Dependence of ionophore- and caffeine-induced calcium release from sarcoplasmic reticulum vesicles on external and internal calcium ion concentrations.

The effects of the ionophore, X537A, and caffeine on ATP-dependent calcium transport by fragmented sarcoplasmic reticulum were studied in the absence (calcium storage) or presence (calcium uptake) of calcium-precipitating anions. The ionophore caused rapid calcium release after calcium storage, the final level of calcium storage being the same whether a given concentration of X537A was added prior to initiation of the reaction or after calcium storage had reached a steady state. Although 10 to 12 muM X537A caused approximately 90% inhibition of oxalate-supported calcium uptake when added prior to the start of the reaction, this ionophore concentration caused only a small calcium release when added after a calcium oxalate precipitate had formed within the vesicles, and only slight inhibition of calcium uptake velocity when added during the calcium uptake reaction. When low initial calcium loads limited calcium uptake to 0.4 mumol of calcium/mg of protein, subsequent calcium additions in the absence of the ionophore led to renewed calcium uptake. Uptake of the subsequent calcium additions was not significantly inhibited by 10 to 12 muM X537A. These phenomena are most readily understood in terms of constraints imposed by fixed Cai (calcium ion concentration inside the vesicles) on the pump-leak situation in sarcoplasmic reticulum vesicles containing a large amount of an insoluble calcium precipitate, where most of the calcium is within the vesicles and Cai is maintained at a relatively low level. These constraints restrict calcium loss after calcium permeability is increased because calcium release can end when the calcium pump is stimulated by the increased Cao (calcium concentration outside the vesicles) so as to compensate for the increased efflux rate. In contrast, an increased permeability in vesicles that have stored calcium in the absence of a calcium-precipitating ion causes a much larger portion of the internal calcium store to be released. Under these conditions calcium storage capacity is low so that release of stored calcium is less able to raise Cao to levels where the calcium pump can compensate for the increased efflux rate. The constraints imposed by anion-supported calcium uptake explain the finding that more calcium is released by X537A or caffeine when these agents are added at higher levels of Cao, and that more calcium leaves the vesicles in response to a given increase in calcium permeability at higher Cai. Although such calcium release is amplified by increased Cao, the amplification is attributable to the constraints described above and does not represent a "calcium-triggered calcium release."     

Palmitylcarnitine inhibition of the calcium pump in cardiac sarcoplasmic reticulum: a possible role in myocardial ischemia.

Palmitylcarnitine is a time-dependent inhibitor of the Ca²⁺-ATPase activity of cardiac sarcoplasmic reticulum isolated from adult dogs. Half-maximal inhibition was obtained at approximately 20 μM (2 μmoles/mg). The extent of inhibition depended on the ratio of palmitylcarnitine to sarcoplasmic reticulum protein. Calcium uptake by cardiac sarcoplasmic reticulum (measured in the presence of sodium oxalate) was found to be even more sensitive to inhibition by palmitylcarnitine and complete inhibition was obtained at concentrations as low as 2.5 μM (0.25 μmole/mg) following preincubation. Calcium binding (measured in the absence of oxalate) was inhibited by palmitylcarnitine and calcium release was stimulated at similar ratios. The level of palmitylcarnitine has been reported to increase several fold in myocardial ischemia and inhibition of the sarcoplasmic reticulum calcium pump could conceivably contribute either to the initial loss of contractility or the subsequent inability to restore full contractile function after prolonged ischemia.     

The ionophore X537A (Lasolocid) transiently increases acetylcholine release from rat brain invitro

The effect of X537A on acetylcholine (ACh) release was examined $\text{in vitro}$ in superfused slices of rat cerebrum and striatum. The ionophore (30 μM) induced a transient release of ACh which was not dependent on calcium in the medium. Also in contrast to K⁺-stimulated release, X537A-induced release was not sustained by 10^?5M choline in the superfusion medium and not inhibited by 5 × 10^?4M pentobarbital. The ionophore did not transport ACh or choline from an aqueous to an organic phase. Both K⁺ and X537A inhibited 1 μM (³H) choline uptake into striatal synaptosomes but this effect of X537A was more extensive and less reversible than that caused by K⁺. X537A did not inhibit choline acetyltransferase activity.     

Lanthanide binding to cardiac and skeletal muscle microsomes. Effects of adenosine triphosphate, cations, and ionophores.

Lanthanide (gadolinium, Gd) binding to cardiac and skeletal muscle microsomes was studied, and high- and low-affinity sites were identified. The high-affinity constant was 10⁶ M^?1, and there were 131 and 107 nmol/mg bound to this site in dog heart and rabbit skeletal muscle, respectively. Zn²⁺, Cd²⁺, Al³⁺, and Ca²⁺ (5 mm) inhibited binding, especially of the high-affinity site. Ionophores X537A (10 μm) and A23187 (1–2 μm) increased lanthanide binding and did not cause release. Addition of ATP in low concentration (20–50 μm) increased the binding of Gd without hydrolysis of the ATP. The extra binding induced by ATP was blocked by heating the microsomes and was reversed by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. High concentrations (10^?4–10^?3, m) of ATP blocked extra Gd binding by competitive chelation. The Ca²⁺-activated ATPase was inhibited by Gd and stimulated by X537A. The Gd did not block the ionophore-stimulated increase in Ca²⁺-ATPase activity. It is postulated that lanthanides bind predominantly to the ionophoric component of the Ca-transport site rather than the hydrolytic site and that ATP may facilitate such binding without being split.     

Effects of the ionophores X537A and A23187 on GABA,glycine, and taurine release from chick retinal subcellular fractions

The ionophore X537A at concentrations of 5–20 M stimulated the release of [³H]GABA and [³⁵S]taurine, from retinal subcellular crude nuclear (P₁) and crude synaptosomal (P₂) fractions. The release of [³H]GABA increased 114% and 136% over control values in P₁ and P₂ fractions, respectively. The efflux of [³⁵S]taurine from P₁ was increased by 45% and that from P₂ by 21%. X537A increased⁴⁵Ca²⁺ uptake in the P₂ fraction but not in the P₁ fraction. The effect of X537A on the amino acid release was not dependent on the presence of exogenous calcium. X537A did not affect [³H]GABA or [³⁵S]taurine uptake by the retinal fractions. A23187 enhanced [³H]GABA release from P₁ and P₂ by 52% and 105%, respectively. The ionophore also increased [¹⁴C]glycine liberation in both P₁ (35%) and P₂ (50%) but failed to stimulate [³⁵S]taurine release. A23187 produced a transient increase of⁴⁵Ca²⁺ uptake of 38% in P₁ and 30% in P₂. The effects of A23187 on the release of amino acids were calcium dependent. The amino acid uptake was not affected by the ionophore. These results are consisent with the suggested neurotransmitter role for GABA at the outer synaptic layer and for GABA and glycine at the inner synaptic layer of the retina. A neurotransmitter role for taurine is not supported by the present results.     

The role of calcium in transferrin and iron uptake by reticulocytes

The possible role of calcium in the uptake of transferrin and iron by rabbit reticulocytes was investigated by altering cellular calcium levels through the use of the chelating agents EDTA and $\text{ethyleneglycol-bis}\text{-(3-}\text{aminoethylether}\text{)-N,N′-}\text{tetraacetic}$ acid (EGTA) and the ionophores, A23187 and X537A. Incubation of reticuloyctes with EDTA or EGTA at 4°C had no effect on transferrin and iron uptake but incubation at 37°C resulted in an irreversible inhibition associated with decreased adsorption of transferrin to the cells and evidence of inactivation or loss of the transferrin receptors. Transferrin and iron uptake were also inhibited when the cells were incubated with A23187 or X537A. In the case of A23187 the action was primarily exerted on the temperature-sensitive stage of transferrin uptake and was associated with loss of cellular K⁺ and decrease in cell size. The effect was greater when Ca²⁺ was added to the incubation medium than its absence. X537A produced relatively greater inhibition of iron uptake than of transferrin uptake, associated with a reduction in cellular ATP concentratio. The action of X537A was unaffected by the presence of Ca²⁺ in the incubation medium.The results obtained with EDTA and EGTA indicate that cell membrane Ca²⁺ is required for the integrity or binding of transferrin receptors to the reticulocyte membrane. No evidence was obtained from the experiments with ionophores that an increase of cellular Ca²⁺ affects transferrin and iron uptake directly. The inhibition caused by A23187 was mainly due to a reduction in cell size resulting from increased membrane permeability to K⁺ and that caused by X537A appeared to result from an inhibition of energy metabolism and ATP production.     

The release of dopamine from synaptosomes from rat striatum by the ionophores X 537A and A 23187

The antibiotics X 537A and A 23187 are negatively charged divalent cation ionophores. X 537A may, in addition, be an ionophore for amines including catecholamines. The effects of these ionophores were examined on the uptake and release of dopamine by synaptosomes prepared from rat corpus striatum. Both X 537A and A 23187, at concentrations less than 0.5 μM, release both endogenous and [³H]-dopamine from synaptosomes. They had virtually no effect on the uptake of exogenous dopamine. These compounds act by different mechanisms. X 537A causes divalent ion-independent release in which a large fraction of the effluent consists of deaminated products. X 537A, in addition, releases [³H]dopamine from rat adrenal medullary chromaffin granules. The results suggest that X 537A causes release of dopamine from intrasynaptosomal storage vesicles and perhaps is acting as a catecholamine carrier across the vesicular membrane. A 23187, on the other hand, causes a Ca²⁺-dependent release in which only a small fraction of the catechol in the effluent is deaminated. A 23187 has little effect on the release of [³H]dopamine from chromaffin granules. These results suggest that A 23187 carries Ca²⁺ into the synaptosomes and thereby initiates exocytotic release.     

Calcium incorporation by smooth muscle microsomes.

The purpose of the present work was to study the factors influencing calcium incorporation into a microsomal fraction prepared from the longitudinal smooth muscle of the guinea-pig ileum. Calcium incorporation required the presence of both ATP and Mg2+ and was unaffected by azide. It was enhanced by oxalate; this effect was pH dependent and it was maximal at pH 6.6. The relation between calcium uptake with oxalate and free Ca2+ concentration in the medium was represented by a curve with an optimum for Ca2+ equal to 3-10-5 M. The threshold concentration was comprised between 5-10-7 and 10-6 7. The optimum calcium uptake rate was 4.5 nmol Ca2+/mg protein per min. In the absence of oxalate, two distinct groups of binding sites were identified. Low affinity sites had a binding constant of 7-104 M-1 and a maximum binding capacity of 0.6-106 M-1 and a binding capacity of 33 nmol Ca2+/mg protein; their capacity was sensitive to pH changes. In the absence of oxalate, Ca2+ binding was depressed by Na+ with respect to K+ or choline. When the medium was supplemented with oxalate, the stimulation of 45Ca incorporation was barely detectable in the presence of choline+ and it was lower in a medium containing Na+ instead of K+. The subcellular distribution profiles of calcium incorporation with and without oxalate indicate the microsomal location of both activities. However, the oxalate-stimulated calcium uptake activity sedimented faster than the calcium binding activity. The subcellular distribution of marker enzyme actvities has been examined. The present results indicate that Ca2+ incorporations with and without oxalate are the result of two processes likely related to two different structures. The role of microsomal calcium uptake in excitation-contraction coupling and its modification by the activity of the sodium pump is discussed.     

Effects of ionophores A23187 and X537A on brain calcium,catecholamines and excitability

The ionophores A23187 and X537A inhibit ⁴⁵Ca uptake by rabbit brain mitochondria and synaptosomes and also stimulate the release of accumulated ⁴⁵Ca from these preparations, but have no effect on ⁴⁵Ca binding by synaptic membranes or on total brain Ca in mice. Both agents inhibit uptake and stimulate release of ³H-norepinephrine by rabbit P₂ synaptosomal preparations, while the NE and serotonin levels of mouse brain are depressed by X537A. The changes in Ca activities may be related both to the elevated thresholds for cortical after-discharge produced in cats by these ionophores, and to the ionophore induced reduction of pentylenetetrazol seizures in mice.     

Differential sensitivity to nickel and SK&F96365 of second messengerm operated and receptormoperated calcium channels in rat submandibular ductal cells

The intracellular concentration of calcium ([Ca²⁺]_i) of rat submandibular ductal cells was measured with the intracellular fluorescent dye Fura-2. Carbachol (100 μM) and ATP (1 mM) both increased the [Ca²⁺]_j. The late response to ATP was blocked by 0.5 mM Ni²⁺. This concentration of Ni²⁺ also blocked the increase of the [Ca²⁺]_i and the uptake of manganese and calcium in response to 2′- and 3′-O-(4-benzoylbenzoyl) adenosine 5′-triphosphate (BzATP, 100 μM), a specific agonist of P2X receptors from salivary glands. The increase of the [Ca²⁺]_i in response to 2-methylthioadenosine 5′-triphosphate (2-McSATP, 100 μM) a specific P2Y agonist in salivary glands or to a muscarinic agonist (carbachol) was not affected by 0.5 mM Ni²⁺. Only higher concentrations of Ni²⁺ (in the millimolar range) inhibited the uptake of extracellular calcium in response to carbachol. SK&F 96365, a blocker of store-operated calcium channels, inhibited the uptake of extracellular calcium in response to carbachol without affecting the response to BzATP. It is concluded that at low concentrations (below 0.5 mM), Ni²⁺ inhibits the non-specific cation channel coupled to P2X receptors. The uptake of extracellular calcium by store-operated calcium channels is inhibited by higher concentrations of Ni²⁺ and by SK&F96365.     

Relationship of prolactin receptors to concanavalin A binding

Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of ¹²⁵I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar α-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 μg/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (K_d) as the controls. The binding of ¹²⁵I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (K_d ≈ 6.6 · 10^?8 M). At high lectin concentrations, low affinity (K_d ≈ 6.7 · 10^?5 M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 μg/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone.Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized ¹²⁵I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by α-methyl-D-mannopyranoside.     

Energy-dependent calcium transport in endoplasmic reticulum of adipocytes.

The endoplasmic reticulum from isolated rat adipocytes has the ability to actively accumulate calcium. The calcium uptake was characterized using the 20,000 X g supernatant (S1 fraction) of total cellular homogenate. Endoplasmic reticulum vesicles isolated from the S1 fraction as a 160,000 X g microsomal pellet prior to testing demonstrated little ability to accumulate calcium. The calcium uptake in the S1 fraction was localized to the endoplasmic reticulum vesicles by morphologic appearance, by the use of selective inhibitors of calcium uptake, and by high speed sedimentation of the accumulated calcium. The uptake was MgATP- and temperature-dependent and was sustained by the oxalate used as the intravesicular trapping agent. Uptake was linear with time for at least 30 min at all calcium concentrations tested (3 to 100 muM) and exhibited a pH optimum of approximately 7.0. The sulfhydryl inhibitor p-chloromercuribenzene sulfonate produced a dose-dependent inhibition of calcium uptake with total inhibition at 0.07 mumol/mg protein. Ruthenium red and sodium azide inhibited less than 5% of the uptake at concentrations (5 muM and 10 mM, respectively) which completely blocked calcium uptake by mitochondria isolated from the same cells. The Km for calcium uptake was 10 muM total calcium which corresponded to approximately 3.6 muM ionized calcium in the assay system. The maximum velocity of the uptake was 5.0 nmol (mg of microsomal protein)-1 (min)-1 at 24 degrees under the assay conditions used and exhibited a Q10 of 1.8. The uptake activity of the endoplasmic reticulum vesicles in the S1 fraction exhibited a marked time- and temperature-dependent lability which might account in part for the lack of uptake in the isolated microsomal fraction. This energy-dependent calcium uptake system would appear to be of physiologic importance to the regulation of intracellular calcium.     

Stimulation of calcium uptake in platelet membrane vesicles by adenosine 3',5'-cyclic monophosphate and protein kinase.

The events involved in platelet shape change, aggregation, the release reaction and contraction are thought to be mediated by the availability of Ca2+. Increased cytoplasmic calcium, released from intracellular stores, triggers platelet activity, and increased concentration of adenosine 3',5'-cyclic monophosphate (cyclic AMP) inhibits platelet alterations. We have studied the hypothesis that cyclic AMP may regulate the level of platelet cytoplasmic calcium by stimulating calcium removal by a membrane system. Such a hypothesis would be consistent with the reversibility of most manifestations of platelet activation. Human platelets were sonicated and unlysed platelets, mitochondria and granules were removed by centrifugation at 19 000 X g. Electron microscopy shows that the sediment, after centrifugation of the supernatant at 40 000 X g consists to a large extent of membrane vesicles. Such preparations actively concentrate calcium, as measured by the uptake of 45Ca, and also have the maximal calcium-stimulated ATPase activity. Optimal calcium uptake requires ATP and oxalate, and release of calcium from loaded vesicles was stimulated by the calcium ionophore A23187 and inhibited by LaCl3. These data indicate that calcium was being actively concentrated within membrane vesicles. After washing of such preparations in the absence of ATP, their capacity to take up Ca2+ is reduced to an initial value of 2.8 nmol/mg protein per min. In the presence of 2 - 10(6) M cyclic AMP to which was added a protein kinase preparation from human platelets, up to a 3-fold increase of this rate of uptake was observed. These results suggest that in platelets, as in muscle, cyclic AMP is a regulatory factor in the control of cytoplasmic calcium. Although the cyclic nucleotide may have still other functions, it appears likely that the well-known inhibition of many platelet activities by high intracellular cyclic AMP concentrations is directly linked to the stimulation of the removal of Ca2+ from the cytoplasm.     

Contrasting calcium localization and speciation in leaves of the Medicago truncatula mutant cod5 analyzed via synchrotron X–ray techniques

Oxalate‐producing plants accumulate calcium oxalate crystals (CaOx_(c)) in the range of 3–80% w/w of their dry weight, reducing calcium (Ca) bioavailability. The calcium oxalate deficient 5 (cod5) mutant of Medicago truncatula has been previously shown to contain similar Ca concentrations to wild‐type (WT) plants, but lower oxalate and CaOx_(c) concentrations. We imaged the Ca distribution in WT and cod5 leaflets via synchrotron X–ray fluorescence mapping (SXRF). We observed a difference in the Ca distribution between cod5 and WT leaflets, manifested as an abundance of Ca in the interveinal areas and a lack of Ca along the secondary veins in cod5, i.e. the opposite of what is observed in WT. X–ray microdiffraction (μXRD) of M. truncatula leaves confirmed that crystalline CaOx_(c) (whewellite; CaC₂O₄·H₂O) was present in the WT only, in cells sheathing the secondary veins. Together with μXRD, microbeam Ca K–edge X–ray absorption near‐edge structure spectroscopy (μXANES) indicated that, among the forms of CaOx, i.e. crystalline or amorphous, only amorphous CaOx was present in cod5. These results demonstrate that deletion of COD5 changes both Ca localization and the form of CaOx within leaflets.     

Inhibition of lipolysis in hamster adipocytes by the cation ionophor X537A.

The present study reports the effects of the lipophylic ionophore X537A on lipolysis and accumulation of cAMP in isolated hamster epidiymal adipocytes. X537A inhibited lipolysis activated with norepinephrine, isoproterenol, dibutyryl cAMP or theophylline but failed to influence basal lipolysis. The minimum effective concentration of X537A required to inhibit lipolysis was between 1 and 3 micrograms/ml; at a concentration of 10 micrograms/ml, X537A inhibited lipolysis by approximately 50%. The antilipolytic effect of X537A does not result from decreased formation of cAMP because the accumulation of cAMP in response to isoproterenol or theophylline was significantly potentiated in the presence of the ionophore. Most of the additional cAMP that accumulated in the presence of X537A was found to be intracellelular, the distribution of cAMP between cells and incubation medium not being influenced by X537A. Neither the basal activity of cAMP dependent protein kinase nor the activity in the presence of isoproterenol or theophylline was influenced by X537A. The effects of X537A on lipolysis and on accumulation of cAMP were found to persist in the absence of extracellular calcium, but adipocytes that were preincubated in a calcium free media containing 4.0 mM EGTA failed to respond to X537A with an increase in cAMP levels. It is concluded that X537A inhibits lipolysis by uncoupling cAMP accumulation from activation of triglyceride lipase by a mechanism unrelated to activation of protein kinase.     

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.     

State of translocated Ca2+ by sarcoplasmic reticulum inferred from kinetic analysis of calcium oxalate precipitation

Uptake of Ca²⁺ by sarcoplasmic reticulum in the presence of oxalate displays biphasic kinetics. An initial phase of normal uptake is followed by a second phase coincident with precipitation of calcium oxalate inside the vesicles. The precipitation rate induced by actively transported Ca²⁺ is depressed by increasing the added Ca²⁺ concentration. This correlates linearly with the reciprocal of precipitation rate. Therefore, a maximal limit rate could be extrapolated at zero Ca²⁺ ($\text{V}{}_0$). The rate of precipitation, also a function of added amount protein, gives a linear correlation in a double reciprocal plot. Thus, it was possible to estimate the maximal precipitation rate occurring at infinite protein concentration ($\text{V}{}^{\mathrm{\infty }}$). With the combined extrapolated values a maximal expected precipitation rate could be calculated ($\text{V}{}_0{}^{\mathrm{\infty }}$). Kinetics of calcium oxalate precipitation was studied in the absence of calcium uptake and empirical equations relating the rate of precipitation with the added Ca²⁺ were established. Entering $\text{V}{}_0{}^{\mathrm{\infty }}$ in the equations, an internal free Ca²⁺ concentration of approx. 2.5 mM was estimated. Additionally, it is shown that the ionophore X-537A does not supress the Ca²⁺ uptake, if added during the oxalate-dependent phase, albeit the uptake proceeds at a slower rate after the release of approx. 70 nmol Ca²⁺/mg protein. This amount presumably equals the internal free Ca²⁺ not sequestered by oxalate, producing a maximal concentration approx. 14 mM. Taking into account low affinity binding of internal binding sites and the transmembrane Ca²⁺ gradients built up during the uptake of Ca²⁺, values of free Ca²⁺ ranging from 3 to 6 mM, approaching those estimated by the precipitation analysis, could be estimated.     

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.     

The effects of copper,cadmium and zinc on particle filtration and uptake of glycine in the pacific oyster Crassostrea gigas

1. The filtration rate (volume of water completely cleared of collodial carbon per unit time) by control oysters is 36.60 ml/g hr ± 7.68 (sd).2. Filtration rates decrease with increasing concentrations of Cd²⁺ and Zn²⁺.3. In 8–16 mg/l Cu²⁺, filtration rates are significantly higher than the control, but in Cu²⁺ concentrations above 32 mg/l, filtration rates are lower than controls.4. Influx of ¹⁴C-glycine is characterized by Michaelis-Menten kinetics with J_max and K_t values of 1.85 ± 0.097 μmol/g hr and 33.7 ± 4.6 μM respectively.5. The uptake rate of glycine from 1 μM solution is 37.79 μmol/g hr.6. In order of degree of inhibition of glycine uptake, Cu²⁺ > Cd²⁺ > Zn²⁺.7. In 128 mg/l Cu²⁺, glycine uptake rate is reduced to 3.96 nmol/g hr or 10.5% of control.8. The rate of glycine uptake by filter feeding bivalves is dependent on rate of water pumping rate.9. The volume specific glycine transport (amount of glycine transported/unit volume of seawater completely cleared of colloidal carbon) by control oysters in 1 μM glycine concentrations is 1.03 μmol/l.10. The volume specific glycine transport remains constant in increasing Zn²⁺ concentrations, and declines in increasing Cu²⁺ concentrations, suggesting differential effects of the metals on particle filtration and the epithelial amino acid carriers.11. The apparent volume specific glycine transport increases to 2.14 μmol/l in 128 mg/l Cd²⁺. This volume specific transport greater than the glycine concentration in the medium suggests that there may be uptake of cadmium complexed glycine by the oysters.     

Oxalate transfer across the membranes of sarcoplasmic reticulum during the uptake of Ca++

The kinetic profile of Ca++ uptake in the presence of oxalate is biphasic. An initial phase independent on oxalate is followed by an oxalate-dependent phase delayed in time. The ionophore X-537A only abolishes the net Ca++ uptake if added before the onset of the oxalate phase. However, during this phase, X-537A suddenly releases an amount of Ca++ similar in quantity to that released in the initial phase. The delay of the oxalate-dependent phase is a function of pH. At pH of about 5.5, the oxalate phase and simultaneous calcium oxalate precipitation would theoretically start at the beginning, with no delay. Ejection of protons during Ca++ uptake is strongly depressed by oxalate, but not by other organic anions which do not trap Ca++. It is suggested that oxalate is transferred to the inside of the vesicles as a monoprotonated species at expense of protons ejected by the Ca++-pump during the uptake of Ca++.