A novel mode of lactate metabolism in strictly anaerobic bacteria

Lactate is a common substrate for major groups of strictly anaerobic bacteria, but the biochemistry and bioenergetics of lactate oxidation is obscure. The high redox potential of the pyruvate/lactate pair of E₀′ = ?190 mV excludes direct NAD⁺ reduction (E₀′ = ?320 mV). To identify the hitherto unknown electron acceptor, we have purified the lactate dehydrogenase (LDH) from the strictly anaerobic, acetogenic bacterium Acetobacterium woodii. The LDH forms a stable complex with an electron‐transferring flavoprotein (Etf) that exhibited NAD⁺ reduction only when reduced ferredoxin (Fd^2?) was present. Biochemical analyses revealed that the LDH/Etf complex of A. woodii uses flavin‐based electron confurcation to drive endergonic lactate oxidation with NAD⁺ as oxidant at the expense of simultaneous exergonic electron flow from reduced ferredoxin (E₀′ ≈ –500 mV) to NAD⁺ according to: lactate + Fd^2? + 2 NAD⁺ → pyruvate + Fd + 2 NADH. The reduced Fd^2? is regenerated from NADH by a sequence of events that involves conversion of chemical (ATP) to electrochemical and finally redox energy (Fd^2? from NADH) via reversed electron transport catalysed by the Rnf complex. Inspection of genomes revealed that this metabolic scenario for lactate oxidation may also apply to many other anaerobes.     

Aralar mRNA and protein levels in neurons and astrocytes freshly isolated from young and adult mouse brain and in maturing cultured astrocytes

Intense glucose-based energy metabolism and glutamate synthesis by astrocytes require malate–aspartate-shuttle (MAS) activity to regenerate NAD⁺ from NADH formed during glycolysis, since brain lacks significant glycerophosphate shuttle activity. Aralar is a necessary aspartate/glutamate exchanger for MAS function in brain. Based on cytochemical immunoassays the absence of aralar in adult astrocytes was repeatedly reported. This would mean that adult astrocytes must regenerate NAD⁺ by producing lactate from pyruvate, eliminating its use by oxidative and biosynthetic pathways. We alternatively used astrocytes and neurons from adult brain, freshly isolated by fluorescence-activated cell sorting, to determine aralar protein by a specific antibody and its mRNA by real-time PCR. Both protein and mRNA expressions were identical in adult neurons and astrocytes and similar to whole brain levels. The same level of aralar expression was reached in well-differentiated astrocyte cultures, but not until late development, coinciding with the late-maturing brain capability for glutamate formation and degradation.     

Aralar mRNA and protein levels in neurons and astrocytes freshly isolated from young and adult mouse brain and in maturing cultured astrocytes

Intense glucose-based energy metabolism and glutamate synthesis by astrocytes require malate–aspartate-shuttle (MAS) activity to regenerate NAD⁺ from NADH formed during glycolysis, since brain lacks significant glycerophosphate shuttle activity. Aralar is a necessary aspartate/glutamate exchanger for MAS function in brain. Based on cytochemical immunoassays the absence of aralar in adult astrocytes was repeatedly reported. This would mean that adult astrocytes must regenerate NAD⁺ by producing lactate from pyruvate, eliminating its use by oxidative and biosynthetic pathways. We alternatively used astrocytes and neurons from adult brain, freshly isolated by fluorescence-activated cell sorting, to determine aralar protein by a specific antibody and its mRNA by real-time PCR. Both protein and mRNA expressions were identical in adult neurons and astrocytes and similar to whole brain levels. The same level of aralar expression was reached in well-differentiated astrocyte cultures, but not until late development, coinciding with the late-maturing brain capability for glutamate formation and degradation.     

Lactate dehydrogenase isoenzymes of sperm cells and testes

1. The kinetic and metabolic properties of lactate dehydrogenase isoenzyme LDH_x from human sperm cells and rat testes were studied. 2. LDH_x shows a sensitivity to inhibition by stilboestrol diphosphate, urea and guanidinium chloride different from that of the LDH-H₄ and LDH-M₄ isoenzymes. 3. About 10 and 20% of the total lactate dehydrogenase activity of testes and sperm cells respectively were associated with particulate fractions. In sperm cells 11% was localized in the middle piece and 18·8% in the head fraction. LDH_x was found in all particulate fractions of sperm cells. The middle piece contained 41·0% of total LDH_x activity and showed high succinate dehydrogenase activity. 5. The pH-dependence of lactate/pyruvate and NAD⁺/NADH concentration ratios were estimated. Lactate dehydrogenase in sperm cells has maximal activity with NADH as coenzyme at pH7·5 and with NADPH as coenzyme at pH6·0. At pH6·0 a 10% greater oxidation of NADPH than of NADH was found. At acid pH lactate hydrogenase may function as an enzyme bringing about transhydrogenation from NADPH to NAD⁺. 6. In agreement with the stoicheiometry of the lactate de- hydrogenase reaction, the lactate/pyruvate concentration ratio decreased with increasing pH. 7. The lactate/pyruvate and NAD⁺/NADH concentration ratios were estimated with glucose, fructose and sorbitol as substrates and as a function of time after addition of these substrates. During a 20min. period after the addition of the substrates, changes in lactate/pyruvate and NAD⁺/NADH concentration ratios were noticed. Increasing concentration of the substrates mentioned gave rise to asymptotic increases in lactate and pyruvate. 8. Sorbitol did not act as a substrate for LDH_x. 9. The findings described are consistent with the idea that LDH_x is different from other known lactate dehydrogenase isoenzymes, but that it has a metabolic function similar to that of the isoenzymes of other tissues.     

Physical and Functional Association of Lactate Dehydrogenase (LDH) with Skeletal Muscle Mitochondria

The intracellular lactate shuttle hypothesis posits that lactate generated in the cytosol is oxidized by mitochondrial lactate dehydrogenase (LDH) of the same cell. To examine whether skeletal muscle mitochondria oxidize lactate, mitochondrial respiratory oxygen flux (JO₂) was measured during the sequential addition of various substrates and cofactors onto permeabilized rat gastrocnemius muscle fibers, as well as isolated mitochondrial subpopulations. Addition of lactate did not alter JO₂. However, subsequent addition of NAD⁺ significantly increased JO₂, and was abolished by the inhibitor of mitochondrial pyruvate transport, α-cyano-4-hydroxycinnamate. In experiments with isolated subsarcolemmal and intermyofibrillar mitochondrial subpopulations, only subsarcolemmal exhibited NAD⁺-dependent lactate oxidation. To further investigate the details of the physical association of LDH with mitochondria in muscle, immunofluorescence/confocal microscopy and immunoblotting approaches were used. LDH clearly colocalized with mitochondria in intact, as well as permeabilized fibers. LDH is likely localized inside the outer mitochondrial membrane, but not in the mitochondrial matrix. Collectively, these results suggest that extra-matrix LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria.     

Purification and Kinetic Characterisation of Lactate Dehydrogenase from Dioscorea cayenensis Tuber

Lactate dehydrogenase from yellow yam tuber (Dioscorea cayenensis Lam.) was isolated and purified using various chromatographic methods and electrophoresis. Only one form of the enzyme obtained, which obeyed Michaelis-Menten kinetics, was activated by Mg²⁺ and Ca²⁺ and inhibited by nucleotides and PEP. AMP, which activated the enzyme in the direction of pyruvate reduction, inhibited it in the direction of lactate oxidation. The enzyme is specific for pyruvate L-lactate and uses only NADH and NAD⁺ as the electron carriers. Polyacrylamide gel electrophoresis showed single band of lactate dehydrogenase activity. The average molecular mass obtained for the enzyme was 160 ± 1.2 kDa, while SDS gel electrophoresis indicated a dimer for the enzyme protein. The enzyme is very stable when frozen but its activity was hardly detectable when the tubers were stored in a well aerated place.     

A Novel Subtype of Astrocytes Expressing TRPV4 (Transient Receptor Potential Vanilloid 4) Regulates Neuronal Excitability via Release of Gliotransmitters

Astrocytes play active roles in the regulation of synaptic transmission. Neuronal excitation can evoke Ca²⁺ transients in astrocytes, and these Ca²⁺ transients can modulate neuronal excitability. Although only a subset of astrocytes appears to communicate with neurons, the types of astrocytes that can regulate neuronal excitability are poorly characterized. We found that ∼30% of astrocytes in the brain express transient receptor potential vanilloid 4 (TRPV4), indicating that astrocytic subtypes can be classified on the basis of their expression patterns. When TRPV4⁺ astrocytes are activated by ligands such as arachidonic acid, the activation propagates to neighboring astrocytes through gap junctions and by ATP release from the TRPV4⁺ astrocytes. After activation, both TRPV4⁺ and TRPV4⁻ astrocytes release glutamate, which acts as an excitatory gliotransmitter to increase synaptic transmission through type 1 metabotropic glutamate receptor (mGluR). Our results indicate that TRPV4⁺ astrocytes constitute a novel subtype of the population and are solely responsible for initiating excitatory gliotransmitter release to enhance synaptic transmission. We propose that TRPV4⁺ astrocytes form a core of excitatory glial assembly in the brain and function to efficiently increase neuronal excitation in response to endogenous TRPV4 ligands.     

低温冷藏提高家蚕滞育卵NAD含量和胞质苹果酸脱氢酶活性

为了调查5℃低温处理是否改变家蚕Bombyx mori卵滞育NAD代谢, 本研究利用HPLC和分光光度法测定了经25℃和5℃分别处理的滞育卵中NADH 含量、 NAD⁺含量、 乳酸脱氢酶（LDH）活性和胞质苹果酸脱氢酶（cMDH）活性。结果表明： 5℃处理的NAD（NADH + NAD⁺）含量和cMDH活性分别增加了106%和53%, 并且显著高于25℃处理（P< 0.01）; 但是两种处理的NADH/NAD⁺比值和LDH活性没有显著差异（P> 0.05）。据此推测, 5℃低温处理加强了家蚕滞育卵NAD⁺合成和再生能力。     

Astrocytes and Synaptosomes Transport and Metabolize Lactate and Acetate Differently

Astrocytes transport the monocarboxylate acetate, but synaptosomes do not. The reason for this is unknown, because both preparations express monocarboxylate transporters (MCT). The transport and metabolism of lactate, another monocarboxylate, was examined in these two preparations, and the results were compared to those for acetate. Lactate transport is more rapid in astrocytes than in synaptosomes, but of lower affinity (Kms of 17 and 4 mM, respectively). Lactate (0.2 mM) is metabolized to CO2 more rapidly in synaptosomes than in astrocytes (rates of 0.37 and 0.07 nmol x mg protein(-1) x min(-1), respectively). The reason for this is unclear, but cellular differences in lactate dehydrogenase isotype expression may be involved. Acetate is metabolized to CO2 more rapidly in astrocytes than in synaptosomes (rates of 0.43 and 0.02 nmol x mg protein(-1) x min(-1), respectively). This is likely due to cellular differences in the expression of monocarboxylate transporter subtypes.     

Exogenous Glutamate-Induced Modulation of Neurosecretory Process in Nerve Terminals Obtained from the Rat Brain

We studied intracellular processes in nerve terminals of neurons of the rat brain in response to application of exogenous glutamate. Using a рН-sensitive fluorescence probe, acridine orange (AO), and labeled gammaaminobutyric acid ([³Н]GABA), we estimated the effect of application of glutamate on the level of acidification of synaptic vesicles and also on the release of GABA from nerve terminals (synaptosomes) obtained from hippocampal tissue. Our experiments showed that glutamate in a dose-dependent manner stimulated the [³Н]GABA release from nerve terminals, and then we observed re-uptake of this neurotransmitter. A selective blocker of GABA transporters, NO-711, completely blocked the uptake of neurotransmitter but did not influence its release; this observation indicates that the glutamate-induced GABA release was from the vesicular, not cytosolic, pool. We confirmed that glutamate stimulates the process of exocytosis in experiments using AO, where we obtained data indicating that this process is two-phase. The first phase, which reflects probably calcium-induced exocytosis, looked like a “burst” of fluorescent signal typical of the response of synaptosomes to the action of KCl applied in depolarization concentration. Both phases of the response were completely blocked or significantly suppressed in calcium-free medium or in the presence of 25 μM Cd²⁺. The second (slow) phase of the response developed after a certain lag period and was characterized by a gradual increase in the intensity of fluorescent signal. This phase was completely dependent on the presence of sodium in the extracellular medium and completely blocked when sodium was replaced by choline or N-methyl-D-glucamine. We hypothesize that the second phase of the response can reflect either spontaneous unstimulated exocytosis or dissipation of the proton gradient in synaptic vesicles induced by the entry of Na⁺ into the nerve terminal.     

Plasmalemmal Na+/Ca2+ exchanger modulates Ca2+-dependent exocytotic release of glutamate from rat cortical astrocytes

Astroglial excitability operates through increases in Ca²⁺_cyt (cytosolic Ca²⁺), which can lead to glutamatergic gliotransmission. In parallel fluctuations in astrocytic Na⁺_cyt (cytosolic Na⁺) control metabolic neuronal-glial signalling, most notably through stimulation of lactate production, which on release from astrocytes can be taken up and utilized by nearby neurons, a process referred to as lactate shuttle. Both gliotransmission and lactate shuttle play a role in modulation of synaptic transmission and plasticity. Consequently, we studied the role of the PMCA (plasma membrane Ca²⁺-ATPase), NCX (plasma membrane Na⁺/Ca²⁺ exchanger) and NKA (Na⁺/K⁺-ATPase) in complex and coordinated regulation of Ca²⁺_cyt and Na⁺_cyt in astrocytes at rest and upon mechanical stimulation. Our data support the notion that NKA and PMCA are the major Na⁺ and Ca²⁺ extruders in resting astrocytes. Surprisingly, the blockade of NKA or PMCA appeared less important during times of Ca²⁺ and Na⁺ cytosolic loads caused by mechanical stimulation. Unexpectedly, NCX in reverse mode appeared as a major contributor to overall Ca²⁺ and Na⁺ homoeostasis in astrocytes both at rest and when these glial cells were mechanically stimulated. In addition, NCX facilitated mechanically induced Ca²⁺-dependent exocytotic release of glutamate from astrocytes. These findings help better understanding of astrocyte-neuron bidirectional signalling at the tripartite synapse and/or microvasculature. We propose that NCX operating in reverse mode could be involved in fast and spatially localized Ca²⁺-dependent gliotransmission, that would operate in parallel to a slower and more widely distributed gliotransmission pathway that requires metabotropically controlled Ca²⁺ release from the ER (endoplasmic reticulum).     

Lactate Dehydrogenase Activity is Inhibited by Methylmalonate in vitro

Methylmalonic acidemia (MMAemia) is an inherited metabolic disorder of branched amino acid and odd-chain fatty acid metabolism, involving a defect in the conversion of methylmalonyl-coenzyme A to succinyl-coenzyme A. Systemic and neurological manifestations in this disease are thought to be associated with the accumulation of methylmalonate (MMA) in tissues and biological fluids with consequent impairment of energy metabolism and oxidative stress. In the present work we studied the effect of MMA and two other inhibitors of mitochondrial respiratory chain complex II (malonate and 3-nitropropionate) on the activity of lactate dehydrogenase (LDH) in tissue homogenates from adult rats. MMA potently inhibited LDH-catalyzed conversion of lactate to pyruvate in liver and brain homogenates as well as in a purified bovine heart LDH preparation. LDH was about one order of magnitude less sensitive to inhibition by MMA when catalyzing the conversion of pyruvate to lactate. Kinetic studies on the inhibition of brain LDH indicated that MMA inhibits this enzyme competitively with lactate as a substrate (K _i=3.02±0.59 mM). Malonate and 3-nitropropionate also strongly inhibited LDH-catalyzed conversion of lactate to pyruvate in brain homogenates, while no inhibition was observed by succinate or propionate, when present in concentrations of up to 25 mM. We propose that inhibition of the lactate/pyruvate conversion by MMA contributes to lactate accumulation in blood, metabolic acidemia and inhibition of gluconeogenesis observed in patients with MMAemia. Moreover, the inhibition of LDH in the central nervous system may also impair the lactate shuttle between astrocytes and neurons, compromising neuronal energy metabolism.S. R. Mirandola and E. N. Maciel contributed equally to this work.     

The depolarization-induced release of cholecystokinin C-terminal octapeptide (CCK-8) from rat synaptosomes and brain slices

Studies on the subcellular distribution of immunoreactive cholecystokinin (CCK) in homogenates of rat cerebral cortex showed that approximately 95% was associated with particulate fractions, including presynaptic terminals (synaptosomes). Chromatography of extracts of tissue and medium from incubated synaptosomes revealed that this material was almost exclusively in the form of COOH-terminal octapeptide (CCK-8), very little CCK-33 being present. There was a wide range of CCK-8 concentrations in synaptosomes from different brain regions (cortex > striatum ? hypothalamus > brain stem). Cerebral cortex synaptosomes were incubated in vitro and showed a complex pattern of CCK-8 release with varying concentrations of tissue: amounts in the medium rose rapidly with increasing synaptosome concentrations, then fell to a plateau at higher tissue values. A mechanism for the rapid disposal of extracellular CCK-8 was associated with synaptosomal fractions. Depolarization-induced (high K⁺) release of CCK-8 was observed with cortex and corpus striatum synaptosomes. A rapid and reversible enhancement of CCK-8 release from cortex slices was observed in response to elevated K⁺. Veratrine also released CCK-8 from cortex slices, although this was not reversible. Stimulus-induced release of CCK-8 from synaptosomes and slices required extracellular Ca²⁺. The storage, release and degradation of CCK-8 by nerve-endings suggest a synaptic function for this peptide.     

Nitric oxide and antioxidant status in glucose and oxygen deprived neonatal and adult rat brain synaptosomes

Nitric oxide (NO) has been implicated in the process of cerebral ischemia/reperfusion injury. We have examined the production of NO, as reflected by nitrite (NO₂ ⁻)+nitrate (NO₃ ⁻) accumulation, from synaptosomes isolated from neonatal or adult rat brain and subjected to a period of glucose and oxygen deprivation. There was a significant increase in the amount of NO₂ ⁻+NO₃ ⁻ production from adult synaptosomes under these conditions, whereas there was no difference compared to control in the production of NO₂ ⁻+NO₃ ⁻ from the neonatal synaptosomes. The total antioxidant status of the synaptosomes at these different stages of brain development was found to be the same. These data suggest that the vulnerability of the adult brain to ischemia/reperfusion injury may be associated with the production of NO from nerve terminals. The ratios of antioxidant capacity to NO production under such conditions have been shown here to be different between the neonatal and adult nerve terminals. Thus the well documented resistance of neonatal brain to ischemia/reperfusion injury may involve the neonatal nerve terminal being under less oxidative stress than the adult.     

Presynaptic malfunction: the neurotoxic effects of cadmium and lead on the proton gradient of synaptic vesicles and glutamate transport

Exposure to Cd²⁺ and Pb²⁺ has neurotoxic consequences for human health and may cause neurodegeneration. The study focused on the analysis of the presynaptic mechanisms underlying the neurotoxic effects of non-essential heavy metals Cd²⁺ and Pb²⁺. It was shown that the preincubation of rat brain nerve terminals with Cd²⁺ (200 μM) or Pb²⁺ (200 μM) resulted in the attenuation of synaptic vesicles acidification, which was assessed by the steady state level of the fluorescence of pH-sensitive dye acridine orange. A decrease in l-[¹⁴C]glutamate accumulation in digitonin-permeabilized synaptosomes after the addition of the metals, which reflected lowered l-[¹⁴C]glutamate accumulation by synaptic vesicles inside of synaptosomes, may be considered in the support of the above data. Using isolated rat brain synaptic vesicles, it was found that 50 μM Cd²⁺ or Pb²⁺ caused dissipation of their proton gradient, whereas the application of essential heavy metal Mn²⁺ did not do it within the range of the concentration of 50-500 μM. Thus, synaptic malfunction associated with the influence of Cd²⁺ and Pb²⁺ may result from partial dissipation of the synaptic vesicle proton gradient that leads to: (1) a decrease in stimulated exocytosis, which is associated not only with the blockage of voltage-gated Ca²⁺ channels, but also with incomplete filling of synaptic vesicles; (2) an attenuation of Na⁺-dependent glutamate uptake.     

How astrocytes feed hungry neurons

For years glucose was thought to constitute the sole energy substrate for neurons; it was believed to be directly provided to neurons via the extracellular space by the cerebral circulation. It was recently proposed that in addition to glucose, neurons might rely on lactate to sustain their activity. Therefore, it was demonstrated that lactate is a preferred oxidative substrate for neurons not only in vitro but also in vivo. Moreover, the presence of specific monocarboxylate transporters on neurons as well as on astrocytes is consistent with the hypothesis of a transfer of lactate from astrocytes to neurons. Evidence has been provided for a mechanism whereby astrocytes respond to glutamatergic activity by enhancing their glycolytic activity, resulting in increased lactate release. This is accomplished via the uptake of glutamate by glial glutamate transporters, leading to activation of the Na⁺/K⁺ ATPase and a stimulation of astrocytic glycolysis. Several recent observations obtained both in vitro and in vivo with different approaches have reinforced this view of brain energetics. Such an understanding might be critically important, not only because it forms the basis of some classical functional brain imaging techniques but also because several neurodegenerative diseases exhibit diverse alterations in energy metabolism.     

Effect of oxygenation and sulfate concentrations on pyruvate and lactate formation inKlebsiella oxytoca ZS growing in chemostat cultures

Klebsiella oxytoca ZS fermented glucose to ethanol and lactic, formic, and acetic acids, but, in contrast to many strains, accumulates pyruvic and acetic acids as the principal end products in aerobic growth conditions. This strain was grown in sulfate-limited chemostat at a fixed low dilution rate (D=0.033 h^–1) with glucose present in excess. When oxygen was supplied at a high level, pyruvate and acetate were produced, and the ratio NADH/NAD⁺ was low (0.04) while the internal pyruvate concentration increased to 100 mol (g dry wt)^–1. A shortage of oxygen supply was accompanied by lactate production, an increase of the ratio NADH/NAD⁺ (0.53), and an undetectable level in internal pyruvate concentration. The observed changes in LDH activity found in vitro in extracts of the cells are not strictly related to those found in vivo. In fact, the specific activity of LDH was essentially stable at 30% of dissolved oxygen tension (d.o.t.) and decreased slightly at 60% of d.o.t., whereas specific lactic acid production decreased rapidly. The in vitro LDH activity was strongly affected by the NADH/NAD⁺ ratio.     

Relations Between Intracellular Ions and Energy Metabolism Under Acidotic Conditions: A Study with Nigericin in Synaptosomes, Neurons, and C6 Glioma Cells

Abstract: Effects of nigericin were investigated in rat brain synaptosomes, cultured neurons, and C6 glioma cells to characterize the relations among ATP synthesis, [Na⁺]_i., [K⁺]_i, and [Ca²⁺]_i, and pH under conditions when [H⁺]_i is substantially increased and transmembrane electrical potential is decreased. Intracellular acidification and loss of K⁺ were accompanied by enhanced oxygen consumption and lactate production and a decrease in cellular energy level. Changes in the last three parameters were attenuated by addition of 1 mM ouabain. In synaptosomes treated with nigericin, neither respiration nor glycolysis was affected by 0.3 μM tetrodotoxin, whereas 1 mM amiloride reduced lactate production by 20% but did not influence respiration. In C6 cells, amiloride decreased the nigericin-stimulated rate of lactate generation by about 50%. The enhancement by nigericin of synaptosomal oxygen uptake and glycolytic rate decreased with time. However, there was only a small reduction in respiration and none in glycolysis in C6 cells. Measurements with ion-selective microelectrodes in neurons and C6 cells showed that nigericin also caused a rise in [Ca²⁺], and [Na⁺]., The increase in [Na⁺], in C6 cells was partially reversed by 1 mM amiloride. It is concluded that nigericin-induced loss of K⁺ and subsequent depolarization lead to an increase in Na⁺ influx and stimulation of the Na⁺/K⁺ pump with a consequent rise in energy utilization; that acidosis inhibits mitochondrial ATP production; that a rise in [H⁺] does not decrease glycolytic rate when the energy state (a fall in [ATP] and rises in [ADP] and [AMP]) is simultaneously reduced; that a fall in [K⁺], depresses both oxidative phosphorylation and glycolysis; and that the nigericin-induced alterations in ion levels and activities of energy-producing pathways can explain some of the deleterious effects of ischemia and hypoxia.