Intracellular Calcium Dynamics and Cellular Energetics in Ischemic NG108-15 Cells Studied by Concurrent 31P/19F and 23Na Double-Quantum Filtered NMR Spectroscopy

Abstract: The role of voltage-sensitive Ca²⁺ channels in mediating Ca²⁺ influx during ischemia was investigated in NG108-15 cells, a neuronal cell line that does not express glutamate-sensitive receptor-mediated Ca²⁺ channels. Concurrent ³¹P/¹⁹F and ²³Na double-quantum filtered (DQF) NMR spectra were used to monitor cellular energy status, intracellular [Ca²⁺] ([Ca²⁺]_i), and intracellular Na⁺ content in cells loaded with the calcium indicator 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5FBAPTA) during ischemia and reperfusion. Cells loaded with 5FBAPTA were indistinguishable from unloaded cells except for small immediate decreases in levels of phosphocreatine (PCr) and ATP. Ischemia induced a steady decrease in intracellular pH and PCr and ATP levels, and a steady increase in intracellular Na⁺ content; however, a substantial increase in [Ca²⁺]_i (about threefold) was seen only following marked impairment of cellular energy status, when PCr was undetectable and ATP content was reduced to 55% of control levels. A depolarization-induced increase in [Ca²⁺]_i could be completely blocked by 1 µM nifedipine, whereas up to 20 µM nifedipine had no effect on the increase in [Ca²⁺]_i seen during ischemia. These data demonstrate that voltage-gated Ca²⁺ channels do not mediate significant Ca²⁺ flux during ischemia in this cell line and suggest an important role for Ca²⁺_i stores, the Na⁺/Ca²⁺ antiporter, or other processes linked to cellular energy status in the increase in cytosolic Ca²⁺ level during ischemia.     

33P translocation in the thallus of the mat-forming lichen Cladonia portentosa

The extent of vertical migration of ³³P in thalli of the heathland lichen Cladonia portentosa was investigated under field conditions. ³³P-labelled orthophosphate was introduced into the bottom 25 mm of podetia cut to a length of 50 mm from the apices. The distribution of label was scanned using a molecular imager immediately after incubation, and after growing for 8 wk and 6 months. Differences in the relative distribution of label between podetia harvested at the beginning and the end of the experiment showed that there had been a significant migration of ³³P upwards out of the labelled 25 mm stratum towards the apex. This was confirmed by statistically significant changes in the median (md) and the 90 percentile of total relative distribution of ³³P label. In a control treatment in which label was introduced into the apical 25 mm of podetia, which were then grown inverted (top down), no upward movement of label was detected. By contrast, a statistically significant reduction in the md of the distribution indicated migration downwards towards the thallus apex. The results are consistent with the hypothesis that P is recycled within podetia of mat-forming lichens, migrating from degrading basal regions upwards to the growing apices following a source–sink relationship.     

THE EFFECT OF DENERVATION ON INCORPORATION OF 32P AND [3H]GLYCEROL BY THE MUSCLE MEMBRANE

Abstract— The incorporation in vivo of ³²P₁ was significantly increased in all glycerophosphatide of preparations of denervated muscle membrane in frogs. There was no increase in incorporation of ³²P₁ into sphingomyelin. Disuse induced by tenotomy did not significantly increase incorporation of ³²P₁ into phospholipids of the muscle membrane. The phospholipid content of muscle membranes remained unchanged as a result of denervation or tenotomy. Denervation produced an increase in the incorporation of [2-³H]glycerol into all glycerophosphatides in parallel with the increase in ³²P₁ incorporation. Although the stimulated incorporation of ³²P₁ was increased in the regions of the muscle membrane rich in endplates, the most marked effect was in the endplate-poor region where activity in phosphatidylserine was most markedly increased.     

Tolerance of Low Intracellular pH During Hypercapnia by Rat Cortical Brain Slices: A 31P/1H NMR Study

Metabolic tolerance of low intracellular pH (pH(i)) was studied in well-oxygenated, perfused, neonatal, rat cerebrocortical brain slices (350 microns thick) by inducing severe hypercapnia. In each of 17 separate experiments 80 brain slices (approximately 3.2 g wet weight) were suspended in an NMR tube, perfused with artificial CSF (ACSF), and studied at 4.7 T with 31P and 1H NMR spectroscopy. Spectra obtained every 5 min monitored relative concentrations of lactate or high-energy phosphate metabolites, from which pH(i) and extracellular pH were determined. Unperturbed slice preparations were metabolically stable for > 10 h, with no significant changes occurring in pHi, ATP, phosphocreatine (PCr), inorganic phosphate, or lactate. Different levels of hypercapnia were produced by sequentially perfusing slices with the following different ACSF batches, each having previously been equilibrated with a specific mixture of CO2 in oxygen: (a) 10% CO2, 15 min of perfusion; (b) 30% CO2, 15 min of perfusion; (c) 50% CO2, 15 min of perfusion; (d) 70% CO2, 30 min of perfusion; (e) 50% CO2, 15 min of perfusion; (f) 30% CO2, 15 min of perfusion; and (g) 10% CO2, 15 min of perfusion. At the completion of this protocol slices were again perfused with fresh ACSF that was equilibrated with a 95% O2/5% CO2 gas mixture. In each of five separate 1H and 31P experiments, brain slices were recovered within 2 h after termination of exposure to high CO2. The pHi was determined from measurements of the chemical shift difference between phosphoethanolamine and PCr, using a calibration curve obtained for our preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Red Light Effects on Uptake of 14C and 32P into Etiolated Corn Leaf Tissue During Photomorphogenic Leaf Opening

Red light effects on the uptake of ¹⁴C and of ³²P was studied by observing leaf sections from 8-day-old etiolated corn leaves that were placed on various substrates following a brief exposure to red radiant energy. There was a general increase of ¹⁴C uptake over dark levels into all metabolite fractions that were prepared. This is in contrast with results obtained previously in which leaf samples were first floated on substrate and then irradiated (Mitrakos et al. 1967, Price et al. 1965). The latter tests resulted in a general decrease in sugar and starch as well as ¹⁴C content of all fractions. However, under both types of experimental conditions the red light effect manifested itself as an increase in hexosemonophosphate turn-over rate and accumulation of radioactivity in the cell wall polysaccharide fraction. The present data further substantiate the previous work in that they demonstrate the regulatory influence of the hexose pool size on the intermediary metabolism and the manifestation of the phytochrome responses. From the data thus far obtained it cannot be determined as to whether or not phytochrome mediates by controlling phosphorylating activity or through control of specific enzymatic processes that lead to observable cell wall polysaccharide synthesis and leaf unrolling.     

Cerebral Acid Buffering Capacity at Different Ages Measured In Vivo by 31P and 1H Nuclear Magnetic Resonance Spectroscopy

Cerebral acidosis occurring during ischemia has been proposed as one determinant of tissue damage. Newborn animals appear to be less susceptible to ischemic tissue damage than adults. One possible component of ischemic tolerance could derive from maturational differences in the extent of acid production and buffering in newborns compared to adults. The purpose of this study was to measure the dependency of acid production on the blood plasma glucose concentrations and acid buffering capacity of piglets at different stages of development. Complete ischemia was induced in 29 piglets ranging in postconceptual age from 111 to 156 days (normal term conception, 115 days). Brain buffering capacity during the first 30 min of ischemia was quantified in vivo, via 31P and 1H nuclear magnetic resonance (NMR) spectroscopy, by measuring the change in intracellular brain pH for a given change in the concentration of compounds that contribute to the production of hydrogen ions. Animals from all four age groups showed a similar linear correlation between preischemia blood glucose concentration and intracellular pH after 30 min of ischemia. For each animal the slope of the plot of intracellular pH versus cerebral buffer base deficit was used to calculate the buffer capacity. Using data obtained over the entire 30 min of ischemia, there was no difference in the mean buffer capacity of the different age groups, nor was there a significant correlation between buffer capacity and age. However, there was a significant increase in buffer capacity for the intracellular pH range 6.6-6.0, compared to 7.0-6.6, for all age groups. No significant differences in buffer capacity for these two pH ranges were observed between any of the age groups. Acid buffering capacity was also measured by performing pH titrations on brain tissue homogenized in the presence of inhibitors of glycolysis and creatine kinase. Plots of homogenate pH versus buffer base deficit showed a nonlinear trend similar to that seen in vivo, indicating an increase in buffer capacity as intracellular pH decreases. A comparison of newborn and 1-month-old brain tissue frozen under control conditions or after 45 min of ischemia revealed no differences that could be attributed to age and a slight decrease in buffer capacity of ischemic brain compared to control brain tissue homogenates. There was no difference between the brain buffering capacity measured in vivo using 31P and 1H NMR and that measured in vitro using brain homogenates.     

Distribution of mineral nutrient from nodal roots of Trifolium repens: Genotypic variation in intra-plant allocation of 32P and 45Ca

To assess genotypic variability in nutrient supply of shoot branches, the distribution of ³²P and ⁴⁵Ca exported from a source nodal root (24-h uptake period) was measured within a genotype of a large-leaved (Kopu) and a small-leaved (Tahora) cultivar of Trifolium repens. Source-sink relationships of plants were modified by root severance, defoliation, and shade treatments. In control plants of both genotypes distribution of ³²P and ⁴⁵Ca closely followed the pathways that could be predicted from the known phyllotactic constraints on the vascular system. As such there was little allocation of radioisotopes (3.1% and 2.5% of exported ³²P and ⁴⁵Ca, respectively) from the source root to branches on the apposite side of the parent axis (far-side branches). However, genotypic differences in nutrient allocation were apparent, when treatments were imposed to alter intra-plant source-sink relationships. In the large-leaved genotype, the imposed treatments had minor effects on the allocation to far-side branches: whereas, in the small-leaved genotype, root severance and defoliation treatments increased lateral transport to far-side branches to 30% (³²P) and 10% (⁴⁵Ca) of exported radioisotopes. Genotypes with low (8–9) and high (12–13) numbers of vascular bundles were selected from within the large-leaved cultivar. Distribution of ³²P was then measured after plants had been pre-treated by removal of all far-side roots two days prior to labelling. Genotypes with low vascular bundle number allocated 20% and those with high vascular bundle number 3.2% of exported ³²P to far-side branches. It was concluded (1) that genotypic variation exists within T. repens for potential to alter intra-plant allocation of mineral nutrients, in response to treatments that modify source-sink relationships within plants; and (2) that this variation is correlated with differences among genotypes in the organisation of the vasculature of their stolons.     

Methyl Mercury Stimulates Protein 32P Phospholabeling in Cerebellar Granule Cell Culture

Cultures of cerebellar granule neurons have been utilized to examine morphological and biochemical consequences of methyl mercury (MeHg). Exposure to MeHg for 24 h was found to exert toxic effects at concentrations below 1 microM characterized by neuron degeneration and neuritic varicosities. Dose-response and time course profiles for cell death were established using the 51Cr release assay, which revealed that 1 microM MeHg produced 15% cell death at 24 h, progressing to 50% at 48 h. Labeling of cultures with [32P]orthophosphate following 24-h exposure to 1 microM MeHg disclosed abnormalities in both protein and lipid phosphorylation. After 24-h exposure to 5 microM MeHg, phospholabeling of protein and lipid increased 174 and 128%, respectively, compared with controls. This stimulation of phosphorylation appeared to be neuron specific since cultures enriched in cerebellar glial cells and devoid of granule neurons displayed dose-dependent inhibition of total phosphorylation. Measurement of 32P labeling of ATP using a cyclic AMP-dependent protein kinase assay in conjunction with the firefly luciferase assay for ATP indicated no significant change in either total ATP levels or [32P]ATP specific activity at 1 or 4 h as a function of [MeHg]. Studies measuring 32P-phosphoprotein turnover indicated that MeHg had no effect on intracellular protein phosphatase activity. We conclude that one of the manifestations associated with in vitro cerebellar granule cell neurotoxicity is an abnormality in protein phosphorylation that is independent of [32P]ATP specific activity and protein phosphatase activity.     

Acid Homeostasis Following Partial Ischemia in Neonatal Brain Measured In Vivo by 31P and 1H Nuclear Magnetic Resonance Spectroscopy

The purpose of this study was to investigate neonatal brain energy metabolism, acid, and lactate homeostasis in the period immediately following partial ischemia. Changes in brain buffering capacity were quantified by measuring mean intracellular brain pH, calculated from the chemical shift of P_i, in response to identical episodes of hypercarbia before and after ischemia. In addition, the relationship between brain buffer base deficit and intracellular pH was compared during and following ischemia. Thus, in vivo ³¹P and ¹H nuclear magnetic resonance spectra were obtained from the brains of seven newborn piglets exposed to sequential episodes of hypercarbia, partial ischemia, and a second episode of hypercarbia in the postischemic recovery period. For the first episode of hypercarbia, brain buffering was similar to values reported for adult animals of other species (percentage pH regulation = 54 ± 16%). During ischemia, the brain base deficit per unit change in pH was ?19 ± 5 mM/pH unit, which is similar to values reported for adult rats. By 20–35 min postischemia, brain acidosis partly resolved in spite of a net increase in lactate concentration. Therefore, the consumption of lactate could not explain acid homeostasis in the first 35 min following ischemia. We conclude that H⁺/HCO^-₃ or other proton equivalent translocation mechanisms must be sufficiently developed in piglet brain to support acid regulation. This is surprising, because a substantial body of evidence implies these processes would be less active in immature brain. The second episode of hypercarbia, from 35 to 65 min postischemia, resulted in a smaller decrease in brain pH compared with the first episode, a result indicating an increase in brain buffering capacity (percentage pH regulation = 79 ± 29%). This was associated with a parallel decrease in brain lactate content, and therefore acid regulation could be attributed to either continued ion translocation or the consumption of lactate. A mild decrease in brain pH and content of energy metabolites was observed, a finding suggesting that the metabolic consequences of severe postischemic hypercarbia are neither particularly dangerous or beneficial.     

IN VITRO INCORPORATION OF 3H-THYMIDINE, 3H-URIDINE, 3H-LEUCINE AND 3H-MELPHALAN BY PLASMACYTOMA PLASMA CELLS

Bone marrow plasma cells from fifteen cases of multiple myeloma, immunologically typed, were incubated with different tritiated compounds. The labelling index with tritiated thymidine is generally low, while the mean grain count is fairly normal in the active cells. The labelling index of ³H-uridine and ³H-leucine was very high, while the mean grain count per cell lies within the normal range. The results obtained with ³H-phenylalanine-mustard (melphalan), which is a drug used in the treatment of the plasmacytoma, show also incorporation values roughly comparable to those of ³H-leucine. The present data seem to support the clinical use of melphalan as a compound that is actively incorporated into the plasma cells of plasmacytoma although inhibition of protein synthesis due to specific binding to protein was not demonstrated.     

Effects of Acute Hyperammonemia on Cerebral Amino Acid Metabolism and pHi In Vivo, Measured by 1H and 31P Nuclear Magnetic Resonance

The effects of an acute intravenous infusion of ammonium acetate on rat cerebral glutamate and glutamine concentrations, energy metabolism, and intracellular pH were measured in vivo with 1H and 31P nuclear magnetic resonance (NMR). The level of blood ammonia maintained by the infusion protocol used in this study (approximately 500 microM, arterial blood) did not cause significant changes in arterial PCO2, PO2, or pH. Cerebral glutamate levels fell to at least 80% of the preinfusion value, whereas glutamine concentrations increased 170% relative to the preinfusion controls. The fall in brain glutamate concentrations followed a time course similar to that of the rise of brain glutamine. There were no detectable changes in the content of phosphocreatine (PCr) or nucleoside triphosphates (NTP), within the brain regions contributing to the sensitive volume of the surface coil, during the ammonia infusion. Intracellular pH, estimated from the chemical shift of the inorganic phosphate resonance relative to the resonance of PCr in the 31P spectrum, was also unchanged during the period of hyperammonemia. 1H spectra, specifically edited to allow quantitation of the brain lactate content, indicated that lactate rose steadily during the ammonia infusion. Detectable increases in brain lactate levels were observed approximately 10 min after the start of the ammonia infusion and by 50 min of infusion had more than doubled. Spectra acquired from rats that received a control infusion of sodium acetate were not different from the spectra acquired prior to the infusion of either ammonium or sodium acetate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of Potential Effectors of Agonal Glycolytic Rate in Developing Brain Measured In Vivo by 31P and 1H Nuclear Magnetic Resonance Spectroscopy

Abstract: Previously we have shown that hypercarbia produces a larger decrease in agonal glycolytic rate in 1-month-old swine than in newborns. In an effort to understand the mechanism responsible for this difference, we tested the hypothesis that hypercarbia produces age-related changes in the concentration of one or more effectors of phosphofructokinase activity. Specifically, in vivo ³¹P and ¹H NMR spectroscopy was used to compare changes in lactate levels, intracellular pH, free magnesium concentration, and content of phosphorylated metabolites for these two age groups at three intervals during the first 1.5 min of complete ischemia in the presence or absence of hypercarbia (P_aco ₂ = 102–106 mm Hg). Hypercarbia produced the same drop in intracellular brain pH for both age groups, but the decrease in phosphocreatine level and increase in inorganic phosphate content were greater in 1-month-olds compared with newborns. During ischemia there was no difference between the magnitude of change in intracellular pH and levels of phosphocreatine and inorganic phosphate in hypercarbic 1-month-olds versus newborns. Under control conditions, i.e., normocarbia and normoxia, the free Mg²⁺ concentration was lower and the fraction of magnesium-free ATP was higher for newborns than 1-month-olds. However, there was no change in these variables for either age group during hypercarbia and early during ischemia. Thus, age-related differences in the relative decrease in agonal glycolytic rate during hypercarbia could not be explained by differences in intracellular pH, inorganic phosphate content, or free magnesium concentration. The [ADP]_free at control was higher in newborns compared with 1-month-olds, and there was no age-related difference in [AMP]_free. These variables did not change for newborns when exposed to hypercarbia, but for 1-month-olds [ADP]_free and [AMP]_free increased during hypercarbia relative to control values. High-energy phosphate utilization during ischemia for hypercarbic 1-month-olds was reduced by 74% compared with normocarbic 1-month-olds during ischemia, whereas the reduction in energy utilization (14%) was not significant for hypercarbic versus normocarbic newborns during ischemia. Because hypercarbia reduces the rate of ATP depletion during ischemia in 1-month-olds to a greater extent than in newborns, the increase in [ADP]_free and [AMP]_free will be slower in the former age group. It follows therefore that for 1-month-olds, the agonal glycolytic rate would not be accelerated by ADP and AMP to the same degree during hypercarbia plus ischemia compared with normocarbic plus ischemia, whereas for newborns hypercarbia has relatively little impact on agonal glycolytic rate.     

TRIETHYLTIN AND THE INCORPORATION OF [33P] PHOSPHATE INTO RAT BRAIN PHOSPHOLIPIDS

By the use of quantitative thin-layer chromatography, it has been shown that triethyltin sulphate 7.5 mglkg body wt. reduces the incorporation of ⁸⁸P₁ into rat brain phospholipids, especially lecithin, when the animals are kept at an environmental temperature of 20°. Triethyltin at this dose also reduces the body temperature by approximately 6°. When the body temperature of the triethyltin-treated animals is maintained at a normal level by placing them in an environmental temperature of 33°, no significant reduction in the incorporation of ³²P₁ into any of the phospholipids is observed.     

Traumatic Brain Injury in the Rat: Alterations in Brain Lactate and pH as Characterized by 1H and 31P Nuclear Magnetic Resonance

Application of both phosphorus (31P) and proton (1H) magnetic resonance spectroscopy (MRS) to the study of brain metabolism permits the noninvasive measurement of intracellular pH and brain lactate level. We have used water-suppression 1H MRS with novel lactate-editing techniques, together with 31P MRS, to characterize sequential changes in brain lactate level and pH in vivo over an 8-h period following fluid-percussion brain injury of graded severity in the rat. A transient fall in intracellular pH (from 7.09 +/- 0.07 at baseline to 6.88 +/- 0.09 at 40 min postinjury) occurred in animals subjected to moderate- (1.5-2.2 atm) and high- (2.5-3.3 atm) but not low-level (0.1-1.2 atm) injury; intracellular pH returned to baseline by 90 min postinjury. Transient elevations in brain lactate level were observed that temporally paralleled and were significantly correlated with the pH changes for all injury levels (r = 0.93, p less than 0.001). Postinjury alterations in intracellular brain pH and lactate level were identical in magnitude in animals subjected to either moderate or high-level injury. However, animals subjected to moderate injury had a moderate chronic neurological deficit that persisted up to 4 weeks postinjury, whereas animals subjected to a high level of injury showed greater histopathological damage and a more severe chronic neurological deficit. These data suggest that the extent of posttraumatic intracellular cerebral acidosis in our model of experimental head injury is not directly related to the severity of functional neurological deficit.     

Observation of Cerebral Metabolites in an Animal Model of Acute Liver Failure In Vivo: A 1H and 31P Nuclear Magnetic Resonance Study

Acute liver failure was induced in rats by a single intragastric dose of carbon tetrachloride. This causes hepatic centrilobular necrosis, as indicated by histological examinations, and produces a large increase in the activity of serum alanine aminotransferase. The plasma NH4+ level (mean +/- SEM) was 123 +/- 10 microM in the control group and 564 +/- 41 microM in animals with acute liver failure (each n = 5). 31P nuclear magnetic resonance (NMR) was used to monitor brain cortical high-energy phosphate compounds, Pi, and intracellular pH. 1H NMR spectroscopy was utilised to detect additional metabolites, including glutamate, glutamine, and lactate. The results show that the forebrain is capable of maintaining normal phosphorus energy metabolite ratios and intracellular pH despite the metabolic challenge by an elevated blood NH4+ level. There was a significant increase in the brain glutamine level and a concomitant decrease in the glutamate level during hyperammonaemia. The brain lactate level increased twofold in rats with acute liver failure. The results indicate that 1H NMR can be used to detect cerebral metabolic changes in this model of hyperammonaemia, and our observations are discussed in relation to compartmentation of NH4+ metabolism.