The dependence of the cytoplasmic pH in aerobic and anaerobic cells of the green algae Chlorella fusca and Chlorella vulgaris on the pH of the medium as determined by 31P in vivo NMR spectroscopy

The pH in the cytoplasm of aerobic and anaerobic cells of the green algae Chlorella fusca and Chlorella vulgaris was determined in dependence on the pH of the external medium, which was varied between pH 3 and pH 10. In aerobic cells of both species the cytoplasmic pH is maintained at a value above 7.2 even at an external pH of 3 and below 7.8 at an external pH of 10. In anaerobic cells the cytoplasmic pH shows linear dependence on external pH in the range of pH 6 to 9 (cytoplasmic pH 6.9 to 7.2), while below an external pH of 6 cytoplasmic pH is maintained at about 6.5.Abbreviations CCCP Carbonylcyanide-m-chlorophenyl-hydrazone - EDTA Ethylendiaminetetraacetic acid - MES 2-(N-Morpholino)-ethanesulfonic acid - MOPSO 3-(N-Morpholino)-2-hydroxy-propanesulfonic acid - NMR Nuclear Magnetic Resonance - pH cyt cytoplasmic pH - pH ex external pH - PIPES Piperazine-N,N-bis(2-ethanesulfonic acid) - PP_i Pyrophosphate - PP₁, PP₂, PP₃ 1st, 2nd, 3rd phosphate group of polyphosphates - PP₄ core phosphate groups of polyphosphates - TRIS Tris-hydroxymethyl-aminomethane     

Assessment of energy metabolism in the developing brain following aglycemic hypoxia by1H and31P NMR

The role played by external calcium and calcium channels in the recovery from aglycaemic hypoxia in cortical brain slices from 10-day old rats was investigated by¹H and³¹P NMR. 30 minutes of aglycaemic hypoxia significantly decreased the levels of phosphocreatine (PCr), ATP, lactate and intracellular pH (pH_i). After a 30 minute recovery period there was incomplete recovery of PCr and ATP with lactate increasing by 50% with pH_i normal. When the aglycemic hypoxia was carried out in media which had no added calcium (≈10 μM) the PCr and ATP recovery was significantly greater. Application of diltiazem or verapamil but not nifedipine significantly improved the recovery from the aglycemic hypoxia. These data suggest that calcium influx through L-type voltagegated calcium channels is involved in the ischemic damage in neonatal brain which manifests itself as a decrease in the energy state and an increase in lactate. Dedication This article is dedicated to our friend and colleague Herman Bachelard. We wish to thank him for his comradeship, advice and support over many years. Our hope for him is a long and fruiful retirement and that he will remain active in the neurosciences for many years, even though the establishment has blown for “full time”.     

Determination of the transmembrane proton gradient in the anaerobic bacterium Desulfovibrio desulfuricans by 31P nuclear magnetic resonance

The transmembrane proton gradient of the sulfate-reducing bacterium Desulfovibrio desulfuricans strain CSN has been determined by in vivo³¹P nuclear magnetic resonance (NMR) spectroscopy in the absence of dioxygen. At pH 7.0 in the medium (pH_ex) the intracellular pH (pH_in) was 7.5. By lowering pH_ex to 5.9 pH_in decreased to 7.1. At pH_ex greater than 7.7 the transmembrane proton gradient (pH) was zero. The uncouplers 3,3,4,5-tetrachlorosalicylanilide (TCS) and carbonylcyanide-m-chlorophenylhydrazone (CCCP), or the permeant anion thiocyanate caused complete dissipation of pH.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - TCS 3,3,4,5-tetrachlorosalicylanilide - MOPS 3-(N-morpholino)-propanesulfonic acid - P _i inorganic phosphate - pH _in (pH_ex) intracellular (extracellular) pH - pH transmembrane proton gradient (pH_in-pH_ex) - electrochemical membrane potential - chemical shift in parts per million - NMR nuclear magnetic resonance     

Effects of vanadate on the ATP content,ATPase activity and phosphate absorption capacity of maize roots

Although the sensitivity of the plasma membrane H⁺-ATPase to vanadate is well known, the metabolic response of plant cells to vanadate is less well characterised in vivo and its use as an inhibitor in whole plant experiments has had mixed success. Experiments with maize (Zea mays, L.) roots and with purified plasma membrane fractions from the same tissues showed that exposure to vanadate caused: (i) a reduction in the capacity for phosphate uptake; (ii) a reduction in the extractable ATPase activity from the tissue; and (iii) a significant increase in the ATP level. The measurements on the extractable ATPase activity and the ATP level showed that the effect of vanadate developed slowly, apparently reflecting the slow accumulation of intracellular vanadate. The marked effect of vanadate on the ATP level-exposure to 500 M vanadate for 5 h doubled the ATP content of the roots tips-indicates that there is no stringent control over the ATP level in the roots and that the plasma membrane H⁺-ATPase activity is likely to have a significant role in determining the ATP level under normal conditions.     

Observation of Two Inorganic Phosphate NMR Resonances in the Perfused Hypothermic Rat Heart

The effect of hypothermia on isolated perfused rat hearts was studied with³¹P NMR. Hearts were continuously perfused with phosphate-free Krebs–Henseleit buffer while the perfusate temperature was adjusted. Perfusate pH was kept at 7.40 ± 0.02 throughout the experiments. Using the chemical shift difference between PCr and P_ithe intracellular pH was estimated. At 36, 20, and 10°C a cytosolic alkalinization at a pH of 7.05 ± 0.04, 7.21 ± 0.05, and 7.40 ± 0.03 was observed, respectively. At 10°C two P_iresonances were observed with a separation of 0.25 ppm. This resonance corresponded to a P_iresonance of a cellular compartment with a local pH of 7.78 ± 0.06, likely mitochondrial. This additional resonance disappeared upon warming of the hearts back to 36°C.     

Respirational activity of Chlorella fusca monitored by in vivo P-31 NMR

Energy metabolism during dark respiration of the green alga Chlorella fusca was investigated by ³¹P NMR spectroscopy. The kinetics of the transition from anaerobic to aerobic conditions (and vice versa) was followed with a temporal resolution of 16 s. This transition is accompanied by a shift of the cytoplasmic pH from 6.8 to 7.4, while the vacuolar pH remains constant. Simultaneously, an increase in the concentration of nucleoside-triphosphates and a decrease in the concentration of cytoplasmic orthophosphate take place, as well as the formation of mobile polyphosphates. The concentration of ATP and P _i reach steady-state levels within 30 s. Upon the reverse transition, from aerobic to anaerobic conditions, steady-state concentrations are obtained only after 3 min.     

Effects of L-carnitine and its acetyl and propionyl esters on ATP and PCr levels of isolated rat hearts perfused without fatty acids and investigated by means of 31P-NMR spectroscopy

³¹P-NMR in vivo spectroscopy is a non-invasive and non-hazardous technique which investigates chemical composition and metabolism of living objects, for example by determining phosphocreatine (PCr) and ATP concentrations. In the present study we investigated the influence of L-carnitine, acetyl-L-carnitine and propionyl-L-carnitine on the energetic state of the Langendorff rat heart subjected to an ischemic period of 20 min followed by a reperfusion period of 60 min. To avoid an overlapping of the effects of fatty acids and glucose, the hearts were perfused with a Tyrode solution containing no fatty acids. Ischemia causes a rapid decrease in the PCr signal, followed by a decrease in the ATP signal after a prolonged period of ischemia. At the same time, a drastic increase in the P_i signal was observed. A partial recovery of the ATP and PCr signals was observed in the reperfusion period. With L-carnitine a markedly improved recovery of the high energy phosphates (e.g. increased PCr/P_i ratios) was found. With acetyl-L-carnitine this effect was enhanced in the first postischemic phase. It was followed, however, by a more rapid decrease in the PCr/P_i ratio in the late reperfusion period. The effect of propionyl-L-carnitine was not significantly improved in the first minutes of the reperfusion period, but during the whole reperfusion phase a stabilization of the PCr/P_i ratio was observed. Intracellular pH can be calculated from determination of the P_i-chemical shift. This shows that L-carnitine and its derivatives have a protective effect against intracellular pH decrease during ischemia. L-carnitine improves the energetic state of the heart, which leads to increased ischemia tolerance. Hearts under L-carnitine were able to tolerate up to four ischemia-reperfusion periods in succession, whereas the controls were not able to do so. These NMR results confirm the hypothesis that L-carnitine and its esters have a protective effect in the reperfusion period of the ischemic rat heart. This could be of importance for the treatment of ischemic cardiac diseases.     

Relationship between intracellular pH and N metabolism in maize (Zea mays L.) roots

In vivo ³¹P nuclear magnetic resonance (NMR) was used to characterize the effect of the N form (NO₃ vs. NH₄) and the external pH (4, 6, and 8), on the intracellular pH of root tips (0–5 mm) and root segments (5–30 mm). Ammonium-grown root tips were the most sensitive to changes in the external pH. In vivo ¹⁵N NMR was used to characterize the pathway of primary ammonium assimilation in the ammonium-grown roots and to compare the activity of the apical and more-basal root parts. The kinetics of ¹⁵NH₄ ⁺ incorporation showed that primary assimilation in both root tips and root segments followed the glutamine synthetase (GS) pathway. In agreement with the reported gradient of GS along the seminal root of maize, incorporation of label into glutamine amide was more rapid in tips than in segments. It is suggested that this higher GS activity increases the endogenous proton production and thus contributes to the greater dependence of the cytoplasmic pH on the external pH in the ammonium-treated root tips.     

Investigation of subcellular acidic compartments using alpha-aminophosphonate 31P nuclear magnetic resonance probes

The ³¹P nuclear magnetic resonance (NMR) characteristics, toxicity, and cellular penetration of five linear or cyclic α-aminophosphonate highly sensitive pH probes were investigated in Dictyostelium discoideum cells and isolated rat hearts and were compared with three phosphonic acid derivatives. The line width broadening at pH pK_a, which was satisfactorily modelized for all compounds, was significantly limited in biological milieu for the new markers, affording a four- to sixfold better accuracy in pH determination. Cellular uptake or washout of nontoxic concentrations (<15 mM) of α-aminophosphonates occurred by rapid passive permeation, whereas standard probes required a much slower fluid-phase pinocytosis and transport processes that could ultimately lead to trapping. Using mild concentrations (<4 mM) three α-aminophosphonates having 6 < pK_a < 7 allowed an easy and simultaneous ³¹P NMR determination of cytosolic, acidic, and extracellular compartments in anoxic–reoxygenated or starving D. discoideum.     

Phosphorylation of C6- and C3-positions of glucosyl residues in starch is catalysed by distinct dikinases

Glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) are required for normal starch metabolism. We analysed starch phosphorylation in Arabidopsis wild-type plants and mutants lacking either GWD or PWD using (31)P NMR. Phosphorylation at both C6- and C3-positions of glucose moieties in starch was drastically decreased in GWD-deficient mutants. In starch from PWD-deficient plants C3-bound phosphate was reduced to levels close to the detection limit. The latter result contrasts with previous reports according to which GWD phosphorylates both C6- and C3-positions. In these studies, phosphorylation had been analysed by HPLC of acid-hydrolysed glucans. We now show that maltose-6-phosphate, a product of incomplete starch hydrolysis, co-eluted with glucose-3-phosphate under the chromatographic conditions applied. Re-examination of the specificity of the dikinases using an improved method demonstrates that C6- and C3-phosphorylation is selectively catalysed by GWD and PWD, respectively.     

31P Nuclear magnetic resonance studies of ethanol inhibition in Zymomonas mobilis

Ethanol inhibition of glucose catabolism in Zymomonas mobilis was investigated using ³¹P NMR spectroscopy in vivo and of perchloric acid extracts from cell suspensions incubated with 0, 5 and 10% (w/v) ethanol. In vivo ³¹P NMR experiments revealed slower glucose utilization and decreased levels of nucleoside triphosphates in the presence of 10% ethanol as compared to controls. Using ³¹P NMR spectroscopy of perchloric acid extracts, intracellular accumulation of 3.4 mM 3-phosphoglycerate was found when 10% ethanol was present in the medium. No accumulation of this metabolite occurred in cells incubated with 0 and 5% ethanol. Enzyme assays confirmed that phosphoglycerate-mutase and enolase were inhibited 31 and 40%, respectively, in the presence of 10% ethanol in the test system. Therefore, under the conditions used the decrease in the fermentative activity of Z. mobilis at high ethanol concentrations is due to inhibition of phosphoglycerate-mutase and enolase.Abbreviation KDPG 2-keto-3-deoxy-6-phosphogluconate     

Using 31P NMR Spectroscopy at 14.1 Tesla to Investigate PARP-1 Associated Energy Failure and Metabolic Rescue in Cerebrocortical Slices

PARP-1 activation by H(2)O(2) in an acute preparation of superfused, respiring, neonatal cerebrocortical slices was assessed from PAR-polymer formation detected with immunohistochemistry and Western blotting. (31)P NMR spectroscopy at 14.1 Tesla of perchloric acid slice extracts was used to assess energy failure in a 1-h H(2)O(2) exposure as well as in a subsequent 4-h recovery period where the superfusate had no H(2)O(2) and specifically chosen metabolic substrates. Although more data are needed to fully characterize different bioenergetic responses, a high NMR spectral resolution (PCr full-width at half-max approximately.01 ppm) and narrow widths for most metabolites (<.2 ppm) permitted accurate quantifications of spectrally resolved resonances for ADP, ATP, NAD(+)/NADH, and other high energy phosphates. It appears possible to use brain slices to quantitatively study PARP-related, NAD-associated energy failure, and rescue with TCA metabolites.     

A phosphorus-31 nuclear magnetic resonance study of elicitor-mediated metabolic changes in Catharanthus roseus suspension cultures

Summary The induction of metabolic changes in suspension cultured cells of Catharanthus roseus upon elicitation has been investigated. Addition of a yeast glucan preparation to the growth medium resulted in induction of phenylalanine ammonia lyase. Phosphate uptake and metabolism of elicited cells was followed by ³¹P nuclear magnetic resonance. The uptake rate of Pi from the medium by oxygenated cells of C. roseus was reduced immediately after elicitation. Despite this reduced Pi uptake elicited cells had significantly increased amounts of ATP (twofold increase within 6 h). Cytoplasmic levels of Pi, phosphomonoesters, and Uridine Diphasphate glucose (UDP-Glc) were unaffected by eliciation. Furthermore, the cytoplasmic and vacuolar pH remained constant after addition of elicitor.     

A 31P NMR study of the internal pH of yeast peroxisomes

The internal pH of peroxisomes in the yeasts Hansenula polymorpha, Candida utilis and Trichosporon cutaneum X4 was estimated by ³¹P nuclear magnetic resonance (NMR) spectroscopy. ³¹P NMR spectra of suspensions of intact cells of these yeasts, grown under conditions of extensive peroxisomal proliferation, displayed two prominent P_i-peaks at different chemical shift positions. In control cells grown on glucose, which contain very few peroxisomes, only a single peak was observed. This latter peak, which was detected under all growth conditions, was assigned to cytosolic P_i at pH 7.1. The additional peak present in spectra of peroxisome-containing cells, reflected P_i at a considerably lower pH of approximately 5.8–6.0. Experiments with the protonophore carbonyl cyanide m-chlorophenylhydrazon (CCCP) and the ionophores valinomycin and nigericin revealed that separation of the two P_i-peaks was caused by a pH-gradient across a membrane separating the two pools. Experiments with chloroquine confirmed the acidic nature of one of these pools. In a number of transfer experiments with the yeast H. polymorpha it was shown that the relative intensity of the P_i-signal at the low pH-position was correlated to the peroxisomal volume fraction. These results strongly suggest that this peak has to be assigned to P_i in peroxisomes, which therefore are acidic in nature. The presence of peroxisome-associated P_i was confirmed cytochemically.Abbreviations CCCP Carbonyl cyanide m-chlorophenylhydrazon - DCCD N,N-dicyclohexylcarbodiimide     

Regional and developmental variations in metabolite concentration in the rat brain and eye: A study using1H NMR spectroscopy and high performance liquid chromatography

Regional and developmental changes in metabolite concentrations were measured by¹H NMR spectroscopy and HPLC of perchloric acid extracts from rat brain and eye. The highest concentrations of N-acetylaspartate were found in grey matter as opposed to white matter with concentration increasing with age from neonate to adult, while the related compound N-acetylaspartylglutamate was highest in adult optic nerve. Creatine and choline-containing compounds were present in all regions throughout development, with higher levels of creatine found in grey matter compared to other regions. Choline-containing compounds were present at the highest concentrations in the eye at all ages examined, and tended to decrease in concentration to minimum values in adulthood in all regions. The presence of hypotaurine in corpus callosum and optic nerve was consistent with the metabolic profiles of O-2A progenitor cells and oligodendrocytes, which are cells composing these tissues. The neurotransmitters glutamate and GABA reached their highest concentrations in the olfactory bulb (higher than in adult cortex). Special issue dedicated to Dr. Herman Bachelard.     

A new experimental approach and signal processing scheme for the detection and quantitation of P brain neurochemicals from in vivo MRS studies using dual tuned (H/P) head coil

Brain ³¹P-neurometabolites play an important role in energy and membrane metabolism. Unambiguous identification and quantification of these neurochemicals in different brain regions would be a great aid in advancing the understanding of metabolic processes in the nervous system. Phosphomonoester (PME), consisting of phosphoethanolamine (PE) and phosphocholine (PC), is the “building block” for membranes, while phosphodiesters (PDE), consisting of glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) metabolites are involved in the membrane breakdown process. In the clinical setting, generating well-resolved spectra for PC, PE, GPC, and GPE could be crucial phospholipids in providing information regarding membrane metabolism. We present here a new experimental approach for generating well-resolved ³¹P spectra for PC and PE as well as for GPC, GPE, and other ³¹P metabolites. Our results (based on uni-dimensional (1D) and multi-voxel ³¹P studies) indicate that an intermediate excitation pulse angle (35°) is best suited to obtain well-resolved PC/PE and GPC/GPE resonance peaks. Our novel signal processing scheme allows generating metabolite maps of different phospholipids include PC/PE and GPC/GPE using the ‘time-domain–frequency-domain’ method as referred to in the MATLAB programming language.     

Locust flight metabolism studied in vivo by 31P NMR spectroscopy

Flight metabolism of locusts has been extensively studied, but biochemical and physiological methods have led to conflicting results. For this reason the non-invasive and non-destructive method of ³¹P NMR spectroscopy was used to study migratory locusts, Locusta migratoria, at rest and during flight.

Nuclear magnetic resonance studies of the location and function of plant nutrients in vivo

The cytoplasmic and vacuolar pools of ammonium, inorganic phosphate and potassium can be studied non-invasively in plant tissues using high resolution nuclear magnetic resonance spectroscopy. The techniques that allow these pools to be discriminated in vivo are described and their application to plants is reviewed with reference to the phosphorus, nitrogen and potassium nutrition of root tissues.     

Ammonia rhythm in Microcystis firma studied by in vivo 15N and 31P NMR spectroscopy

Cultures of the cyanobacterium Microcystis firma show rhythmic uptake and release of ammonia under conditions of carbon limitation. The massive removal of ammonia from the medium during the first light phase has little impact on the intracellular pH: a pH shift of less than 0.2 U towards the alkaline can be measured by in vivo ³¹P NMR. Furthermore, the energy status of the cells remains regulated. In vivo ¹⁵N NMR of M. firma, cultivated either with labelled nitrate or ammonia as the sole nitrogen source, reveals only gradual differences in the pool of free amino acids. Additionally both cultivation types show -aminobutyric acid, acid amides and yet unassigned secondary metabolites as nitrogen storing compounds. Investigating the incorporation of nitrogen under carbon limitation, however, only the amide nitrogen of glutamine is found permanently labelled in situ. While transamination reactions are blocked, nitrate reduction to ammonia can still proceed. Cation exchange processes in the cell wall are considered regarding the ammonia disappearance in the first phase, and the control of ammonia uptake is discussed with respect to the avoidance of intracellular toxification.Abbreviations GABA -aminobutyric acid - GOGAT glutamate synthase - GS glutamine synthetase - MDP methylene diphosphonate - MOPSO 3-(N-morpholino)-2-hydroxy-propanesulfonic acid - NDPS nucieoside diphosphosugars - NOE nuclear Overhauser effect - NMR nuclear magnetic resonance For convenience, the term ammonia is used throughout to denote ammonia or ammonium ion when there is no good evidence as to which chemical species is involved