca2+. Under these conditions EGTA completely prevented Mg2+ and K+ effluxes, while they were only partially inhibited by Ruthenium red and NEM. It is assumed that Mg2+ efflux, occurring at low diamide concentrations or in the presence of phosphate, is dependent on a cyclic in-and-out movement of Ca2+ across the inner mitochondrial membrane, in which the passive efflux is compensated by a continuous energy linked reuptake. This explains the dependence of Mg2+ efflux on coupled respiration, as well as the increased rate of state 4 respiration. The dependence of Mg2+ efflux on phosphate transport is explained by the phosphate requirement for Ca2+ movement.

Magnesium efflux in dialyzed squid axons.

The efflux of Mg++ from squid axons subject to internal solute control by dialysis is a function of ionized [Mg], [Na], [ATP], and [Na]o. The efflux of Mg++ from an axon with physiological concentrations of ATP, Na, and Mg inside into seawater is of the order of 2-4 pmol/cm2s but this efflux is strongly inhibited by increases in [Na]i, by decreases in [ATP]i, or by decreases in [Na]o. The efflux of Mg++ is largely independent of [Mg]i when ATP is at physiological levels, but in the absence of ATP reaches half the value of Mg efflux in be presence of ATP when [Mg]i is about 4 mM and [Na] 40 mM. Half-maximum responses to ATP occur at about 350 micronM ATP into seawater with Na either present or absent. The Mg efflux mechanism has many similarities to the Ca efflux system in squid axons especially with respect to the effects of ATP, Nao, and Na on the flux. The concentrations of free Mg and Ca in axoplasm differ, however, by a factor of 10(5) while the observed fluxes differ by a factor of 10(2).

The efflux of magnesium from single crustacean muscle fibres.

1. In the large single muscle fibres from the barnacle Balanus nubilus, the total fibre Mg concentration was estimated as 15.1 m-mole/kg wet wt., of which about 3-3.5 m-mole/kg wet wt. was extracellular. The diffusible Mg, measured by internal sampling, was 11.5 m-mole/kg wet wt., of which at least half may be complexed to larger diffusible molecules. The free ionized Mg level was estimated as < 5 m-mole/kg wet wt.2. The loss of [(28)Mg]MgCl(2) from both Maia and Balanus muscle fibres following axial micro-injection approximated to first-order kinetics. The maximum rate constant for the loss was 1.51 +/- 0.20 (S.E.) x 10(-5) sec(-1) for Balanus (sixty-seven fibres) and 1.06 +/- 0.46 (S.E.) x 10(-5) sec(-1) for Maia (seven fibres) at 20-25 degrees C.3. The calculated Mg efflux was in the range 6-12 p-mole/cm(2).sec based on this rate constant, assuming isotopic equilibration internally and that the surface area of the fibres approximated to that of a simple cylinder. If account was taken of the area of the cleft system the efflux was reduced by about fifteen times.4. The diffusion coefficient for injected (28)Mg was estimated as 2-3 x 10(-6) cm(2) sec(-1), about half the value in free solution. Injections of 2 M-MgCl(2) or 200 mM-EDTA subsequent to the injection of the isotope caused about a 30% reduction in the tracer efflux.5. External application of salines containing 100 mM-Ca or Mg caused a rapid but reversible inhibition of the magnesium efflux. Similar effects were observed with salines containing 32 mM-Co or Mn chlorides or 1-2 mM-La or Gd chlorides. Polyarginine (200 mug/ml.) had no effect.6. The Mg efflux had a Q(10) of 3-4 over the temperature range of about 5-20 degrees C. It was irreversibly inhibited by the sulphydryl reagent NEM (1 mM), but PCMBS (0.2-2 mM) had no effect. Contractile agents (5 mM caffeine or 200 mM-K salines) and a variety of inhibitors of ion movement or active transport had no appreciable effect on the Mg efflux. Lowering the pH of the saline from 7 to 5 produced a 70% reduction in the efflux which was reversible over short periods of application.7. Replacement of external Na, but not Ca or Mg, with Li, choline or sucrose caused a rapid and partially reversible reduction of the Mg efflux, but increasing the internal Na by micro-injection in zero Na salines had no consistent effect. It is suggested that the extrusion of Mg from these muscle cells is largely dependent upon the inward movement of Na.

Intravenous triamterene in the treatment of acute digitalis intoxication.

It was recently found that the “potassium‐sparing” diuretic triamterene, given prophylactically, increases tolerance to digitalis intoxication. This study demonstrated that triamterene is also of therapeutic value in dogs acutely intoxicated with ouabain. Not only was the digitalis‐induced myocardial potassium efflux reversed, but also the efflux of magnesium was reversed. In the doses employed the clinical benefit was definite but not always permanent. Triamterene also seemed to stop the ouabain‐induced hypementilation and retching. A significant technical problem was the poor solubility and relatively low pH of the drug for intravenous administration. Triamterene has theoretic advantages over intravenous potassium chloride, as it affects membrane Na‐K dependent ATPase and may favorably influence the Ki/Ko ratio. The implications in man are clear.

A23187: A Divalent Cation Ionophore

A23187 is a carboxylic acid antibiotic which simultaneously uncouples oxidative phosphorylation and inhibits ATPase of rat liver mitochondria incubated in a magnesium-free medium. The antibiotic acts as a freely mobile carrier to transport calcium and magnesium, but not potassium, from an aqueous medium buffered at pH 7.4 into a bulk organic phase. A23187 causes a progressive release of endogenous magnesium and inorganic phosphate from mitochondria incubated with substrate, but little change in calcium or potassium content. If EDTA is added, A23187 severely depletes intramitochondrial calcium and magnesium in Release of State 4 succinate oxidation by A23187 is inhibited by lanthanum chloride, ruthenium red, ((ethylene bis)oxyethylene nitrilo)tetraacetic acid (EGTA), EDTA, MgCl2, and ATP. Inhibition by EGTA (or EDTA) of A23187′s uncoupling is reversed readily by calcium but not magnesium. In the presence of A23187 plus EGTA, mitochondria retain only 2 to 3 nmoles of Mg2+ per mg of protein but remain coupled and phosphorylate ADP at 80 to 85% of control rates with β-hydroxybutyrate or succinate as substrate. Under these conditions, coupled phosphorylation supported by glutamate, and ATP hydrolysis are severely depressed, but increasing concentrations of magnesium in the incubating medium prevent these inhibitions. It is proposed that a cyclic, energy-dissipating flux of mitochondrial calcium accounts for uncoupling by A23187, whereas inhibition of ATPase and glutamate oxidation and increased potassium permeability of mitochondria result from antibiotic-mediated magnesium efflux.

Thyroxine-induced stimulation of hepatic cell transport of calcium and magnesium.

The effect of L-thyroxine on the bidirectional transport of calcium and magnesium in rat liver was assessed in vitro. An increase of 34% in the fractional coefficient for calcium influx was observed 24 hr after the administration of 500 mug of thyroxine. Chronic treatment with thyroxine for 1 and 3 wk at a dose of 750 mug/wk resulted in increases in calcium influx of 57 and 51%, respectively. Calcium efflux was increased irregularly, by 14-26%. Magnesium transport measured in a similar system was not altered by 24 or 48 hr of treatment with thyroxine, but continuation of treatment for 1-3 wk resulted in increases in magnesium influx of 47-49%. Magnesium efflux was not significantly affected. Neither increased cellular binding of divalent cations nor enhanced protein synthesis could be incriminated in the stimulatory effect of thyroxine on divalent cation transport. Actinomycin-D and D,L-ethionine, inhibitors of protein synthesis, stimulated calcium and magnesium transport in liver independently of the effects of thyroxine. These data present the possibility that certain actions of thyroid hormone may be mediated or modulated by associated, direct changes in the cellular transport and intracellular concentrations of divalent cations.

An effect of penetrating ions on magnesium efflux from rat small intestine, in vitro

Incubation of isolated intestinal segments in varying concentrations of sodium, potassium, and lithium showed magnesium-ion displacement from the mucosal cells during and in the absence of glucose absorption. The extent of this displacement appears to be a function of monovalent cation entry into the epithelial surface as no loss of magnesium occurred when relatively non-permeable substitutes for electrolytes were used. It is suggested that a possible role of penetrating ions on the intestinal epithelium may be the unbinding of membrane magnesium for participation in regulating passive permeability, Magnesium unbinding may also be associated with activation of mucosal ATPase, possibly necessary for increased activity of the sodium pump.

Tissue distribution and transport of electrolytes Mg 28 and Ca 47 in hypermagnesemia

The effect of acute hypermagnesemia (9–14 mEq./L.) on the distribution of electrolytes Mg 28 and Ca 47 in various tissues was studied in renal-ligated dogs. The sodium content of 5 of 8 tissues was decreased by 10–30 per cent, the potassium content of 3 tissues was increased 5–30 per cent and the acid-soluble phosphorus concentration of 2 tissues was decreased by 15 per cent. Total and/or exchangeable cellular concentrations of calcium were decreased by 25–35 per cent in skeletal muscle, myocardium, brain and pancreas. Total and/or exchangeable cellular concentrations of magnesium were increased by 8–50 per cent in skeletal muscle, nerve, pancreas and aorta and were unchanged in liver, myocardium and brain. These data suggest that magnesium has inhibitory effects on the cellular transport of electrolytes which are similar to those of calcium. The cellular influx of calcium is inhibited by magnesium. The stability of the cellular concentrations of magnesium in relation to the degree of hypermagnesemia is compatible with in vitro studies in rat liver indicating that the fractional rate of cellular influx of magnesium is decreased and the fractional rate of cellular efflux of magnesium is increased during extreme hypermagnesemia.

The effect of cold shock and deep-freezing on the concentration of major cations in spermatozoa.

Summary. Cold-shocking and deep-freezing bull and ram semen caused an influx of sodium to and an efflux of potassium and magnesium from the spermatozoa. The movement of these cations was accentuated when the semen was diluted 1 : 8 in sodium phosphate buffers. Calcium is actively accumulated in the spermatozoa of these two species after cold-shocking. No significant changes in the cation content of human, dog, rabbit or fowl spermatozoa were found after cold-shocking. There was no great disturbance of the cation status of human and fowl spermatozoa, even after deep-freezing. Lecithin prevented calcium accumulation in bull and ram spermatozoa on cold-shocking, and glycerol also had this effect on cold-shocked bull spermatozoa. An egg-yolk-citrate/glycerol-fructose diluent protected bull and ram spermatozoa to a considerable extent from the influx of sodium and the efflux of potassium that occurs after deep-freezing to −79°C.

The effect of dilution on the concentration of sodium, potassium, calcium and magnesium in ram and bull spermatozoa.

Summary. Mild dilution (<1·8) of ram and bull semen in isotonic sodium phosphate diluent of pH 7·0 caused an influx of sodium and an efflux of potassium, calcium and magnesium from the spermatozoa. Equilibrium was reached soon after dilution and little further change in the concentration of these cations was observed over 3 hr. Similar changes occurred when ram and bull semen was diluted in tris and veronal diluents of pH 7; however, calcium was found to accumulate in ram spermatozoa diluted in tris. Diluting semen in buffers of pH range 8·5 to 5·5 indicated that potassium loss from the spermatozoa increased with increasing pH. Greater dilution (1:100) and exhaustive washing (four times) had a similar but more severe effect on the cation concentration of ram and bull spermatozoa. A large influx of sodium was accompanied by a marked depletion of potassium which tended to be further depleted over 3 hr.