Regulation of serotonin transporter activity by adenosine in intestinal epithelial cells

Serotonin plays a critical role in the regulation of intestinal physiology. The serotonin transporter (SERT) expressed in the intestinal epithelium determines 5-HT availability and activity. The serotoninergic system and SERT activity have been described as being altered in chronic intestinal pathologies such as inflammatory diseases. Adenosine has also been shown to be involved in a variety of intestinal functions and to play a central role in the regulation of inflammatory responses of injured tissue. Since the modulation of SERT by adenosine in the intestine remains unknown, the aim of the present work was to study the effect of adenosine on SERT activity and expression and to determine the molecular mechanism involved. The study has been carried out using human enterocyte-like Caco-2 cells which endogenously express SERT. The results show that adenosine diminishes SERT activity in both the apical and basal membranes by acting in the intrinsic molecule with no alteration of either SERT mRNA or protein levels. The effect of adenosine appears to be mediated by A₂ receptors and activation of the cAMP/PKA signalling pathway. Moreover, the adenosine effect did not seem to involve the activation of AMP activated protein kinase. Adenosine effects are reached at high concentrations, which suggests that adenosine modulation of SERT may be significant under conditions of inflammation and tissue injury.     

Adenosine and adenosine analogs inhibit phosphodiesterase activity in the heart

Summary Adenosine and its analogs (-)-N⁶-phenylisopropyladenosine and 5-N-ethylcarboxamideadenosine inhibit cAMP and cGMP phosphodiesterase activity in guinea-pig atrial and ventricular preparations at concentrations of 100 mol l^–1 and higher. These effects are probably unrelated to the inotropic effects of these substances. However, inhibition of cAMP breakdown may compensate for the adenosine-induced inhibition of adenylate cyclase and may thus at least partially explain why with this drug no changes in cAMP or cGMP content have previously been observed in intact cardiac tissue.     

Partial adenosine A(1) receptor agonists inhibit sarin-induced epileptiform activity in the hippocampal slice

Organophosphate poisoning can result in seizures and subsequent neuropathology. One possible therapeutic approach would be to employ adenosine A(1) receptor agonists, which have already been shown to have protective effects against organophosphate poisoning. Using an in vitro model of organophosphate-induced seizures, we have investigated the ability of several adenosine A(1) receptor agonists to inhibit epileptiform activity induced by the organophosphate sarin, in the CA1 stratum pyramidale of the guinea pig hippocampal slice. Application of the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) or the partial adenosine A(1) receptor agonists 2-deoxy-N(6)-cyclopentyladenosine (2-deoxy-CPA) and 8-butylamino-N(6)-cyclopentyladenosine (8-butylamino-CPA) abolished epileptiform activity in a concentration-related manner. The rank order of potency was CPA (IC(50) 4-5 nM) >2-deoxy-CPA (IC(50) 113-119 nM)=8-butylamino-CPA (IC(50) 90-115 nM). These data suggest that partial adenosine A(1) receptor agonists, which have fewer cardiovascular effects, should be further evaluated in vivo as potential treatments for organophosphate poisoning.     

Comparison of the behavioral effects of adenosine agonists and dopamine antagonists in mice

The adenosine agonists 5-N-ethylcarboxamidea-denosine (NECA), 2-chloroadenosine (2-CLA), N⁶-cyclohexyladenosine (CHA), N⁶-cyclopentyladenosine (CPA), 2-(phenylamino)adenosine (CV-1808) and R and S isomers of N⁶-phenylisopropyladenosine (R-PIA and S-PIA) decreased spontaneous locomotor activity in mice and, except for CPA, did so at doses that did not impair motor coordination, a profile shared by dopamine antagonists. CV-1808, the only agent with higher affinity for A₂ as compared with A₁ adenosine receptors, displayed the largest separation between locomotor inhibitory and ataxic potency. Like dopamine antagonists, NECA and CV-1808 also decreased hyperactivity caused by d-amphetamine at doses that did not cause ataxia whereas A₁-selective adenosine agonists reduced amphetamine's effects only at ataxic doses. Unlike dopamine antagonists, adenosine agonists inhibited apomorphine-induced cage climbing only at doses that caused ataxia. Involvement of central adenosine receptors in these effects was suggested by the significant correlation obtained between potency for locomotor inhibition after IP and ICV administration. Affinity for A₁ but not A₂ adenosine receptors was significantly correlated with potency for inducing ataxia. These results suggest that the behavioral profile of adenosine agonists in mice is related to their affinity for A₁ and A₂ adenosine receptors and indicate that adenosine agonists produce certain behavioral effects that are similar to those seen with dopamine antagonists.     

The coexistence of adenosine A1 and A2 receptors in guinea-pig aorta

The effects of adenosine, 5'-(N-ethyl)carboxamidoadenosine (NECA), 2-chloroadenosine (2-CA), No-cyclohexyladenosine (CHA) and N6(R-2-phenylisopropyl)-adenosine (R-PIA) on the tone of phenylephrine-constricted guinea-pig isolated aorta have been examined. For aortic relaxation the analogues exhibited the following rank order of potency: NECA > adenosine > 2-CA > R-PIA > CHA. This is consistent with previous reports that relaxation of this tissue is mediated by the adenosine A₂ receptor. An unexpected finding was that R-PIA, 2-CA and CHA all induced contractions at concentrations lower than were required for relaxation, giving a biphasic dose-response curve. Neither NECA nor adenosine contracted the aorta. This is consistent with activation of vascular A₁ receptors. An A₁-selective concentration of the antagonist l,3-dipropyl-8-cyclopentyl xanthine abolished the contraction elicited by R-PIA in the guinea-pig aorta. This further suggests that the contraction is mediated by a₁ receptors.     

4-Caffeoyl-1,5-quinide in roasted coffee inhibits [<Superscript>3</Superscript>H]naloxone binding and reverses anti-nociceptive effects of morphine in mice

Rationale Cinnamoylquinides are formed from the corresponding chlorogenic acids during coffee roasting. Instant coffee has been shown to displace binding of the mu opioid receptor antagonist, [³H]naloxone, but the putative active agent, feruloylquinide, has not been characterized.Objectives The goal was to identify the active agent(s) in coffee by measuring the binding affinity of individual cinnamoyl-1,5-quinides to the human mu opioid receptor, and determine the effects of these compounds on morphine-induced anti-nociceptive behavior in mice.Methods Cinnamoyl-1,5-quinides in extracts of decaffeinated instant coffee were quantified by reverse-phase HPLC comparisons with synthetic samples of 3-coumaroyl-1,5-quinide and 4-coumaroyl-1,5-quinide, 3-caffeoyl-1,5-quinide and 4-caffeoyl-1,5-quinide (4-CQL) 3-feruloyl-1,5-quinide and 4-feruloyl-1,5-quinides and 3,4-dicaffeoyl-1,5-quinide (DICAQ). Affinities of the cinnamoyl-1,5-quinides and decaffeinated instant coffee extract were determined by displacement of [³H]naloxone binding in cultured HEK-MOR cells. Inhibition of the anti-nociceptive activity of morphine (1 mg/kg IP) was determined in C57BL/6J mice using the hot plate test at 52°C.Results Extract of decaffeinated instant coffee produced a displacement K_i of 42±16 mg/l, while the K_i of a synthetic sample of 4-CQL was 4.4±0.4 M. Compounds with a cinnamoyl substituent in the 4-position of the quinide, i.e. 4-CQL, DICAQ, 3,4-diferuloyl-1,5-quinide, and 3,4-dicoumaroyl-1,5-quinide, had affinities for the mu opioid receptor in the low micromolar range. In the hot plate test, coffee extract, containing 0.78% of 4-CQL, reversed the anti-nociceptive effect of morphine at 10 mg/kg IP. Two cinnamoyl-1,5-quinides found in roasted coffee, DICAQ, and 4-CQL, were active at 1 and 0.1 mg/kg IP, respectively.Conclusions These results suggest that the previously reported anti-opioid activity of instant coffee is caused primarily by the presence of 4-CQL, and to lesser extent by other cinnamoyl-1,5-quinides.     

Reduced appetite for caffeine in adenosine A(2A) receptor knockout mice

Adenosine A(2A) receptor knockout mice (A(2A)R KO) were compared to wild-type controls (A(2A)R WT) in a caffeine intake paradigm. When mice had ad libitum access to caffeine (0.3 g/l) and water in a two-bottle paradigm for 12 consecutive days, adenosine A(2A)R KO mice drank less caffeinated solution, demonstrating a reduced appetite for caffeine as compared to adenosine A(2A)R WT mice. These data reveal an important role for the adenosine A(2A) receptor in the appetitive properties of caffeine.     

Rate of binding of various inhibitors at the dopamine transporter in vivo

The rate of entry of drugs into brain is thought to be a factor in their abuse liability. In this investigation, we have examined the rate of entry and binding at dopamine transporters in mouse striatum for a variety of dopamine transporter inhibitors. The method utilized was based on measuring the displacement of³H-WIN 35,428 from striatal dopamine transporter sites in vivo at different times. Eleven cocaine analogs (RTI-31, RTI-32, RTI-51, RTI-55, RTI-113, RTI-114, RTI-117, RTI-120, RTI-121, WIN 35,065-2, and WIN 35,428) as well as other dopamine uptake site blockers (bupropion, nomifensine, and methylphenidate) were compared with (–) cocaine for their rates of displacement of³H-WIN 35,428 binding in vivo. The drugs that displayed the fastest occupancy rates were bupropion, (–) cocaine, nomifensine, and methylphenidate. RTI-51, RTI-121, RTI-114, RTI-117, RTI-120, RTI-32, RTI-55, and RTI-113, showed intermediate rates, whereas RTI-31, WIN 35,065-2, and WIN 35,428 exhibited the slowest rates of displacement. While many of the cocaine analogs have proven to be behaviorally and pharmacologically more potent than (–) cocaine, their rates of entry and binding site occupancy were slower than that for (–) cocaine. Earliest times of transporter occupancy by the different drugs were correlated (although weakly) with their degree of lipophilicity (r=0.59;P<0.02). Kinetic effects and metabolism of the compounds could complicate the interpretations of these data. There was no obvious correlation between rate of occupancy in this animal model and abuse liability in humans, which is consistent with the notion that other factors are critical as well.     

Involvement of adenosine receptor activities in aggressive responses produced by clonidine in mice

A behavioral study was made of the mechanisms underlying the aggressive behavior induced by high doses of clonidine in mice. The frequency of clonidine-induced aggressive responses such as attacking and biting was increased dose-dependently from 10 to 50 mg/kg. Aggressive behavior induced by clonidine at doses of 10–30 mg/kg was potentiated under conditions of isolation and food deprivation for 24 h. Clonidine (30 mg/kg)-induced aggressive behavior was attenuated by adenosine (10 mg/kg IP) or dipyridamole (10 mg/kg IP), but markedly antagonized by combined pretreatment with both drugs. The behavior was strongly reduced by potent adenosine analogs, such as N⁶-cyclohexyl adenosine (CHA, 0.1 and 0.2 mg/kg IP) and N⁶-(l-phenyl isopropyl) adenosine (l-PIA, 0.2 mg/kg IP), but conversely was potentiated by phentolamine (10 mg/kg IP) or theophylline (10 mg/kg IP). Diazepam (2.5 mg/kg IP) and Ro 15-1788 (2.5 mg/kg IP), a benzodiazepine receptor antagonist, also blocked the aggressive behavior. The inhibition by CHA (0.2 mg/kg IP) or diazepam (2.5 mg/kg) of clonidine-induced aggression was not antagonized by additional pretreatment with bicuculline (2 mg/kg IP). The aggressive response to apomorphine (8 mg/kg IP) was not affected by those drugs which inhibited the response to clonidine. The results suggest that the aggressive behavior evoked by high doses of clonidine, but not that by apomorphine, involves a blockade of adenosine receptors.     

The coexistence of adenosine A1 and A2 receptors in guinea-pig aorta

The effects of adenosine, 5'-(N-ethyl)carboxamidoadenosine (NECA), 2-chloroadenosine (2-CA), No-cyclohexyladenosine (CHA) and N6(R-2-phenylisopropyl)-adenosine (R-PIA) on the tone of phenylephrine-constricted guinea-pig isolated aorta have been examined. For aortic relaxation the analogues exhibited the following rank order of potency: NECA > adenosine > 2-CA > R-PIA > CHA. This is consistent with previous reports that relaxation of this tissue is mediated by the adenosine A₂ receptor. An unexpected finding was that R-PIA, 2-CA and CHA all induced contractions at concentrations lower than were required for relaxation, giving a biphasic dose-response curve. Neither NECA nor adenosine contracted the aorta. This is consistent with activation of vascular A₁ receptors. An A₁-selective concentration of the antagonist l,3-dipropyl-8-cyclopentyl xanthine abolished the contraction elicited by R-PIA in the guinea-pig aorta. This further suggests that the contraction is mediated by a₁ receptors.     

Functional characterization of adenosine transport across the BBB in mice

We investigated transport characteristics of adenosine across the blood–brain barrier (BBB) in mice. Uptake clearance across the BBB was measured by using an in situ mouse brain perfusion technique and cultured mouse brain capillary endothelial cell line (MBEC4 cells). Nucleoside transporter was cloned by RT-PCR and expressed on Xenopus laevis oocyte. Both in situ and in vitro studies revealed that the adenosine uptake is concentration-dependent, Na⁺-independent and S-(p-nitrobenzyl)-6-thioinosine (NBMPR)-sensitive. The K_t values of in situ and in vitro studies were 31.7 ± 13.8 μM and 11.9 ± 2.84 μM, respectively. A good correlation was found for the inhibitory effects of nucleoside analogs to adenosine uptake between in situ and in vitro studies. RT-PCR revealed the expression of RNA of mouse equilibrative nucleoside transporter (mENT1) in mouse brain capillary and MBEC4 cells. In mENT1 expressed on X. laevis oocyte, K_t value of adenosine transport was 6.9 ± 2.7 μM (and comparable to those in situ and in vitro studies). In conclusion, we characterized the adenosine transport across the BBB in mice by using in situ brain perfusion technique and MBEC4 cells and found that these transports share common characteristics with mENT1-mediated transport. Transport of adenosine across the BBB in mice may be attributable to mENT1.     

Quantitative [3H] dipyridamole autoradiography: evidence for adenosine transporter heterogeneity in guinea pig brain

Summary [³H] Dipyridamole binding in guinea pig brain slices has been characterized. Binding of [³H] dipyridamole to guinea pig forebrian slices was found to be rapid, reversible and saturable. Saturation experiments revealed a class of high affinity binding sites with a B _max value of 592 ± 118 fmol/mg protein and K _d value of 10.8 nM ± 2.1 nM in the analysed concentration range. In competition experiments, the adenosine transport inhibitors hexobendine and dipyridamole itself were the most potent displacers (inhibition constants of 4.6 nM ± 1 nM and 11.5 nM ± 3 nM) with pseudo-Hill coefficients close to 1. Competition curves with nitrobenzylthioinosine, another adenosine transport inhibitor, however, showed a biphasic profile with a pseudo-Hill coefficient of 0.33 ± 0.04. Just 42% ± 4% of [³H] dipyridamole binding were inhibited by nanomolar concentrations of nitrobenzylthioinosine and only micromolar concentrations displaced the remainder. Subsequent quantitative autoradiography demonstrated regional differences in the inhibition of [³H] dipyridamole binding by submicromolar concentrations of nitrobenzylthioinosine. While in cortical areas of cerebrum and cerebellum 500 nM nitrobenzylthioinosine displaced binding of [³H] dipyridamole to only about one-third of its sites (in the Purkinje cell layer less than 10%), it showed similar potency as dipyridamole in various areas of the brainstem and hypothalamus. This biphasic and regionally heterogenous interaction of nitrobenzylthioinosine with [³H] dipyridamole binding sites in guinea pig brain slices strongly suggests heterogeneity of adenosine transporters.     

Cerebral ischemia in gerbils: effects of acute and chronic treatment with adenosine A2A receptor agonist and antagonist

Despite significant progress in understanding of the potential of adenosine A₁ receptor-based therapies in treatment of cerebral ischemia and stroke, very little is known about the effect of selective stimulation of adenosine A_2A receptors on the outcome of a cerebrovascular arrest. In view of a major role played by adenosine A₂ receptors in the regulation of cerebral blood flow, we have investigated the effect of both acute and chronic administration of the selective adenosine receptor agonist 2-[(2-aminoethylamino)-carbonylethylphenylethylamino]-5′-N-ethylcarboxoamidoadenosine (APEC) and antagonist 8-(3-chlorostyryl)caffeine (CSC) on the outcome of 10 min ischemia in gerbils. Acute treatment with APEC improved recovery of postischemic blood flow and survival without affecting neuronal preservation in the hippocampus. Acute treatment with CSC had no effect on the cerebral blood flow but resulted in a very significant protection of hippocampal neurons. Significant improvement of survival was present during the initial 10 days postischemia. Due to subsequent deaths of animals treated acutely with CSC, the end-point mortality (14 days postischemia) in this group did not differ statistically from that seen in the controls. It is, however, possible that the late mortality in the acute CSC group was caused by the systemic effects of brain ischemia that are not subject to the treatment with this drug. Chronic treatment with APEC resulted in a statistically significant improvement in all studied measures. Although chronic treatment with CSC improved postischemic blood flow, its effect on neuronal preservation was minimal and statistically insignificant. Mortality remained unaffected. The results indicate that the acute treatment with adenosine A_2A receptor antagonists may have a limited value in treatment of global ischemia. However, since administered CSC has no effect on the reestablishment of postischemic blood flow, treatment of stroke with adenosine A_2A receptor antagonists may not be advisable. Additional studies are necessary to elucidate whether chronically administered drugs acting at adenosine A₂ receptors may be useful in treatment of stroke and other neurodegenerative disorders.     

Differential regulation of adenosine A(1) and A(2A) receptors in serotonin transporter and monoamine oxidase A-deficient mice.

The serotonin (5HT) transporter (5HTT) removes 5HT from the synaptic cleft and is thus critical to the control of serotonergic neurotransmission. Mice with a targeted inactivation of the 5HTT represent a novel and unique tool to study serotonergic system functioning. Because the release of 5HT is regulated by adenosine, we investigated 5HTT-deficient mice for possible adaptive changes of adenosine A₁ and A_2A receptors. A₁ and A_2A receptors were studied by means of quantitative autoradiography using the radioligands [³H]8-cyclopentyl-1,3-dipropylxanthine and [³H]CGS 21680, respectively. A comparison of 5HTT knockout versus control mice revealed upregulation of A₁ receptors in the dorsal raphe nucleus (DRN, +21%), but not in any of the serotonergic projection areas, and downregulation of A_2A receptors in basal ganglia. The adaptive changes of A₁ and A_2A receptors in 5HTT-deficient mice are likely to represent a compensatory neuroprotective effect mediated by the adenosinergic modulatory system. For comparison, these receptors were also studied in monoamine oxidase A (MAOA) knockout mice and in 5HTT/MAOA double knockout mice. 5HTT/MAOA double knockout mice showed adaptive changes of adenosine A₁ and A_2A receptors similar to 5HTT knockout mice, while investigation of MAOA-deficient mice revealed an upregulation of A_2A receptors, which may relate to a role of both MAOA and adenosine A_2A receptors in anxiety.     

In vivo inhibition of adenosine triphosphate (ATP) synthesis associated with thiabendazole-induced teratogenesis in mice and rats

Ogata et al. (1984) reported that thiabendazole (TBZ) was teratogenic in mice when olive oil was used as a vehicle, but not teratogenic when administered in gum arabic. Results of investigations into the possible association between TBZ-induced teratogenicity and adenosine triphosphate (ATP) are reported here. ATP levels in the limb buds were measured at day 10 of gestation in controls and mice treated with 1300 mg/kg TBZ 24 h previously. The results showed that there was a correlation between the dosage of TBZ and the ATP levels of fore and hind limb buds (r=–0.827 and r=–0.799, respectively). ATP levels in mouse limbs were reduced to 22–31% of control values when TBZ was given in olive oil, but the ATP reduction was only 6–9% when TBZ was given in gum arabic. This suggests that there may be a relationship between the teratogenic and ATP-depressing action of TBZ on the limb buds of mouse embryos.Pregnant rats were also treated with TBZ. TBZ at a dose level of 1000 mg/kg was given orally on day 11 of gestation. After 24 h, ATP levels in fetal rat limbs were still 82% of control values.These results confirm the observations of Ogata et al. on the vehicle effect and the difference in response between mice and rats.     

Lanthanides inhibit the human noradrenaline, 5-hydroxytryptamine and dopamine transporters

Transporters for the monoamine neurotransmitters, including noradrenaline, 5-hydroxytryptamine [5-HT] and dopamine, have twelve transmembrane spanning regions and cotransport Na⁺ and Cl^– ions. Another family of Na⁺-dependent transporters is that containing the Na⁺/glucose and Na⁺/proline cotransporters that are found in the epithelial cells of renal and intestinal brush border membranes. It has been shown that various trivalent lanthanides can substitute for Na⁺ for transport of glucose and proline. The aim of this study was to determine the effects of lanthanides on the activities of the human noradrenaline, 5-HT and dopamine transporters. Cultured cells were incubated for 2min with 10nM ³H-noradrenaline (SK-N-SH-SY5Y human neuroblastoma cells), ³H-5-HT (JAR human placental choriocarcinoma cells) or ³H-dopamine (COS-7 cells transfected with the cDNA of the human dopamine transporter). Specific amine uptake was determined as the difference between accumulation of the amine in the cells in the absence and presence of a corresponding uptake inhibitor. Under both isotonic (150mM NaCl or LiCl or 90mM lanthanide salt) and hypertonic (150mM NaCl + 100mM LiCl, 250mM LiCl or 150mM lanthanide salt) conditions, replacement of Na⁺ by Li⁺, La³⁺, Eu³⁺ or Sm³⁺ abolished the specific uptake of noradrenaline in SK-N-SH-SY5Y cells and replacement of Na⁺ by Li⁺ or Eu³⁺ decreased the specific uptake of 5-HT in JAR cells by 94–100% and that of dopamine in transfected COS-7 cells by 95–99%. The direct effects of Eu³⁺ (with Na⁺ present) on the human noradrenaline transporter in SK-N-SH-SY5Y cells were also examined. Eu³⁺ inhibited noradrenaline uptake into the cells (IC₅₀ 2.6mM) and nisoxetine binding to crude membranes of SK-N-SH-SY5Y cells (IC₅₀ 4.7mM) with similar potencies. Further experiments showed that 4.5mM EuCl₃ in the presence of 150mM Na⁺ caused a 3.5-fold increase in the K_m of noradrenaline and no change in the maximal rate of noradrenaline uptake. EuCl₃ (4.5mM) also caused a pronounced inhibition of the Na⁺-dependent stimulation of noradrenaline uptake by SK-N-SH-SY5Y cells. It can be concluded from these data that, in contrast with the Na⁺/glucose and Na⁺/proline cotransporters, the lanthanides cannot substitute for Na⁺ in the transport of substrates by the monoamine neurotransmitter transporters and that the lanthanides inhibit the latter transporters by interacting with sites of the transporters involved in amine and Na⁺ binding. Received: 15 July 1996 / Accepted: 28 January 1997     

HPLC determination of adenosine in human synovial fluid

A high-performance liquid chromatographic method has been developed for the quantitative determination of adenosine in human synovial fluid. The method is simple, rapid and, overall, selective. No interference with the components of the biological matrix was observed in these chromatographic conditions. An ODS (250×4.6 mm) 5 μm column was used with an isocratic elution of a phosphate buffer–acetonitrile mobile phase. Detection was carried out on a UV detector at 260 nm. Calibration curve was found to be linear in the 0.7–70 μg ml⁻¹ range. Linear regression analysis of the data demonstrates the efficacy of the method in terms of precision and accuracy. The precision of this method, calculated as the relative standard deviation (RSD) of the recoveries (1.57–2.21%), was excellent. The limits of quantitation (LOQ) and detection (LOD) were respectively 0.7 and 0.2 μg ml⁻¹. The method was applied to some samples of synovial effusion from patients affected by rheumatoid arthritis. The concentrations of adenosine which were found were included in the range of the calibration curve.     

Astrocytic adenosine kinase regulates basal synaptic adenosine levels and seizure activity but not activity-dependent adenosine release in the hippocampus

Adenosine is an endogenous inhibitor of excitatory synaptic transmission with potent anticonvulsant properties in the mammalian brain. Given adenosine's important role in modulating synaptic transmission, several mechanisms exist to regulate its extracellular availability. One of these is the intracellular enzyme adenosine kinase (ADK), which phosphorylates adenosine to AMP. We have investigated the role that ADK plays in regulating the presence and effects of extracellular adenosine in area CA1 of rat hippocampal slices. Inhibition of ADK activity with 5′-iodotubercidin (IODO; 5 μM) raised extracellular adenosine, as measured with adenosine biosensors, and potently inhibited field excitatory post-synaptic potentials (fEPSPs) in an adenosine A₁R-dependent manner. In nominally Mg²⁺-free aCSF, which facilitated the induction of electrically-evoked epileptiform activity, adenosine biosensor recordings revealed that seizures were accompanied by the transient release of adenosine. Under these conditions, IODO also inhibited the fEPSP and greatly suppressed epileptiform activity evoked by brief, high-frequency stimulation. During spontaneous seizures evoked by the A₁R antagonist CPT, adenosine release was unaffected by IODO. This suggests that ADK activity does not limit activity-dependent adenosine release. On the basis of strong ADK immunoreactivity in GFAP-positive cells, astrocytes are likely to play a key role in regulating basal adenosine levels. It is this action of ADK on the basal adenosine tone that is permissive to seizure activity, and, by extension, other forms of activity-dependent neuronal activity such as synaptic plasticity.     

Dual actions of adenosine on rat peritoneal mast cells

Summary The effects of adenosine and its analogues on cAMP-responses and histamine release of rat peritoneal mast cells were investigated. The adenosine analogue 5-N-ethylcarboxamidoadenosine (NECA') activates the adenylate cyclase of the mast cell membranes and elevates the cAMP-levels of the intact mast cells. Both effects are antagonized by methylxanthines, suggesting that they are mediated via an A₂ adenosine receptor. Adenosine and its analogues enhance the release of histamine from these cells, when the release is stimulated either by the calcium ionophore A 23187 or by concanavalin A. However, this effect is not antagonized by theophylline or 8-phenyltheophylline. In contrast, it is antagonized by the adenosine uptake blockers S-(p-nitrobenzyl)-6-thioinosine (NBTI) and S-(p-nitrobenzyl)-6-thioguanosine (NBTG). It is concluded that adenosine has two different effects on mast cells: it activates adenylate cyclase via an A₂ adenosine receptor, and it enhances histamine release via an action at an intracellular site.Abbreviations NECA 5-N-ethylcarboxamidoadenosine - NBTI S-(p-nitrobenzyl)-6-thioinosine - NBTG S-(p-nitrobenzyl)-6-thioguanosine - PIA N⁶-phenylisopropyladenosine - Con A concanavalin A Send offprint requests to M. J. Lohse at the above address     

Opioid receptors on bone marrow neutrophils modulate chemotaxis and CD11b/CD18 expression

The effect of adenosine agents on amnesia induced by pentylenetetrazole was examined in mice. Post-training administration of pentylenetetrazole (50 and 60 mg/kg) disrupted 24-h retention of a single-trial passive avoidance task. The adenosine receptor antagonists, theophylline (2.5–25 mg/kg) and 8-phenyltheophylline (0.5–2 mg/kg), administered 30 min before and just after training at doses which did not affect retention, reduced the amnestic effect of pentylenetetrazole in a dose-dependent manner. Post-training administration of the adenosine A₁ receptor agonists, N⁶-cyclohexyladenosine (CHA, 0.1 and 0.5 mg/kg) and N⁶-phenylisopropyladenosine (R-PIA, 0.03 and 0.1 mg/kg), but not the adenosine A₂ receptor agonist, 5′-N-ethylcarboxamidoadenosine (NECA, 0.01 and 0.001 mg/kg), impaired retention. Nonamnestic doses of CHA and R-PIA potentiated the disruption induced by a lower dose of pentylenetetrazole (40 mg/kg). NECA did not induce any response in this respect. It is suggested that an adenosine A₁ receptor mechanism is involved in amnesia induced by pentylenetetrazole.