H. Ebel

Magnesium Metabolism: A Review

The main literature concerning the physiology and biochemistry as well as the pathophysiology and pathobiochemistry of magnesium is reviewed, including: Distribution and physico-chemical state of magnesium in the extracellular and intracellular fluid as well as in the subcellular organelles (membranes, mitochondria, microsomes, ribosomes). Intestinal resorption, transport across membranes and excretion by the kidney. Hormonal regulation of magnesium distribution and its clinical disturbances. Biochemical mechanism and the clinical effects of hypo- and hypermagnesemia.

Role of Magnesium in Cardiac Disease

Some aspects of the pathogenesis of cardiac disease are reviewed in the light of current knowledge of the physiological and biochemical actions of magnesium (Mg2+) on heart function. Several authors have reported a reduction of the human myocardial Mg2+ content in areas with (Mg2+ poor) soft water, without a significant alteration of Mg2+ in serum or skeletal muscle. In human during myocardial infarction, there is a reduction of Mg2+ content even in noninfarcted areas of the myocardium, followed by a transient reduction of serum Mg2+ concentration. These effects are explained by the action of catecholamines on the myocardial cells, resulting in a loss of Mg2+ accompanied by a catecholamine-induced urinary Mg2+ loss and/or increased lipolysis which binds Mg2+ as Mg2+ soaps in the adipocytes. As a consequence serum Mg2+ may be decreased. A reduced serum Mg2+ concentration may enhance the action of catecholamines on the heart muscle as well as the action of vasopressive hormones, thus provoking contraction of coronary artery smooth muscle cells and favouring the development of arrhythmia.

Isolation of the basal and lateral plasma membranes of rat kidney tubule cells.

A method was developed to isolate renal basolateral membranes from cortical kidney tubule cells of single rats. The isolated membrane fraction was characterized by the measurement of marker enzyme activities and by electron microscopy. 1. After centrifugation of crude plasma membranes on a discontinuous sucrose density gradient the basolateral membranes accumulated at a sucrose density of p= 1.14-1.15 g/ml. The yield was 147 mug membrane protein/g kidney wet weight. Protein recovery was 0.1%. 2. (Na+ + K+)-ATPase was enriched 22-fold from the homogenate. The recovery was 2.6%. The (Na+ + K+)/Mg2+-ATPase ratio was 4.1. 3. The contamination by brush borders was small. Alkaline phosphatase was 1.6-fold enriched and 0.2% was recovered. Aminopeptidase was 1-fold enriched with a recovery of 0.1%. The contamination by mitochondria, lysosomes and endoplasmic reticulum was negligible. 4. In electron micrographs the basolateral membranes showed a typical triple layered profile and were characterized by the presence of junctional complexes, gap junctions or tight junctions.

Characterization of Mg2+ transport in brush border membrane vesicles of rabbit ileum studied with mag-fura-2

Mg2+ transport in rabbit ileal brush border membrane vesicles (BBMV) was characterized by means of a modified mag-fura-2 technique. In the presence of an i>o Na+ gradient, BBMV showed a saturable Mg2+ uptake with a Km of 1.64 mmol l-1. There was no evidence of an overshoot. K+, Li+, and choline+ were as effective as Na+ in stimulating Mg2+ transport. In contrast, only a small amount of Mg2+ transport was observed in the presence either of an o>i Na+ gradient, or in an Na+ equilibrium or in the absence of Na+. Moreover, the findings that Na+ efflux was not stimulated but inhibited by outside Mg2+ and that the nonfluorescent amiloride-analogues DMA and EIPA did not affect Mg2+ transport do not favour the idea of an Mg2+/Na+ antiport system. At Cl- equilibrium, independent of the Na+ gradient, the rate of Mg2+ transport was markedly suppressed compared with the transport rate noted in the presence of an i>o Cl- gradient. The stimulating effect of inside anions could be enhanced by SCN- and decreased by SO2-4. Furthermore, nonfluorescent anion transport antagonist H2-DIDS stimulated Mg2+ transport. These findings indicate that Mg2+ transport can be modulated by inside anions. Mg2+ transport appeared to be electroneutral because it was not dependent on membrane potential. Mg2+ transport was neither stimulated by Bay K8644, a Ca2+ channel agonist, nor inhibited by verapamil, diltiazem, nifedipine and imipramine, the Ca2+ channel antagonists. It, therefore, seems unlikely that Mg2+ uses the Ca2+ transport system.

Plasma cell membranes of the rat kidney

SummaryA fraction enriched with plasma cell membranes (PMF) was isolated from rat kidney homogenate by differential centrifugation. Before NaJ treatment electron micrographs of the preparation showed a membraneous fraction with only a small contamination of mitochondria. After treatment with NaJ the residual PMF exhibited a low microsomal glucose 6-phosphatase activity. Marker enzymes of other subcellular fractions were not detected. The NaJ extracted PMF revealed a high specific activity of ATPase, 91% of which was stimulated by Na+ and K+. The MgNaK-ATPase was characterized by Michaelis Menten kinetics. In contrast, Hill coefficients (“n”) of NaK-ATPase for the activation by Na+, K+ and Mg-ATP were greater than one. Experiments with various nucleotide tri-, di- and monophosphates revealed a high substrate specificity of the NaK-ATPase. The pH optimum was in the range of 7.2. SH-reagents and ouabain depressed the Na+ and K+ stimulated enzyme activity. PMF isolated from rat kidneys exhibited an acylphosphatase and a nitrophenylphosphatase activity, both of which were stimulated by K+. Furthermore 5′-nucleotidase and leucine aminopeptidase activities were present in the fraction.—Thus, NaK-ATPase of the PMF revealed the typical properties of the NaK-ATPase demonstrated in the microsomal preparations, which has been referred to by Skou (1965) as the enzymatic basis of active cation transport.

(Na+K+)-activated ATPase in human cornea

SummaryDistribution and principal characteristics of (Na+K+)-activated ATPase in human cornea were investigated.(Na+K+)-ATPase was present in both epithelium and endothelium, whereas the corneal stroma did not exhibit significant enzyme activity.In homogenates specific activity of the (Na+K+)-ATPase was 2.3-fold higher in endothelium than in epithelium. Calculation of total enzyme activity revealed a 6.1-fold higher content of (Na+K+)-ATPase in the epithelium.In the epithelium a 7-fold enrichment of (Na+K+)-ATPase compared to the homogenate was obtained in the 150–1500×gav fraction. Maximum enrichment in the endothelium was 3.5-fold and was achieved in the 1500–2500×gav fraction. Both fractions showed, however, the same specific activity.The pH-optimum of (Na+K+)-ATPase in the 150–1500×gav fraction ranged from 8.0–8.2 in both epithelium and endothelium.In the epithelial 150–1500×gav fraction the apparentKm-values were 4.0 mM for Na+, 2.8 mM for K+ and 0.12 mM for Mg2+ · ATP in equimolar concentrations.The inhibition constant of epithelial (Na+K+)-ATPase for ouabain was determined asKi=3.3×10−7 M.The present data support the view that control of corneal hydration in man is a function of both endothelium and epithelium.

Properties of adenylate cyclase in mucosal cells of the rabbit ileum and the effect of cholera toxin

A study of the properties of adenylate cyclase in mucosal epithelial cells of rabbit ileum has been performed under control conditions and after stimulation by cholera toxin. A 3–5 fold stimulation of enzyme activity occurred after treatment with cholera toxin. Optimal conditions for activity were attained with Mg2+ concentrations between 5 and 25 mM at a pH of 8.0–9.0. Linearity of reaction rate was achieved over 25 min at protein concentrations of the homogenate of up to 40 μg per 50 μl incubation mixture when an ATP regeneration system was employed. Mn2+ at 3·10−4 to 1·10−3 M could replace Mg2+ but was inhibitory at 5 mM. Ca2+ was inhibitory at all concetrations above 10−5 M. Michaelis-Menten plots were linear with regard to ATP concentrations under both sets of of conditions, V was 28 pmoles·mg−1 protein·min−1 for control and 90 pmoles·mg−1 protein·min−1 for toxin-treated enzyme. Km for both conditions were 4·10−4 M. No evidence of cooperativity was detected. The mechanisms of activation by F− and cholera toxin were presumed different from an analysis of experiments involving combinations of F− and toxin and by a study of pyrophosphate inhibition. Isoproterenol at 10−5 M was stimulatory to the control enzyme, while no effect of epinephrine or propranolol was detected. In contrast, isoproterenol, epinephrine and propranolol were inhibitory to the enzyme after stimulation by cholera toxin.

Role of the choline exchanger in Na+-independent Mg2+ efflux from rat erythrocytes

Two types of Na(+)-independent Mg(2+) efflux exist in erythrocytes: (1) Mg(2+) efflux in sucrose medium and (2) Mg(2+) efflux in high Cl(-) media such as KCl-, LiCl- or choline Cl-medium. The mechanism of Na(+)-independent Mg(2+) efflux in choline Cl medium was investigated in this study. Non-selective transport by the following transport mechanisms has been excluded: K(+),Cl(-)- and Na(+),K(+),Cl(-)-symport, Na(+)/H(+)-, Na(+)/Mg(2+)-, Na(+)/Ca(2+)- and K(+)(Na(+))/H(+) antiport, Ca(2+)-activated K(+) channel and Mg(2+) leak flux. We suggest that, in choline Cl medium, Na(+)-independent Mg(2+) efflux can be performed by non-selective transport via the choline exchanger. This was supported through inhibition of Mg(2+) efflux by hemicholinum-3 (HC-3), dodecyltrimethylammonium bromide (DoTMA) and cinchona alkaloids, which are inhibitors of the choline exchanger. Increasing concentrations of HC-3 inhibited the efflux of choline and efflux of Mg(2+) to the same degree. The K(d) value for inhibition of [(14)C]choline efflux and for inhibition of Mg(2+) efflux by HC-3 were the same within the experimental error. Inhibition of choline efflux and of Mg(2+) efflux in choline medium occurred as follows: quinine>cinchonine>HC-3>DoTMA. Mg(2+) efflux was reduced to the same degree by these inhibitors as was the [(14)C]choline efflux.

Characterization of Mg2+ efflux from rat erythrocytes non-loaded with Mg2+

Non-Mg(2+)-loaded rat erythrocytes with a physiological level of Mg(2+)(i) exhibited Mg(2+) efflux when incubated in nominally Mg(2+)-free media. Two types of Mg(2+) efflux were shown: (1) An Na(+)-dependent Mg(2+) efflux in NaCl and Na gluconate medium, which was inhibited by amiloride and quinidine, as was Na(2+)/Mg(2+) antiport in Mg(2+)-loaded rat erythrocytes; and (2) an Na(+)-independent Mg(2+) efflux in sucrose medium and choline Cl medium, which may be differentiated into SITS-sensitive Mg(2+) efflux at low Cl(-)(o) (in sucrose) and into SITS-insensitive Mg(2+) efflux at high Cl(-)(o) (in 150 mmol/l choline Cl).

Plasma membranes of the kidney

SummaryThe effects of furosemide, amiloride and ethacrynic acid on ATPase activity in rat renal plasma membranes were studiedin vivo andin vitro. Furosemide increased volume and electrolyte content of the urine but had no influence on ATPase activity. Amiloride produced a moderate natriuresis and antikaliuresis and inhibited both Mg-ATPase and NaK-ATPase activity. Ethacrynic acid, that was active as a diuretic only after intravenous injection had no influence on NaK-ATPase activity.In vitro at concentrations of 1 mM, furosemide had no influence on ATPase activity, amiloride inhibited Mg-ATPase, NaK-ATPase and K+-activated AcPase activity. In kinetic studies the mechanism of inhibition of total ATPase activity was further characterized. No consistent relationship was observed between inhibition of Na+ reabsorption and inhibition of plasma membrane NaK-ATPase. Thus, it was concluded that these diuretics do not act by inhibiting plasma membrane NaK-ATPase.