Involvement of intracellular calcium in the phosphate efflux from mammalian nonmyelinated nerve fibers

Summary Phosphate efflux was measured as the fractional rate of loss of radioactivity from desheathed rabbit vagus nerves after loading with radiophosphate. The effects of strategies designed to increase intracellular calcium were investigated. At the same time, the exchangeable calcium content was measured using⁴⁵Ca. Application of calcium ionophore A23187 increased phosphate efflux in the presence of external calcium in parallel with an increase in calcium content. In the absence of external calcium, there was only a late, small increase in phosphate efflux. For nerves already treated with the calcium ionophore, the phosphate efflux was sensitive to small changes in external calcium, in the range 0.2 to 2mm calcium, whereas similar increases in calcium in absence of ionophore gave much smaller increases in phosphate efflux. Removal of external sodium (choline substitution) produced an initial increase in phosphate efflux followed by a fall. The initial increase in phosphate efflux was much larger in the presence of calcium, than in its absence. The difference was again paralleled by an increase in calcium content of the preparation, thought to be due to inhibition of Na/Ca exchange by removal of external sodium. Measurements of ATP content and ATP, ADP, phosphate and creatine phosphate ratios did not indicate significant metabolic changes when the calcium content was increased. Stimulation of phosphate efflux by an increase in intracellular calcium may be due to stimulation of phospholipid metabolism. Alternatively, it is suggested that stimulation of phosphate efflux is associated with the stimulation of calcium efflux, possibly by cotransport of calcium and phosphate.     

Calcium efflux and intracellular exchangeable calcium in mammalian nonmyelinated nerve fibers

Summary Calcium efflux was measured in desheathed rabbit vagus nerves loaded with⁴⁵Ca²⁺. The effects of extracellular calcium, sodium, phosphate, potassium and lanthanum ions on the calcium efflux were investigated and the distribution of intracellular calcium determined by kinetic analysis of⁴⁵Ca²⁺ efflux profiles. The⁴⁵Ca²⁺ desaturation curve can be adequately described by three exponential terms. The rate constant of the first component (0.2 min^–1) corresponds to an efflux from an extracellular compartment. The two slow components had rate constants of 0.03 and 0.08 min^–1 and represent the efflux from two intracellular pools. The amounts of exchangeable calcium in these two pools, after a loading period of 150 min, were 0.170 and 0.102 mmol/kg wet weight, respectively. The total calcium efflux in physiological conditions amounted to about 24 fmol cm^–2 sec^–1. The magnitude of the two intracellular compartments as well as the total calcium efflux were markedly affected by extracellular phosphate, sodium and lanthanum, whereas the corresponding rate constants remained almost unchanged. Phosphate reversed the effect of sodium withdrawal on the calcium efflux: in the absence of phosphate, sodium withdrawal increased the calcium efflux to 224%, but in the presence of phosphate, sodium withdrawal decreased calcium efflux to 44%. Phosphate also affected the increase in calcium efflux produced by inhibitors of mitochondrial calcium uptake, suggesting that two different mitochondrial pools contribute to the control and regulation of intracellular calcium and of the transmembrane calcium transport.Deceased 18 April 1988     

Spin label studies on rat liver and heart plasma membranes: Effects of temperature,calcium, and lanthanum on membrane fluidity

The structures of rat liver and heart plasma membranes were studied with the 5-nitroxide stearic acid spin probe, I(1 2,3). The polarity-corrected order parameters (S) of liver and heart plasma membranes were independent of probe concentration only if experimentally determined low I(1 2,3)/lipid ratios were employed. At higher probe/lipid ratios, the order parameters of both membrane systems decreased with increasing probe concentration, and these effects were attributed to enhanced nitroxide radical interactions. Examination of the temperature dependence of approximate and polarity-corrected order parameters indicated that lipid phase separations occur in liver (between 19° and 28°C) and heart (between 21° and 32°C) plasma membranes. The possibility that a wide variety of membrane-associated functions may be influenced by these thermotropic phase separations is considered. Addition of 3.9 mM CaCl₂ to I(1 2,3)-labeled liver plasma membrane decreased the fluidity as indicated by a 5% increase in S at 37°C. Similarly, titrating I(1 2,3)-labeled heart plasma membranes with either CaCl₂ or LaCl₃ decreased the lipid fluidity at 37°C, although the magnitude of the La³⁺ effect was larger and occurred at lower concentrations than that induced by Ca²⁺; addition of 0.2 mM La³⁺ or 3.2 mM Ca²⁺ increased S by approximately 7% and 5%, respectively. The above cation effects reflected only alterations in the membrane fluidity and were not due to changes in probe–probe interactions. Ca²⁺ and La³⁺ at these concentrations decrease the activities of such plasma membrane enzymes as Na⁺, K⁺-ATPase and adenylyl cyclase, and it is suggested that the inhibition of these enzymes may be due in part to cation-mediated decreases in the lipid fluidity.     

Continuous measurement of calcium influx in mammalian nonmyelinated nerve fibers: Effects of Na o,Ca o,and electrical activity

Summary Efflux of⁴²K⁺ was measured in frog sartorius muscles equilibrated in hyperosmotic depolarizing solutions. At the internal potentials obtained, K⁺ passes mainly through the inward rectifier potassium channels.Inhibition of K⁺ efflux by external Zn²⁺ (0.25 to 15mm) differs in three significant ways from inhibition by Ba²⁺. (1) The dose-response relation does not correspond to action at a single site. (2) The Zn²⁺-sensitivity of K⁺ efflux does not depend on [K⁺] _o at constant internal potential. (3) Zn²⁺ inhibition is reduced by hydrogen ions, while Ba²⁺ inhibition is unaffected. Further, the Ba²⁺-sensitivity of K⁺ efflux is not altered by a half-inhibiting Zn²⁺ concentration, suggesting that the two ions do not interact at a common site.The histidine-modifying reagent diethylpyrocarbonate (DEPC) reduces Zn²⁺ inhibition. After DEPC treatment Zn²⁺ inhibition is further reduced by low pH. DEPC has little effect on Ba²⁺ inhibition. Zn²⁺ inhibition is not altered by treatment with the sulfhydryl reagents 5,5-dithio-bis(2-nitrobenzoic acid) or dithiothreitol.The results can be described by either of two models in which two sites can bind Zn²⁺ and one or both of the sites may also bind H⁺. When both sites bind Zn²⁺, K⁺ efflux is inhibited, and a third site may then bind H⁺. The effects of DEPC can be accounted for by a decrease in H⁺ affinity of the first two sites by a factor of 50, and a decrease in Zn²⁺ affinity of these sites and of the H⁺ affinity of the third site by about one order of magnitude.     

Role of potassium in the phosphate efflux from mammalian nerve fibers

Summary The efflux of phosphate was measured in rabbit vagus nerve loaded with radiophosphate. The efflux was found to depend on the K concentration of the bathing solutions; increasing the K from 5.6 up to 150mm produced a maximal lowering of 28%; K-free solution produced a transient increase whose peak was 86% above the normal efflux. In the presence of Na, the K-free effect could be repeated; in Na-free solution, it was found only for the first application of the K-free solution. The phosphate efflux was not altered when K was replaced by Rb; replacement with Cs showed that this ion only partially mimics the effect of K.The results suggest that the transient increase in phosphate efflux is due to release of label from a K-dependent saturable binding site, which is distinct from the main intracellular pool. The binding site appears to be labeled from the inside by the Na-dependent phosphate efflux previously described. It may correspond to the phosphorylation of membrane phospholipids. A mathematical model of this system is developed and curves simulated by an analog computer are compared to the experimental results.Measurements of the membrane potential and the internal inorganic phosphate showed that the effect of K on the phosphate efflux could not be explained by changes in the membrane potential or in the internal phosphate pool.     

聚合磷酸钙骨水泥理化性能研究

将磷酸四钙与磷酸氢钙与丙烯酸－衣康酸共聚酸制成磷酸钙骨水泥体系。结果显示：在共聚酸浓度为３０％时，该体系的凝固时间为８分钟，抗压强度为３８．３１Ｍｐａ，溶解率为１．０２％，最终产物为羟磷灰石。本材料是一种有较大应用前景的粘接、垫底、根管充填和骨缺损修复替代物。     

Chemotherapy drug delivery from calcium phosphate nanoparticles

Calcium phosphate nanoparticles (nanoCaP) conjugated with cis-diamminedichloroplatinum (CDDP, cisplatin) were prepared through the electrostatic binding of an aquated species of cisplatin to the nanoCaP in a chloride-free solution. The agglomeration of the nanoCaP that typically occurs during synthesis of CaP was controlled through the addition of DARVAN 811 immediately after precipitation and before drug conjugation. In vitro drug release studies were completed and showed a sustained release of CDDP from the nanoconjugates over time. The cytotoxicity of the nanoCaP/CDDP was compared to that of the free drug in an in vitro cell proliferation assay using the CDDP resistant A2780cis human ovarian cancer cell line. The CDDP released from the nanoconjugates was equally effective as the free drug against the A2780cis cell line. Direct addition cytotoxicity studies revealed that the sterically-stabilized, negatively-charged drug nanoconjugates are unable to overcome drug resistance and had an increased IC50 value as compared to the free drug.     

Dent disease: A window into calcium and phosphate transport

This review examines calcium and phosphate transport in the kidney through the lens of the rare X‐linked genetic disorder Dent disease. Dent disease type 1 (DD1) is caused by mutations in the CLCN5 gene encoding ClC‐5, a Cl^?/H⁺ antiporter localized to early endosomes of the proximal tubule (PT). Phenotypic features commonly include low molecular weight proteinuria (LMWP), hypercalciuria, focal global sclerosis and chronic kidney disease; calcium nephrolithiasis, nephrocalcinosis and hypophosphatemic rickets are less commonly observed. Although it is not surprising that abnormal endosomal function and recycling in the PT could result in LMWP, it is less clear how ClC‐5 dysfunction disturbs calcium and phosphate metabolism. It is known that the majority of calcium and phosphate transport occurs in PT cells, and PT endocytosis is essential for calcium and phosphorus reabsorption in this nephron segment. Evidence from ClC‐5 KO models suggests that ClC‐5 mediates parathormone endocytosis from tubular fluid. In addition, ClC‐5 dysfunction alters expression of the sodium/proton exchanger NHE3 on the PT apical surface thus altering transcellular sodium movement and hence paracellular calcium reabsorption. A potential role for NHE3 dysfunction in the DD1 phenotype has never been investigated, either in DD models or in patients with DD1, even though patients with DD1 exhibit renal sodium and potassium wasting, especially when exposed to even a low dose of thiazide diuretic. Thus, insights from the rare disease DD1 may inform possible underlying mechanisms for the phenotype of hypercalciuria and idiopathic calcium stones.     

Phosphate transport,membrane potential,and movements of calcium in rat liver mitochondria

The membrane potential and calcium accumulation of mitochondria were followed by ion-specific electrodes in the presence of the proton-donor anions phosphate, acetate, glutamate, and beta-hydroxybutyrate. Phosphate was the only anion which allowed rapid and complete restoration of both the membrane potential and the steady-state extramitochondrial calcium concentration after the uptake of 100–200 nmol calcium per mg protein. If there was no influx of any proton-donor anion, the extent of calcium uptake depended on the intramitochondrial phosphate content. Both the fall of the membrane potential and the increase of the external calcium concentration brought about by a given amount of uncoupler were counteracted by phosphate transported into the mitochondria.     

Effects of lanthanum and calcium on photoelectron transport activity and the related protein complexes in chloroplast of cucumber leaves

The effects of lanthanum and calcium ions on electron transport, dichlorephenol indophenol (DCIP) photoreduction, and oxygen evolution activities in chloroplast from cucumber (Cucumis satives L.) were determined. The lanthanum inhibited the whole electron chain-transport activity of chloroplast. DCIP photoreduction and oxygen evolution activities of the photosystem I (PSII) also decrease after treatment with La³⁺. But the diminished activities of PSII and chloroplast caused by La³⁺ could be reversed by Ca²⁺ and even became higher than the control level. The concentration analysis of related protein complexes to photoelectron transport in chloroplast included that La³⁺ induced the concentration of chlorophyll protein complexes increasing but caused some nonchlorophyll protein complexes to decompose partially. This increasing effect of La³⁺ on chlorophyll protein complexes results in the improvement of chlorophyll content, which will improve the absorption of photoelectron and energy transport in the process of photosynthesis.     

Occurrence of adrenergic nerve fibers in human thymus during immune response

The adrenergic nerve fibers (ANF), the neuropeptide Y-like immunoreactive nerve fibers (NPY-NF) and the noradrenaline (NA) amount were studied in the human thymus in subjects previously treated or not treated with interferon therapy with the aim to identify the changes due to the interferon therapy. This therapy has been used in patients affected by multiple sclerosis (MS). Biochemical and morphological methods were used associated with quantitative analysis of images. The whole thymuses were removed during autopsies in young and adult patients not treated with interferon. Moreover, samples of thymus were removed from patients, either young or adult who had previously been treated with interferon therapy, and subjected, for diagnostic reasons, to thymic biopsy. All samples of thymus were weighed, measured and dissected. Thymic slices were stained with Eosin-orange for detection of the microanatomical details, or with Bodian's reaction for recognition of nervous structures. Histofluorescence microscopy was used for detection of ANF, and immunofluorescence microscopy for recognition of NPY-like immunoreactive structures. All morphological results were subjected to quantitative analysis of images. Noradrenaline contained in thymic structures was measured by biochemical methods. Our results only concerned the effects of the therapy and suggested that treatment with interferon therapy induces many changes in the thymic structures: (1) The protein content of thymus is significantly increased; (2) the NA content in the thymus is also significantly increased; (3) NPY-like immunoreactive structures in the thymus are significantly increased; (4) occurrence of NPY-like immunoreactivity is particularly and significantly increased both in thymic microenvironment and in structures resembling nerve fibers; (5) ANF are significantly increased in the same thymic structures in which NPY-like immunoreactivity is also increased (i.e. thymic microenvironment and structures resembling nerve fibers). The morphological and biochemical changes observed can also explain the immunological changes induced in the thymus after immunostimulating therapy.     

Immunohistochemical demonstration of serotonin-containing nerve fibers in the cerebellum

Summary The localization of serotonin (5-HT)-immunoreactive nerve fibers in the cerebellum of the rat and cat was investigated by means of the peroxidase-anti-peroxidase (PAP) method using highly specific antibodies to 5-HT.Serotonin-containing nerve fibers were distributed throughout the entire cerebellum including the deep cerebellar nuclei, while 5-HT-positive neuronal somata were not detected in the cerebellum of either species. A different pattern of 5-HT innervation was found among the three layers of the cerebellar cortex. There were also interspecific differences in the pattern of distribution of 5-HT. In the rat, the pool of 5-HT nerve fibers mainly consisted of tangential elements, which were predominant in the molecular layer, while in the cat only a few 5-HT fibers were found in the molecular layer of the cerebellar cortex; dense networks of 5-HT nerve fibers were present in the granular layer. Some differences are evident in the pattern of distribution of 5-HT fibers in cerebellar regions classified on an anatomical and functional basis.This work was supported by a grant (No. 56440022) from the Ministry of Education, Science and Culture, Japan     

Calcium regulation of skeletal myogenesis: IV A defined culture medium permissive for myotube formation and the use of the calcium antagonist lanthanum

Summary Lanthanum has been used effectively in studies of calcium physiology in experiments of short duration. In experiments of longer duration, we report that solutions, such as cell culture medium, containing lanthanum (La⁺⁺) undergo a decrease in pH on the time scale of hours. Presumaly, the decrease in pH is a consequece of the hydrolysis of water by the solution-active La⁺⁺⁺ ions. We have devised a defined culture medium without serum and chick embryo extract which is permissive for myotube formation. This defined medium is also useful for studies of La⁺⁺⁺ as a calcium antagonist. with Ca⁺⁺ to low-Ca⁺⁺ fusion-blocked cultures. This study was supported in part by NIH grants NS 10196 and AM 25202 and The Muscular Dystrophy Association.     

Effects of weak amplitude-modulated microwave fields on calcium efflux from awake cat cerebral cortex

Calcium (45Ca2+) efflux was studied from preloaded cortex in cats immobilized under local anesthesia, and exposed to a 3.0-mW/cm2 450-MHz field, sinusoidally amplitude modulated at 16 Hz modulation depth 85%). Tissue dosimetry showed a field of 33 V/m in the interhemispheric fissure (rate of energy deposition 0.29 W/kg). Field exposure lasted 60 min. By comparison with controls, efflux curves from field exposed brains were disrupted by waves of increased 45Ca2+ efflux. These waves were irregular in amplitude and duration, but many exhibited periods of 20-30 min. They continued into the postexposure period. Binomial probability analysis indicates that the field-exposed efflux curves constitute a different population from controls at a confidence level of 0.96. In about 70% of cases, initiation of field exposure was followed by increased end-tidal CO2 excretion for about 5 min. However, hypercapnea induced by hypoventilation did not elicit increased 45Ca2+ efflux. Thus this increase with exposure does not appear to arise as a secondary effect of raised cerebral CO2 levels. Radioactivity measurements in cortical samples after superfusion showed 45Ca2+ penetration at about 1.7 mm/hr, consistent with diffusion of the ion in free solution.     

Effects of calcium and phosphate on the corpuscles of Stannius of the teleost fish,Oreochromis mossambicus

Summary Removal of the corpuscles of Stannius (CS) in Oreochromis mossambicus leads to hypercalcemia and hypophosphatemia. The effects on CS size and ultrastructure of different calcium and phosphate concentrations of the ambient water and of the food were investigated. A six-fold increase of the calcium concentration of the water leads to a four-fold increase in CS volume; this is mainly caused by an increase in the size and number of the type-1 cells. The effect of external calcium is most probably mediated by the calcium concentration of the blood plasma. Plasma ionic calcium may be the relevant factor. Changes in the calcium concentration of the food had no effect on the CS. Similarly, hyperphosphatemia or hypophosphatemia induced by high phosphate concentrations of the water or the food, or by a phosphate-deficient diet, had no noticeable effect on the CS. The results support the hypothesis that the type-1 cells produce the hypocalcemic factor of the CS. There is no evidence for the production by the CS of an endocrine factor involved in the control of phosphate metabolism.     

ATP-dependent calcium transport by a Golgi-enriched membrane fraction from mouse mammary gland

Summary Crude particulate preparations from the mammary glands of lactating mice were shown to transport calcium against a concentration gradient in the presence of ATP and mitochondrial inhibitors. Density gradient centrifugation with both sucrose and Percoll gradients indicated the presence of ATP-dependent transport in more than one membrane fraction. A Golgi-enriched membrane fraction possessed the highest specific activity of calcium transport. Digitonin, which increases the permeability of plasma membranes to calcium, did not affect this process. The Golgi fraction contained a 100,000 Dalton protein whose phosphorylation by -[³²P]-ATP was enhanced by a micromolar concentrations of free calcium. The phosphorylation was acid-stable and hydroxylamine-sensitive. These properties suggest that Golgi membranes in an actively secreting mammary epithelium possess a calcium transport system which resembles the calcium ATPase present in the sarcoplasmic reticulum of skeletal muscle.     

Phosphate transport in human red blood cells: Concentration dependence and pH dependence of the unidirectional phosphate flux at equilibrium conditions

Summary The concentration dependence and the pH dependence of the phosphate transport across the red cell membrane were investigated. The unidirectional phosphate fluxes were determined by measuring the³²P-phosphate self-exchange in amphotericin B (5 mol/liter) treated erythrocytes at 25°C.The flux/concentration curves display anS-shaped increase at low phosphate concentrations, a concentration optimum in the range of 150 to 200mm phosphate and a self-inhibition at high phosphate concentrations. The apparent half-saturation concentrations,P _(0.5), range from 50 to 70mm and are little affected by pH. The self-inhibition constants, as far as they can be estimated, range from 400 to 600mm. The observed maximal phosphate fluxes exhibit a strong pH dependence. At pH 7.2, the actual maximal flux is 2.1×10^–6 moles·min^–1·g cells^–1. The ascending branches of the flux/concentration curves were fitted to the Hill equation. The apparent Hill coefficients were always in the range of 1.5–2.0. The descending branches of the flux/concentration curves appear to follow the same pattern of concentration response.The flux/pH curves were bell-shaped and symmetric with regard to their pH dependence. The pH optimum is at approximately pH 6.5–6.7. The apparent pK of the activator site is in the range of 7.0 to 7.2, while the apparent pK for the inactivating site is in the range of 6.2 to 6.5. The pK-values were not appreciably affected by the phosphate concentration.According to our studies, the transport system possesses two transport sites and probably two modifier sites as indicated by the apparent Hill coefficients. In addition, the transport system has two proton binding sites, one with a higher pK that activates and one with a lower pK that inactivates the transport system. Since our experiments were executed under self-exchange conditions, they do not provide any information concerning the location of these sites at the membrane surfaces.     

Ex vivo detection of calcium phosphate and calcium carbonate in rat blood serum

The total calcium (tCa) in blood serum comprises free Ca²⁺ ions (fCa), protein-bound calcium (prCa), and complexed calcium by small anions (cCa). The cCa fraction, in addition to fCa, has been indicated to have some physiological activity. However, there is little evidence for the structure of its constituents. Here we report an ex vivo detection of the cCa constituents by synchrotron X-ray absorption near-edge structure spectroscopy. We collected the data directly on rat blood serum and, by making use of the reference samples, derived a spectrum that exhibits the features of cCa constituents. Among the features are those of the complexes of calcium phosphate and calcium carbonate. The detected complexes in the cCa fraction are mainly Ca(η²-HPO₄)(H₂O)₄ and Ca(η¹-HCO₃)(H₂O)₅⁺, in which HPO₄^2? and HCO₃^? serve as bidentate and unidentate ligands, respectively. The remained H₂O molecules on the coordination sphere of Ca²⁺ enable these complexes to behave partially like aquated Ca²⁺ ions in protein-binding. Besides, as the dominant part of prCa, albumin-bound calcium (albCa) exhibits a spectrum that closely resembles that of fCa, indicating weak interactions between the protein carboxyl groups and calcium. The weak-bound cCa and albCa, along with fCa and the relevant anions, compose a local chemical system that could play a role in maintaining the calcium level in blood.     

On the Protection by Inorganic Phosphate of Calcium-Induced Membrane Permeability Transition

The role of inorganic phosphate as inhibitor of mitochondrial membrane permeability transition was studied. It is shown that in mitochondria containing a high phosphate concentration, i.e., 68 nmol/mg, Ca²⁺ did not activate the pore opening. Conversely, at lower levels of matrix phosphate, i.e., 38 nmol/mg, Ca²⁺ was able to induce subsequent pore opening. The inhibitory effect of phosphate was apparent in sucrose-based media, but it was not achieved in KCl media. The matrix free Ca²⁺ concentration and matrix pH were lowered by phosphate, but they were always higher in K⁺-media. In the absence of ADP, phosphate strengthened the inhibitory effect of cyclosporin A on carboxyatractyloside-induced Ca²⁺ efflux. Acetate was unable to replace phosphate in the induction of the aforementioned effects. It is concluded that phosphate preserves selective membrane permeability by diminishing the matrix free Ca²⁺ concentration.     

Short-time pre-washing of brushite-forming calcium phosphate cement improves its in vitro cytocompatibility

A pre-washing protocol was developed for resorbable, brushite-forming calcium phosphate cements (CPCs) to avoid harmful in vitro effects on cells. CPC discs (JectOS+, Kasios; self-developed CPC) were pre-washed with repeated changes of phosphate-buffered saline (PBS; 24 h total). Unwashed or PBS-pre-washed discs were incubated in culture medium (5% fetal calf serum; up to 10 days) and then tested for their influence on pH/calcium/phosphate levels in H₂O extracts. Effects on pH/calcium/phosphate levels in culture supernatants, and morphology, adherence, number, and viability of ATDC5 cells and adipose-tissue derived stem cells were analyzed in co-culture. Pre-washing did not alter CPC surface morphology or Ca/P ratio (scanning electron microscopy; energy-dispersive X-ray spectroscopy). However, acidic pH of unwashed JectOS+ and self-developed CPC (5.82; 5.11), and high concentrations of Ca (2.17; 2.40 mM) and PO₄ (38.15; 49.28 mM) in H₂O extracts were significantly counteracted by PBS-pre-washing (pH: 7.92; 7.92; Ca: 0.64; 1.11 mM; PO₄: 5.39–5.97 mM). Also, PBS-pre-washing led to physiological pH (approx. 7.5) and PO₄ levels (max. 5 mM), and sub-medium Ca levels (0.5–1 mM) in supernatants and normalized cell morphology, adherence, number, and viability. This CPC pre-washing protocol improves in vitro co-culture conditions without influencing its structure or chemical composition.