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.
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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.
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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.
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Project description:The presence of phosphate from different origins (inorganic, bioorganic) is found more and more in calcium carbonate-based biominerals. Phosphate is often described as being responsible for the stabilization of the transient amorphous calcium carbonate phase. In order to specify the composition of the mineral phase deposited at the onset of carbonated shell formation, the present study investigates, down to the nanoscale, the growing shell from the European abalone Haliotis tuberculata, using a combination of solid state nuclear magnetic resonance, scanning transmission electron microscope and spatially-resolved electron energy loss spectroscopy techniques. We show the co-occurrence of inorganic phosphate with calcium and carbonate throughout the early stages of abalone shell formation. One possible hypothesis is that this first-formed mixed mineral phase represents the vestige of a shared ancestral mineral precursor that appeared early during Evolution. In addition, our findings strengthen the idea that the final crystalline phase (calcium carbonate or phosphate) depends strongly on the nature of the mineral-associated proteins in vivo.
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Project description:Diets containing inorganic phosphate additives are unbalanced with respect to calcium and these diets have been linked to the development of altered bone metabolism. Using 2 randomized cross-over studies in healthy humans, we (1) characterized the hormonal and urinary response to 2 meals with the same reported phosphorus amount (562-572 mg), where one was manufactured with inorganic phosphate additives and a comparatively lower Ca:P molar ratio (0.26 vs 0.48), and (2) assessed how acute homeostatic mechanisms adapt following 5-d exposure to recommended dietary phosphorus amount (~700 mg P/d) compared to a diet enriched with inorganic phosphate additives (~1100 mg P/d). Participants were then challenged with 500 mg of oral phosphorus in the form of inorganic phosphate after an overnight fast following each diet condition. Measurements included serum calcium, phosphate, PTH, and fibroblast growth factor 23 , vitamin D metabolites, and urine calcium and phosphate excretion. Following the meal containing inorganic phosphate additives with a low Ca:P ratio, serum phosphate was higher and more phosphate was excreted in the urine compared to the low additive meal. Although the Ca:P and calcium content was lower in the high additive meal, the same amount of calcium was excreted into the urine. Subsequently, increasing only dietary phosphate through additives resulted in lower 24-h excretion of calcium. The oral phosphate challenge promoted urinary calcium excretion, despite no consumption of calcium, which was attenuated when pre-acclimated to a high phosphate diet. These data suggest that ingestion of inorganic phosphate promotes calcium excretion, but homeostatic mechanisms may exist to reduce calcium excretion that are responsive to dietary intake of phosphate. Future studies are required to evaluate potential implication of diets enriched with inorganic phosphate additives on bone health.
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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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