Chloride-Dependent Uncoupling of Oxidative Phosphorylation by Triethyllead and Triethyltin Increases Cytosolic Free Calcium in Guinea Pig Cerebral Cortical Synaptosomes

Metabolically competent isolated cerebral cortical nerve terminals were used to determine the effects of triethyllead (TEL) and triethyltin (TET) on cytosolic free calcium ([Ca2+]c), on plasma and mitochondrial membrane potentials, and on oxidative metabolism. In the presence of physiological concentrations of extracellular ions, 20 microM TEL and 20 microM TET increase [Ca2+]c from 185 nM to 390 and 340 nM, respectively. A simultaneous depolarization of plasma membrane potential (delta psi p) by only 3-4 mV occurs, a drop which is insufficient to open the voltage-sensitive Ca2+ channels. In contrast, an instant and substantial depolarization of mitochondrial membrane potential (delta psi m) upon addition of TEL and TET is evident, as monitored with safranine O fluorescence. At the same concentration, TEL and TET stimulate basal respiration of synaptosomes by 45%, induce oxidation of endogenous NAD(P)H, and reduce the terminal ATP/ADP ratio by 45%. Thus, TEL and TET inhibit ATP production of intrasynaptosomal mitochondria by a mechanism consistent with uncoupling of oxidative phosphorylation. This bioenergetic effect by TEL and TET can be prevented by omitting external chloride, and a concomitant reduction of the increase in [Ca2+]c by about 60% is observed. Uncoupling of mitochondrial ATP synthesis from oxidation by TEL and TET, [corrected] a process that is dependent on external chloride, is the main mechanism by which they [corrected] increase [Ca2+]c.     

Multigradient method for optimization of slow biotechnological processes

A new method (named a "jumping spider") is introduced for the optimization of slow biotechnological processes. The more traditional sequential experimentation (i.e., gradient search, simplex, etc.) is not well suited for slow dynamic processes, e.g., plant cell culture and differentiation. Therefore, a more simultaneous approach is proposed. A large number of initial experiments are performed, on the basis of which several of the initial experiments are selected as starting points. A search is then performed simultaneously from several gradient directions and the optimum is estimated by a quadratic approximation. In simulations, the spider generally climbs up the slopes quickly and the final estimator yields good maximum point estimates even on a complex topography. The spider may even approach more than one local maximum point simultaneously. As a model application, the average xylitol conversion rate of Candida guilliermondii was optimized in relation to cultivation volume (oxygen availability) and the concentration of nitrogen and phosphorus in the medium. A threefold increase in xylitol production was obtained with three experimental steps. (c) 1993 John Wiley & Sons, Inc.     

Regulation of Intracellular pH in Guinea Pig Cerebral Cortex Ex Vivo Studied by 31P and 1H Nuclear Magnetic Resonance Spectroscopy: Role of Extracellular Bicarbonate and Chloride

Abstract: The role of transmembrane processes that are dependent on external anions in the regulation of cerebral intracellular pH (pH_i), high-energy metabolites, and lactate was investigated using ³¹P and ¹H NMR spectroscopy in an ex vivo brain slice preparation. During oxygenated superfusion, removal of external HCO₃^?/CO₂ in the presence of Na⁺ led to a sustained split of the inorganic phosphate (P_i) peak so that the pH_i indicated by one part of the peak was 0.38 pH units more alkaline and by the other part 0.10 pH units more acidic at 5 min than in the presence of HCO₃^?. The pH in the compartment with a higher pH_i value returned to 7.29 ± 0.04 by 10.5 min of superfusion in a HCO₃^?-free medium, whereas the pH_i in an acidic compartment was reduced to 7.02. In the presence of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid or the absence of external Cl^?, removal of HCO₃^? caused alkalinization without split of the P_i peak. Both treatments reduced the rate of pH_i normalization following alkalinization. Simultaneous omission of external HCO₃^? and Na⁺ did not inhibit alkalinization of the pH_i following CO₂ exit. All these data show that the acid loading mechanism at neutral pH_i is mediated by an Na⁺-independent anion transport. During severe hypoxia, pH_i dropped from 7.29 ± 0.05 to 6.13 ± 0.16 and from 7.33 ± 0.03 to 6.67 ± 0.05 in the absence and presence of HCO₃^?, respectively, in Na⁺-containing medium. Lactate accumulated to 18.7 ± 2.8 and 19.6 ± 1.5 mmol/kg under the respective conditions. In the HCO₃^?-free medium supplemented with 1 mM amiloride, the pH_i fell only to 6.94 ± 0.08 despite the lactate concentration of 18.9 ± 2.4 mmol/kg. Acidification caused by hypoxia was also small in the slice preparations superfused in the absence of both HCO₃^? and Cl^?, as the pH_i was 7.01 ± 0.12 at a lactate concentration of 24.5 ± 2.4 mmol/kg. These data indicate that apart from anaerobic glucose metabolism, separate acidifying mechanisms are functioning during hypoxia under these conditions. Recovery of phosphocreatine levels following reoxygenation was >75% relative to the prehypoxic level in the slice preparations superfused in the absence of HCO₃^? but <47% in those preparations superfused without HCO₃^? and Cl^?. This indicates that either neutral pH_i or absence of Cl^? during hypoxia was deleterious to the energy metabolism. The present data indicate that Cl^?/HCO₃^? exchange mechanisms have distinct roles in cerebral H⁺ homeostasis depending on the level of pH_i and energy state.     

Studies on the optimization of growth and indole alkaloid production by hairy root cultures of Catharanthus roseus

Catharanthus roseus hairy root cultures, genetically transformed with Agrobacterium rhizogenes, produce a wide variety of indole alkaloids. The effect of sucrose, phosphate, nitrate, and ammonia concentrations on growth and indole alkaloid production of C. roseus hairy root cultures were studied by using statistical experimental designs and linear regression analysis. Contradictory effects of these nutrients on growth and indole alkaloid production were found. The maximal growth was obtained by having 77. 8 mg NaH(2)PO(4) . H(2)O/L and 1. 311 g KNO(3)/L in the medium, whereas the specific production of alkaloids was highest at the lowest levels of all the nutrients studied. The maximal dry weight was obtained with high values of sucrose and ammonia, but clear optimum concentrations could not be found. When having enough nutrients to support reasonable growth, it appeared difficult to affect the specific alkaloid production rates considerably. The growth (dry wt.) with the optimized nutrient concentrations in the medium was more than 50% better than in the control medium with about the same alkaloid production.     

Nuclear Magnetic Resonance Spectroscopy Study on Energy Metabolism, Intracellular pH, and Free Mg2+ Concentration in the Brain of Transgenic Mice Overexpressing Human Ornithine Decarboxylase Gene

We have generated a transgenic mouse line strikingly overexpressing the human ornithine decarboxylase (ODC) gene in their brain. Brain ODC activity was increased in the transgenic animals by a factor of 70 in comparison with their nontransgenic littermates. The content of brain putrescine, the product of ODC, was greater than 60 mumol/g of tissue in the transgenic mice, whereas in the normal animals it was below the level that could be detected by an HPLC method. The concentrations of the higher polyamines (spermidine and spermine) were not significantly different from control values. 31P nuclear magnetic resonance (31P NMR) spectroscopy analyses revealed a significantly reduced (40%) free Mg2+ concentration as calculated from the chemical shift differences of the nucleoside triphosphate alpha and beta peaks in the brains of the transgenic animals. The lower free Mg2+ concentration in the brains of ODC transgenic mice was not a consequence of altered intracellular pH or changes in cellular high-energy metabolites. 1H NMR showed no differences in brain choline/N-acetylaspartate and total creatine/N-acetylaspartate ratios between the two animal groups. These ODC transgenic animals may serve as models in vivo for studies on cerebral postischemic events and on epilepsy, as polyamines are supposed to be involved in these processes.     

Indole alkaloid production by hairy root cultures ofCatharanthus roseus: Growth kinetics and fermentation

Summary Growth kinetics and indole alkaloid production ofCatharanthus roseus hairy root cultures were studied in shake flasks and in a small scale fermenter. A logistic growth model commonly used for microbes described well the growth of hairy roots. Of the several parameters analyzed during the cultivation of hairy roots, a linear relationship between sucrose consumption and dry weight increase was obtained. This suggests the validity of sugar analysis as a means in monitoring the growth of hairy roots in fermenters.     

Interrelationships (Between Glucose Metabolism, Energy State, and the Cytosolic Free Calcium Concentration in Cortical Synjaptosomes from the Guinea Pig

The stoichiometries of glycolysis and pyruvate oxidation were determined in cortical synaptosomes under varying rates of ATP consumption. Glycolysis was measured by using D-3-[3H]glucose as a marker and pyruvate oxidation by using D-3,4-[14C]glucose, which has to be metabolized to 1-[14C]pyruvate before being decarboxylated by the pyruvate dehydrogenase complex of intrasynaptosomal mitochondria. Cytosolic free Ca2+ concentration [( Ca2+]c) was determined in parallel and was manipulated by using EGTA in the incubation. The results show that in nonstimulated synaptosomes glycolysis and pyruvate oxidation are tightly coupled and stoichiometric. In the absence of Ca2+, when [Ca2+]c drops from 260 nM to 40 nM, glucose utilization increases, following the increase in energy demand, which has been shown to be due to elevated Na+ cycling. KCl depolarization, veratridine, and a mitochondrial uncoupler, carbonyl cyanide m-chlorophenylhydrazone, all stimulate glycolysis and pyruvate oxidation stoichiometrically, independently of the presence of external Ca2+. A rise in [Ca2+]c, therefore, is not required to regulate mitochondrial pyruvate metabolism. It is concluded that synaptosomes exhibit a high degree of respiratory control, that they rely on glucose oxidation for their energetics, and that stimulation of energy production can be achieved independently of changes in [Ca2+]c.     

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.     

Mitochondrial membrane potential, transmembrane difference in the NAD redox potential and the equilibrium of the glutamate-aspartate translocase in the isolated perfused rat heart

The distribution of glutamate and aspartate and the mitochondrial membrane potential (Δψ) were studied in isolated rat heart mitochondria and in the intact perfused rat heart. The diffusion potential imposed by the glutamate-aspartate exchange through mediation of the electrogenic glutamate-aspartate translocator attained a value close to the mitochondrial Δψ measured from the distribution of triphenylmethylphosphonium ion (TPMP⁺) both in isolated mitochondria and in intact myocardium. Distributions of the Δψ probe and metabolites were determined by subcellular fractionation of the heart muscle in a non-aqueous medium. The results indicate that the glutamate-aspartate translocator is in near equilibrium in the myocardium. The diffusion potential of the glutamate-aspartate exchange, and the mitochondrial/cytosolic difference in the redox potentials of the free NAD⁺/NADH pools are equal allowing for experimental error. These data obtained from intact tissue can therefore be interpreted as supporting the notion of the transmembrane uphill transport of reducing equivalent from the cytosolic free NAD⁺/NADH pool being driven by the malate-aspartate cycle energized by the mitochondrial Δψ.     

Glucose Metabolism in the Developing Cerebral Cortex as Detected by 1H{13C} Nuclear Magnetic Resonance Spectroscopy Ex Vivo

Abstract: Metabolism of [1-¹³C]glucose was monitored in superfused cerebral cortex slice preparations from 1-, 2-, and 5-week-old rats using ¹H-observed/¹³C-edited (¹H{¹³C}) NMR spectroscopy. The rate of label incorporation into glutamate C-4 did not differ among the three age groups: 0.52–0.67% of total ¹H NMR-detected glutamate/min. This was rather unexpected, as oxygen uptake proceeded at 1.1 ± 0.1, 1.9 ± 0.1, and 2.0 ± 0.1 µmol/min/g wet weight in brain slices prepared from 1-, 2-, and 5-week-old animals, respectively. Steady-state glutamate C-4 fractional enrichments in the slice preparations were ∼23% in all age groups. In the acid extracts of slices glutamate C-4 enrichments were smaller, however, in 1- and 2-week-old (17.8 ± 1.7 and 16.8 ± 0.8%, respectively) than in 5-week-old rats (22.7 ± 0.7%) after 75 min of incubation with 5 m M [1-¹³C]glucose. We add a new assignment to the ¹H{¹³C} NMR spectroscopy, as acetate C-2 was detected in slice preparations from 5-week-old animals. In the acid extracts of slice preparations acetate C-2 was labeled by ∼30% in 5-week-old rats but by 15% in both 1- and 2-week-old animals, showing that the turnover rate was increased in 5-week-old animals. In the extracts 3–4% of the C-6 of N -acetyl-aspartate (NAA; CH₃ of the acetyl group) contained label as determined by both NMR and mass spectrometry, which indicated that there was no significant labeling to other carbons in NAA. NAA accumulated label from [1-¹³C]glucose but not from [2-¹³C]acetate, and the rate of label incorporation increased by threefold on cerebral maturation.