用~（31）P磁共振谱对艾氏腹水癌和肉瘤S－180细胞代谢的研究

艾氏腹水癌细胞和肉瘤Ｓ－１８０细胞是抗肿瘤药物筛选常用细胞株．实验采用取自小鼠腹腔的第７～８天的艾氏腹水癌和Ｓ－１８０细胞,用３１Ｐ磁共振谱测得了细胞内小分子含磷代谢成分;计算了细胞内ｐＨ值;还用３１Ｐ谱探讨了作用机制不同的三种抗代谢物：碘乙酸、２,４－二硝基苯酚及棉酚对艾氏腹水癌细胞代谢的影响     

31P NMR Identification of Metabolites and pH Determination in the Cyanobacterium Synechocystis sp. PCC 6308

The identity of a number of phosphorus-containing metabolites present in Synechocystis sp. PCC 6308 has been confirmed by ³¹P NMR spectroscopy. The presence of D-ribulose 1,5-bisphosphate (RuBP); DL-glyceraldehyde 3-phosphate (GlyP); D(−) 3-phosphoglyceric acid (3PGA); D-ribulose 5-phosphate (Ru5P); 6-phosphogluconic acid (6PGA); phosphoenolpyruvate (PEP); inorganic phosphate (Pi); uridine diphosphoglucose (UDPG); ADP and ATP were demonstrated by the pH dependence of their ³¹P NMR chemical shifts in spectra of perchloric acid cell extracts. Intracellular pH of cells was determined to be 7.5–7.7. Received: 20 September 1996 / Accepted: 26 October 1996     

Fluoride effects on 31P NMR spectra of macrophages

31P High resolution nuclear magnetic resonance studies have been carried out on the P388D1 tumoral cell line and the BCG elicited alveolar rabbit macrophages both in sedimented cells and in perfused agarose-embedded cells. When the cells were sufficiently oxygenated, the phosphorylated sugars and ATP concentrations attained high levels. The intensity of the peak representing phosphorylated sugars varied inversely with ATP level when macrophagic cells were treated by NaF. The identities of the phosphorylated sugars were revealed by 1H and 31P NMR studies of the P 388D1 cells perchloric extracts.     

Comparative 31P and 1H NMR studies on rat astrocytes and C6 glioma cells in culture.

Rat astroglial cells in primary culture (95% enrichment) and C6 glioma cells were adapted to grow on microcarrier beads. In vivo 31P NMR spectra were collected from cell-covered beads perfused in the NMR tube. The NMR-visible phosphorylated metabolite contents of both cell types were determined using saturation factors calculated from the values of longitudinal relaxation times determined for C6 cells using progressive saturation experiments. On the other hand, the amounts of phosphorylated metabolites in cells were determined from proton decoupled 31P NMR spectra of cell perchloric acid extracts. The results indicate that the NTP and Pi contents of the normal and tumoral cells were similar, whereas the PCr level was higher in C6 cells and the NDP and phosphomonoester levels higher in astrocytes. The comparison of 1H NMR spectra of cell perchloric acid extracts evidenced larger inositol and alanine contents in C6 cells, whereas larger taurine and choline (and choline derivatives) contents were found in astrocytes. The Glu/Gln ratio was very different, 3.5 and 1 in C6 cells and astrocytes, respectively. In both cases, the more intense resonance in the 1H NMR spectrum was assigned to glycine. Based on the comparison of the metabolite content of a tumoral and a normal cell of glial origin, this work emphasizes the usefulness of a multinuclear NMR study in characterizing intrinsic differences between normal and tumoral cells.     

Developmental regulation of energy metabolism in Caenorhabditis elegans

Changes in energy metabolism during larval development in Caenorhabditis elegans have been investigated using phosphorus nuclear magnetic resonance (31P NMR). The relative concentrations of ATP, ADP, AMP, sugar phosphates, and other metabolites were observed to change during larval development, producing stage-specific spectra. These spectra are consistent with enzyme assays for isocitrate dehydrogenase and isocitrate lyase, indicating that high activity of the glyoxylate pathway during embryonic development decreases during the first larval (L1) stage, and respiration during the L2, L3, and L4 stages occurs preferentially through the TCA cycle. Metabolic strategies were further studied using mutants that are predisposed to enter the dauer stage, a developmentally arrested third-stage larva formed under conditions of overcrowding and limited food. After the L1 molt, energy metabolism in animals destined to become dauer larvae diverges from that of animals committed to growth. Relative to the L1, the L2 larvae committed to growth exhibit increased isocitrate dehydrogenase activity as well as increases in ATP and other high-energy phosphates, but predauer (L2d) larvae exhibit declining enzyme activities and declining levels of high-energy phosphates. The predominant phosphorus NMR signal in dauer larva extracts corresponds to inorganic phosphate. We conclude that metabolism is regulated during C. elegans larval development, with a major transition apparent after the L1 stage. This transition does not occur in larvae destined to form dauer larvae.     

Analysis of sugar metabolism in an EPS producing Lactococcus lactis by 31P NMR

Sugar metabolism and exopolysaccharide (EPS) production was analysed in Lactococcus lactis by in vivo 31P NMR. Transient production of several sugar phosphates, transient depletion of intracellular phosphate, transient production of ATP and UTP, transient acidification of the medium and alkalinisation of the cytoplasm could be observed in a period of 20 min upon energization by the addition of glucose. EPS and non-EPS producing variants showed similar NMR spectra, the exception being two pH-dependent resonances observed in the former. They were already observed before addition of glucose and their response to glucose incubation reflected exposure to the medium. They are presumably phosphorylated poly- or oligosaccharides being loosely adhered to cell walls. By freezing and perchloric acid extraction of the cell material, different types of phosphorylated compounds could be recognised in the NMR spectra such as fructose-1-6-diphosphate, nucleotides (like ADP, ATP, UTP and TDP) and several nucleotide sugars. The ongoing work is focused on identifying the unknown peaks and quantifying the differences between wild-type cells and the EPS producing variant.     

31P-NMR studies of phosphate transfer rates in T47D human breast cancer cells

The concentration of phosphates and the kinetics of phosphate transfer reactions were measured in the human breast cancer cell line, T47D, using 31P-NMR spectroscopy. The cells were embedded in agarose filaments and perifused with oxygenated medium during the NMR measurements. The following phosphates were identified in spectra of perifused cells and of cell extracts: phosphorylcholine (PC), phosphorylethanolamine (PE), the glycerol derivatives of PC and PE, inorganic phosphate (Pi), phosphocreatine (PCr), nucleoside triphosphate (primarily ATP) and uridine diphosphate glucose. The rates of the transfers: PC----gamma ATP (0.2 mM/s), Pi----gamma ATP (0.2 mM/s) and the conversion beta ATP----beta ADP (1.3 mM/s) were determined from analysis of data obtained in steady-state saturation transfer and inversion recovery experiments. Data from spectrophotometric assays of the specific activity of creatine kinase (approx. 0.1 mumol/min per mg protein) and adenylate kinase (approx. 0.4 mumol/min per mg protein) suggest that the beta ATP----beta ADP rate is dominated by the latter reaction. The ratio between the rate of ATP synthesis from Pi and the rate of consumption of oxygen atoms (4 X 10(-3) mM/s) was approx. 50. This high value and preliminary measurements of the rate of lactate production from glucose, indicated that aerobic glycolysis is the main pathway of ATP synthesis.     

31P-NMR studies of the metabolisms of the parasitic helminths Ascaris suum and Fasciola hepatica

31P-NMR has been applied to the study of the metabolisms of the intact parasitic helminths Ascaris suum (the intestinal roundworm) and Fasciola hepatica (the liver fluke). After calibration of the chemical shift of Pi in muscle extracts the internal pH of adult Ascaris worms and the effect of the pH of the external medium on the organism's internal pH were measured. Assignments of nearly all of the observable 31P resonances could be made. A large resonance from glycerophosphorylcholine whose function is unclear was observed but no signals from energy storage compounds such as creatine phosphate were detected. The profiles of the phosphorus-containing metabolites in both organisms were monitored as a function of time. Changes in sugar phosphate distributions but not ATP/ADP were observed. Studies of the drug closantel on Fasciola hepatica were performed. Initial effects of the drug were a decrease in glucose 6-phosphate and an increase in Pi with no substantial change in ATP levels as observed by 31P-NMR. Studies involving treatment with closantel followed by rapid freezing, extraction, and analytical determination of glycolytic intermediates confirmed NMR observations. This NMR method can serve as a simple noninvasive procedure to study parasite metabolism and drug effects on metabolism.     

Phosphate Compounds of Scenedesmus C-2A' in Darkness or Blue Light as Measured by 31P NMR

Phosphorus-31 nuclear magnetic resonance (³¹P NMR) spectra ofintact cells of Scenedesmus mutant C-2A' and of their perchloricacid extracts are presented. Sugarphosphates, including glucose-6-phosphateand fructose-1,6-bisphosphate, orthophosphate, nucleotide di-and triphosphates, NAD(P), UDPG and—in the case of intactcells—also polyphosphates were identified. Blue light,which is known to stimulate the carbohydrate breakdown of greenalgae, leads to a transient drop in P_i, a pronounced decreasein the ATP/ADP ratio, and an increase in sugarphosphates, givingrise to the idea that the enhancement of phosphorolytic starchbreakdown is a primary response to blue light. Addition of glucoseto Scenedesmus mutant cells leads to comparable changes (besidesan additionally enhanced glucose-6-phosphate level), which thussupport the view that blue light stimulates dark-type respiration.Altogether the results demonstrate the applicability of ³¹PNMR spectroscopy to the study of the metabolism of green algae. (Received December 7, 1984; Accepted February 12, 1985)     

Mapping hypoxia-induced bioenergetic rearrangements and metabolic signaling by 18O-assisted 31P NMR and 1H NMR spectroscopy

Brief hypoxia or ischemia perturbs energy metabolism inducing paradoxically a stress-tolerant state, yet metabolic signals that trigger cytoprotection remain poorly understood. To evaluate bioenergetic rearrangements, control and hypoxic hearts were analyzed with 18O-assisted 31P NMR and 1H NMR spectroscopy. The 18O-induced isotope shift in the 31P NMR spectrum of CrP, betaADP and betaATP was used to quantify phosphotransfer fluxes through creatine kinase and adenylate kinase. This analysis was supplemented with determination of energetically relevant metabolites in the phosphomonoester (PME) region of 31P NMR spectra, and in both aromatic and aliphatic regions of 1H NMR spectra. In control conditions, creatine kinase was the major phosphotransfer pathway processing high-energy phosphoryls between sites of ATP consumption and ATP production. In hypoxia, creatine kinase flux was dramatically reduced with a compensatory increase in adenylate kinase flux, which supported heart energetics by regenerating and transferring beta- and gamma-phosphoryls of ATP. Activation of adenylate kinase led to a build-up of AMP, IMP and adenosine, molecules involved in cardioprotective signaling. 31P and 1H NMR spectral analysis further revealed NADH and H+ scavenging by alpha-glycerophosphate dehydrogenase (alphaGPDH) and lactate dehydrogenase contributing to maintained glycolysis under hypoxia. Hypoxia-induced accumulation of alpha-glycerophosphate and nucleoside 5'-monophosphates, through alphaGPDH and adenylate kinase reactions, respectively, was mapped within the increased PME signal in the 31P NMR spectrum. Thus, 18O-assisted 31P NMR combined with 1H NMR provide a powerful approach in capturing rearrangements in cardiac bioenergetics, and associated metabolic signaling that underlie the cardiac adaptive response to stress.     

Combined 1H and 31P NMR studies of cerebral metabolism in vivo

NMR spectroscopic methods have recently been developed for measurement of several concentrated cerebral metabolites in vivo. At present, 31P spectra from the brain permit detection of ATP, PCr, Pi, and certain sugar and lipid phosphates. The resonant frequency of Pi also provides a measure of cerebral pHi, and under some conditions ADP concentration can be calculated from information available in the 31P spectrum. The 1H spectrum of brain provides measurements of lactate, creatine, and several amino acids and choline-containing compounds. Both kinds of spectra can be obtained from the same subject. Our group at Yale used combined 31P and 1H methods to demonstrate that loss and recovery of phosphate energy stores and concomitant changes in cerebral amino acids during hypoglycemic coma in rodents could be observed in vivo. We then used the same methods to show that cerebral pHi can be normal while lactate is elevated in status epilepticus. NMR spectroscopy performed in vivo provides an array of chemically specific measurements unavailable by any other non-invasive method. It is thought to be entirely free of deleterious biological effects; hence, its potential for use in humans is considerable.     

A phosphorus-31 nuclear magnetic resonance study of phosphate uptake and storage in cultured Catharanthus roseus and Daucus carota plant cells

High resolution 31P NMR spectra (103.2 MHz) of oxygenated Catharanthus roseus and Daucus carota cells grown in suspension cultures were obtained using a solenoidal perfusion probe. The spectra showed resonances for various phosphorylated metabolites such as ATP, ADP, NAD(P)(H), nucleoside diphosphoglucose, and sugar phosphates. The relative levels of the phosphorylated metabolites remained constant throughout the growth curve. No resonances for storage compounds such as polyphosphates, pyrophosphate, or phytates were observed. Two resolved resonances for Pi indicated an intracellular pH of 7.3 and 5.7 (or below) for the cytoplasm and vacuoles, respectively. The time course of Pi uptake and storage during growth in fresh culture medium was followed by studying the level of vacuolar Pi with 31P NMR (145.7 MHz). Simultaneously, the level of Pi in the culture medium was followed with radioactive 32P. C. roseus quickly takes up all the Pi from the culture medium (maximum rate 1.7 mumol min-1 g-1 (dry weight of cells]. The Pi is first stored in the vacuoles; subsequently, one part of this pool is used to keep a constant cytoplasmic Pi level while another part is apparently accumulated as an NMR invisible Pi store, probably in another cell organelle. In contrast, D. carota does not accumulate Pi in the vacuoles and consequently it takes up Pi from the medium at a much slower rate (0.05 mumol min-1 g-1 (dry weight of cells].     

31P NMR measurements of the ADP concentration in yeast cells genetically modified to express creatine kinase

Rabbit muscle creatine kinase has been introduced into the yeast Saccharomyces cerevisiae by transforming cells with a multicopy plasmid containing the coding sequence for the enzyme under the control of the yeast phosphoglycerate kinase promoter. The transformed cells showed creatine kinase activities similar to those found in mammalian heart muscle. 31P NMR measurements of the near-equilibrium concentrations of phosphocreatine and cellular pH together with measurements of the total extractable concentrations of phosphocreatine and creatine allowed calculation of the free ADP/ATP ratio in the cell. The calculated ratio of approximately 2 was considerably higher than the ratio of between 0.06 and 0.1 measured directly in cell extracts.     

Development of a small-scale bioreactor: application to in vivo NMR measurement

A perfused bioreactor allowing in vivo NMR measurement was developed and validated for Eschscholtzia californica cells. The bioreactor was made of a 10-mm NMR tube. NMR measurement of the signal-to-noise ratio was optimized using a sedimented compact bed of cells that were retained in the bioreactor by a supporting filter. Liquid medium flow through the cell bed was characterized from a mass balance on oxygen and a dispersive hydrodynamic model. Cell bed oxygen demand for 4 h perfusion required a minimal medium flow rate of 0.8 mL/min. Residence time distribution assays at 0.8-2.6 mL/min suggest that the cells are subjected to a uniform nutrient environment along the cell bed. Cell integrity was maintained for all culture conditions since the release of intracellular esterases was not significant even after 4 h of perfusion. In vivo NMR was performed for (31)P NMR and the spectrum can be recorded after only 10 min of spectral accumulation (500 scans) with peaks identified as G-6P, F-6P, cytoplasmic Pi, vacuolar Pi, ATP(gamma) and ADP(beta), ATP(alpha) and ADP(alpha), NADP and NDPG, NDPG and ATP(beta). Cell viability was shown to be maintained as (31)P chemical shifts were constant with time for all the identified nuclei, thus suggesting constant intracellular pH.     

Uridine diphospho sugars and related hexose phosphates in the liver of hexosamine-treated rats: identification using 31P-[1H] two-dimensional NMR with HOHAHA relay

The effects of administration of galactosamine (GalN) and glucosamine (GlcN) on the levels of UDP-sugars and hexose monophosphates in rat livers were studied by a variety of 31P NMR methods. The flux of metabolites in the liver was monitored by in vivo NMR and showed elevated levels of UDP-sugars, and even greater increases in resonances at 4.6 ppm for GlcN treatment and at 2.0 ppm for GalN treatment. The individual compounds corresponding to these changes were identified in PCA liver extracts by 31P-[1H] two-dimensional relay spectroscopy with a HOHAHA-type 1H spin-lock. This method of transferring proton magnetization allows for nearly all of the proton chemical shifts to be observed for the hexose moiety of a UDP-sugar present in a complex mixture. The UDP-sugars in the extracts from treated rats were predominantly UDP-hexosamines. Relay spectra were also used to determine that GalN-1-P was the major component (16.0 mumol/g of liver) of the GalN-treated liver, while both alpha and beta anomers of GlcNAc-6-P were readily identified as the major hexose monophosphates in the GlcN experiment. Spectra from the 1H dimension of relay experiments conducted on extracts were nearly superimposable on relay spectra obtained under the same conditions for mixtures of standard compounds of known structure. UDP-GlcN and UDP-GalN were not commercially available, but their presence was established in the extracts after GalN treatment by obtaining relay spectra for a mixture of the compounds produced in situ enzymatically, without purification.(ABSTRACT TRUNCATED AT 250 WORDS)     

A convenient NMR method for in situ observation of aerobically cultured cells

We observed P31-NMR signals of intracellular phosphorylated metabolites, i.e. ATP, NAD(H) and UDPG, in the aerobically cultured cells of AJ13375 (a derivative of E. coli K-12) without a cell culture circulating system, which needs much more cells. Each NMR spectrum of 40 ml cell-culture was obtained with a 25 mm Phi sample tube mini-fermenter equipped with a pH electrode and three supply routes for O2 gas, aqueous ammonia and glucose. Spectra were measured at 15-min intervals. When glucose in the culture medium was consumed, P31-NMR signals of ATP disappeared first and then those of ADP decreased to below the limit of detection. The intracellular concentration of ATP was estimated to be approximately 7 mM.     

Effects of the anti-cancer drug adriamycin on the energy metabolism of rat heart as measured by in vivo 31P-NMR and implications for adriamycin-induced cardiotoxicity

In vivo 31P-NMR was used to measure the effects of the anti-tumor drug adriamycin on the energy metabolism of rat heart. The exclusive acquisition of NMR signal from cardiac muscle was assured by positioning a solenoidal radio-frequency NMR coil around the heart. Appropriate control experiments verified that 31P-NMR spectra solely originated from this organ. Acute effects occurring shortly after adriamycin administration are expressed in 31P spectra as a dose-dependent decline in the cardiac levels of phosphocreatine, after which stabilization at a new steady-state level occurs. These acute effects of a single dose are complete in 30-60 min and no significant further changes take place within 150 min after drug introduction. Longer-term effects of single high doses and of multiple lower doses were measured up to a week after the initiation of treatment. It seemed that at a total dose of 20 mg/kg, drug-induced interference with cardiac energy metabolism was more pronounced than at the same dose in the acute phase. These 31P-NMR data demonstrate that adriamycin treatment is accompanied by a decrease of the cardiac phosphocreatine/ATP ratio which might be an expression of the well-established cardiotoxicity of the drug.     

In vivo 31P- and 13C-NMR studies of ATP synthesis and methane formation by Methanosarcina barkeri

Carbon and phosphorus metabolism of cell suspensions of Methanosarcina barkeri strain MS (DSM 800), grown on methanol, were probed in vivo by NMR. The experimental conditions, which involved thick cell suspensions, did not significantly affect the efficiency of the rate of methanol uptake by cells. Following exposure to methanol an acidification of both the intracellular and the extracellular spaces was observed and a gradient of 0.5 pH units across the cytoplasmic membrane was determined from the 31P-NMR data. High levels of intracellular ATP up to 4 mM were detected. The ADP concentration determined in a suspension of starved cells was only 2 mM, suggesting that a significant amount of ADP may be immobilized and is thus not detectable by NMR. In the presence of the protonophore, 3,3',4',5-tetrachlorosalicylanilide, the proton gradient was dissipated and the synthesis of ATP stopped. The inhibitor of the ATP synthase, N,N'-dicyclohexylcarbodiimide, was rather inefficient in inhibiting ATP synthesis. High concentrations of N,N'-dicyclohexylcarbodiimide (corresponding to 300 nmol/mg protein-1) were required to decrease the ATP content by approximately 60%, and, under these conditions, formation of acetyl phosphate was detected. However, the methanol consumption rate was not affected.     

Phosphorus-31 nuclear magnetic resonance of C6 glioma cells and rat astrocytes. Evidence for a modification of the longitudinal relaxation time of ATP and Pi during glucose starvation

31P-NMR spectroscopy has been used to study the energy metabolism and the NMR visibility of ATP and intracellular Pi of the C6 glioma cell line and rat astrocyte grown on microcarrier beads with the following results. 1. In vivo NMR spectra of C6 glioma cells and rat astrocytes indicate that these cells were able to maintain their level of ATP resonances during a long anoxic period (more than an hour). Both cell types were sensitive to ischemia which induced a loss of ATP resonances within 40 min. Glucose starvation induced by 40% decrease in ATP resonances correlated to a 50% increase in the intensity of the Pi signal. These changes corresponded to a new steady state which could be reversed by reperfusing the cells with a glucose-containing medium. 2. In contrast to in vivo data, 31P-NMR analyses of perchloric acid extracts of cells incubated in a glucose-free medium showed that their ATP and Pi contents were unchanged during starvation. The changes of NMR visibility of the metabolites in living C6 cells were correlated to modifications of their macroscopic longitudinal relaxation times, evolving from 0.30 +/- 0.08 s and 6.6 +/- 1.5 s in the presence of glucose to 0.68 +/- 0.26 s and 3.2 +/- 0.9 s in the absence of glucose for ATP and Pi, respectively. The changes of the NMR detectability of ATP and Pi indicate that changes in their microenvironment occur during glucose starvation, suggesting the existence of different pools of these metabolites within the cells. 3. Under various experimental conditions, i.e. anoxia, ischemia and glucose starvation, rat astrocytes in primary culture showed a very similar behavior to that of C6 cells, suggesting a similar adaptability to the nature of the energy supply for both the normal and the malignant cell.     

Characterization of the renal epithelial cell line, PKE 5, using 31P- and 13C-NMR spectroscopy

In order to characterize intracellular pH regulation and cellular metabolism in PKE 5 cells, a mutant of the renal epithelial cell line LLC-PK1 supposed to lack Na+-H+ exchanger activity, 31P and 13C-NMR studies were conducted. The 31P studies on intact cell suspensions revealed that these cells have an ATP content and an ATP/ADP ratio similar to the parent cell line. Their intracellular pH, in the presence of 5 mM HCO3-, was 7.17 +/- 0.04 (n = 5) - identical to that of LLC-PK1 cells. After acid loading the cells with 15% CO2, the initial rate of realkalinization was 0.027 pH units/min (n = 6), 50% lower than in the parent cells. The recovery rate was not affected by the removal of extracellular sodium or by the addition of 1 mM amiloride. These results indicate that PKE 5 cells are devoid of Na+-H+ exchange activity, but are able to regulate their intracellular pH by amiloride-insensitive, sodium-independent mechanisms. Extracts prepared from PKE 5 cells incubated with [13C]lactate showed 13C spectra identical to those of the parent cell line. In particular, no synthesis of 13C-labeled D-glucose was observed.