Myocardial adenosine salvage rates and restoration of ATP content following ischemia.

The isolated perfused rat heart was utilized to determine the maximum rate of adenosine incorporation into adenine nucleotides and the effect of ischemia on this rate. In aerobic hearts, the rates of [8-14C]adenosine incorporation into nucleotides in nanomoles/minute per gram dry tissue were ATP 34 +/- 2, ADP 6 +/- 0.4, AMP 3 +/- 0.3, and IMP, 1 +/- 0.2. Following ischemia these values were not significantly different except for the rate of incorporation into IMP, which doubled. The extent of adenosine deamination with one pass through the coronary vasculature was the same in aerobic and postischemic hearts: 2% and 7% of the perfusate adenosine was converted to hypoxanthine and inosine, respectively. These percentages were similar at 50, 100, and 200 micron adenosine. Perfusion of aerobic hearts for 5 h with adenosine did not change ATP concentrations. Therefore, [8-14C]adenosine incorporation into ATP in these hearts appeared to represent ATP turnover. In contrast, 5 h perfusion of postischemic hearts with adenosine restored ATP concentrations to control values. The synthesis rate calculated from the increase in ATP concentration was comparable to the synthesis rate calculated from [8-14C]adenosine incorporation. Thus, incorporation of [8-14C]adenosine into ATP in postischemic hearts represented net ATP synthesis.     

Uptake and release of adenosine in isolated rat fat cells.

Radioactively labelled adenosine and adenine were rapidly taken up by isolated rat fat cells, and incorporated into nucleotides, of which ATP dominated. The overall process had an apparent Km of 1--5 micrometers. During incubation, especially in the presence of lipolytic agents, there was a reduction in labelled ATP with a compensatory increase in ADP, AMP, cAMP and nucleosides. The build-up of adenosine during incubation was inhibited by theophylline, which inhibits 5'-nucleotidase. Radioactivity released from perifused fat cells consisted mainly of nucleoside material, of which adenosine predominated. Lipolytic stimulation caused no significant increase in nucleoside outflow from perifused cells, whereas oxygenation was capable of reducing this outflow. It is concluded that adenosine is formed by fat cells as a consequence of ATP breakdown. Stimulation of lipolysis during activation of the sympathetic nerves leads to reversible ATP breakdown and adenosine release. Adenosine might therefore act as a modulator of lipolysis in vivo under these conditions, even though it does not serve as a feed back regulator in the proper sense.     

Uptake and release of adenosine in isolated rat fat cells

Radioactively labelled adenosine and adenine were rapidly taken up by isolated rat fat cells, and incorporated into nucleotides, of which ATP dominated. The overall process had an apparent K_m of 1–5 μM. During incubation, especially in the presence of lipolytic agents, there was a reduction in labelled ATP with a compensatory increase in ADP, AMP, cAMP and nucleosides. The build-up of adenosine during incubation was inhibited by theophylline, which inhibits 5′-nucleotidase. Radioactivity released from perifused fat cells consisted mainly of nucleoside material, of which adenosine predominated. Lipolytic stimulation caused no significant increase in nucleoside outflow from perifused cells, whereas oxygenation was capable of reducing this outflow. It is concluded that adenosine is formed by fat cells as a consequence of ATP breakdown. Stimulation of lipolysis during activation of the sympathetic nerves leads to reversible ATP breakdown and adenosine release. Adenosine might therefore act as a modulator of lipolysis in vivo under these conditions, even though it does not serve as a feed back regulator in the proper sense.     

Delayed phytohemagglutinin-stimulated production of adenosine triphosphate by aged human lymphocytes: Possible relation to mitochondrial dysfunction

The decreased immune response associated with aging may, in part, reflect intrinsic age-related biochemical alterations in lymphocytes from older animals. We measured levels of lymphocyte adenosine triphosphate (ATP) and continuous [³H]thymidine incorporation in phytohemagglutinin-stimulated lymphocytes from young and old humans, and the effects thereon of inhibitors of mitochondrial oxidative phosphorylation and protein synthesis. No difference was found in adenine nucleotide content between young and old subjects. After 24 hours of culture there was a decrease in ATP, with recovery and 2–3-fold increase at 48 hours in young cells after phytohemagglutinin stimulation. We observed a clearcut delay in older lymphocytes of the increase in ATP and [³H]thymidine incorporation following phytohemagglutinin stimulation. We found no evidence for decreased viability or diminished number of responding units in aged cultures. The evidence suggests that mitochondrial dysfunction may play a role in the immunodeficiency of aging.     

Adenine nucleotide levels and adenosine metabolism in cultured calvarial bone

Adenine nucleotide levels were measured in extracts of murine calvaria after different periods of culture with or without parathyroid hormone (PTH; 10(-8) M) or PGE2 (10(-7) M). In control calvaria the energy charge, (ATP + 1/2 ADP)/(ATP + ADP + AMP), remained at close to 0.7 over a 24 hour culture period. However, bones cultured with PTH or PGE2 showed a transient fall in the energy charge down to 0.5. This was not associated with a fall in total adenine nucleotides. The rate of adenosine metabolism in cultured bone in vitro was studied by determining the contents of adenosine, inosine, 2-deoxyadenosine, 2-deoxyinosine and hypoxanthine in the culture medium. There was a continuous increase in adenosine, inosine and hypoxanthine as well as a disappearance of medium 2-deoxyadenosine that was accounted for by appearance of 2-deoxyinosine. The deaminating activity could only partly be accounted for by activity in the medium and thus probably mainly resides in the bone cells. PTH (10(-8) M) did not alter the rate of disappearance of 2-deoxyadenosine or adenosine deaminase activity determined in bone extracts. The results demonstrate that two substances that increase calcium mobilization from bone alter ATP utilization and/or synthesis without significantly influencing adenosine production or metabolism.     

Adenosine 5'-triphosphate synthesis and metabolism localized in neurites of cultured sympathetic neurons

Adenosine triphosphate synthesis and metabolism in cultured sympathetic neurons was studied after the incorporation of [2-3H]adenine into intact or microdissected neurites to determine whether ATP is provided locally during neurite outgrowth, when and where it is synthesized and how its levels are regulated at rest and following depolarization. Neurites maintained an independent capability for synthesis of ATP at any stage of growth: [3H]ATP levels increased in neurites in direct proportion to neurite length and equivalent amounts of [3H]ATP were synthesized by intact neurites, by neurites separated from cell bodies or by neurites further segmented into sections. Thus, metabolic labelling of cultured neurons with [3H]adenine provides a simple method to measure relative neurite outgrowth. Neurite ATP was maintained mainly by respiration but also by glycolysis and [3H]ATP levels were stable for at least 14 h after adenine withdrawal when cells were at rest. Depolarization overcame respiratory control, causing a quantitative conversion of ATP to adenosine monophosphate (AMP) and inosine monophosphate (IMP) and the release of nucleosides (adenosine and inosine) and nucleotides [adenosine diphosphate (ADP) and adenosine monophosphate (AMP)]. Release of nucleosides, but not of nucleotides or [3H]noradrenaline, was enhanced by NaN3 or 2-deoxyglucose under nondepolarizing conditions and was prevented by the adenosine transport inhibitor p-nitrobenzyl-6-thioinosine. It is concluded that neurites can use local mechanisms for ATP synthesis that do not depend on a functional connection to the cell body. Any metabolic stress which causes ATP breakdown causes these cells to express a transient purinergic phenotype involving release of adenosine and inosine by facilitated diffusion. To promote the release of purine nucleotides, however, more specific stimuli are required.     

Binding of adenosine diphosphate to intact human platelets.

1. Human platelet-rich plasma was incubated at 37 degrees C with [8-14C]ADP and with human serum albumin labelled with 125I. The platelets were rapidly separated by centrifugation through silicone oil. From radioactivity determinations of plasma and platelet pellets the uptake of ADP, without or with break-down products, by the platelets was calculated on the assumption that the 125I radioactivity in the pellet represented trapped plasma. 2. ADP radioactivity was taken up by platelets within 10 sec and increased with time of incubation. The uptake of other nucleotide diphosphates was less initially and increased much more slowly. 3. Radioactivity added as ADP was recovered as ATP to the extent of 60%; as ADP of 30%; and as AMP of 10%. 4. Prostaglandin E1 which inhibited platelet aggregation had no effect on the initial or subsequent uptake or on this distribution of radioactivity. 5. The rate of rise in uptake was much slower when platelets were resuspended in plasma heated to 56 degrees C for 30 min. 6. Unlabelled adenosine inhibited the later, but not the initial, uptake while unlabelled ADP inhibited both. Dipyridamole, which blocks adenosine uptake, prevented the later but not the initial uptake. 7. [alpha-32P]ADP radioactivity was taken up at the earliest sampling time and the extent of uptake did not further increase. 8. Guinea-pig platelets, which do not take up adenosine, took up [8-14C]ADP radioactivity from purine-labelled ADP initially. 9. It was concluded that the initial uptake represented binding of ADP and that the later uptake represented labelled adenosine originating as a break-down product of ADP. 10. A Scatchard plot of ADP uptake indicated more than one type of binding site. There were approximately 88,000 high affinity sites per platelet which had an affinity constant of 5-41 X 10(5) M-1.     

Myocardial adenosine stimulates release of cyclic adenosine monophosphate from capillary endothelial cells in guinea pig heart

Activation of coronary endothelial cell adenylate cyclase was studied in the isolated guinea pig heart by prelabelling endothelial adenine nucleotides using intracoronary infusion of [³H]-adenosine, and measuring the coronary efflux of [³H]-cyclic adenosine monophosphate (cAMP). Hypoxia (30 % O₂) caused a 4-fold increase in coronary release of [³H]-cAMP, which was decreased by 63 % by infusion of the adenosine receptor antagonist, theophylline (50 M). During normoxic control conditions, degrading adenosine to non-vasoactive inosine by intracoronary infusion of adenosine deaminase (1.7 U/ml) caused a 20 % decrease in the release of [³H]-cAMP. The effect of adenosine deaminase was reversed by a specific enzyme inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride. Coronary efflux of [³H]-cAMP during intracoronary infusion of 1 M adenosine triphosphate (ATP), adenosine diphosphate or adenosine monophosphate (AMP) (plus adenosine deaminase 8 U/ml) was only 13 % of that due to 1 M adenosine. Adenosine receptor blockers theophylline and CGS 15943A caused equivalent inhibition of the coronary vasodilator actions of adenosine and ATP. Intracoronary infusion of prostaglandin E₁ and the ₂-adrenergic agonist procaterol caused parallel, dose-dependent increases in coronary conductance and the venous release of [³H] cAMP. It is concluded that (1) under both normoxic and hypoxic conditions, adenosine formed by the heart may activate endothelial cell adenylate cyclase via membrane adenosine receptors, (2) coronary receptors for adenosine and ATP share common ligand affinities but ATP receptors are not coupled to adenylate cyclase, and (3) other vasodilators known to activate endothelial adenylate cyclase in vitro cause parallel increases in coronary conductance and adenylate cyclase activity in the beating heart.     

Action of tubercidin and other adenosine analogs on Schistosoma mansoni schistosomules

The incorporation of the radiolabeled adenosine analogs tubercidin, formycin A, 9-deaza-adenosine, and adenine arabinoside into nucleotides of Schistosoma mansoni schistosomules was studied in vitro. Of the four analogs, only tubercidin and formycin A were incorporated into the nucleotide pool, at rates respectively one-tenth and one-fiftieth the rate of adenosine incorporation. Tubercidin inhibited schistosomule motility in vitro with an approximate IC50 value of 1 microM, whereas formycin A exerted no visible effect even when more of it than of tubercidin was incorporated into the nucleotides and nucleic acids. Formycin A thus acts like a nontoxic adenosine analog. 7-Deaza-adenine, the purine base of tubercidin, was not incorporated into nucleotides. 7-Deaza-adenine, 9-deaza-adenosine, and adenine arabinoside all had no effect on schistosomule motility at concentrations up to 100 microM. Formycin A blocked the incorporation of tubercidin and of adenosine with equal effectiveness, as did p-nitrobenzyl-6-mercaptopurine ribonucleoside, a specific inhibitor of nucleoside transport in many mammalian cells. Thus, formycin A, tubercidin, and adenosine appear to have a common mechanism of cellular uptake. The significant levels of adenosine phosphorylase and adenine phosphoribosyl transferase activity found in schistosomule extracts suggests that most of the transported adenosine is converted to adenine before conversion to AMP. The levels of adenosine kinase and tubercidin kinase, while low, can more than account for the rate of tubercidin incorporated into intact schistosomules. The kinase(s) may also represent a minor pathway for direct adenosine incorporation. It may have a rather unusual substrate specificity because it is able to recognize adenosine, tubercidin, and formycin A as substrates, but not 9-deaza-adenosine or adenine arabinoside.     

Regulatory effects of adenosine and adenine nucleotides on oxygen radical responses of neutrophils

Our recent studies have indicated that release of ATP/ADP from platelets causes enhanced O2-. responses in stimulated neutrophils. The current investigations were designed to provide further details of this phenomenon, to determine the structure-function correlates of the adenine compounds, and to assess if the results might be explained by the formation of a single metabolic product of ATP. ATP, ADP, AMP and adenosine enhanced O2-. responses of rat neutrophils stimulated with immune complexes or formyl chemotactic peptide (FMLP) but had no effect on responses of phorbol ester-stimulated neutrophils. Similar results were obtained in human neutrophils stimulated with immune complexes; when FMLP was the agonist, the results were divergent: ATP and ADP enhanced the responses, whereas AMP and adenosine were inhibitory. In structure-function studies, hydrolytically resistant forms of ATP (and other adenine nucleotides) containing blocked or cross-linked phosphate groups were active, suggesting that hydrolysis of these compounds to a common metabolic product is not required for their effects on O2-. responses. In contrast, other chemical modifications of the ribose ring or adenine base of ATP resulted in greatly diminished activity. To further pursue the question of whether metabolism of the adenine compounds via the adenosine pathway was related to the observed effects on O2-. responses, addition to rat neutrophils of inhibitors of adenosine deaminase, S-adenosyl homocysteine hydrolase, or xanthine oxidase failed to reproduce or augment the enhancement effects of the adenine compounds on O2-. responses, suggesting that metabolism of the adenine compounds to a common product may not be a requirement for the observed effects. Although the manner by which the adenine compounds affect O2-. responses is not known, the data suggest that adenosine and adenine nucleotides have important regulatory effects on oxygen radical responses of stimulated neutrophils.     

Characterization of a mutant Leishmania donovani deficient in adenosine kinase activity

From a mutagenized population of wildtype Leishmania donovani promastigotes, a clonal cell line, TUBA2, was isolated by virtue of its ability to survive and grow in 20 microM tubercidin (7-deazaadenosine). The TUBA2 clone was also 1000-fold less sensitive than the parental line to growth inhibition by formycin A, another cytotoxic adenosine analog. Parental and mutant cells, however, were equally sensitive to growth inhibition by formycin B, allopurinol riboside, and 6-thioguanosine. Mutant cell extracts, unlike those prepared from wildtype cells, did not phosphorylate radiolabelled adenosine, tubercidin, or formycin A. Intact adenosine kinase-deficient cells did not accumulate exogenous tubercidin or formycin A but incorporated [14C]adenosine at rates 25% of those found for parental cells. The uptake data suggest that adenosine kinase plays an important role in the metabolism of adenosine but indicate alternative metabolic pathways for this nucleoside. The metabolism of adenosine to the nucleotide level in TUBA2 cells appears to be initiated via deribosylation to adenine. Significant amounts of both adenosine hydrolytic and adenosine phosphorylytic activities have been detected in L. donovani promastigotes. Furthermore, L. donovani extracts could slowly catalyze the deamination of formycin A. The isolation and characterization of adenosine kinase-deficient cells has provided considerable insight into the function of the purine pathway in L. donovani.     

Nucleotide uptake by isolated cholinergic synaptic vesicles: Evidence for a carrier of adenosine 5'-triphosphate

The in vitro uptake of [³H]nucleotides was studied using cholinergic syaptic vesicles isolated from Torpedo electric organ, with a resting membrane potential of 50–60 mV. The osmotically sensitive uptake of [³H]adenosine 5'-triphosphate (ATP) was markedly influenced by temperature and external pH, and was maximal after 40–50 min; longer incubation resulted in loss of accumulated radiolabel. Similar characteristics were also observed for adenosine 5'-mono- and diphosphate and guanosine and uridine triphosphates, all of which acted as competitive substrates for the saturable system which transported ATP (K_T 1.15 mM). Breakdown of [³H]nucleotides in the medium was not a significant factor, and adenosine, guanosine and adenine were very poorly incorporated. Under conditions of V_max, vesicle to medium ratios of [³H]ATP of 20–25 were observed; the amount of radiolabel was equivalent to 20–50% of the initial endogenous amount of ATP in the vesicles. Atractyloside specifically inhibited nucleotide transport with no modification of hemicholinium-3 sensitive acetylcholine uptake. Antisera raised (a), to whole Torpedo vesicle extract, and (b), to a single purified vesicle polypeptide, greatly stimulated ATP uptake without effect on simultaneous influx of either acetylcholine or glucose.It is concluded that isolated vesicles contain a nucleotide carrier of wide pharmacological specificity (possibly the 34,000 molecular weight protein of Stadler & tashiro [1979]), which is likely to be of physiological relevance. Implications for vesicular refilling mechanisms are discussed.     

Uptake of adenosine by dispersed chich embryonic cardiac cells.

Adenosine is involved in the regulation of coronary blood flow, but its mechanism of action is not clear. The present investigation is an attempt to understand the mechanism(s) of uptake of adenosine in dispersed chick embryonic cardiac cells and its relationship to the adenosine hypothesis. Adenosine is readily taken up by these cardiac cells, and a small fraction is incorporated into adenine nucleotides, whereas a major fraction is deaminated to inosine. The mechanism of uptake is different in 12- to 15-day-old chick embryos compared to 16- to 22-day-old embryos. The younger embryo heart cells show the incorporation of adenosine into adenine mononucleotides of the incubation medium as well as all the adenine nucleotides of the cells, whereas the older embryo heart cells show incorporation of adenosine only into the adenine nucleotides of the cells. The isolated cells used in the present study do not leak any significant amounts of adenosine kinase and/or nucleotides, and free adenosine was not found in the cells, even with extracellular concentrations as high as 1 mM. The absence of free adenosine in isolated dispersed cells reflects the activities of adenosine kinase and adenosine deaminase and is compatible with the adenosine hypothesis for the regulation of coronary blood flow.     

Purine salvage at the cholinergic nerve endings of the Torpedo electric organ: the central role of adenosine

The uptake and metabolism of adenosine, adenine, inosine and hypoxanthine were studied at the cholinergic nerve endings of the Torpedo electric organ. In isolated synaptosomes there is a linear uptake (measured up to 60 min) for adenosine and adenine at concentrations of 0.3 μM Uptake of adenosine exceeds that of adenine by a factor of 10. Adenosine is transported into synaptosomes via a saturable uptake system (K_m, 2 μM;V_max, ～- 30 pmols/min/mg protein). 2′-Deoxyadenosine is a competitive inhibitor of synaptosomal adenosine uptake. The nerve terminal possesses anabolic pathways for the formation of adenosine 5′-triphosphate from both adenosine and adenine. Adenosine becomes phosphorylated rapidly after entry into synaptosomes to form adenosine 5′-monophosphate; adenosine 5′-diphosphate and adenosine 5′-triphosphate were also major metabolites (70%). Adenine, inosine and hypoxanthine first accumulate in the synaptosomes. However, adenine leads to major formation of nucleotides (41% adenosine 5′-triphosphate after 60 min). Only traces of adenosine-3′:5′ cyclic monophosphate are formed from both adenosine and adenine. If adenosine 5′-triphosphate is added to a suspension of intact synaptosomes it becomes degraded to adenosine.We conclude that cholinergic nerve endings in the Torpedo electric organ possess an effective purine salvage system. Adenosine 5′-triphosphate released from either a pre- or a postsynaptic source would become degraded to adenosine in the extra-cellular medium and be re-used via an uptake system for renewed synthesis of adenosine 5′-triphosphate in nerve terminals.     

Effect of dipyridamole on adenosine incorporation into hypoxanthine nucleotides of fresh human red cells

Fresh human red cells were incubated for 2 hours in a medium containing adenosine, pyruvate and inorganic phosphate (APP medium), or in APP medium supplemented with 10(-4) M dipyridamole (APPD medium). No measureable amount of ITP was found in fresh red cells, and the average IMP content in these cells was 0.18 +/- 0.09 mumol/g Hb. After 2 hours incubation in APP medium, the IMP content increased almost 8.5-fold to 1.52 +/- 0.78 mumol/g Hb. Under these conditions the ITP level also increased to 1.40 +/- 0.84 mumol/g Hb. After 2 hours incubation of red cells in APPD medium, the average IMP content increased to 5.30 +/- 2.33 mumol/g Hb, about 3.5 times that found in APP medium. At the same time ITP content was about 53.6% lower, that is 0.65 mumol/g Hb. In red cells incubated in APPD medium, penetration of 8-14C-adenosine decreased by 50%, and incorporation of this nucleotide into the pool of all free nucleotides also decreased by 18.2% as compared to red cells incubated in APP medium. It is concluded that IMP is probably formed directly from AMP gained by the phosphorylation of adenosine during its penetration.     

Adenine Nucleotide Degradation by the Obligate Intracellular Bacterium Rickettsia typhi

Adenosine 5′-triphosphate (ATP) was catabolized by whole cells and cell-free extracts of Rickettsia typhi to adenosine 5′-diphosphate (ADP) and then to adenosine 5′-monophosphate (AMP), the end product of ATP catabolism under the experimental conditions used. The only intermediate of the pathway from ATP to AMP which was identified by thin-layer chromatography and quantitated by the ¹⁴C content was ADP, whereas products such as adenine, adenosine, hypoxanthine, inosine, and inosine 5′-monophosphate were not detected. The enzymes which could be theoretically responsible for the catabolism or the anabolism of AMP were not detected by standard assay procedures. Most importantly, 5′-nucleotidase or nonspecific phosphatase and AMP nucleosidase activities were undetectable under a variety of experimental conditions. Although these two enzymes remove AMP from the adenylate pool in other cells, they are apparently nonfunctional in R. typhi. The biosynthesis of ATP was initiated by adenylate kinase because no adenine phosphoribosyltransferase or adenosine kinase could be detected. Furthermore, AMP was transported intact without prior dephosphorylation. These observations suggest that for R. typhi the in vivo activity of adenine nucleotide interconversion was limited to the nucleotides, with AMP being the end product of ATP catabolism, and that the salvage of purine bases and nucleosides was not an essential feature of purine metabolism. These results elucidate the findings of a previous study which showed that in the absence of glutamate as a source of energy, the adenylate energy charge of resting cells of R. typhi is drastically lowered by the high proportion of AMP.     

Neuromodulation by adenine nucleotides, as indicated by experiments with inhibitors of nucleotide inactivation

The adenine nucleotides AMP, ADP and ATP (3 X 10(-7) M and above) inhibited contractile responses to transmural nerve stimulation in guinea-pig ileum longitudinal muscle via a prejunctional action. Nucleotides assumed to inhibit the degradation of adenine nucleotides were employed to determine whether inhibition of contractile responses was elicited by adenine nucleotides per se, or required breakdown to adenosine. The IMP or 2'-deoxy AMP enhanced the prejunctional inhibitory effect elicited by AMP. A similar enhancement of the inhibitory effect of ADP and ATP was seen after administration of IDP and ITP, respectively. The inhibitory effect of adenosine was not enhanced by inosine, IMP or IDP. The 5'-nucleotidase inhibitor, TDP enhanced inhibition elicited by ADP. In contrast, alpha, beta-meADP did not influence the prejunctional inhibitory effect elicited by the adenine nucleotides. However, the combination of alpha, beta-meADP and IMP enhanced the inhibitory effect of ATP. The postjunctional contractile effect elicited by ADP and ATP was enhanced by pretreatment with inosine nucleotides, alpha, beta-meADP or TDP, indicating decreased inactivation of ADP and ATP during concurrent nucleotide administration. The fact that the prejunctional effect of adenine nucleotides can be enhanced by forms of pretreatment known to antagonize the breakdown of adenine nucleotides, constitutes strong evidence for prejunctional action per se by adenine nucleotides.     

Immunoregulatory effects of adenosine 5'-triphosphate on cytokine release from stimulated whole blood

In vitro studies suggest that extracellular nucleotides and nucleosides may be important regulators of inflammatory and immune responses. Most studies with adenosine 5'-triphosphate (ATP) have been performed in cell lines, which are remote from the human situation. The purpose of the present study was to determine the effects of ATP on TNF-alpha, IL-6 and IL-10 release in stimulated whole blood. Blood samples were drawn from healthy volunteers and incubated with ATP and lipopolysaccharide (LPS) + phytohemagglutinin (PHA) for 24 h. Contrary to expectations, ATP at 100 microM and 300 microM induced a reduction in TNF-alpha secretion by 32+/-8% (mean +/- SEM) and 65+/-4%, respectively. Furthermore, these ATP concentrations induced an increase in IL-10 secretion by 48+/-5% and 62+/-7% in whole blood. The ATP analogue adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S) and adenosine 5'-diphosphate (ADP) also inhibited TNF-alpha release, but only ADP showed a stimulatory effect on IL-10. Co-treatment with adenosine deaminase did not reverse the ATP effect on TNF-alpha and IL-10. These results show, for the first time, that ATP inhibits the inflammatory response in stimulated whole blood as indicated by inhibition of TNF-alpha and stimulation of IL-10 release and that this effect is predominantly mediated by ATP and not by adenosine.     

Potassium transport and nucleoside metabolism in human red cells

1. A study has been made of active cation transport in relation to the metabolism of nucleosides and deoxynucleosides in human red blood cells.2. Lactate production from inosine depended on the cellular inorganic phosphate available for phosphorolysis and was raised to some 4 times the normal rate from glucose. Inosine inhibited glucose metabolism according to the amount of glucose-6-phosphate produced. Guanosine behaved similarly, but glucose-6-phosphate was not formed from adenosine, deoxyinosine and deoxyguanosine.3. Glucose metabolism was related to the ATP content of the cells, which was raised by incubation with adenosine, or with inosine plus adenine.4. Adenosine and deoxyadenosine raised the potassium influx above the values found with inosine and deoxyinosine under conditions when the lactate production was constant. Active potassium influx was correlated with the ATP concentration in the cells, but independent of the lactate production when the latter was raised above normal.     

Enzyme immunoassays for the estimation of adenosine 3',5' cyclic monophosphate and guanosine 3',5' cyclic monophosphate in biological fluids.

Simple, rapid and sensitive competitive enzyme immunoassays for the estimation of adenosine 3',5' cyclic monophosphate (cAMP) and guanosine 3',5' cyclic monophosphate (cGMP) in human plasma and urine are described. Specific antisera to each nucleotide were raised in rabbits by immunization with succinyl cyclic nucleotide--human serum albumin conjugates. For the assay, specific antibodies were incubated with a mixture of succinyl cyclic nucleotide labelled with horseradish peroxidase together with unlabelled standard or sample. The antibody-bound enzyme conjugate was separated from free hapten by anti-rabbit (IgG) sera immobilized to a microtitre plate. Activity of the bound enzyme conjugate was determined with tetramethylbenzidine. The assays were capable of detecting levels as low as 2 fmol of cAMP and cGMP. Good correlations were obtained between values generated by enzyme immunoassay and radioimmunoassay.