Intracellular Citrate Research Articles

Inositol triphosphate produces different patterns of cytoplasmic Ca 2+ spiking depending on its concentration

In single mouse pancreatic acinar cells the effects of intracellular infusion of inositol 1,4,5-trisphosphate (InsP 3) or the non-metabolizable InsP 3 analogue inositol 1,4,5-triphosphorothioate (InsPS 3) have been investigated using a wide range of concentrations. Different types of cytosolic Ca 2+ fluctuation patterns (monitored as Ca 2+-dependent Cl − current in patch-clamp whole-cell recording experiments) could be generated by InsP 3 or InsPS 3, dependent on concentration, resembling those previously shown to be evoked by varying degrees or receptor activation in these cells. Low InsPS 3 concentrations evoked repetitive local Ca 2+ spikes whereas at relatively high concentrations repetitive Ca 2+ waves were produced. In the presence of intracellular citrate a much lower messenger level was sufficient to generate waves. The InsP 3 concentration determines whether the cytosolic Ca 2+ signals are local or global.

Renal handling of citrate

Intracellular citrate is a central component of the tricarboxylic acid cycle; its excretion in the urine was utilized by Krebs and coworkers to demonstrate this biochemical pathway in whole animals. Subsequently, urinary citrate has been viewed as a window on renal metabolism [1]. Additionally, renal physiologists and biochemists have been intrigued for decades by the dramatic changes in urinary citrate which occur with changes in acid-base homeostasis. In recent years, renal handling of citrate and citrate excretion in the urine have attracted renewed interest because of several considerations. First, modern techniques in renal physiology (such as, transport studies in brush border membrane vesicles and perfused proximal tubules) have provided insights into the cellular and molecular mechanisms of citrate transport [2]. Also, excretion of urinary citrate (and other organic anions) has been recognized to influence systemic acid-base status, at least in certain species [3]. And perhaps most importantly, urinary citrate has been increasingly recognized as an important endogenous inhibitor of calcium nephrolithiasis [4]. This review will focus on the renal handling of citrate, particularly the mechanisms of citrate transport in the proximal tubule, and briefly discuss other aspects of citrate metabolism.

End products of glucose and glutamine metabolism by cultured cell lines.

Rates of CO2 production from glucose and glutamine, intracellular metabolite levels, and release of metabolic end products into the culture medium were determined for 13 cultured cell lines, including a glycolysis-defective mutant. All the non-mutant lines synthesized pyruvate, lactate, alanine, proline, aspartate, and citrate, so that the metabolism of glucose and glutamine resulted mainly in the production of these compounds and only to a lesser extent in complete oxidation to CO2. These data and the pattern of metabolites produced by the mutant line were consistent with a model characterized by incomplete glutamine oxidation leading to end product accumulation. Multiple linear regression analysis identified the metabolite levels most highly correlated with the intracellular citrate level and with the amount of citrate released into the medium. The analysis also showed that the rates of CO2 production from glucose and glutamine were themselves positively correlated, suggesting that the oxidation of the two substrates is coordinately controlled under normal culture conditions.

Citrate regulation of the change in carbohydrate degradation during the initial phase of the citric acid production by Aspergillus niger

A change from pentose phosphate pathway to glycolysis has been shown to occur in the early stages of Aspergillus niger growth in a high citric acid yielding medium. The decrease of pentose phosphate metabolism was shown to be due to inhibition of 6-phosphogluconate dehydrogenase by intracellular citrate. The regulatory enzyme of glycolysis, 6-phosphofructokinase, whose activity could not be detected immediately after spore germination, showed a progressive increase in activity thereafter.

Mechanism for the oleate stimulation of gluconeogenesis from dihydroxyacetone by hepatocytes from fasted rats

Oleate stimulates glucose production and concomitantly decreases lactate and pyruvate production by rat hepatocyte suspensions incubated with dihydroxyacetone as substrate. The actions of oleate could be blocked by d-(+)dodecanoylcarnitine, which inhibits transport of the fatty acid into the mitochondria and the subsequent oxidation. β-Hydroxybutyrate, but not acetoacetate, also stimulated glucose synthesis and inhibited lactate and pyruvate production. Furthermore, both β-hydroxybutyrate and oleate stimulated oxygen consumption to the same extent. This suggests that oleate stimulates glucose production by the provision of energy subsequent to mitochondrial β-oxidation of the fatty acids. The content of ATP itself did not appear to be responsible for the effects of oleate. Crossover analysis of the gluconeogenic intermediates implicated a site of oleate action between fructose 1,6-bisphosphate and fructose 6-phosphate, suggesting phosphofructokinase and/or fructose-bisphosphatase as possible regulatory sites. Coupled with the finding that intracellular citrate accumulates upon addition of oleate or β-hydroxybutyrate, but not acetoacetate, the results suggest that citrate inhibition of phosphofructokinase accounts for the redirection of carbon flow from lactate and pyruvate formation and towards that of glucose.

Inhibition of citric acid accumulation by manganese ions in Aspergillus niger mutants with reduced citrate control of phosphofructokinase.

Mutant strains of Aspergillus niger with reduced citrate control of carbohydrate catabolism (cic mutants) grow faster than the parent strain on media containing 5% (wt/vol) citrate. The mutants tolerated a higher intracellular citrate concentration than the parent strain. One mutant (cic-7/3) contained phosphofructokinase activity significantly less sensitive towards citrate than the enzyme from the parent strain. When this mutant was grown under citrate-accumulating conditions, acidogenesis was far less sensitive to inhibition by Mn2+ than in the parent strain. Some of the cic mutants also showed altered citrate inhibition of NADP-specific isocitrate dehydrogenase.

Effect of Nitrogen Source on Lipid Accumulation in Oleaginous Yeasts

The effect of various nitrogen sources on lipid accumulation by 17 species and strains of yeast was examined. Organic nitrogen sources resulted in considerably increased lipid contents only in strains of Rhodosporidium toruloides. Lipid accumulation in Rs. toruloides CBS 14 increased from 18% (w/w), with NH4Cl as nitrogen source, to above 50% (w/w) when glutamate, urea or arginine was used. Stimulation of lipid production by glutamate was not observed when the yeast was grown in continuous culture with nitrogen-limiting medium. The increase in lipid content of glutamate-grown cells in batch culture was accompanied by a marked increase in the intracellular citrate concentration and its excretion from the cells. The pattern of citrate accumulation in glutamate-grown cells was mirrored by the accumulation of other metabolites, especially 2-oxoglutarate and NH+ 4 ions, which were produced as a result of the increased catabolism of glutamate. It is proposed that the products of glutamate metabolism in Rs. toruloides play a major role in regulating the flux of carbon to precursors of lipid biosynthesis, such as citrate.

Citrate excretion: a window on renal metabolism.

The rate of intracellular metabolism of citrate plays a major role in determining the amount of citrate excreted in the urine. Fractional excretion of citrate can be increased either by increasing intracellular citrate synthesis from precursors or by inhibiting mitochondrial citrate metabolism. Increased excretion secondary to increased synthesis of citrate occurs when citric acid cycle precursors such as malate or succinate are infused. Increased excretion resulting from inhibition of citrate metabolism occurs when malonate, maleate, or fluorocitrate is administered. Systemic acid-base changes cause striking changes in citrate clearance and metabolism. Recent evidence suggests that the effects of acid-base changes are mediated by alteration in the pH gradient across the inner mitochondrial membrane. Metabolic alkalosis causes cytoplasmic pH and bicarbonate to increase, resulting in a decrease in the mitochondrial pH gradient. This change inhibits the tricarboxylate carrier, slowing entry of citrate into the mitochondrial matrix compartment. The level of citrate in the cytoplasm increases, tubular and peritubular citrate uptake are reduced, and citrate clearance increases. Opposite changes occur in acidosis. Change in the mitochondrial pH gradient provides a sensitive mechanism for regulating renal substrate metabolism.

Citrate accumulation by a Golgi apparatus-rich fraction from lactating bovine mammary gland

1. 1. Golgi apparatus-rich fractions from lactating bovine mammary gland rapidly accumulated citrate from incubation medium. Characteristics of this process suggested that a citrate transport system may be present in Golgi apparatus membranes. 2. 2. Endoplasmic reticulum fractions accumulated citrate at nearly the same rate as Golgi apparatus; secretory vesicle fractions displayed lower ability to accumulate citrate. Intact epithelial cells (acini) from lactating mammary gland did not accumulate citrate. 3. 3. Citrate accumulation by Golgi apparatus was pH and temperature sensitive but was not altered by metabolic inhibitors. 4. 4. These observations suggest a role for Golgi apparatus in packaging intracellular citrate for secretion into milk.

Citrate-dependent iron transport system in Escherichia coli K-12.

Induction of the citrate-dependent iron transport system of Escherichia coli K-12 required 0.1 mM citrate and 0.1 micrometer iron in the growth medium. Five--ten-times more iron than citrate was taken up into the cells which suggests that citrate was largely excluded from the transport. Fluorocitrate and phosphocitrate induced the citrate-dependent iron transport system although they supported iron uptake only very poorly. An outer membrane protein (FecA), belonging to the transport system, was induced in fecB mutants which were devoid of citrate-dependent iron transport. The intracellular citrate and iron concentrations were 10--100-times higher than the external concentrations required for induction of the transport system. It is concluded that only exogenous ferric citrate induced the transport system, and that citrate did not have to enter the cytoplasm. The Tn10 transposon, conferring tetracycline resistance, was inserted near the fec gene region which controls the expression of the citrate-dependent iron transport system. The determination of the cotransduction frequencies of Tn10 with the fecA and fecB markers suggested the gene order fecA fecB Tn10.

Circadian plasma citrate rhythms in juvenile diabetics.

Postabsorptive plasma concentrations of citrate, glucose, lactate, free fatty acids (FFA), ketone bodies and free insulin were measured once weekly for 5 weeks in 18 male juvenile diabetics. The circadian rhythms of the same substances were followed in 12 male diabetics. In 8 of them, daily rhythms were measured twice, before and after plasma glucose was lowered by increasing insulin doses. In the postabsorptive state, the mean plasma citrate concentration of the diabetics, 117 mumol/l (range 65--160), did not differ from that of non-diabetics despite two- to threefold higher levels of plasma glucose, FFA and ketone bodies in diabetics. Daily plasma citrate profile in diabetics showed peak concentrations in the morning and late afternoon. Citrate level throughout the day fell after increased insulin administration, whereas the diurnal pattern remained unchanged. Both the week-to-week coefficient of variation (mean 10%) and the diurnal coefficient of variation (mean 17%) of plasma citrate were below those of any other substances measured (p less than 0.001). Postabsorptive citrate concentration correlated negatively to the diurnal variation of plasma glucose whether diabetic control was apparently good or poor. The results support the idea that plasma citrate level reflects intracellular citrate regulation of glucose utilization. In spite of an interindividual range of 100%, individual citrate level was remarkably constant. Postabsorptive plasma citrate concentration is proposed as a control marker of lability of circulating glucose in insulin-treated diabetics.

Intracellular citrate or externaly applied tetraethylammonium ions produce calcium-dependent action potentials in an insect motoneurone cell body.

1. Electrophysiological observations have been made upon the cell body of an identified motoneurone of the cockroach, Periplaneta americana. Normal responses were compared with those observed after intracellular injection of citrate anions or when the preparation was bathed in solutions containing tetraethylammonium ions (TEA+).2. Normally when depolarized, the motoneurone soma gave a series of damped oscillations; the amplitude of these responses increased with increase in the applied current.3. After citrate ions had been injected into the neurone soma, all-or-none action potentials were evoked by depolarization; such responses appeared about 5-10 min after the onset of citrate injection. Injection of EGTA produced similar effects. Citrate and EGTA probably produce their effect through a reduction in the intracellular free calcium concentration.4. When preparations were bathed in saline solution containing 50 mM-TEA+, soma depolarization produced prolonged all-or-none action potentials (up to approximately 100 msec duration).5. The action potentials produced by citrate injection or externally applied TEA+ appeared to have a similar ionic mechanism; they were not depressed by sodium-free solutions or by tetrodotoxin (4 x 10(-6)M) but were reversibly blocked in saline solution containing 40 mM-manganous chloride.6. The overshoot amplitude of action potentials recorded after injection of citrate anions or in solutions containing TEA+ showed a 22.5 mV change for a ten-fold change in the external calcium concentration.7. Both intracellular citrate and external TEA+ caused a significant increase in the input resistance and membrane time constant of the motoneurone.8. It is concluded that action potentials generated under various experimental conditions in the soma of this insect motoneurone map have differing ionic mechanisms.

Acetyl-CoA carboxylase. Evidence for polymeric filament to protomer transition in the intact avian liver cell.

Digitonin treatment of chick liver cells in monolayer culture perforates the plasma membrane, causing release of acetyl-CoA carboxylase and other cytosolic enzymes. The rate of carboxylase release is affected by conditions known to alter the position of the protomer-polymer (filament) equilibrium of the enzyme. Citrate, an allosteric activator of the carboxylase, induces polymerization of the protomeric avidin-sensitive form giving rise to the avidin-insensitive polymeric filamentous form. When cells are exposed to N6,O2-dibutyryl cyclic adenosine 3':5'-monophosphate which lowers intracellular citrate levels, the rate of carboxylase release from digitonin-treated cells is greatly accelerated. The presence of avidin, which rapidly enters the cell during digitonin treatment, inactivates carboxylase under conditions that promote depolymerization and rapid release, but not under conditions which promote polymerization and slow release. These findings indicate that carboxylase filaments exist in the intact chick liver cell when the cytoplasmic citrate level is high and undergo depolymerization when citrate levels fall.

Isolation and characterization of adult rat heart cells

Adult rat heart muscle cells were isolated after simultaneous perfusion of multiple (two to eight) hearts with buffered salt solutions containing collagenase and hyaluronidase. Yields (35 to 50% of ventricular weight with approximately 70% viability) are quantitatively suitable for metabolic studies. Viability has been determined by the ability of intact cells to exclude trypan blue and the inability of intact cells to oxidize exogenous succinate. Micrographs show that the fine structure of the isolated cells is well ordered. Cell concentrations of glycogen, glucose 6-phosphate, citrate, and various enzymes were similar to those of intact heart. ATP and creatine phosphate concentrations were lower than in whole hearts. Adenosine 3′,5′-monophosphate concentrations were somewhat elevated. Deoxyribonucleic acid was lower than in whole tissue. The isolated cells retain certain metabolic control mechanisms. The uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, increased oxygen consumption severalfold, whereas exogenous ADP had no effect on respiration. Under anaerobic conditions the rates of glucose utilization and lactate production were faster than in the presence of oxygen, indicating retention of the Pasteur effect. The addition of glucose and insulin caused a decrease in oxygen uptake or the Crabtree effect. Exogenously added pyruvate decreased glycolytic flux and produced a pronounced increase in intracellular citrate and glucose 6-phosphate. Isoproterenol stimulated adenylate cyclase activity of the isolated cells at the same concentrations effective with intact heart preparations. Isoproterenol and glucagon caused the activation of phosphorylase. The cells deteriorated as a function of incubation time, as indicated by a decrease in ATP content and a loss of lactate dehydrogenase into the medium. Cell deterioration was greatly accelerated by Ca 2+ at concentrations greater than 10 −5 m.

Selection for citrate synthase deficiency in icd mutants of Escherichia coli.

Cultures of isocitrate dehydrogenase-deficient (icd) mutants were overgrown by double mutants (icd glt) lacking citrate synthase activity also. The icd mutants grew more slowly than wild-type cells or the double mutants because they accumulated an inhibitory metabolite (possibly citrate). Intracellular citrate levels were several hundred-fold higher in icd cells than in wild-type or icd glt cells. Final growth yields of the wild type and the icd mutant on limiting glucose were equivalent and greater than the growth yield of icd glt double mutants. The icd gene mapped between 60 and 74 min. icd mutants were resistant to nalidixic acid, but glt and icd glt mutants and wild-type cells were sensitive, indicating that resistance results from accumulation of isocitrate, citrate, or a derivative of these compounds.

Transport of citric acid by Aerobacter aerogenes

The kinetics of citrate transport by induced cells of Aerobacter aerogenes was measured and an apparent K m of 2.5 × 10 −6 was determined. Citrate was transported optimally at pH 7 and 30–37 °. Osmotic shock decreased total citrate uptake by 75% but the initial rate of transport was unaffected. Shocked cells incubated with citrate recovered their ability to take up citrate, but chloramphenicol prevented this recovery. Aconitase activity was 20-fold higher in extracts from citrate-grown cells of A. aerogenes than from glucose-grown cells. Aconitase activity was diminished by osmotic shock. Aconitase activity was recovered by incubation of shocked cells with citrate concomitant with the recovery of citrate uptake. There was no accumulation of label from uptake of [6- 14C]citrate and 14CO 2 was evolved immediately. Intracellular citrate could not be detected after a 10-sec pulse with [1,5- 14C]citrate or [2,4- 14C]citrate; instead, the major intracellular product was glutamate. Citrate lyase was not detected in cell-free extracts of citrate-grown cells. These results indicated that A. aerogenes metabolized citrate as rapidly as it entered the cells and that aerobic citrate catabolism proceeded via the tricarboxylic acid cycle.

The nature of the inhibitory action of H + and fluoroacetic acid on metabolism in yeast cells

The concentration of intracellular citrate of non-dividing yeast cells has been shown to fluctuate with variations in environmental pH and glucose concentration. Environmental conditions which lead to a low intracellular citrate favor poisoning by fluoroacetic acid (HFA), but a high concentration of citrate does not always protect against HFA toxicity. It is suggested that yeast cells possess two metabolic pathways in which citrate occurs as an intermediate, but only one of these is blocked by HFA. A high H + ion concentration limits the input into one of these pathways and therefore limits the total amount of citrate that accumulates in HFA poisoned cells. While the addition of K + ions stimulates citrate production, this does not appear to take place in the HFA blocked pathway.