Characterization and localization of two forms of active Ca2+ transport in vesicles derived from rat submandibular glands

Energy-dependent Ca2+ uptake was characterized in vesicles derived from rat submandibular salivary glands. Ca2+ transport was stimulated by submicromolar levels of Ca2+, reached a plateau at 1-20 microM Ca2+ then again increased as the Ca2+ concentration rose to millimolar levels. Ruthenium red (2.5 microM) was used to resolve this pattern of uptake into two components: ruthenium red-insensitive Ca2+ transport occurs in the presence of the dye, is stimulated by submicromolar Ca2+ concentrations and reaches a maximum steady state at about 1 microM Ca2+. The distribution of ruthenium red-insensitive Ca2+ uptake in membrane subfractions obtained by differential centrifugation is positively (r = 0.717) and significantly (p = 0.001) correlated with the distribution of membrane-bound RNA in the same subfractions. Ca2+ uptake which is abolished by ruthenium red is greatest at millimolar Ca2+ concentrations. Its distribution is positively (r = 0.828) and significantly (p = 0.0001) correlated with the cytochrome-c oxidase activity of the membrane subfractions but is unrelated to the distribution of particulate RNA and is negatively correlated with Na+-K+ ATPase activity. We conclude that vesicles derived from the endoplasmic reticulum of rat submandibular glands actively transport Ca2+ by a ruthenium red-insensitive mechanism which is stimulated at Ca2+ concentrations typical of the cytosol. Membranes derived from mitochondria also sequester Ca2+ but by a mechanism which is inhibited by ruthenium red and which reaches its maximum steady state capacity at relatively high Ca2+ concentrations.     

Subcellular fractionation of pig coronary artery smooth muscle

A detailed procedure for subcellular fractionation of the smooth muscle from pig coronary arteries based on dissection of the proper tissue, homogenization, differential centrifugation and sucrose density gradient centrifugation is described. A number of marker enzymes and Ca2+ uptake in presence or absence of oxalate, ruthenium red and azide were studied. The ATP-dependent oxalate-independent azide- or ruthenium red-insensitive Ca2+ uptake, and the plasma membrane markers K+-activated ouabain-sensitive p-nitrophenylphosphatase, 5'-nucleotidase and Mg2+-ATPase showed maximum enrichment in the F2 fraction (15-28% sucrose) which was also contaminated with the endoplasmic reticulum marker NADPH: cytochrome c reductase, and to a small extent with the inner mitochondrial marker cytochrome c reductase, and also showed a small degree of oxalate stimulation of the Ca2+ uptake. F3 fraction (28-40% sucrose) was maximally enriched in the ATP- and oxalate-dependent azide-insensitive Ca2+ uptake and the endoplasmic reticulum marker NADPH: cytochrome c reductase but was heavily contaminated with the plasma membrane and the inner mitochondrial markers. The mitochondrial fraction was enriched in cytochrome c oxidase and azide- or ruthenium red-sensitive ATP-dependent Ca2+ uptake but was heavily contaminated with other membranes. Electron microscopy showed that F2 contained predominantly smooth surface vesicles and F3 contained smooth surface vesicles, rough endoplasmic reticulum and mitochondria. The ATP-dependent azide-insensitive oxalate-independent and oxalate-stimulated Ca2+ uptake comigrated with the plasma membrane and the endoplasmic reticulum markers, respectively, and were preferentially inhibited by digitonin and phosphatidylserine, respectively. This study establishes a basis for studies on receptor distribution and further Ca2+ uptake studies to understand the physiology of coronary artery vasodilation.     

Different Subunit Location of the Inhibition and Transport Sites in the Mitochondrial Calcium Uniporter

The mitochondrial calcium uniporter behaves as a cooperative mechanism, where the velocity is dependent on [Ca2+]ex. Transport kinetics follows a sigmoidal behavior with a Hill coefficient near 2.0, indicating the binding of at least two calcium molecules. Calcium transport in mitochondria is dependent on a negative inner membrane potential and is inhibited by policationic ruthenium compounds. In this study, calcium uptake activity was reconstituted into cytochrome oxidase vesicles by incorporating solubilized mitochondrial proteins. Calcium accumulation plotted against increasing Ca2+ concentrations followed a sigmoidal behavior with a Hill coefficient of 1.53. The uptake was sensitive to ruthenium policationic inhibitors, e.g. ruthenium red and Ru360. After mitochondrial proteins were separated by preparative isoelectrofocusing and incorporated into cytochrome oxidase vesicles, two peaks of calcium uptake activity were recovered. One of the activities was inhibited by Ru360, while the second activity was insensitive to Ru360 and was associated with proteins focused at very acidic isoelectric points. By using a thiol-group crosslinker and radiolabeled Ru360, we proposed a scheme of partial dissociation of the uniporter inhibitor-binding subunit under acidic conditions.     

Characterization of Ca2+ uptake in plasma membrane vesicles isolated from guinea pig ileum smooth muscle

We have characterized ATP-dependent Ca2+ transport into highly purified plasma membrane fraction isolated from guinea pig ileum smooth muscle. The membrane fraction contained inside-out sealed vesicles and was enriched 30-40-fold in 5'-nucleotidase and phosphodiesterase I activity as compared to post nuclear supernatant. Plasma membrane vesicles showed high rate (76 nmol/mg/min) and high capacity for ATP dependent Ca2+ transport which was inhibited by addition of Ca2+ ionophore A23187. The inhibitors of mitochondrial Ca2+ transport, i.e., sodium azide, oligomycin and ruthenium red did not inhibit ATP-dependent Ca2+ uptake into plasma membrane vesicles. The energy dependent Ca2+ uptake into plasma membranes showed very high specificity for ATP as energy source and other nucleotide triphosphates were ineffective in supporting Ca2+ transport. Phosphate was significantly better as Ca2+ trapping anion to potentiate ATP-dependent Ca2+ uptake into plasma membrane fraction as compared to oxalate. Orthovanadate, an inhibitor of cell membrane (Ca2+-Mg2+)-ATPase activity, completely inhibited ATP-dependent Ca2+ transport and the Ki was approximately 0.6 microM. ATP-dependent Ca2+ transport and formation of alkali labile phosphorylated intermediate of (Ca2+-Mg2+)-ATPase increased with increasing concentrations of free Ca2+ in the incubation mixture and the Km value for Ca2+ was approximately 0.6-0.7 microM for both the reactions.     

Ba2+ ions inhibit the release of Ca2+ ions from rat liver mitochondria

The release of Ca2+ from respiring rat liver mitochondria following the addition of either ruthenium red or an uncoupler was measured by a Ca2+-selective electrode or by 45Ca2+ technique. Ba2+ ions are asymmetric inhibitors of both Ca2+ release processes. Ba2+ ions in a concentration of 75 microM inhibited the ruthenium red and the uncoupler induced Ca2+ release by 80% and 50%, respectively. For the inhibition, it was necessary that Ba2+ ions entered the matrix space: Ba2+ ions did not cause any inhibition of Ca2+ release if addition of either ruthenium red or the uncoupler preceded that of Ba2+. The time required for the development of the inhibition of the Ca2+ release and the time course of 140Ba2+ uptake ran in parallel. Ba2+ accumulation is mediated through the Ca2+ uniporter as 140Ba2+ uptake was competitively inhibited by extramitochondrial Ca2+ and prevented by ruthenium red. Due to the inhibition of the ruthenium red insensitive Ca2+ release, Ba2+ shifted the steady-state extramitochondrial Ca2+ concentration to a lower value. Ba2+ is potentially a useful tool to study mitochondrial Ca2+ transport.     

Swelling-induced Ca2+ release from intracellular calcium stores in rat submandibular gland acinar cells

The effects of osmotically-induced cell swelling on cytoplasmic free Ca2+ concentration ([Ca2+]i) were studied in acinar cells from rat submandibular gland using microspectrofluorimetry. Video-imaging techniques were also used to measure cell volume. Hypotonic stress (78% control tonicity) caused rapid cell swelling reaching a maximum relative volume of 1.78 +/- 0.05 (n = 5) compared to control. This swelling was followed by regulatory volume decrease, since relative cell volume decreased significantly to 1.61 +/- 0.08 (n = 5) after 10 min exposure to hypotonic medium. Osmotically induced cell swelling evoked by medium of either 78% or 66% tonicity caused a biphasic increase of [Ca2+]i. The rapid phase of this increase in [Ca2+]i was due to release of Ca2 + from intracellular stores, since it was also observed in cells bathed in Ca2+-free solution. The peak increase of [Ca2+]i induced by cell swelling was 3.40 +/- 0.49 (Fura-2 F340/F380 fluorescence ratio, n = 11) and 3.17 +/- 0.43 (n = 17) in the presence and the absence of extracellular Ca2+, respectively, corresponding to an absolute [Ca2+]i of around 1 microm. We found that around two-thirds of cells tested still showed some swelling-induced Ca2+ release (SICR) even after maximal concentrations (10(-5) M - 10(-4) M) of carbachol had been applied to empty agonist-sensitive intracellular Ca2+ stores. This result was confirmed and extended using thapsigargin to deplete intracellular Ca2+ pools. Hypotonic shock still raised [Ca2+]i in cells pretreated with thapsigargin, confirming that at least some SICR occurred from agonist-insensitive stores. Furthermore, SICR was largely inhibited by pretreatment of cells with carbonyl cyanide m-cholorophenyl hydrazone (CCCP) or ruthenium red, inhibitors of mitochondrial Ca2+ uptake. Our results suggest that the increase in [Ca2+]i, which underlies regulatory volume decrease in submandibular acinar cells, results from release of Ca2+ from both agonist-sensitive and mitochondrial Ca2+ stores.     

Developmental status of sympathetic innervation in relation to calcium accumulation by submandibular gland following reserpine, surgical sympathectomy or cyclocytidine

Sympathectomy (Sx) of the submandibular gland was induced at various postnatal ages by ip administration of a single dose of reserpine or by unilateral excision of a superior cervical ganglion. If animals were 12 days old or less at the time of drug administration, [Ca] of the submandibular gland was not measurably increased 24 hr later; if rats were 14 days of age or older, [Ca] of the gland 24 hr after reserpine injection was nearly double that of untreated controls. Two days after surgical Sx, [Ca] of the denervated submandibular gland was unchanged from that of the innervated member of a pair if animals were less than 14 days of age at the time of denervation; [Ca] was twice that of glands of control rats if animals were older than 14 days of age when the denervation was performed. The anti-tumor agent, cyclocytidine (CC), given daily for 3 days in an ip dose of 500 mg/kg, also caused a two- to threefold increase in [Ca] of the submandibular gland when rats were more than 12 days of age at the time of the initial injection of the drug, but in rats younger than this age, CC caused no change in the [Ca] of the submandibular gland. Present data show that there are age-related differences in the ability of the submandibular gland to accumulate calcium following sympathetic denervation or treatment with a norepinephrine-releasing drug. These differences may be attributed either to incomplete development of calcium transport mechanisms, or incomplete development of the sympathetic innervation before 14 days of age.     

Novel ATP-dependent calcium transport component from rat liver plasma membranes. The transporter and the previously reported (Ca2+-Mg2+)-ATPase are different proteins

An ATP-dependent calcium transport component from rat liver plasma membranes was solubilized by cholate and reconstituted into egg lecithin vesicles by a cholate dialysis procedure. The uptake of Ca2+ into the reconstituted vesicles was ATP-dependent and the trapped Ca2+ could be released by A23187. Nucleotides, including ADP, UTP, GTP, CTP, GDP, AMP, and adenyl-5'-yl beta, gamma-imidophosphate, and p-nitrophenylphosphate did not substitute for ATP. The concentration of ATP required for half-maximal stimulation of Ca2+ uptake into the reconstituted vesicles was 6.2 microM. Magnesium was required for calcium uptake. Inhibitors of mitochondrial calcium-sequestering activities, i.e. oligomycin, sodium azide, ruthenium red, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and valinomycin did not affect the uptake of Ca2+ into the vesicles. In addition, strophanthidin and p-chloromercuribenzoate did not affect the transport. Calcium transport, however, was inhibited by vanadate in a concentration-dependent fashion with a K0.5 of 10 microM. A calcium-stimulated, vanadate-inhibitable phosphoprotein was demonstrated in the reconstituted vesicles with an apparent molecular weight of 118,000 +/- 1,300. These properties of Ca2+ transport by vesicles reconstituted from liver plasma membranes suggest that this ATP-dependent Ca2+ transport component is different from the high affinity (Ca2+-Mg2+)-ATPase found in the same membrane preparation (Lotersztajn, S., Hanoune, J. and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215; Lin, S.-H., and Fain, J.N. (1984) J. Biol. Chem. 259, 3016-3020). When the entire reconstituted vesicle population was treated with ATP and 45Ca in a buffer containing oxalate, the vesicles with Ca2+ transport activity could be separated from other vesicles by centrifugation in a density gradient and the ATP-dependent Ca2+ transport component was purified approximately 9-fold. This indicates that transport-specific fractionation may be used to isolate the ATP-dependent Ca2+ transport component from liver plasma membrane.     

Effects of Taurine and Mitochondrial Metabolic Inhibitors on ATP-Dependent Ca2+ Uptake in Synaptosomal and Mitochondrial Subcellular Fractions of Rat Retina

ATP-dependent Ca2+ uptake was investigated at low Ca2+ concentrations (10 microM) in rat retinal synaptosomal and mitochondrial preparations obtained by differential centrifugation on Ficoll gradients. Ca2+ uptake in the synaptosomal and mitochondrial subcellular preparations was stimulated by ATP and additionally stimulated by ATP plus taurine. The ATP-dependent and taurine-stimulated ATP-dependent Ca2+ uptakes were inhibited by mitochondrial metabolic inhibitors (atractyloside, oligomycin, and ruthenium red). These metabolic inhibitors had a greater effect on the ATP-dependent and taurine-stimulated ATP-dependent Ca2+ uptake activities in the mitochondrial preparation than in the synaptosomal preparation. ATP-dependent Ca2+ uptake in a synaptosomal subfraction obtained by osmotic shock was only partially inhibited by atractyloside. ATP-dependent Ca2+ uptake in the synaptosomal subfraction was also stimulated by taurine but to a lesser extent than in either the synaptosomal or mitochondrial preparation. These studies suggest that mitochondria are primarily responsible for taurine-stimulated ATP-dependent Ca2+ uptake in synaptosomal preparations.     

Effects of adenine nucleotide translocase inhibitors on dinitrophenol-induced Ca2+ efflux from pig heart mitochondria

Bongkrekic acid and atractyloside, inhibitors of adenine nucleotide translocase, do not inhibit Ca2+ uptake and H+ production by pig heart mitochondria. However, bongkrekic acid, but not atractyloside, inhibits dinitrophenol-induced Ca2+ efflux and H+ uptake. Conversely, ruthenium red blocks Ca2+ uptake and H+ production but does not prevent dinitrophenol-induced Ca2+ efflux and H+ uptake by mitochondria. These results suggest that mitochondrial Ca2+ uptake and release exist as two independent pathways. The efflux of Ca2+ from mitochondria is mediated by a bongkrekic acid sensitive component which is apparently not identical to the ruthenium red sensitive Ca2+ uptake carrier.     

Correlation between Ca2+-stimulated adenosine triphosphatase activity and Ca2+ uptake in microsomal fraction of rat submandibular gland

Both the Ca2+-ATPase activity and the Ca2+ uptake in a microsomal fraction of rat submandibular gland were inhibited by the addition of indomethacin in vitro. The decrease of both the Ca2+-ATPase activity and the Ca2+ uptake caused by the drug closely paralleled each other (r = 0.97). The inhibitory manner of indomethacin on Ca2+-ATPase and Ca2+ uptake was noncompetitive for Ca2+. These results suggest that the Ca2+-ATPase in the microsomal fraction of rat submandibular gland is a Ca2+ pump in this tissue.     

Relationships between Ca2+ release, Ca2+ cycling, and Ca2+-mediated permeability changes in mitochondria

Ruthenium red and/or EGTA prevent cyclic uptake and release of Ca2+ in mitochondria. These compounds inhibit but do not prevent the swelling of liver mitochondria induced by Ca2+ plus t-butyl hydroperoxide or Ca2+ plus N-ethylmaleimide. Ruthenium red and/or EGTA have complex effects on the release rate of Ca2+ and other cations induced by t-butyl hydroperoxide or N-ethylmaleimide. To determine the relationship between permeability changes and Ca2+ release in the absence of Ca2+ cycling, a novel method of data collection and analysis is developed which allows the relative time courses of Ca2+ release and Mg2+ release or swelling to be accurately and quantitatively compared. This method eliminates errors in time course comparisons which arise from the aging of mitochondrial preparations and allows data from different preparations to be directly contrasted. Using the method, it is shown that permeability changes caused by Ca2+-releasing agents are not secondary effects arising from Ca2+ cycling between uptake and release carriers. In the absence of Ca2+-cycling inhibitors, Ca2+ release induced by t-butyl hydroperoxide or N-ethylmaleimide is, in part, carrier-mediated. In the presence of EGTA and ruthenium red, Ca2+ release induced by either agent is mediated solely by the permeability pathway. No differences are apparent in the solute selectivity of the inner membrane permeability defect induced by Ca2+ plus t-butyl hydroperoxide or Ca2+ plus N-ethylmaleimide. A novel type of Ca2+ release from energized liver mitochondria is reported. This release is induced by EGTA, occurs in the absence of other releasing agents or nonspecific permeability changes, and is rapid (greater than or equal to 50 nmol/min/mg protein).     

Characterization of Ca2+ release from heterogeneous Ca2+ stores in sarcoplasmic reticulum isolated from arterial and gastric smooth muscle

Ca2+ transport was investigated in vesicles of sarcoplasmic reticulum subfractionated from bovine main pulmonary artery and porcine gastric antrum using digitonin binding and zonal density gradient centrifugation. Gradient fractions recovered at 15-33% sucrose were studied as the sarcoplasmic reticulum component using Fluo-3 fluorescence or 45Ca2+ Millipore filtration. Thapsigargin blocked active Ca2+ uptake and induced a slow Ca2+ release from actively loaded vesicles. Unidirectional 45Ca2+ efflux from passively loaded vesicles showed multicompartmental kinetics. The time course of an initial fast component could not be quantitatively measured with the sampling method. The slow release had a half-time of several minutes. Both components were inhibited by 20 microM ruthenium red and 10 mM Mg2+. Caffeine, inositol 1,4,5-trisphosphate, ATP, and diltiazem accelerated the slow component. A Ca2+ release component activated by ryanodine or cyclic adenosine diphosphate ribose was resolved with Fluo-3. Comparison of tissue responses showed that the fast Ca2+ release was significantly smaller and more sensitive to inhibition by Mg2+ and ruthenium red in arterial vesicles. They released more Ca2+ in response to inositol 1,4,5-trisphosphate and were more sensitive to activation by cyclic adenosine diphosphate ribose. Ryanodine and caffeine, in contrast, were more effective in gastric antrum. In each tissue, the fraction of the Ca2+ store released by sequential application of caffeine and inositol 1,4,5-trisphosphate depended on the order applied and was additive. The results indicate that sarcoplasmic reticulum purified from arterial and gastric smooth muscle represents vesicle subpopulations that retain functional Ca2+ channels that reflect tissue-specific pharmacological modulation. The relationship of these differences to physiological responses has not been determined.     

Ruthenium red inhibits the mitochondrial Ca2+ uptake in intact bovine spermatozoa and increases the cytosolic Ca2+ concentration

The uptake and cycling of Ca2+ by ejaculated bovine spermatozoa are almost completely abolished by ruthenium red, antimycin A or FCCP. The inhibitory effect of ruthenium red is also observed after washing of the dye-pretreated cells followed by addition of digitonin or filipin. In contrast, the inhibition is overcome by A23187 treatment. It is concluded that ruthenium red penetrates into intact spermatozoa, inhibits the mitochondrial Ca2+ uptake 'in situ', and causes the observed increase of the cytosolic free Ca2+ concentration.     

Ruthenium red and caffeine affect the Ca2+-ATPase of the sarcoplasmic reticulum

Effects of ruthenium red and caffeine (a Ca2+ release blocker and an inducer, respectively) on Ca2+ uptake by sarcoplasmic reticulum (SR) vesicles and formation of the phosphorylated intermediate (EP) of the Ca2+-ATPase were studied using fast-kinetic techniques. Ruthenium red increased the rate and the maximum level of EP formation, while caffeine decreased both. Similarly, ruthenium red accelerated rapid Ca2+ uptake, while caffeine inhibited it. These drugs affected EP formation also with detergent solubilized Ca2+-ATPase. The concentrations required for half maximal effects on these functions (0.2 microM ruthenium red, 1.0 mM caffeine) are about the same as those for altering Ca2+ release. These results indicate that these reagents affect both the Ca2+-pump as well as the Ca2+ release mechanism, suggesting that the Ca2+-pump and Ca2+ release have some mechanisms in common.     

Ca2+ transport by coupled Trypanosoma cruzi mitochondria in situ

The use of digitonin to permeabilize Trypanosoma cruzi plasma membrane enabled us to study Ca2+ transport and oxidative phosphorylation in mitochondria in situ. Addition of Ca2+ to these preparations evoked a cycle of respiratory stimulation. Ca2+ uptake was partially inhibited by ruthenium red, almost totally inhibited by antimycin A, and stimulated by inorganic phosphate. Addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone to digitonin-permeabilized T. cruzi epimastigotes under steady-state conditions was followed by Ca2+ release. Antimycin A- and carbonyl cyanide p-trifluoromethoxyphenylhydrazonein-sensitive Ca2+ uptake was also detected in digitonin-permeabilized epimastigotes. Accordingly, ATP stimulated Ca2+ uptake by preparations de-energized by oligomycin and antimycin A. In conclusion, in contrast to previous reports indicating that a Ca2+ transport system occurs only in mitochondria from vertebrate tissues, T. cruzi epimastigotes also possess a similar system. In addition, these protozoan mitochondria have an extremely high resistance to the deleterious effects of massive Ca2+ loads in comparison with most types of mammalian mitochondria.     

Mitochondrial free radical production induced by glucose deprivation in cerebellar granule neurons

Using a fluorescent probe for superoxide, hydroethidine, we have demonstrated that glucose deprivation (GD) activates production of reactive oxygen species (ROS) in cultured cerebellar granule neurons. ROS production was insensitive to the blockade of ionotropic glutamate channels by MK-801 (10 microM) and NBQX (10 microM). Inhibitors of mitochondrial electron transport, i.e. rotenone (complex I), antimycin A (complex III), or sodium azide (complex IV), an inhibitor of mitochondrial ATP synthase--oligomycin, an uncoupler of oxidative phosphorylation--CCCP, a chelator of intracellular Ca2+--BAPTA, an inhibitor of electrogenic mitochondrial Ca2+ transport--ruthenium red, as well as pyruvate significantly decreased neuronal ROS production induced by GD. GD was accompanied by a progressive decrease in the mitochondrial membrane potential and an increase in free cytosolic calcium ions, [Ca2+](i). Pyruvate, BAPTA, and ruthenium red lowered the GD-induced calcium overload, while pyruvate and ruthenium red also prevented mitochondrial membrane potential changes induced by GD. We conclude that GD-induced ROS production in neurons is related to potential-dependent mitochondrial Ca2+ overload. GD-induced mitochondrial Ca2+ overload in neurons in combination with depletion of energy substrates may result in the decrease of the membrane potential in these organelles.     

Adrenal cortex hormones and small intestine of adult rat: morphology, purity and enzymatic activities of isolated microvillous vesicles

An electron microscopic and enzymatic investigation was carried out to assess the purity as well as the morphologic integrity of small intestine microvillous membrane vesicles prepared from the following groups of adult rats: untreated (normals); adrenalectomized and pair-fed controls; treated for 8 days with corticosterone (1 and 10 mg X 100 g-1 body weight X day-1), and relative controls. Electron microscopy observation of ultrathin sections of ruthenium red stained vesicles from all groups showed that the microvillous membranes were structurally intact and virtually uncontaminated by material from other subcellular structures. The vesicles presented a fuzzy, intensely ruthenium red positive layer in their outer membranes. Apparently no alteration was induced in microvillous membrane by adrenalectomy, semistarvation or corticosterone treatment. The values of the enrichment factors for alkaline phosphatase and sucrase were similar for all the groups, indicating a high grade of purity of the preparations. Adrenalectomy and high dose of corticosterone decreased the vesicles content of alkaline phosphatase; sucrase was increased by semistarvation and adrenalectomy.     

Naproxen affects Ca(2+) fluxes in mitochondria, microsomes and plasma membrane vesicles

There is substantial evidence that nonsteroidal anti-inflammatory drugs (NSAIDs) affect cellular processes regulated by Ca(2+) ions, including the metabolic responses of the liver to Ca(2+)-dependent hormones. The aim of the present study was to determine whether the effects of naproxen are mediated by a direct action on cellular Ca(2+) fluxes. The effects of naproxen on 45Ca(2+) fluxes in mitochondria, microsomes and inside-out plasma membrane vesicles were examined. Naproxen strongly impaired the mitochondrial capacity to retain 45Ca(2+) and inhibited also ATP-dependent 45Ca(2+) uptake by microsomes. Naproxen did not modify 45Ca(2+) uptake by inside-out plasma membrane vesicles, but it inhibited the hexokinase/glucose-induced Ca(2+) efflux from preloaded vesicles. Additional assays performed in isolated mitochondria revealed that naproxen causes mitochondrial uncoupling and swelling in the presence of Ca(2+) ions. These effects were prevented by EGTA, ruthenium red and cyclosporin A, indicating that naproxen acts synergistically with Ca(2+) ions by promoting the mitochondrial permeability transition. The experimental results suggest that naproxen may impair the metabolic responses to Ca(2+)-dependent hormones acting by at least two mechanisms: (1) by interfering with the supply of external Ca(2+) through a direct action on the plasma membrane Ca(2+) influx, and (2) by affecting the refilling of the agonist-sensitive internal stores, including endoplasmic reticulum and mitochondria.     

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.