Pi Accumulation Research Articles

Regulatory role of phosphatidylinositol 3-kinase on TNF-α-induced cyclooxygenase 2 expression in colonic epithelial cells

Cyclooxygenase (COX)-2 is up-regulated in most colonic cancers and in inflammatory bowel disease in which tumor necrosis factor (TNF)-alpha is believed to play a central role. There has been recent speculation on the activation of phosphatidylinositol 3-kinase (PI 3-kinase) by TNF-alpha and its role in the regulation of genes controlled by NF-kappaB. We investigated the regulatory role of PI 3-kinase on COX-2 expression in colonic epithelial cells. In HT-29 and Caco-2 colonic epithelial cells, COX-2 expression was induced by either TNF-alpha or interleukin (IL)-1alpha as observed by Northern and Western analyses. COX-2 activity was assessed by measuring prostaglandin E(2) (PGE2) production by enzyme-linked immunosorbent assay. NF-kappaB binding activity was assessed by electrophoretic mobility shift assay. PI 3-kinase activity was measured by quantifying the accumulation of PI 3-kinase-dependent D-3 lipid products by high-performance liquid chromatography. The PI 3-kinase inhibitor wortmannin up-regulated induced COX-2 expression in a concentration-dependent manner in both HT-29 and Caco-2 cells. An alternative PI 3-kinase inhibitor, LY294002, caused up-regulation of induced COX-2 messenger RNA (mRNA) in HT-29 cells at concentrations of < or =1 micromol/L. IL-4 and IL-13, which are known to activate PI 3-kinase, down-regulated HT-29 COX-2 mRNA, protein, and PGE2 production. NF-kappaB binding activity was unaltered by PI 3-kinase inhibition in HT-29 cells, in which TNF-alpha was shown to activate PI 3-kinase directly. COX-2 is negatively regulated by PI 3-kinase; we propose that the inhibitory effect of IL-4 and IL-13 is mediated via a PI 3-kinase-dependent pathway. This mechanism does not appear to involve NF-kappaB because PI 3-kinase inhibition did not alter NF-kappaB binding activity. TNF-alpha can activate PI 3-kinase directly in addition to inducing COX-2.

Phosphate and sulfate uptake in the halotolerant alga Dunaliella are driven by Na +-symport mechanism

Summary Phosphate and sulfate uptake were measured in the halotolerant alga Dunaliella salina under anion-sufficient or anion-deficient conditions. Both phosphate and sulfate uptake were strictly and specifically dependent on Na+ ions. Pi accumulation was inhibited by Na+ ionophores and by inhibitors of Na+ extrusion. Pi uptake was associated with a stoichiometric influx of Na+ ions at a ratio of 1 Na+/1 Pi. Depletion of phosphate or sulfate from growth media induced a 20-fold or a 2.5-fold stimulation of the corresponding rate of uptake, respectively. The enhancements were associated with an increase in Vmax values, but not in the Km for anion substrates (Km(Pi)∼1 μmol/L, Km(SO4)∼15 μmol/L) or for Na+ (Km(Na+)∼35 mmol/L for Pi, ∼170 mmol/L for SO42-). These results suggest that uptake of phosphate and of sulfate in D. salina are mediated by Na+/anion symporters and are driven by δμNa+ across the plasma membrane. The implications of these results on the availability of phosphate and sulfate in hypersaline solutions are discussed.

Growth Factor-Stimulated Phosphorylation of Akt and p70S6K Is Differentially Inhibited by LY294002 and Wortmannin*

The phosphoinositide 3-kinase (PI3K) inhibitors, LY294002 (LY) and wortmannin (WM), are widely used to examine the role of PI3K in growth factor signaling. These compounds inhibit the kinase action of PI3K, thus preventing the accumulation of PI(3,4,5)P3 and PI(3,4)P2 (PIs) and subsequent phosphorylation and activation of the downstream effectors of PI3K, Akt and p70(S6K). The efficacy of these inhibitors has been demonstrated by measuring cellular levels of PIs or the kinase activity of immunoprecipitated PI3K. However, their effects on activation of Akt and p70(S6K), more widely used markers of PI3K activation, has not been formally tested. We have examined the effects of LY and WM on phosphorylation of Akt and p70(S6K) by insulin-like growth factor-I, insulin, and platelet-derived growth factor in skeletal muscle cells. LY is much less effective in blocking the phosphorylation of Akt than p70(S6K); at concentrations which completely inhibit phosphorylation of p70(S6K), phosphorylation of Akt is only partially inhibited by LY. WM also inhibits IGF-I-stimulated phosphorylation of Akt and p70(S6K) with unequal potency but is equally effective in blocking insulin-stimulated phosphorylation of these peptides. Our data demonstrate that inhibiting PI3K signaling through one of its downstream mediators (p70(S6K)) may not indicate complete blockage of the PI3K pathway which may be signaling through an alternate downstream branch (Akt). These findings indicate that the efficacy of LY and WM in blocking PI3K-activation of Akt and p70(S6K) must be tested within the context of every experiment, and that the results obtained with the use of these inhibitors must be interpreted according to their specific effects on the PI3K/Akt and PI3K/p70(S6K) signaling pathways.

Growth Factor-Stimulated Phosphorylation of Akt and p70S6K Is Differentially Inhibited by LY294002 and Wortmannin

The phosphoinositide 3-kinase (PI3K) inhibitors, LY294002 (LY) and wortmannin (WM), are widely used to examine the role of PI3K in growth factor signaling. These compounds inhibit the kinase action of PI3K, thus preventing the accumulation of PI(3,4,5)P3 and PI(3,4)P2 (PIs) and subsequent phosphorylation and activation of the downstream effectors of PI3K, Akt and p70S6K. The efficacy of these inhibitors has been demonstrated by measuring cellular levels of PIs or the kinase activity of immunoprecipitated PI3K. However, their effects on activation of Akt and p70S6K, more widely used markers of PI3K activation, has not been formally tested. We have examined the effects of LY and WM on phosphorylation of Akt and p70S6K by insulin-like growth factor-I, insulin, and platelet-derived growth factor in skeletal muscle cells. LY is much less effective in blocking the phosphorylation of Akt than p70S6K; at concentrations which completely inhibit phosphorylation of p70S6K, phosphorylation of Akt is only partially inhibited by LY. WM also inhibits IGF-I-stimulated phosphorylation of Akt and p70S6K with unequal potency but is equally effective in blocking insulin-stimulated phosphorylation of these peptides. Our data demonstrate that inhibiting PI3K signaling through one of its downstream mediators (p70S6K) may not indicate complete blockage of the PI3K pathway which may be signaling through an alternate downstream branch (Akt). These findings indicate that the efficacy of LY and WM inblocking PI3K-activation of Akt and p70S6K must be tested within the context of every experiment, and that the results obtained with the use of these inhibitors must be interpreted according to their specific effects on the PI3K/Akt and PI3K/p70S6K signaling pathways.

Chilling Temperature Storage Changes the Inorganic Phosphate Pool Distribution in Cherimoya (Annona cherimola) Fruit

Phophorous nuclear magnetic resonance (31P-NMR) spectroscopy was used to study the vacuolar and cytoplasmic pH and the inorganic phosphate (Pi) pool distribution in `Fino de Jete' cherimoya (Annona cherimola Mill.) fruit stored at a chilling temperature (6 °C). Fruit stored at the ripening temperature (20 °C) for 3 days were used as a control. 31P-NMR results confirmed that 6 °C storage caused cytoplasmic acidosis (a decrease of 0.72 ± 0.08 pH units) and a notable increase in the amount of Pi in the cytoplasm. Spectra of perchloric acid extracts also revealed that storage at 6 °C was associated with an increase in the total amount of Pi and phosphorylated metabolites. Moreover, perfusion experiments with a phosphate medium confirmed the preferential accumulation of Pi in the cytoplasm in chilled tissues. Specific activation of phosphoenolpyruvate carboxylase (PEPC) (32.1 ± 1.7 μmol·min-1·mg-1) was observed in those fruit. In chilled fruit the amount of ADP was held at steady-state levels and ATP levels increased, contrary to observations for ripe fruit, where the pool of total nucleotides decreased beyond the point of NMR detection. Fruit stored at 6 °C exhibited a low respiration rate, but metabolism was not arrested and an increase in total soluble solid contents was also observed.

Mitochondrial adaptation to in vivo polyunsaturated fatty acid deficiency: increase in phosphorylation efficiency.

Polyunsaturated fatty acid (PUFA) deficiency affects respiratory rate both in isolated mitochondria and in hepatocytes, an effect that is normally ascribed to major changes in membrane composition causing, in turn, protonophoriclike effects. In this study, we have compared the properties of hepatocytes isolated from PUFA-deficient rats with those from control animals treated with concentrations of the protonophoric uncoupler 2,4-dinitrophenol (DNP). Despite identical respiratory rate and in situ mitochondrial membrane potential (delta psi), mitochondrial and cytosolic ATP/ADP-Pi ratios were significantly higher in PUFA-deficient cells than in control cells treated with DNP. We show that PUFA-deficient cells display an increase of phosphorylation efficiency, a higher mitochondrial ATP/ADP-Pi ratio being maintained despite the lower delta psi. This is achieved by (1) decreasing mitochondrial Pi accumulation, (2) increasing ATP synthase activity, and (3) by increasing the flux control coefficient of adenine nucleotide translocation. As a consequence, oxidative phosphorylation efficiency was only slightly affected in PUFA-deficient animals as compared to protonophoric uncoupling (DNP). Thus, the energy waste induced by PUFA deficiency on the processes that generate the proton motive force (pmf) is compensated in vivo by powerful adaptive mechanisms that act on the processes that use the pmf to synthesize ATP.

New components of a system for phosphate accumulation and polyphosphate metabolism in Saccharomyces cerevisiae revealed by genomic expression analysis.

The PHO regulatory pathway is involved in the acquisition of phosphate (P(i)) in the yeast Saccharomyces cerevisiae. When extracellular P(i) concentrations are low, several genes are transcriptionally induced by this pathway, which includes the Pho4 transcriptional activator, the Pho80-Pho85 cyclin-CDK pair, and the Pho81 CDK inhibitor. In an attempt to identify all the components regulated by this system, a whole-genome DNA microarray analysis was employed, and 22 PHO-regulated genes were identified. The promoter regions of 21 of these genes contained at least one copy of a sequence that matched the Pho4 recognition site. Eight of these genes, PHM1-PHM8, had no previously defined function in phosphate metabolism. The amino acid sequences of PHM1 (YFL004w), PHM2 (YPL019c), PHM3 (YJL012c), and PHM4 (YER072w) are 32-56% identical. The phm3 and phm4 single mutants and the phm1 phm2 double mutant were each severely deficient in accumulation of inorganic polyphosphate (polyP) and P(i). The phenotype of the phm5 mutant suggests that PHM5 (YDR452w) is essential for normal catabolism of polyP in the yeast vacuole. Taken together, the results reveal important new features of a genetic system that plays a critical role in P(i) acquisition and polyP metabolism in yeast.

Effects of loaded breathing and hypoxia on diaphragm metabolism as measured by (31)P-NMR spectroscopy.

Diaphragm fatigue may contribute to respiratory failure. (31)P-nuclear magnetic resonance spectroscopy is a useful tool to assess energetic changes within the diaphragm during fatigue, as indicated by P(i) accumulation and phosphocreatine (PCr) depletion. We hypothesized that loaded breathing during hypoxia would lead to diaphragm fatigue and inadequate aerobic metabolism. Seven piglets were anesthetized by using halothane inhalation. Diaphragmatic contractility was assessed by transdiaphragmatic pressure (Pdi) at end expiration with the airway occluded. A nuclear magnetic resonance surface coil placed under the right hemidiaphragm measured P(i) and PCr during four conditions: control, inspiratory resistive breathing (IRB), IRB with hypoxia, and recovery (IRB without hypoxia). IRB alone resulted in hypercarbia (32 +/- 7 to 61 +/- 21 Torr) and respiratory acidosis but no change in diaphragm force output or aerobic metabolism. Combined IRB and hypoxia resulted in decreased force output (Pdi decreased by 40%; from 30 +/- 17 to 19 +/- 11 mmHg) and evidence of metabolic stress (ratio of P(i) to PCr increased by 290%; from 0.19 +/- 0.09 to 0.74 +/- 0.27). We conclude that diaphragm fatigue associated with inadequate aerobic oxidative metabolism occurs in the setting of loaded breathing and hypoxia. Conversely, aerobic metabolism and force output of the diaphragm remain unchanged from control during loaded normoxic or hyperoxic breathing despite the onset of respiratory failure.

Myocardial oxygenation at high workstates in hearts with left ventricular hypertrophy.

High cardiac workloads produced by catecholamine infusion result in loss of myocardial phosphocreatine (PCr) and accumulation of inorganic phosphate (Pi) which are more prominent in heart with left ventricular hypertrophy (LVH) than in normal hearts. Since ischemia can cause changes in phosphorylated compounds similar to those during catecholamine stimulation, this study tested the hypothesis that the exaggerated depletion of PCr and accumulation of Pi during high workloads in LVH is the result of impaired myocyte oxygenation. 31P- and 1H-NMR spectroscopy were used to determine myocardial high energy phosphate levels and myoglobin desaturation, respectively, in eight normal dogs and nine dogs with LVH produced by ascending aortic banding. The mean LV weight/body weight ratio was approximately twice normal in the LVH group. Infusion of dobutamine (15 and 30 micrograms/kg/min), and dobutamine + dopamine (each 20 micrograms/kg/min) caused progressive similar increases in the heart rate x systolic LV pressure product to a maximum of 57.4 +/- 3.3 x 10(3) in normal and 63.9 +/- 2.7 x 10(3) in LVH animals, while myocardial oxygen consumption increased from 0.09 +/- 0.01 to 0.24 +/- 0.04 in normals and from 0.10 +/- 0.02 to 0.25 +/- 0.03 ml/min/g in LVH. PCr/ATP ratios during basal conditions were lower in LVH hearts (1.73 +/- 0.10, 1.61 +/- 0.09 and 1.51 +/- 0.09 in subepicardium, midwall and subendocardium, respectively) as compared with normals (2.24 +/- 0.09, 2.01 +/- 0.08 and 1.89 +/- 0.07; each p < 0.01 normal vs. LVH). Catecholamine infusions caused dose-related decreases in PCr/ATP and appearance of Pi which was more marked in LVH than in normal hearts. 1H-NMR spectroscopy did not detect deoxymyoglobin in either normal or LVH hearts even during the highest workloads. In contrast, occlusion of the anterior descending coronary artery resulted in a large deoxymyoglobin signal. Increases of cardiac work produced by catecholamine stimulation resulted in greater decreases of PCr and greater increases of Pi in hypertrophied than in normal hearts. These abnormalities were not the result of inadequate intracellular oxygen availability and consequently cannot be ascribed to demand ischemia.

Some physiological characteristics of a phosphate-removing bacterium, microlunatus phosphovorus, and a simplified isolation and identification method for phosphate-removing bacteria

Some physiological characteristics of a phosphate (Pi)-removing bacterium, Microlunatus phosphovorus, are investigated using aerobically grown cells and cells exhibiting excess Pi accumulation (EPA) in order to determine a simplified isolation and identification method for other Pi-removing bacteria. Such a method would save on the amount of sterile equipment needed, and reduce the number of experimental steps and labor time. The EPA activity of the isolate reached a plateau 13 hours into the anaerobic incubation time, but it reached 70% of that level after only 5 hours. The EPA activity of the cells during the stationary growth phase was higher than that during the exponential growth phase. Polyphosphate (polyP) accumulated in the cells was shown to be used as an energy storage material (a phosphagen) under both aerobic and anaerobic conditions. During aerobic starvation, the rate of decrease in the ATP concentration of the suspension of cells that contained polyP was markedly less than that of the suspension of cells without polyP. Therefore, bacterial cells rich in polyP survive longer than bacterial cells lacking polyP.

Energy metabolism and mechanical recovery after cardioplegia in moderately hypertrophied rats.

It is well established that severe hypertrophy induces metabolic and structural changes in the heart which result in enhanced susceptibility to ischemic damage during cardioplegic arrest while much less is known about the effect of cardioplegic arrest on moderately hypertrophied hearts. The aim of this study was to elucidate the differences in myocardial high energy phosphate metabolism and in functional recovery after cardioplegic arrest and ischemia in mildly hypertrophied hearts, before any metabolic alterations could be shown under baseline conditions. Cardiac hypertrophy was induced in rats by constriction of the abdominal aorta resulting in 20% increase in heart weight/body weight ratio (hypertrophy group) while sham operated animals served as control. In both groups, isolated hearts were perfused under normoxic conditions for 40 min followed by infusion of St.Thomas' Hospital No. 1 cardioplegia and 90 min ischemia at 25 degrees C with infusions of cardioplegia every 30 min. The changes in ATP, phosphocreatine (PCr) and inorganic phosphate (Pi) were followed by 31P nuclear magnetic resonance (NMR) spectroscopy. Systolic and diastolic function was assessed with an intraventricular balloon before and after ischemia. Baseline concentrations of PCr, ATP and Pi as well as coronary flow and cardiac function were not different between the two groups. However, after cardioplegic arrest PCr concentration increased to 61.8+/-4.9 micromol/g dry wt in the control group and to 46.3+/-2.8 micromol/g in hypertrophied hearts. Subsequently PCr, pH and ATP decreased gradually, concomitant with an accumulation of Pi in both groups. PCr was transiently restored during each infusion of cardioplegic solution while Pi decreased. PCr decreased faster after cardioplegic infusions in hypertrophied hearts. The most significant difference was observed during reperfusion: PCr recovered to its pre-ischemic levels within 2 min following restoration of coronary flow in the control group while similar recovery was observed after 4 min in the hypertrophied hearts. A greater deterioration of diastolic function was observed in hypertrophied hearts. Moderate hypertrophy, despite absence of metabolic changes under baseline conditions could lead to enhanced functional deterioration after cardioplegic arrest and ischemia. Impaired energy metabolism resulting in accelerated high energy phosphate depletion during ischemia and delayed recovery of energy equilibrium after cardioplegic arrest observed in hypertrophied hearts could be one of the underlying mechanisms.

Some physiological characteristics of a phosphate-removing bacterium, Microlunatus phosphovorus, and a simplified isolation and identification method for phosphate-removing bacteria

Some physiological characteristics of a phosphate (Pi)-removing bacterium, Microlunatus phosphovorus, are investigated using aerobically grown cells and cells exhibiting excess Pi accumulation (EPA) in order to determine a simplified isolation and identification method for other Pi-removing bacteria. Such a method would save on the amount of sterile equipment needed, and reduce the number of experimental steps and labor time. The EPA activity of the isolate reached a plateau 13 hours into the anaerobic incubation time, but it reached 70% of that level after only 5 hours. The EPA activity of the cells during the stationary growth phase was higher than that during the exponential growth phase. Polyphosphate (polyP) accumulated in the cells was shown to be used as an energy storage material (a phosphagen) under both aerobic and anaerobic conditions. During aerobic starvation, the rate of decrease in the ATP concentration of the suspension of cells that contained polyP was markedly less than that of the suspension of cells without polyP. Therefore, bacterial cells rich in polyP survive longer than bacterial cells lacking polyP.

Metabolic events with spontaneous malignant hyperthermia crisis in an anaesthetized pig.

To analyze metabolic changes associated with a fulminant malignant hyperthermia (MH) crisis developed spontaneously in an MH susceptible pig which was part of 12 pigs undergoing metabolic investigation (six MH susceptible and six controls) and had been anaesthetized with a non-triggering agent (pentobarbitone). The pig was placed in a cradle and then inserted into a 4.7 T magnet bore. The semi-membranous muscle was submitted to three repetitive stimulation-recovery sessions. 31-P magnetic resonance spectra and mechanical data were recorded. The pig developed a non-rigid MH crisis during recovery from the second set of experiments. Although no mechanical work was performed, dramatic metabolic changes were noted. Twitch tension decreased progressively reaching zero while mouth temperature continuously increased to 44.5 degrees C. Phosphocreatine (PCr) consumption was coupled to Pi accumulation. Also, a marked intracellular acidosis and a large accumulation of phosphomonoesters (PME) were observed, probably as a result of massive glycolysis activation. Interestingly, ATP level remained constant. These irreversible mechanisms may constitute a metabolic dead-end coupling calcium pumping ATP-consuming processes and ATP synthesis through PCr breakdown and anaerobic glycolysis. They do not differ from metabolic changes previously reported in rigid forms of MH crisis.

Muscle contraction and fatigue. The role of adenosine 5'-diphosphate and inorganic phosphate.

Though many explanations are offered for the fatigue process in contracting skeletal muscle (both central and peripheral factors), none completely explain the decline in force production capability because fatigue is specific to the activity being performed. However, one needs to look no further than the muscle contraction crossbridge cycle itself in order to explain a major contributor to the fatigue process in exercise of any duration. The byproducts of adenosine 5'-triphosphate (ATP) hydrolysis, adenosine 5'-diphosphate (ADP) and inorganic phosphate (Pi) are released during the crossbridge cycle and can be implicated in the fatigue process due to the requirement of their release for proper crossbridge activity. Pi release is coupled to the powerstroke of the crossbridge cycle. The accumulation of Pi during exercise would lead to a reversal of its release step, therefore causing a decrement in force production capability. Due to the release of Pi with both the immediate (phosphagen) energy system and the hydrolysis of ATP, Pi accumulation is probably the largest contributor to the fatigue process in exercise of any duration. ADP release occurs near the end of the crossbridge cycle and therefore controls the velocity of crossbridge detachment. Therefore, ADP accumulation, which occurs during exercise of extended duration (or in ischaemic conditions), causes a slowing of the rate constants (and therefore a decrease in the maximal velocity of shortening). in the crossbridge cycle and a reduced oscillatory power output. The combined effects of these accumulated hydrolysis byproducts accounts for a large amount of the fatigue process in exercise of any intensity or duration.

Bacterial phosphonate degradation, phosphite oxidation and polyphosphate accumulation

Large quantities of xenobiotic phosphonates (Pn), which are used as pesticides, detergent additives, antibiotics, and flame retardants, are released into the environment. These C-P compounds are generally resistant to chemical hydrolysis and recalcitrant to biodegradation. We have cloned the Klebsiella aerogenes phn gene cluster which is required for degrading Pn and also for oxidizing phosphite (Pt) to phosphate (Pi). Recombinant plasmids containing the phn gene cluster allowed E. coli MV1184 to grow with aminoethylphosphonate (AEPn), ethylphosphonate (EPn), methylphosphonate (MPn) and Pt as a sole source of phosphorus. Pi, which is generated by Pn breakdown and Pt oxidation, is also a priority target for controlling the eutrophication in natural bodies of water. We examined the genetic improvement of bacterial Pi accumulation by manipulating the genes involved in the key steps of Pi transport and polyphosphate (polyP) formation.

A Peroxovanadium Compound InducesXenopusOocyte Maturation: Inhibition by a Neutralizing Anti-insulin Receptor Antibody

Synthetic peroxovanadium compounds are a new class of potent inhibitors of protein phosphotyrosine phosphatases. These compounds exhibit insulin-like activity bothin vitroand in experimental animals. However, the molecular mechanism by which these compounds exert their biological effect is not well defined. We demonstrate here that several of these compounds induceXenopusoocyte maturationin vitro,as indicated by germinal vesicle breakdown. Using one of these compounds for further studies, we show that the induction is dose-dependent and is accompanied by activation of maturation promoting factor as well as activation ofXenopusMAP kinase. Like insulin, bpV(pic) causes an acute accumulation of PI(3,4,5)P3(phosphotidylinositol-3,4,5-trisphosphate), a product of PI 3-kinase. More importantly, bpV(pic)-induced oocyte maturation was abolished by microinjection of a neutralizing monoclonal anti-insulin receptor antibody (17A3) into oocytes or preincubation of oocytes with a PI 3-kinase inhibitor (wortmannin). These results suggest that bpV(pic) acts upstream of theXenopusIGF-1 receptor in the induction of meiotic maturation, presumably by neutralizing an inhibitory protein tyrosine phosphatase(s) that may regulate the receptor. Finally, using an oocyte–follicle cell complex that responded to human chorionic gonadotropin (hCG) to undergo GVBD, we showed that injection of 17A3 anti-insulin receptor antibody into oocytes did not affect hCG-induced oocyte maturation.

Adaptation to chronic hypoxia alters cardiac metabolic response to β stimulation: Novel face of phosphocreatine overshoot phenomenon

The dynamics of the changes in myocardial phosphorylated compound contents (inorganic phosphate: Pi; phosphocreatine: PCr; ATP) induced by 10(-6)M isoprenaline administration was studied, using 31P-NMR spectroscopy, in hearts isolated from rats adapted for three weeks to normobaric hypoxia (10% of oxygen). When compared with the behaviour of control hearts, the inotropic response to Ca2+ and isoprenaline was larger in the hearts from hypoxic rats, while the oxygen consumption was similar. During administration of isoprenaline, a decrease in the myocardial contents of high energy phosphates (ATP and PCr) and an accumulation of Pi was observed in both groups. After action of isoprenaline, the hearts from hypoxic animals showed significant overshoot of PCr, that was not seen in hearts from normoxic rats. The mechanisms of these alterations are analysed and the phosphocreatine overshoot, as well as the increased rate pressure product to oxygen consumption ratio, are assumed to indicate more efficient energy conversion in the heart from animals adapted to chronic hypoxia.

Lack of Control in Inorganic Phosphate Uptake by Catharanthus roseus (L.) G. Don Cells (Cytoplasmic Inorganic Phosphate Homeostasis Depends on the Tonoplast Inorganic Phosphate Transport System?).

Inorganic phosphate (Pi) uptake by Catharanthus roseus (L.) G. Don cells was studied in relation to its apparent uncontrolled uptake using 31P-nuclear magnetic resonance spectroscopy. Kinetics of Pi uptake by the cells indicated that apparent Km and Vm were about 7 [mu]M and 20 [mu]mol g-1 fresh weight h-1, respectively. Pi uptake in Murashige-Skoog medium under different Pi concentrations and different initial cell densities followed basically the same kinetics. When supplied with abundant Pi, cells absorbed Pi at a constant rate (Vm) for the first hours and accumulated it in the vacuole. As the endogenous pool expanded, the rate of Pi uptake gradually decreased to nil. Maximum Pi accumulation was 100 to 120 [mu]mol g-1 fresh weight if cell swelling during Pi uptake (about 2-fold in cell volume) was not considered. Results indicated that (a) the rate of Pi uptake by Catharanthus cells was independent of initial cell density and was constant over a wide range of Pi concentrations (2 mM to about 10 [mu]M) unless the cells were preloaded with excess Pi, and (b) there was no apparent feedback control over the Pi uptake process in the plasma membrane to avoid Pi toxicity. The importance of the tonoplast Pi transport system in cytoplasmic Pi homeostasis is discussed.

Continuous monitoring of energy metabolites using microdialysis during myocardial ischaemia in the pig.

A diagonal branch of the left anterior descending artery was occluded for 80 min. One microdialysis probe was inserted in the ischaemic area and two in the non-ischaemic areas. In six animals radiolabelled microspheres were injected before and during ischaemia and after reperfusion to monitor flow. Arterial and pulmonary artery pressures, pulmonary capillary wedge pressure and cardiac output were followed-up. Radiolabelled microsphere distribution confirmed the reduction of myocardial blood flow during coronary artery occlusion, with flow returning to normal values upon reperfusion. Rate pressure product as an index of heart work was stable throughout the 80 min ischaemic period, but increased at reperfusion. Dialysates from non-ischaemic tissue showed stable baseline values. In ischaemic tissue, lactate increased by 300% and pyruvate levels decreased by 50% within 15 min. Adenosine increased rapidly five-fold, but started to decline after 15 min of ischaemia. Inosine showed a slower but a marked 20-fold increase, decreasing at the end of the ischaemic period. Hypoxanthine increased five-fold during the first 30 min. Finally, guanosine showed a slow increase to about four times above basic values. Microdialysis was an excellent tool for the continuous monitoring of myocardial metabolites during ischaemia. Adenosine appeared to be a more sensitive marker of early ischaemia than lactate, although interestingly, adenosine levels had decreased already after 15 min, most probably due to intracellular accumulation of protons and Pi.

Reduced purine catabolite production in the postischemic rat heart: a31P NMR assessment of cytosolic metabolites

Ischemia can cause release of adenosine and purine catabolites from the heart, through the breakdown of ATP. If repeated periods of ischemia are induced, the efflux of purines is markedly reduced, although it is not clear if this is beneficial for the long-term survival of the heart. We have investigated changes in high-energy phosphates and purine release in the isolated perfused rat heart using31P NMR spectroscopy and high-performance liquid chromatography. Hearts were subjected to one of the following protocols: Group A—1 min of total global ischemia (TGI) after 40 min, 60 min, and 85 min of perfusion (a total of 3 × 1 min ischemia); Group B—1 min of TGI after 40 min of perfusion, 10 min of TGI after 50 min of perfusion, and a final 1 min of TGI after 85 min of perfusion. The profile of high-energy phosphate metabolites, Pi accumulation and purine release was similar for each 1-min period of TGI in Group A, whereas phosphocreatine content was increased and ATP content reduced by an extended period of TGI in Group B, leading to a less severe acidosis and purine efflux in the final 1 min of TGI at 85 min of perfusion. In conclusion, the reduced purine release observed in Group B may be related to the preischemic ATP pool size and accessibility and the increased myocardial energy reserve in the form of phosphocreatine.