Effects of calcium-EGTA buffers on active calcium transport in inside-out red cell membrane vesicles

Summary In inside-out red cell membrane vesicles, the free calcium concentration half-maximally stimulating active calcium uptake is about 2 orders of magnitude smaller in a calcium-EGTA buffer than in media containing unbuffered calcium. In calcium-EGTA buffer, the maximum rate of calcium uptake is determined by the total calcium concentration present. A possible model for explaining these findings is presented.Acknowledgments. This work was supported by the Scientific Research Council, Ministry of Health, Hungary (6-03-0306-01-1/Gá). We thank for the valuable comments of Dr Ilma Szász and for the skilful technical assistance of Mrs M. Sarkadi.     

Long-chain fatty acid uptake by skeletal myocytes: a confocal laser scanning microscopy study

Studies of regulation of free fatty acid (FFA) utilization by skeletal muscles have focused on plasma FFA delivery and on intracellular factors affecting FFA metabolism. The present study was conducted to directly analyse the uptake process of fatty acids into single myocytes. Cells were isolated from the rat flexor digitorum brevis muscle. Confocal laser scanning microscopy was utilized to analyse the uptake of the fluorescent fatty acid derivative 12-NBD-stearate, which is not metabolized by muscle tissue. Uptake represented a saturable function of the unbound fatty acid concentration in the medium (K _m 366 ± 118 nM, V _max 2.1 ± 0.3 AU/s) and depended on the medium sodium concentration. Reduced buffer pH increased initial uptake rates, whereas lactate (10 mM) had no effect. Membrane hyper- and depolarization decreased uptake rates. This study demonstrates for the first time kinetic data from isolated myocytes with evidence for a carrier-mediated transport mechanism for long-chain fatty acids. Received 31 March 1998; accepted 8 May 1998     

Sodium/calcium exchange in ventricular muscle

Ventricular cells possess two Ca extrusion mechanisms, a Na/Ca exchange system and a Ca pump. Reversing the exchanger by extracellular Na removal causes [Na]i to decrease, and the cells take up mmolar quantities of calcium. Since [Ca]i shows only a marginal increase the calcium load must be buffered. The capacity of the SR is limited so the mitochondria probably buffer a large part of this load. However, when Ca uptake into the mitochondria is blocked, the gain in Ca is still mmolar and the increase in [Ca]i still marginal, suggesting an additional buffering site. Measurements of the Na/Ca stoichiometry on sarcolemmal vesicles gave a value of 3, but in ventricle values of around 2.5 or 3 are found. Reasons for this are discussed, as are the differences amongst the different methods of Ca measurement. The interaction of the sarcolemmal Ca pump and the exchanger are considered and it is suggested they could interact via [Na]i. At rest both systems could remove Ca from the cell but on a large perturbation the Na/Ca exchange would be the more important of the two.     

Der Einfluss von Acetylcholin auf den Calciumumsatz ruhender und kontrahierender Vorhofmuskulaturin vitro

Summary Acetylcholine 5 × 10^–8 g/ml reduces the Ca⁴⁵ uptake of the beating left atria of guinea-pig; the tissue calcium is not altered. In resting atria, acetylcholine 5 × 10^–7 g/ml has no influence upon the calcium content and Ca⁴⁵ uptake. It is concluded that acetylcholine acts by shortening the action potential duration and thereby reduces the release of cellular calcium per excitation.

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Mit grosszügiger Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Inhibitory action of serum from a Laron dwarf on normal cellular function

Glucose uptake and O2 consumption of confluent glial cells grown in culture were measured in the presence of serum-free buffer and compared with those measured in the presence of serum from a normal volunteer, from an hGH-deficient dwarf and from a Laron dwarf. Cellular glucose uptake and respiration in the absence or presence of insulin or hGH are inhibited by Laron serum.     

Modulation of calcium sensitivity in guinea pig taenia coli: skinned fiber studies

In summary then, skinned fiber experiments have provided evidence showing that the relationship between force development and free calcium ion concentration may be variable. The cyclic-nucleotides c-GMP (cf 38) and c-AMP in particular might be important in modulating calcium sensitivity of skinned fibers possibly via an alteration of myosin phosphorylation. Additionally, the coupling between myosin phosphorylation and the ATPase activity of actomyosin or tension generation may itself be modulated in actomyosin contractile systems. The recognition of the physiological relevance of these modulating mechanisms however must await experiments in which the relationship between force development, free calcium ion concentration and myosin phosphorylation is studied in intact fibers. With the advent of more sophisticated calcium measuring techniques, such information is now available.     

Chronic overcrowding decreases cytoplasmic free calcium levels in T lymphocytes of aged CBA/CA mice

Intracellular calcium concentration is a sensitive marker of the homeostasis of living cells, and its increase is an essential step of T lymphocyte activation. Changes in the environment provoke an adaptive stress-response of the organism. In our present work we have investigated the effect of chronic overcrowding on resting and lectin-stimulated cytoplasmic free calcium concentration of splenic T lymphocytes from young and aged CBA/CA mice (50 animals total). The animals were kept under ‘normal’ (68 cm²/animal) or ‘overcrowded’ (22 cm²/animal) conditions for 3 months. Young animals showed no change in resting and stimulated calcium after overcrowding. T cells from aged mice, however, displayed significantly smaller levels of both resting and lectin-stimulated intracellular calcium concentration (p<0.01 each), as compared to those of the non-stressed, aged animals. This inadequate adaptation in the calcium metabolism of T lymphocytes may significantly contribute to the diminished immune response of the aged in stress.     

Failure of calcium to stimulate Na,K-ATPase in the presence of EDTA

The effect of calcium on Na,K-ATPase activity of rat brain homogenates and its modification by the chelating agent EDTA has been investigated. In the absence of EDTA, free calcium (approximately 10(-6) mol/l) stimulates Na,K-ATPase activity; in the presence of EDTA the same concentration of free calcium is without effect on the enzyme. In the absence of EDTA the stimulation by calcium of Na,K-ATPase activity is enhanced by the additional presence of calmodulin but in the presence of EDTA, even when calmodulin is added to excess, calcium still fails to stimulate the enzyme. The possibility that EDTA interferes with an interaction between a calcium-calmodulin complex and Na,K-ATPase is discussed.     

Die Wirkung von Herzglykosiden und Kalzium auf den Kaliumtransport im Herzmuskel

Summary In isolated perfused guinea pig hearts, both strophanthin and calcium produced positive inotropic effects and contracture increasing with the concentration of the drugs. Strophanthin caused a net loss of potassium from the heart to the perfusing fluid whereas calcium did not interfer in the same way with potassium exchange. The data are consistent with the view that the positive inotropic effect of cardiac glycosides depends mainly on the increased intracellular calcium concentration, perhaps due to inhibition of the active potassium and sodium transport.     

The effect of calcitonin on calcium uptake in mouse molars in vitro

Summary Mouse maxillary second molars were removed at either 24 or 96 h of age and maintained in vitro. Half of the teeth, of each age group, were treated with 50 m-units of synthetic salmon calcitonin. By comparing the initial and final calcium concentrations in the medium, the net uptake or release of calcium was inferred. The treated molars took up significantly more calcium than the untreated groups.     

Ca2+ uptake and distribution in alloxan-diabetic rat arterial and venous smooth muscle

Summary This report demonstrates that in experimental diabetes mellitus (DM) calcium uptake and its distribution is altered in rat aortic but not in portal venous smooth muscle. Results are interpreted as consequences of increased calcium binding by aortic smooth muscle in experimental DM, which could account for the hyporeactivity of alloxan diabetic rat aorta reported previously.Supported by NIH grant No.HL-18015 and NIDA grant No. DA-02339.     

Failure of calcium to stimulate Na,K-ATPase in the presence of EDTA

Summary The effect of calcium on Na, K-ATPase activity of rat brain homogenates and its modification by the chelating agent EDTA has been investigated. In the absence of EDTA, free calcium (approximately 10^–6mol/l) stimulates Na,K-ATPase activity; in the presence of EDTA the same concentration of free calcium is without effect on the enzyme. In the absence of EDTA the stimulation by calcium of Na,-K-ATPase activity is enhanced by the additional presence of calmodulin but in the presence of EDTA, even when calmodulin is added to excess, calcium still fails to stimulate the enzyme. The possibility that EDTA interferes with an interaction between a calcium-calmodulin complex and Na,K-ATPase is discussed.The expert technical assistance of Mrs Paula Jarvie is gratefully acknowledged. Thanks are due also to Professor Philip Kuchel for assistance with the calculations to determine the concentrations of metal-ligand complexes in the experimental media.     

Über die Wirkung blutzuckersenkender Sulfonylharnstoffe auf das isolierte Rattenzwerchfell

Summary The action of blood sugar depressing sulfonylureas on glucose and oxygen uptake, as well as on glycogen content and formation of C¹⁴O₂ from uniformly labelled C¹⁴-glucose was investigated in rat hemidiaphragms incubated in phosphate buffer. The following results were obtained: (1) Tolbutamide and Carbutamide increased the glucose uptake. (2) Tolbutamide decreased the glycogen-content. (3) Oxygen uptake as well as formation of C¹⁴O₂ were increased by Tolbutamide. (4) The action of Tolbutamide and insuline was equal with respect to glucose uptake but different as regarding the glycogen content, oxygen uptake and CO₂-formation.It is concluded that sulfonylureas increase glucose oxidation in the rat hemidiaphragm probably without increasing insulin sensitivity. To our knowledge this mechanism of action has hitherto not been described.     

Presence of NAD pyrophosphorylase in skeletal muscle in dystrophic mice

Summary NAD pyrophosphorylase (ATP:NMN adenylyltransferase) activity has been measured in the skeletal muscle of dystrophic mice. The amount of this enzyme in the dystrophic mice, as determined by three different methods, was about one half of that in the controls. In addition, the concentration of ATP was too low to be detected in crude extracts of dystrophic mouse skeletal muscle, which were prepared using Tris buffer alone or Tris buffer containing either 3 M KCl, or 1 mM PMSF.     

Presence of NAD pyrophosphorylase in skeletal muscle in dystrophic mice

NAD pyrophosphorylase (ATP:NMN adenylyltransferase) activity has been measured in the skeletal muscle of dystrophic mice. The amount of this enzyme in the dystrophic mice, as determined by three different methods, was about one half of that in the controls. In addition, the concentration of ATP was too low to be detected in crude extracts of dystrophic mouse skeletal muscle, which were prepared using Tris buffer alone or Tris buffer containing either 3 M KCl, or 1 mM PMSF.     

Diltiazem prevents the damage to cultured aortic smooth muscle cells induced by hyperlipidemic serum

Diltiazem, a calcium antagonist, significantly reduced the increased 45Ca uptake and the number of dead cells in cultured aortic smooth muscle cells induced by hyperlipidemic serum.     

Use of 3H-QNB in the isolation of plasma membrane from smooth muscle of the urinary bladder: effect of oxalate on calcium uptake by the membrane fractions

Specific binding of tritiated quinuclidinyl benzilate (3H-QNB) to surface membrane muscarinic receptors was utilized to identify plasma membrane (PM) fractions from smooth muscle of the rabbit urinary bladder. Accumulation of 3H-QNB in the PM fraction was 4-5-fold higher than that in fractions of endoplasmic reticulum (EM) or mitochondria (M). A similar pattern of distribution was found for 5'-nucleotidase. 3H-QNB binding therefore appears to be a suitable marker for plasma membrane of the urinary bladder. Data on ATP-dependent calcium uptake by PM and ER fractions showed that oxalate highly potentiated calcium uptake by both fractions and consequently this feature cannot be used to identify ER fractions specifically.     

Calcium uptake by sarcoplasmic reticulum from nerve-intact and standard skeletal muscle grafts

A freely grafted rat soleus muscle exhibits a decrease in velocity and capacity of SR calcium uptake. This deficit is not prevented by maintaining neural connections (nerve-intact graft) during grafting. Thus the greater mechanical capability of nerve-intact grafts, relative to standard grafts, is not accompanied by any enhancement of the SR tubules.     

Reduction of high affinity glutamate uptake in rat hippocampus by two polyamine-like toxins isolated from the venom of the predatory wasp Philanthus triangulum F

Two components of the venom of the predatory wasp Philanthus triangulum F. significantly reduce--to a greater or less extent--the high affinity uptake of glutamate in rat hippocampus. A concentration of 10 microM delta-PTX caused a reduction of 74%, while the other component, beta-PTX, at the same concentration, caused a reduction of 18%. Hence the effect of delta-PTX on high affinity glutamate uptake in the hippocampus is comparable with its effect on high affinity glutamate uptake in insect neuromuscular junctions. Contrary to our previous findings that beta-PTX has no effect on high affinity glutamate uptake in insect glutamatergic terminal axons, however, beta-PTX significantly reduces high affinity glutamate uptake in the hippocampus, albeit less effectively than delta-PTX.