2-Chloroadenosine inhibits complement-induced reactive oxygen metabolite production and recovery of human polymorphonuclear leucocytes attacked by complement

Luminol-enhanced chemiluminescence demonstrated that 2-chloroadenosine inhibited complement-induced reactive oxygen metabolite production in human polymorphonuclear leucocytes. This inhibition was reversed by 8-phenyltheophylline. Binding of a [125I]-labelled monoclonal antibody against C9 to human PMN demonstrated removal of membrane attack complexes from the cell membranes, and in addition indicated inhibition of this process by 2-chloroadenosine. This compound also increased the percentage of complement-induced cell lysis of polymorphonuclear leucocytes.     

Pholasin--a bioluminescent indicator for detecting activation of single neutrophils

Pholasin is the protein-bound luciferin from the bivalve mollusc Pholas dactylus which reacts with its luciferase and molecular oxygen to produce light. Pholasin was purified 226-fold with a yield of 58% from P. dactylus to give a preparation free from luciferase contamination. The ratio (k) of endogenous pholasin chemiluminescence to that when maximally stimulated by luciferase was 8.12 X 10(-6) +/- 0.87 X 10(-6) (mean +/- SD, n = 6), equivalent to a t 1/2 of 23.7 h at pH 9. Pholasin could detect reactive oxygen metabolite production from neutrophils stimulated by the chemotactic peptide N-formyl-Met-Leu-Phe, in the presence and absence of 2-chloroadenosine or cytochalasin B, and by latex beads in the presence and absence of cytochalasin B. Pholasin was also able to detect a longer-lived oxidative activity distinct from myeloperoxidase, and released from neutrophils activated by latex beads or chemotactic peptide; luminol could not. Under optimal conditions pholasin produced a signal some 50-100 times that of luminol in the presence of activated neutrophils. This enabled activation of a single neutrophil by chemotactic peptide (1 microM) to be detected, giving a signal of 194 +/- 21 chemiluminescent counts per second, some six times that of the background signal (mean +/- SD, n = 2). Pholasin thus provides an indicator sufficiently sensitive to establish whether neutrophil activation occurs through thresholds in individual cells.     

Two distinct mechanisms for stimulation of oxygen-radical production by polymorphonuclear leucocytes.

Oxygen-radical production stimulated from rat polymorphonuclear leucocytes by either unopsonized latex particles (diameter = 1.01 microM) or chemotactic peptide (N-formyl-Met-Leu-Phe) was monitored by using luminol-dependent chemiluminescence. Azide inhibited by more than 80% the luminescence response induced by chemotactic peptide whether added before or after stimulation. However, the luminescence response to latex particles was progressively less susceptible to azide inhibition if the azide was added after the stimulus. Cytochalasin B, which was shown to abolish phagocytosis of the latex beads, also abolished the chemiluminescence response. However, the same cells showed a greatly enhanced response to chemotactic peptide. Cytochalasin B-treated cells secreted approx. 45% of total cellular myeloperoxidase in response to chemotactic peptide, but there was no detectable secretion in response to unopsonized latex particles. Microperoxidase equivalent to 20% of cellular peroxidase activity added to the cells before addition of the stimulus had no effect on the response to latex particles but increased approx. 2-fold the peak rate of chemiluminescence induced by chemotactic peptide. It was concluded that the unopsonized latex particles stimulated oxygen-radical production by the mechanism that involved endocytosis, whereas chemotactic peptide stimulated production by a mechanism that involved exocytosis of myeloperoxidase, the latter mechanism requiring an increase in intracellular free [Ca2+].     

The effect of adenosine on the fluorescence responses of chlorotetracycline-loaded human polymorphonuclear leukocytes

Chlorotetracycline has been used in human polymorphonuclear leukocytes as a probe to investigate the state of membrane-bound calcium. We examined the effect of adenosine on the fluorescence responses of CTC-loaded PMNs stimulated with the synthetic chemotactic peptide, formyl-methionyl-leucyl- phenylalanine. Adenosine inhibited the decrease in CTC fluorescence in a dose-dependent fashion and its effect was reversed by theophylline, an adenosine receptor antagonist. Removal of extracellular adenosine by incubating PMNs with adenosine deaminase abolished the effect of adenosine. These data suggest that adenosine inhibits the release of membrane-bound calcium in PMNs that normally occurs in response to chemotactic stimuli, acting via PMN surface adenosine receptors.     

Adenosine and its analogues, possibly acting at A2 receptors, stimulate mouse sperm fertilizing ability during early stages of capacitation

Earlier studies have provided indirect evidence that the availability of endogenous adenosine can modulate the fertilizing ability of mouse spermatozoa during capacitation. More direct evidence has been sought by evaluating the effect of exogenous adenosine present during the early stages of capacitation. A concentration-dependent stimulation of in-vitro fertilizing ability was observed, with 10 microM- and 100 microM-adenosine significantly increasing the proportion of eggs fertilized compared with drug-free controls. The adenosine-induced stimulation was observed in the presence of 0.01 microM- and 0.1 microM-dipyridamole, an inhibitor of adenosine uptake, suggesting that adenosine is acting at an external site. Comparison of adenosine with its analogues 2'-deoxyadenosine and 2-chloroadenosine indicated that the analogues at 10 microM were able to stimulate fertilization in a manner similar to adenosine. While neither adenosine nor 2'-deoxyadenosine was consistently effective at 1 microM, 2-chloroadenosine significantly stimulated fertilization at both 1 microM and 0.1 microM. In addition, 5'-N-ethylcarboxamidoadenosine (NECA) and (R)-N6-phenylisopropyladenosine (R-PIA), potent analogues in somatic cell systems, proved to be so with mouse sperm suspensions, NECA being stimulatory at greater than or equal to 0.01 microM and R-PIA at greater than or equal to 0.1 microM. Subjective evaluation of motility patterns indicated that more cells exhibited hyperactivated motility in the presence of stimulatory concentrations of adenosine or analogues. Assessment of capacitation state using chlortetracycline fluorescence patterns indicated that incubation in 2'-deoxyadenosine resulted in significantly fewer cells expressing the uncapacitated F pattern and significantly more cells with the capacitated AR (acrosome-reacted) pattern, compared with drug-free counterparts. It is concluded that adenosine promotes capacitation by interacting with externally-directed receptors, possibly on adenylate cyclase to increase the intracellular availability of cyclic adenosine monophosphate (cAMP); cAMP is known to stimulate mouse sperm fertilizing ability. The greater sensitivity to NECA, 2-chloroadenosine and R-PIA, relative to adenosine and 2'-deoxyadenosine, is consistent with interaction at stimulatory A2 adenosine receptors.     

Stimulation of glycogenolysis and vasoconstriction by adenosine and adenosine analogues in the perfused rat liver.

Infusion of adenosine into perfused rat livers resulted in transient increases in glucose output, portal-vein pressure, the effluent perfusate [lactate]/[pyruvate] ratio, and O2 consumption. 8-Phenyltheophylline (10 microM) inhibited adenosine responses, whereas dipyridamole (50 microM) potentiated the vasoconstrictive effect of adenosine. The order of potency for adenosine analogues was: 5'-N-ethylcarboxamidoadenosine (NECA) greater than L-phenylisopropyladenosine greater than cyclohexyladenosine greater than D-phenylisopropyladenosine greater than 2-chloroadenosine greater than adenosine, consistent with adenosine actions modulated through P1-purine receptors of the A2-subtype. Hepatic responses exhibited homologous desensitization in response to repeated infusion of adenosine. Adenosine effects on the liver were attenuated at lower perfusate Ca2+ concentrations. Indomethacin decreased hepatic responses to both adenosine and NECA. Whereas adenosine stimulated glycogen phosphorylase activity in isolated hepatocytes, NECA caused no effect in hepatocytes. The response to adenosine in hepatocytes was inhibited by dipyridamole (50 microM), but not 8-phenyltheophylline (10 microM). The present study indicates that, although adenosine has direct effects on parenchymal cells, indirect effects of adenosine, mediated through the A2-purinergic receptors on another hepatic cell type, appear to play a role in the perfused liver.     

Adenosine; a physiologic modulator of superoxide anion generation by human neutrophils. Adenosine acts via an A2 receptor on human neutrophils

Adenosine specifically inhibits superoxide anion generation by N-formyl-methionyl-leucyl-phenylalanine-stimulated neutrophils without affecting either degranulation or "aggregation." We present data that also supports the hypothesis that adenosine engages a specific cell surface receptor to mediate inhibition of stimulated neutrophils. Theophylline (10 and 100 mu M), a competitive antagonist at adenosine receptors, reversed the effects of adenosine (0.1 mu M) on superoxide anion generation by stimulated neutrophils. The adenosine analogue 5'N-ethylcarboxamidoadenosine (NECA) was a more potent inhibitor of superoxide anion generation than either N6-phenylisopropyladenosine (PIA) or adenosine, an order of potency consistent with that previously demonstrated for adenosine A2 receptors. 2-Chloroadenosine inhibited superoxide anion generation at concentrations similar to NECA. [3H]-NECA and [3H]-2-chloroadenosine bound to a single receptor on intact neutrophils. The characteristics of the receptors for [3H]-NECA and [3H]-2-chloroadenosine were similar (Kd = 0.22 and 0.23 mu M, respectively; number of binding sites = 9.31 and 11.1 X 10(3) sites/cell, respectively). NECA, 2-chloroadenosine, adenosine, and PIA inhibited binding of [3H]-NECA with a rank order similar to that for inhibition of superoxide anion generation (NECA = 2-chloroadenosine greater than adenosine greater than PIA). There was 50% inhibition of superoxide anion generation by NECA at approximately 20% receptor occupancy. Adenosine, derived from damaged tissues, may serve as a specific, endogenous modulator of superoxide anion generation by activated neutrophils through interaction at this newly described receptor on human neutrophils.     

Forskolin Stimulation of Cyclic AMP Accumulation in Rat Brain Cortex Slices Is Markedly Enhanced by Endogenous Adenosine

Stimulation of cyclic AMP (cAMP) accumulation in rat cortex slices by 1 microM forskolin (F) was markedly reduced (96%) by treatment with adenosine deaminase (ADA). The effect of ADA was progressively less at higher concentrations of F, but still inhibited the response by 50% at 100 microM F. ADA-mediated inhibition of the cAMP response to 1 microM F was completely reversed by 5 microM 2-chloroadenosine (CA), an ADA-resistant analogue. Stimulation by F (controls) and F plus CA (ADA treated) in cortex slices was significantly inhibited by 200 microM caffeine (CAF) and by 10 microM 8-phenyltheophylline. cAMP accumulation in ADA-treated cortex slices stimulated with CA at concentrations from 5 to 100 microM was markedly enhanced by 1 microM F. Neither ADA treatment nor 200 microM CAF significantly affected cAMP accumulation in slices stimulated by 1 microM vasoactive intestinal polypeptide or adenylate cyclase in membranes stimulated by 1 microM F. CAF (1 mM) did not significantly increase basal cAMP levels in cortex slices, whereas 1 mM 3-isobutyl-1-methylxanthine caused a significant 80% increase and 100 microM rolipram enhanced cAMP levels by 4.5-fold. F-stimulated cAMP accumulation (1 microM) in cortex slices was inhibited 98% by 1 mM CAF and 49% by 1 mM 3-isobutyl-1-methylxanthine, and was enhanced 2.5-fold by 100 microM rolipram. These data have been interpreted to indicate that the stimulation of cAMP accumulation in rat cortex slices by 1 microM F is predominantly due to synergistic interaction with endogenous adenosine and that the inhibition of this response by CAF is largely due to blockade of adenosine receptors.     

Oxygen-radical production during inflammation may be limited by oxygen concentration.

The relationship between oxygen-radical production by rat polymorphonuclear leucocytes and O2 concentration was established by the measurement of luminol-dependent chemiluminescence at defined O2 concentrations. The O2 concentration that gave 50% of the maximum stimulated oxygen-radical production was 31 +/- 9 microM for non-opsonized latex beads and 22 +/- 9 microM for chemotactic peptide. The O2 concentration in rheumatoid synovial fluid was approx. 30 microM. It is therefore proposed that radical production at an inflammatory site may be limited by O2 concentration.     

腺苷及其衍生物的心血管效应和作用机制

在实验中观察了腺苷及其衍生物的心血管效应和作用机制,结果表明：（１）腺苷和２－氯腺苷先引起由颈动脉体化学感受器内的Ａ２受体所中介的血压短暂升高,随之为心血管系统Ａ１和Ａ２受体中介的持久而明显的血压降低;（２）腺苷受体激动剂环戊腺苷抑制窦房结起搏细胞的电生理活动;（３）环戊腺苷减弱异丙肾上腺素诱发的早发和迟发性后除极及触发电活动;（４）内源性腺苷参与无氧所致的心率减慢;（５）预缺血时腺苷受体的激活及     

Regulation of Glutamate and Aspartate Release from Slices of the Hippocampal CA1 Area: Effects of Adenosine and Baclofen

Glutamate and/or aspartate is the probable transmitter released from synaptic terminals of the CA3-derived Schaffer collateral, commissural, and ipsilateral associational fibers in area CA1 of the rat hippocampal formation. Slices of the CA1 area were employed to test the effects of adenosine- and gamma-aminobutyrate (GABA)-related compounds on the release of glutamate and aspartate from this projection. Under the conditions of these experiments, the release of glutamate and aspartate evoked by 50 mM K+ was more than 90% Ca2+-dependent and originated predominantly from the CA3-derived pathways. Adenosine reduced the K+-evoked release of glutamate and aspartate by a maximum of about 60%, but did not affect the release of GABA. This action was reversed by 1 microM 8-phenyltheophylline. The order of potency for adenosine analogues was as follows: L-N6-phenylisopropyladenosine greater than N6-cyclohexyladenosine greater than D-N6-phenylisopropyladenosine approximately equal to 2-chloroadenosine greater than adenosine much greater than 5'-N-ethylcarboxamidoadenosine. 8-Phenyltheophylline (10 microM) by itself enhanced glutamate/aspartate release, whereas dipyridamole alone depressed release. These results support the view that adenosine inhibits transmission at Schaffer collateral-commissural-ipsilateral associational synapses mainly by reducing transmitter release and that these effects involve the activation of an A1 receptor. Neither adenosine, L-N6-phenylisopropyladenosine, nor 8-phenyltheophylline affected the release of glutamate or aspartate evoked by 10 microM veratridine. The differing effects of adenosine compounds on release evoked by K+ and veratridine suggest that A1 receptor activation either inhibits Ca2+ influx through the voltage-sensitive channels or interferes with a step subsequent to Ca2+ entry that is coupled to the voltage-sensitive Ca2+ channels in an obligatory fashion. Neither baclofen nor any other agent active at GABAB or GABAA receptors affected glutamate or aspartate release evoked by elevated K+ or veratridine. Therefore, either baclofen does not inhibit transmission at these synapses by depressing transmitter release or else it does so in a way that cannot be detected when a chemical depolarizing agent is employed.     

The role of C-kinase in the physiological activation of the neutrophil oxidase. Evidence from using pharmacological manipulation of C-kinase activity in intact cells.

The role of C-kinase in the triggering of the neutrophil oxidase by two stimuli (latex beads and the chemotactic peptide fMet-Leu-Phe), representative of endocytotic and exocytotic routes of activation, were investigated by using experimental agents that activate, or inhibit C-kinase, in intact cells. The activation by the phagocytotic stimulus latex beads was mimicked by C-kinase activators giving the same characteristic lag (20-30s), followed by a constant oxygen consumption rate with the same maximum rate and affinity for oxygen (Km approx. 13 microM), competed with activation by PMA (4 beta-phorbol 12-myristate 13-acetate) in a simple common-target manner, and was inhibited by retinal, an inhibitor shown to inhibit activation by PMA. In contrast, activation by chemotactic peptide was not mimicked by C-kinase activation alone, chemotactic peptide inducing biphasic oxygen consumption with a Km for oxygen of the second prolonged phase of 3.9 microM, did not compete with activation by PMA, and was not inhibited by retinal. However, PMA and retinal produced slight enhancements of activation by chemotactic peptide and production of monophasic oxygen consumption. It was concluded that C-kinase activation plays a simple central transducing role in activation of the oxidase by latex beads, but that its role in activation by chemotactic peptide is a part of a more complex set of interactions that involve other Ca2+-activated and non-Ca2+-activated processes.     

Antagonism of presynaptic adenosine receptors by theophylline 9-beta-D-riboside and 8-phenyltheophylline

Theophylline 9-beta-D-riboside and 8-phenyltheophylline were evaluated as presynaptic adenosine receptor antagonists in the rat vas deferens in vitro. Stimulation of presynaptic adenosine receptors, which results in an inhibition of the twitch response to electrical field stimulation, was achieved with 2-chloroadenosine, an adenosine analogue that appears not to be a substrate for the adenosine transport system. The presynaptic inhibitory action of 2-chloroadenosine was antagonized by theophylline (10 and 100 microM) and by 8-phenyltheophylline (10 microM) but not by theophylline 9-beta-D-riboside (100 microM). It is concluded that the addition of a ribose moiety to theophylline does not enhance the antagonist potency of the molecule but actually renders the compound inactive. However, 8-phenyltheophylline is approximately three times more potent than theophylline at presynaptic adenosine receptors.     

Stimulation of Ca2+-activated human platelet phospholipase A2 by diacylglycerol.

The adenosine analogues 5'-(N-ethyl)carboxamidoadenosine (NECA) and N6-(phenylisopropyl)adenosine (PIA) activate glycogen phosphorylase 5-fold and 4.2-fold respectively in rat hepatocytes incubated in the absence of endogenous adenosine. Half-maximally effective concentrations are 0.5 microM for NECA and 20 microM for PIA, demonstrating the presence of A2-adenosine receptors. Exogenous adenosine activates phosphorylase 4.6-fold, but high rates of adenosine disappearance from the medium render estimates of its half-maximally effective concentration unreliable. These effects of NECA and adenosine are inhibited by 0.1 mM-caffeine. Activation of phosphorylase by a physiological concentration of adenosine (3.3 microM) was 50% inhibited by a physiological concentration of caffeine (35 microM).     

The binding of [3H]adenosine to synaptosomal and other preparations from the mammalian brain.

1. A high-affinity adenosine-binding site with Kd(adenosine) 0.5-1.3 microM was demonstrated in particulate and synaptosomal fractions isolated from the cerebral cortex of guinea pig, rat and ox. 2. Binding of [3H]adenosine to this site was inhibited by theophylline and by 2-chloroadenosine, but not by four other adenosine analogues. 3. Endogenous adenosine, found to be present in some preparations at approx. 1 pmol/mg of protein, diminished the binding capacity of the preparations for [3H]adenosine. 4. Addition of the adenosine deaminase inhibitor erythro-9-[1-(1-hydroxyethyl)heptyl]-adenine revealed the presence of a second lower affinity binding site with Kd (adenosine) 5-9 microM and a higher maximal adenosine-binding capacity. The inhibitor partially blocked binding to the high-affinity site in preparations from which adenosine deaminase had been removed by washing. 5. To preparations of particulate fractions maintained under iso-osmotic conditions, adenosine attachment was non-saturable and temperature-dependent, indicating the existence of an active uptake process. 6. The location and binding constant of the high-affinity adenosine-binding site suggest that it corresponds to the receptor site for adenosine-activated adenylate cyclase.     

Identification of Adenosine Receptor in Rat Pineal Gland: Evidence for A-2 Selectivity

We have examined the binding of the adenosine agonist radioligands [3H]cyclohexyladenosine [( 3H]CHA), R-N6-[3H]phenylisopropyladenosine [( 3H]R-PIA), and 5'-N-ethylcarboxamido[3H]adenosine [( 3H]NECA) to membranes prepared from rat pineal gland. The results showed that the A-1-selective ligands (CHA and R-PIA) had less than or equal to 10% specific binding. By contrast, [3H]NECA, a nonselective A-1/A-2 ligand, gave 72% specific binding of the total binding. This specific binding was insensitive to cyclopentyladenosine (50 nM) or R-PIA (50 microM). To characterize this binding, we used the N-ethylmaleimide pretreatment method. Under these conditions, this binding was of high affinity with a KD of 51 +/- 10 nM and an apparent Bmax of 1,060 +/- 239 fmol/mg of protein. Specific binding was unaffected by the presence of MgCl2 (10 mM) but was sensitive to guanylylimidodiphosphate (100 microM) (-25%), a result suggesting the involvement of an N-protein mechanism in the coupling of the adenosine receptor labeled by [3H]NECA to other components of the receptor complex. The rank of activity of adenosine analogues in displacing specific [3H]NECA binding was NECA greater than 2-chloroadenosine greater than S-adenosyl-L-homocysteine greater than CHA. Binding was also displaced by 3-isobutyl-1-methylxanthine (IC50 = 23.6 microM). These findings are consistent with the selective labeling by [3H]NECA of an A-2-type adenosine receptor in rat pineal membranes.     

The ability of adenosine to decrease the concentration of fructose 2,6-bisphosphate in isolated hepatocytes. A cyclic AMP-mediated effect.

The presence of adenosine (25-250 microM) or of 2-chloroadenosine (2.5-100 microM) in the incubation medium caused a marked decrease in the concentration of fructose 2,6-bisphosphate in isolated hepatocytes. This effect was accompanied by an increase in the concentration of cyclic AMP, an activation of phosphorylase and of fructose 2,6-bisphosphatase, and an inactivation of pyruvate kinase and of 6-phosphofructo-2-kinase. As a rule, the changes in the fructose 2,6-bisphosphate-modifying system were slower but more persistent than those in the activities of phosphorylase and pyruvate kinase. The effect of the nucleoside to decrease the concentration of fructose 2,6-bisphosphate was not affected by an inhibitor of adenosine transport and could not be obtained in a liver high-speed supernatant. These data indicate that the effect of adenosine to decrease the concentration of fructose 2,6-bisphosphate is mediated by the stimulation of adenylate cyclase, secondary to the binding of adenosine to membranous receptors. Like glucagon, 2-chloroadenosine stimulated gluconeogenesis in isolated hepatocytes, whereas adenosine had an opposite effect.     

Role of adenosine deaminase, ecto-(5''-nucleotidase) and ecto-(non-specific phosphatase) in cyanide-induced adenosine monophosphate catabolism in rat polymorphonuclear leucocytes.

1. The role of adenosine deaminase (EC 3.5.4.4), ecto-(5'-nucleotidase) (EC 3.1.3.5) and ecto-(non-specific phosphatase) in the CN-induced catabolism of adenine nucleotides in intact rat polymorphonuclear leucocytes was investigated by inhibiting the enzymes in situ. 2. KCN (10mM for 90 min) induced a 20-30% fall in ATP concentration accompanied by an approximately equimolar increase in hypoxanthine, ADP, AMP and adenosine concentrations were unchanged, and IMP and inosine remained undetectable ( less than 0.05 nmol/10(7) cells). 3. Cells remained 98% intact, as judged by loss of the cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27). 4. Pentostatin (30 microM), a specific inhibitor of adenosine deaminase, completely inhibited hypoxanthine production from exogenous adenosine (55 microM), but did not black CN-induced hypoxanthine production or cause adenosine accumulation in intact cells. This implied that IMP rather than adenosine was an intermediate in AMP breakdown in response to cyanide. 5. Antibodies raised against purified plasma-membrane 5'-nucleotidase inhibited the ecto-(5'-nucleotidase) by 95-98%. Non-specific phosphatases were blocked by 10 mM-sodium beta-glycerophosphate. 6. These two agents together blocked hypoxanthine production from exogenous AMP and IMP (200 microM) by more than 90%, but had no effect on production from endogenous substrates. 7. These data suggest that ectophosphatases do not participate in CN-induced catabolism of intracellular AMP in rat polymorphonuclear leucocytes. 8. A minor IMPase, not inhibited by antiserum, was detected in the soluble fraction of disrupted cells.     

Inhibition of IMP-specific cytosolic 5''-nucleotidase and adenosine formation in rat polymorphonuclear leucocytes by 5''-deoxy-5''-isobutylthio derivatives of adenosine and inosine.

1. The partially purified IMP-specific cytosolic 5'-nucleotidases from rat liver, polymorphonuclear leucocytes and heart were inhibited by 50% by 2-6 mM-5'-deoxy-5'-isobutylthioadenosine (IBTA) or 7-10 mM-5'-deoxy-5'-isobutylthioinosine (IBTI). IBTA and IBTI inhibited the rat liver and polymorphonuclear-leucocyte enzymes non-competitively. IBTA, but not IBTI, also inhibited the ecto-5'-nucleotidase of polymorphonuclear leucocytes. IBTI was, by contrast, a more potent inhibitor than IBTA of the AMP-specific soluble 5'-nucleotidase from pigeon heart. 2. During 2-deoxyglucose-induced ATP-catabolism in rat polymorphonuclear leucocytes, adenosine formation was inhibited by approx. 80% by 3 mM-IBTA and by approx. 70% by 7 mM-IBTI. 3. The results show that 5'-modified nucleosides are inhibitors of cytosolic 5'-nucleotidases and that they penetrate to inhibit their target enzymes in intact cells. Such inhibitors may be useful to clarify the mechanisms of adenosine formation and to prevent mononucleotide hydrolysis during ATP breakdown.     

Adenosine and its derivatives inhibit the cAMP signaling response in Dictyostelium discoideum

In developmentally competent Dictyostelium discoideum amoebae, binding of cAMP to high-affinity surface receptors produces a rapid activation of adenylate cyclase which adapts within minutes. The result is a transient increase in intracellular cAMP which is rapidly secreted. Adenosine inhibited this cAMP signaling response with an apparent Ki of 300 microM. The apparent Ki's for 2'-O-methyladenosine and 2-chloroadenosine were approximately 30 and 100 microM, respectively. Inhibition by adenosine was rapid, reversible, and depended on the cAMP stimulus concentration. In addition, the adaptation of the cAMP signaling response was blocked by adenosine. As has been previously reported, adenosine inhibits cAMP binding to intact cells. Under the same developmental conditions as in the perfusion studies, we find the binding inhibition depends on both the cAMP and adenosine concentrations, with an apparent Ki of 100 microM. The apparent Ki's for 2'-O-methyl- and 2-chloroadenosine were approximately 8 and 35 microM, respectively. However, with cells developed for short times and with an axenic strain, inhibition was independent of cAMP concentration or cells showed mixed-type binding inhibition. The effect of adenosine on the cAMP signaling response is consistent with the reported effects of adenosine on other cAMP-mediated processes such as chemotaxis and the increase in intracellular cGMP, and may provide an explanation for the reported inhibition of center formation.