MgATP-dependent accumulation of calcium ions and inorganic phosphate in a liver reticular pool.
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ABSTRACT:
SUBMITTER: Fulceri R
PROVIDER: S-EPMC1149738 | biostudies-other | 1990 Dec
REPOSITORIES: biostudies-other
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Project description:The effect of CoA and fatty acyl-CoA esters on Ca2+ fluxes has been studied in isolated liver microsomes and in digitonin-permeabilized hepatocytes. When microsomes were loaded with increasing concentrations of Ca2+ (6-29 nmol/mg of protein), the extent to which CoA and palmitoyl-CoA released Ca2+ increased. At 23 nmol of Ca2+/mg of protein, half-maximal [CoA] and [palmitoyl-CoA] were 35 and 50 microM respectively. Under conditions of minimal Ca2+ loading, net release of Ca2+ was absent, but Ca2+ translocation from a CoA-sensitive to a CoA-insensitive pool took place. The effect of CoA required the presence of fatty acids, probably to form fatty acyl esters. In permeabilized hepatocytes, the pool(s) mobilized by CoA (or by palmitoyl-CoA) appeared to be different from that mobilized by Ins(1,4,5)P3.
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Project description:The regulation of the activity of blowfly flight-muscle phosphorylase b kinase by P(i) and Ca(2+) was studied, and the actions of these effectors on the kinases from insect flight and rabbit leg muscles were compared. Preincubation of blowfly kinase with P(i) increased activity severalfold. The effect was concentration-dependent, with an apparent K(m) of about 20mm, and time-dependent, requiring at least 10min for maximal activation. Neither ATP nor cyclic AMP was needed, suggesting that a protein kinase may not be involved. Maximal activation of the insect kinase required Mg(2+) in addition to P(i). The apparent K(m) for Mg(2+) was 3mm. Rabbit leg-muscle phosphorylase b kinase was slightly inhibited, rather than stimulated, by P(i), and was strongly inhibited by K(+), Na(+) and Li(+). At physiological concentrations, Ca(2+) activated the phosphorylase b kinases from both blowfly flight and rabbit leg muscles. However, the responses to Ca(2+) of the enzymes from the two tissues were different. The mammalian kinase had virtually no activity in the absence of Ca(2+), and showed a large increase in activity over a narrow range of Ca(2+) concentrations. Flight-muscle kinase had appreciable activity in the absence of Ca(2+), and had a smaller increase over a wide range of Ca(2+) concentration. The concentrations of Ca(2+) required for half-activation were 0.1 and 1mum for the blowfly and rabbit enzymes respectively. The pH-activity profiles of the non-activated, phosphate- and Ca(2+)-activated kinase revealed considerable enhancement of activity with little, if any, increase in the ratio of activities at pH6.8 to those at 8.2. These results are discussed in relation to the mechanism coupling contraction to glycogenolysis and to the biochemical distinction between asynchronous and synchronous types of muscle.
| S-EPMC1166154 | biostudies-other
Project description:Traditional analyses of calcium homeostasis have separately quantified either calcium accumulation or release mechanisms. To define the system as a whole, however, requires multiple experimental techniques to examine both accumulation and release. Here we describe a technique that couples the simultaneous quantification of radio-labeled calcium accumulation in endoplasmic reticulum (ER) microsomes with the release of inorganic phosphate (Pi) by the hydrolytic activity of sarco-endoplasmic reticulum calcium ATPase (SERCA) all in the convenience of a 96-well format.
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Project description:In the exchange of metabolites and ions between the mitochondrion and the cytosol, the voltage-dependent anion channel (VDAC) is a key element, as it forms the major transport pathway for these compounds through the mitochondrial outer membrane. Numerous experimental studies have promoted the idea that VDAC acts as a regulator of essential mitochondrial functions. In this study, using a combination of molecular dynamics simulations, free-energy calculations, and electrophysiological measurements, we investigated the transport of ions through VDAC, with a focus on phosphate ions and metabolites. We showed that selectivity of VDAC towards small anions including monovalent phosphates arises from short-lived interactions with positively charged residues scattered throughout the pore. In dramatic contrast, permeation of divalent phosphate ions and phosphate metabolites (AMP and ATP) involves binding sites along a specific translocation pathway. This permeation mechanism offers an explanation for the decrease in VDAC conductance measured in the presence of ATP or AMP at physiological salt concentration. The binding sites occur at similar locations for the divalent phosphate ions, AMP and ATP, and contain identical basic residues. ATP features a marked affinity for a central region of the pore lined by two lysines and one arginine of the N-terminal helix. This cluster of residues together with a few other basic amino acids forms a "charged brush" which facilitates the passage of the anionic metabolites through the pore. All of this reveals that VDAC controls the transport of the inorganic phosphates and phosphate metabolites studied here through two different mechanisms.
| S-EPMC4393092 | biostudies-literature
Project description:1. MgATP-dependent 45Ca2+ uptake by microsomes obtained from various non-hepatic tissues, namely rat brain, rat solid Morris hepatoma 3924A and human platelets, was measured in the presence of P(i) at low, cytosol-like, concentrations. 2. Increasing P(i) concentrations (0.5-3 mM) caused a progressive enlargement of the 45Ca(2+)-storage capacity of all the microsomal fractions. 3. As a result of P(i) stimulation of Ca2+ uptake, 45Ca2+ and [32P]P(i) were co-accumulated by the three microsomal fractions. 4. The time course for 45Ca2+ and [32P]P(i) accumulation in brain microsomes revealed a biphasic 45Ca2+ uptake: a rapid phase was followed by a second, slower, phase, which depended on the presence of P(i). During the P(i)-dependent phase, the uptake of 45Ca2+ was paralleled by the uptake of [32P]Pi. 5. The passive efflux of Ca2+ was paralleled by the efflux of P(i) and vice versa. In fact, the inhibition of active Ca2+ uptake by excess EGTA, or lowering the P(i) concentration of the incubation system by dilution, caused the release of 45Ca2+ and [32P]P(i) from 45Ca2+ or [32P]P(i) pre-loaded brain microsomes. The Ca2+ ionophore A23187 also released 45Ca2+ and [32P]P(i). 6. Ca2+ efflux by A23187 was rapid (t 1/2 approx. 2 s) and independent of the extent of intravesicular Ca2+ loading, which indicates that Ca2+ and P(i) do not form intravesicular insoluble complexes. 7. The progressive increase in Ca2+ accumulation, depending on P(i) stimulation, resulted in a proportional increase in the amount of Ca2+ releasable by InsP3 in the three non-hepatic microsomal fractions and in digitonin-permeabilized platelets. 8. Concomitantly to Ca2+, microsomal P(i) was also released by InsP3.
| S-EPMC1132164 | biostudies-other
Project description:Phosphorus (P) in plants is taken from soil as an inorganic phosphate (Pi) and is one of the most important macroelements in growth and development. Plants actively react to Pi starvation by the induced expression of Pi transporters, MIR399, MIR827, and miR399 molecular sponge - IPS1 genes and by the decreased expression of the ubiquitin-conjugating enzyme E2 (PHOSPHATE2 - PHO2) and Pi sensing and transport SPX-MFS genes. The PHO2 protein is involved in the degradation of Pi transporters PHT1;1 (from soil to roots) and PHO1 (from roots to shoots). The decreased expression of PHO2 leads to Pi accumulation in shoots. In contrast, the pho1 mutant shows a decreased level of Pi concentration in shoots. Finally, Pi starvation leads to decreased Pi concentration in all plant tissues. Little is known about plant Pi homeostasis in other abiotic stress conditions. We found that, during the first hour of heat stress, Pi accumulated in barley shoots but not in the roots, and transcriptomic data analysis as well as RT-qPCR led us to propose an explanation for this phenomenon. Pi transport inhibition from soil to roots is balanced by lower Pi e?ux from roots to shoots directed by the PHO1 transporter. In shoots, the PHO2 mRNA level is decreased, leading to an increased Pi level. We concluded that Pi homeostasis in barley during heat stress is maintained by dynamic changes in Pi-related genes expression.
| S-EPMC4919326 | biostudies-literature
Project description:Erythrocytes from several different species were exposed to Ca2+ and the bivalent-cation ionophore A23187. The lipid composition, morphology and K+ permeability of the treated cells were investigated. Erythrocytes from human, rat, guinea pig and rabbit (a) showed an increased concentration of 1,2-diacyl-sn-glycerol and enhanced labelling of phosphatidate with 32P, (b) underwent echinocytosis and outward vesiculation, and (c) rapidly released much of their intracellular K+. Pig cells showed only the K+ loss, and ox and sheep (high-K+) cells showed none of these Ca2+-evoked effects. All of the cells underwent stomatocytosis and inward vesiculation when treated externally with Clostridium perfringens phospholipase C. These results support the idea that there is a correlation between the asymmetric insertion of diacylglycerol (or ceramide) into the membrane and the shape-changes leading to microvesiculation, but they indicate that Ca2+-triggered K+ efflux and diacylglycerol production are unrelated events. Erythrocytes of chicken and turkey showed no Ca2+-stimulated K+ efflux. They showed slight ionophore A23187-stimulated vesiculation, but this appeared to be associated with the appearance in the membrane of ceramide rather than of diacylglycerol. Phospholipase C treatment caused very similar changes in morphology and phosphatidate labelling to those seen in mammalian erythrocytes.
| S-EPMC1165033 | biostudies-other
Project description:Primary renal inorganic phosphate (Pi) wasting leads to hypophosphatemia, which is associated with skeletal mineralization defects. In humans, mutations in the gene encoding the type IIc sodium-dependent phosphate transporter lead to hereditary hypophophatemic rickets with hypercalciuria, but whether Pi wasting directly causes the bone disorder is unknown. Here, we generated Npt2c-null mice to define the contribution of Npt2c to Pi homeostasis and to bone abnormalities. Homozygous mutants (Npt2c(-/-)) exhibited hypercalcemia, hypercalciuria, and elevated plasma 1,25-dihydroxyvitamin D(3) levels, but they did not develop hypophosphatemia, hyperphosphaturia, renal calcification, rickets, or osteomalacia. The increased levels of 1,25-dihydroxyvitamin D(3) in Npt2c(-/-) mice compared with age-matched Npt2c(+/+) mice may be the result of reduced catabolism, because we observed significantly reduced expression of renal 25-hydroxyvitamin D-24-hydroxylase mRNA but no change in 1alpha-hydroxylase mRNA levels. Enhanced intestinal absorption of calcium (Ca) contributed to the hypercalcemia and increased urinary Ca excretion. Furthermore, plasma levels of the phosphaturic protein fibroblast growth factor 23 were significantly decreased in Npt2c(-/-) mice. Sodium-dependent Pi co-transport at the renal brush border membrane, however, was not different among Npt2c(+/+), Npt2c(+/-), and Npt2c(-/-) mice. In summary, these data suggest that Npt2c maintains normal Ca metabolism, in part by modulating the vitamin D/fibroblast growth factor 23 axis.
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Project description:BackgroundSex differences in calcium and phosphate have been observed. We aimed to assess a relation with age.MethodsWe used the laboratory values of serum calcium, phosphate and albumin from three different samples ( 2005, 2010 and 2014 years) using the hospital information system of Erasmus MC, Rotterdam. The samples were divided into three age groups: 1-17, 18-44 and ≥45 years. Sex differences in calcium and phosphate were analyzed using ANCOVA, adjusting for age and serum albumin. Furthermore, sex by age interactions were determined and we analyzed differences between age groups stratified by sex.ResultsIn all three samples there was a significant sex × age interaction for serum calcium and phosphate, whose levels were significantly higher in women compared to men above 45 years. No sex differences in the younger age groups were found. In men, serum calcium and phosphate levels were highest in the youngest age group compared to age groups of 18-44 and ≥45 years. In women, serum calcium levels were significantly higher in the age group 1-17 and the age group ≥45 years compared to the 18-44 years age group. In women, serum phosphate was different between the three different age groups with highest level in the group 1-17 years and lowest in the group 18-44 years.ConclusionThere are age- dependent sex differences in serum calcium and phosphate. Furthermore, we found differences in serum calcium and phosphate between different age groups. Underlying mechanisms for these age- and sex- differences are not yet fully elucidated.
| S-EPMC8052581 | biostudies-literature
Project description:Amorphous calcium phosphate (ACP) has been widely found during bone and tooth biomineralization, but the meta-stability and labile nature limit further biomedical applications. The present study found that the chelation of polyacrylic acid (PAA) molecules with Ca2+ ions in Mg-ACP clusters (~2.1 ± 0.5 nm) using a biomineralization strategy produced inorganic-organic Mg-ACP/PAA hybrid nanoparticles with better thermal stability. Mg-ACP/PAA hybrid nanoparticles (~24.0 ± 4.8 nm) were pH-responsive and could be efficiently digested under weak acidic conditions (pH 5.0-5.5). The internalization of assembled Mg-ACP/PAA nanoparticles by MC3T3-E1 cells occurred through endocytosis, indicated by laser scanning confocal microscopy and cryo-soft X-ray tomography. Our results showed that cellular lipid membranes remained intact without pore formation after Mg-ACP/PAA particle penetration. The assembled Mg-ACP/PAA particles could be digested in cell lysosomes within 24 h under weak acidic conditions, thereby indicating the potential to efficiently deliver encapsulated functional molecules. Both the in vitro and in vivo results preliminarily demonstrated good biosafety of the inorganic-organic Mg-ACP/PAA hybrid nanoparticles, which may have potential for biomedical applications.
| S-EPMC7841502 | biostudies-literature