Presence Of DPCPX Research Articles

Adenosine modulates synaptic plasticity in hippocampal slices from aged rats

Adenosine is known to modulate synaptic plasticity in the hippocampus of young animals through activation of adenosine A 1 receptors. The objective of the present study is to investigate whether the modulatory role of adenosine on phenomena of synaptic plasticity is maintained or modified in the hippocampus of aged animals. We compared the effects of the selective adenosine A 1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 50 nM), on paired-pulse facilitation (PPF), long-term depression (LTD), long-term potentiation (LTP) and depotentiation elicited in hippocampal slices taken from young adult (5–6 weeks) and old (2 years old) male Wistar rats. DPCPX attenuated PPF both in young (1.64±0.05 vs. 1.76±0.05%, n=6) and in old rats (1.33±0.05 vs. 1.55±0.1%, n=6). LTD was only observed in the presence of DPCPX in both young (21.3±0.6%, n=4) and old rats (14.4±0.9%, n=6). LTP induced by high-frequency stimulation (HFS) was not significantly different in young and old animals, in the presence or in the absence of DPCPX. A larger depotentiation was observed in the presence of DPCPX in young rats (27.6±4.4% vs. 16.8±4.7%, n=7) as well as in old rats (41.3±5.1% vs. 16.1±2.7%, n=6). LTP induced by θ-burst stimulation was observed only in the presence of DPCPX (53.9±4.9%, n=5) in young rats, but could be obtained either in the control solution (81.8±17.9%, n=7) or in the presence of DPCPX (98.5±24.2%, n=7) in old rats. The modulatory role of endogenous adenosine on synaptic plasticity is generally maintained in aged animals. Drugs interfering with adenosine A 1 receptor effects could then be used in old animals to modify synaptic plasticity with relevant behavioural consequences.

Extracellular adenosine concentrations during in vitro ischaemia in rat hippocampal slices.

1. The application of an ischaemic insult in hippocampal slices results in the depression of synaptic transmission, mainly attributed to the activation of A1 adenosine receptors by adenosine released in the extracellular space. 2. To estimate the concentration of endogenous adenosine acting at the receptor level during an ischaemic episode, we recorded field e.p.s.ps (fe.p.s.ps) from hippocampal slices, and evaluated the ability of the selective A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), to reverse the fe.p.s.p. depression induced by in vitro ischaemia. A relationship between the IC50 of an antagonist and the endogenous concentration of a neurotransmitter has been used for pharmacological analysis. 3. The complete and reversible depression of fe.p.s.p. in the CA1 region induced by 5 min ischaemia was decreased in the presence of DPCPX (50-500 nM). 8-Phenyltheophylline (10 microM) abolished the depression of fe.p.s.ps during the ischaemic period, while a small (peak effect 12 +/- 4%) decrease in fe.p.s.ps was observed during the initial phase of reperfusion. 4. In the time-interval of maximal depression of fe.p.s.ps., IC50 and adenosine concentration changed as function of time with a good degree of correlation. The maximal value of adenosine concentration was 30 microM. 5. Our data provide an estimation of the adenosine concentration reached at the receptor level during an ischaemic episode, with a higher time discrimination (15 s) than that achieved with any biochemical approach. This estimation may be useful in order to establish appropriate concentrations of purinergic compounds to be tested for their pharmacological effects during an ischaemic episode.

A1 and A2 adenosine receptor modulation of contractility in the cauda epididymis of the guinea-pig.

1. The effects of adenosine receptor agonists upon phenylephrine-stimulated contractility and [3H]-cyclic adenosine monophosphate ([3H]-cyclic AMP) accumulation in the cauda epididymis of the guinea-pig were investigated. The alpha1-adrenoceptor agonist, phenylephrine elicited concentration dependent contractile responses from preparations of epididymis. In the absence or presence of the L-type Ca2+ channel blocker, nifedipine (10 microM) the non-selective adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA, 1 microM) shifted phenylephrine concentration-response curves to the left (4 and 5 fold respectively). Following the incubation of preparations with pertussis toxin (200 ng ml(-1) 24 h) NECA shifted phenylephrine concentration-response curves to the right (5.7+/-0.9 fold). 2. In the presence of phenylephrine (1 microM), NECA and the A1 adenosine receptor selective agonists, N6-cyclopentyladenosine (CPA) and (2S)-N6-[2-endo-norbornyl]adenosine ((S)-ENBA) elicited concentration-responses dependent contractions from preparations of epididymis (pEC50 values 8.18+/-0.19, 7.79+/-0.29 and 8.15+/-0.43 respectively). The A3 adenosine receptor agonists N6-iodobenzyl-5'-N-methylcarboxamido adenosine (IBMECA) and N6-2-(4-aminophenyl) ethyladenosine (APNEA) mimicked this effect (but only at concentrations greater than 10 microM). In the presence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 30 nM) CPA concentration-response curves were shifted, in parallel to the right (apparent pKB 8.75+/-0.88) and the maximal response to NECA was reduced. 3. In the presence of DPCPX (100 nM) the adenosine agonist NECA and the A2A adenosine receptor selective agonist, CGS 21680 (2-p-(2-carboxyethyl)-phenethylamino-N-ethylcarboxamido adenosine), but not CPA, inhibited phenylephrine (20 microM) stimulated contractions (pIC50 7.15+/-0.48). This effect of NECA was blocked by xanthine amine congener (XAC, 1 microM) and the A2A adenosine receptor-selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-++ +ylamino]ethyl)phenol (ZM 241385; 30 nM). 4. (S)-ENBA (in the absence and presence of ZM 241385, 100 nM), but not NECA or CPA inhibited the forskolin (30 microM)-stimulated accumulation of [3H]-cyclic AMP in preparations of the epididymis of the guinea-pig (by 17+6% of control). In the presence of DPCPX (100 nM) NECA and CGS 21680, but not (S)-ENBA, increased the accumulation of [3H]-cyclic AMP in preparations of epididymis (pEC50 values 5.35+/-0.35 and 6.42+/-0.40 respectively), the NECA-induced elevation of [3H]-cyclic AMP was antagonised by XAC (apparent pKB 6.88+/-0.88) and also by the A2A adenosine receptor antagonist, ZM 241385 (apparent pKB 8.60+/-0.76). 5. These studies are consistent with the action of stable adenosine analogues at post-junctional A1 and A2 adenosine receptors in the epididymis of the guinea-pig. A1 Adenosine receptors potentiate alpha1-adrenoceptor contractility, an effect blocked by pertussis toxin, but which may not be dependent upon an inhibition of adenylyl cyclase. The epididymis of the guinea-pig also contains A2 adenosine receptors, possibly of the A2A subtype, which both inhibit contractility and also stimulate adenylyl cyclase.

A role for adenosine in the regulation of long-term depression in the adult rat hippocampus in vitro

We describe how endogenous adenosine can prevent the induction of homosynaptic long-term depression (LTD) in the CA1 region of slices of adult rat hippocampus. Neither of two consecutive periods of prolonged low frequency stimulation (LFS; 1 Hz, 900 stimuli) of the Schaffer collateral-commissural fibres resulted in the induction of LTD in the CA1 region of hippocampal slices from adult (8–30 week) animals. However, in the presence of adenosine deaminase or the selective adenosine A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), LTD was induced by each of the first and second of two periods of LFS. The first period of LFS did not, but the second period of LFS did, induce LTD in the presence of DPCPX and the NMDA receptor antagonist, d-2-amino-5-phosphonopentanoate (AP5). The present results show that A1 receptor activation by endogenous adenosine can prevent the induction of LTD in the adult hippocampus.

Endogenous Adenosine Attenuates Long-term Depression and Depotentiation in the CA1 Region of the Rat Hippocampus

This study tested the hypothesis that endogenous adenosine, a neuromodulator which is known to modify long-term potentiation (LTP), might also affect other forms of long-lasting synaptic plasticity, namely long-term depression (LTD) and depotentiation, in the hippocampus. Long-term depression was induced by applying low-frequency stimulation (LFS; 1 Hz, 900 stimuli, test intensity) to the Schaffer collateral-commissural fibres in hippocampal slices taken from young (12–14-day old) animals. Depotentiation was induced by delivering LFS to a pathway in which LTP had previously been saturated. Under control conditions, LTD induced in two distinct pathways was similar. However, low-frequency stimulation, applied in either pathway in the presence of the selective adenosine A 1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 10 nM), resulted in LTD which was larger than in control conditions. In a similar way, while under control conditions depotentiation induced in two distinct pathways was similar, when LFS was applied in the presence of DPCPX (10 nM) facilitation of depotentiation was observed. These results suggest that endogenous adenosine, acting through adenosine A 1 receptors, is able to attenuate long-term depression and depotentiation in the hippocampus. © 1997 Elsevier Science Ltd. All rights reserved.

125I]4-Aminobenzyl-5′- N-methylcarboxamidoadenosine ([ 125I]AB-MECA) labels multiple adenosine receptor subtypes in rat brain

Adenosine modulates neuronal activity and neurotransmitter release through interaction with cell surface receptors. Four adenosine receptor subtypes, A 1, A 2A, A 2B, and A 3 receptors, have been cloned and characterized. The agonist ligand, [ 125I]AB-MECA ([ 125I]4-aminobenzyl-5′- N-methylcarboxamidoadenosine) has high affinity for recombinant A 1 and A 3 receptors [Olah et al., Mol. Pharmacol., 45 (1994) 978–982]. Rodent A 3 receptors are relatively insensitive to xanthines; inhibition of A 1 receptors with xanthines allows selective detection of A 3 receptors despite the lack of selectivity of the ligand. We studied whether [ 125I]AB-MECA is useful for localization and characterization of A 3 receptors in rat brain. The autoradiographic distribution of total [ 125I]AB-MECA (400 pM) binding closely resembled the pattern of A 1 receptor binding, with highest levels in cerebellum, hippocampus, and thalamus, and moderate levels in cortex and striatum. Drug competition studies confirmed that almost all [ 125I]AB-MECA binding could be attributed to labeling of A 1 receptors. Xanthine amine congener (1 μM) reduced specific [ 125I]AB-MECA binding by >95%, indicating that xanthine-resistant A 3 receptors represent a quantitatively minor subtype. Despite the use of a radioligand with high affinity and high specific activity, the low density of A 3 receptors in rat brain appears insufficient to allow localization, or even consistent detection, of this receptor subtype. In the presence of DPCPX (50 nM, to block A 1 receptors), residual [ 125I]AB-MECA binding to A 2A receptors was observed in the striatum. Thus, [ 125I]AB-MECA labels primarily A 1 and A 2A adenosine receptors in rat brain.

Adenosine A2A receptor stimulation increases release of acetylcholine from rat hippocampus but not striatum, and does not affect catecholamine release.

Rat striatal and hippocampal slices, preincubated with [3H] dopamine (DA) ¿or [3H] noradrenaline (NA)] and [14C] choline, were superfused continuously and stimulated electrically. 2-chloroadenosine (2-CADO 0.001-100 microM), a non-selective adenosine receptor agonist, produced a concentration-dependent inhibition of the electrically evoked DA and acetylcholine (ACh) release from the striatal slices and of the electrically evoked NA and ACh release from the hippocampal slices. 8-cyclopentyl-1,3-dipropylxanthine (DPCPX 3, 30 and 200 nM), a selective adenosine A1 receptor antagonist, caused a concentration-dependent, parallel, rightward shift of the 2-CADO concentration-response curve, consistent with competitive antagonism. The pA2 values ranged between 8.4 and 8.8. In the case of ACh release from the hippocampus, but in no other case, was there an increase in release of radioactivity at low concentrations of 2-CADO in the presence of DPCPX. The stimulation in the hippocampus could be blocked by a selective adenosine A2A receptor antagonist KF 17837. By itself KF 17837 (0.1-100 microM) had no effect on electrically evoked NA release from hippocampal slices, but decreased electrically evoked ACh release. This inhibition was counteracted by DPCPX (1 microM). These results show that, under the conditions used, DA release in the striatum, and NA release in the hippocampus, as well as ACh release from the striatum are regulated by adenosine A1 but not by adenosine A2A receptors. By contrast, ACh release from the hippocampus is tonically regulated both by adenosine A1 receptors, which inhibit release, and by adenosine A2A receptors which stimulate release.

Adenosine A 2 receptor activation facilitates 45Ca 2+ uptake by rat brain synaptosomes

Adenosine has been shown to increase the release of neurotransmitters by stimulation of adenosine A 2 receptors. This effect probably depends on Ca 2+ entry into presynaptic nerve terminals. In the present work the ability of the mixed adenosine A 1 A 2 agonist, 2-chloroadenosine, to stimulate Ca 2+ uptake into rat brain synaptosomes was investigated. 45Ca 2+ uptake was induced by 20 μM veratridine. In the absence of other drugs, 2-chloroadenosine (1 μM) decreased 45Ca 2+ uptake into synaptosomes. Blocking the adenosine A 1 receptor with 100 nM of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), 2-chloroadenosine (1 μM) increased rather than decreased the uptake of 45Ca 2+ into synaptosomes. The excitatory effect of 2-chloroadenosine observed in the presence of DPCPX was reversed by 200 nM of ω-agatoxin-IVA, a specific P-type Ca 2+ channel antagonist, but not by L-type (nifedipine, 100 nM to 1 μM ; methoxyverapamil 1–10 μM) or N-type (ω-conotoxin GVIA, 500 nM) Ca 2+ channel antagonists. The adenosine A 2A selective agonist, 2- p-(2-carboxyethyl)-phenethylamino-5′- N-ethyl-carboxamido-adenosine (CGS 21680), did not significantly modify Ca 2+ uptake induced by veratridine. In contrast, the selective adenosine A 2 receptor agonist, N 6-(2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl)-adenosine (DPMA), in concentrations ranging from 10 nM to 1 μM increased Ca 2+ uptake induced by veratridine. The selective adenosine A 2 receptor antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) at a concentration of 10 μM antagonized the stimulatory effect of DPMA (0.1 μM) on 45Ca 2+ uptake. In conclusion, activation of adenosine A 2 receptors increases Ca 2+ uptake by synaptosomes depolarized by veratridine, which could explain the increase of neurotransmitter release observed when A 2 receptors are activated.

Adenosine receptor modulation of sympathetic neurotransmission in rat isolated kidney

In the present study we set out to define, using discriminatory agonists and antagonists, the adenosine receptors modulating sympathetic neurotransmission in the rat kidney. Isolated kidneys from male Wistar rats were perfused with modified Krebs-Henseleit buffer solution at constant flow. The neuronal noradrenaline stores were labeled with 3 H-noradrenaline and the renal nerves stimulated electrically (2 Hz, 3 msec, 9 mA, during 20 sec at intervals of 6 min). 3 H overflow was taken as an index of 3 H-noradrenaline release. The A 1 receptor selective agonists N 6 -cyclopentyladenosine (CPA), N6-cyclohexyladenosine (CHA), and N 6 -[R(-)-1-phenyl-2-propyl] adenosine (R-PIA), and the mixed A 1 /A 2A receptor agonists 5'-N-ethylcarboxamidoadenosine (NECA) and 2-chloroadenosine (CADO) inhibited evoked 3 H outflow concentration-dependently. The selective A 2A receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680), at concentrations selective for A 2A receptors, failed to modify 3 H outflow, whereas at higher concentrations it induced inhibition. The rank order of potency of agonists, CPA > CHA = R-PIA > NECA > CADO >> CGS 21680, is typical for an interaction with the A 1 receptor. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), at concentrations selective for blockade of A 1 receptors, blocked concentration-dependently the inhibitory effects of CPA and NECA ; no evidence of an increase in outflow was seen with NECA in the presence of DPCPX. The selective A 2A receptor antagonist 9-chloro-2-(2-furanyl)[1,2,4]triazol[1,5-c] quinazoline-5-amine (CGS 15943) did not influence the agonist effects at concentrations interacting selectively with A 2A receptors but antagonized them concentration-dependently at higher, non-selective concentrations. Taken together, our data establish the presence of inhibitory adenosine A 1 receptors on the terminal sympathetic neurons of rat kidney. No evidence was obtained for the presence of functional A 2A receptors in this preparation.

Structure-activity relationship of a pyrimidine receptor in the rat isolated superior cervical ganglion.

1. The effects of pyrimidines and purines on the d.c. potential of the rat isolated superior cervical ganglion (SCG) have been examined by a grease-gap technique to determine the structure-activity requirements of the receptor activated by pyrimidines, i.e. a pyrimidinoceptor. 2. 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl (ZTP), the pyrimidines, cytidine 5'-triphosphate (CTP), uridine 5'-triphosphate (UTP) and thymidine 5'-triphosphate (TTP) and the purines, adenosine 5'-triphosphate (ATP; in the presence of an A1-purinoceptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (1 microM)), adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), guanosine 5'-triphosphate (GTP), inosine 5'-triphosphate (1TP) depolarized ganglia in a concentration-dependent manner. The relative order of ZTP and purine 5'-triphosphates in depolarizing ganglia was ZTP > or = ATP gamma S > > ATP > or = ITP = GTP, and for the pyrimidine 5'-triphosphates UTP > TTP > or = CTP. Depolarizations evoked by ATP gamma S were followed by concentration-dependent hyperpolarizations at 100 and 1000 microM. 3. At concentrations of between 0.1 microM and 1 mM, uridine 5'-diphosphate (UDP), uridine 5'-diphosphoglucose (UDPG) and uridine 5'-diphosphoglucuronic acid (UDPGA) evoked significant and concentration-dependent depolarizations, whereas uridine 5'-monophosphate (UMP), uridine and uracil were inactive or produced small (< 45 microV) depolarizations. The relative order of potency of uridine analogues in depolarizing ganglia was UDP > or = UTP > UDPG > UDPGA > > uracil > or = UMP = pseudouridine > or = uridine. At 3 and 10 mM, uridine produced concentration-dependent hyperpolarizations. Nikkomycin Z, a nucleoside resembling UTP (viz. the triphosphate chain at the 5'-position on the ribose moiety being replaced by a peptide), was inactive between 1 microM and 1 mM. Generally, a concentration of 10 mM was required before thymidine, 6-azathymine, 6-azauracil or 6-azauridine depolarized ganglia. 4. Suramin (300 microM), a P2-purinoceptor antagonist, significantly depressed depolarizations evoked by alpha, beta-methylene-ATP (alpha, beta-MeATP; 100 microM), ATP gamma S (100 microM), CTP (1 mM), GTP (1 mM), ZTP (30 microM) and ATP (300 microM) in the presence of DPCPX (1 microM). Suramin reversed a small depolarization evoked by UMP (1 mM) into a small hyperpolarization. In contrast depolarizations evoked by UDP, UTP, UDPG (all at 100 microM) and TTP (300 microM) were unaltered or enhanced by suramin. 5. It is concluded that the rat SCG contains distinct nucleotide receptors including a P2-purinoceptor (activated by alpha, beta-MeATP, ATP, GTP, ITP and ZTP) and a pyrimidinoceptor (activated by UTP, UDP, UDPG, UDPGA and TTP). The pyrimidinoceptor on rat SCG neurones had specific structure activity requirements with the di- and triphosphates of uridine being the most effective depolarizing agonists examined.

Endogenous adenosine reduces depression of cardiac function induced by β-adrenergic stimulation during low flow perfusion

High levels of norepinephrine in the heart are cardiotoxic resulting in contractile dysfunction and arrhythmic activity via beta-adrenoceptor mediated mechanisms. A low flow heart model perfused with physiological saline containing glucose and bubbled with an O2 gas mixture was used to determine whether adenosine, a nucleoside with antiadrenergic properties, could reduce the functional manifestations of catecholamine cardiotoxicity. Isolated rat hearts were treated with dipropylcyclopentylxanthine (DPCPX; 0.1 microM; A1 receptor antagonist) to block endogenous adenosine. In DPCPX-treated hearts stimulated with isoproterenol (ISO; 1 microM) during 45 min of low flow (0.5 ml/min) perfusion, the recovery of contractile function (ConF) at 15 min after the restoration of normal flow was 64% of control (before low flow) values as compared to 110% recovery of ConF in the absence of ISO. The incidence of arrhythmias observed upon restoration of control flow was increased by ISO when the action of endogenous adenosine was blocked with DPCPX. In the absence of DPCPX both the functional depression and arrhythmias induced by ISO were prevented in the presence of phenylisopropyladenosine (PIA; 1 microM; A1 receptor agonist). At 15 min after normal flow was restored. ConF in ISO-treated hearts with PIA was 53% greater than in the absence of PIA and presence of DPCPX. This enhancement of ConF by PIA was significantly reduced by DPCPX. By 30 min after flow restoration, these significant differences were absent. DPCPX reversed the PIA-induced reduction in arrhythmias observed upon restoration of normal flow. PIA and DPCPX alone in the absence of ISO, and ISO in the absence of PIA and DPCPX, did not result in altered ConF upon restoration of normal flow. These findings indicate that intense beta-adrenergic stimulation of the heart during low-flow perfusion in the absence of adenosine A1 receptor activity induces contractile depression and arrhythmicity subsequent to restoration of control perfusion. It is concluded that endogenous adenosine protects the heart against catecholamine toxicity via stimulation of adenosine A1 receptors.

Co-expression of the adenosine A2A receptor and the stimulatory G-protein alpha subunit in a stable cell line using a novel poly-cistronic expression vector

The effects of different adenosine agonists on cAMP levels in isolated adult guinea pig ventricular myocytes were investigated. 2-Chloro-N6-cyclopentyladenosine (CCPA), R-N6-phenylisopropyl-adenosine (R-PIA), S-N6-phenylisopropyladenosine (S-PIA) and 5′-N-ethylcarboxolamidoadenosine (NECA) reduced isoprenaline-stimulated cAMP levels. Both the nonselective adenosine antagonist 8-{4-[([{(2-aminoethyl)amino}carbonyl]methyl)oxy]-phenyl}-1,3-dipropylxanthine (‘xanthine amine congener’; XAC) and the A1-selective adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) blocked the inhibitory effect of NECA. Basal cAMP levels were not altered by NECA or the highly A2-selective 2-[p-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamidoadenosine (CGS 21680), either alone or in the presence of DPCPX or XAC. These results provide evidence against the existence of A2 receptors on the ventricular myocyte but support the presence of A1 receptors.

Evidence for P2-purinoceptor-mediated inhibition of noradrenaline release in rat brain cortex.

1. Some postganglionic sympathetic axons possess P2Y-like P2-purinoceptors which, when activated, decrease the release of noradrenaline. We examined the question of whether such receptors also occur at the noradrenergic axons in the rat brain cortex. Slices of the brain cortex were preincubated with [3H]-noradrenaline, then superfused with medium containing desipramine (1 microM) and stimulated electrically, in most experiments by trains of 4 pulses/100 Hz. 2. The selective adenosine A1-receptor agonist, N6-cyclopentyl-adenosine (CPA; 0.03-3 microM) as well as the non-subtype-selective agonist 5'-N-ethylcarboxamido-adenosine (NECA; 0.3-3 microM) reduced the evoked overflow of tritium, whereas the adenosine A2a-receptor agonist, 2-p-(2-carbonylethyl)-phenethylamino-5'-N-ethylcarboxamido-a denosine (CGS-21680; 0.003-30 microM) and the adenosine A3-receptor agonist N6-2-(4-aminophenyl)ethyl-adenosine (APNEA; 0.03-3 microM) caused no change. Of the nucleotides tested, ATP (30-300 microM), adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S; 30-300 microM), adenosine-5'-O-(2-thiodiphosphate) (ADP beta S; 30-300 microM), P1,P4-di(adenosine-5')-tetraphosphate (Ap4A; 30-300 microM) and the preferential P2Y-purinoceptor agonist, 2-methylthio-ATP (300 microM) decreased the evoked overflow of tritium. The P2X-purinoceptor agonist, alpha,beta-methylene-ATP (3-300 microM) caused no change. 3. The A1-selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 10 nM) attenuated the effects of the nucleosides CPA (apparent pKB value 9.8) and NECA as well as of the nucleotides ATP (apparent pKB 9.3), ATP gamma S (apparent pKB 9.2) and ADP beta S (apparent pKB 8.7). CGS-21680 and APNEA were ineffective also in the presence of DPCPX. The A2-selective antagonist 1,3-dipropyl-8-(3,4-dimethoxystyryl)-7-methylxanthine (KF-17837) reduced the effects of CPA, NECA and ATP gamma S only when given at a concentration of 300 nM but not at 1O nM.4. The P2-purinoceptor antagonists, suramin (300 micro M), reactive blue 2 (30 micro M) and cibacron blue 3GA(30 micro M) did not change the effect of CPA. Suramin and cibacron blue 3GA shifted the concentration response curve of ATP gamma S to the right (apparent pKB values 3.7 and 5.0, respectively). Reactive blue 2 also attenuated the effect of ATPyS, and cibacron blue 3GA attenuated the effect of ATP, but in these cases the agonist concentration-response curves were not shifted to the right. There was no antagonistic effect of suramin against ATP and ADP beta S.5. The results indicate that rat cerebrocortical noradrenergic axons possess, in addition to the knownadenosine Al-receptor, a separate purinoceptor for nucleotides (P2) which, in contrast to the Al-receptor,is blocked by suramin, reactive blue 2 and cibacron blue 3GA. Nucleotides such as ATP and ATP gamma S activate both receptors. Inconsistencies in antagonist effects against nucleotides are probably due to this activation of two receptors. The presynaptic P2-purinoceptor is P2Y-like, as it is in the peripheral sympathetic nervous system.

Adenosine receptor‐mediated modulation of acetylcholine release from rat striatal synaptosomes

1. The effects of A1 and A2a adenosine receptor agonists on the veratridine-evoked release of [3H]-acetylcholine ([3H]-ACh) from rat striatal synaptosomes was investigated by use of the A1-selective agonist, R-PIA and the 185 fold selective A2a agonist, CGS 21680. The effects of NECA, which is equipotent at both receptor subtypes, were also studied. 2. The evoked release of [3H]-ACh was significantly enhanced by the A2a agonist CGS 21680 but decreased by the A1 agonist, R-PIA. The effects of NECA were dependent on the concentration used, with high concentrations inhibiting and low concentrations enhancing the evoked release of [3H]-ACh. In the absence of any antagonists, the rank order of potency for these three drugs on increasing [3H]-ACh release was CGS 21680 > NECA > R-PIA. 3. The stimulatory effects of CGS 21680 and low NECA concentrations on evoked [3H]-ACh release were antagonized by the A2a receptor antagonists, CP66,713 (300 nM) and CGS 15943A (50 nM) whilst the inhibitory effects of R-PIA were reversed by the selective A1 antagonist, DPCPX (4 nM). In the presence of DPCPX, NECA greatly enhanced the evoked release of [3H]-ACh at all concentrations studied when, during such A1 receptor blockade, the rank order of potency was NECA >> CGS 21680 > R-PIA. 4. These results demonstrate that both A1 and A2a adenosine receptors modulate the veratridine-evoked release of [3H]-ACh from rat striatal synaptosomes.

Adenosine A1 receptors mediate inhibition of tachykinin release from perifused enteric nerve endings.

A perifused preparation of guinea pig myenteric nerve varicosities (synaptosomes) was used to determine the characteristics of evoked tachykinin release and the inhibition of such release by adenosine analogues. Release of substance P-like immunoreactivity (SP-LI) and neurokinin A-like immunoreactivity (NKA-LI) was evoked by elevated extracellular [K+] in a reversible and repeatable manner. This release was completely abolished in the absence of extracellular Ca2+. Perifusion in the presence of 5'-N-ethylcarboxamidoadenosine (NECA), a nonselective A1/A2 adenosine receptor agonist, decreased K(+)-evoked release of SP-LI and NKA-LI compared with that in the absence of the nucleoside. Similar decrements in peptide release were obtained with N6-cyclopentyl adenosine (CPA), a selective A1 agonist, and 2-[p-(2-carboxyethyl)]phenethylamino-5'-N-ethyl-carboxamidoadenosi ne (CGS 21680), a selective A2 agonist. Response to all nucleosides was graded. Potency order of adenosine analogues was CPA greater than NECA much greater than CGS 21680. Inhibition due to the nucleosides was diminished in the presence of the highly selective A1-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) while perifusion in the presence of DPCPX alone did not alter evoked release of either peptide. These findings provide direct measurements of inhibitory effects of adenine nucleosides on the release, from enteric nerve endings, of endogenous neuromediators SP and NKA. The findings also directly demonstrate the presence of functional adenosine receptors of the A1 subtype on enteric nerve endings coupled negatively to release of tachykinins. The presence of A2 receptors on enteric nerve endings is neither supported nor excluded.

Excitatory effects of adenosine 5′-triphosphate on rat locus coeruleus neurones

Pontine slices of the rat brain were used for extracellular recording of the frequency of spontaneous action potentials of locus coeruleus (LC) neurones. In the absence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), α,β-methyleneadenosine 5′-triphosphate (α,β-meATP; 0.3–30 μmol/1) and 2-methylthio ATP (0.3–100 μmol/1), but not ATP (1–100 μmol/1) increased the firing rate. In the presence of DPCPX 0.1 μmol/1, all three purinoceptor agonists were active, the potency order being α,β-meATP > 2-methylthio ATP = ATP. Preincubation of the slices with tetrodotoxin (TTX) 0.5 μmol/l decreased the spike discharge but did not alter the percent facilitatory effect of α,β-meATP 30 μmol/l. There was no desensitization to α,β-meATP 10 μmol/l on repeated or continuous application. Suramin 100 μmol/l selectively depressed the effect of α,β-meATP 30 μmol/l without interfering with the effect of equiactive concentrations (10–100 μmol/l) of glutamic acid. The concentration-response curve of α,β-meATP was shifted in a parallel manner to the right by suramin 10 μmol/l. While DPCPX 0.1 μmol/l facilitated firing, suramin 100 μmol/l did not change it. In conclusion, LC neurones may possess P 2-purinoceptors of an unidentified type, which share some P 2x characteristics.

Expression and pharmacological characterization of a stimulatory subtype of adenosine receptor in fetal chick ventricular myocytes.

Ventricular and atrial myocytes cultured from chick embryos 14 days in ovo were used as model systems to study cardiac adenosine receptors. In membranes of ventricular cultures, blocking of the A1-adenosine receptor pathway by the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or by pertussis toxin treatment of the myocyte resulted in a significant adenosine agonist-mediated stimulation of the adenylate cyclase activity. The maximal increases in adenylate cyclase activity caused by the equipotent or the A2-adenosine receptor-selective agonists (from 52.1 +/- 3% to 63 +/- 10% [mean +/- SEM]) were significantly greater than those caused by the A1-selective agonists (from 11 +/- 5% to 34.6 +/- 7%) (p less than 0.01, by t test, n = 4-8). However, in membranes of atrial myocytes, when A1-subtype had been blocked, the various adenosine agonists had no effect on the adenylate cyclase activity. Whether the stimulatory adenylate cyclase-coupled adenosine receptor is also capable of stimulating contractility in the intact ventricular myocyte was next investigated. In ventricular but not in atrial cells, the various adenosine agonists caused an increase in the contractile amplitude in the presence of DPCPX or in myocytes preexposed to pertussis toxin. The increase in contraction amplitude caused by each agonist was expressed as percent of maximum (maximum is the increase in contractility caused by 2.4 mM calcium). In the pertussis toxin-treated myocyte, the maximal increases caused by the equipotent or A2-agonists (NECA, MECA, CV-1808, and CGS21680, from 49.6 +/- 3% to 52.5 +/- 6%, n = 8-12) were significantly greater than those elicited by the A1-agonists (2-CADO, S-PIA, R-PIA, and DCCA, from 12 +/- 4% to 37 +/- 3%, n = 8) (p less than 0.05, by t test). These data demonstrated that a stimulatory adenosine receptor, likely the A2-adenosine receptor, was present on the ventricular but not the atrial myocytes and was linked directly to a stimulation of the cardiac contractility. The functional effects mediated by the A1-subtype became manifested in the presence of isoproterenol, as evidence by an inhibition of the isoproterenol-stimulated increases in adenylate cyclase activity and in cardiac contractility by adenosine agonists. Thus, both subtypes of adenosine receptors, each mediating opposing responses, were present on the ventricular myocytes, whereas only the A1-subtype was found in the atria. The presence of a stimulatory functional A2-adenosine receptor may help explain the absence of a direct negative inotropic response to adenosine in the ventricle.

Inhibitory and excitatory effects of adenosine receptor agonists on evoked transmitter release from phrenic nerve endings of the rat

1. The effects of the adenosine analogues, 5'-N-ethyl-carboxamide adenosine (NECA), R-N6-phenylisopropyladenosine (R-PIA), 2-chloroadenosine (CADO), and CGS 21680C on electrically evoked tritium outflow from preparations loaded with [3H]-choline and on evoked endplate potentials (e.p.ps), as well as the ability of the xanthines, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) and PD 115,199 to antagonize the effects of the adenosine analogues, were investigated in phrenic nerve-diaphragm preparations. 2. NECA, R-PIA and CADO decreased, in a concentration-dependent manner, the evoked tritium outflow from preparations loaded with [3H]-choline. NECA and R-PIA were about equipotent and more potent than CADO. 3. DPCPX shifted to the right in a near parallel fashion the concentration-response curve for the inhibitory effect of R-PIA on evoked tritium outflow. 4. In the presence of DPCPX, NECA increased, rather than decreased, evoked tritium outflow. PD 115,119 antagonized, in a concentration-dependent manner, this excitatory effect of NECA. 5. CGS 21680C, in low nanomolar concentrations, increased evoked tritium outflow, an effect also antagonized by PD 115,119. 6. CGS 21680C increased, and R-PIA decreased, the amplitude of e.p.ps recorded from preparations paralysed with tubocurarine. Both effects could be observed in the same endplate. 7. It is concluded that both inhibitory (probably A1) and excitatory (probably A2) adenosine receptors coexist at the rat neuromuscular junction, modulating the evoked release of acetylcholine.

Evidence against the presence of A 2 adenosine receptors on guinea pig ventricular myocytes

The effects of different adenosine agonists on cAMP levels in isolated adult guinea pig ventricular myocytes were investigated. 2-Chloro-N 6-cyclopentyladenosine (CCPA), R-N 6-phenylisopropyl-adenosine (R-PIA), S-N 6-phenylisopropyladenosine (S-PIA) and 5′-N-ethylcarboxolamidoadenosine (NECA) reduced isoprenaline-stimulated cAMP levels. Both the nonselective adenosine antagonist 8-{4-[([{(2-aminoethyl)amino}carbonyl]methyl)oxy]-phenyl}-1,3-dipropylxanthine (‘xanthine amine congener’; XAC) and the A 1-selective adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) blocked the inhibitory effect of NECA. Basal cAMP levels were not altered by NECA or the highly A 2-selective 2-[p-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamidoadenosine (CGS 21680), either alone or in the presence of DPCPX or XAC. These results provide evidence against the existence of A 2 receptors on the ventricular myocyte but support the presence of A 1 receptors.

Adenosine receptor coupling to adenylate cyclase of rat ventricular myocyte membranes

The effects of adenosine analogues on beta-adrenergic receptor and receptor-independent elicited increases in adenylate cyclase activity were investigated using membranes obtained from primary cultures of adult rat ventricular myocytes. Phenylisopropyladenosine, an A1-receptor agonist, at concentrations of 0.1, 1.0, and 10 microM, maximally inhibited isoproterenol-stimulated adenylate cyclase activity by 35, 55, and 41%, respectively. The inhibition by phenylisopropyladenosine was antagonized by 10 microM theophylline. One micromolar phenylisopropyladenosine was much less effective at attenuating forskolin-stimulated activity, such that the maximum inhibition was 26%. Phenylisopropyladenosine had no effect on adenylate cyclase stimulation by 5'-guanylylimidodiphosphate. Phenylaminoadenosine, an A2 agonist, at 10 microM or greater stimulated adenylate cyclase activity. This effect was not significantly inhibited by theophylline or 0.1 microM 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), which antagonized phenylisopropyladenosine inhibition of isoproterenol-stimulated adenylate cyclase activity. Additionally, N-ethylcarboxamidoadenosine, a nonselective adenosine-receptor agonist, had no effect on adenylate cyclase activity in the absence of DPCPX but stimulated adenylate cyclase activity in the presence of DPCPX. These results indicate that both A1 and A2 receptors exist on the ventricular myocyte sarcolemma. More importantly, the findings suggest that adenosine inhibition of catecholamine-stimulated adenylate cyclase activity is primarily due to an alteration in beta-adrenergic receptor-mediated transduction and perhaps in part by a direct inhibition of the catalytic component.