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PubMed1992

Regulation of intracellular pH values in higher plant cells. Carbon-13 and phosphorus-31 nuclear magnetic resonance studies.

The regulation of the cytoplasmic and vacuolar pH values (pHc and pHv) in sycamore (Acer pseudoplatanus L.) cells was analyzed using 31P and 13C nuclear magnetic resonance spectroscopy. Suspension-cultured cells were compressed in the NMR tube and perfused with the help of an original arrangement enabling a tight control of the pH (external pH, pHe) of the carefully oxygenated circulating nutrient medium. Intracellular pH values were measured from the chemical shifts of: CH2-linked carboxyl groups of citric acid below pH 5.7; orthophosphate between pH 5.7 and 8.0; 13C-enriched bicarbonate over pH 8.0. pHc and pHv were independent of pHe over the range 4.5-7.5. In contrast intracellular pH values decreased rapidly below pHe 4.5 and increased progressively at pHe over 7.5. There was an acceleration in the rate of O2 consumption accompanied with a decrease in cytoplasmic ATP concentration as pHe decreased. When the rate of O2 consumption was approaching the uncoupled O2 uptake rate, a loss of pHc control was observed. It is concluded that as pHe decreased, the plasma membrane ATPase consumed more and more ATP to reject the invading H+ ions in order to maintain pHc at a constant value. Below pHe 4.5 the efficiency of the H+ pump to react to back leakage of H+ ions became insufficient, leading to an acidification of pHc and to an alkalinization of pHe. On the other hand, over pHe 7.5 a passive influx of OH- ions was observed, and pHc increased proportionally to the increase of pHe. Simultaneously appreciable amounts of organic acids (malate and citrate) were synthesized by cells during the course of the alkalinization of the cytoplasmic compartment. The synthesis of organic acids which partially counteract the alkalinization of the cytoplasmic compartment may result from a marked activation of the cytoplasmic phosphoenolpyruvate carboxylase induced by an increase in cytoplasmic bicarbonate concentration. The fluctuations of pHv followed a similar course to that of pHc. It is concluded that the vacuole, which represents a potentially large H+ ions reservoir, can counteract H+ (or OH-) ion invasion observed at acidic (or alkaline) pHe contributing to the homeostasis of pHc.

Carbon Isotopes↗
PubMed1993

Alterations in alveolar type II cell metabolism induced by tetrandrine and other alkaloids.

Tetrandrine (TT) and other bisbenzylisoquinoline alkaloids have been used in China as a treatment for fibrotic lung diseases. Because of their potential use as pulmonary therapeutic agents, we studied the effects of some of these compounds on energy metabolism in isolated rat alveolar type II cells, i.e., cells which play a critical role in maintaining normal lung function. Incubation of type II cells with most of the alkaloids produces a reduction in cellular ATP content. However, there is no effect of the alkaloids on cellular oxygen consumption. All of the alkaloids which produce reductions in cell ATP levels cause increases in internal calcium levels of type II cells. Incubation of the cells with the calcium ionophore, 4-bromo A-23187, leads to increased amounts of intracellular calcium and reductions in ATP levels, but has no effect on oxygen consumption. Exposure of isolated lung mitochondria to calcium produces a concentration-dependent reduction in ATP synthesis with no effect on mitochondrial oxygen consumption. Direct exposure of mitochondria to TT has no effect on ATP synthesis. These results are consistent with the notion that the alkaloids produce an increase in type II cell internal calcium levels which, in turn, leads to reduced rates of mitochondrial ATP synthesis.

Adenosine Triphosphate↗
PubMed2001

Further evidence for mitochondrial dysfunction in progressive supranuclear palsy.

Recent data from our laboratory have identified a role for mitochondrial dysfunction in the pathogenesis of progressive supranuclear palsy (PSP). To extend this finding, we measured key parameters of mitochondrial function in platelet-derived cytoplasmic hybrid (cybrid) cell lines expressing mitochondrial genes from patients with PSP. We observed significant decreases in aconitase activity, cellular ATP levels, and oxygen consumption in PSP cybrids as compared to control cybrids, further suggesting a contributory role of impaired mitochondrial energy metabolism in PSP, possibly due to genetic abnormalities of mitochondrial DNA.

Adenosine Triphosphate↗
PubMed2003

Mitochondrial Dysfunction in Sepsis.

Sepsis is an increasingly common problem, particularly among critically ill patients. Mechanisms by which sepsis induces organ dysfunction have not been elucidated. The coexisting findings (unique to sepsis) of metabolic acidosis yet increased tissue oxygen tensions suggest cellular availability but decreased use of oxygen (tissue dysoxia). Because mitochondria use more than 90% of total body oxygen consumption for adenosine triphosphate (ATP) generation, a bioenergetic abnormality is implied. Cell and animal data have shown that nitric oxide (and its metabolites), produced in considerable excess in patients with sepsis, can affect oxidative phosphorylation by inhibiting several of its component respiratory enzymes. Human data are scarce. However, in skeletal muscle biopsies taken from patients with sepsis, we have recently demonstrated a relationship between increased nitric oxide production, antioxidant depletion, reduced respiratory chain complex I activity, and low ATP levels. These findings correlated with severity of disease and outcome and support the notion that mitochondrial dysfunction resulting in bioenergetic failure may be an important factor in the pathophysiology of sepsis-associated multiorgan failure. However, a reasonable argument can be made that the reduction in energy supply could represent a last-ditch adaptive response to ongoing inflammation, resulting in a cellular shutdown analogous to hibernation that allows eventual restoration of organ function and long-term survival in patients fit enough to survive the acute phase.

Journal Article↗
PubMed1990

Assessment of S-(1,2-dichlorovinyl)-L-cysteine induced toxic events in rabbit renal cortical slices. Biochemical and histological evaluation of uptake, covalent binding, and toxicity.

A renal cortical slice system was utilized to investigate the events leading to site-specific nephrotoxicity induced by S-(1,2-dichlorovinyl)-L-cysteine (DCVC). DCVC uptake into renal cortical slices was shown to be rapid (5-15 min) as well as time- and concentration-dependent. Of the total amount taken up at 1 h, 40% was subsequently covalently bound. These observations were confirmed by autoradiography, illustrating uptake and binding in the proximal tubule cells. Following these events, toxicity was evidenced by alterations in ATP content and O2 consumption between 4 and 8 h as well as leakage of the brush border enzymes (gamma glutamyl transpeptidase and alkaline phosphatase) as early as 4 h. Light microscopy provided a sequence of histopathological changes from an initial S3 lesion between 4 and 8 h to a lesion encompassing all proximal tubule segments (by 12 h). Electron microscopy demonstrated not only the specificity of DCVC toxicity (at 6 h) but also illustrated mitochondrial damage and loss of brush borders. A comparison of continuous versus short-term exposure to DCVC indicated that an irreversible sequence of events was initiated as early as 30 min. By utilizing an in vitro model which allows correlation of biochemical and histological changes, a sequence of events leading to DCVC induced toxicity was established.

Animals↗
PubMed1986

Protection against oxygen-induced reperfusion injury of the isolated canine heart by superoxide dismutase and catalase.

While oxygen-derived free radicals have been implicated in the pathogenesis of myocardial injury, the exact nature of this injury is still unclear. To test the hypothesis that oxygen-induced injury may influence the recovery of cardiac function from ischemic damage, we used an oxygen free radical scavenger, superoxide dismutase (SOD), together with catalase, during the reperfusion of isolated canine heart which had been subjected to 15 min of normothermic ischemic arrest followed by 2 hr of hypothermic cardioplegic preservation using a modified Collins solution. Determinations of thiobarbituric acid reactive substances and coenzyme Q10 within the myocardium showed that the treatment with SOD and catalase was capable of inhibiting lipid peroxidation induced by reperfusion. This inhibition was apparently associated with the improvement of myocardial energy metabolism and cardiac performance. Coronary flow was significantly higher in the heart treated with SOD and catalase during the working stage with a corresponding increase in oxygen consumption. Myocardial adenosine triphosphate (ATP) was partially, but significantly restored during reperfusion in these hearts whereas no restoration was observed in the heart without the enzymes. The treatment with SOD and catalase also improved left ventricular stroke work index and left ventricular maximum dp/dt at an early stage of the working mode. These results suggest that the use of SOD and catalase during reperfusion can protect the ischemic heart against reperfusion injury by scavenging oxygen-derived free radicals.

Animals↗
PubMed1989

Metabolic effects of forskolin in chick chondrocytes.

The effects of forskolin on parameters of energy metabolism and proteoglycan synthesis have been investigated in chick embryo sternal chondrocyte cultures. After 8 h exposure to 100 microM forskolin, ATP levels and oxygen consumption were unaltered. Protein synthesis was unaffected up to 50 microM forskolin and protein degradation was unaffected by forskolin up to 100 microM. In contrast, incorporation of the proteoglycan precursors, 35SO4 and [3H]glucosamine, was more sensitive to forskolin. Inhibition was linear with dose between 10 and 100 microM, reaching 70% at 100 microM. Incorporation of 35SO4 into glycosaminoglycan chains initiated on an artificial beta-xyloside acceptor was inhibited in the same manner. cAMP accumulation was maximal at 10 microM forskolin, a concentration which did not alter proteoglycan synthesis. We conclude that a major, acute effect of forskolin in these short-term experiments is inhibition of proteoglycan synthesis in a cAMP-independent manner.

Adenosine Triphosphate↗
PubMed2004

The role of oxidative stress, impaired glycolysis and mitochondrial respiratory redox failure in the cytotoxic effects of 6-hydroxydopamine in vitro.

The neurotoxin, 6-hydroxydopamine (6-OHDA) has been implicated in the neurodegenerative process of Parkinson's disease. The current study was designed to elucidate the toxicological effects of 6-OHDA on energy metabolism in neuroblastoma (N-2A) cells. The toxicity of 6-OHDA corresponds to the total collapse of anaerobic/aerobic cell function, unlike other mitochondrial toxins such as MPP+ that target specific loss of aerobic metabolism. The toxicity of 6-OHDA paralleled the loss of mitochondrial oxygen (O2) consumption (MOC), glycolytic activity, ATP, H+ ion gradients, membrane potential and accumulation of the autoxidative product, hydrogen peroxide (H2O2). Removing H2O2 with nonenzymatic stoichiometric scavengers, such as carboxylic acids, glutathione and catalase yielded partial protection. The rapid removal of H2O2 with pyruvate or catalase restored only anaerobic glycolysis, but did not reverse the loss of MOC, indicating mitochondrial impairment is independent of H2O2. The H2O2 generated by 6-OHDA contributed toward the loss of anaerobic glycolysis through lipid peroxidation and lactic acid dehydrogenase inhibition. The ability of 6-OHDA to maintain oxidized cytochrome c (CYT-C-OX) in its reduced form (CYT-C-RED), appears to play a role in mitohondrial impairment. The reduction of CYT-C by 6-OHDA, was extensive, occurred within minutes, preceded formation of H2O2 and was unaffected by catalase or superoxide dismutase. At similar concentrations, 6-OHDA readily altered the valence state of iron [Fe(III)] to Fe(II), which would also theoretically sustain CYT-C in its reduced form. In isolated mitochondria, 6-OHDA had negligible effects on complex I, inhibited complex II and interfered with complex III by maintaining the substrate, CYT-C in a reduced state. 6-OHDA caused a transient and potent surge in isolated cytochrome oxidase (complex IV) activity, with rapid recovery as a result of 6-OHDA recycling CYT-C-OX to CYT-C-RED. Typical mitochondrial toxins such as MPP+, azide and antimycin appeared to inhibit the catalytic activity of ETC enzymes. In contrast, 6-OHDA alters the redox of the cytochromes, resulting in loss of substrate availability and obstruction of oxidation-reduction events. Complete cytoprotection against 6-OHDA toxicity and restored MOC was achieved by combining catalase with CYT-C (horse heart). In summary, CYT-C reducing properties are unique to catecholamine neurotransmitters, and may play a significant role in selective vulnerability of dopaminergic neurons to mitochondrial insults.

Animals↗
PubMed2005

Nitric oxide stimulates PC12 cell proliferation via cGMP and inhibits at higher concentrations mainly via energy depletion.

We investigated the mechanisms by which nitric oxide (NO) from an NO donor (DETA/NO) regulates proliferation of pheochromocytoma PC12 cells. The NO donor stimulated proliferation at low concentrations, but reversibly and completely inhibited proliferation at higher concentrations. The stimulation (but not the inhibition) of proliferation was apparently due to NO stimulation of soluble guanylate cyclase to produce cGMP, as it was prevented by a specific cyclase inhibitor (ODQ), and replicated by a cell-permeable form of cGMP. The NO-induced cytostasis was not reversed by inhibitors of MEK kinase or poly(ADP-ribose)polymerase, or by treatments that bypass inhibition of ribonucleotide reductase or ornithine decarboxylase. Cytostatic concentrations of DETA/NO strongly inhibited respiration of PC12 cells, and specific respiratory inhibitors (rotenone, myxothiazol, or azide) caused complete cytostasis. Uridine and pyruvate reversed the cytostasis induced by the specific respiratory inhibitors, but not that induced by DETA/NO. However, the combination of uridine, pyruvate, and N-acetyl-cysteine did reverse DETA/NO-induced cytostasis. DETA/NO strongly and progressively inhibited glycolysis measured by glucose consumption, lactate production, and ATP level, and a specific glycolytic inhibitor (5 mM 2-deoxy-d-glucose) caused complete cytostasis. Our results indicate that NO at low concentrations increases cell proliferation via cGMP, while high concentrations of NO block proliferation via inhibition of both glycolysis and respiration, causing energy depletion.

Acetylcysteine↗
PubMed2005

Differential inhibitory effect of long-chain acyl-CoA esters on succinate and glutamate transport into rat liver mitochondria and its possible implications for long-chain fatty acid oxidation defects.

Long-chain fatty acid beta-oxidation defects are associated with a series of clinical and biochemical abnormalities, including accumulation of long-chain acyl-CoA esters which have been shown to inhibit several enzymes and transport systems that may disturb energy metabolism. Using isolated rat liver mitochondria incubated under state 3 conditions, we observed that long-chain acyl-CoA esters and their beta-oxidation intermediates inhibit ATP synthesis and oxygen consumption, both with succinate (plus rotenone) and l-glutamate as respiratory substrates. When an uncoupler (2,4-dinitrophenol) was used instead of ADP, to stimulate respiration maximally, the various CoA esters showed differential effects on the oxidation of succinate and l-glutamate, respectively. With succinate as substrate, there was a strong inhibition of oxygen consumption by palmitoyl-CoA, 2,3-unsaturated, 3-hydroxy, and 3-keto-palmitoyl-CoA, in coupled as well as uncoupled mitochondria. On the other hand, with l-glutamate as substrate, inhibition was only observed under coupled conditions. The finding that acyl-CoA esters inhibit the uncoupler-induced respiration with succinate as substrate but not with glutamate, indicates that the observed inhibitory effect is most probably at the level of the transport of succinate across the mitochondrial membrane as mediated by the mitochondrial dicarboxylate carrier. This conclusion was substantiated by mitochondrial swelling studies, which showed inhibition of succinate transport by the different CoA esters whereas no effect was observed on the phosphate/hydroxyl and glutamate/hydroxyl carriers. Furthermore, long-chain acyl-CoA esters were found to potentiate the inhibitory effect of N-butylmalonate, a known inhibitor of the dicarboxylate carrier, upon oxygen consumption driven by succinate (plus rotenone). We conclude that the inhibitory effects of long-chain acyl-CoA esters on oxidative phosphorylation are dependent on the type of substrate used with the ATP/ADP carrier and the dicarboxylate carrier as targets for inhibition.

Acyl Coenzyme A↗
PubMed1989

The contribution of ATP turnover by the Na+/K+-ATPase to the rate of respiration of hepatocytes. Effects of thyroid status and fatty acids.

In less than 1 min ouabain maximally inhibits oxygen consumption due to gramicidin-induced ATP turnover by the Na+/K+-ATPase in hepatocytes. Ouabain rapidly inhibits respiration on palmitate or glucose by only 6-10% indicating that the Na+/K+-ATPase plays a minor role in cell ATP turnover. 29% of the extra oxygen consumption of hepatocytes isolated from hyperthyroid rats was inhibited by ouabain showing that the Na+/K+-ATPase is responsible for some but not the majority of the stimulation of respiration induced by thyroid hormone.

Adenosine Triphosphate↗
PubMed1998

Polyamine modulation of mitochondrial calcium transport. II. Inhibition of mitochondrial permeability transition by aliphatic polyamines but not by aminoglucosides.

In this study, the effects of polyamines and analogous compounds on mitochondrial permeability transition were characterized to distinguish between these effects and those on mitochondrial Ca2+ uptake, which are described in an accompanying report (Rustenbeck et al., Biochem Pharmacol 8: 977-985, 1998). When a transitional Ca2+ release from Ca2+-loaded mitochondria was induced by an acute increase in Ca2+ concentration in a cytosol-adapted incubation medium (Ca2+ pulse), this process was inhibited, but not abolished by spermine in the concentration range of 0.4 to 20 mM. The aminoglucoside, gentamicin, and the basic polypeptide, poly-L-lysine, which like spermine are able to enhance mitochondrial Ca2+ accumulation (preceding paper), had no or only a minimal inhibitory effect, while the aliphatic polyamine, bis(hexamethylene)triamine, which is unable to enhance mitochondrial Ca2+ accumulation, achieved a complete inhibition at 4 mM. The conclusion that the Ca2+ efflux was due to opening of the permeability transition pore was supported by measurements of mitochondrial membrane potential, ATP production, and oxygen consumption. Mg2+, a known inhibitor of mitochondrial membrane permeability transition, did not mimic the effects of spermine on mitochondrial Ca2+ accumulation, while ADP, the main endogenous inhibitor, showed both effects. However, a combination of spermine and ADP was significantly more effective than ADP alone in restoring low Ca2+ concentrations after a Ca2+ pulse. Two different groups of spermine binding sites were found at intact liver mitochondria, characterized by dissociation constants of 0.5 or 4.7 mM and maximal binding capacities of 4.6 or 19.7 nmol/mg of protein, respectively. In contrast to aminoglucosides, the aliphatic polyamine bis(hexamethylene)triamine did not displace spermine from mitochondrial binding sites. The total intracellular concentration of spermine in hepatocytes was measured to be ca. 450 microM and the free cytoplasmic concentration was estimated to be in the range of 10-100 microM. In conclusion, the enhancement of mitochondrial Ca2+ uptake by spermine is not an epiphenomenon of the inhibition of permeability transition. The physiological role of spermine appears to be that of an enhancer of mitochondrial Ca2+ accumulation rather than an inhibitor of permeability transition.

Aminoglycosides↗
PubMed1997

Effects of carbonylcyanide-m-chlorophenylhydrazone (CCCP) and acetate on Escherichia coli O157:H7 and K-12: uncoupling versus anion accumulation.

Non-growing cells of Escherichia coli O157:H7 and K-12 that were incubated anaerobically in sodium phosphate buffer at pH 6.5 consumed glucose at a rate of approximately 8 mumol.(mg protein)-1.h-1 and had intracellular pH values of 7.3 and 7.5, respectively. The uncoupler, carbonylcyanide-m-chlorophenylhydrazone (CCCP), caused a marked decrease in intracellular pH, ATP and potassium of both strains. Low concentrations of CCCP stimulated glucose consumption rate, but higher concentrations were inhibitory. Acetate also caused a decrease in intracellular pH, but it never caused a large decrease in glucose consumption rate. Acetate decreased the intracellular ATP of E. coli K-12, but it had no effect on the ATP of O157:H7. Acetate had no effect on the intracellular potassium of E. coli O157:H7, and acetate-treated K-12 cells had even more potassium than untreated controls. Based on these results, acetate and CCCP appear to have different effects on E. coli. The comparison of E. coli O157:H7 and K-12 indicated that intracellular pH, acetate accumulation and intracellular potassium were related. E. coli K-12 maintained a higher intracellular pH than O157:H7, accumulated more acetate and had a greater intracellular potassium.

Acetates↗
PubMed2001

Glutathione depletion, lipid peroxidation and mitochondrial dysfunction are induced by chronic stress in rat brain.

Damage to the mitochondrial electron transport chain has been suggested to be an important factor in the pathogenesis of a range of neurodegenerative disorders. We have previously demonstrated that chronic stress induced an increase in nitric oxide (NO) production via an expression of inducible NO synthase (iNOS) in brain. Since it has been demonstrated that NO regulates mitochondrial function, we sought to study the susceptibility of the mitochondrial respiratory chain complexes to chronic restrain stress exposure in brain cortex. In adult male rats, stress (immobilization for six hours during 21 days) inhibits the activities of the first complexes of the mitochondrial respiratory chain (inhibition of 69% in complex I-III and of 67% in complex II-III), without affecting complex IV activity, ATP production and oxygen consumption. The mitochondrial marker citrate synthase is not significantly affected by stress after 21 days, indicating that at this time the mitochondrial structure is still intact. Moreover, the administration of the preferred inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine (400 mg/kg i.p. daily from days 7 to 21 of stress) protects against the inhibition of the activity of complexes of the mitochondrial respiratory chain as well as prevents NO(x)(-) accumulation, lipid peroxidation and glutathione depletion induced by stress. These results suggest that a sustained overproduction of NO via iNOS is responsible, at least in part, of the inhibition of mitochondrial respiratory chain caused by stress and that this pathway also accounts for the oxidative stress found in this situation.

Adenosine Triphosphate↗
PubMed2002

A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity.

We report a systematic RNA interference (RNAi) screen of 5,690 Caenorhabditis elegans genes for gene inactivations that increase lifespan. We found that genes important for mitochondrial function stand out as a principal group of genes affecting C. elegans lifespan. A classical genetic screen identified a mutation in the mitochondrial leucyl-tRNA synthetase gene (lrs-2) that impaired mitochondrial function and was associated with longer-lifespan. The long-lived worms with impaired mitochondria had lower ATP content and oxygen consumption, but differential responses to free-radical and other stresses. These data suggest that the longer lifespan of C. elegans with compromised mitochrondria cannot simply be assigned to lower free radical production and suggest a more complex coupling of metabolism and longevity.

Adenosine Triphosphate↗
PubMed2003

Metabolic therapy in the treatment of ischaemic heart disease: the pharmacology of trimetazidine.

The primary result of myocardial ischaemia is reduced oxygen consumption and adenosine triphosphate (ATP) formation in the mitochondria, and accelerated anaerobic glycolysis, lactate accumulation and cell acidosis. Classic pharmacotherapy for demand-induced ischaemia is aimed at restoring the balance between ATP synthesis and breakdown by increasing the oxygen delivery (i.e. with long acting nitrates or Ca2+ channel antagonist) or by decreasing cardiac power by reducing blood pressure and heart rate (i.e. with beta-blocker or Ca2+ channel antagonist). Animal studies show that fatty acids are the primary mitochondrial substrate during moderate severity myocardial ischaemia, and that they inhibit the oxidation of carbohydrate and drive the conversion of pyruvate to lactate. Drugs that partially inhibit myocardial fatty acid oxidation increase carbohydrate oxidation, which results in reduced lactate production and a higher cell pH during ischaemia. Trimetazidine (1-[2,3,4-trimethoxibenzyl]-piperazine) is the first and only registered drug in this class, and is available in over 90 countries world-wide. Trimetazidine selectively inhibits the fatty acid beta-oxidation enzyme 3-keto-acyl-CoA dehydrogenase (3-KAT), and is devoid of any direct haemodynamic effects. In double-blind placebo-controlled trials trimetazidine significantly improved symptom-limited exercise performance in stable angina patients when used either as monotherapy or in combination with beta-blockers or Ca2+ channel antagonists. Given available evidence, trimetazidine is an excellent alternative to classic haemodynamic agents, and is unique in its ability to reduce symptoms of angina when used in patients resistant to a haemodynamic treatment as vasodilators, beta-blockers or Ca2+ channel antagonists.

Angina Pectoris↗
PubMed2003

Evidence that tyrphostins AG10 and AG18 are mitochondrial uncouplers that alter phosphorylation-dependent cell signaling.

Receptor agonists that initiate fluid secretion in salivary gland epithelial cells also increase protein phosphorylation. To assess contributions of tyrosine phosphorylation to secretion, changes in muscarinic receptor-initiated secretion (estimated from sodium pump-dependent increases in oxygen consumption) were measured in parotid acinar cells exposed to tyrosine kinase inhibitors. However, like the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, tyrphostins AG10 and AG18 increased the rate of oxygen consumption and reduced cellular ATP by approximately 90% in the absence of the muscarinic agonist carbachol, indicating that these tyrphostins uncouple mitochondria. Exposure of isolated mitochondria to five structurally related tyrphostins demonstrated that their relative potencies as uncouplers differed from their in vitro kinase-inhibitory potencies due to different molecular requirements for the two effects. AG10 and AG18 blocked parotid phosphorylation events only at concentrations that reduced ATP content. The tyrosine kinase inhibitor genistein reduced ATP content by 15-20% and weakly uncoupled isolated mitochondria, but its inhibition of carbachol-mediated protein kinase Cdelta tyrosine phosphorylation and ERK1/2 activation appeared attributable to blocking tyrosine kinases directly. Carbachol itself rapidly reduced ATP content by 15-20%. Carbachol, 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (P2X(7) receptor agonist), AG10, AG18, and carbonyl cyanide p-trifluoromethoxyphenyl hydrazone rapidly activated the fuel sensor AMP-activated protein kinase (AMPK); however, only AMPK activation by carbachol and BzATP was due to sodium pump stimulation. AG10 and AG18 also activated AMPK and/or uncoupled mitochondria in PC12, HeLa, and HEK293 cells. These studies demonstrate that some tyrosine kinase inhibitors produce cellular effects that are mechanistically different from their primary in vitro characterizations and, as do salivary secretory stimuli, promote rapid metabolic alterations that initiate secondary signaling events.

Animals↗
PubMed2006

Analysis of growth of Lactobacillus plantarum WCFS1 on a complex medium using a genome-scale metabolic model.

A genome-scale metabolic model of the lactic acid bacterium Lactobacillus plantarum WCFS1 was constructed based on genomic content and experimental data. The complete model includes 721 genes, 643 reactions, and 531 metabolites. Different stoichiometric modeling techniques were used for interpretation of complex fermentation data, as L. plantarum is adapted to nutrient-rich environments and only grows in media supplemented with vitamins and amino acids. (i) Based on experimental input and output fluxes, maximal ATP production was estimated and related to growth rate. (ii) Optimization of ATP production further identified amino acid catabolic pathways that were not previously associated with free-energy metabolism. (iii) Genome-scale elementary flux mode analysis identified 28 potential futile cycles. (iv) Flux variability analysis supplemented the elementary mode analysis in identifying parallel pathways, e.g. pathways with identical end products but different co-factor usage. Strongly increased flexibility in the metabolic network was observed when strict coupling between catabolic ATP production and anabolic consumption was relaxed. These results illustrate how a genome-scale metabolic model and associated constraint-based modeling techniques can be used to analyze the physiology of growth on a complex medium rather than a minimal salts medium. However, optimization of biomass formation using the Flux Balance Analysis approach, reported to successfully predict growth rate and by product formation in Escherichia coli and Saccharomyces cerevisiae, predicted too high biomass yields that were incompatible with the observed lactate production. The reason is that this approach assumes optimal efficiency of substrate to biomass conversion, and can therefore not predict the metabolically inefficient lactate formation.

Culture Media, Conditioned↗