Evidence that IgE receptor stimulation increases adenosine release from rat basophilic leukaemia (RBL-2H3) cells.

Adenosine may play a role in asthma by enhancing inflammatory mediator release from lung mast cells. In this study, we investigated whether adenosine is released from cultured rat basophilic leukaemia (RBL-2H3) cells in response to antigen challenge and whether released adenosine enhances mediator release. RBL-2H3 cells closely resemble mucosal mast cells, the most common type of mast cell in lung tissue, and they express adenosine A3 receptors (which have been associated with asthma). Measurement of adenosine in RBL-2H3 cell incubation medium was possible if adenosine metabolism was inhibited by EHNA (10 microM; an adenosine deaminase inhibitor) and 5-iodotubericidin (5-IT; 10 microM; an adenosine kinase inhibitor). Basal adenosine concentration increased up to 1.0 microM during a 90 min incubation; after antigen challenge, adenosine concentration was increased by 0.3-0.4 microM above basal. Antigen-induced adenosine release ranged from 30-70 nmol/1.25x10(6) cells. Antigen-induced mediator release (beta-hexosaminidase and [3H]5-hydroxytryptamine) was increased by APNEA, an adenosine A3 receptor agonist (EC50 approximately 20 nm) but inhibited by EHNA and 5-IT, despite increased adenosine levels. This inhibition was not blocked by the adenosine A1/A2 receptor antagonist DPSPX (5 microM). Therefore, it is unlikely to be related to adenosine receptor activation. In conclusion, although our data provide no direct support for a positive feedback effect of adenosine on mast cell mediator release, the observation that IgE receptor stimulation increases adenosine production in cells which express stimulatory A3 receptors is consistent with this hypothesis.

Release of adenosine from human sensitized lung fragments and its effect on antigen-induced mediator release.

Adenosine may play a role in asthma as a pro-inflammatory mediator. In this study, the release of adenosine from human sensitized lung fragments and its effect on antigen-induced histamine and leukotriene release has been explored. Antigen challenge increased histamine and leukotriene release five-fold but was without effect on adenosine release. In contrast, the adenosine deaminase inhibitor EHNA (10 microM) and the adenosine kinase inhibitor 5-iodotubericidin (10 microM) increased adenosine concentration 45-fold (P < or = 0.001; n = 4 patients). Of major interest was the finding that the non-selective, cell impermeant, adenosine antagonist pSPT (100 microM) decreased histamine and leukotriene release by 25% (P < or = 0.001) and 40%, respectively (P < or = 0.05; n = 9 patients). Additionally, the non-selective adenosine agonist NECA (10 microM) markedly inhibited antigen-induced leukotriene release by 80-90% (P < or = 0.001) and marginally inhibited histamine release by approximately 10% (P < or = 0.05; n = 9); the A2a-selective agonist DPMA (10 microM) was without effect on either histamine or leukotriene release. These results are consistent with adenosine having a biphasic effect on antigen-induced mediator release with low concentrations potentiating release and high concentrations inhibiting release. The overall stimulatory effect of endogenous adenosine supports the proposal that adenosine may act as a pro-inflammatory mediator in asthma.

Involvement of adenosine deaminase and adenosine kinase in regulating extracellular adenosine concentration in rat hippocampal slices.

In this study the relative importance of adenosine deaminase and adenosine kinase in regulating extracellular adenosine concentration was investigated in rat hippocampal slices labelled with [3H]-adenine. The release of [3H]-purines evoked by electrical stimulation or energy depletion (oxygen and glucose deprivation) was measured and, using high-performance liquid chromatography (HPLC), the proportion of [3H]-label in the form of [3H]-adenosine, [3H]-inosine and [3H]-hypoxanthine was determined. In addition, endogenous purine release was measured by HPLC with UV detection. 10 microM 5-iodotubericidin (5-IT), an inhibitor of adenosine kinase, significantly increased endogenous adenosine release and altered the pattern of [3H]-purine release by increasing the proportion released as [3H]-adenosine, under basal conditions and after electrical stimulation or energy depletion. 5 microM erythro-9-(2-hydroxy-3-nonyl) adenosine (EHNA), an inhibitor of adenosine deaminase, also increased endogenous adenosine release and altered the pattern of [3H]-purine release evoked by energy depletion by decreasing the proportion of [3H]-label released as [3H]-hypoxanthine and [3H]-inosine, whilst approximately doubling that of [3H]-adenosine. In contrast, adenosine release was not altered by EHNA under basal conditions or electrical stimulation. It is concluded that under conditions which provide adequate oxygen and glucose, adenosine kinase plays a much greater role than adenosine deaminase in regulating the extracellular concentration of adenosine. However, adenosine deaminase becomes important in regulating extracellular adenosine concentration when adenosine formation is increased by energy depletion.

The early events of oxygen and glucose deprivation: setting the scene for neuronal death?

It is generally thought that neuronal death caused by a reduction in oxygen or glucose supply, or both, occurs as a result of massive increases in the extracellular concentrations of excitatory amino acid neurotransmitters, particularly glutamate. A pertinent question is what happens before this increase, because measures which prevent extracellular accumulation of glutamate could have potential for clinical use in, for example, management of acute stroke. This article will review the major pathophysiological responses which occur up until the time of accumulation of glutamate. Withdrawal of energy substrate quickly leads to modest changes in membrane potential and intracellular and extracellular ion concentrations. Depression of action-potential-dependent synaptic transmission occurs a little later and might, in part, reflect actions of adenosine. Increases in the extracellular concentration of excitatory amino acids to neurotoxic levels take place only as membrane potential falls rapidly towards 0mV, coincident with massive changes in ion gradients.

Adenosine metabolism in the guinea pig heart: the role of cytosolic S-adenosyl-L-homocysteine hydrolase, 5'-nucleotidase and adenosine kinase.

This study was conducted to elucidate the role of S-adenosyl-L-homocysteine (SAH) hydrolase, 5'-nucleotidase and adenosine kinase in the production and removal of adenosine in the isolated guinea pig heart during normoxic (95% O2) and hypoxic (30% O2) perfusion. Using an adenosine kinase inhibitor (5'-amino-5'-deoxy-adenosine; 50 microM) and an adenosine deaminase inhibitor (EHNA; 5 microM) the total steady-state production rate of adenosine in the heart was estimated to be greater than 1.2 nmol.min-1 per g wet wt., during normoxia. Most (95%) of the SAH-derived adenosine is salvaged by adenosine kinase action. The rate of adenosine phosphorylation increased 3-fold when isolated hearts were perfused with hypoxic medium, suggesting that adenosine kinase is not substrate-saturated under normoxic conditions. The steady-state production of adenosine was also estimated during hypoxia (5.9 nmol-min-1 per g wet wt.) and compared with previously determined transmethylation rate during hypoxia (1.12 nmol.min-1 x g wet wt.). In an attempt to assess the in-vivo activity of cytosolic 5'-nucleotidase, the 5'-AMP pool was labelled by perfusing the isolated hearts with tricyclic nucleoside (TCN) which became phosphorylated (TCN-P). The release rate of both adenosine and TCN in the post-labelling phase was increased by hypoxic perfusion, suggesting that the increased rate of 5'-AMP hydrolysis may be due to increased availability of substrate, as well as activation of 5'-nucleotidase. Our findings suggest that during normoxic perfusion a significant amount of adenosine is derived from an apparently oxygen-independent mechanism (cellular transmethylation) whereas during hypoxic perfusion hydrolysis of adenine nucleotides to adenosine prevails.

Synaptosomal amino acid release: effect of inhibiting protein phosphatases with okadaic acid.

The protein phosphatase inhibitor okadaic acid was used to investigate the role of protein phosphatases in regulating the release of amino acids from synaptosomes. Okadaic acid increased the basal release of the amino acids glutamate, aspartate and GABA. The effect was specific in that taurine was not released by either KCl or okadaic acid and there was no synaptosomal lysis or change in ATP/ADP ratios in the presence of okadaic acid. The okadaic acid-stimulated release of amino acids was, however, only a small proportion of that produced by KCl depolarisation. Since okadaic acid raised synaptosomal protein phosphorylation levels to those equivalent to that produced by KCl depolarisation, it is unlikely therefore that there is a direct causal relationship between protein phosphorylation and the release of amino acids. Nevertheless, that release of amino acids from synaptosomes can be elevated under basal conditions by okadaic acid treatment does suggest that okadaic acid-sensitive protein phosphatases have a modulatory role in this process.

Intracellular formation and release of adenosine from rat hippocampal slices evoked by electrical stimulation or energy depletion.

In this study, the basal and evoked release of [3H]- and endogenous adenosine, inosine and hypoxanthine from rat hippocampal slices, labelled with [3H]adenine, was investigated. Evoked release was brought about by either electrical stimulation or energy depletion. The aim was to determine whether adenosine is formed intracellularly, and released as adenosine or extracellularly, from sequential extracellular hydrolysis of released ATP. All measurements were made in the presence of 5 microM erythro-9-(2-hydroxy-3-nonyl) adenosine (EHNA) to inhibit the enzyme adenosine deaminase. It was found that electrical field stimulation (5 min) increased the release of endogenous adenosine from hippocampal slices 10-fold and increased the proportion of [3H]-label associated with adenosine from approx 7% of the total released to 13% after the first stimulation and 20% after the second stimulation. Removal of oxygen and glucose from the superfusion medium (energy depletion) increased the release rate of endogenous adenosine 16-fold and increased the proportion of [3H]-label associated with [3H]adenosine from approx 10% of the total released to 50%. In order to prevent extracellular formation of adenosine, experiments were carried out in the presence of 50 microM alpha, beta-methylene ADP (AOPCP), an inhibitor of ecto-5'-nucleotidase. AOPCP was found to be without effect on either the basal or evoked release of adenosine. In contrast, L-homocysteine thiolactone (0.1-1.0 mM) which was used to "trap" intracellular adenosine reduced both the basal and evoked release of adenosine by 70-85%. This effect of L-homocysteine thiolactone also occurred in the presence of adenosine uptake inhibitors. It is concluded from these results that adenosine is formed predominantly intracellularly in hippocampal slices and is released as adenosine as a result of either tissue depolarisation or energy depletion. Furthermore, the finding that during energy depletion there is a proportionally greater release of adenosine than other ATP breakdown products, such as inosine and hypoxanthine, indicates that energy depletion is both a potent and selective stimulus for adenosine formation and release.

Contribution of S-adenosylhomocysteine to cardiac adenosine formation.

The S-adenosylhomocysteine (SAH) hydrolase inhibitor adenosine dialdehyde was used in isolated guinea pig hearts to determine the contribution of the transmethylation pathway to cardiac adenosine formation. This inhibitor did not alter cardiac hemodynamics but effectively inhibited SAH-hydrolase activity under in vitro and in vivo conditions. In normoxic perfused hearts adenosine dialdehyde (10 microM) caused tissue levels of SAH to linearly increase at a rate of 160 pmol/g/min over 60 min. At the same time adenosine dialdehyde decreased release of adenosine into the coronary effluent perfusate by 16 pmol/min (34%). Hypoxic perfusion (30% O2) of guinea-pig hearts increased release of adenosine from 43 to 3700 pmol/min. However, rate of SAH formation in the presence of adenosine dialdehyde was only slightly enhanced from 160 to 200 pmol/g/min and adenosine dialdehyde did not significantly alter the hypoxia induced adenosine release. Since all experiments were performed in the presence of the adenosine deaminase inhibitor EHNA (5 microM) the results demonstrate: (1) the transmethylation pathway of the heart contributes one third to global cardiac adenosine production under normoxic conditions and provides a constant source of adenosine independent of tissue oxygenation. (2) The majority of SAH-derived adenosine is salvaged most likely via adenosine kinase. (3) The hypoxia induced adenosine production is predominantly derived from enhanced 5' AMP hydrolysis.

Effect of magnesium on depression of the monosynaptic reflex induced by 2-chloroadenosine or hypoxia in the isolated spinal cord of neonatal rats.

Superfusion of the isolated spinal cord of neonatal rats (4-9 days postpartum) with physiological medium containing 2-chloroadenosine (2-CA) or anoxic medium (equilibrated with 95% N2-5% CO2) depressed the evoked monosynaptic reflex (MSR) recorded extracellularly from a ventral spinal root. The effectiveness of 2-CA or anoxic medium in depressing the MSR was significantly reduced when the concentration of Mg2+ in the physiological medium was lowered from 1.25 X 10(-3) M to zero. The absence of Mg2+ resulted in a 7-fold shift to the right of the concentration-response curve to 2-CA and a reduction in the maximal depression of the MSR from 100% to 65 +/- 4% (mean +/- S.E.M.) of control. A 10 min exposure to anoxic medium containing 1.25 X 10(-3) M Mg2+ decreased the amplitude of the MSR to 23 +/- 6% of control, whilst in zero Mg2+ a decrease to only 50 +/- 5% of control was observed. These data provide further evidence that the response to adenosine, at the A1-receptor, is sensitive to Mg2+ ion concentration and suggest that there is an absolute requirement for Mg2+ in order to obtain full expression of the adenosine effect. Furthermore, the data are consistent with the hypothesis that adenosine is an important mediator of hypoxia-induced depression of the evoked MSR in the spinal cord, and suggest a potential role for Mg2+ during or after exposure to hypoxia in altering the actions of adenosine on neuronal activity or synaptic events.

Involvement of adenosine in synaptic depression induced by a brief period of hypoxia in isolated spinal cord of neonatal rat.

The monosynaptic reflex (MSR), recorded extracellularly from the ventral root isolated, superfused spinal cords of neonatal rats (6-10 days post-partum), was rapidly depressed to 35-45% of control values by either cessation of superfusion (4 min stop-flow period) or by superfusion with anoxic medium (95% N2-5% CO2; 4 min). The depression was reversible, 85-115% recovery occurring after 15 min of restoration of flow or normoxic (95% O2-5% CO2) superfusion. 2-Chloroadenosine, a metabolically stable adenosine analogue, also reversibly inhibited the MSR, an effect which was antagonised by 10(-6) M 8-cyclopentyltheophylline (8-CPT). The depression of the MSR, caused by 4 min of hypoxia (either stop-flow or anoxic superfusion), was prevented by 10(-6) M 8-CPT. These results provide strong evidence for a critical involvement of adenosine in mediating early synaptic depression evoked by a brief period of hypoxia.

The transmethylation pathway as a source for adenosine in the isolated guinea-pig heart.

In order to quantify adenosine production from the transmethylation pathway [S-adenosylmethionine (AdoMet)----S-adenosylhomocysteine (AdoHcy) in equilibrium adenosine + L-homocysteine] in the isolated guinea-pig heart under basal conditions (normoxic perfusion with 95% O2) and during elevated adenosine production (hypoxic perfusion with 30% O2), two methods were used. (1) Hearts were perfused with normoxic medium containing [2,5,8-3H]adenosine (5 microM) and L-homocysteine thiolactone (0.1 mM), which brings about net AdoHcy synthesis via reversal of the AdoHcy hydrolase reaction and labels the intracellular pool of AdoHcy. From the decrease in AdoHcy pool size and specific radioactivity of AdoHcy in the post-labelling period, the rate of transmethylation, which is equivalent to the rate of adenosine production, was calculated to be 0.98 nmol/min per g. Adenosine release from the hearts was 40-50 pmol/min per g. (2) Hearts were perfused with hypoxic medium containing [35S]homocysteine (50 microM). Owing to the hypoxia-induced increase in adenosine production, this procedure also results in expansion and labelling of the AdoHcy pool. From the dilution of the specific radioactivity of AdoHcy relative to that of [35S]homocysteine, the rate of AdoHcy synthesis from AdoMet (transmethylation) was calculated to be 1.12 nmol/min per g. It is concluded that in the oxygenated heart the transmethylation pathway is quantitatively an important intracellular source of adenosine, which exceeds the rate of adenosine wash-out by the coronary system by about 15-fold. Most of the adenosine formed by this pathway is re-incorporated into the ATP pool, most likely by adenosine kinase. The transmethylation pathway is essentially O2-independent, and the known hypoxia-induced production of adenosine must be derived from an increase in 5'-AMP hydrolysis.

Effect of copper on the binding and electrophysiological actions of cyclohexyladenosine.

Since copper ions irreversibly reduce the binding of cyclohexyladenosine (CHA) to rat brain membranes and copper levels are elevated in the brains of seizure-prone mice, the binding of CHA was compared in seizure-prone DBA/2 and control C57 mice. No strain difference was detected in the binding of CHA or the reduction of that binding by copper, although copper was more potent than in rats (IC50 12 microM). In rat hippocampal slices copper did not diminish the inhibitory effects of adenosine or CHA. The results suggest that the seizure sensitivity of DBA/2 mice is not due to an effect of copper on purine receptors and that the copper binding domain of purine receptors is inaccessible to extracellular copper.

Cyclohexyladenosine binding in rat striatum.

N6-Cyclohexyl-[3H]adenosine [( 3H]CHA) binds specifically to rat brain membranes prepared from the caudate-putamen complex with a Kd value of 2.50 +/- 0.39 nM and Bmax of 458 +/- 51 fmol/mg protein. Lesioning the nigrostriatal dopaminergic pathway using 6-hydroxydopamine failed to alter [3H]CHA binding characteristics. Intrastriatal kainate lesions reduced the binding capacity of [3H]CHA by 28% though this was not statistically significant (0.1 less than P greater than 0.05). In kainate-lesioned striata, however, 2-deoxyglucose uptake was reduced by only 39%.

Inhibition of adenosine accumulation by a CNS benzodiazepine antagonist (Ro 15-1788) and a peripheral benzodiazepine receptor ligand (Ro 05-4864).

Although benzodiazepines can inhibit adenosine uptake into central neurones, this effect is not antagonized by behaviourally effective 'benzodiazepine antagonists' such as Ro 15-1788. We now report that Ro 15-1788 and the 'peripheral' benzodiazepine ligand Ro 05-4864 themselves inhibit adenosine accumulation by rat brain synaptosomes. The inhibition of adenosine accumulation may thus underlie those behavioural effects of benzodiazepines which are mimicked but not antagonized by Ro 15-1788.

Benzodiazepine inhibition of adenosine uptake is not prevented by benzodiazepine antagonists.

Uptake of [3H]adenosine into rat cerebral cortex synaptosomes was studied. Hexobendine (10(-5) M) and the benzodiazepine agonists diazepam (10(-5) M) and flurazepam (10(-4) M) significantly inhibited this uptake, but only if the compounds were pre-incubated for 10 min in the case of the benzodiazepines. The benzodiazepine antagonists Ro15-1788 (10(-5) M) and CGS 8216 (10(-5) M) failed to reverse the action of benzodiazepine agonists or hexobendine on [3H]adenosine uptake. The results add weight to the view that inhibition of adenosine uptake processes by benzodiazepines do not contribute to their behavioural effects.

Ethylenediamine as a specific releasing agent of gamma-aminobutyric acid in rat striatal slices.

Following incubation with [14C]gamma-aminobutyric acid (GABA) or [3H]dopamine, slices of rat striatum were superfused with media containing 36 mM K+ or ethylenediamine (EDA), 1 or 5 mM. Both K+ and EDA induced a release being largely Ca2+-dependent, while the EDA-induced release was not. Whereas K+ also evoked a Ca2+-dependent release of [3H]dopamine, EDA evoked no release of dopamine. EDA may therefore have potential as a specific GABA releasing agent.

Uptake and calcium-dependent release of ethylenediamine (1,2-diaminoethane) by rat brain slices.

The uptake of [14C]ethylenediamine into slices of rat brain and its subsequent evoked release have been studied. An active uptake process was demonstrated by comparing uptake at 37 and 4 degrees C. This uptake showed a Km of 1.36 mM, was partly sodium-dependent and was reduced by nipecotic acid. Release could be readily evoked by 30 mM potassium, and by electrical stimulation, the release in both cases being calcium-dependent. In view of these findings and the reported interactions of ethylenediamine with gamma-aminobutyric acid-related mechanisms, it might be of interest to determine whether this simple diamine occurs endogenously in the mammalian brain.

Chronic methylxanthine treatment in rats: a comparison of Wistar and Fischer 344 strains.

Caffeine, theophylline or aminophylline were administered chronically to rats of both sexes, in the weight range 30-245 g. Self-injurious behaviour was noted only rarely in Wistar rats allowed free access to food, but developed over 3 to 4 weeks in half of the animals given a restricted diet of about one third of the intake of control rats. Fischer rats showed self-injurious behaviour more readily, 87% of animals showing signs within 9 days even on an ad lib diet. It is suggested that Fischer rats treated with methylxanthines may provide a model of the Lesch-Nyhan syndrome. Behavioural observations made during the period of methylxanthine treatment suggest that an activation of both the dopamine and 5-hydroxytryptamine neurone systems may be produced. Further work will seek a relationship between these systems and self-injurious behaviour.