Effect of bed rest on the adenine nucleotides concentration in human blood platelets.

The effect of immobilization in bed on metabolism and function of human blood platelet was studied. Blood platelets taken from patients with bone fractures after long term bed rest (14 days and 28 days) demonstrated significantly reduced concentration of total adenine nucleotides (after 28 days reduction about 30%). This decrease of total platelet adenine nucleotides after immobilization in bed is probably caused by stimulation of platelet secretory process. Thrombin which released from control platelets 58.2% +/- 1.5% of total adenine nucleotides liberated decreased amounts (only 23.1% +/- 3.3% of total) of nucleotides from patient platelets isolated after 28 days of immobilization in bed. Loss of nucleotides from platelets was accompanied by slightly increased extent of platelet aggregation. It is concluded that during bed rest the reactivity of blood platelets (aggregation and release reaction) is stimulated.     

The effect of resveratrol on the platelet secretory process induced by endotoxin and thrombin

The effect of resveratrol (trans-3,4',5-trihydroxystilbene) on the release of adenine nucleotides and proteins from blood platelets activated by lipopolysaccharide (LPS), from Proteus mirabilis and by thrombin, were studied. Thrombin stimulated the release of adenine nucleotides from dense granules and proteins from alpha-granules. The LPS (0.3 microg/10(8) platelets, 5 min, 37 degrees C), like thrombin (2.5 U/10(8) platelets, 5 min, 37 degrees C) was found to cause a release of adenine nucleotides and proteins (p <0.05). Resveratrol (6.25-100 microg/ml, 30 min, 37 degrees C) had a different effect on the platelet release reaction caused by either LPS or thrombin. The results indicated that resveratrol inhibited, in dose-dependent manner, the secretory process (release of adenine nucleotides and proteins) induced by thrombin (p <0.05), but it significantly stimulated the liberation of proteins from blood platelets activated by LPS (p <0.05).     

Fluorometric determination of adenine nucleotides and adenosine by ion-paired, reverse-phase, high-performance liquid chromatography

A sensitive and specific assay for measurement of adenine nucleotides and adenosine by paired-ion high-performance liquid chromatography is described. The 1,N6-ethenoderivatives of ATP (epsilon-ATP), ADP (epsilon-ADP), AMP (epsilon-AMP), and adenosine (epsilon-Ado), formed by reaction with chloroacetaldehyde at 37 degrees C, were separated under isocratic conditions in 20 min. These compounds are strongly fluorescent at an emission wavelength of 280 nm, rendering a lowest detection limit of 2-5 pmol per injection. The detector responded linearly over the measured ranges (5-100 pmol for epsilon-Ado and 5-4000 pmol for nucleotides). Specificity was confirmed enzymatically. alpha, beta-Methyleneadenosine 5'-diphosphate could be used as an internal standard for measurement of the nucleotides. Significant amounts of NADH appeared as a separate peak in hypoxic tissue. Recoveries from snap-frozen kidney were 88, 92, 76, and 63% for AMP, ADP, ATP, and adenosine, with SD for recovery of 1.0, 10.5, 8.3, and 5.6%, respectively. This method was successfully used to measure adenine nucleotides and adenosine in oxygenated and hypoxic perfused rat kidneys.     

In vitro alteration of the size of the liver mitochondrial adenine nucleotide pool: Correlation with respiratory functions

Mitochondrial respiration was studied as a function of the total adenine nucleotide content of rat liver mitochondria. The adenine nucleotide content was varied by treating isolated mitochondria with pyrophosphate or by incubating pyrophosphate-treated mitochondria with ATP. Mitochondria with at least 4 nmol adenine nucleotides/mg protein maintained at least 80% of the State 3 activity of control mitochondria, which had approximately 10 nmol/mg protein. However, State 3 decreased rapidly once the adenine nucleotide content fell below 4 nmol/mg protein. Between 2 and 4 nmol adenine nucleotides/mg, State 3 was not limited by the maximal capacity of electron flow as measured by the uncoupled respiration. However, at very low adenine nucleotide levels (<2 nmol/mg), the uncoupled rates of respiration were markedly depressed. State 4 was not affected by changes in the mitochondrial adenine nucleotide content. Adenine translocase activity varied in almost direct correlation with changes in the adenine nucleotide content. Therefore, adenine translocase activity was more sensitive than State 3 to changes in total adenine nucleotides over the range of 4 to 10 nmol/mg protein. The results suggest that (i) State 3 is dependent on the level of intramitochondrial adenine nucleotides, particularly in the range below 4 nmol/mg protein, (ii) adenine translocase activity is not rate-limiting for oxidative phosphorylation in mitochondria with the normal complement of adenine nucleotides, however, at low adenine nucleotide levels, depressed State 3 rates may be explained in part by the low rate of ADP translocation, and (iii) a mechanism of net ATP uptake exists in mitochondria with low internal adenine nucleotides.     

Secretory mechanisms. Behaviour of adenine nucleotides during the platelet release reaction induced by adenosine diphosphate and adrenaline

1. Platelets containing adenine nucleotides labelled with ³H and ¹⁴C in vitro were aggregated biphasically with ADP and adrenaline. Amounts of ATP and ADP as well as the radioactivity of ATP, ADP, AMP, IMP, hypoxanthine and adenine were determined in platelets and plasma at different stages of aggregation. 2. ATP and ADP were released during the second aggregation phase and had a low specific radioactivity compared with the ATP and ADP retained by the cells. The specific radioactivity of intracellular nucleotides increased during release. The parameters observed with ADP and adrenaline as release inducers were the same as for collagen and thrombin. 3. Release induced by all four inducers was accompanied by conversion of cellular [³H]ATP into extracellular [³H]-hypoxanthine. By variation of temperature, inducer concentration, time after blood withdrawal and use of acetylsalicylic acid, the aggregation pattern caused by adrenaline and ADP could be made mono- or bi-phasic. Release or second-phase aggregation was intimately connected with the ATP–hypoxanthine conversion, whereas first phase aggregation was not. 4. The [³H]ATP–hypoxanthine conversion started immediately after ADP addition. With adrenaline it usually started with the appearance of the second aggregation phase. The conversion was present during first phase of ADP-induced aggregation only if a second phase were to follow. 5. When secondary aggregation took place while radioactive adenine was being taken up by the platelets, increased formation of labelled hypoxanthine still occurred, but there was either no change or an increase in the concentration of labelled ATP. 6. Biphasically aggregated platelets converted [³H]adenine more rapidly into [³H]-ATP and -hypoxanthine than non-aggregated platelets. Addition of [³H]adenine at different stages of biphasic aggregation showed that more [³H]hypoxanthine was formed during than after the release step. 7. We conclude that ADP and adrenaline, like thrombin and collagen, cause extrusion of non-metabolic granula-located platelet adenine nucleotides. During release metabolic ATP breaks down to hypoxanthine, and this process might reflect an ATP-requiring part of the release reaction.     

Decrease in mitochondrial levels of adenine nucleotides and concomitant mitochondrial dysfunction in ischemic rat liver

The process of mitochondrial dysfunction in ischemic rat liver was studied. A close correlation was found between decrease in the mitochondrial adenine nucleotide content and deterioration of oxidative phosphorylation capacity. The level of total adenine nucleotides, which was 15--20 nmol/mg protein in mitochondria isolated from normal liver, fell to 1--2 nmol/mg protein with concomitant loss of oxidative phosphorylation capacity after anoxic incubation in vitro or in vivo for 120 min. However, neither the permeability barrier to adenine nucleotides nor matrix enzymes were affected under these conditions. The loss of adenine nucleotides was ascribed to degradation of AMP to adenosine and then leakage of the latter. Conventional procedures for maintenance of oxidative phosphorylation capacity of isolated mitochondria, preservation in the cold and addition of ATP or a respiratory substrate under aerobic conditions, were very effective in maintaining the intramitochondrial levels of adenine nucleotides. Of the three species of adenine nucleotides, only AMP was ineffective in maintaining mitochondrial function; mitochondria containing more than 5 nmol of ATP plus ADP/mg protein exhibited normal activity of oxidative phosphorylation, but with less than 2 nmol they showed no activity.     

Some effects of ionophores for divalent cations on blood platelets Comparison with the effects of thrombin

The ionophores A 23187 and X-537 A induce an uptake of ⁴⁵Ca by human blood platelets. They induce the release of adenine nucleotides and of serotonin. A 23187 also induces platelet aggregation and the retraction of a clot formed in platelet-rich plasma by reptilase. These results suggest that an increase of the concentration of Ca²⁺ in the cytoplasm plays a role in the activation of blood platelets.     

Nucleotide profiles of normal human blood cells determined by high-performance liquid chromatography

An anion-exchange high-performance liquid chromatography method has been used to quantitate the intracellular purine and pyrimidine nucleotides in extracts of pure lymphocytes, monocytes, neutrophils, eosinophils, erythrocytes, and platelets isolated from the blood of healthy human donors. For accurate and reproducible measurements of the nucleotide profiles in different types of pure leukocytes, the cell suspensions have to be free of platelets and erythrocytes. Incubation of the purified leukocytes for 1 h at 0 degrees C did not alter the nucleotide concentrations but reduced the interdonor variation to 10%. Incubation of purified lymphocytes for 1 h at 37 degrees C caused considerable changes in the relative concentrations of the adenine, guanine, uracil, and cytosine nucleotides. During this incubation the cell viability, the cell number, and the ATP:ADP ratio decreased. Incubation of monocytes and granulocytes for 1 h at 37 degrees C caused considerable loss of cells and/or cell death. For erythrocytes and platelets reproducible nucleotide concentrations were obtained after extraction of freshly isolated cells. During storage of erythrocytes, both at 0 degrees C and at 37 degrees C, a decrease in the ATP:ADP ratio was detected. In all cell types the predominant nucleotides were purine nucleotides, especially adenosine triphosphate. The relative concentrations of the adenine, guanine, uracil, and cytosine nucleotides were very reproducible per cell type and appeared to be characteristic for each cell type. The total nucleotide content was nearly the same for all cell types except erythrocytes, when expressed per microgram of protein. The described methods for purification and storage of blood cells will be useful for comparison of blood cells from healthy donors with those of patients, for example, leukemia patients, in which deviations of the purine and pyrimidine metabolic enzymes have already been described.     

UV light-induced changes in washed pig platelets

The effect of ultraviolet radiation (UV-A, 360 nm) on the thrombin-induced aggregation of washed pig platelets as well as on the release of adenine nucleotides and proteins was studied. The level in platelets of adenine nucleotides, mainly ADP and ATP, decreased rapidly following the exposure of platelets to a high dose of UV-A (0.5 W/cm2, 30 min). Through thrombin-induced aggregation of irradiated platelets was inhibited, the release reaction occurred. The amount of the released adenine nucleotides and proteins was, however, dependent on the dose of UV light. These findings suggest that UV-A light can stimulate the platelet release reaction.     

Primary dysfunction in aggregation and secretion of SHRSP platelets: not secondary to the circulation of "exhausted" platelets

The thrombin-induced secretion of [14C]-serotonin and adenine nucleotides from stroke-prone spontaneously hypertensive rats (SHRSP) platelets was markedly reduced with the development of hypertension accompanying hypo-aggregability compared with that from age-matched Wistar Kyoto rats (WKY) platelets. Calcium Ionophore A23187-induced secretion and aggregation were also attenuated in SHRSP platelets. Additionally, an enhancement of platelet secretion as well as aggregation by extracellular Ca2+ was less in SHRSP platelets than in WKY platelets. The platelet contents of adenine nucleotides and serotonin were not different between SHRSP and WKY at 5-16 weeks of age whereas they became significantly lower in SHRSP beginning at 22 weeks. The serotonin content in SHRSP platelets at 36 weeks of age was only 55% of that in WKY platelets. It is suggested that the reduced platelet aggregation and secretion observed in SHRSP platelets at ages lower than approximately 20 weeks are not secondary phenomena to the circulation of degranulated platelets, but the primary defect of SHRSP platelets appears to be an impaired function of Ca2+.     

The effect of ebselen on adenine nucleotide hydrolysis by platelets from adult rats

The extracellular hydrolysis of adenine nucleotides by intact rat blood platelets occurs by the action of a cascade of enzymes constituted by an NTPDase 3 (CD39, EC 3.6.1.5, apyrase) and a 5'-nucleotidase (CD73, EC 3.5.7.3), whose final product is adenosine. Ebselen is a seleno-organic compound that possesses low toxicity and exhibits antioxidant, anti-inflammatory, anti-atherosclerotic, and cytoprotective properties. The main objective of this study was to evaluate if the anti-inflammatory drug ebselen can modulate the extracellular adenine nucleotide hydrolysis by platelets from rats. Our results showed that ebselen, at final concentrations of 30 and 100 microM, inhibits in vitro ATP extracellular hydrolysis by 48 and 60%, respectively. Ebselen, at final concentrations of 100 and 130 microM, also inhibited the in vitro extracellular hydrolysis of ADP by 28 and 35%, respectively. However, this drug did not alter AMP hydrolysis by platelets in the appropriate assay conditions. Kinetic analysis showed that the inhibition of ADP and ATP hydrolysis by ebselen, in rat platelets, is of the uncompetitive type. The IC50 calculated from the results were 99 +/- 10 and 186 +/- 47 microM (mean +/- S.D., n = 3) for ATP and ADP hydrolysis, respectively.     

A simple assay for adenylate cyclase in intact cells by affinity elution chromatography.

A method using the principle of affinity elution chromatography is described for the assay of adenylate cyclase in intact human platelets. By incubating platelet-rich plasma in the presence of radioactively labelled adenine, the ATP pool of the cells was prelabelled. Formation of labelled cyclic AMP from ATP was determined by extracting the platelets with HC1O4. After removal of the latter as KC1O4, the extract containing cyclic AMP and other adenine nucleotides was adsorbed in a NN-diethyl-N-2-hydroxypropylamino (QAE)-cellulose column. The column was washed, and subsequently cyclic AMP was specifically eluted with a cyclic AMP-dependent protein kinase and the radioactivity of the eluate was determined.     

Reversible electropermeabilisation of human and rat blood platelets: evaluation of morphological and functional integrity 'in vitro' and 'in vivo'

A high-voltage discharge procedure has been developed for permeabilising the plasma membranes of both human and rat blood platelets. The cells can be resealed by incubation at 37 degrees C, show less than 4% loss of lactate dehydrogenase (LDH) implying minimal cell lysis and also have well maintained morphological and functional integrity. The prototype apparatus used at field strengths between 6 and 8 kV/cm produces membrane pores which allow free diffusion of low molecular weight substances such as adenine nucleotides, inositol phosphate and fluorescent dyes. Two properties, namely Ca2+-induced secretion of granule stored 5-hydroxytryptamine (5HT) and inositol 1,4,5-trisphosphate (IP3)-induced release of intracellularly sequestered 45Ca, which are both well expressed immediately after permeabilisation, are essentially abolished after resealing. The efficiency of permeabilisation and resealing can be simply monitored by shifts in 'apparent platelet volume' using a resistive particle counter (Coulter). Permeabilised platelets show a shift in modal volumes from a control range 4-7 fl to 10-15 fl. Resealing restores these modal volumes to the original control range. Encapsulation of the fluorochrome, Lucifer yellow (Mr 550), during permeabilisation revealed that after resealing greater than 85% of rat platelets, and close to 100% human platelets, contained the encapsulated dye. The initial rates and % aggregation responses of both human and rat platelets to collagen, thrombin and the thromboxane A2-mimetic U46619 remained essentially normal after permeabilisation and resealing further illustrating the maintenance of functional competence following treatment. Resealed rat platelets reinfused into the circulation after labelling with [111In]indium oxine gave survival curves similar to those of control platelets. Therefore, this reversible permeabilisation procedure may allow the use of autologous or heterologous platelets as carrier vehicles for the delivery of drugs and other agents 'in vivo'.     

Stimulatory effects of endotoxin on the platelet secretory process

The effects of the lipopolysaccharides (LPS) of Proteus mirabilis (smooth and rough types), differing significantly in their composition on the release of compounds stored in specific platelet granules, were studied. There are two main types of secretory granules in blood platelets. Dense granules contain adenine nucleotides, and in alpha-granules different proteins are stored. The LPS were found to cause a dose-dependent release of proteins and adenine nucleotides. In the extracellular medium LDH activity was not present. The results presented in this study indicate that LPS from P. mirabilis act directly on blood platelets and induce the platelet secretory process. In comparison with thrombin, a strong platelet agonist, the action of the endotoxins tested was weak.     

Determination of chloroacetaldehyde, a metabolite of oxazaphosphorine cytostatic drugs, in plasma

A derivatization high-performance liquid chromatographic method with ultraviolet detection to monitor the plasma concentration of chloroacetaldehyde, a neurotoxic metabolite of oxazaphosphorine drugs, is presented. To prevent the rapid degradation of chloroacetaldehyde, the plasma samples are stabilized with formaldehyde. The method is linear in the concentration range 1–250 nmol/ml. Blood samples from a patient who was treated with a ten-day continuous infusion of ifosfamide were assayed. The chloroacetaldehyde concentrations did not exceed 10 nmol/ml.     

Transfer of adenine nucleotides between the releasable and nonreleasable compartments of rabbit blood platelets

The metabolic pool of adenine nucleotides in platelets can be labeled by incubating platelets for 1 h in vitro with [14C]adenosine or [32P]orthophosphate. When these platelets are treated with thrombin, the adenine nucleotides released are not labeled. Because of this, Holmsen's suggestion of a metabolically inert pool of granule nucleotides has been generally accepted. We have found that upon incubation of labeled rabbit platelets for longer times (up to 6 h) in vitro, or upon reinjection and reharvesting at times up to 66 h, the releasable pool of adenine nucleotides becomes labeled. Because the rates of 32p and 14C incorporation into this releasable pool are similar, it seems likely that these labels enter the granules as ATP. Equilibrium between the metabolic and granule pools is complete by 18 h. When rabbit platelets are labeled in vivo by intravenous injection of [32P]orthophosphate, peak labeling occurs between 60 and 70 h; this corresponds to their maturation time. The platelets probably incorporate 32P during their production in the megakaryocytes. The specific radioactivity of the adenine nucleotides in the releasable (granule) pool of these platelets is the same as the specific radioactivity in the nonreleasable (metabolic) pool. Since inorganic phosphate in platelets (and undoubtedly in the megakaryocytes) exchanges with inorganic phosphate in plasma, and since the radioactivity of the latter decreases rapidly, the adenine nucleotides in the two pools must exchange to maintain the same specific radioactivity. Transfer of adenine nucleotides into storage granules may represent a general phenomenon because it has been observed in the chromaffin cells of the adrenal medulla also.     

Elevation of the adenylate pool in rat cardiomyocytes by S-adenosyl-L-methionine

Rapid resynthesis of the adenylate pool in cardiac myocytes is important for recovery of contractility and normal function of regulatory mechanisms in the heart. Adenosine and adenine are thought to be the most effective substrates for nucleotide synthesis, but the possibility of using other compounds has been studied very little in cardiomyocytes. In the present study, the effect of S-adenosyl-L-methionine (SAM) on the adenylate pool of isolated cardiomyocytes was investigated and compared to the effect of adenine and adenosine. Adult rat cardiomyocytes were isolated using the collagenase perfusion technique. The cells were incubated in the presence of adenine derivatives for 90 min followed by nucleotide determination by HPLC. The concentrations of adenine nucleotides expressed in nmol/mg of cell protein were initially 22.1 +/- 1.4, 4.0 +/- 0.3 and 0.70 +/- 0.08 for ATP, ADP and AMP, respectively (n = 10, +/- S.E.M.), and the total adenylate pool was 26.8 +/- 1.6. In the presence of 1.25 mM SAM in the medium, the adenylate pool increased by 5.2 +/- 0.4 nmol/mg of cell protein, but only if 1 mM ribose was additionally present in the medium. No changes were observed with SAM alone. A similar increase (by 4.9 +/- 0.6 nmol/mg protein) was observed after incubation with 1.25 mM adenine plus 1 mM ribose, but no increase was observed if ribose was omitted. Adenosine at 0.1 or 1.25 mM concentrations also caused an increase in the adenylate pool (by 5.2 +/- 1.0 and 5.2 +/- 0.9 nmol/mg protein, respectively), which in contrast to the SAM or adenine was independent of the additional presence of ribose. Thus, S-adenosyl-L-methionine could be used as a precursor of the adenylate pool in cardiomyocytes, which is as efficient in increasing the adenylate pool after 90 min of incubation as adenosine or adenine. Nucleotide synthesis from SAM involves the formation of adenine as an intermediate with its subsequent incorporation by adenine phosphoribosyltransferase.     

The role of blood platelets in nucleoside metabolism: regulation of platelet thymidine phosphorylase

Blood platelets are the smallest cellular elements in mammalian blood. Because of their small size, platelets have an usually large surface area: volume ratio and are exquisitely sensitive to a multitude of physiologica and environmental stimuli. Platelets lack nuclei, but most possess functional mitochondria and remain capable of both anaerobic and aerobic energy metabolism, for which they utilise a variety of substrates including many which are cytotoxic and genotoxic for other (nucleated) cells. Nucleic acid precursors are amongst the potentially genotoxic compounds for which platelets have an apparently insatiable appetite. In particular platelets actively scavenge adenine and adenosine, which they convert to nucleotides and use in energy metabolism, but they also rapidly phosphorylyse thymidine and liberate thymine into the extracellular medium. In addition, platelets contain non-metabolisable membrane-bound pools of adenine nucleotides which they secrete in response to strong agonists. Taken together, these observations suggest that blood platelets play an important role in nucleic acid precursor metabolism.

In the previous paper we have shown that most thymidine phosphorylase activity present in normal human blood resides in the cytoplasm of platelets. Here we demonstrate that this enzyme activity can be modulated in a dose-dependent fashion, not only by substances recognised as platelet agonists and antagonists, but also by some compounds which are considered to be toxic, mutagenic and/or carcinogenic. The data which we present provide additional support for our previous suggestion that platelets regulate thymidine homeostasis and further imply that this is the normal, physiological, platelet function. Preliminary results suggest that assays of blood platelet thymidine metabolism may provide data with a wide variety of applications.     

Preservation of red blood cells with purines and nucleosides. III. Synthesis of adenine, guanine, and hypoxanthine nucleotides

Purine nucleotides of fresh human red cells and of red cells during storage at 4 degrees and 25 degrees C with additions of adenine, guanine, guanosine and inosine were estimated by HPLC. Six nucleotides were found in red cells: ATP, ADP, AMP, GTP, GDP, and IMP. The adenine nucleotides represented 92 per cent of the total purine nucleotides, guanine nucleotides 7 per cent and IMP less than 1 per cent. In red cells stored with adenine the total concentration of purine nucleotides increased to 125 per cent of the normal value. An adenine-free but guanine and guanine + inosine containing medium caused a decrease of the concentration of purine nucleotides by 10 to 20 per cent. When red cells were stored without adding guanine or guanosine the content of the guanine nucleotides decreased from 0.32 to 0.17 mumol/g Hb due to the decrease in the GTP content, but the GDP concentration increased slightly. In CPD-AG blood, however, the concentration of guanine nucleotides increased considerably up to 0.6 mumol/g Hb. IMP was estimated in all investigated stored red cells. In CPD-A and in CPD-AG blood 0.4 mumol/g Hb were produced during 3 weeks of storage, but twice of that in CPD-AI blood. The principles of the synthesis and the degradation of purine nucleotides in stored red cells are discussed in detail.     

Investigation of the dependence of the intramitochondrial [ATP]/[ADP] ratio on the respiration rate

The relationship between the respiration rate and the intra- and extramito-chondrial adenine nucleotides was investigated in isolated rat liver mitochondria.

For the determination of adenine nucleotide patterns in both compartments a new procedure was developed, based on the evaluation of these metabolites from incubation of various amounts of mitochondria under identical stationary states of oxidative phosphorylation. These identical states were adjusted by addition of appropriate amounts of hexokinase to a glucose-containing incubation mixture.

Adenine nucleotides were measured in aliquots of the total extract of the incubation mixture without any separation. The concentrations of the adenine nucleotides in both compartments were obtained from a plot of the total concentration of these species versus mitochondrial protein. Disturbances of this method by unspecific efflux of adenine nucleotides could be excluded.

The results obtained for the total adenine nucleotide content (12 nmol · mg⁻¹ protein) and the intramitochondrial [ATP]/[ADP] ratio (about 4 in the resting state) are in good agreement with data obtained by other methods.

Strong evidence is provided for a decrease of the intramitochondrial [ATP]/[ADP] ratio with increasing rate of oxygen consumption. Therefore it is not necessary to assume a microcompartmentation of the intramitochondrial adenine nucleotide pool in respect to the ATPase reaction and the adenine nucleotide translocation.