Maria Erecińska

Regulation of cellular energy metabolism.

One of the characteristic features of living matter is its ability to generate metabolic energy which is required for growth and the maintenance of multiple cellular functions. The major pathways whereby eukaryotic organisms produce energy are glycolysis and mitochondrial oxidative phosphorylation; the latter process, which involves complete combustion of glucose to carbon dioxide and water, yields approximately 17 times as much useful energy as does anaerobic production of lactic acid (glycolysis). Owing to its high energy yield, mitochondrial oxidative phosphorylation is responsible for supplying over 95% of the total ATP requirement in eukaryotic cells. All living organisms are steady-state systems in which the rate of energy production (ATP synthesis) equals the rate of energy utilization (except in rapid transient conditions in which this may not necessarily be true). This means that precise dynamic balance is maintained between the reactions which produce ATP and those which utilize it in which the concentration of ATP in the cell remains at an essentially constant level. Any decline in the usage of ATP causes an immediate decrease in the rate of its synthesis and vice versa. The range of activities over which mitochondrial oxidative phosphorylation operates in vivo is best illustrated by the observation that in man the rate of ATP synthesis varies from 0.4 g ATP/min/kg body weight at rest to 9.0 g ATP/min/kg body weight during strenuous exercise. This high flux through the ATP synthesizing pathway and its large dynamic range ensures that oxidative phosphorylation plays a paramount role in cellular homeostasis. In eukaryotic cells, the enzymes of oxidative phosphorylation are intimately associated with or form an integral part of the mitochondrial inner membrane (for review see Lehninger, 1966; Wainio, 1970). The site of ATP synthesis (ATP synthase) is located on the internal surface of the inner membrane and communicates with the matrix space. It is well known that the mitochondrial matrix houses but a few reactions which require ATP whereas the cytosol is the main site of ATP utilization. Mechanisms must, therefore, be present which allow the mitochondrial matrix to sense the need of the cytosol for an appropriate rate of ATP generation. Moreover, ATP has to be supplied under conditions for which its hydrolysis is sufficiently exergonic to do the required metabolic work (i.e., its hydrolysis has to provide a negative free energy change of sufficient magnitude). Since the maximum work that ATP hydrolysis can do is dependent on the [ATP]/[ADP] [Pi], variations in the cellular concentrations of ATP, ADP, and inorganic phosphate provide a very sensitive means through which homeostatic mechanisms can regulate energy production. The mitochondrial respiratory chain is a multienzyme complex which accepts reducing equivalents from NADH and FADH z and transfers them in a sequence of oxidation-reduction reactions to molecular oxygen with the formation of water and concomitant synthesis of ATP. In cells in vivo, reducing equivalents are provided through dehydrogenation of various substrates which are generated by the metabolic feeder pathways such as the tricarboxylic acid cycle or fatty acid oxidation system. Since mitochondrial dehydrogenases utilize either NAD or FAD as reducible coenzymes the amounts of NADH and FADH2 formed by the particular tissue depend on relative activities of these pathways, but in most cells NADH is the

The oxygen dependence of cellular energy metabolism.

Abstract Suspensions of cultured C 1300 neuroblastoma cells, sarcoma 180 ascites tumor cells, and Tetrahymena pyriformis cells were used to study the oxygen dependence of cellular energy metabolism. Cellular respiration was found to be almost independent of oxygen tension to values of less than 20 μ m with an apparent Km for oxygen of less than 1 μ m . In contrast, the reduction of mitochondrial cytochrome c was found to be dependent on oxygen tension at all values from 240 μ m downward. Oxygen dependence was also observed in terms of cellular energy metabolism expressed as adenosine triphosphate and adenosine diphosphate concentrations. These data provide direct evidence that in intact cells mitochondrial oxidative phosphorylation is oxygen dependent throughout the physiological range of oxygen tension (air saturation and below). The respiratory rate is maintained constant when the oxygen tension is lowered by decreasing values of the cytosolic [ATP] [ADP] [P i ] and intramitochondrial [NAD] + ] [NADH] because these regulatory parameters adjust to maintain a constant rate of ATP synthesis. The lack of oxygen dependence in the respiratory rate means that the rate of cellular ATP utilization is essentially oxygen independent until the mitochondria can no longer synthesize ATP at the required rate and [ATP] [ADP] [P i ] .

Effects of Hypothermia on Energy Metabolism in Mammalian Central Nervous System

This review analyzes, in some depth, results of studies on the effect of lowered temperatures on cerebral energy metabolism in animals under normal conditions and in some selected pathologic situations. In sedated and paralyzed mammals, acute uncomplicated 0.5- to 3-h hypothermia decreases the global cerebral metabolic rate for glucose (CMRglc) and oxygen (CMRO2) but maintains a slightly better energy level, which indicates that ATP breakdown is reduced more than its synthesis. Intracellular alkalinization stimulates glycolysis and independently enhances energy generation. Lowering of temperature during hypoxia–ischemia slows the rate of glucose, phosphocreatine, and ATP breakdown and lactate and inorganic phosphate formation, and improves recovery of energetic parameters during reperfusion. Mild hypothermia of 12 to 24-h duration after normothermic hypoxic–ischemic insults seems to prevent or ameliorate secondary failures in energy parameters. The authors conclude that lowered head temperatures help to protect and maintain normal CNS function by preserving brain ATP supply and level. Hypothermia may thus prove a promising avenue in the treatment of stroke and trauma and, in particular, of perinatal brain injury.

Interactions of bioactive glasses with osteoblasts in vitro : effects of 45S5 Bioglass®, and 58S and 77S bioactive glasses on metabolism, intracellular ion concentrations and cell viability

In a cell culture model of murine osteoblasts three particulate bioactive glasses were evaluated and compared to glass (either borosilicate or soda-lime-silica) particles with respect to their effect on metabolic activity, cell viability, changes in intracellular ion concentrations, proliferation and differentiation. 45S5 Bioglass caused extra- and intracellular alkalinization, a rise in [Ca2+]i and [K+]i, a small plasma membrane hyperpolarization, and an increase in lactate production. Glycolytic activity was also stimulated when cells were not in direct contact with 45S5 Bioglass particles but communicated with them only through the medium. Similarly, raising the pH of culture medium enhanced lactate synthesis. 45S5 Bioglass had no effect on osteoblast viability and, under most conditions, did not affect either proliferation or differentiation. Bioactive glasses 58S and 77S altered neither the ion levels nor enhanced metabolic activity. It is concluded that: (1) some bioactive glasses exhibit well-defined effects in osteoblasts in culture which are accessible to experimentation; (2) 45S5 Bioglass causes marked external and internal alkalinization which is, most likely, responsible for enhanced glycolysis and, hence, cellular ATP production; (3) changes in [H+] could contribute to alternations in concentrations of other intracellular ions; and (4) the rise in [Ca2+]i may influence activities of a number of intracellular enzymes and pathways. It is postulated that the beneficial effect of 45S5 on in vivo bone growth and repair may be due to some extent to alkalinization, which in turn increases collagen synthesis and crosslinking, and hydroxyapatite formation.

Ion Homeostasis in Rat Brain in vivo: Intra- and Extracellular [Ca2+] and [H+] in the Hippocampus during Recovery from Short-Term, Transient Ischemia

Changes in intra- and extracellular [Ca2+] and [H+], together with alterations in tissue Po2 and local blood flow, were measured in areas CA1 and CA3 of the hippocampus during recovery (up to 8 h) after an 8-min period of low-flow ischemia. Restoration of blood supply was followed by an immediate rise in flow and tissue Po2 above normal, with large fluctuations in both persisting for up to 4 h. In area CA1, [Ca2+]i decreased rapidly from an ischemic mean value of 30 μM to a control mean level of 73.1 nM in 20–30 min, whereas normalization of [Ca2+]e took ∼1 h. Recovery of [Ca2+]i was accelerated by preischemic administration of a calcium antagonist, nifedipine, and a free radical scavenger, N-tert-butyl-α-phenylnitrone (PBN), but not by MK-801, a blocker of N-methyl-d-aspartate receptors. There was a secondary rise in [Ca2+]i in many cells beginning ∼2 h after reperfusion. This was attenuated somewhat by PBN but not clearly influenced by either nifedipine or MK-801. Changes of [Ca2+]i in area CA3 were much smaller and slightly slower than in area CA1 and were not affected by the drugs mentioned above. In both areas CA1 and CA3, pHe and pHi fell during ischemia to an average value of 6.2, from which there was a rapid initial recovery in the first 5–10 min when blood flow was restored. Thereafter tissue pH rose slowly and did not reach control levels for ∼1 h, and in some microareas not at all. It is concluded that (a) effective mechanisms for restoring normal [Ca2+]i remain intact after 8 min of low-flow ischemia; (b) in neurons of area CA1, some insidious change in the homeostasis of calcium triggers a secondary rise in its free cytosolic concentration, which may be causally related to activation of irreversible cell damage; and (c) the changes in [Ca2+]i and [Ca2+]e during and following 8 min of ischemia can be adequately accounted for by movements of a fixed pool of Ca between intra- and extracellular compartments, and possible mechanisms are discussed.

Toxicity of Dopamine to Striatal Neurons In Vitro and Potentiation of Cell Death by a Mitochondrial Inhibitor

Abstract: Intrastriatal injections of the mitochondrial toxins malonate and 3‐nitropropionic acid produce selective cell death similar to that seen in transient ischemia and Huntingtons disease. The extent of cell death can be attenuated by pharmacological or surgical blockade of cortical glutamatergic input. It is not known, however, if dopamine contributes to toxicity caused by inhibition of mitochondrial function. Exposure of primary striatal cultures to dopamine resulted in dose‐dependent death of neurons. Addition of medium supplement containing free radical scavengers and antioxidants decreased neuronal loss. At high concentrations of the amine, cell death was predominantly apoptotic. Methyl malonate was used to inhibit activity of the mitochondrial respiratory chain. Neither methyl malonate (50 µM) nor dopamine (2.5 µM) caused significant toxicity when added individually to cultures, whereas simultaneous addition of both compounds killed 60% of neurons. Addition of antioxidants and free radical scavengers to the incubation medium prevented this cell death. Dopamine (up to 250 µM) did not alter the ATP/ADP ratio after a 6‐h incubation. Methyl malonate, at 500 µM, reduced the ATP/ADP ratio by ∼30% after 6 h; this decrease was not augmented by coincubation with 25 µM dopamine. Our results suggest that dopamine causes primarily apoptotic death of striatal neurons in culture without damaging cells by an early adverse action on oxidative phosphorylation. However, when combined with minimal inhibition of mitochondrial function, dopamine neurotoxicity is markedly enhanced.

Ion homeostasis in brain cells: differences in intracellular ion responses to energy limitation between cultured neurons and glial cells.

Intracellular concentrations of sodium, potassium and calcium together with membrane potentials were measured in cultured murine cortical neurons and glial cells under conditions which mimicked in vivo hypoxia, ischemia and hypoglycemia. These included; glucose omission with and without added pyruvate, addition of rotenone in the presence and absence of glucose and substitution of 2-deoxyglucose for glucose with and without rotenone. Cellular energy levels ([ATP], [ADP], [phosphocreatine], [creatine]) were measured in suspensions of C6 cells incubated in parallel under identical conditions. [Na+]i and [Ca2+]i rose while [K+]i fell and plasma membrane depolarized when energy production was limited. Intracellular acidification was observed when glycolysis was the sole source for ATP synthesis. There was a positive correlation between the extent of energy depletion in glial cells and the magnitude and velocity of alterations in ion levels. Neither glycolysis alone nor oxidative phosphorylation alone were able to ensure unaltered ion gradients. Since oxidative phosphorylation is much more efficient in generating ATP than glycolysis, this finding suggests a specific requirement of the Na pump for ATP generated by glycolysis. Changes in [Na+]i and [K+]i observed during energy depletion were gradual and progressive whereas those in [Ca2+]i were initially slow and moderate with large elevations occurring only as a late event. Increases in [Na+]i were usually smaller than reductions in [K+]i, particularly in the glia, suggestive of cellular swelling. Glia were less sensitive to identical insults than were neurons under all conditions. Results presented in this study lead to the conclusion that the response to energy deprivation of the two main types of brain cells, neurons and astrocytes, is a complex function of their capacity to produce ATP and the activities of various pathways which are involved in ion homeostasis.

Mitochondrial responses to carbon monoxide toxicity

CO not only bonds hematin iron in blood hemoglobin but also ligands hematin iron atoms of cytochrome a/sub 3/. Nonuniform distribution of CO means that rapidly metabolizing tissue are more susceptible to CO toxicity. Mitochondria are protected by branching and cushioning mechanism so CO toxicity response is not linear as in COHb formation. O/sub 2/ flux in respiratory change is not altered by CO. In these in vitro experiments, as little as 100 ppM CO transiently disrupted cytochrome system in transition from anoxia to nonmoxia, slowing the oxidaton of the chain. Pigeon mitochondria were extremely sensitive to CO when in metabolically-active (uncoupled) state. Increased binding of CO to cytochrome a/sub 3/ during anoxia was observed.

Quantitative dependence of mitochondrial oxidative phosphorylation on oxygen concentration: A mathematical model☆

Abstract The model of Wilson and co-workers ( D. F. Wilson, C. S. Owen, and A. Holian, 1977 , Arch. Biochem. Biophys . 182 , 749–762) for the regulation of mitochondrial oxidative phosphorylation has been extended to include the dependence on oxygen tension. The derived rate expression correctly describes the observed dependence of cellular energy metabolism on oxygen tension, including the oxygen dependence at “normoxic” physiological values. Experimental evidence is presented that oxidative phosphorylation by suspensions of isolated rat liver mitochondria is also dependent on oxygen concentration up to values of at least 100 μ M .

Oxygen and Ion Concentrations in Normoxic and Hypoxic Brain Cells

The goal of the present contribution is to discuss the relationships among brain oxygen tension, energy (ATP) level, and ion gradients and movements. The function of the CNS, the generation and transmission of impulses, is determined to a large extent by the movements of ions. Hence elucidation of these relationships is necessary to the understanding of how brain works. Moreover, such knowledge is indispensable for the design of rational therapies for treatment of a large group of pathological states caused by lack of oxygen. This paper is partly a review and partly an original contribution although the former involves to a considerable extent, results obtained in our laboratories. It is divided into 3 parts: a) a very brief general introduction which reminds the reader some well-known facts; b) presentation and discussion of data; and c) conclusions and/or predictions.