Evolution of carbohydrate metabolic pathways

Current studies of hyperthermophilic archaea and bacteria, the phylogenetically deepest-rooted and slowest-evolving extant organisms known, are allowing new insights into the nature of presumably ancient metabolic pathways. The apparent common occurrence of modified non-phosphorylated Entner-Doudoroff (ED) pathways among saccharolytic archaea and the absence of the conventional Embden-Meyerhof-Parnas (EMP) mode of glycolysis indicate that the ED pathway is the older route of carbohydrate dissimilation. However, gluconeogenesis via the "reversed" EMP route has been found in archaea. Thus, the EMP pathway was probably an anabolic pathway to begin with; its catabolic role came later, with the evolution of fructose phosphate kinases, using ATP, ADP or pyrophosphate as phosphate donors. Similarly, the presence of reductive reactions of the citric acid cycle in anaerobic archaea and the most deeply rooted bacteria, including autotrophs, indicates that the citric acid cycle was originally a reductive biosynthetic pathway.     

Characteristics and efficiency of glutamine production by coupling of a bacterial glutamine synthetase reaction with the alcoholic fermentation system of baker's yeast

Glutamine production with bacterial glutamine synthetase (GS) and the sugar-fermenting system of baker's yeast for ATP regeneration was investigated by determining the product yield obtained with the energy source for ATP regeneration (i.e., glucose) for yeast fermentation. Fructose 1,6-bisphosphate was accumulated temporarily prior to the formation of glutamine in mixtures which consisted of dried yeast cells, GS, their substrate (glucose and glutamate and ammonia), inorganic phosphate, and cofactors. By an increase in the amounts of GS and inorganic phosphate, the amounts of glutamine formed increased to 19 to 54 g/liter, with a yield increase of 69 to 72% based on the energy source (glucose) for ATP regeneration. The analyses of sugar fermentation of the yeast in the glutamine-producing mixtures suggested that the apparent hydrolysis of ATP by a futile cycle(s) at the early stage of glycolysis in the yeast cells reduces the efficiency of ATP utilization. Inorganic phosphate inhibits phosphatase(s) and thus improves glutamine yield. However, the analyses of GS activity in the glutamine-producing mixtures suggested that the higher concentration of inorganic phosphate as well as the limited amount of ATP-ADP caused the low reactivity of GS in the glutamine-producing mixtures. A result suggestive of improved glutamine yield under the conditions with lower concentrations of inorganic phosphate was obtained by using a yeast mutant strain that had low assimilating ability for glycerol and ethanol. In the mutant, the activity of the enzymes involved in gluconeogenesis, especially fructose 1,6-bisphosphatase, was lower than that in the wild-type strain.     

Compartmentation of phosphoglycerate kinase in Trypanosoma brucei plays a critical role in parasite energy metabolism

African trypanosomes compartmentalize glycolysis in a microbody, the glycosome. When growing in the mammalian bloodstream, trypanosomes contain only a rudimentary mitochondrion, and the first seven glycolytic enzymes, including phosphoglycerate kinase, are located in the glycosome. Procyclic trypanosomes, growing in the gut of tsetse flies, possess a fully developed mitochondrion that is active in oxidative phosphorylation. The first six glycolytic enzymes are still glycosomal, but phosphoglycerate kinase is now found in the cytosol. We demonstrate here that bloodstream trypanosomes are killed by expression of cytosolic phosphoglycerate kinase. The toxicity depends on both enzyme activity and cytosolic location. One possible explanation is that cytosolic phosphoglycerate kinase creates an ATP-generating shunt in the cytosol, thus preventing full ATP regeneration in the glycosome and ultimately inhibiting the first, ATP-consuming, steps of glycolysis.     

Calculation of biochemical net reactions and pathways by using matrix operations

Pathways for net biochemical reactions can be calculated by using a computer program that solves systems of linear equations. The coefficients in the linear equations are the stoichiometric numbers in the biochemical equations for the system. The solution of the system of linear equations is a vector of the stoichiometric numbers of the reactions in the pathway for the net reaction; this is referred to as the pathway vector. The pathway vector gives the number of times the various reactions have to occur to produce the desired net reaction. Net reactions may involve unknown numbers of ATP, ADP, and Pi molecules. The numbers of ATP, ADP, and Pi in a desired net reaction can be calculated in a two-step process. In the first step, the pathway is calculated by solving the system of linear equations for an abbreviated stoichiometric number matrix without ATP, ADP, Pi, NADred, and NADox. In the second step, the stoichiometric numbers in the desired net reaction, which includes ATP, ADP, Pi, NADred, and NADox, are obtained by multiplying the full stoichiometric number matrix by the calculated pathway vector.     

Probes of initial phosphorylation events in ATP synthesis by chloroplasts

Rapid mixing and quenching techniques have been used with chloroplasts activated by an acid-base transition or by light to assess the nature and characteristics of the substances initially labeled by inorganic [32P]phosphate during ATP synthesis. With light-activated chloroplast fragments, but not with acid-base-activated preparations, an initial rapid labeling of a small amount of ADP is observed. With the acid-base activated preparations a slower continued labeling of ADP occurs that is uncoupler-sensitive, that does not proceed via [gamma-32]ATP of the medium and for which medium ADP furnishes the AMP moiety. The results point to ADP as the initial acceptor of phosphate for ATP synthesis, with a slow side reaction in which bound ATP phosphorylates bound AMP to give a bound ADP. The phosphorylation of bound ADP by medium [32P]phosphate in the absence of added ADP is confirmed, but the reaction is too slow to serve as an intermediate in photophosphorylation. The appearance of label from [32P]phosphate in ATP in the acid-base transition at 25 degrees shows a lag of only about 3 to 7 ms, consistent with the absence of any phosphorylated intermediate. The lag is followed by a linear rate of [gamma-32]ATP formation that is about as fast as that observed in steady photophosphorylation, consistent with a proton gradient serving for transmission of energy from electron transfer reactions to the ATP-synthesizing complex.     

Performance and cost-effectiveness of selective screening criteria for Chlamydia trachomatis infection in women. Implications for a national Chlamydia control strategy

At 9 mM glucose, experimental results show that mitochondrial phosphate depletion (induced by glucose phosphorylation, catalyzed by mitochondrial hexokinase) reduces the activities of the respiratory chain, oxidative phosphorylation, and glutaminase. Consequently, the 14C-lactate oxidation to 14CO2 is lowered in the presence of glucose. The fall of ATP level triggers a high aerobic glycolysis by deinhibiting fructose-6-P kinase. NADH, generated by enhanced glyceraldehyde-3-P dehydrogenase activity, increases the reducing power. Moreover, the lactate dehydrogenase (LDH) system is shifted toward lactate formation, while NAD+ is regenerated and the oligomycin-inhibited ATP production is replaced by the iodoacetate-inhibited ATP production. From 14CO2 production and lactate accumulation it is calculated that about 60% of 14C-glucose which disappears is channelled into extraglycolytic reactions. On the contrary, 82% of glucose below l mM is metabolized through non-glycolytic reactions. The pyruvate kinase-M2 (PK-M2) inhibition does not limit the glycolytic flow from 9 mM glucose, but it may cause sustained gluconeogenesis.     

Quick-freeze deep-etch electron microscopy of the actin-heavy meromyosin complex during the in vitro motility assay

Since mica is a substitute for glass in the in vitro actin motility assay, I examined the structure of heavy meromyosin (HMM) crossbridges supporting actin filaments by quick-freeze deep-etch replica electron microscopy. This method was capable of resolving the inter-domain cleft of the monomeric actin molecule. HMM heads that are not bound to actin, when observed by this technique, were straight and elongated in the absence of ATP but strongly kinked upon addition of ATP or ADP.inorganic vanadate to produce the putative long-lived analog of HMM-ADP.inorganic phosphate. The low-magnification image of the ATP-containing acto-HMM preparation showed features characteristic of sliding actin filaments on glass coverslips. At high magnification, all the HMM molecules were found attached to actin by one head with the majority projecting perpendicular to the filament axis, whereas in the absence of ATP, HMM exhibited two-head binding with a preponderance of molecules tilted at 45 degrees. Detailed examination of the shape of HMM heads involved in sliding showed a rounded, and flat appearance of the tip and comparatively thin neck portion as if the heads grasp actin filament, in contrast to rigor crossbridges which have a pear-shaped configuration with more gradual taper. Such configurations of HMM heads were essentially the same as I observed previously on acto-myosin subfragment-1 (S1) by the same technique, except for the presence of an additional neck portion of HMM which makes interpretaion of the images easier. Interestingly, under actively sliding conditions, very few heads were tilted in the rigor configuration. At first glance, the addition of ADP to the rigor-complex gave images rather like those obtained with ATP, but they turned out to be different. The contribution of the structural change of crossbridges to the force development is discussed.     

Notion of metabolic adaptation to exercise

During muscular exercise, adenosine triphosphate (ATP) must be resynthesized in order to allow subsequent muscle contraction. ATP can be immediately resynthesized from adenosine diphosphate (ADP) and from creatine phosphate (CP). However, such resynthesis from phosphorylated compounds is not possible in very short-duration exercise. For further exercise an energy source, mainly carbohydrates and lipids, is needed. While anaerobic (glycolysis and glycogenolysis) and aerobic (mitochondrial oxidation) pathways are used for carbohydrates, lipids can only undergo oxidation. Carbohydrates are used preferentially with rising exercise intensity because of increased muscle uptake, progressive recruitment of type II fibers and catecholamine release. Lipid oxidation rises with the duration of exercise and falls with increasing intensity. The observation that training plays an important role in the use of these energy substrates has led to the development of the crossover theory by which the use of carbohydrates and lipids in all individuals depends at all times on the combined effect of training and exercise intensity. Training facilitates lipid oxidation but, by allowing higher intensity, also implies inevitable use of carbohydrates. Thus there is an exercise level, or a crossover point, at which carbohydrate energy predominates over lipid energy. Beyond this point, increasing energy requirements are satisfied by preferential use of carbohydrates. Finally, although it was incorrectly thought that proteins play a very minor role, actually amino acids coming from the voluminous tissue mass provide an important source of energy during prolonged exercise.     

Rifampin, a useful drug for nonmycobacterial infections

Hexokinase I, the pacemaker of glycolysis in brain tissue and red blood cells, is comprised of two similar domains fused into a single polypeptide chain. The C-terminal half of hexokinase I is catalytically active, whereas the N-terminal half is necessary for the relief of product inhibition by phosphate. A crystalline complex of recombinant human hexokinase I with glucose and phosphate (2.8 A resolution) reveals a single binding site for phosphate and glucose at the N-terminal half of the enzyme. Glucose and phosphate stabilize the N-terminal half in a closed conformation. Unexpectedly, glucose binds weakly to the C-terminal half of the enzyme and does not by itself stabilize a closed conformation. Evidently a stable, closed C-terminal half requires either ATP or glucose 6-phosphate along with glucose. The crystal structure here, in conjunction with other studies in crystallography and directed mutation, puts the phosphate regulatory site at the N-terminal half, the site of potent product inhibition at the C-terminal half, and a secondary site for the weak interaction of glucose 6-phosphate at the N-terminal half of the enzyme. The relevance of crystal structures of hexokinase I to the properties of monomeric hexokinase I and oligomers of hexokinase I bound to the surface of mitochondria is discussed.     

Exercise metabolism in Duchenne muscular dystrophy: a biochemical and [31P]-nuclear magnetic resonance study of mdx mice

Intracellular pH, ratios of phosphocreatine (PCr) to ATP and PCr to inorganic phosphate (Pi) as well as isometric tension were measured during 1 Hz sciatic nerve stimulation and during recovery in the calf muscles of mdx (a model of Duchenne muscular dystrophy) and control mice. Tension did not decline significantly in either strain. The ratio of PCr/(PCr + Pi) was significantly reduced in mdx as against control muscle during exercise and recovery, but the ratio of PCr/ATP and the half-time for PCr recovery were similar in both strains. A reduction in the maximal activities of succinate dehydrogenase and succinate-cytochrome c reductase suggests that mitochondrial metabolism may be impaired. The similarity in PCr recovery times suggests that the muscle has adapted, making any impairment of oxidative metabolism negligible in the intact system. The rate of pH recovery is prolonged in mdx muscle and provides strong evidence for a decline in the capacity of dystrophic muscle to extrude proton equivalents. These data are compared with a previous study which used 10 Hz stimulation and also observed a slow pH recovery. The slow pH recovery could be explained by an elevation in intracellular sodium.     

Apyrase functions in plant phosphate nutrition and mobilizes phosphate from extracellular ATP

ATP, which is present in the extracellular matrix of multicellular organisms and in the extracellular fluid of unicellular organisms, has been shown to function as a signaling molecule in animals. The concentration of extracellular ATP (xATP) is known to be functionally modulated in part by ectoapyrases, membrane-associated proteins that cleave the gamma- and beta-phosphates on xATP. We present data showing a previously unreported (to our knowledge) linkage between apyrase and phosphate transport. An apyrase from pea (Pisum sativum) complements a yeast (Saccharomyces cerevisiae) phosphate-transport mutant and significantly increases the amount of phosphate taken up by transgenic plants overexpressing the gene. The transgenic plants show enhanced growth and augmented phosphate transport when the additional phosphate is supplied as inorganic phosphate or as ATP. When scavenging phosphate from xATP, apyrase mobilizes the gamma-phosphate without promoting the transport of the purine or the ribose.     

Oxidative phosphorylation in intact hepatocytes: quantitative characterization of the mechanisms of change in efficiency and cellular consequences

Two mechanisms may affect the yield of the oxidative phosphorylation pathway in isolated mitochondria: (i) a decrease in the intrinsic coupling of the proton pumps (H+/2e- or H+/ATP), and (ii) an increase in the inner membrane conductance (proton or cation leak). Hence three kinds of modifications can occur and each of them have been characterized in isolated rat liver mitochondria (see preceding chapter by Rigoulet et al.). In intact isolated hepatocytes, these modifications are linked to specific patterns of bioenergetic parameters, i.e. respiratory flux, mitochondrial redox potential, DY, and phosphate potential. (1) The increase in H+/ATP stoichiometry of the mitochondrial ATP synthase, as induced by almitrine [20], leads to a decrease in mitochondrial and cytosolic ATP/ADP ratios without any change in the protonmotive force nor in the respiratory rate or redox potential. (2) In comparison to carbohydrate, octanoate metabolism by beta-oxidation increases the proportion of electrons supplied at the second coupling site of the respiratory chain. This mimics a redox slipping. Octanoate addition results in an increased respiratory rate and mitochondrial NADH/NAD ratio while protonmotive force and phosphate potential are almost unaffected. The respiratory rate increase is associated with a decrease in the overall apparent thermodynamic driving force (2deltaE'o - ndeltap) which confirms the 'redox-slipping-like' effect. (3) An increase in proton conductance as induced by the protonophoric uncoupler 2,4-dinitrophenol (DNP) leads to a decrease, as expected, in the mitochondrial NADH/NAD and ATP/ ADP ratios and in deltapsi while respiratory rate is increased. Thus, each kind of modification (proton leak, respiratory chain redox slipping or increase in H+/ATP stoichiometry of ATPase) is related to a specific set of bioenergetic parameters in intact cells. Moreover, these patterns are in good agreement with the data found in isolated mitochondria. From this work, we conclude that quantitative analysis of four bioenergetic parameters (respiration rate, mitochondrial NADH/ NAD ratio, protonmotive force and mitochondrial phosphate potential) gives adequate tools to investigate the mechanism by which some alterations may affect the yield of the oxidative phosphorylation pathway in intact cells.     

Phosphorus nuclear magnetic resonance: a non-invasive technique for the study of muscle bioenergetics during exercise

Phosphorus nuclear magnetic resonance (31P NMR) spectroscopy is a non-destructive analytical laboratory technique that, due to recent technical advances, has become applicable to the study of high-energy phosphate metabolism in both animal and human extremity muscles (in vivo). 31P NMR can assay cellular phosphocreatine, ATP, inorganic phosphate, the phosphorylated glycolytic intermediates, and intra-cellular pH in either resting or exercising muscle, in a non-invasive manner. NMR uses non-perturbing levels of radio-frequency energy as its biophysical probe and can therefore safely study intact muscle in a repeated fashion while exerting no artifactual influence on ongoing metabolic processes. Compared with standard tissue biopsy and biochemical assay techniques, NMR possesses the advantages of being non-invasive, allowing serial in situ studies of the same tissue sample, and providing measurements of only active (unbound) metabolites. NMR studies of exercising muscle have yielded information regarding fatigue mechanisms at the cellular level and are helping resolve long-standing questions regarding the metabolic control of glycolysis, oxidative phosphorylation, and post-exercise phosphocreatine re-synthesis. NMR is also being utilized to measure enzymatic reaction rates in vivo. In the near future, other forms of NMR spectroscopy may also permit the non-invasive measurement of tissue glycogen and lactate content.     

The danger of metabolic pathways with turbo design

Many catabolic pathways begin with an ATP-requiring activation step, after which further metabolism yields a surplus of ATP. Such a 'turbo' principle is useful but also contains an inherent risk. This is illustrated by a detailed kinetic analysis of a paradoxical Saccharomyces cerevisiae mutant; the mutant fails to grow on glucose because of overactive initial enzymes of glycolysis, but is defective only in an enzyme (trehalose 6-phosphate synthase) that appears to have little relevance to glycolysis. The ubiquity of pathways that possess an initial activation step, suggests that there might be many more genes that, when deleted, cause rather paradoxical regulation phenotypes (i.e. growth defects caused by enhanced utilization of growth substrate).     

Impaired aerobic glycolysis in muscle phosphofructokinase deficiency results in biphasic post-exercise phosphocreatine recovery in 31P magnetic resonance spectroscopy

Using 31P magnetic resonance spectroscopy, energy metabolism in calf muscles of two patients with biochemically and genetically proven muscular phosphofructokinase deficiency, and an asymptomatic heterozygote was monitored during isometric foot plantarflexion performed under aerobic and anaerobic conditions and in the aerobic recovery phases. In the heterozygote only a moderate alteration from normal was found in terms of an elevated ATP demand during exercise. In the homozygote, hexose phosphates, indicated as phosphomonoesters, increased dramatically during contraction. Phosphomonoester accumulation resulted in consumption of free inorganic phosphate (P(i)). During ischemic exercise the absence of glycolytic ATP formation resulted in a linear time course of phosphocreatine breakdown and a moderate alkalinization. During the recovery, phosphocreatine resynthesis showed a biphasic time course, indicating that mitochondrial function itself was not directly affected. At first glance, the early depletion of P(i) below initial resting levels and the rate of phosphate splitting from sugar phosphates seemed to become the limiting factor for the rate of the oxidative phosphorylation and creatine kinase reaction. However, the actual concentrations of P(i) and ADP estimated at the onset of delay were too high to exclusively explain the dramatic delay in PCr resynthesis. For this reason, a reduced turnover of the citric acid cycle was assumed, which was caused by the complete absence of glycolysis in PFK deficiency patients. Furthermore, results from PFK deficiency patients were compared with previous findings from myophosphorylase deficiency patients in the literature.     

Glucose metabolism in photoreceptor outer segments. Its role in phototransduction and in NADPH-requiring reactions

Glucose metabolism in the photoreceptor rod outer segment produces both ATP (GTP) and NADPH to support phototransduction and NADPH-requiring processes in this organelle. Glycolysis in isolated bovine rod outer segments produces 44.0 +/- 6.4 nmol of ATP/min/mg of protein or 5.7 mM ATP/min. This rate of ATP production is more than sufficient to maintain the basal rate of cGMP synthesis (0.86 mM cGMP/min) in the dark requiring 1.7 mM ATP/min. Following photoexcitation, the 4.5-fold increase in the turnover of cGMP requires an ATP synthesis rate of up to 7.7 mM ATP/min (Ames, A., Walseth, T. F., Heyman, R. A., Barad, M., Graeff, R. M., and Goldberg, N. D. (1986) J. Biol. Chem. 261, 13034-13042). Under these conditions the rate of ATP production by glycolysis as measured in isolated rod outer segments is not sufficient for the regeneration of cGMP. Additional energy is most likely provided by the phosphocreatine shuttle which transports high energy phosphate groups in the form of creatine phosphate from the rod inner segment to the rod outer segment for conversion to ATP. The hexose monophosphate pathway in bovine rod outer segments can produce up to 39.8 +/- 2.2 nmol of NADPH/min/mg of protein. This rate of NADPH production is sufficient to support both the reduction of retinal to retinol (1.2 +/- 0.2 nmol of NADPH/min/mg of protein) following the photobleaching of rhodopsin and glutathione reduction (1.1 +/- 0.1 nmol of NADPH/min/mg of protein) for the protection of rod outer segments from oxidative damage. These studies provide insight into the contribution of anaerobic glycolysis and the hexose monophosphate pathway in providing energy and nucleotides for phototransduction and other outer segment processes.     

Mechanism of clathrin basket dissociation: separate functions of protein domains of the DnaJ homologue auxilin

Auxilin was recently identified as cofactor for hsc70 in the uncoating of clathrin-coated vesicles (Ungewickell, E., H. Ungewickell, S.E. Holstein, R. Lindner, K. Prasad, W. Barouch, B. Martin, L.E. Greene, and E. Eisenberg. 1995. Nature (Lond.). 378: 632-635). By constructing different glutathione-S-transferase (GST)-auxilin fragments, we show here that cooperation of auxilin's J domain (segment 813-910) with an adjoining clathrin binding domain (segment 547-814) suffices to dissociate clathrin baskets in the presence of hsc70 and ATP. When the two domains are expressed as separate GST fusion proteins, the cofactor activity is lost, even though both retain their respective functions. The clathrin binding domain binds to triskelia like intact auxilin with a maximum stoichiometry of 3 and concomitantly promotes their assembly into regular baskets. A fragment containing auxilin's J domain associates in an ATP-dependent reaction with hsc70 to form a complex with a half-life of 8 min at 25 degrees C. When the clathrin binding domain and the J domain are recombined via dimerization of their GST moieties, cofactor activity is partially recovered. The interaction between auxilin's J domain and hsc70 causes rapid hydrolysis of bound ATP. Release of inorganic phosphate appears to be correlated with the disintegration of the complex between auxilin's J domain and hsc70. We infer that the metastable complex composed of auxilin, hsc70, ADP, and P(i) contains an activated form of hsc70, primed to engage clathrin that is brought into apposition with it by the DnaJ homologue auxilin.     

Formyl phosphate: a proposed intermediate in the reaction catalyzed by Escherichia coli PurT GAR transformylase

The Escherichia coli purT encoded glycinamide ribonucleotide transformylase (GAR transformylase) serves as an alternate enzyme in the production of formyl GAR for use in de novo purine biosynthesis. This enzyme differs from the previously known purN encoded enzyme in size, sequence, and substrates; ATP and formate are required as opposed to formyl tetrahydrofolate. Kinetic studies of the wild-type PurT enzyme described here demonstrate that formyl phosphate behaves as a chemically and kinetically competent intermediate. The requirement for ATP and GAR in these reactions is consistent with previous steady-state kinetic results, which demonstrated that all substrates must be bound before catalysis. Kinetic characterization of a mutant, which releases formyl phosphate into solution, and positional isotope exchange studies also support the assignment of formyl phosphate as a plausible intermediate.     

Functional recovery after human heart transplantation is related to the metabolic condition of the hypothermic donor heart

BACKGROUND: Although strict selection criteria are being used for the acceptance of human donor hearts for transplantation, problems with respect to functional recovery on reperfusion sometimes still occur. Therefore, evaluation of the viability of a human donor heart before implantation during heart transplantation may be of great value. METHODS AND RESULTS: In the present study, the energy metabolism of 25 excised human donor hearts arrested with St Thomas' Hospital No. 2 cardioplegic solution was evaluated noninvasively by use of 31P magnetic resonance spectroscopy (MRS) before implantation and was correlated with myocardial function measured with thermodilution in heart transplant patients. No significant correlation was observed between the cardiac index of heart transplant patients during the first hours after transplantation and the phosphocreatine/ATP (r = .13, P = .54), inorganic phosphate/ATP (r = .26, P = .21), phosphomonoesters/ATP (r = .02, P = .92), or phosphocreatine/inorganic phosphate (r = .16, P = .44) ratio or the intracellular pH (r = .06, P = .78) at the time of reperfusion. However, 1 week after transplantation, a significant correlation was observed between the cardiac index and the phosphocreatine/ATP (r = .49, P = .01), phosphomonoesters/ATP (r = .45, P = .02), and phosphocreatine/inorganic phosphate (r = .40, P = .05) ratios at the time of reperfusion. In contrast, the inorganic phosphate/ATP (r = .10, P = .63) ratio and pH (r = .31, P = .13) at the time of reperfusion showed a poor correlation with the cardiac index 1 week after transplantation. CONCLUSIONS: Functional recovery after human heart transplantation is related to the metabolic condition of the hypothermic donor heart.     

Toxicity assessment of 16 inorganic environmental pollutants by six bioassays

ATP and ADP are simultaneously released from activated platelets in equimolar concentrations. Micromolar concentrations of ATP inhibit platelet aggregation by both competitive and non-competitive mechanisms. The current studies addressed the question of how platelets respond to agonists in the presence of nanomolar and micromolar concentrations of ATP and ADP alone or in combination. This is a significant issue since the concentration of ATP +/- ADP may vary widely within a microenvironment depending upon the source and cause for the release of the nucleotides. ATP (1-10 nM) was found to significantly enhance the thromboxane A2 analog, U44619-, collagen- and thrombin-induced platelet aggregations. Conversely, ATP at 1-100 microM inhibited these same reactions. ADP, in general, behaved exactly opposite to ATP. When equal amounts of ATP and ADP were added together the ADP response appeared to predominate. The observed ATP-induced response was not due to a hydrolytic product as evidenced by an unaltered response to ATP in the presence of adenosine deaminase or the ATP generating system, creatine phosphate plus creatine phosphokinase. Adenosine (1-10 nM), like ADP, inhibited agonist-induced platelet aggregation. The stimulation of agonist-induced platelet aggregation by 1-10 nM extracellular ATP appears to depend upon the phosphorylation of platelet membrane ecto proteins. The ATP analog, beta gamma-methylene ATP, that is incapable of serving as a phosphate donor for protein kinases, inhibited rather than stimulated agonist-induced platelet aggregation. The dual response of platelets to low and high concentrations of extracellular ATP +/- ADP may play a physiological role in hemostasis and thrombosis under normal and pathological conditions.