EARLY CHANGES IN RESPIRATION, AEROBIC GLYCOLYSIS AND CELLULAR NAD(P)H IN SLICES OF RAT CEREBRAL CORTEX EXPOSED TO ELEVATED CONCENTRATIONS OF POTASSIUM

Abstract— The initial effects of an elevated potassium concentration (30 m m ) on the energy metabolism of incubated slices of rat cerebral cortex have been examined using spectrophotometric and polarographic techniques. Respiratory responses to additions of potassium were found to be definitely limited in time. This response was followed by an increase in the rate of aerobic glycolysis. Slice NAD(P)H and cytochrome b paralleled this metabolic sequence by exhibiting an initial oxidation followed by a net increase in the steady-state levels of reduced intermediates, particularly in the case of NAD(P)H. Substitution of pyruvate (10 m m ) for glucose in the standard incubation media produced significant alterations in the respiratory responses to the addition of potassium. Although the period of increased oxygen consumption was again limited it was somewhat greater in magnitude and significantly prolonged in time relative to changes observed with glucose as substrate. Changes in steady-state levels of NAD(P)H were altered similarly and the net increase of NAD(P)H was not observed with pyruvate as substrate. We suggest that the metabolic responses of brain slices to increased potassium do not involve simultaneous activation of the respiratory and glycolytic pathways as has been previously assumed. Rather, a distinctly biphasic response is observed reminiscent of the Crabtree effect observed in other systems.     

Aggregate-formation by Clostridium butyricum

Summary Clostridium butyricum was grown in a glucose-limited chemostat culture at a dilution rate of 0.1 h^–1 at pH 6.0. With 0.9% w/v input glucose in the medium the cells were found to grow in suspension and glucose was fermented completely to acetate and butyrate. An increase in the input concentration of glucose resulted in increased concentrations of end-products, but not all extra glucose was consumed. It could be demonstrated that this was due to a lowering of the maximal growth rate by elevated levels of butyric acid. However, prolonged growth in the presence of high glucose concentrations led to an increase in biomass. This was caused by the selection of a variant that was less sensitive to butyrate. This variant was able to form aggregates in an anaerobic gas-lift reactor at high dilution rates. Inoculation of these aggregates in a conventional chemostat culture with high glucose input resulted in an aggregated culture that remained stable for at least 6 months, and in which all glucose was consumed. Whether the organisms grew in suspension or in aggregates was found to be determined by the concentration of butyrate. The isolation of aggregate-forming variants from chemostat cultures leads to a very simple and new type of immobilization technique.Offprint requests to: G. R. Zoutberg     

Inhibition of stomatal opening during uptake of carbohydrates by guard cells in isolated epidermal tissues

The uptake of glucose and other carbohydrates into the guard cells of Commelina communis L. was found to inhibit the opening of the stomata. The concentration of glucose necessary to achieve about 50% inhibition was of the same order of magnitude as the potassium concentration required for opening; the uptake systems for potassium and glucose appear to be competitive and to exhibit the same degree of affinity. It is suggested that the uptake of glucose occurs via a proton cotransport, which, depolarizing the membrane potential, slows down the electrogenic import of potassium ions. The process of stomatal closure, in contrast, appears not to be affected by carbohydrate uptake. In guard cells of Tulipa gesneriana L. and Vicia faba L., which do not possess subsidiary cells, import of glucose or other carbohydrates did not interfere with the regulation of stomatal movements.     

Effect of glucose on pH and potassium ion balance in suspensions of ascites tumor cells subjected to x-irradiation

Glucose (2.14 microM) was introduced into nonirradiated and irradiated (6 and 60 Gy) ascites tumor cell suspension. Irradiation was shown to suppress the consumption of potassium by cells having no influence on the rate of protoxidation of the suspension, induced by glucose, and on the passive release of potassium from cells.     

The functional significance of glucose dehydrogenase in Klebsiella aerogenes

In order to assess the functional significance of the quinoprotein glucose dehydrogenase recently found to be present in K⁺-limited Klebsiella aerogenes, a broad study was made of the influence of specific environmental conditions on the cellular content of this enzyme. Whereas high activities were manifest in cells from glucose containing chemostat cultures that were either potassium- or phosphate-limited, only low activities were apparent in cells from similar cultures that were either glucose-, sulphate- or ammonia-limited. With these latter two cultures, a marked increase in glucose dehydrogenase activity was observed when 2,4-dinitrophenol (1 mM end concentration) was added to the growth medium. These results suggested that the synthesis of glucose dehydrogenase is not regulated by the level of glucose in the growth medium, but possibly by conditions that imposed an energetic stress upon the cells. This conclusion was further supported by a subsequent finding that K⁺-limited cells that were growing on glycerol also synthesized substantial amounts of glucose dehydrogenase.The enzyme was found to be membrane associated, and preliminary evidence has been obtained that it is located on the periplasmic side of the cytoplasmic membrane and functionally linked to the respiratory chain. This structural and functional orientation is consistent with glucose dehydrogenase serving as a low impedance energy generating system.Abbreviations D dilution rate - DNP 2,4-dinitrophenol - PQQ 2,7,9-tricarboxy-1H-pyrrolo(2,3-f)quinoline-4,5-dione - PTS phosphoenolpyruvate: glucose phosphotransferase - WB Wurster's Blue     

Acidification power: Indicator of metabolic activity and autolytic changes inSaccharomyces cerevisiae

Acidification power, defined as the sum of the spontaneous pH change determined after suspending yeast cells in water and the substrate-induced pH change after addition of glucose to the resulting suspension, reflects the level of cellular energy sources. Its use as an indicator of metabolic state of the cells was tested during a 120-h aerobic starvation. Its changes coincided with changes in cell viability, initial rate of endogenous oxygen consumption rate, cell ATP, extra- and intracellular buffering capacity, and the ability of cell-free extract to produce acidity by glucose fermentation. It was used as a sensitive marker of metabolic changes occurring during starvation, on treatment with glycolytic and respiratory inhibitors, and at elevated temperature.     

Response of the respiratory rate of Daphnia magna to changing food conditions

Summary The respiratory rate of the cladoceran Daphnia magna was measured at varying concentrations of a green alga in a flow-through respirometer. Daphnids were either preconditioned to the respective food concentration or the food concentration was suddenly changed during the experiment and the response of the respiratory rate monitored. Previously starved animals were provided with food or prefed animals were deprived of food. The respiratory rate increased considerably with increasing concentrations of algae until a maximum rate was reached at a critical algal concentration corresponding to the incipient limiting level for feeding. The response of the respiratory rate to changing food conditions was fast, lagging only a few minutes behind the change in food. The respiratory rate of starved daphnids increased quickly when they received food, even at low concentrations. Prefed daphnids responded to the replacement of the food suspension by filtered water with reductions in their respiratory rates. A linear relationship between the assimilation rate and the respiration rate was found, indicating that the more than twofold range of the respiratory rate was due to specific dynamic action. The SDA coefficients of 0.15–0.24 found for Daphnia are consistent with values for marine zooplankton.     

Involvement of ATP-sensitive potassium (K(ATP)) channels in the loss of beta-cell function induced by human islet amyloid polypeptide

Islet amyloid polypeptide (IAPP) is a major component of amyloid deposition in pancreatic islets of patients with type 2 diabetes. It is known that IAPP can inhibit glucose-stimulated insulin secretion; however, the mechanisms of action have not yet been established. In the present work, using a rat pancreatic beta-cell line, INS1E, we have created an in vitro model that stably expressed human IAPP gene (hIAPP cells). These cells showed intracellular oligomers and a strong alteration of glucose-stimulated insulin and IAPP secretion. Taking advantage of this model, we investigated the mechanism by which IAPP altered beta-cell secretory response and contributed to the development of type 2 diabetes. We have measured the intracellular Ca(2+) mobilization in response to different secretagogues as well as mitochondrial metabolism. The study of calcium signals in hIAPP cells demonstrated an absence of response to glucose and also to tolbutamide, indicating a defect in ATP-sensitive potassium (K(ATP)) channels. Interestingly, hIAPP showed a greater maximal respiratory capacity than control cells. These data were confirmed by an increased mitochondrial membrane potential in hIAPP cells under glucose stimulation, leading to an elevated reactive oxygen species level as compared with control cells. We concluded that the hIAPP overexpression inhibits insulin and IAPP secretion in response to glucose affecting the activity of K(ATP) channels and that the increased mitochondrial metabolism is a compensatory response to counteract the secretory defect of beta-cells.     

Calorimetric measurement of free energy utilization by Nitrosomonas and Nitrobacter

Summary Calorimetric estimates of the utilization efficiency of the free-energy derived from substrate oxidation by cell suspensions of two nitrifying bacteria, Nitrosomonas and Nitrobacter, provided two ranges of values: 11 to 27% and 15 to 51%, respectively. About 15 to 30% of the utilized free-energy is used for driving endergonic reactions other than CO₂ fixation, probably the synthesis of polyphosphates.The molar heat of substrate oxidation does not seem to be influenced by the age of cells harvested during growth or by the length of the incubation period during which cells have been kept in a buffer suspension in a starved condition. The loss of respiratory activity measured either by oxygen uptake or heat evolution in the presence of the specific substrate, nitrite or ammonium, decreases according to kinetics which are influenced by the aerobiosis of the suspension. The viability of the starved cells decreases in a way which is similar to that of the respiratory activity. It seemed impossible to obtain cells which had lost their viability but kept the ability to oxidize their substrate.Two inhibitors of the respiratory chain, quinacrine and cyanide, are without effect on the molar heat of substrate oxidation and consequently on the free-energy utilization efficiency. 2.4 dinitrophenol did decrease the rate of heat evolution during substrate oxidation at concentrations at which the rate of oxygen uptake was not depressed, with the consequences that free-energy efficiency was apparently increased.     

Co-ordinated induction of mRNAs for extracellular invertase and a glucose transporter in Chenopodium rubrum by cytokinins

Extracellular or cell wall invertase is regarded as crucial to supply sink tissues with carbohydrates via an apoplastic pathway. A cell wall invertase from Chenopodium rubrum was purified to homogeneity and the corresponding cDNA encoding CIN1 was identified via peptide sequences. The CIN1 mRNA was found to be highly induced by physiological concentrations of both adenine- and phenylurea-derived cytokinins in suspension culture cells. This was paralleled both by a higher steady-state protein level and a higher enzyme activity of the extracellular invertase. The cytokinin-inducible accumulation of CIN1 mRNA in tissues of C. rubrum plants supports the physiological significance of this regulatory mechanism. In contrast to the extracellular sucrose cleaving enzyme, the mRNA levels of the two putative intracellular invertases CIN2 and CIN3 and of sucrose synthase were not elevated. In addition, it has been found that the accumulation of mRNA for one out of three hexose transporters present in the suspension culture cells is induced co-ordinately with the mRNA for extracellular invertase by cytokinins. It has been shown that this regulatory mechanism results in higher uptake rates both for sucrose, via the hexose monomers, and for glucose. The increased level of both extracellular invertase and hexose transporters and the resulting higher carbohydrate supply are discussed with respect to the control of carbohydrate partitioning by plant hormones and the molecular basis for known physiological cytokinin responses such as the stimulation of cell division.     

Methylmalonate-semialdehyde dehydrogenase is induced in auxin-stimulated and zinc-stimulated root formation in rice

Proteins induced in rice by auxin and zinc were determined by proteome analysis. Cultured suspension cells of rice were treated with 2,4-dichlorophenoxyacetic acid and ZnSO₄ and then proteins were separated by two-dimensional polyacrylamide gel electrophoresis; seven proteins were found to be induced by auxin and zinc. Of these seven, methylmalonate-semialdehyde dehydrogenase (MMSDH) was elevated by treatment with auxin alone. MMSDH was detected in cultured suspension cells, root and leaf sheath, but not in leaf blades. MMSDH responded to auxin and gibberellin, but did not respond to brassinolide and cytokinin. Furthermore, the amount of MMSDH in slr1, a constitutive gibberellin response mutant, was 2-fold that of wild type. MMSDH mRNA and protein were stimulated in root formation induced by auxin and/or zinc over a 4-week period. These results suggest that MMSDH may be necessary for root formation in rice induced by auxin and/or zinc.Abbreviations BA 6-Benzylaminopurine - BL Brassinolide - CBB Coomassie Brilliant Blue - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA Gibberellic acid - IAA Indole-3-acetic acid - MALDI-TOF MS Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry - MMSDH Methylmalonate-semialdehyde dehydrogenase - 2D-PAGE Two-dimensional polyacrylamide gel electrophoresis - PVDF Polyvinylidene difluoride     

Effects of pH on the growth rate, motility and photosynthesis inEuglena gracilis

The influence of pH 3–10 on the growth, motility and photosynthesis inEuglena gracilis was demonstrated during a 7-d cultivation. The cells did not survive at pH<4 and >8, highest growth rate being detected at pH 7. Motility followed a similar patterns as growth rate. Photosynthetic response curves were shown to be of the same type over the whole pH range. High respiration was characteristic for cells grown at pH 5 and 6, the lowest one at 7. At high and also at low pH more active respiration was found which can be considered as a protective response on proton stress. Respiration was not completely inhibited with potassium cyanide. Photosynthesis was the most effective at pH 6; lower and higher pH decreased photosynthetic efficiency. pH affected more the growth rate than the photosynthesis.     

The Proteomic Response of Bacillus pumilus Cells to Glucose Starvation

Since starvation for carbon sources is a common condition for bacteria in nature and it can also occur in industrial fermentation processes due to mixing zones, knowledge about the response of cells to carbon starvation is beneficial. The preferred carbon source for bacilli is glucose. The response of Bacillus pumilus cells to glucose starvation using metabolic labeling and quantitative proteomics was analyzed. Glucose starvation led to an extensive reprogramming of the protein expression pattern in B. pumilus. The amounts of proteins of the central carbon metabolic pathways (glycolysis and TCC) remained stable in starving cells. Proteins for gluconeogenesis were found in higher amounts during starvation. Furthermore, many proteins involved in acquisition and usage of alternative carbon sources were present in elevated amounts in starving cells. Enzymes for fatty acid degradation and proteases and peptidases were also found in higher abundance when cells entered stationary phase. Among the proteins found in lower amounts were many enzymes involved in amino acid and nucleotide synthesis and several NRPS and PKS proteins.     

Effects of potassium sorbate on growth patterns, morphology, and heat resistance of Zygosaccharomyces rouxii at reduced water activity.

A study was made of the effects of potassium sorbate on growth, morphology, and heat sensitivity of an osmotolerant yeast, Zygosaccharomyces rouxii, grown in media (water activity (aw) 0.93) supplemented with glucose and sucrose. Growth patterns of Z. rouxii in YM broth supplemented with glucose (YMBG) and sucrose (YMBS) were similar, although increased potassium sorbate concentration in both media resulted in decreased growth rates. Growth in YMBS containing potassium sorbate was not as prolific as that in YMBG containing potassium sorbate. Inhibition of growth was indicated by decreased absorbance (at 600 nm) of cells grown in YMBS and in YMBG and YMBS supplemented with potassium sorbate at 600 or 1000 micrograms/mL. Slight decreases in cell size and alteration of cellular morphology were associated with increased potassium sorbate concentration. Plasmolysis increased as potassium sorbate concentration was elevated in YMBS but not in YMBG. Tolerance of Z. rouxii to potassium sorbate was enhanced by previous adaptation of cells in media with elevated potassium sorbate concentrations. Heat resistance of cells unadapted to potassium sorbate showed little or no increase regardless of culture age, but increased substantially in cells grown in media containing potassium sorbate, particularly YMBS.     

Glucose,Lactate and Glutamine but not Glutamate Support Depolarization-Induced Increased Respiration in Isolated Nerve Terminals

Synaptosomes prepared from various aged and gene modified experimental animals constitute a valuable model system to study pre-synaptic mechanisms. Synaptosomes were isolated from whole brain and the XFe96 extracellular flux analyzer (Seahorse Bioscience) was used to study mitochondrial respiration and glycolytic rate in presence of different substrates. Mitochondrial function was tested by sequentially exposure of the synaptosomes to the ATP synthase inhibitor, oligomycin, the uncoupler FCCP (carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone) and the electron transport chain inhibitors rotenone and antimycin A. The synaptosomes exhibited intense respiratory activity using glucose as substrate. The FCCP-dependent respiration was significantly higher with 10 mM glucose compared to 1 mM glucose. Synaptosomes also readily used pyruvate as substrate, which elevated basal respiration, activity-dependent respiration induced by veratridine and the respiratory response to uncoupling compared to that obtained with glucose as substrate. Also lactate was used as substrate by synaptosomes but in contrast to pyruvate, mitochondrial lactate mediated respiration was comparable to respiration using glucose as substrate. Synaptosomal respiration using glutamate and glutamine as substrates was significantly higher compared to basal respiration, whereas oligomycin-dependent and FCCP-induced respiration was lower compared to the responses obtained in the presence of glucose as substrate. We provide evidence that synaptosomes are able to use besides glucose and pyruvate also the substrates lactate, glutamate and glutamine to support their basal respiration. Veratridine was found to increase respiration supported by glucose, pyruvate, lactate and glutamine and FCCP was found to increase respiration supported by glucose, pyruvate and lactate. This was not the case when glutamate was the only energy substrate.     

The course of respiration during the life cycle of Chlorella cells

Endogenous and glucose respiration were studied during the life history of Chlorella pyrenoidosa. A generalized picture of the course of respiration during the life cycle is suggested. At the liberation of daughter cells from the wall of the mother cell, or soon after, the respiration rate reaches its lowest level. If the daughter cells are placed in light the respiration rate rapidly increases with time, soon reaches a maximum, and then declines slowly. Two factors are important in the initial increase—the early developmental stage of the cells and the influence of light. In autotrophically developing algae the parts played by developmental processes and by light have not been separated. Direct activation of respiratory enzymes by light, in addition to the level of respiratory substrate, cannot be excluded. The decline of respiration rate over most of the cell history seems to have no connection with light and is probably bound to the developmental processes per se. Darkening the suspension interrupts growth and induces liberation of daughter cells, with concomitant faster decrease in respiration rate. The rate of respiration of small daughter cells decreases in darkness only slowly with time. Illumination seems necessary to bring these cells back to a high level of respiratory activity.     

Glucose and lactic acid trends in suspension cultures of two established mammalian cell strains in chemically defined media

In replicated 30 to 40-ml suspension cultures of rapidly proliferating monkey kidney cells of a comparatively fragile strain, the rates of glucose utilization and lactic acid accumulation averaged about 400 micrograms and 110 micrograms per 10⁶ cells per day respectively, with average molar La/Gl ratios of 0.48. These two rates of glucose utilization and lactic acid accumulation were about 4 × and 10 × as high as the corresponding rates in comparable cultures of the hardier strain 2071-L mouse fibroblasts under the same conditions, with average molar La/Gl ratios of 0.16. In comparable but nonproliferating suspension cultures of the same strain of monkey kidney cells, during about 3 weeks the rates were extremely high, with about 710 micrograms glucose utilized and 445 micrograms lactic acid accumulated per 10⁶ cells per day, with average molar La/Gl ratios of 1.37. The rates of glucose uptake and lactic acid accumulation were higher in the nonproliferating cultures aerated with 5% CO₂ in air than in those aerated with 10% CO₂ in air. This difference was associated with pH, which was higher in the former group. It was concluded that with this fragile strain of monkey Kidney cells(1) in nonproliferating cultures the cells were metabolizing actively but with a marked tendency to higher La/Gl ratios, (2) in the proliferating cultures the high rates of glucose utilization and lactic acid accumulation were definitely not directly correlated with the rate of growth, and (3) in none of the cultures was the amount of glucose remaining in the fluid at fluid changes so low as to have been a limiting factor. Information in the literature concering glucose utilization and lactic acid production by cells vitro is voluminous and in some respects contradictory. In the present study the rates were unexpectedly high for the monkey kidney cells, particularly those in the otherwise apparently inactive nonproliferating cultures. The data seem to be unique, in that an established strain of cells in chemically defined medium in suspension cultures has been characterized for these metabolic parameters in both proliferating cultures and in equivalent nonproliferating cultures under directly comparable conditions. The concept was developed that since these monkey kidney cells are obviously more fragile than the other cells examined, the complex physical stresses imposed upon these cells in agitated cultures can be modified and lessened in order to permit growth. Lessening of such mechanical stress waa brought about in several ways, of which only the smaller flask size seemed to be at least partly effective. Increasing either the concentration or the viscosity type of Methocel waa not effective.     

14C-glucose utilization byRhizobium lupini bacteroids

Summary The ability of bacteroids, isolated fromLupinus luteus L. nodules at the stage of active nitrogen fixation, to assimilate (1-¹⁴C)-glucose and (2-¹⁴C)-glucose was being studied. The label is incorporated into all the Krebs cycle metabolites, amino acids and sugars after 5 min of glucose insertion into cell suspension. High activity of glucose phosphorylation was found in bacteroidsin vitro, the reaction rate being the highest at a glucose concentration of over 100 mM.In lupine nodules sugars can be essential carbon substrate delivered to the bacteroids from host-plant cells. This point of view is discussed.     

Differential sugar uptake by cell suspension cultures of <Emphasis Type="Italic">Rudgea jasminoides</Emphasis>, a tropical woody Rubiaceae

The primary utilization of carbohydrates by cell suspension cultures of Rudgea jasminoides, a native woody Rubiaceae from tropical forests, was investigated. Sucrose, glucose + fructose, glucose, or fructose were supplied as carbon sources. The growth curves of R. jasminoides cultured in glucose + fructose, glucose, or fructose showed similar patterns to that observed when sucrose was supplied to the cells, except that an increase in dry mass was observed at the beginning of the stationary growth phase in the media containing only one monosaccharide. The increase in hexose levels in the media during the early stages of the cultures indicated extracellular hydrolysis of sucrose, which was further supported by the increase in the activity of acid invertase bound to the cell wall. Glucose was preferentially taken up, whereas uptake of fructose was delayed until glucose was nearly depleted from the medium. Measurements of intracellular sucrose content and cytoplasmatic and vacuolar invertases indicate that the enzymatic activity seems to be correlated with a decrease in the hexose flux into the cells of R. jasminoides. Our results indicate that the behavior of cell suspension cultures of R. jasminoides regarding sugar utilization seems to be similar to other dicotyledonous undifferentiated cell suspension cultures.