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PubMed1998

Regulation of ATP supply during muscle contraction: theoretical studies.

The dynamic computer model of oxidative phosphorylation developed previously and successfully tested for large-scale changes in fluxes and metabolite concentrations was used to study the question of how the rate of ATP production by oxidative phosphorylation is adjusted to meet the energy demand during muscle contraction, which causes a great increase in ATP consumption in relation to the resting state. The changes in the respiration rate and ATP/ADP ratio after the onset of maximal work measured experimentally were compared with simulated changes in the respiration rate and ATP/ADP in several different cases, assuming direct activation of different steps by an external effector. On the basis of the computer simulations performed, it was possible to conclude which enzymes/metabolic blocks should be directly activated to cause the experimentally observable changes in fluxes and metabolite concentrations. The theoretical results obtained suggest that the parallel direct activation of actinomyosin-ATP-ase and oxidative phosphorylation by an external effector (for example calcium ions) is the main mechanism responsible for fitting of ATP production to ATP consumption, while the negative feedback via an increase in ADP concentration (decrease in ATP/ADP), which indirectly activates the ATP supply, plays only a minor role. Additionally, the conclusion is drawn that most of the oxidative phosphorylation steps should be directly activated in order to explain the observed changes in the respiration rate and ATP/ADP ratio (and also in other parameters) during muscle contraction. It is suggested that there should exist a universal external activator/regulatory mechanism which causes a parallel stimulation of different enzymes/processes. A possible nature of such an activator is shortly discussed.

Adenosine Diphosphate↗
PubMed2000

The distribution of rest periods affects performance and adaptations of energy metabolism induced by high-intensity training in human muscle.

The effect of the distribution of rest periods on the efficacy of interval sprint training is analysed. Ten male subjects, divided at random into two groups, performed distinct incremental sprint training protocols, in which the muscle load was the same (14 sessions), but the distribution of rest periods was varied. The 'short programme' group (SP) trained every day for 2 weeks, while the 'long programme' group (LP) trained over a 6-week period with a 2-day rest period following each training session. The volunteers performed a 30-s supramaximal cycling test on a cycle ergometer before and after training. Muscle biopsies were obtained from the vastus lateralis before and after each test to examine metabolites and enzyme activities. Both training programmes led to a marked increase (all significant, P < 0.05) in enzymatic activities related to glycolysis (phosphofructokinase - SP 107%, LP 68% and aldolase - SP 46%, LP 28%) and aerobic metabolism (citrate synthase - SP 38%, LP 28.4% and 3-hydroxyacyl-CoA dehydrogenase - SP 60%, LP 38.7%). However, the activity of creatine kinase (44%), pyruvate kinase (35%) and lactate dehydrogenase (45%) rose significantly (P < 0.05) only in SP. At the end of the training programme, SP had suffered a significant decrease in anaerobic ATP consumption per gram muscle (P < 0.05) and glycogen degradation (P < 0.05) during the post-training test, and failed to improve performance. In contrast, LP showed a marked improvement in performance (P < 0.05) although without a significant increase in anaerobic ATP consumption, glycolysis or glycogenolysis rate. These results indicate that high-intensity cycling training in 14 sessions improves enzyme activities of anaerobic and aerobic metabolism. These changes are affected by the distribution of rest periods, hence shorter rest periods produce larger increase in pyruvate kinase, creatine kinase and lactate dehydrogenase. However, performance did not improve in a short training programme that did not include days for recovery, which suggests that muscle fibres suffer fatigue or injury.

Adaptation, Physiological↗
PubMed2006

Measurement of activation energy and oxidative phosphorylation onset kinetics in isolated muscle fibers in the absence of cross-bridge cycling.

This study utilized N-benzyl-p-toluene sulfonamide (BTS), a potent inhibitor of cross-bridge cycling, to measure 1) the relative metabolic costs of cross-bridge cycling and activation energy during contraction, and 2) oxygen uptake kinetics in the presence and absence of myosin ATPase activity, in isolated Xenopus laevis muscle fibers. Isometric tension development and either cytosolic Ca2+ concentration ([Ca2+]c) or intracellular Po2 (PiO2) were measured during contractions at 20 degrees C in control conditions (Con) and after exposure to 12.5 microM BTS. BTS attenuated tension development to 5+/-0.4% of Con but did not affect either resting or peak [Ca2+]c during repeated isometric contractions. To determine the relative metabolic cost of cross-bridge cycling, we measured the fall in PiO2) (DeltaPiO2; a proxy for Vo2) during contractions in Con and BTS groups. BTS attenuated DeltaP(iO2) by 55+/-6%, reflecting the relative ATP cost of cross-bridge cycling. Thus, extrapolating DeltaPiO2 to a value that would occur at 0% tension suggests that actomyosin ATP requirement is approximately 58% of overall ATP consumption during isometric contractions in mixed fiber types. BTS also slowed the fall in PiO2) (time to 63% of overall DeltaPiO2) from 75+/-9 s (Con) to 101+/-9 s (BTS) (P<0.05), suggesting an important role of the products of ATP hydrolysis in determining the Vo2 onset kinetics. These results demonstrate in isolated skeletal muscle fibers that 1) activation energy accounts for a substantial proportion (approximately 42%) of total ATP cost during isometric contractions, and 2) despite unchanged [Ca2+]c transients, a reduced rate of ATP consumption results in slower Vo2 onset kinetics.

Adenosine Triphosphate↗
PubMed1986

ATP keeps exocytosis sites in a primed state but is not required for membrane fusion: an analysis with Paramecium cells in vivo and in vitro.

We have tried to specify a widespread hypothesis on the requirement of ATP for exocytosis (membrane fusion). With Paramecium tetraurelia cells, synchronously (approximately 1 s) exocytosing trichocysts, ATP pools have been measured in different strains, including wild type cells, "non-discharge" (nd), "trichless" (tl), and other mutations. The occurrence of a considerable and rapid ATP consumption also in nd and tl mutations as well as its time course (with a maximum 3-5 s after exocytosis) in exocytosis-competent strains does not match the actual extent of exocytosis performance. However, from in vivo as well as from in vitro experiments, we came to the conclusion that ATP might be required to keep the system in a primed state and its removal might facilitate membrane fusion. (For the study of exocytosis in vitro we have developed a new system, consisting of isolated cortices). In vivo as well as in vitro exocytosis is inhibited by increased levels of ATP or by a nonhydrolyzable ATP analogue. In vitro exocytosis is facilitated in ATP-free media. In vivo-microinjected ATP retards exocytosis in response to chemical triggers, whereas microinjected apyrase triggers exocytosis without exogenous trigger. Experiments with this system also largely exclude any overlaps with other processes that normally accompany exocytosis. Our data also explain why it was frequently assumed that ATP would be required for exocytosis. We conclude that membrane fusion during exocytosis does not require the presence of ATP; the occurrence of membrane fusion might involve the elimination of ATP from primed fusogenic sites; most of the ATP consumption measured in the course of exocytosis may be due to other effects, probably to recovery phenomena.

Adenosine Triphosphate↗
PubMed1999

Minoxidil-induced cardiac hypertrophy in guinea pigs.

To investigate whether during cardiac hypertrophy changes occur in contractile protein composition and in mechanical and energetic properties of the myocardium, contractile protein composition, isometric force and adenosine triphosphate (ATP) consumption were studied in control and hypertrophied guinea-pig hearts. Cardiac hypertrophy was induced by adding minoxidil (120 or 200 mg/l) to the drinking water. Protein analysis was performed by one-dimensional gel electrophoresis. The myosin heavy-chain (MHC) composition was determined in an enzyme-linked immunosorbent assay (ELISA). ATP consumption and force development were simultaneously measured during isometric contraction in chemically skinned trabeculae. Histochemical analysis of cross-sectional area of cardiomyocytes and interstitial space was performed on the left ventricular tissue of 200 mg/l minoxidil-treated and control guinea pigs. Minoxidil treatment (120 and 200 mg/l) significantly increased left ventricular dry weight normalized for body weight by 19 +/- 4 and 24 +/- 4%, respectively. No significant differences were found in the cellular cross-sectional area, while interstitial space was slightly decreased in minoxidil-treated hearts. In left ventricular trabeculae of 200 mg/l minoxidil-treated guinea pigs, ATPase activity was slightly less than in those of control guinea pigs, whereas force did not differ significantly. Calcium sensitivity of force and ATPase activity were not affected by minoxidil treatment. Gel electrophoresis revealed no difference in contractile protein composition, but a tendency towards a lower amount of alpha-MHC in the minoxidil-treated hearts was found in ELISA.

Adenosine Triphosphate↗
PubMed1982

Accumulation and disposal of tricarboxylic acid cycle intermediates during propionate oxidation in the isolated perfused rat heart.

The role of the metabolite disposal mechanisms in the regulation of the tricarboxylic acid cycle pool size was studied in isolated perfused rat hearts oxidizing 2 mM propionate. Malate and succinate accumulated during the propionate metabolism. A further 118% increase in the malate concentration and 600% increase in the succinate concentration and a slight inhibition of the propionate uptake were observed during a subsequent KCl-induced arrest of the heart metabolizing propionate. When the mechanical activity of the heart was restored, the malate and succinate concentrations returned to the same levels as before the arrest of the heart, but the propionate uptake did not rise significantly. The mean disposal rates of the tricarboxylic acid cycle metabolites during the cardiac arrest and subsequent restoration of the activity were 1.4 and 2.4 mumol/min per g dry weight, respectively during cardiac arrest the malate carbon disposed was almost totally recovered as C3 compounds, whereas after the increase in the ATP-consumption most of it was oxidized. The result show that propionate is oxidized by heart muscle at an appreciable rate but the disposal rate of the tricarboxylic acid cycle intermediates is not tightly regulated by the cellular energy state. Although the metabolite pool size of the tricarboxylic acid cycle responds to change in the ATP consumption, the energy state appears to have a greater effect on the fate of the C3 compounds formed than on the actual rate of C4 compound disposition.

Alanine↗
PubMed2005

Myocardial contraction is 5-fold more economical in ventricular than in atrial human tissue.

OBJECTIVE: Cardiac energetics and performance depend on the expression level of the fast (alpha-) and slow (beta-) myosin heavy chain (MHC) isoform. In ventricular tissue, the beta-MHC isoform predominates, whereas in atrial tissue a variable mixture of alpha- and beta-MHC is found. In several cardiac diseases, the slow isoform is upregulated; however, the functional implications of this transition in human myocardium are largely unknown. The aim of this study was to determine the relation between contractile properties and MHC isoform composition in healthy human myocardium using the diversity in atrial tissue. METHODS: Isometric force production and ATP consumption were measured in chemically skinned atrial trabeculae and ventricular muscle strips, and rate of force redevelopment was studied using single cardiomyocytes. MHC isoform composition was determined by one-dimensional SDS-gel electrophoresis. RESULTS: Force development in ventricular tissue was about 5-fold more economical, but nine times slower, than in atrial tissue. Significant linear correlations were found between MHC isoform composition, ATP consumption and rate of force redevelopment. CONCLUSION: These results clearly indicate that even a minor shift in MHC isoform expression has considerable impact on cardiac performance in human tissue.

Adenosine Triphosphate↗
PubMed1981

Energy and heat production of human erythrocytes in different media.

Variation in ATP consumption of human erythrocytes was achieved by incubation in different media. Addition of ouabain to a conventional isotonic medium or elimination of the ionic Na+-K+ gradient across the membrane decreases lactate production of the cells by about 40%. Applying a calcium-gradient of 1 mmol . l-1 across the membrane increased glycolysis by about 25%. The various introduced changes in the medium and thus in ATP-consumption cause similar alterations in heat production and lactate production. The experimental results give no suggestions explaining the origin of the part of the heat production that is unrelated to glycolysis.

Adenosine Triphosphate↗
PubMed1998

Calcium handling by the sarcoplasmic reticulum during oscillatory contractions of skinned skeletal muscle fibres.

Isometric ATP consumption and force were investigated in mechanically skinned fibres from iliofibularis muscle of Xenopus laevis. Measurements were performed at different [Ca2+], in the presence and absence of caffeine (5 nM). In weakly Ca2+-buffered solutions without caffeine, spontaneous oscillations in force and ATPase activity occurred. The repetition frequency was [Ca2+]-and temperature-dependent. The Ca2+ threshold (+/- SEM) for the oscillations corresponded to a pCa of 6.5 +/- 0.1. The maximum ATP consumption associated with calcium uptake by the sarcoplasmic reticulum (SR) reached during the oscillations was similar to the activity under steady-state conditions at saturating calcium concentrations in the presence of caffeine. Maximum activity was reached when the force relaxation was almost complete. The calculated amount of Ca2+ taken up by the SR during a complete cycle corresponded to 5.4 +/ 0.4 mmol per litre cell volume. In strongly Ca2+-buffered solutions, caffeine enhanced the calcium sensitivity of the contractile apparatus and, at low calcium concentrations, SR Ca uptake. These results suggest that when the SR is heavily loaded by net Ca uptake, there is a massive calcium-induced calcium release. Subsequent net Ca uptake by the SR then gives rise to the periodic nature of the calcium transient.

Adenosine Triphosphatases↗
PubMed1971

Reduction in adipocyte ATP by lipolytic agents: relation to intracellular free fatty acid accumulation.

Epinephrine, norepinephrine, ACTH, and dibutyryl 3',5'-cyclic AMP reduced adipocyte ATP levels during 60 min incubation; glucose displayed a protective effect. The reduction in adipocyte ATP levels could not be attributed solely to: a direct hormone effect, deficiency in metabolic substrate, activation of adenyl cyclase with ATP consumption, loss of adenine nucleotide from the cell or loss of cells during incubation, lipolytic rate per se, or extracellular accumulation of FFA or glycerol. To determine whether intracellular FFA accumulation was a causative factor, intracellular FFA levels were measured during hormone-stimulated lipolysis. This was accomplished by using sucrose-U-(14)C as a marker for the extracellular space to correct for contamination of cells by extracellular albumin-bound FFA. These experiments showed that the fall in adipocyte ATP correlated with FFA saturation of medium albumin and progressive accumulation of FFA within the adipocyte. Furthermore, the protective effect of glucose noted above was associated with a marked reduction in intracellular FFA as compared to the extracellular FFA pool. On the basis of these studies, combined with those in the literature, it is concluded that in vitro effects of lipolytic agents on adipocyte ATP levels are the net result of imparied ATP synthesis (uncoupled oxidative phosphorylation) in the face of normal or augmented ATP consumption.

Adenosine Triphosphate↗
PubMed1978

Effects of pent-4-enoate on cellular redox state, glycolysis and fatty acid oxidation in isolated perfused rat heart.

The metabolic effects of pent-4-enoate were studied in beating and potassium-arrested perfused rat hearts. The addition of 0.8mm-pent-4-enoate to the fluid used to perfuse a potassium-arrested heart resulted in a 70% increase in the O(2) consumption and a 66% decrease in the glycolytic flux as measured in terms of the de-tritiation of [3-(3)H]glucose, although the proportion of the O(2) consumption attributable to glucose oxidation decreased from an initial 30% to 10%. The pent-4-enoate-induced increase in O(2) consumption was only 15% in the beating heart. In the potassium-arrested heart, pent-4-enoate stimulated palmitate oxidation by more than 100% when measured in terms of the production of (14)CO(2) from [1-(14)C]palmitate, but in the beating heart palmitate oxidation was inhibited. Perfusion of the heart with pent-4-enoate had no effect on the proportion of pyruvate dehydrogenase found in the active form, in spite of large changes in the CoASH and acetyl-CoA concentrations and changes in their concentration ratios. The effects of pent-4-enoate on the cellular redox state were dependent on the ATP consumption of the heart. In the beating heart, pent-4-enoate caused a rapid mitochondrial NAD(+) reduction that subsequently faded out, so that the final state was more oxidized than the initial state. The arrested heart, however, remained in a more reduced state than initially, even after the partial re-oxidation that followed the initial rapid NAD(+) reduction. The ability of pent-4-enoate to increase or decrease fatty acid oxidation can be explained on the basis of the differential effects of pent-4-enoate on the concentration of citric acid-cycle intermediates under conditions of high or low ATP consumption of the myocardial cell. The proportion of the fatty acids in the fuel consumed by the heart is probably primarily determined by the regulatory mechanisms of glycolysis. When pent-4-enoate causes an increase in the citric acid-cycle intermediates, feedback inhibition of glycolysis results in an increase in the oxidation of fatty acids.

Animals↗
PubMed2004

Two simple and generic antibody-independent kinase assays: comparison of a bioluminescent and a microfluidic assay format.

In this study, the authors have compared the performance of 2 high-throughput screening assays for a serin/threonine kinase: a microplate-based, bioluminescent assay that uses the luciferin/luciferase system to monitor ATP consumption, and a microfluidic assay that measures the change in mobility in an electric field of a fluorescently labeled peptide upon phosphorylation. Both assays are homogeneous, nonradioactive, antibody independent and could be miniaturized to a reaction volume of 4 microl. The robustness of both formats was demonstrated by Z' values > 0.8. Screening of a small library (2133 compounds) showed that the results obtained with both technologies correlate very well. Although the threshold for hits was set to a comparably low value-22.2% and 13.7% inhibition for the ATP consumption and microfluidic assay, respectively, corresponding to mean plus 3 standard deviations-the overlap of active compounds identified with the 2 assay formats was greater than 94%. Thus, both assays allow the identification of even low potency inhibitors with a high level of confidence.

Adenosine Triphosphate↗
PubMed1986

Primary active sodium transport, oxygen consumption, and ATP: coupling and regulation.

Several metabolic aspects of primary active transport have been explored in this communication. One emphasized theme entailed the need to investigate the properties of the mitochondria and the active transport systems within the intact cell. Several methodological and conceptual approaches were described that permitted such an analysis. The answers provided were sometimes qualitative or quantitative. Qualitative information was provided regarding the cytosolic signal linking active transport with respiration, suggesting that the cytosolic ADP concentration was an important element in that link. The intact renal cell was found to work normally at 50 to 60% of its maximal respiratory capacity, indicating that sufficient reserve capacity was present for increased metabolic demands. Several examples were described in which a combination of qO2 measurements and/or optical techniques were used to differentiate between effects of agents which act primarily on transport or metabolic events. Finally, the control of transport by metabolism was discussed, primarily emphasizing the role of ATP and Pi. One of the overall conclusions from these studies is that, in general, the mitochondria and the transport systems seem to display similar properties in the intact cell as they do in isolated form. However, uncertainties concerning the cellular microenvironment surrounding the mitochondria and the plasma membrane transporters have produced some interesting surprises concerning their function in the intact cell. More quantitative information on the energy compartmentation of the renal cell would be helpful to clarify numerous aspects of metabolic function.

Adenosine Diphosphate↗
PubMed2000

Electron flow to oxygen in higher plants and algae: rates and control of direct photoreduction (Mehler reaction) and rubisco oxygenase.

Linear electron transport in chloroplasts produces a number of reduced components associated with photosystem I (PS I) that may subsequently participate in reactions that reduce O2. The two primary reactions that have been extensively studied are: first, the direct reduction of O2 to superoxide by reduced donors associated with PS I (the Mehler reaction), and second, the rubisco oxygenase (ribulose 1,5-bisphosphate carboxylase oxygenase EC 4.1.1.39) reaction and associated peroxisomal and mitochondrial reactions of the photorespiratory pathway. This paper reviews a number of recent and past studies with higher plants, algae and cyanobacteria that have attempted to quantify O2 fluxes under various conditions and their contributions to a number of roles, including photon energy dissipation. In C3 and Crassulacean acid metabolism (CAM) plants, a Mehler O2 uptake reaction is unlikely to support a significant flow of electron transport (probably less than 10%). In addition, if it were present it would appear to scale with photosynthetic carbon oxidation cycle (PCO) and photosynthetic carbon reduction cycle (PCR) activity This is supported by studies with antisense tobacco plants with reduced rubisco at low and high temperatures and high light, as well as studies with potatoes, grapes and madrone during water stress. The lack of significant Mehler in these plants directly argues for a strong control of Mehler reaction in the absence of ATP consumption by the PCR and PCO cycles. The difference between C3 and C4 plants is primarily that the level of light-dependent O2 uptake is generally much lower in C4 plants and is relatively insensitive to the external CO2 concentration. Such a major difference is readily attributed to the operation of the C4 CO2 concentrating mechanism. Algae show a range of light-dependent O2 uptake rates, similar to C4 plants. As in C4 plants, the O2 uptake appears to be largely insensitive to CO2, even in species that lack a CO2 concentrating mechanism and under conditions that are clearly limiting with respect to inorganic carbon supply. A part explanation for this could be that many algal rubsicos have considerably different oxygenase kinetic properties and exhibit far less oxygenase activity in air. This would lead to the conclusion that perhaps a greater proportion of the observed O2 uptake may be due to a Mehler reaction and less to rubisco, compared with C3 plants. In contrast to algae and higher plants, cyanobacteria appear to have a high capacity for Mehler O2 uptake, which appears to be not well coupled or limited by ATP consumption. It is likely that in all higher plants and algae, which have a well-developed non-photochemical quenching mechanism, non-radiative energy dissipation is the major mechanism for dissipating excess photons absorbed by the light-harvesting complexes under stressful conditions. However, for cyanobacteria, with a lack of significant non-photochemical quenching, the situation may well be different.

Animals↗
PubMedDate not supplied

Relation between energy metabolism, glycolysis, noradrenaline release and duration of ischemia.

We studied the effect of 12-36 min of global ischemia followed by 36 min of reperfusion in Langendorff perfused rabbit hearts (n = 26). Metabolism was determined in terms of peak and total release of purines (adenosine, inosine, hypoxanthine), lactate and noradrenaline during reperfusion; and myocardial content of nucleotides (ATP, ADP, AMP), glycogen and noradrenaline at the end of reperfusion. An inverse relationship (r = -0.79) existed between duration of ischemia and developed pressure post-ischemia. Early during reperfusion, after 12 min of ischemia, the purine concentration (peak release) increased 100x (p < 0.01), that of lactate and noradrenaline 10x (p < 0.05). Total purine release rose with progression of the ischemic period (30x after 36 min of ischemia; p < 0.01), concomitant with a reduction in nucleotide content. Lactate release was independent from the duration of ischemia, although glycogen had declined by 30% (p < 0.01) after 36 min of ischemia. The acid insoluble glycogen fraction, which presumably contains proglycogen, increased substantially during short-term ischemia. Peak noradrenaline increased 100x, and 200x, (p < 0.05) after 24 and 36 min of ischemia, respectively. Total noradrenaline release due to various periods of ischemia mirrored its peak release. Function recovery was inversely related to total purine and noradrenaline efflux (both r = -0.81); it correlated with tissue nucleotide content (r = 0.84). In conclusion, larger amounts of noradrenaline are released only after a substantial drop in myocardial ATP. During severe ischemia ATP consumption more than limited ATP production by anaerobic glycolysis, is a key factor affecting recovery on subsequent reperfusion. In contrast to lactate efflux, purine and noradrenaline release are useful markers of ischemic and reperfusion damage.

Adenosine↗
PubMed2002

The role of adenosine in the anoxic survival of the epaulette shark, Hemiscyllium ocellatum.

The epaulette shark (Hemiscyllium ocellatum) is among the few vertebrates that can tolerate extreme hypoxia for prolonged periods and, as shown here, anoxia. We examined how anoxia affected this shark's level of responsiveness, concentration of brain ATP and adenosine -- an endogenous neuronal depressant. In addition, we investigated how these variables were affected by aminophylline, an adenosine receptor antagonist. Epaulette sharks placed in an anoxic environment (<0.02 mg O2 l(-1)) lost their righting reflex after 46.3 +/- 2.8 min, but immediately regained vigilance upon return to normoxia. Then 24 h later, the same sharks were injected with either saline or aminophylline (30 mg kg(-1)) in saline and re-exposed to anoxia. In this second anoxic episode, controls sharks showed a 56% decrease in the time taken to lose their righting reflex but maintained their brain ATP levels; conversely, aminophylline-treated epaulette sharks displayed a 46% increase in the time to loss of righting reflex and had significantly lower brain ATP levels. Since anoxia also caused a 3.5-fold increase in brain adenosine levels, these results suggest that adenosine receptor activation had a pre-emptive role in maintaining brain ATP levels during anoxia. Perhaps because adenosine receptor activation initiates metabolic depression, indicated by the early loss of responsiveness (righting reflex), such a mechanism would serve to reduce ATP consumption and maintain brain ATP levels.

Adenosine↗
PubMed2000

Polyene antibiotics: relative degrees of in vitro cytotoxicity and potential effects on tubule phospholipid and ceramide content.

Polyene antibiotic administration is limited by dose-dependent nephrotoxicity. The latter is believed to be mediated by polyene anchoring to plasma membrane cholesterol, resulting in pore formation, abnormal ion/solute flux, adenosine triphosphate (ATP) declines, and, ultimately, a loss of tubule viability. The relative nephrotoxicity of these agents and their liposomal preparations has remained poorly defined. Thus, freshly isolated mouse proximal tubules or cultured human proximal tubule (HK-2) cells were exposed to either nystatin, amphotericin B, or three different polyene liposomal preparations (Nyotran, AmBisome, or Abelcet; 4 to 64 microg/mL). The impact of these agents on (1) plasma membrane injury (sodium-driven ATP consumption, assessed by ATP-adenosine diphosphate [ADP] ratios); (2) cellular susceptibility to superimposed injury (chemical hypoxia or ferrous ammonium sulfate-mediated oxidative stress; assessed by lactate dehydrogenase release); and (3) membrane cholesterol, phospholipid, and ceramide expression was assessed. Amphotericin B was more cytotoxic than nystatin (approximately 25% to 50% greater ATP-ADP ratio declines). Most of this toxicity could be eliminated by polyene liposomal formulation. Nevertheless, the liposomal polyenes still fully sensitized tubule cells to superimposed chemical hypoxic (antimycin/deoxyglucose), but not oxidant, attack. Nystatin and amphotericin B caused acute increments in tubule sphingomyelin-phosphatidylcholine ratios and ceramide content (indicating an impact on the plasma membrane extending beyond the classic view of pore formation with ion flux). In conclusion, (1) nystatin is seemingly less cytotoxic than amphotericin B (in contrast to the prevailing clinical view); (2) liposomal formulation markedly decreases this cytotoxicity; (3) despite this reduced toxicity, liposomal polyenes are still able to render tubule cells more vulnerable to selected forms of superimposed injury; and (4) acute alterations in plasma membrane phospholipid and ceramide expression are previously unrecognized consequences and potential mediators of polyene-mediated tubular cell attack.

Adenosine Diphosphate↗
PubMed1994

Galactose Expulsion during Lactose Metabolism in Lactococcus lactis subsp. cremoris FD1 Due to Dephosphorylation of Intracellular Galactose 6-Phosphate.

In Lactococcus lactis subsp. cremoris FD1, galactose and lactose are both transported and phosphorylated by phosphotransferase systems. Lactose 6-phosphate (lactose-6P) is hydrolyzed intracellularly to galactose-6P and glucose. Glucose enters glycolysis as glucose-6P, whereas galactose-6P is metabolized via the tagatose-6P pathway and enters glycolysis at the tagatose diphosphate and fructose diphosphate pool. Galactose would therefore be a gluconeogenic sugar in L. lactis subsp. cremoris FD1, but since fructose 1,6-diphosphatase is not present in this strain, galactose cannot serve as an essential biomass precursor (glucose-6P or fructose-6P) but only as an energy (ATP) source. Analysis of the growth energetics shows that transition from N limitation to limitation by glucose-6P or fructose-6P gives rise to a very high growth-related ATP consumption (152 mmol of ATP per g of biomass) compared with the value in cultures which are not limited by glucose-6P or fructose-6P (15 to 50 mmol of ATP per g of biomass). During lactose metabolism, the galactose flux through the tagatose-6P pathway (r(max) = 1.2 h) is lower than the glucose flux through glycolysis (r(max) = 1.5 h) and intracellular galactose-6P is dephosphorylated; this is followed by expulsion of galactose. Expulsion of a metabolizable sugar has not been reported previously, and the specific rate of galactose expulsion is up to 0.61 g of galactose g of biomass h depending on the lactose flux and the metabolic state of the bacteria. Galactose excreted during batch fermentation on lactose is reabsorbed and metabolized when lactose is depleted from the medium. In vitro incubation of galactose-6P (50 mM) and permeabilized cells (8 g/liter) gives a supernatant containing free galactose (50 mM) but no P(i) (less than 0.5 mM). No organic compound except the liberated galactose is present in sufficient concentration to bind the phosphate. Phosphate is quantitatively recovered in the supernatant as P(i) by hydrolysis with alkaline phosphatase (EC 3.1.3.1), whereas inorganic pyrophosphatase (EC 3.6.1.1) cannot hydrolyze the compound. The results indicate that the unknown phosphate-containing compound might be polyphosphate.

Journal Article↗