The effect of calcium on the respiratory responses of corn mitochondria

Tightly coupled respiring corn mitochondria (Zea mays L.) respond to calcium addition with a transitory respiratory increase, proton extrusion, and Ca²⁺ binding. The extent of response is dependent upon the level of endogenous phosphate, and a large sustained respiratory increase can be obtained with addition of phosphate. However, calcium does not act as a permeant cation in that it will not penetrate with acetate. It appears that the transitory respiratory increase must be linked to the uptake of a calcium phosphate complex, but there is no evidence that transport of the complex serves to produce an electrophoretic calcium uniport. It is believed that calcium phosphate transport in corn is a constitutive property, and not produced by membrane damage.     

Calcium and pancreatic β-cell function: 4. Evidence that glucose and phosphate stimulate calcium-45 incorporation into different intracellular pools

β-Cell-rich pancreatic islets were microdissected from ob/ob-mice and used for studies of ⁴⁵Ca uptake and washout. Irrespective of whether the experiments were performed at 21 or 37°C both glucose and phosphate stimulated the net uptake of lanthanum-nondisplaceable ⁴⁵Ca. The stimulatory effect of phosphate was additive to that produced by glucose. ⁴⁵Ca incorporated in response to phosphate differed from that taken up in the presence of 20 mM glucose in being easily washed out although it was not affected by the glucose concentration of the washing medium. The efflux of ⁴⁵Ca was reduced after introducing phosphate into a medium used to perifuse islets which had accumulated ⁴⁵Ca in response to 20 mM glucose. This suggests that the outward calcium transport can be influenced also by intracellular trapping of the cation. The glucose-stimulated insulin release was inhibited by phosphate; an effect reversed by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. It is concluded that a common effect of glucose and phosphate is to trap calcium in the pancreatic β-cells but that there are fundamental differences between their effects on intracellular distribution of calcium and on insulin release.     

Calcium Uptake and Associated Adenosine Triphosphatase Activity of Isolated Platelet Membranes

A platelet subcellular fraction, sedimenting between 14,000 and 40,000 g and consisting primarily of membrane vesicles, accumulates up to 200–400 nmoles calcium/mg protein in the presence of ATP and oxalate. Steady-state levels of calcium accumulation are attained in 40–60 min. Calcium uptake requires adenosine triphosphate (ATP), is enhanced by oxalate, and is accompanied by the release of inorganic phosphate. Calcium accumulation and phosphate release require magnesium and are inhibited by Salyrgan (10 µM) and adenosine diphosphate (ADP) (1 mM), but not by ouabain (0.1 mM). The ATPase activity is stimulated by low concentrations of calcium (5–10 µM) and is inhibited by 2 mM EGTA. Electron microscopic histochemistry using lead nitrate to precipitate released phosphate results in lead precipitates localized primarily at the inner surface of membrane vesicles. These results provide evidence for a membrane ATPase that is stimulated by low concentrations of calcium and may be involved in the transport of calcium across the membrane. It is postulated that the observed calcium uptake activity is an in vitro manifestation of a calcium extrusion pump in the intact platelet.     

Effects of Polypeptide and Protein Hormones on Lipid Monolayers : I. Effect of insulin and parathyroid hormone on monomolecular films of monooctadecyl phosphate and stearic acid

Insulin in low concentrations inhibits the uptake of Ca⁺⁺ by the monooctadecyl (stearyl) phosphate monolayer (at air-water interface) and facilitates the release of Ca⁺⁺ adsorbed to the monolayer. These effects of insulin are more pronounced at higher insulin concentrations. Evidence is presented that a relatively intact insulin molecule competes with Ca⁺⁺ for the free phosphate group of the monolayer. Albumin has a slight inhibitory action on calcium uptake and parathyroid hormone has no observable action on calcium uptake or release.     

Calcium-activated phosphate uptake in contracting corn mitochondria

The phosphate inhibition of succinate-powered contraction in corn mitochondria can be reversed with calcium. Associated with this reversal is an accumulation of phosphate and calcium. Both ions are essential for accumulation, although strontium will partially substitute for calcium. Arsenate does not substitute for phosphate except in producing the inhibition of contraction.

The antibiotics oligomycin and aurovertin do not block the phosphate inhibition of contraction or the calcium-activated phosphate uptake associated with the release of the inhibition. Dinitrophenol uncouples the phosphate uptake but permits full contraction.

Calcium promotes inorganic phosphate accumulation in root tissue as well as in mitochondria.

The results are discussed from the viewpoint of theories of calcium reaction with high energy intermediates of oxidative phosphorylation. It is concluded that calcium probably reacts with X~P in corn mitochondria, rather than with X~I as with animal mitochondria.

     

Fundamental Mechanisms of Phosphate Removal by Anaerobic/Aerobic Activated Sludge in Treating Municipal Wastewater

Acetate is thought to be an important substrate for phosphate removal in anaerobic/aerobic activated sludge (AS) processes. The acetate content in municipal wastewater is low, and the main organic compounds in such wastewater are particulate organic matters (POMs) that are converted to endogenous substrates in AS processes when municipal wastewater is introduced into AS reactors. The question which then arises is which substrate, acetate or POM, is important for phosphate removal in full‐scale AS plants. The rates of phosphate release and substrate uptake were determined using AS harvested from a full‐scale anaerobic/aerobic AS plant and also AS acclimated to peptone under alternate anaerobic and aerobic conditions for 26 months. The rate of phosphate release upon POM addition per AS concentration per unit of time was about 0.84 mg PO₄‐P/(g MLSS·h) irrespective of the wastewater quality. This value was about 0.05 in the case of AS acclimated to peptone for 26 months. When the AS concentration is 2.5 g/L and the mixed liquor retention time is 2 h in the anaerobic zone, about 4.2 mg/L PO₄‐P is released upon POM addition. Hence, phosphate can be removed from municipal wastewater using full‐scale AS plants running under these conditions.     

Calcium and pancreatic beta-cell function. 4. Evidence that glucose and phosphate stimulate calcium-45 incorporation into different intracellular pools.

beta-Cell-rich pancreatic islets were microdissected from ob/ob-mice and used for studies of 45Ca uptake and washout. Irrespective of whether the experiments were performed at 21 or 37 degrees C both glucose and phosphate stimulated the net uptake of lanthanum-nondisplaceable 45Ca. The stimulatory effect of phosphate was additive to that produced by glucose. 45Ca incorporated in response to phosphate differed from that taken up in the presence of 20 mM glucose in being easily washed out although it was not affected by the glucose concentration of the washing medium. The efflux of 45Ca was reduced after introducing phosphate into a medium used to perifuse islets which had accumulated 45Ca in response to 20 mM glucose. This suggests that the outward calcium transport can be influenced also by intracellular trapping of the cation. The glucose-stimulated insulin release was inhibited by phosphate; an effect reversed by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. It is concluded that a common effect of glucose and phosphate is to trap calcium in the pancreatic beta-cells but that there are fundamental differences between their effects on intracellular distribution of calcium and on insulin release.     

Calcium dependent neurotransmitter release and protein phosphorylation in synaptic vesicles.

A highly purified preparation of synaptic vesicles was prepared to study the relationship between calcium-dependent neurotransmitter release and protein phosphorylation. Calcium ions simultaneously produced significant increases in both the endogenous release of norepinephrine from the synaptic vesicles and the endogenous incorporation of [³²p] phosphate into specific synaptic vesicle proteins. The results are compatible with the hypothesis that the action of calcium on the phosphorylation of specific synaptic vesicle proteins is the molecular mechanism mediating some of the effects of calcium on neurotransmitter release and synaptic vesicle function.     

Kinetics of growth and phosphate uptake in pure culture studies of Acinetobacter species

Acinetobacter has been found to be the major species responsible for mediating biological phosphate removal. The growth kinetics and phosphate uptake were investigated for an isolated Acinetobacter strain growing in a defined medium. The phosphate uptake is dependent on growth rate, temperature, and pH. Polyphosphate granules occurred in a balanced growth stage. The maximum phosphorus content in cells was 4.8% at the dilution rate of 12 day(-1). The specific phosphate uptake rate was found to be a quadratic polynomial function of the dilution rate. Increased calcium (up to 36 mg/L) and magnesium (up to 15 mg/L), and the addition of yeast extract (100 mg/L), primary effluent (20%), and fluoride (10 mg/L) did not affect phosphate uptake. Anaerobic conditioning (N(2) stripping), low pH (CO(2) stripping), and addition of sodium acetate under anaerobic conditions failed to stimulate immediate phosphate release. Nevertheless, After 21-24 h, the phosphate release was ca. 3, 5, and 15 mg P/g cell, respectively, for N(2) purging, the addition of acetate, and CO(2) purging. For two-stage completely stirred reactor operation, there was negligible phosphate overplus at the second reactor when phosphate was added, when the first reactor was subjected to phosphate limitation. When both phosphate and carbon limited the growth in the first reactor, there was slight phosphate accumulation under endogenous respiration conditions in the second reactor.     

Vincristine-sulphate–loaded liposome-templated calcium phosphate nanoshell as potential tumor-targeting delivery system

Vincristine-sulfate–loaded liposomes were prepared with an aim to improve stability, reduce drug leakage during systemic circulation, and increase intracellular uptake. Liposomes were prepared by the thin-film hydration method, followed by coating with calcium phosphate, using the sequential addition approach. Prepared formulations were characterized for size, zeta potential, drug-entrapment efficiency, morphology by transmission electron microscopy (TEM), in vitro drug-release profile, and in vitro cell cytotoxicity study. Effect of formulation variables, such as drug:lipid ratio as well as nature and volume of hydration media, were found to affect drug entrapment, and the concentration of calcium chloride in coating was found to affect size and coating efficiency. Size, zeta potential, and TEM images confirmed that the liposomes were effectively coated with calcium phosphate. The calcium phosphate nanoshell exhibited pH-dependent drug release, showing significantly lower release at pH 7.4, compared to the release at pH 4.5, which is the pH of the tumor interstitium. The in vitro cytotoxicity study done on the lung cancer cell line indicated that coated liposomes are more cytotoxic than plain liposomes and drug solution, indicating their potential for intracellular drug delivery. The cell-uptake study done on the lung cancer cell line indicated that calcium-phosphate–coated liposomes show higher cell uptake than uncoated liposomes.     

Transport mechanism for calcium and phosphate in ram spermatozoa

Calcium uptake into ejaculated ram spermatozoa is highly enhanced by the addition of extracellular phosphate. Under identical conditions, extracellular calcium stimulates the uptake of phosphate by the cells. Both calcium and phosphate uptake are comparably inhibited by the sulfhydryl reagent mersalyl. The I50 was found to be 6.36 and 10.14 nmol mersalyl per mg protein for phosphate and calcium uptake, respectively. Calcium uptake is inhibited by mersalyl whether phosphate is present or not. Extracellular fructose causes a 5-fold increase in calcium uptake. When fructose and phosphate are present in the cell's medium, there is an additive effect, which indicates that two independent systems are involved in calcium transport into the cell. Ruthenium red, which blocks Ca2+ transport into the mitochondria, causes 70% and 95% inhibition of calcium uptake in the absence or in the presence of fructose, respectively. Ruthenium red does not affect phosphate uptake unless calcium was present in the incubation medium. The stimulatory effect of fructose upon calcium uptake can be mimicked by L-lactate and can be inhibited by the glycolytic inhibitor 2-deoxyglucose. Fructose and L-lactate stimulate mitochondrial respiration in a comparable way. Oligomycin, which inhibits mitochondrial ATP synthesis, does not inhibit Ca2+ uptake. This indicates that ATP is not involved in the mechanism by which mitochondrial respiration stimulates Ca2+ uptake. The calcium channel blocker, verapamil, inhibits Ca2+ uptake in the presence or absence of extracellular phosphate. The phosphate-dependent calcium transport mechanism is more sensitive to verapamil than is the phosphate-independent transporter. In summary, the data indicate that the plasma membrane of mammalian spermatozoa contains a calcium/phosphate symporter, a phosphate-independent calcium carrier and a calcium-independent phosphate carrier.     

Transient Mitochondrial Depolarizations Reflect Focal Sarcoplasmic Reticular Calcium Release in Single Rat Cardiomyocytes

Digital imaging of mitochondrial potential in single rat cardiomyocytes revealed transient depolarizations of mitochondria discretely localized within the cell, a phenomenon that we shall call “flicker.” These events were usually highly localized and could be restricted to single mitochondria, but they could also be more widely distributed within the cell. Contractile waves, either spontaneous or in response to depolarization with 50 mM K⁺, were associated with propagating waves of mitochondrial depolarization, suggesting that propagating calcium waves are associated with mitochondrial calcium uptake and consequent depolarization. Here we demonstrate that the mitochondrial flicker was directly related to the focal release of calcium from sarcoplasmic reticular (SR) calcium stores and consequent uptake of calcium by local mitochondria. Thus, the events were dramatically reduced by (a) depletion of SR calcium stores after long-term incubation in EGTA or thapsigargin (500 nM); (b) buffering intracellular calcium using BAPTA-AM loading; (c) blockade of SR calcium release with ryanodine (30 μM); and (d) blockade of mitochondrial calcium uptake by microinjection of diaminopentane pentammine cobalt (DAPPAC), a novel inhibitor of the mitochondrial calcium uniporter. These observations demonstrate that focal SR calcium release results in calcium microdomains sufficient to promote local mitochondrial calcium uptake, suggesting a tight coupling of calcium signaling between SR release sites and nearby mitochondria.     

Increased Calcium Absorption in Nephrolithiasis Explained by Uptake Studies in Ileal Brush Border Membrane Vesicles

We previously showed that recurrent calcium renal stone formers have enhanced urinary excretions of calcium and oxalate resulting from malabsorption of citrate. In the present investigation, the mechanism of the citrate-induced increased calcium uptake was studied using guinea pig ileal brush border membrane vesicles. In this model, calcium is absorbed in a concentration dependent, single mechanism uptake with a K_m of 275 ± 30 umol/liter (SD) and a V_max of 4.0 ± 0.5 nmol/min · mg protein. Under conditions of maximal calcium uptake, both citrate and phosphate inhibited calcium absorption into brush border membrane vesicles (BBMVs). In contrast, when phosphate and citrate were added together, calcium absorption normalized. Citrate inhibition of calcium absorption appeared to be due to free citrate ions, and phosphate ions overcame this inhibition. Phosphate inhibition was mostly due to decreased concentrations of ionized calcium and partly to precipitation of insoluble calcium phosphate. These studies confirm that the effects of citrate in humans in enhancing calcium absorption occur in the lumen of the gut and are not related to further biochemical conversions of citrate by the gut cells, to effects of citrate on calcium-related hormones, or to the renal handling of calcium. Also, the effects of citrate on increasing calcium absorption should be increased or attenuated in patients who malabsorb citrate, and this explains the increased urinary calcium and oxalate excretions reported for recurrent calcium stone formers.     

On the state of calcium ions in isolated rat liver mitochondria. II. Effects of phosphate and pH on Ca2+-induced Ca2+ release

At high K+ concentration, the effect of phosphate on Ca2+ uptake and release was studied in isolated rat liver mitochondria. Phosphate stimulated uptake at moderately high Ca2+ concentration, and inhibited release at high pH. At low pH, phosphate accelerated Ca2+ release. Ca2+ was released after a lag phase. The time of onset and the velocity of Ca2+ release depended on Ca2+ concentration. Ca2+ release was associated with mitochondrial swelling and destruction of the permeability barrier for sucrose and for chloride. Mg2+ inhibited Ca2+ release and the accompanying events. Ruthenium red and EGTA protected mitochondria from the destructive Ca2+ release and induced an immediate, slow release of Ca2+ and phosphate. Destructive Ca2+ release depended on the time of preincubation of respiration-inhibited mitochondria in the presence of Ca2+, prior to respiration-initiated Ca2+ uptake. The presence of phosphate and mitochondrial energization antagonized the destructive effect of calcium ions. Ca2+ release by acetoacetate also depended on pH. At pH 6.8, phosphate-stimulated Ca2+ release by acetoacetate, while it inhibited the acetoacetate effect at pH 7.6. The results suggest that an essential cause for the destruction of mitochondrial integrity is an increase in the intramitochondrial concentration of free calcium ions under the influence of phosphate.     

Studies of mitochondrial calcium movements using chlorotetracycline

1. The association of calcium with isolated rat liver mitochondrial membranes under various metabolic conditions was monitored using the fluorescent chelate probe, chlorotetracycline. Chlorotetracycline fluorescence increased markedly during energized calcium uptake in the absence of a permeant anion. Uncoupler and a respiratory chain inhibitor caused a rapid decrease in chlorotetracycline fluorescence when added either before or after calcium. During calcium uptake experiments concentrations of calcium exceeding 100 μM caused a transient fluorescence increase followed by an extensive decrease in fluorescence.

2. Changes in the chlorotetracycline-associated fluorescence of the mitochondrial suspensions were correlated with the uptake of exogenous ⁴⁵Ca. A positive correlation was observed between fluorescence and energized ⁴⁵Ca uptake in the absence of permeant anions. Addition of the permeant anion, phosphate, caused an extensive decrease in chlorotetracycline fluorescence but an enhanced uptake of exogenous ⁴⁵Ca.

3. The interaction of endogenous mitochondrial calcium with the fluorescent chelate probe was studied under a number of experimental conditions using mitochondria labeled during preparation with ⁴⁵Ca. Endogenous ⁴⁵Ca was lost rapidly from the mitochondria upon treatment with uncoupler, antimycin A, and A23187. Potassium phosphate and EGTA had no effect on the endogenous calcium as measured by either the ⁴⁵Ca content of the mitochondria or the fluorescence of the probe.

4. Mitochondria treated with antimycin A lost most of their endogenous ⁴⁵Ca within 3 min; subsequent energization of the mitochondria resulted in a partial uptake of the released ⁴⁵Ca but caused nearly a complete return of the chlorotetracycline fluorescence to the original level. Addition of phosphate did not change the fluorescence level but resulted in an almost complete accumulation of the ⁴⁵Ca previously released.

5. Following this energized uptake of ⁴⁵Ca, EGTA, p-trifluoromethoxyphenyl hydrazone of carbonyl cyanide, A23187 and calcium chloride all caused a nearly complete loss of the ⁴⁵Ca from the mitochondria and, with the exception of calcium chloride, caused an extensive decrease in the fluorescence level. Hence, the apparent location and/or properties of the endogenous calcium in this rat liver mitochondrial system were altered significantly by manipulation of the energetic state of the mitochondrial membrane.     

Effect of pH on calcium accumulation and release by isolated fragments of cardiac and skeletal muscle sarcoplasmic reticulum.

The ability of a sudden increase in pH to initiate a release of calcium from isolated skeletal and cardiac muscle sarcoplasmic reticulum following calcium accumulation in the absence of a precipitating anion (calcium binding) is described. In skeletal sarcoplasmic reticulum a sudden increase in pH caused a rapid release of accumulated calcium. In cardiac sarcoplasmic reticulum a sudden increase in pH before the calcium binding process was complete caused the release of a small amount of calcium at a relatively slow rate. A sudden change in pH after the completion of calcium binding failed to trigger a release of calcium. The effect of pH on oxalate supported calcium uptake and on unidirectional calcium efflux rate by cardiac sarcoplasmic reticulum was also studied. Both the rate of calcium uptake and of unidirectional calcium efflux increased as the pH was raised from 6.4 to 7.2, reflecting an increased permeability of the sarcoplasmic reticulum membrane to calcium. These results indicate that in cardiac muscle a sudden increase in pH is unlikely to be the in vivo signal for calcium release from the sarcoplasmic reticulum. However, the effect of pH on calcium uptake and efflux by cardiac sarcoplasmic reticulum may contribute to the negative inotropic effect of an acidosis on the heart.     

Role of calcium in the secretion of leptin from white adipocytes

The mechanism by which calcium regulates leptin secretion was studied in adipocytes isolated from rat white adipose tissue. Incubation of adipocytes in a medium containing glucose, but no calcium, markedly inhibited insulin-stimulated leptin secretion (ISLS) and synthesis, without affecting basal leptin secretion or lipolysis. However, when pyruvate was used as a substrate, ISLS was insensitive to the absence of calcium. Likewise, the stimulatory effects of insulin were completely prevented by phloretin, cytochalasin B, and W-13 (3 agents that interfere with early steps of glucose metabolism) in the presence of glucose, but not in the presence of pyruvate. Thus calcium appears to be specifically required for glucose utilization. On the other hand, (45)Ca uptake and leptin secretion were not affected by insulin or by inhibitors of L-type calcium channels. However, agents increasing plasma membrane permeability to calcium (high calcium concentrations, A-23187, and ATP) increased (45)Ca uptake and concomitantly inhibited ISLS. Similarly, release of endogenous calcium stores by thapsigargin inhibited ISLS in a dose-dependent manner. ATP, A-23187, calcium, and thapsigargin inhibited ISLS, even in the presence of pyruvate. These results show that 1) extracellular calcium is necessary for ISLS, mainly by affecting glucose uptake, 2) insulin does not affect extracellular calcium uptake, and 3) increasing cytosolic calcium by stimulating its uptake or its release from endogenous stores inhibits ISLS at a level independent of glucose metabolism. Thus calcium regulates leptin secretion from adipocytes in a manner that is markedly different from its role in the exocytosis of many other polypeptidic hormones.     

The effect of calcium on the respiratory responses of corn mitochondria.

Tightly coupled respiring corn mitochondria (Zea mays L.) respond to calcium addition with a transitory respiratory increase, proton extrusion, and Ca2+ binding. The extent of response is dependent upon the level of endogenous phosphate, and a large sustained respiratory increase can be obtained with addition of phosphate. However, calcium does not act as a permeant cation in that it will not penetrate with acetate. It appears that the transitory respiratory increase must be linked to the uptake of a calcium phosphate complex, but there is no evidence that transport of the complex serves to produce an electrophoretic calcium uniport. It is believed that calcium phosphate transport in corn is a constitutive property, and not produced by membrane damage.     

The effect of external calcium and lanthanum on platelet calcium content and on the release reaction

Calcium compartments in calf platelets were studied using a lanthanum washout procedure to distinguish between surface-bound calcium and intracellular calcium. The calcium content of calf platelets ranges from 20 to 60 nmol/10⁹ platelets and is sensitive to the calcium concentration of the suspending medium. With 1 mM calcium in the medium, calcium uptake is rapid and reaches steady state within 1–2 min. Results obtained with the lanthanum procedure indicate that it is the surface compartment which is most affected by the extracellular calcium concentration. The surface compartment appears to be saturable and is highly exchangeable. Although the total calcium as well as the calcium content of the surface and internal compartments are variable, the ratio of calcium in either compartment to the total saturated calcium is quite constant. The data indicate that 68–85% of the platelet calcium is located internally. Thrombin produces an immediate release of platelet calcium and labeled serotonin and an increase in the ⁴⁵Ca²⁺ uptake of both the surface and internal compartments. The release reaction is not dependent upon exogenous calcium or an influx of exogenous calcium since it occurs even in the presence of $\text{ethyleneglycol-bis-(β-aminoethylether)}\text{-N,N′-}\text{tetraacetic}$ acid. Lanthanum, however, inhibits the release reaction possibly by blocking surface calcium site and reducing the mobility of endogenous platelet calcium.     

Age-related impairment in rat parotid cell α1-adrenergic action at the level of inositol trisphosphate responsiveness

α₁-Adrenergic-stimulated calcium efflux from rat parotid cell aggregates declines approx. 40% between 3 and 24 months of age, with the bulk of the reduction occurring between 12 and 24 months. Intracellular free calcium levels following α₁-adrenoceptor stimulation are also reduced about 40% between 3 and 24 months. No significant age differences in stimulation of inositol mono-, bis- or trisphosphate production are observed. However, the ability of inositol trisphosphate to directly stimulate calcium efflux is reduced by about 50% with increasing age. Concentrations of this inositol phosphate required for maximal calcium release do not change between 3 and 24 months. Differences in response are not due to a reduction in uptake of inositol trisphosphate into older cells, but suggest an age-related defect in the ability of inositol trisphosphate to liberate calcium from intracellular stores. Such dysfunction may be at least partially responsible for impaired α₁-adrenergic responsiveness during aging.