ATP-dependent Ca2+-uptake by the plasma membrane fraction of the myometrium

The specific activities of Mg2+, Ca2+-ATPase in the plasma membrane fraction of rabbit and cattle myometrium are 8.30 +/- 0.80 and 2.36 +/- 0.48 mkmoles of Pi per mg of protein, respectively. This fraction possesses a higher (in comparison with other subcellular fractions) capacity for ATP-dependent uptake of 45Ca2+ (9.37 +/- 1.66 and 6.86 +/- 0.96 nmoles of 45Ca2+ per mg of protein in 15 min for rabbit and cattle myometrium, respectively); the ratio of ATP-dependent uptake of Ca2+ to adsorbed Ca2+ is also high. Phosphate increases Ca2+ uptake in the presence of ATP and Mg2+. The ionophore A-23187 added to the incubation mixture without ATP and Mg2+ sharply increases Ca2+ binding. An addition of the ionophore at the 15th min of the ATP-dependent Ca2+ uptake causes a complete and rapid release of the accumulated Ca2+. The release of Ca2+ can be also caused by an addition of Na-DS or EGTA to the incubation mixture. This suggests that Ca2+ is accumulated through the plasma membrane inside the closed structures. It was assumed that myometrial sarcolemma plays an essential role in regulation of intracellular Ca2+ concentration in the uterus at rest and that the active Ca2+ efflux from the cells is controlled by the Mg2+, Ca2+-ATPase system.     

Effects of in vivo ethanol administration on Ca2+/Mg2+ ATPase and ATP-dependent Ca2+ uptake activity in synaptosomal membranes

1. Acute administration of ethanol (4 g/kg, i.p.) to mice inhibits the sequestration of calcium into endoplasmic reticulum-like organelles in synaptosomal membranes.
2. Ethanol administration inhibits both Ca²⁺-stimulated adenosine triphosphate hydrolysis and ATP-dependent calcium uptake in the vesicles at time of loss of righting reflex.
3. At recovery of righting reflex, the Ca²⁺-ATPase activity returns to normal levels, while the ATP-dependent uptake remains inhibited.
4. The effect of ethanol is specific for the sequestration (active transport) of calcium since calcium binding to synaptic membranes is not altered.
5. Alteration in mechanisms responsible for synaptosomal buffering of cytosolic Ca²⁺ levels by in vivo ethanol may contribute to altered transmitter release rates following ethanol adminstration.

     

Ca2+ transport by the synaptosomal plasma membrane Ca2+-ATPase and the effect of thioridazine

Thioridazine inhibits the activity of the synaptic plasma membrane Ca(2+)-ATPase from pig brain and slightly decreases the rate of Ca(2+) accumulation by synaptic plasma membrane vesicles in the absence of phosphate. However, in the presence of phosphate, thioridazine increases the rate of Ca(2+) accumulation into synaptic plasma membrane vesicles. Phosphate anions diffuse through the membrane and form calcium phosphate crystals, reducing the free Ca(2+) concentration inside the vesicles and the rate of Ca(2+) leak. The higher levels of Ca(2+) accumulation obtained in the presence of thioridazine could be explained by a reduction of the rate of slippage on the plasma membrane ATPase.     

Inhibition of Ca2+-transport ATPase from synaptosomal vesicles by flavonoids

The inhibitory action of the flavonoid quercetin has been examined on the calcium-transport ATPase of synaptosomal vesicles and compared to that of two other flavonoids, morin and rutin. We have found that while quercetin caused a 50% inhibition of calcium transport at a concentration of 15 microM, morin and rutin had similar effects at concentrations of about 200 microM. A similar order of potency was observed also for ATP hydrolysis, though at higher concentrations. Quercetin also strongly inhibited phosphorylation of membrane proteins by ATP in synaptosomal vesicles. Rutin and morin had an almost negligible effect on membrane protein phosphorylation. The order of inhibitory potency of the flavonoids on the Ca2+-transport ATPase from synaptosomal vesicles: quercetin greater than morin greater than rutin, could be linked to their possible solubility in the membrane lipid phase since: (1) it paralleled their partitioning between a mixture of oil and water; (2) it paralleled their uptake from the reaction mixture by synaptosomal vesicles and phosphatidylcholine liposomes; (3) they had almost equal potency as inhibitors of the water soluble system of histone phosphorylation by protein kinase.     

ATP-dependent interaction of propranolol and local anaesthetic with sarcoplasmic reticulum. Stimulation of Ca2+ efflux.

Preincubation of sarcoplasmic reticulum (SR) with propranolol or tetracaine inhibits Ca2+ accumulation and stimulates ATPase activity by more than 2-fold. This effect is obtained only when the preincubation is carried out in the presence of ATP or other nucleoside triphosphates. The (ATP + drug)-induced inhibition of Ca2+ accumulation is pH-dependent, increasing as the pH rises above 7.5. The presence of micromolar concentrations of Ca2+ or Mg2+ during the preincubation prevents the inhibitory effect of ATP plus drug on Ca2+ accumulation or ATPase activity. The (ATP + drug) modification of SR vesicles resulted in stimulation of a rapid Ca2+ efflux from passively loaded vesicles. The ATP-dependent inhibition of Ca2+ accumulation by the drug is obtained with other local anaesthetics. The drug concentration required for 50% inhibition was 0.15 mM for dibucaine and 0.4 mM for both propranolol and tetracaine, whereas it was 5 mM, 8 mM and greater than 10 mM for lidocaine, benzocaine and procaine respectively. The heavy SR vesicles were only slightly affected by the incubation with propranolol or tetracaine in the presence of ATP, but their sensitivity increased markedly after storage at 0 degrees C for 24-48 h. These results suggest that propranolol and some local anaesthetics, in the presence of ATP, stimulate Ca2+ efflux by modifying a protein factor(s) rather than the phospholipid bilayer.     

Estradiol affect Na-dependent Ca2+ efflux from synaptosomal mitochondria

The effects of gonadal steroid hormone, 17beta-estradiol (E2), in vitro on rat brain mitochondria Ca2+ movement were investigated. Intrasynaptosomal mitochondria Ca2+ uptake via an energy-driven Ca2+ uniporter have Km = 112.73 +/- 7.3 micromol x l(-1) and Vmax = 21.97 +/- 1.7 nmol 45Ca2+ mg(-1). Ca2+ release trough a Na+/Ca2+ antiporter was measured with a Km for Na+ of 43.7 +/- 2.6 mmol x l(-1), and Vmax of 1.5 +/- 0.3 nmol 45Ca2+ mg(-1). Addition of estradiol in preincubation mixture did not affect the uptake of Ca2+ mediated by the ruthenium red-sensitive uniporter, while it produced biphasic effect on Na-dependent Ca2+ efflux. Estradiol at concentrations up to 1 nmol x l(-1) decreased the efflux significantly (63% inhibition with respect to the control), and at concentrations above 10 nmol x l(-1) increased it exponentially. The maximum inhibiting concentration of estradiol (0.5 nmol x l(-1)) increased the affinity of the uniporter (Km reduced by about 30%), without affecting significantly the capacity (Vmax) for Na+. The results presented suggest that estradiol inhibits Na-dependent Ca2+ efflux from mitochondria and acts on mitochondrial retention of Ca2+, which may modulate mitochondrial and consequently synaptosomal content of Ca2+, and in this way exerts its role in the homeostasis of calcium in nerve terminals.     

Characterization of ATP-dependent Ca2+ uptake by canine brain microsomes with saponin

ATP-dependent Ca2+ uptake by brain microsomes was classified into two fractions according to the sensitivity to saponin. Properties of each fraction of Ca2+ uptake were examined and compared with those of inside-out membrane vesicles of erythrocyte and cardiac sarcoplasmic reticulum. The concentration of saponin for 50% inhibition (IC50) of major saponin-sensitive Ca2+ uptake was 11 micrograms/ml, and this uptake was enhanced by calmodulin. The minor saponin-insensitive Ca2+ uptake fraction (IC50; 90 micrograms/ml) was not affected by calmodulin but was enhanced by oxalate or 0.1 M KCl. The IC 50 of saponin for inside-out membrane vesicles of erythrocyte and cardiac sarcoplasmic reticulum was 11.3 and 114.8 micrograms/ml, respectively. A characteristic ring-like saponin-cholesterol micellar structure was observed electron microscopically in most membrane vesicles of brain microsomes and erythrocyte membrane vesicles but not in the cardiac sarcoplasmic reticulum. These observations indicate that saponin-sensitive and insensitive Ca2+ uptake was derived from plasma membranes and endoplasmic reticulum, respectively. Saponin proved useful for distinguishing the Ca2+ transport activity of plasma membrane from the Ca2+ uptake of other cellular organelles in the membrane preparations.     

N-ethylmaleimide inhibits Ca2+ influx induced by collagen or arachidonate on rabbit platelets

N-Ethylmaleimide dose dependently inhibited platelet aggregation induced by collagen or arachidonate but did not inhibit the aggregation by thrombin or ionophore A23187 within the concentrations tested. [3H]Arachidonate release from membrane phospholipids of the collagen-stimulated platelets was inhibited by N-ethylmaleimide in parallel with the inhibition of aggregation, but not in response to A23187. N-Ethylmaleimide prevented 45Ca2+ influx into platelet cells from outer medium induced by collagen, and also inhibited the increase in the concentration of cytoplasmic free Ca2+, which probably results from Ca2+ influx, as monitored by quin2 fluorescence, under stimulation with arachidonate. The concentration of N-ethylmaleimide giving a complete inhibition of Ca2+ influx was consistent with that required to inhibit collagen- or arachidonate-induced aggregation. Prostaglandin metabolism from arachidonate to thromboxane A2 was not disturbed by N-ethylmaleimide, while phosphatidate formation induced by arachidonate was slightly inhibited by it at concentrations at which aggregation was completely inhibited. These data suggest that N-ethylmaleimide preferentially suppresses increase in cytoplasmic free Ca2+ which is linked to thromboxane A2-receptor occupation in collagen- or arachidonate-stimulated platelets, probably due to blockage of Ca2+ influx through Ca2+-channel protein, thereby inhibiting aggregation induced by these agonists.     

Calmodulin-sensitive ATP-dependent Ca2+ transport across adipocyte plasma membranes

An ATP-dependent transport system which is active at concentrations of free Ca2+ in the submicromolar range has been identified in adipocyte plasma membranes. The system appears to represent the functional component of the high affinity insulin-sensitive calcium-stimulated, magnesium-dependent adenosine triphosphatase preveiously described in the same preparation (Pershadsingh, H. A., and McDonald, J. M. (1979) Nature 281, 495-497). This ATP-dependent Ca2+ transport pump was stimulated approximately 3-fold by the Ca2+-dependent regulatory protein, calmodulin. This effect was confined to the plasma membrane since a similar effect was undetectable in the fraction enriched in endoplasmic reticulum. Calmodulin stimulation was dose-dependent but saturable with half-maximal activation occurring at 0.72 microgram/ml (43 nM). Calmodulin appeared to stimulate the system primarily by decreasing the apparent half-maximal saturation constant for free Ca2+ from 0.20 +/- 0.04 microM to 0.07 +/- 0.01 microM (n = 3). The Hill coefficient increased from 1.6 +/- 0.2 to 3.2 +/- 0.6 (n = 3), thus showing an increased positive cooperativity which allows the pump to be activated by an exceedingly narrow Ca2+ threshold in the presence of calmodulin. The calmodulin stimulation of the plasma membrane Ca2+ extrusion pump in adipocytes, working in opposition to metabolic signals which increase cytoplasmic Ca2+, could constitute a self-regulating negative feedback device for maintaining a low steady state level of intracellular Ca2+. This feedback system may be of critical importance in regulation of cellular metabolism by insulin.     

Fusion of proteoliposomes and cells. ATP-dependent Ca2+ uptake into erythrocytes catalyzed by Ca2+-ATPase from skeletal muscle

Purified Ca2+-ATPase from rabbit skeletal muscle has been incorporated into intact erythrocyte membranes by a two-step procedure. The isolated protein was reconstituted into proteoliposomes composed of phosphatidylethanolamine, phosphatidylcholine, and cardiolipin (50:20:30%, respectively). The resulting proteoliposomes were fused with erythrocytes in presence of La3+, Ca2+, or Mg2+. Subsequently, 45Ca uptake into the cells could be demonstrated. It was dependent on externally added ATP, inhibited by N-ethylmaleimide and p-hydroxymercuribenzoate, and enhanced by inactivation of the endogenous Ca2+-ATPase which catalyzes Ca2+ extrusion from the cells. The insertion of the protein did not induce cell lysis, but the cells did become more fragile. Functional insertion of isolated membrane proteins into cell membranes allows a new approach to research of plasma membranes.     

Ca2+ binding,ATP-dependent Ca2+ transport,and total tissue Ca2+ in embryonic and adult avian dystrophic pectoralis

Summary Avian muscular dystrophy is an autosomal recessive genetic disease characterized by early hypertrophy and loss of function of the pectoralis major. The disease is progressive, ultimately resulting in atrophy and heavy lipid deposition.Previous investigators have noted a decrease in the ability of the dystrophic sarcoplasmic reticulum to concentrate Ca²⁺. More recently, other investigators have shown an abnormal calcium uptake in avian dystrophic sarcoplasmic reticulum. They indicated, using freeze-fracture techniques, that a 90 Å particle of the vesicle membrane exhibited a decreased population and suggested that they might be the ATPase involved in calcium transport.Our studies confirm the earlier observations of a decreased rate of Ca²⁺ uptake and Ca²⁺ binding capacity of dystrophic fragmented sarcoplasmic reticulum vesicles which are isolated from both embryonic and adult pectoralis. These observations correlate in turn with a 75% drop in the Ca: ATP transport efficiency of the dystrophic sarcoplasmic reticulum determined by measuring the rate of³²P_i liberation from -ATP³² during active calcium transport by the isolated sarcoplasmic reticulum SR.In addition, we have found a quantitative deficiency in a 65,000 dalton component of the dystrophic fragmented SR at the time of myoblast fusion by measuring³⁵S-Methionine incorporation into the SR, coupled to high resolution polyacrylamide gel electrophoresis and radioautography. Analysis of total tissue calcium by atomic absorption spectroscopy revealed a decrease in the total calcium content of dystrophic muscle.     

Heart sarcolemmal Ca2+ transport in endotoxin shock: I. Impairment of ATP-dependent Ca2+ transport

Effects of endotoxin administration on the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma were investigated. The results show that the sidedness of the sarcolemmal vesicles was not affected but the ATP-dependent Ca²⁺ transport in cardiac sarcolemma was decreased by 22 to 46% (p < 0.05) at 4 h following endotoxin administration. The kinetic analysis indicates that the V_max for ATP and for Ca²⁺ were decreased by 50% (p < 0.01) and 32% (p < 0.01), respectively, while the Km values for ATP and Ca²⁺ were not significantly affected after endotoxin administration. Magnesium (1–5 mM) stimulated while vanadate (0.25–3.0 M) inhibited the ATP-dependent Ca²⁺ transport, but the Mg²⁺-stimulated and the vanadate-inhibitable activities remained significantly lower in the endotoxin-treated animals. These data demonstrate that endotoxin administration impairs the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma and that the impairment is associated with a mechanism not affecting the affinity towards ATP and Ca²⁺. Additional experiments show that the Ca²⁺ sensitivity of the Ca²⁺-ATPase activity was indifferent between the control and endotoxic groups suggesting that endotoxic injury impairs Ca²⁺ pumping without affecting Ca²⁺-ATPase activity. Since sarcolemmal ATP-dependent Ca²⁺ transport plays an important role in the regulation of cytosolic Ca²⁺ homeostasis, an impairment in the sarcolemmal ATP-dependent Ca²⁺ transport induced by endotoxin administration may have a pathophysiological significance in contributing to the development of myocardial dysfunction in endotoxin shock.     

Modulation of brain mitochondrial membrane permeability and synaptosomal Ca2+ transport by dopamine oxidation

Effects of dopamine on the membrane permeability transition, thioredoxin reductase activity, production of free radicals and oxidation of sulfhydryl groups in brain mitochondria and the Ca²⁺ uptake by Na⁺-Ca²⁺ exchange and sulfhydryl oxidation in brain synaptosomes were examined. The brain mitochondrial swelling and the fall of transmembrane potential were altered by pretreatment of dopamine in a dose dependent manner. Depressive effect of dopamine on mitochondrial swelling was reversed by 10 g/ml catalase, and 10 mM DMSO. The activities of thioredoxin reductase in intact or disrupted mitochondria were decreased by dopamine (1-100 M), 25 M Zn²⁺ and 50 M Mn²⁺. Dopamine-inhibited enzyme activity was reversed by 10 g/ml SOD and 10 g/ml catalase. Pretreatment of dopamine decreased Ca²⁺ transport in synaptosomes, which was restored by 10 g/ml SOD and 10 mM DMSO. Dopamine (1-100 M) in the medium containing mitochondria produced superoxide anion and hydrogen peroxide, while its effect on nitrite production was very weak. The oxidation of sulfhydryl groups in mitochondria and synaptosomes were enhanced by dopamine with increasing incubation times. Results suggest that dopamine could modulate membrane permeability in mitochondria and calcium transport at nerve terminals, which may be ascribed to the action of free radicals and the loss of reduced sulfhydryl groups.     

Histamine stimulated synaptosomal Ca2+ uptake through activation of calcium channels

Histamine stimulated Ca2+ uptake in synaptosomes was completely inhibited by the slow Ca2+ channel antagonists verapamil, cinnarizine and flunarizine, and slightly inhibited by nifedipine and diltiazem. Ca2+ uptake in synaptosomes depolarized or predepolarized with varying K+ concentrations was increased by histamine, in both conditions, until 30mM K+. At higher K+ concentrations histamine was not able to alter K+ effects in either conditions. 30mM K+ stimulated uptake of Ca2+ in the absence or presence of histamine was not inhibited by verapamil and diltiazem. However nifedipine slightly inhibited K+ and K+ +histamine effects. 3-Isobutyl-1-methyl-xanthine and dibutyryl cyclicAMP potentiated (10%) the uptake of Ca2+ in synaptosomes induced by histamine. Dibutyryl cyclicAMP alone however decreased the basal Ca2+ uptake in a concentration-dependent manner. Verapamil, but not diltiazem, antagonized the effects elicited by 3-isobutyl-1-methyl-xanthine and dibutyryl cyclicAMP in the presence of histamine. The data suggest that the increase in synaptosomal Ca2+ uptake induced by histamine is mediated by the activation of the voltage sensitive calcium channels, and possibly a cyclicAMP-dependent protein kinase phosphorylation can modulate the opening of Ca2+ channels.     

Modulation of ATP-dependent Ca2+ transport in rat parotid basolateral membrane vesicles by K+ + Cl- flux

In basolateral membrane vesicles (BLMV) isolated from rat parotid glands, the initial rate of ATP-dependent Ca2+ transport, in the presence of KCl, was approx. 2-fold higher than that obtained with mannitol, sucrose or N-methyl-D-glucamine (NMDG)-gluconate. Only NH4+, Rb+, or Br- could effectively substitute for K+ or Cl-, respectively. This KCl activation was concentration dependent, with maximal response by 50 mM KCl. An inwardly directed KCl gradient up to 50 mM KCl had no effect on Ca2+ transport, while equilibration of the vesicles with KCl (greater than 100 mM) increased transport 15-20%. In presence of Cl-, 86Rb+ uptake was 2.5-fold greater than in the presence of gluconate. 0.5 mM furosemide inhibited 86Rb+ flux by approx. 60% in a Cl- medium and by approx. 20% in a gluconate medium. Furosemide also inhibited KCl activation of Ca2+ transport with half maximal inhibition either at 0.4 mM or 0.05 mM, depending on whether 45Ca2+ transport was measured with KCl (150 mM) equilibrium or KCl (150 mM) gradient. In a mannitol containing assay medium, potassium gluconate loaded vesicles had a higher (approx. 25%) rate of Ca2+ transport than mannitol loaded vesicles. Addition of valinomycin (5 microM) to potassium gluconate loaded vesicles further stimulated (approx. 30%) the Ca2+ transport rate. These results suggest that during ATP dependent Ca2+ transport in parotid BLMV, K+ can be recycled by the concerted activities of a K+ and Cl- coupled flux and a K+ conductance.     

ATP-dependent H+ transport in synaptosomal membrane vesicles of the rat brain

H+ transport into synaptosomal membrane vesicles of the rat brain was stimulated by ATP and to a lesser extent by GTP, but not by ITP, CTP, UTP, ADP, AMP or beta, gamma-methylene ATP. ATP at concentrations up to 200 mM concentration-dependently stimulated the rate of H+ transport with a Km value of 0.6 mM, but at higher concentrations of this nucleotide the rate decreased. Other nucleotides such as CTP, UTP, GTP and AMP, or products of ATP hydrolysis i.e. ADP and Pi also reduced the ATP-stimulated H+ transport. The inhibition by GTP and ADP was not affected by the ATP concentration. These findings suggest that plasma membranes of nerve endings transport H+ from inside to outside of the cells utilizing energy from ATP hydrolysis, and that this transport is regulated by the intracellular concentration of nucleotides and Pi on sites other than those involved in substrate binding.     

Effects of Ca2+ and Mg2+ on ATP-dependent 45Ca2+ influx in A-431 human epidermoidal carcinoma cells

Upon stimulation with 10(-6) -10(-3) M ATP, A-431 human epidermoidal carcinoma cells incorporated radioactive calcium from their medium in a temperature-dependent manner. The rate of incorporation of 45Ca2+ was rapid for the initial 5 min, but decreased immediately thereafter. The preincubation of cells for 2 h in medium depleted of both Ca2+ and Mg2+ abolished the ATP-dependent 45Ca2+ incorporation, irrespective of whether or not the subsequent incubation medium contained Mg2+ ions. ATP-dependent 45Ca2+ incorporation could be restored by a second preincubation (1 h) in medium containing 1 mM Mg2+, but no Ca2+. The Mg2+ ions in the second preincubation medium could be replaced by Ca2+, Co2+, or Cu2+ for restoration of such activity. Elevation of inositol trisphosphate (InsP3) was observed in cells depleted of either Ca2+ or Mg2+, but not in cells depleted of both ions. A parallel effect was observed in changes in [Ca2+]i. Since the concentration of cytosolic calcium ions does not change by incubation of cells in medium depleted of and (or) restored with calcium ions, we conclude that either calcium or magnesium ions associated with some cellular component(s) are responsible for production of InsP3, which then supposedly mobilizes Ca2+ and provokes 45Ca2+ influx.     

Inhibition by orthovanadate of ATP-dependent Ca2+ transport in microsomes isolated from rat liver

A technique employing sucrose-density centrifugation for the enrichment of rat liver microsomes and rat liver plasma membranes in separate subcellular fractions is described. The fractions are enriched in glucose 6-phosphatase and 5'-nucleotidase, respectively, and are free of cytochrome oxidase activity. Vanadate-sensitive Ca2+ transport activity (half-maximal inhibition at approximately 10 microM vanadate, corresponding to approximately 12 nmol/mg of protein) was detected in only that fraction enriched in microsomal membranes. Inhibition by vanadate of ATP-dependent Ca2+ transport is noncompetitive with respect to added Ca2+ but competitive with respect to added ATP. Because it inhibits ATP-dependent Ca2+ transport in rat liver microsomes but not in rat liver plasma membranes, vanadate becomes a useful tool to distinguish in vitro between these two transport systems.