Status of mitochondria in living human fibroblasts during growth and senescence in vitro: use of the laser dye rhodamine 123

Rhodamine 123, a fluorescent laser dye that is selectively taken up into mitochondria of living cells, was used to examine mitochondrial morphology in early-passage (young), late-passage (old), and progeric human fibroblasts. Mitochondria were readily visualized in all cell types during growth (mid-log) and confluent stages. In all cell strains at confluence, mitochondria became shorter, more randomly aligned, and developed a higher proportion of bead-like forms. Treatment of cells for six days with Tevenel, a chloramphenicol analog that inhibits mitochondrial protein synthesis, brought about a marked depletion of mitochondria and a diffuse background fluorescence. Cyanide produced a rapid release of preloaded mitochondrial fluorescence followed by detachment and killing of cells. Colcemid caused a random coiling and fragmentation of mitochondria particularly in the confluent stage. No gross differences were discernible in mitochondria of the three cell strains in mid-log and confluent states or after these treatments. Butanol-extractable fluorescence after loading with rhodamine 123 was lower in all cell strains in confluent compared to mid-log stages. At confluence all three cell strains had similar rhodamine contents at zero-time and after washout up to 24 h. At the mid-log stage, young cells contained more rhodamine initially and lost it more rapidly than old or progeria cells, in that order. The data indicate no gross derangement in the morphology or number of mitochondria in old and progeria fibroblasts but there is a reduction of protonmotive force evident in these cells at the mid-log stage that may be growth limiting.     

Cell energy metabolism: An update

In living cells, growth is the result of coupling between substrate catabolism and multiple metabolic processes that take place during net biomass formation and maintenance processes. During growth, both ATP/ADP and NADH/NAD⁺ molecules play a key role. Cell energy metabolism hence refers to metabolic pathways involved in ATP synthesis linked to NADH turnover. Two main pathways are thus involved in cell energy metabolism: glycolysis/fermentation and oxidative phosphorylation. Glycolysis and mitochondrial oxidative phosphorylation are intertwined through thermodynamic and kinetic constraints that are reviewed herein. Further, our current knowledge of short-term and long term regulation of cell energy metabolism will be reviewed using examples such as the Crabtree and the Warburg effect.     

Phenylalanyl synthetase function in cultured fibroblasts from subjects with progeria.

Since progeria cells contain a diversity of altered proteins, some aspects of phenylalanyl synthetase function were examined in semipurified extracts of cultured skin fibroblasts using mixed rabbit tRNA as acceptor. No significant differences were found in the Km and Vmax for phenylalanine or ATP in progeria cells compared with controls. Initial velocities of both progeria and control synthetases were lower at late passage owing to either reduced enzyme content or reduced catalytic efficiency. Reverse phase 5 chromatography of tRNAs acylated by progeria and control synthetases gave a single peak of labeled phenylalanine tRNA in all cases with no secondary peaks evident. Total activity of phenylalanyl synthetase in progeria cells was similar to that of control cells at early passage while late-passage control cells had lower specific activities of these synthetases per unit protein.     

Changes of serum-induced ornithine decarboxylase activity and putrescine content during aging of IMR-90 human diploid fibroblasts

The roles of ornithine decarboxylase (ODC, EC 4.1.1.17) and polyamines in cellular aging were investigated by examining serum-induced changes of these parameters in quiescent IMR-90 human diploid fibroblasts as a function of their population doubling level (PDL) and in human progeria fibroblasts. Serum stimulation caused increases of ODC and DNA synthesis in IMR-90 human diploid fibroblasts, with maximal values occurring, respectively, 10 hr and 22 hr after serum stimulation. Both serum-induced ODC activity and DNA synthesis in IMR-90 cells were found to be inversely related to their PDL. Maximal ODC activity and DNA synthesis in young cells (PDL = approximately 18-22) were, respectively, five-fold and six-fold greater than that in old cells (PDL = approximately 50-55), which in turn were comparable or slightly higher than that in progeria fibroblasts. Polyamine contents (putrescine, spermidine, and spermine) in quiescent IMR-90 cells did not show significant PDL-dependency. The putrescine and spermine contents in quiescent progeria cells were comparable to those in quiescent IMR-90 cells. The spermidine content in quiescent progeria cells, however, was extremely low, less than half of that in quiescent IMR-90 cells. Serum stimulation caused a marked increase in putrescine content in young cells but not in old cells or in progeria cells. The spermidine and the spermine content in IMR-90 cells, either young or old, and in progeria cells did not change significantly after serum stimulation. Our study indicated that aging of IMR-90 human diploid fibroblasts was accompanied by specific changes of polyamine metabolism, namely, the serum-induced ODC activity and putrescine accumulation. These changes were also observed in progeria fibroblasts derived from patients with Hutchinson-Gilford syndrome.     

Studies of DNA polymerases alpha and beta from cultured human cells in various replicative states

DNA polymerase activities from HeLa cells and from cultured diploid human fibroblasts in various growth states were compared. alpha-Polymerase activities from log phase fibroblasts treated with sodium butyrate and from stationary phase HeLa cells had DEAE-cellulose elution patterns that differed from those of polymerases from dividing cells. Moreover, alpha- and beta-polymerases from nondividing cells replicated synthetic polymers less faithfully. Although similar changes were observed previously for polymerases from late-passage and postconfluent early passage fibroblasts, amounts of alpha-polymerase activity recovered from nondividing cells in this study did not dramatically decline as they had in the former cases. The alpha-polymerase activities from HeLa cells and fibroblasts in various growth states sedimented near 7.5S in 0.4 M KCI and could be inhibited by a monoclonal IgG fraction prepared against KB cell alpha-polymerase. By several criteria, there was no significant differences in levels of UV-stimulated repair synthesis observed in early or late-passage postconfluent fibroblasts or in log phase fibroblasts treated with sodium butyrate. In summary, levels of alpha-polymerase do not necessarily correlate either with replicative activity or with apparent levels of repair synthesis. However, cells with decreased replicative activity always yielded enzyme with decreased fidelity in vitro and altered chromatographic behavior. It appears, therefore, that the alterations observed for alpha-polymerase from late-passage cells may be attributed more generally to the nondividing nature of these cells.     

Energetic and Morphological Plasticity of C6 Glioma Cells Grown on 3-D Support; Effect of Transient Glutamine Deprivation

The energetic metabolism of rat C6 glioma cells has been investigated as a function of the proliferative and differentiation states under three-dimensional (3-D) growing conditions on microcarrier beads. First, the transient deprivation of glutamine from the culture medium induced a marked decrease in the growth rate and a differentiation of C6 cells through the oligodendrocytic phenotype. Second, the respiratory capacity of the C6 cells during short-term subcultures with or without glutamine continuously declined as a function of the cell density, in part due to the mitochondrial content decrease. During the transition from the early exponential to the plateau growth phase in glutamine-containing medium, the oxygen consumption rate per single cell decreased concomitantly with a decrease in the glucose consumption and lactate production rates. This phenomenon led to a sixfold decrease in the total ATP production flux, without significantly affecting the cellular ATP/ADP ratio, thus indicating that some ATP-consuming processes were simultaneously suppressed during C6 proliferation. In glutamine-free medium, the cellular ATP/ADP ratio transiently increased due to growth arrest and to a reduced ATP turnover. Moreover, the results indicated that glutamine is not an essential respiratory substrate for rat C6 glioma under short-term glutamine deprivation. Worth noting was the high contribution of the mitochondrial oxidative phosphorylation toward the total ATP synthesis (about 80%), regardless of the proliferation or the differentiation status of the C6 cells.     

Interdependence of mitochondrial ATP production and extramitochondrial ATP utilization in intact spermatozoa

The dependence of both respiration and total activity of ATP-consuming reactions on the cellular adenine nucleotide pattern was investigated in intact bovine spermatozoa. ATP consumption was manipulated by inhibition with vanadate and activation with caffeine, leading to a decrease or increase in the rate of respiration up to 70% or 20%, respectively. Oligomycin blocked the respiration to the same extent as did vanadate, suggesting that the total extramitochondrial ATP-consuming activity is vanadate-sensitive. The major part of ATP utilization must be linked to dynein ATPase, since inhibition of (Na⁺, K⁺) ATPase by ouabain showed only a small effect on respiration (−17%). Being a potent inhibitor of dynein ATPase, vanadate drastically reduced the amount of motile cells, whereas caffeine tended to increase the intensity of motion. The effects of vanadate or caffeine on respiration were paralleled by changes in cellular ATP, reflecting the response of mitochondrial respiration on the cellular ATP/ADP ratio. Respiration was found to depend on changes in the ATP/ADP ratio in the range from about 3 (+ caffeine) to 9 (+ vanadate). The range of response of ATP consumption to the ATP/ADP ratio was determined by varying the mitochondrial ATP production via the concentration of lactate which was used as substrate. The measured effects on both respiratory rate and ATP/ADP ratio suggested that ATP consumption was markedly dependent on ATP/ADP ratios below 5. It is concluded that lactate concentrations above 1 mM sufficiently supply bovine spermatozoa with substrate and the energy turnover is mainly limited by the activity of dynein ATPase rather than by the capacity of mitochondrial oxidative phosphorylation.     

Effects of age on energy status and redox state of lympocytes during blastogenesis

The concentrations of the adenine and pyridine nucleotides were determined during mitogen-induced blastogenesis of human peripheral lymphocytes from young and old subjects. Significant differences were found in the energy status (ATP/ADP) and levels of the pyridine nucleotides. While lymphocytes from young subjects maintained a relatively constant energy status over 72 hours, the energy status decreased in both stimulated and control cells from old subjects. In addition, lymphocytes from young subjects show a marked increase in both oxidized and reduced NAD during blastogenesis. In contrast, cells from old subjects are not capable of increasing NAD levels.     

Age-related changes in ATP-producing pathways in human skeletal muscle in vivo.

Energy for muscle contractions is supplied by ATP generated from 1) the net hydrolysis of phosphocreatine (PCr) through the creatine kinase reaction, 2) oxidative phosphorylation, and 3) anaerobic glycolysis. The effect of old age on these pathways is unclear. The purpose of this study was to examine whether age may affect ATP synthesis rates from these pathways during maximal voluntary isometric contractions (MVIC). Phosphorus magnetic resonance spectroscopy was used to assess high-energy phosphate metabolite concentrations in skeletal muscle of eight young (20-35 yr) and eight older (65-80 yr) men. Oxidative capacity was assessed from PCr recovery after a 16-s MVIC. We determined the contribution of each pathway to total ATP synthesis during a 60-s MVIC. Oxidative capacity was similar across age groups. Similar rates of ATP synthesis from PCr hydrolysis and oxidative phosphorylation were observed in young and older men during the 60-s MVIC. Glycolytic flux was higher in young than older men during the 60-s contraction (P < 0.001). When expressed relative to the overall ATP synthesis rate, older men relied on oxidative phosphorylation more than young men (P = 0.014) and derived a smaller proportion of ATP from anaerobic glycolysis (P < 0.001). These data demonstrate that although oxidative capacity was unaltered with age, peak glycolytic flux and overall ATP production from anaerobic glycolysis were lower in older men during a high-intensity contraction. Whether this represents an age-related limitation in glycolytic metabolism or a preferential reliance on oxidative ATP production remains to be determined.     

An increase in the ATP levels occurs in cerebellar granule cells en route to apoptosis in which ATP derives from both oxidative phosphorylation and anaerobic glycolysis

Although it is recognized that ATP plays a part in apoptosis, whether and how its level changes en route to apoptosis as well as how ATP is synthesized has not been fully investigated. We have addressed these questions using cultured cerebellar granule cells. In particular, we measured the content of ATP, ADP, AMP, IMP, inosine, adenosine and L-lactate in cells undergoing apoptosis during the commitment phase (0-8 h) in the absence or presence of oligomycin or/and of citrate, which can inhibit totally the mitochondrial oxidative phosphorylation and largely the substrate-level phosphorylation in glycolysis, respectively. In the absence of inhibitors, apoptosis was accompanied by an increase in ATP and a decrease in ADP with 1:1 stoichiometry, with maximum ATP level found at 3 h apoptosis, but with no change in levels of AMP and its breakdown products and with a relatively low level of L-lactate production. Consistently, there was an increase in the cell energy charge and in the ratio ([ATP][AMP])/[ADP](2). When the oxidative phosphorylation was completely blocked by oligomycin, a decrease of the ATP content was found both in control cells and in cells undergoing apoptosis, but nonetheless cells still died by apoptosis, as shown by checking DNA laddering and by death prevention due to actinomycin D. In this case, ATP was provided by anaerobic glycolysis, as suggested by the large increase of L-lactate production. On the other hand, citrate itself caused a small decrease in ATP level together with a huge decrease in L-lactate production, but it had no effect on cell survival. When ATP level was further decreased due to the presence of both oligomycin and citrate, death occurred via necrosis at 8 h, as shown by the lack of DNA laddering and by death prevention found due to the NMDA receptor antagonist MK801. However, at a longer time, when ATP level was further decreased, cells died neither via apoptosis nor via glutamate-dependent necrosis, in a manner similar to something like to energy catastrophe. Our results shows that cellular ATP content increases in cerebellar granule cell apoptosis, that the role of oxidative phosphorylation is facultative, i.e. ATP can also derive from anaerobic glycolysis, and that the type of cell death depends on the ATP availability.     

An increase in the ATP levels occurs in cerebellar granule cells en route to apoptosis in which ATP derives from both oxidative phosphorylation and anaerobic glycolysis

Although it is recognized that ATP plays a part in apoptosis, whether and how its level changes en route to apoptosis as well as how ATP is synthesized has not been fully investigated. We have addressed these questions using cultured cerebellar granule cells. In particular, we measured the content of ATP, ADP, AMP, IMP, inosine, adenosine and l-lactate in cells undergoing apoptosis during the commitment phase (0-8 h) in the absence or presence of oligomycin or/and of citrate, which can inhibit totally the mitochondrial oxidative phosphorylation and largely the substrate-level phosphorylation in glycolysis, respectively. In the absence of inhibitors, apoptosis was accompanied by an increase in ATP and a decrease in ADP with 1:1 stoichiometry, with maximum ATP level found at 3 h apoptosis, but with no change in levels of AMP and its breakdown products and with a relatively low level of l-lactate production. Consistently, there was an increase in the cell energy charge and in the ratio ([ATP][AMP])/[ADP]². When the oxidative phosphorylation was completely blocked by oligomycin, a decrease of the ATP content was found both in control cells and in cells undergoing apoptosis, but nonetheless cells still died by apoptosis, as shown by checking DNA laddering and by death prevention due to actinomycin D. In this case, ATP was provided by anaerobic glycolysis, as suggested by the large increase of l-lactate production. On the other hand, citrate itself caused a small decrease in ATP level together with a huge decrease in l-lactate production, but it had no effect on cell survival. When ATP level was further decreased due to the presence of both oligomycin and citrate, death occurred via necrosis at 8 h, as shown by the lack of DNA laddering and by death prevention found due to the NMDA receptor antagonist MK801. However, at a longer time, when ATP level was further decreased, cells died neither via apoptosis nor via glutamate-dependent necrosis, in a manner similar to something like to energy catastrophe. Our results shows that cellular ATP content increases in cerebellar granule cell apoptosis, that the role of oxidative phosphorylation is facultative, i.e. ATP can also derive from anaerobic glycolysis, and that the type of cell death depends on the ATP availability.     

Brown adipose tissue mitochondria: recoupling caused by substrate level phosphorylation and extramitochondrial adenosine phosphates.

1. Uncoupled oxidative phosphorylation in isolated guinea pig brown-adipose-tissue mitochondria is reflected by a low phosphorylation state of adenosine phosphates in the mitochondrial matrix and in the extramitochondrial space during oxidation of succinate or glycerol 1-phosphate in the presence of serum albumin and 100 muM ADP. Recoupling of respiration and phosphorylation in the mitochondria is indicatdd by a dramatic increase in the phosphorylation state of adenine nucleotides in both compartments, when substrates inducing substrate level phosphorylation are respired. In this case ATP/ADP ratios in the extramitochondrial compartment are 10-15 times higher than in the mitochondrial matrix. 2. Recoupling mediated by substrate level phosphorylation depends on the presence of extramitochondrial adenosine phosphate and on intact adenine nucleotide translocation. In the presence of substrate level phosphorylation the amount of extramitochondrial ADP required to restore energy coupling can be extremely low (20 muM ADP or 10 nmol ADP/mg mitochondrial protein respectively). If substrate level phosphorylation is prevented by rotenone or in the presence of atractyloside, 20-50 times higher amounts of extramitochondrial adenine nucleotides are necessary to cause coupled oxidative phosphorylation. The recoupling effect of ATP is significantly stronger than that of ADP. 3. GDP (100 muM) causes a rapid increase of the ATP/ADP ratio in both compartments which is independent of substrate level phosphorylation as well as of the extramitochondrial adenosine phosphate concentration and the adenine nucleotide carrier. 4. The amount of extramitochondrial adenosine phosphate in guinea pig brown-adipose-tissue (18 nmol/mg mitochondrial protein or 2.5 mM respectively) would suffice for recoupling of oxidative phosphorylation mediated by substrate level phosphorylation under conditions in vitro; this suggests that substrate level phosphorylation is of essential importance in brown fat in vivo with respect to energy conditions in the tissue during different states of thermogenesis.     

Energy metabolism transition in multi-cellular human tumor spheroids

It is thought that glycolysis is the predominant energy pathway in cancer, particularly in solid and poorly vascularized tumors where hypoxic regions develop. To evaluate whether glycolysis does effectively predominate for ATP supply and to identify the underlying biochemical mechanisms, the glycolytic and oxidative phosphorylation (OxPhos) fluxes, ATP/ADP ratio, phosphorylation potential, and expression and activity of relevant energy metabolism enzymes were determined in multi-cellular tumor spheroids, as a model of human solid tumors. In HeLa and Hek293 young-spheroids, the OxPhos flux and cytochrome c oxidase protein content and activity were similar to those observed in monolayer cultured cells, whereas the glycolytic flux increased two- to fourfold; the contribution of OxPhos to ATP supply was 60%. In contrast, in old-spheroids, OxPhos, ATP content, ATP/ADP ratio, and phosphorylation potential diminished 50-70%, as well as the activity (88%) and content (3 times) of cytochrome c oxidase. Glycolysis and hexokinase increased significantly (both, 4 times); consequently glycolysis was the predominant pathway for ATP supply (80%). These changes were associated with an increase (3.3 times) in the HIF-1alpha content. After chronic exposure, both oxidative and glycolytic inhibitors blocked spheroid growth, although the glycolytic inhibitors, 2-deoxyglucose and gossypol (IC(50) of 15-17 nM), were more potent than the mitochondrial inhibitors, casiopeina II-gly, laherradurin, and rhodamine 123 (IC(50) > 100 nM). These results suggest that glycolysis and OxPhos might be considered as metabolic targets to diminish cellular proliferation in poorly vascularized, hypoxic solid tumors.     

Adenylate energy charge of rat and human cultured hepatocytes

Summary A simple and rapid method for the assay of adenine nucleotides (ATP, ADP, and AMP) was established to evaluate the adenylate energy charge (ATP+ADP/2)/(ATP+ADP+AMP) of cultured hepatocytes. The effects of inhibitors of glycolysis, fatty acid oxidation, or oxidative phosphorylation on the energy charge were examined. The energy charges of cultured hepatocytes in rats and human were almost identical and were maintained at a high level between 6 and 24 h after changing the media (rat: 0.908±0.008n=9, human: 0.918±0.014n=6, mean ± SD). Inhibition of glycolysis with sodium fluoride or oxidative phosphorylation with antimycin A irreversibly reduced both the adenine nucleotide contents and the energy charge. However, the inhibition of fatty acid oxidation with 2-tetradecylglycidic acid did not affect the nucleotide contents, and the energy charge only decreased transiently to recover within 8 h. When the inhibitor of oxidative phosphorylation was removed, the recovery in the energy charge preceded the recovery in the adenine nucleotide contents. These findings suggest that the adenylate energy charge is a more sensitive measure of the changes in energy metabolism than the adenine nucleotide contents. Furthermore, energy charge regulates adenine nucleotide contents in cultured hepatocytes. It is important to confirm that the high energy charge of the cultured hepatocytes is maintained when these cells are used for metabolic studies.     

Uridine, but not cytidine can sustain growth of Ehrlich ascites tumor cells in glucose-deprived medium with altered proliferation kinetics

Cell cycle kinetics and energy metabolism of Ehrlich ascites tumor cells grown in glucose-deprived medium supplemented with uridine, were investigated in order to extend our knowledge about the significance of the metabolic conversion of glucose for cell cycle progression of these cells. Viability (dye exclusion test) of uridine supplemented cells was not affected, although growth was reduced to 50 to 60% of the controls. Uridine did not significantly impair growth of controls in standard medium up to 20 mM. Studies on cell cycle progression using flow cytometry (BrdU-H33258 technique) revealed an accumulation of cells in G2M phase, which can be explained by a delay in the passage of G2M-compartment of about 12 +/- 2 h. After a 24 h culture period, 60% of the cell populations were found in G2M (30% in control cultures). This fraction increased to about 70% in the following passage. Protein and DNA synthesis corresponded to the proliferation rate. Oxygen uptake was increased by about 50%, glutamine consumption by 30%, lactate production was reduced to below 10% of the controls. The ATP/ADP concentration ratio was found in a physiological range of 4 to 6. It was calculated that cells grown in standard medium produced 60% of ATP via oxidative pathways and 40% via glycolysis; however, in glucose-free, uridine-supplemented medium the values are more than 90% and less than 10%. No significant differences in total ATP production were observed. Growth of the cells in glucose-deprived medium could not be sustained by cytidine. All our data substantiate the present concept that glycolytic ATP-production is not essential for maintenance of viability and growth of these cells.     

Hexokinase of rat brain mitochondria: relative importance of adenylate kinase and oxidative phosphorylation as sources of substrate ATP, and interaction with intramitochondrial compartments of ATP and ADP

Interactions between intramitochondrial ATP-generating, ADP-requiring processes and ATP-requiring, ADP-generating phosphorylation of glucose by mitochondrially bound hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) have been investigated using well-coupled mitochondria isolated from rat brain. ADP generated by mitochondrially bound hexokinase was more effective at stimulating respiration than was ADP generated by hexokinase dissociated from the mitochondria, and pyruvate kinase was less effective as a scavenger of ADP generated by the mitochondrially bound hexokinase than was the case with ADP generated by the dissociated enzyme. These results indicate that ADP generated by the mitochondrially bound enzyme is at least partially sequestered and directed toward the mitochondrial oxidative phosphorylation apparatus. Under the conditions of these experiments, the maximum rate of ATP production by oxidative phosphorylation was approximately 10-fold greater than the maximum rate of ATP generation by the adenylate kinase reaction. Moreover, during periods of active oxidative phosphorylation, adenylate kinase made no detectable contribution to ATP production. Thus, adenylate kinase does not represent a major source of ATP for hexokinase bound to actively phosphorylating brain mitochondria. With adenylate kinase as the sole source of ATP, a steady state was attained in which ATP formation was balanced by utilization in the hexokinase reaction. In contrast, when oxidative phosphorylation was the source of ATP, a steady state rate of Glc phosphorylation was attained, but it was equivalent to only about 40-50% of the rate of ATP production and thus there was a continued net increase in ATP concentration in the system. Rates of Glc phosphorylation with ATP generated by oxidative phosphorylation exceeded those seen with equivalent levels of exogenously added ATP. Moreover, at total ATP concentrations greater than approximately 0.2 mM, hexokinase bound to actively phosphorylating mitochondria was unresponsive to continued slow increases in ATP levels; acute increase in ATP (by addition of exogenous nucleotide) did, however, result in increased hexokinase activity. The relative insensitivity of mitochondrially bound hexokinase to extramitochondrial ATP suggested dependence on an intramitochondrial pool (or pools) of ATP during active oxidative phosphorylation. Two intramitochondrial compartments of ATP were identified based on their selective release by inhibitors of electron transport or oxidative phosphorylation. These compartments were distinguished by their sensitivity to inhibitors and the kinetics with which they were filled with ATP generated by oxidative phosphorylation. Exogenous glycerol kinase competed effectively with mitochondrially bound hexokinase for extramitochondrial ATP, with relatively low levels of glycerol kinase completely inhibiting phosphorylation of Glc.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of 2-tetradecylglycidic acid on rat platelet energy metabolism and aggregation.

We investigated the role of energy supplied by long-chain fatty acid oxidation in rat platelet function. Inhibition of the mitochondrial uptake of long-chain fatty acids was achieved by treating rats with 2-tetradecylglycidic acid (TDGA), a potent inhibitor of the overt form of carnitine palmitoyltransferase (CPT-I). The maximum aggregation rate (MAR), CPT-I activity, lactate production, oxygen consumption and adenine nucleotide content of isolated rat platelets were then studied in vitro. 4 h after the in vivo administration of TDGA, the CPT-I activity in saponin-permeabilized platelets was nearly completely inhibited along with a significant reduction in the MAR induced by ADP, thrombin and ionophore A23187. The ATP level, adenylate energy charge (ATP + 1/2 ADP)/(ATP + ADP + AMP) and ATP/ADP ratio in the platelet cytoplasmic pool were also reduced. Platelets from TDGA-treated rats showed lower oxygen consumption rates in both the basal respiratory and oxygen burst states. These results indicate that mitochondrial long-chain fatty acid oxidation coupled to oxidative phosphorylation is an important energy source in rat platelets and is probably involved in the maintenance of platelet function. Enhanced in vitro lactate production in platelets from TDGA-treated rats may have resulted from a compensatory increase in glycolysis which only partly compensated for impaired long-chain fatty acid oxidation.     

A critical appraisal of the association between energy charge and cell damage

The association between the energy charge and cellular damage caused by metabolic inhibitors was investigated in a cellular system of quiescent fibroblasts. The cell damage was assessed by the release of lactate dehydrogenase (LDH) which indicates a severe change of membrane integrity. Inhibition of glycolysis resulted in release of LDH when the energy charge decreased below 0.5 at an ATP level of 10% of the original level. If oxidative phosphorylation was inhibited, the energy charge decreased to 0.1-0.35 (dependent on the type of inhibitor) a long time before release of LDH, and no change occurred in the energy charge when release of LDH started. The ATP level was 0.5-2% of the original at this time. Even a decrease of the energy charge to 0.1 could be reversed to a normal level, and at the same time the morphological cellular changes were fully reversed and no release of LDH occurred. The conclusion is that no simple correlation between energy charge and cell survival exists. The different levels of ATP at which release of LDH started after inhibition of glycolysis and oxidative phosphorylation indicate a special role of glycolysis in maintaining the membrane function and integrity. This was emphasized by measuring the potassium loss of the cells which was much more marked after inhibition of glycolysis.