Stimulated Phosphate Uptake Research Articles

Stimulation of phosphate uptake in human platelets by thrombin and collagen. Changes in specific 32P labeling of metabolic ATP and polyphosphoinositides.

The uptake of [32P]phosphate by human, gel-filtered blood platelets and its incorporation into cytoplasmic ATP and polyphosphoinositides was studied. In unstimulated platelets, uptake was Na+o-dependent and saturable at approximately 20 nmol/min/10(11) cells with a half-maximal rate at 0.5 mM extracellular phosphate. Upon stimulation with thrombin or collagen, net influx of [32P]Pi was accelerated 5- to 10-fold. With thrombin, [32P]Pi efflux was also increased. After the first 2 min, efflux exceeded influx, resulting in the net release of [32P]Pi from the platelets. Since the stimulus-induced burst in [32P]Pi uptake paralleled the secretory responses, it might be an integral part of stimulus-response coupling in platelets. The stimulus-induced burst in net [32P]Pi uptake led to an enhanced labeling of metabolic ATP, which was already detectable at 5 s after stimulation with thrombin. Concomitantly, the incorporation of [32P]Pi into phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was accelerated. The thrombin-induced increase in specific 32P radioactivity of cytoplasmic ATP fully accounted for the simultaneous increase in specific 32P radioactivity of these phosphoinositides. In studying the extent of 32P labeling of phosphorylated compounds in response to a cellular stimulus, it is therefore essential to measure the effect of the stimulus on the specific radioactivity of cytoplasmic ATP.

Increased phosphate incorporation into phospholipids accompanying the substrate-mediated stimulation of phosphate uptake in chick-kidney proximal-tubule cells

Increased phosphate incorporation into phospholipids accompanying the substrate-mediated stimulation of phosphate uptake in chick-kidney proximal-tubule cells

Early effect of growth factors (EGF+insulin) upon ATP turnover in 3T3 cells

Various early biochemical events have been observed after the addition of growth factors to quiescent cultures of 3T3 cells; however, the cascade of events which take place in the cells after growth-factor addition is not yet entirely known. Our results show that the addition of a mixture of two growth factors, i.e., Epidermal Growth Factor (EGF) and insulin, to quiescent cultures of 3T3 cells rapidly stimulated phosphate uptake and ATP turnover. Our present and previous results suggest that the increase in phosphate uptake is the consequence of the stimulation of ATP synthesis. This stimulation was not simply a consequence of an increase in oxidative phosphorylation or in glucose transport and metabolism. The change in ATP turnover was an early event observed as soon as 5 min after growth-factor addition; furthermore, it was not dependent on protein synthesis. This change may therefore be the result of post-synthetic modification of enzymes by phosphorylation. We do not know what cellular process is responsible for the increase in ATP turnover. Since growth-factor addition rapidly enhanced ATP degradation in quiescent 3T3 cell cultures, we assumed that this increase is the result of an increase in ATP degradation. We know that it was not due to a stimulation of an oligomycin-sensitive ATPase. We verified that it was not the consequence of early biochemical events like an increase in Na + K + ATPase or a stimulation of RNA or protein synthesis. However, it is of interest to note that the stimulation of ATP turnover due to the growth-factor addition was inhibited by quercetin.

Light stimulation of phosphate uptake in marine phytoplankton

Light stimulation of phosphate uptake in marine phytoplankton

Light stimulation of phosphate uptake in marine phytoplankton

A phosphate uptake system responding positively to light would offer the algae a competitive advantage over bacteria, particularly when phosphorus is limiting. Effects of light on phosphate uptake in phytoplankton were investigated in view of conflicting literature reports, to test the hypothesis that both the P status and the light history of the population controlled the response. Maximum stimulation, to about 2.7x dark uptake, was observed at a Strait of Georgia, British Columbia, Canada station where phytoplankton was phosphorus sufficient but light limited. Stimulation was similar, about 1.8 x the dark rate, in the Northern Sargasso Sea, where phytoplankton was probably limited by phosphate but not by light. Minimal stimulation was observed in the Sheepscot Estuary, Maine, USA, where the population appeared to be neither phosphorus nor light limited.

Sodium gradient-dependent phosphate transport in renal brush border membrane vesicles. Effect of an intravesicular greater than extravesicular proton gradient.

A H+ gradient (intravesicular greater than extravesicular), in the absence of a Na+ gradient (extravesicular greater than intravesicular) stimulated phosphate uptake by renal brush border membrane vesicles and provided the driving force to effect the transient accumulation of phosphate against its concentration gradient. The H+ gradient-dependent uptake of phosphate had an absolute requirement for Na+. The rates of uptake and peak accumulation were functions of the delta pH and the concentration of H+ in the intravesicular medium. The H+ gradient-energized Na+-phosphate cotransport system was not affected by valinomycin- or carbonyl cyanide p-fluoromethoxyphenylhydrazone-induced ion diffusion potentials. Therefore, it was independent of the membrane potential, i.e. an electroneutral process. Amiloride, which inhibited the H+-Na+ exchange reaction and prevented the efflux of H+ from the intravesicular medium, enhanced the uptake of phosphate. A model is proposed by which the H+ gradient mediates the uphill transport of phosphate. It is suggested that a similar process may operate in more physiologically intact preparations and may provide one mechanism by which acid-base balance regulates renal phosphate transport.

Relationship between Energy-dependent Phosphate Uptake and the Electrical Membrane Potential in Lemna gibba G1

High rates of phosphate uptake into phosphate-starved Lemna gibba L. G1 were correlated with a high membrane potential (pd = -220 millivolts). In plants maintaining a low pd (-110 millivolts), the uptake rate was only 20% of that of high-pd plants. At the onset of phosphate transport, the membrane of high-pd plants was transiently depolarized. This effect was much smaller in low-pd plants. Light stimulated phosphate uptake and the repolarization upon phosphate-induced depolarization, especially in plants grown without sucrose. The phosphate uptake rate was optimal at pH 6 and decreased with increasing pH, corresponding to the phosphate-induced pd changes. Phosphate starvation stimulated the uptake and increased the phosphate-induced depolarization, thus indicating that phosphate uptake depends on the intracellular phosphate level. It is suggested that uptake of monovalent phosphate in Lemna gibba proceeds by an H(+) cotransport dependent on the proton electrochemical potential difference and, hence, on the activity of an H(+) -extrusion pump.

Phosphate uptake and translocation by roots of powdery mildew infected barley

Effects of powdery mildew, Etysiphe graminis f.sp. hordei Marchai, infection on the ability of roots of barley to absorb phosphate and translocate it to the shoot were examined using plants grown and infected in solution culture. Infection stimulated rates of uptake of 32 P-labelled phosphate per unit fresh weight of root tissue, and also stimulated total phosphate uptake per plant, in spite of reducing growth. Stimulation occurred within 48 h of infection and was found whether plants were infected 2 or 4 weeks after sowing and were starved or not starved of nutrients for 5 days before uptake was measured. Examination of phosphate uptake by isolated, 0·8 cm long regions of intact seminal roots showed that uptake in infected plants was stimulated in newly formed root tips as well as in older mid and basal regions formed before inoculation. Infection reduced the respiration rate of all regions of the root, the most marked reduction occurring in root tips. The proportion of the total phosphate absorbed that was translocated to shoots was greater in mildewed than in healthy plants. It is proposed that increased upward transport is the primary cause of stimulated phosphate uptake by roots. Phosphate uptake was stimulated in healthy but not in mildewed plants when they were allowed to accumulate ammonium ions from the external medium before treatment with 32 P. It is proposed that, since ammonium ions accumulate in roots of mildewed plants without special pretreatment, ammonium accumulation may be a secondary cause of stimulated phosphate uptake in those plants.

Uptake of phosphate by symbiotic and free-living dinoflagellates

Uptake of phosphate in the light by Amphidinium carterae, Amphidinium klebsii, cultured and symbiotic Gymnodinium microadriaticum conformed to Michaelis-Menten type saturation kinetics with all organisms showing similar Km values, namely 0.005 to 0.016 μM phosphorus. Vmax values were 0.009–0.32 nmol phosphorus · 105 cells-1 · 10 min-1. Phosphate uptake by all the dinoflagellates was greater in the dark than in the light. The metabolic inhibitor 3-(3,4-dichlorophenyl) 1,1-dimethylurea stimulated phosphate uptake in the light by A. carterae and A. klebsii, but inhibited uptake by cultured and symbiotic G. microadriaticum. Carbonylcyanide 3-chlorophenylhydrazone (CCCP) inhibited phosphate uptake by A. carterae and A. klebsii under both light and dark conditions. Uptake of phosphate by cultured and symbiotic G. microadriaticum in the light, but not in the dark, was inhibited by CCCP. Low concentrations of arsenate (5 μg As · l-1) stimulated phosphate by A. carterae and A. klebsii, but inhibited uptake by cultured and symbiotic G. microadriaticum. High concentrations of arsenate (100 μg As · l-1) did not affect uptake of phosphate by A. carterae and A. klebsii.

Effect of dietary phosphate on transport properties of pig renal microvillus vesicles.

Dietary phosphate manipulation results in stable adaptive changes in the transport functions of microvillus membrane vesicles isolated from pig renal cortex. When assayed under sodium gradient conditions, phosphate uptake is enhanced 200--400% in vesicles prepared from animals maintained on a low-phosphate diet (0.22%) compared to high-phosphate diet controls (0.82%). When transport is assayed in sodium preequilibrated vesicles, a 100% enhancement of phosphate uptake is demonstrable. Stimulation of phosphate uptake into low-phosphate diet vesicles after the imposition of a sodium chloride gradient is equivalent if uptake is measured at pH 6.0 or 8.0 and can be kinetically characterized as resulting from a Vmax alteration in the phosphate transport system. Microvillus membrane vesicle phosphate transport is maximally stimulated after only 2 days of dietary deprivation. Although a longer period (1 and 2 wk) of phosphate restriction does not further stimulate phosphate transport, it does result in an inhibtion of other sodium gradient-dependent transport systems (glucose, alanine).

PHOSPHATE UPTAKE BY SYNECHOCOCCUS LEOPOLIENSIS (CYANOPHYCEAE): ENHANCEMENT BY CALCIUM ION1

ABSTRACTThe influence of metallic, cations (added at 10 μM‐1 mM) on the uptake of orthophosphate from 0.2–10 μM solution by Synechococcus leopoliensis (Racib.) Komarek was investigated. All cations tested except Mg2+ and Zn2+ stimulated phosphate uptake. The most pronounced stimulation of phosphate uptake was caused by Ca2+·Ca2+ markedly decreased the half‐saturation concentration for orthophosphate uptake, apparently by acting upon the metabolic processes of phosphate transport into the cell. Phosphate did not influence Ca2+ fluxes across the cell‐surface.

Hydrocortisone and Vitamin D3Stimulation of 32Pi-Phosphate Accumulation by Organ-Cultured Chick Embryo Duodenum

Either vitamin D3 (or 1 alpha,25--(OH)2-D3) or hydrocortisone (HC) stimulated phosphate accumulation by organ-cultured embryonic chick duodenum. In combination, these two steroids stimulated phosphate uptake synergistically. Phosphate accumulation appeared to be independent of other vitamin D3-stimulated processes: CaBP concentration, cAMP concentration, or alkaline phosphatase activity. L-phenylalanine, a reported alkaline phosphatase inhibitor, when added to the culture medium progressively inhibited either D3- or HC-stimulated phosphate uptake subsequent to culture, but did not inhibit the synergistic action. Under these conditions L-phenylalanine had no consistent effect on alkaline phosphatase activity but unexpectedly, greatly inhibited vitamin D3-stimulated CaBP concentration, but only in the absence of HC. Some limited suggestion of an intestinal phosphoprotein sensitive to either vitamin D3 or HC was observed.

Cotransport of phosphate and sodium by yeast

Phosphate uptake by yeast at pH 7.2 is mediated by two mechanisms, one of which has a K m of 30 μM and is independent of sodium, and a sodium-dependent mechanism with a K m of 0.6 μM, both K m values with respect to monovalent phosphate. The sodium-dependent mechanism has two sites with affinity for Na +, with affinity constants of 0.04 and 29 mM. Also lithium enhances phosphate uptake; the affinity constants for lithium are 0.3 and 36 mM. Other alkali ions do not stimulate phosphate uptake at pH 7.2. Rubidium has no effect on the stimulation of phosphate uptake by sodium. Phosphate and arsenate enhance sodium uptake at pH 7.2. The K m of this stimulation with regard to monovalent orthophosphate is about equal to that of the sodium-dependent phosphate uptake. The properties of the cation binding sites of the phosphate uptake mechanism and those of the phosphate-dependent cation transport mechanism have been compared. The existence of a separate sodium-phosphate cotransport system is proposed.

Energy Relations of Phosphate Uptake and Distribution in Barley Leaf Slices as Affected by Cutting and Adaptive Ageing

Summary CO 2 -dependent photosynthetic O 2 evolution of young barley leaves ( Hordeum vulgare L.) decreased and mitochondrial respiration increased upon cutting the leaves into 1 mm wide slices and during subsequent adaptive ageing of these slices. During the same time a high-affinity phosphate uptake system developed. Analysis of P distribution suggests that P in the inorganic phosphate fraction is mobile and exchanges more rapidly than P in the organic and insoluble phosphate fractions. Light under no conditions stimulated phosphate uptake, neither in freshly cut slices nor after adaptive ageing in CaSO 4 solution. It is concluded that in air even in the light and already in freshly cut slices active phosphate uptake is maintained predominantly by mitochondrial respiration. Other energy sources (noncyclic and cyclic photophosphorylation) can also contribute depending on the possibilities or restraints imposed on the tissue experimentally by varying the gas supply and by using DCMU as inhibitor.

Prostaglandin F 2α and insulin stimulate phosphate uptake and (Na +, K +)ATPase activity in resting mouse fibroblast cultures

The (Na +, K +)ATPase transport system in resting 3T3 Swiss mouse fibroblasts is rapidly activated by prostaglandin F 2α and insulin, which initiate DNA synthesis in these cells. Prostaglandin F 2α, but not insulin, promotes a rapid increase in P i uptake which is partially coupled to the Na + pump. This rapid activation of both transport systems occurs by a mechanism which does not require fluctuation of cyclic AMP levels or new protein synthesis. A subsequent protein synthesis-dependent increase in P i uptake is stimulated by insulin and prostaglandin F 2α. These results suggest that different types of control of membrane transport occur during growth stimulation.

Serum stimulation of phosphate uptake into 3T3 cells.

The stimulation by calf serum of phosphate uptake into 3T3 cells results from a change in maximum velocity of the transport process with no change in the Michaelis constant. Only arsenate among a series of inorganic structural analogs of phosphate inhibited phosphate uptake indicating a high specificity for the process. The arsenate inhibition was competitive in nature. Papaverine, theophylline, and protaglandin E1, drugs known to maintain high intracellular levels of cAMP, had little effect on serum stimulated phosphate uptake. The phosphate uptake stimulating factor(s) in serum could be distinguised from the 3T3 cell survival and migration factors by stability characteristics, but this factor(s) could not be completely separated from a uridine uptake stimulation activity or growth promoting activity using a variety of serum fractionation procedures. Only partial stimulation of the uptake process was achieved with any one serum fraction indicating a multiplicity of serum components is probably involved in this process. Because of the rapidity of serum activation of phosphate uptake and its apparent independence of intracellular cyclic nucleotide levels, it is suggested that serum factors may stimulate phosphate uptake by inducing structural changes in the phosphate carrier system.

Phosphate uptake and its pH-dependence in halophytic and glycophytic algae and higher plants.

Phosphate uptake in various organs of higher plants, i.e. Hordeum roots Avena coleoptiles, leaves of Elodea, and seedlings of the halophytes Cakile maritima, Cochlearia anglica, and Plantago maritima is optimal in the acidic pH range and is not stimulated by Na+ when tested in comparison to K+.In certain algae, i.e. the thermophilic blue-green alga Anacystis nidulans, the freshwater red alga Porphyridium aerugineum and the marine red alga Porphyridium cruentum, phosphate uptake is optimal in the alkaline pH range. In the red algae phosphate uptake is enhanced by Na+; in the marine species the increase is up to 100 times the rate in the presence of K+. It is suggested that the Na+-stimulated phosphate uptake has some phylogenetic and ecological significance.

The Relation between the Synthesis of Inorganic Polyphosphate and Phosphate Uptake byChlorella vulgaris

During a period of phosphate starvation, the phosphate content of cells of Chlorella vulgaris which had been grown in phosphate-rich solution, decreased. The levels of most phosphate fractions declined, especially those of inorganic polyphosphates, which at first accounted for about 5 per cent of the total phosphate and virtually disappeared after 36 h starvation. On return to a phosphate medium, phosphate was taken up at a much faster rate than before starvation, with a striking increase in acid-soluble polyphosphate. The stimulated phosphate uptake and polyphosphate increase have been shown to be specific effects of phosphate starvation, occurred only when excess phosphate was supplied and required light or air for the provision of energy. There was relatively little change in the concentrations of other phosphate fractions, including orthophosphate. Inorganic polyphosphate was found to be synthesized solely from phosphate absorbed from the medium. It is argued that polyphosphate synthesis is a consequence of the stimulation of phosphate uptake, induced by the starvation period.

Phosphate uptake in an obligately marine fungus. II. Role of culture conditions, energy sources, and inhibitors.

Phosphate uptake in the obligately marine fungus, Thraustochytrium roseum, is maximal at pH 7.5 to 7.8, is dependent on temperature, and varies with phosphate concentration. Pyruvate and succinate stimulate phosphate uptake, although they do not increase respiration. The uncoupling agents, 2,4-dinitrophenol and dicoumerol, inhibit phosphate uptake but stimulate oxygen consumption only in the presence of NaCl. Oligomycin inhibits both processes. Among the inhibitors of protein synthesis, chloramphenicol reduces phosphate uptake without affecting respiration. Puromycin is unique in that it greatly enhances phosphate uptake and abolished the lag period associated with this phenomenon. It does not affect respiration.

Synthesis of polyphosphate by rat liver mitochondria

The synthesis and hydrolysis of acid insoluble polyphosphates has been observed in many lower species and the enzymes involved have been partially purified by Kornberg and Hughes. Data also exists indicating that in bacteria under experimental conditions of biosynthetic blockade, e.g., nitrogen deficiency etc., polyphosphates accumulate within cells. Lehninger and others have noted that mammalian mitochondria swell in the presence of mitochondrial substrates and in the absence of ADP but shrink when ATP concentration is maintained at a high level. A reasonable explanation for these observations could be that excess substrate in the absence of adequate concentrations of nucleotide acceptor, activates the turnover of high energy phosphate into a “phosphagen” or polyphosphate pool at a very rapid rate, thus allowing ATP concentration to fall. The data in this report indicate that substrate does markedly stimulate phosphate uptake into a high molecular weight polyphosphate of rat liver mitochondria with a concomitant decrease in ATP concentration.