Relationship of adenosine-3', 5'-monophosphate to other adenine nucleotides in human platelets

Labeled cyclic AMP and other adenine nucleotides in human platelets were distinctly separated by means of thin-layer chromatography. In lysates of human platelets, ATP was decomposed following the major route: ATP→ADP→AMP→IMP→inosine→hypoxanthine. In contrast, cyclic AMP synthesis occurred rapidly following the breakdown of ATP and leveled off after 30–60 min of incubation. Cyclic AMP synthesis in platelet lysates was 1.02 ± 0.39 nanomoles/hr/mg protein. The level of cyclic AMP formed was related to the 5′-AMP level, although the former did not exceed 5 % of the latter.     

Inhibition of adenylate cyclase in human blood platelets by 9-substituted adenine derivatives.

A series of 9-substituted adenine derivatives inhibited adenylate cyclase activity (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) of a particulate preparation of human blood platelets. A 3--6 fold elevation of adenylate cyclase activity by prostaglandin E1 (PGE1) was inhibited in a concentration-related manner by 9-(tetrahydro-5-methyl-2-furyl) adenine (SQ 22,538), 9-(tetrahydro-2-furyl) adenine (SQ 22,536), 9-cyclopentyladenine (SQ 22,534), 9-furfuryladenine (sQ 4647) and 9-benzyladenine (SQ 218611). The I50 values ranged from 21 microM for SQ 22,538 to 140 microM for SQ 21,611. These same adenine derivatives reversed the inhibition by PGE1 of ADP-induced aggregation and the PGE1-stimulated elevation of adenosine 3':5'-monophosphate (cyclic AMP). The reversal of platelet aggregation inhibition by SQ 22,536 and SQ 4647 was concentration-related with I50 values of 30 microM in each case, whereas SQ 22,534 and SQ 21,611 reversed inhibition by 30% at 100 microM. SQ 22,536, SQ 22,534 and SQ 21,611 also blocked the increase in cyclic AMP levels in a concentration-related manner with I50 values of 1, 4 and 60 microM, respectively. SQ 4647 inhibited the elevation of cyclic AMP by more than 85% at 1000 microM. The adenine derivatives had no effect on platelet aggregation or on cyclic AMP levels in the absence of PGE1. These results provide additional evidence that the inhibition of platelet aggregation by PGE1 is mediated by cyclic AMP.     

A new chromatographic method using immobilized acriflavin for measuring cyclic AMP in cells prelabeled with radioactive adenine.

A method using the principle of charge-transfer chromatography has been developed for the determination of cyclic AMP levels in intact prelabeled cells. The ATP pool was prelabeled by incubating the cells in the presence of radioactive adenine. The cyclic AMP formed from ATP was extracted with HC10(4) and separated from adenine and other adenosine-related nucleotides by chromatography on acriflavin-Sephadex G-25. This method provides a rapid and sensitive isolation of cyclic AMP with high recovery (95-100%) and low blnks. Further, no contamination of the cyclic AMP fractions was found by either adenine or adenosine nucleotides such as ATP, ADP or AMP. This procedure is applicable to a variety of cell or tissue systems.     

A separation method for the assay of adenylylcyclase, intracellular cyclic AMP, and cyclic-AMP phosphodiesterase using tritium-labeled substrates.

A method for the separation of cyclic AMP from adenosine and polyvalent adenine nucleotides is described. The method consists of the sequential elution of adenosine and cyclic AMP from a single column of acidic aluminum oxide (alumina) with dilute hydrochloric acid and ammonium acetate. Adenosine, adenine, xanthine, and hypoxanthine are rapidly eluted with the application of 0.005 N hydrochloric acid while cyclic AMP remains adsorbed to the alumina. A subsequent application of 0.1 M ammonium acetate elutes more than 90% of the cyclic AMP. Under these conditions, polyvalent nucleotides (AMP, ADP, and ATP) remain adsorbed to the alumina. The method permits the measurement of adenylylcyclase activity using [3H]ATP as the labeled substrate. The same technique can be used to measure the accumulation of cyclic AMP in intact cells after labeling the ATP pool with [3H]adenine. With slight modification, the technique can be used to measure the activity of cyclic-AMP phosphodiesterase using [3H]cyclic AMP as the substrate. The proposed technique provides rapid, highly reproducible assays using inexpensive, disposable columns.     

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.     

Inhibition of adenylate cyclase by adenosine analogues in preparations of broken and intact human platelets. Evidence for the unidirectional control of platelet function by cyclic AMP.

Whereas adenosine itself exerted independent stimulatory and inhibitory effects on the adenylate cyclase activity of a platelet particulate fraction at low and high concentrations respectively, 2-substituted and N6-monosubstituted adenosines had stimulatory but greatly decreased inhibitory effects. Deoxyadenosines, on the other hand, had enhanced inhibitory but no stimulatory effects. The most potent inhibitors found were, in order of increasing activity, 9-(tetrahydro-2-furyl)adenine (SQ 22536), 2',5'-dideoxyadenosine and 2'-deoxyadenosine 3'-monophosphate. Kinetic studies on prostaglandin E1-activated adenylate cyclase showed that the inhibition caused by either 2',5'-dideoxyadenosine or compound SQ 22536 was non-competitive with MgATP and that the former compound, at least, showed negative co-operativity; 50% inhibition was observed with 4 micron-2',5'-dideoxyadenosine or 13 micron-SQ 22536. These two compounds also inhibited both the basal and prostaglandin E1-activated adenylate cyclase activities of intact platelets, when these were measured as the increases in cyclic [3H]AMP in platelets that had been labelled with [3H]adenine and were then incubated briefly with papaverine or papaverine and prostaglandin E1. Both compounds, but particularly 2',5'-dideoxyadenosine, markedly decreased the inhibition by prostaglandin E1 of platelet aggregation induced by ADP or [arginine]vasopressin as well as the associated increases in platelet cyclic AMP, so providing further evidence that the effects of prostaglandin E1 on platelet aggregation are mediated by cyclic AMP. 2'-Deoxyadenosine 3'-monophosphate did not affect the inhibition of aggregation by prostaglandin E1, suggesting that the site of action of deoxyadenosine derivatives on adenylate cyclase is intracellular. Neither 2',5'-dideoxyadenosine nor compound SQ 22536 alone induced platelet aggregation. Moreover, neither compound potentiated platelet aggregation or the platelet release reaction when suboptimal concentrations of ADP, [arginine]vasopressin, collagen or arachidonate were added to heparinized or citrated platelet-rich plasma in the absence of prostaglandin E1. These results show that cyclic AMP plays no significant role in the responses of platelets to aggregating agents in the absence of compounds that increase the platelet cyclic AMP concentration above the resting value.     

Effects of activation of protein kinase C on the agonist-induced stimulation and inhibition of cyclic AMP formation in intact human platelets.

Jakobs, Bauer & Watanabe [(1985) Eur. J. Biochem. 151, 425-430] reported that treatment of platelets with phorbol 12-myristate 13-acetate (PMA) prevented GTP- and agonist-induced inhibition of adenylate cyclase in membranes from the platelets. This was attributed to the phosphorylation of the inhibitory guanine nucleotide-binding protein (Gi) by protein kinase C. In the present study, the effects of PMA on cyclic [3H]AMP formation and protein phosphorylation were studied in intact human platelets labelled with [3H]adenine and [32P]Pi. Incubation mixtures contained indomethacin to block prostaglandin synthesis, phosphocreatine and creatine kinase to remove ADP released from the platelets, and 3-isobutyl-1-methylxanthine to inhibit cyclic AMP phosphodiesterases. Under these conditions, PMA partially inhibited the initial formation of cyclic [3H]AMP induced by prostaglandin E1 (PGE1), but later enhanced cyclic [3H]AMP accumulation by blocking the slow decrease in activation of adenylate cyclase that follows addition of PGE1. PMA had more marked and exclusively inhibitory effects on cyclic [3H]AMP formation induced by prostaglandin D2 and also inhibited the action of forskolin. Adrenaline, high thrombin concentrations and, in the absence of phosphocreatine and creatine kinase, ADP inhibited cyclic [3H]AMP formation induced by PGE1. The actions of adrenaline and thrombin were attenuated by PMA, but that of ADP was little affected, suggesting differences in the mechanisms by which these agonists inhibit adenylate cyclase. sn-1,2-Dioctanoylglycerol (diC8) had effects similar to those of PMA. The actions of increasing concentrations of PMA or diC8 on the modulation of cyclic [3H]AMP formation by PGE1 or adrenaline correlated with intracellular protein kinase C activity, as determined by 32P incorporation into the 47 kDa substrate of the enzyme. Parallel increases in phosphorylation of 20 kDa and 39-41 kDa proteins were also observed. Platelet-activating factor, [Arg8]vasopressin and low thrombin concentrations, all of which inhibit adenylate cyclase in isolated platelet membranes, did not affect cyclic [3H]AMP formation in intact platelets. However, the activation of protein kinase C by these agonists was insufficient to account for their failure to inhibit cyclic [3H]AMP formation. Moreover, high thrombin concentrations simultaneously activated protein kinase C and inhibited cyclic [3H]AMP formation. The results show that, in the intact platelet, the predominant effects of activation of protein kinase C on adenylate cyclase activity are inhibitory, suggesting actions additional to inactivation of Gi.     

FUNCTIONAL COMPARTMENTS OF ADENINE NUCLEOTIDES SERVING AS PRECURSORS OF ADENOSINE 3'',5''-MONOPHOSPHATE IN MOUSE CEREBRAL CORTEX

Cyclic AMP accumulates in cerebral cortical slices from the C57B1/6J mouse incubated with the following stimulatory agents: norepinephrine, adenosine, veratridine and adenosine-biogenic amine combinations. The results with slices labelled with radioactive adenine or adenosine provide evidence for the existence of distinct functional compartments of adenine nuclcotides which serve as precursors of cyclic AMP on stimulation with specific agents. Thus, in slices labelled with [¹⁴C]adenine or [³H]adenosine the ratio of [¹⁴C] to [³H]cyclic AMP was dependent on the stimulatory agent; with veratridinc the ratio was 1.4 while with adenosine the ratio was 3.0. In addition, a greater than 2-fold difference in the ratio of endogenous/radioactive cyclic AMP was observed in adenine or adenosine-labelled slices after incubation with veratridine, norepinephrine, adenosine or adenosine-amine combinations; the lowest ratios after stimulation with veratridine and the highest after adenosine or adenosine-amine combinations. The high ratio observed with adenosine was in part due to a quite marked incorporation of the stimulant, adenosine, into the accumulating cyclic AMP. Such distinct functional compartments of cyclic AMP precursors may represent different cell types and/or morphological entities within one cell type.     

Effect of prostaglandin E1 alone and in combination with theophylline or aspirin on collagen-induced platelet aggregation and on platelet nucleotides including adenosine 3′:5′-cyclic monophosphate

1. Human platelet nucleotides were labelled by incubating platelet-rich plasma with [U-(14)C]adenine. With such platelets, the effects of prostaglandin E1, theophylline and aspirin were determined on collagen-induced platelet aggregation and release of platelet ATP and ADP. Intracellular changes of platelet radioactive nucleotides, particularly 3':5'-cyclic AMP, were also determined both with and without collagen treatment. 2. Prostaglandin E1, theophylline and aspirin inhibited collagen-induced aggregation of platelets in a dose-dependent manner. Collagen-induced release of ATP and ADP and breakdown of radioactive ATP were also inhibited in a dose-dependent manner. 3. Prostaglandin E1 stimulated the formation of platelet radioactive 3':5'-cyclic AMP in a dose-dependent manner. With a given dose of prostaglandin E1, maximum formation of radioactive 3':5'-cyclic AMP occurred by 10-30s and thereafter the concentrations declined. The degree of inhibition of aggregation produced by prostaglandin E1, however, increased with its time of incubation in platelet-rich plasma before addition of collagen, so that there was an inverse relationship between the radioactive 3':5'-cyclic AMP concentration measured at the time of collagen addition and the subsequent degree of inhibition of aggregation obtained. 4. Neither theophylline nor aspirin at a concentration in platelet-rich plasma of 1.7mm altered platelet radioactive 3':5'-cyclic AMP contents. In the presence of prostaglandin E1, theophylline increased the concentration of radioactive 3':5'-cyclic AMP over that noted with prostaglandin E1 alone, but aspirin did not. 5. Mixtures of prostaglandin E1 and theophylline had a synergistic effect on inhibition of platelet aggregation. The same was true to a lesser extent with mixtures of prostaglandin E1 and aspirin. Such mixtures also inhibited collagen-induced release of platelet ATP and ADP and breakdown of platelet radioactive ATP. 6. Certain concentrations of either theophylline or aspirin and mixtures of small concentrations of prostaglandin E1 with either theophylline or aspirin caused little or no increase of radioactive 3':5'-cyclic AMP at the time of collagen addition, but inhibited aggregation to a marked degree, whereas higher concentrations of prostaglandin E1 alone caused a much greater increase of radioactive 3':5'-cyclic AMP at the time of collagen addition but inhibited aggregation to a lesser extent. With these compounds there does not appear to be a correlation between these parameters.     

INTERRELATIONSHIPS AMONG THE LEVELS OF ATP, ADENOSINE AND CYCLIC AMP IN INCUBATED SLICES OF GUINEA-PIG CEREBRAL CORTEX: EFFECTS OF DEPOLARIZING AGENTS, PSYCHOTROPIC DRUGS AND METABOLIC INHIBITORS

—Adenine nucleotides of guinea-pig cerebral cortical slices were labelled during a 40 min incubation with [¹⁴C]adenine. Subsequent incubation of cortical slices with depolarizing agents, such as veratridine, ouabain, batrachotoxin and high concentrations of potassium ions, or with certain psychotropic drugs such as chlorpromazine, chlorimipramine or prenylamine resulted in a reduction in both endogenous and radioactive ATP, accompanied by a marked increase in levels of both endogenous and radioactive cyclic AMP. Reduction of ATP levels during incubation with depolarizing agents, such as veratridine, is probably associated with increased activity of membranal Na⁺-K⁺-activated ATPase, while the reduction elicited by psychotropic drugs is proposed to be due to inhibition of mitochondrial synthesis of ATP. With both classes of compounds reduction of ATP levels results in enhanced formation and efflux of adenosine which stimulates formation of cyclic AMP from intracellular ATP in the compartments of brain slices which contain the cyclic AMP-generating systems. Certain classical metabolic inhibitors such as 2,4-dinitrophenol, azide, 1,2-naphthoquinone-8-sulfonate and cyanide also reduce ATP levels and in the case of 2,4-dinitrophenol, cyanide, and azide elicit small but significant accumulations of cyclic AMP. With certain metabolic inhibitors reduction of ATP within the cyclic AMP generating compartments would appear to prevent or reduce the accumulation of cyclic AMP elicited by amines, adenosine or veratridine.     

Human platelet secretion and aggregation induced by calcium ionophores. Inhibition by PGE1 and dibutyryl cyclic AMP

Ca2+, Mg2+-ionophores X537A and A23,187 (10(-7)-10(-6) M) induced the release of adenine nucleotides adenosine diphosphate (ADP, adenosine triphosphate (ATP), serotonin, beta-glucuronidase, Ca2+, and Mg2+ from washed human platelets. Enzymes present in the cytoplasm or mitochondria, and Zn2+ were not released. The rate of ATP and Ca2+ release measured by firefly lantern extract and murexide dye, respectively, was equivalent to that produced by the physiological stimulant thrombin. Ionophore-induced release of ADP, and serotonin was substantially (approximately 60%) but not completely inhibited by EGTA, EDTA, and high extracellular Mg2+, without significant reduction of Ca2+ release. The ionophore-induced release reaction is therefore partly dependent upon uptake of extracellular Ca2+ (demonstrated using 45Ca), but also occurs to a significant extent due to release into the cytoplasm of intracellular Ca2+. The ionophore-induced release reaction and aggregation of platelets could be blocked by prostaglandin E1 (PGE1) or dibutyryl cyclic AMP. The effects of PGE1, and N6, O2-dibutyryl adenosine 3':5'-cyclic monophosphoric acid (dibutyryl cAMP) were synergistically potentiated by the phosphodiesterase inhibitor theophylline. It is proposed that Ca2+ is the physiological trigger for platelet secretion and aggregation and that its intracellular effects are strongly modulated by adenosine 3':5'-cyclic monophosphoric acid (cyclic AMP).     

Mechanism of action of ATP on intestinal epithelial cells. Cyclic AMP-mediated stimulation of active ion transport

ATP, ADP and AMP but not adenosine increased cyclic AMP in dispersed enterocytes prepared from guinea pig small intestine. This action of ATP was augmented by IBMX and was reproduced by App(NH)p or App(CH2)p. ATP also increased the formation of cyclic [14C]AMP in enterocytes that had been preincubated with [14C]adenine. Gpp(NH)p and NaF each caused persistent activation of adenylate cyclase in plasma membranes from enterocytes and ATP caused significant augmentation of this persistent activation. In addition to increasing cellular cyclic AMP and augmenting Gpp(NH)p and NaF-stimulated persistent activation of adenylate cyclase, ATP increased the Isc across mounted strips of small intestine and inhibited net absorption of fluid and electrolytes in segments of everted small intestine. These results indicate that intestinal epithelial cells possess a receptor that interacts with ATP and other adenine nucleotides and that receptor occupation by ATP causes activation of adenylate cyclase, increased cyclic AMP and changes in active ion transport across intestinal mucosa.     

Simultaneous analysis of adenosine 3',5'-cyclic monophosphate accumulation and adenosine 5'-triphosphate metabolism in cultured cells preincubated with [2-3H]adenine.

A simple and sensitive method based on metabolic labeling was developed for the simultaneous analysis of cyclic AMP accumulation and ATP metabolism in small numbers of cultured cells. Cells are preincubated overnight with [2-3H]adenine to label the ATP pool to a high specific activity. After cell stimulation the metabolites are extracted in a small volume of aqueous acetic acid and chloroform and separated without further manipulation by one-dimensional thin-layer chromatography and the radioactivity incorporated is determined by liquid scintillation counting. With ATP labeled to about 6 Ci/mmol, the lower limit of cyclic AMP detection is 2 fmol, a sensitivity that is comparable to the radioimmunoassay of acetylated cyclic AMP. In primary neurons and a neural cell line, for example, levels of ATP and its metabolites change when large amounts of cyclic AMP are generated, each with its unique pattern. ATP is also depleted when metabolic energy is consumed concomitantly with stimulation of cyclic AMP production by agonists, probably as a result of an increase in ion pump activity following cation influx. As ATP is utilized for cyclic AMP production and simultaneously for many other processes, an assessment of its metabolism in parallel with that of cyclic AMP is critical. We suggest that the method described here is particularly advantageous over other methods for this purpose.     

Adenine nucleotide metabolism in isolated chicken hepatocytes.

The turnover of the adenine nucleotide pool, the pathway of the degradation of AMP and the occurrence of recycling of adenosine were investigated in isolated chicken hepatocytes, in which the adenylates had been labelled by prior incubation with [14C]adenine. Under physiological conditions, 85% of the IMP synthesized by the 'de novo' pathway (approx. 37 nmol/min per g of cells) was catabolized directly via inosine into uric acid, and 14% was converted into adenine nucleotides. The latter were found to turn over at the rate of approx. 5 nmol/min per g of tissue. Inhibition of adenosine deaminase by 1 microM-coformycin had no effect on the formation of labelled uric acid, indicating that the initial degradation of AMP proceeds by way of deamination rather than dephosphorylation. Inhibition of adenosine kinase by 100 microM-5-iodotubercidin resulted in a loss of labelled ATP, demonstrating that adenosine is normally formed from AMP but is recycled. Unexpectedly, 5-iodotubercidin did not decrease the total concentration of ATP, indicating that the loss of adenylates caused by inhibition of adenosine kinase was nearly completely compensated by formation of AMP de novo. Anoxia induced a greatly increased catabolism of the adenine nucleotide pool, which proceeded in part by dephosphorylation of AMP. On reoxygenation, the formation of AMP de novo was increased 8-fold as compared with normoxic conditions. The latter results indicate the existence of adaptive mechanisms in chick liver allowing, when required, channelling of the metabolic flux through the 'de novo' pathway, away from the uricotelic catabolic route, into the synthesis of adenine nucleotides.     

Stimulation of glycogenolysis by adenine nucleotides in the perfused rat liver.

Infusion of adenine nucleotides and adenosine into perfused rat livers resulted in stimulation of hepatic glycogenolysis, transient increases in the effluent perfusate [3-hydroxybutyrate]/[acetoacetate] ratio, and increased portal vein pressure. In livers perfused with buffer containing 50 microM-Ca2+, transient efflux of Ca2+ was seen on stimulation of the liver with adenine nucleotides or adenosine. ADP was the most potent of the nucleotides, stimulating glucose output at concentrations as low as 0.15 microM, with half-maximal stimulation at approx. 1 microM, and ATP was slightly less potent, half-maximal stimulation requiring 4 microM-ATP. AMP and adenosine were much less effective, doses giving half-maximal stimulation being 40 and 20 microM respectively. Non-hydrolysed ATP analogues were much less effective than ATP in promoting changes in hepatic metabolism. ITP, GTP and GDP caused similar changes in hepatic metabolism to ATP, but were 10-20 times less potent than ATP. In livers perfused at low (7 microM) Ca2+, infusion of phenylephrine before ATP desensitized hepatic responses to ATP. Repeated infusions of ATP in such low-Ca2+-perfused livers caused homologous desensitization of ATP responses, and also desensitized subsequent Ca2+-dependent responses to phenylephrine. A short infusion of Ca2+ (1.25 mM) after phenylephrine infusion restored subsequent responses to ATP, indicating that, during perfusion with buffer containing 7 microM-Ca2+, ATP and phenylephrine deplete the same pool of intracellular Ca2+, which can be rapidly replenished in the presence of extracellular Ca2+. Measurement of cyclic AMP in freeze-clamped liver tissue demonstrated that adenosine (150 microM) significantly increased hepatic cyclic AMP, whereas ATP (15 microM) was without effect. It is concluded that ATP and ADP stimulate hepatic glycogenolysis via P2-purinergic receptors, through a Ca2+-dependent mechanism similar to that in alpha-adrenergic stimulation of hepatic tissue. However, adenosine stimulates glycogenolysis via P1-purinoreceptors and/or uptake into the cell, at least partially through a mechanism involving increase in cyclic AMP. Further, the hepatic response to adenine nucleotides may be significant in regulating hepatic glucose output in physiological and pathophysiological states.     

Mechanism of action of ATP on intestinal epithelial cells: Cyclic AMP-mediated stimulation of active ion transport

ATP, ADP and AMP but not adenosine increased cyclic AMP in dispersed enterocytes prepared from guinea pig small intestine. This action of ATP was augmented by IBMX and was reproduced by App(NH)p or App(CH₂)p. ATP also increased the formation of cyclic [¹⁴C]AMP in enterocytes that had been preincubated with [¹⁴C]adenine. Gpp(NH)p and NaF each caused persistent activation of adenylate cyclase in plasma membranes from enterocytes and ATP caused significant augmentation of this persistent activation. In addition to increasing cellular cyclic AMP and agumenting Gpp(NH)p and NaF-stimulated persistent activation of adenylate cyclase, ATP increased the I_sc across mounted strips of small intestine and inhibited net absorption of fluid and electrolytes in segments of everted small intestine. These results indicate that intestinal epithelial cells possess a receptor that interacts with ATP and other adenine nucleotides and that receptor occupation by ATP causes activation of adenylate cyclase, increased cyclic AMP and changes in active ion transport across intestinal mucosa.     

Effects of antimycin and 2-deoxyglucose on adenine nucleotides in human platelets. Role of metabolic adenosine triphosphate in primary aggregation, secondary aggregation and shape change of platelets

1. Human platelet-rich plasma prelabelled with [(3)H]adenine was incubated at 37 degrees C with antimycin A and 2-deoxy-d-glucose. Variations in the amounts of ATP, ADP and P(i), and in the radioactivity of ATP, ADP, AMP, IMP, hypoxanthine+inosine and adenine were determined during incubation. Adrenaline- and ADP-induced platelet aggregation and the ADP-induced shape change of the platelets were determined concurrently. 2. 2-Deoxyglucose caused conversion of [(3)H]ATP to [(3)H]hypoxanthine+inosine. The rate of this conversion increased with increasing 2-deoxyglucose concentration and was markedly stimulated by addition of antimycin, which had no effect alone. At maximal ATP-hypoxanthine conversion rates, the IMP radioactivity remained at values tenfold higher than control, whereas [(3)H]ADP and [(3)H]AMP radioactivity gave variations typical for product/substrates in consecutive reactions. The specific radioactivityof ethanol-soluble platelet ATP decreased during incubation to less than one-tenth of its original value. The amounts and radioactivity of ethanol-insoluble ADP did not vary during incubation with the metabolic inhibitors. 3. The rate of ADP- and adrenaline-induced primary aggregation decreased as the amount of radioactive ATP declined, and complete inhibition of aggregation was obtained at a certain ATP concentration (metabolic ATP threshold). This threshold decreased with increasing concentration of inducer ADP. 4. Secondary platelet aggregation (release reaction) had a metabolic ATP threshold markedly higher than that of primary aggregation. 5. Shape change was gradually inhibited as the ATP radioactivity decreased, and had a metabolic ATP threshold distinctly lower than that of primary aggregation, and which decreased with increasing concentration of ADP. 6. A small but distinct fraction of [(3)H]ATP disappeared rapidly during the combined shape change-aggregation process induced by ADP in platelets incubated with metabolic inhibitors, whereas no ATP disappearance occurred during aggregation in their absence.     

An adenosine 3':5'-monophosphate-adenosine binding protein from mouse liver. Factors affecting the activation of the binding protein by adenosine 5'-triphosphate.

A cyclic AMP-adenosine binding protein, whose binding sites are activated by preincubation in the presence of Mg⁺-ATP, has been purified to apparent homogeneity from mouse liver (P.M. Ueland and S.O. Døskeland, 1977, J. Biol. Chem.,252, 677–686). The degree of activation of both the cyclic AMP binding site and a high-affinity site for adenosine depends on the concentration of ATP during the preincubation. The velocity and the degree of activation are dependent on the temperature and the presence of Mg²⁺ and K⁺. The NH₄⁺ ion can be substituted for K⁺, whereas Na⁺ is inefficient. Low pH promotes the conversion from the inactive to the active form. The apparent affinity for adenosine to the high-affinity site for this adenine derivative and the affinity for cyclic AMP to the site specific for this nucleotide are independent of the degree of activation as judged from the slope of Scatchard plots. The activation of the cyclic AMP binding site by ATP (6 mm) was determined at pH 7 in the presence of 10 μm cyclic AMP, AMP, ADP, or adenosine. Adenosine specifically inhibits the activation and does not promote the inactivation of the binding protein. The possibility that the apparent inhibition of activation was effected by interference with cyclic AMP binding by adenosine was ruled out.     

Energy metabolism and lipid peroxidation of human erythrocytes as a function of increased oxidative stress.

To study the influence of oxidative stress on energy metabolism and lipid peroxidation in erythrocytes, cells were incubated with increasing concentrations (0.5-10 mM) of hydrogen peroxide for 1 h at 37 degrees C and the main substances of energy metabolism (ATP, AMP, GTP and IMP) and one index of lipid peroxidation (malondialdehyde) were determined by HPLC on cell extracts. Using the same incubation conditions, the activity of AMP-deaminase was also determined. Under nonhaemolysing conditions (at up to 4 mM H2O2), oxidative stress produced, starting from 1 mM H2O2, progressive ATP depletion and a net decrease in the intracellular sum of adenine nucleotides (ATP + ADP + AMP), which were not paralleled by AMP formation. Concomitantly, the IMP level increased by up to 20-fold with respect to the value determined in control erythrocytes, when cells were challenged with the highest nonhaemolysing H2O2 concentration (4 mM). Efflux of inosine, hypoxanthine, xanthine and uric acid towards the extracellular medium was observed. The metabolic imbalance of erythrocytes following oxidative stress was due to a dramatic and unexpected activation of AMP-deaminase (a twofold increase of activity with respect to controls) that was already evident at the lowest dose of H2O2 used; this enzymatic activity increased with increasing H2O2 in the medium, and reached its maximum at 4 mM H2O2-treated erythrocytes (10-fold higher activity than controls). Generation of malondialdehyde was strictly related to the dose of H2O2, being detectable at the lowest H2O2 concentration and increasing without appreciable haemolysis up to 4 mM H2O2. Besides demonstrating a close relationship between lipid peroxidation and haemolysis, these data suggest that glycolytic enzymes are moderately affected by oxygen radical action and strongly indicate, in the change of AMP-deaminase activity, a highly sensitive enzymatic site responsible for a profound modification of erythrocyte energy metabolism during oxidative stress.     

Binding of 3',4'-dideoxykanamycin B to ATP and its related compounds

The interactions of aminoglycoside, 3',4'-dideoxykanamycin B(DKB) with ATP and its related compounds were investigated. ATP, ADP, cyclic AMP and FAD bound to the DKB-conjugated Sepharose 4B column. The binding of DKB to ATP was also confirmed by equilibrium gel filtration. In the acidic pH region, the fluorescence of nucleotides was quenched by DKB. The Stern-Volmer plots showed that the molar ratios of the complexes were 1:1. The apparent stability constant was dependent on the number of the phosphate groups of nucleotides and was in the order of ATP greater than ADP greater than AMP.