N-ethylcarboxamidoadenosine Research Articles

Adenosine receptor expression and function in rat striatal cholinergic interneurons.

Cholinergic neurons were identified in rat striatal slices by their size, membrane properties, sensitivity to the NK(1) receptor agonist (Sar(9), Met(O(2))(11)) Substance P, and expression of choline acetyltransferase mRNA. A(1) receptor mRNA was detected in 60% of the neurons analysed, and A(2A) receptor mRNA in 67% (n=15). The A(1) receptor agonist R-N(6)-(2-phenylisopropyl)adenosine (R-PIA) hyperpolarized cholinergic neurons in a concentration dependent manner sensitive to the A(1) antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 100 nM). In dual stimulus experiments, the A(2A) receptor antagonist 8-(3-chlorostyryl)caffeine (CSC, 500 nM) decreased release of [(3)H]-acetylcholine from striatal slices (S2/S1 0.78+/-0.07 versus 0.95+/-0.05 in control), as did adenosine deaminase (S2/S1 ratio 0.69+/-0.05), whereas the A(1) receptor antagonist DPCPX (100 nM) had no effect (S2/S1 1.05+/-0.14). In the presence of adenosine deaminase the adenosine A(2A) receptor agonist 2-p-((carboxyethyl)phenylethylamino)-5'-N-ethylcarboxamidoadeno sin e (CGS21680, 10 nM) increased release (S2/S1 ratio 1.03+/-0.05 versus 0.88+/-0.05 in control), an effect blocked by the antagonist CSC (500 nM, S2/S1 0.68+/-0.05, versus 0.73+/-0.08 with CSC alone). The combined superfusion of bicuculline (10 microM), saclofen (1 microM) and naloxone (10 microM) had no effect on the stimulation by CGS21680 (S2/S1 ratio 0.99+/-0.04). The A(1) receptor agonist R-PIA (100 nM) inhibited the release of [(3)H]-acetylcholine (S2/S1 ratio 0.70+/-0.03), an effect blocked by DPCPX (S2/S1 ratio 1.06+/-0.07). It is concluded that both A(1) and A(2A) receptors are expressed on striatal cholinergic neurons where they are functionally active.

Further characterization of an adenosine transport system in the mitochondrial fraction of rat testis

Previous work from our laboratory has demonstrated the presence of high-affinity binding sites for [ 3H]nitrobenzylthioinosine ([ 3H]NBTI), a marker of adenosine uptake systems, in the mitochondrial fraction of rat testis. Here, we characterize this system functionally through [ 3H]adenosine uptake assays. This system ( K m=2±1.3 μM; V max=86.2±15.5 pmol/mg protein/min) was found to be saturable, non sodium-dependent and sensitive to temperature, pH and osmolarity. [ 3H]Adenosine incorporation was potently inhibited by hydroxynitrobenzylthioguanosine (HNBTG, IC 50=3 nM) although NBTI inhibited this uptake weakly (IC 50=72.7±37.1 μM). Dilazep>dipyridamole≥hexobendine inhibited [ 3H]adenosine incorporation at low micromolar concentrations. The nucleosides inosine and uridine were weak inhibitors of this system. The adenosine receptor ligands N 6-phenylisopropyladenosine (PIA) and 2-chloroadenosine inhibited the uptake only at micromolar concentrations. Neither 5′-( N-ethylcarboxamido)-adenosine (NECA) nor theophylline inhibited adenosine uptake by more than 60% but the mitochodrial benzodiazepine receptor ligands 4′-chloro-diazepam (Ro 5-4864) and 1-(2-chlorophenyl)- N-methyl- N-(1-methyl-propyl) isoquinoline carboxamide (PK 11195) were able to inhibit it. The lack of inhibition by the blockers of the mitochondrial adenine-nucleotide carrier, atractyloside and α,β-methylene-ATP, indicates that [ 3H]adenosine uptake occurs via a transporter other than this carrier. All these results support the existence of an equilibrative adenosine transport system, which might mediate the passage of adenosine formed in the mitochondria to the cytoplasm.

Binding of [ 3H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-( m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes — a new, selective antagonist radioligand for A 2A adenosine receptors

The present study describes the preparation and binding properties of a new, potent, and selective A 2A adenosine receptor (AR) antagonist radioligand, [ 3H]3-(3-hydroxypropyl)-7-methyl-8-( m-methoxystyryl)-1-propargylxanthine ([ 3H]MSX-2). [ 3H]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [ 3H]MSX-2 labeled a single class of binding sites with high affinity ( K d =8.0 nM) and limited capacity ( B max =1.16 fmol·mg −1 of protein). The presence of 100 μM GTP, or 10 mM magnesium chloride, respectively, had no effect on [ 3H]MSX-2 binding. AR agonists competed with the binding of 1 nM [ 3H]MSX-2 with the following order of potency: 5′- N-ethylcarboxamidoadenosine (NECA)>2-[4-(carboxyethyl)phenylethylamino]-5′- N-ethylcarboxamidoadenosine (CGS-21680)>2-chloroadenosine (2-CADO)> N 6-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-( m-bromostyryl)-3,7-dimethyl-1-propargylxanthine (BS-DMPX)>1,3-dipropyl-8-cyclopentylxanthine (DPCPX)>( R)-5,6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7 H-pyrrolo[2,3- d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K i values for antagonists were in accordance with data from binding studies with the agonist radioligand [ 3H]CGS21680, while agonist affinities were 3–7-fold lower. [ 3H]MSX-2 is a highly selective A 2A AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20–30%, at 1 nM.

Involvement of mast cells, sensory afferents and sympathetic mechanisms in paw oedema induced by adenosine A 1 and A 2B/3 receptor agonists

Both the adenosine A 1 receptor agonist N 6-cyclopentyladenosine and the adenosine A 2B/3 receptor agonist N 6-benzyl-5′- N-ethylcarboxamido adenosine ( N 6-B-NECA) produce an acute paw oedema response following local s.c. injection into the rat hindpaw. This study characterized aspects of the mechanisms by which these responses occur by determining the effect of compound 48/80 (mast cell depleting agent), capsaicin (sensory neurotoxin) and 6-hydroxydopamine (sympathetic nervous system neurotoxin) on the paw oedema response produced by these agents. Compound 48/80 markedly reduced the increase in paw volume produced by both N 6-cyclopentyladenosine and N 6-B-NECA. Capsaicin significantly reduced paw oedema induced by N 6-cyclopentyladenosine but not N 6-B-NECA. In contrast, 6-hydroxydopamine reduced paw oedema induced by N 6-B-NECA but not N 6-cyclopentyladenosine. These results indicate an involvement of mast cells in paw oedema produced by both adenosine A 1 and A 2B/3 receptor agonists. For N 6-cyclopentyladenosine, this involvement may be a secondary involvement due to activation of a neurogenic mechanism, but for N 6-B-NECA, it may be a direct effect on mast cells. The nature of the involvement of the sympathetic nervous system in the action of N 6-B-NECA is not entirely clear.

Molecular identification and pharmacological characterization of adenosine receptors in the guinea-pig colon.

The aim of this study is to elucidate the role of adenosine in the motor function of the guinea-pig distal colon.2 To determine whether adenosine A(1) receptors and A(2B) receptors are expressed in the guinea-pig colon, we employed the reverse transcription-polymerase chain reaction (RT - PCR). The gene expression of A(1) receptor and A(2B) receptor was found for the first time in the guinea-pig proximal and distal colon.3 Adenosine A(1) agonist N(6)-cyclopentyladenosine (CPA), and A(1)/A(2) agonist 5'-N-ethylcarboxamidoadenosine (NECA) concentration-dependently inhibited neurogenic responses to electrical field stimulation (EC(50)=1.07x10(-8) and 2.12x10(-8) M) in the longitudinal muscle, but A(2A) agonist 2-p-(2-carboxyethyl)phenylethylamino-5'-N-ethycarboxamido-ad enosine (CGS21680) had only a slight inhibitory effect (25.9%, 1 microM). A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 10 nM: A(1) selective concentration) antagonized responses to CPA and NECA. Furthermore, the affinity order of antagonists at inhibiting the effect NECA was: DPCPX>8-phenyltheophylline (8-PT: A(1)/A(2) antagonist).3 In the presence of tetrodotoxin (TTX, 0.3 microM), CPA and NECA relaxed myogenic precontraction induced by KCl (50 mM) (EC(50)=1.26x10(-5) and 1.04x10(-5) M, respectively), but CGS21680 (1 microM) did not cause any relaxation. DPCPX did not affect responses to CPA and NECA at a concentration of 10 nM, but a higher concentration (1 microM) of DPCPX and 10 microM of 8-PT antagonized those responses.5 These data lead us to the hypothesis that adenosine may mediate relaxation through two different inhibitory receptor subtypes; A(1) receptors on the enteric neuron and A(2B) receptor on the smooth muscle in the guinea-pig distal colon.

Role of adenosine A(2B) receptors in vasodilation of rat pial artery and cerebral blood flow autoregulation.

This study was aimed to investigate the underlying mechanism of vasodilation induced by the activation of A(2B) adenosine receptors in relation to cerebral blood flow (CBF) autoregulation. Changes in pial arterial diameters were observed directly through a closed cranial window. N(omega)-nitro-L-arginine methyl ester (L-NAME, nitric oxide synthase inhibitor) significantly suppressed the concentration-dependent vasodilations induced by adenosine and 5'-N-ethylcarboxamido-adenosine (NECA) but not the vasodilation by CGS-21680 (A(2A)-receptor agonist). Moreover, NECA-induced vasodilation was suppressed by alloxazine (1 micromol/l) but not by ZM-241385 (1 micromol/l, A(2A) antagonist), which suggests mediation by A(2B)- receptor activation. Otherwise, the level of nitrite/nitrate was concentration dependently increased in the artificial cerebrospinal fluid (CSF) when adenosine and NECA were suffused over the cortical surface. L-NAME and alloxazine, but not ZM-241385, largely inhibited their releases. The lower limit of CBF autoregulation was little affected following pretreatment with L-NAME or alloxazine. Thus it is suggested that adenosine-induced vasodilation via activation of A(2B)-adenosine receptors of the rat pial artery is coupled to the production of nitric oxide, which contributes little to CBF autoregulation.

Sympathoinhibition by adenosine A 1 receptors, but not P2 receptors, in the hamster mesenteric arterial bed

The aim of the present study was to determine whether there are prejunctional inhibitory P2 purine receptors on sympathetic nerves in the hamster isolated perfused mesenteric arterial bed. Adenosine 5′- O-(3-thiotriphosphate (ATPγS; 10 μM), adenosine 5′- O-(2-thiodiphosphate) (ADPβS; 100 μM) and AMP (10 μM) had no significant effect on neurogenic contractions to electrical field stimulation. In contrast, P1 receptor agonists attenuated sympathetic vasoconstriction with a potency order of N 6-cyclopentyladenosine (CPA)>5′-( N-ethylcarboxamido)-adenosine (NECA)>adenosine. The pEC 50 value for CPA was 7.5±0.1 ( n=7). The concentration-inhibitory effect curve to CPA was shifted to the right by the adenosine A 1 receptor antagonist, 8-cyclopentyl-1,3-dipropyl-xanthine (DPCPX; 10 nM; apparent p K B 9.6; n=6–7). In methoxamine raised-tone mesenteries CPA (0.001–10 μM) did not elicit vasorelaxation, and NECA and adenosine were only weak vasorelaxants. These results indicate that adenosine A 1 receptors, but not P2 receptors, inhibit prejunctionally sympathetic neurotransmission in the hamster mesenteric arterial bed.

Ligand-activation of the adenosine A2a receptors inhibits IL-12 production by human monocytes.

Adenosine (ADO) exerts potent anti-inflammatory and immunosuppressive effects. In this paper we address the possibility that these effects are partly mediated by inhibition of the secretion of IL-12, a proinflammatory cytokine and a major inducer of Th1 responses. We demonstrate that 5'-N-ethylcarboxamidoadenosine (NECA), a nonspecific ADO analogue, and 2-p-(2-carbonyl-ethyl)phenylethylamino-5'-N-ethylcarboxamidoadenos ine (CGS-21680), a specific A2a receptor agonist, dose-dependently inhibited, in whole blood ex vivo and monocyte cultures, the production of human IL-12 induced by LPS and Stapholococcus aureus Cowan strain 1. However, the A1 receptor agonist 2-Chloro-N6-cyclopentyladenosine and the A3 receptor agonists N6-Benzyl-NECA and 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-be ta-d -ribofuranuronamide expressed only weak inhibitory effects. On the other hand, NECA and CGS-21680 dose-dependently potentiated the production of IL-10. The differential effect of these drugs on monocyte IL-12 and IL-10 production implies that these effects are mediated by A2a receptor signaling rather than by intracellular toxicity of ADO analogue's metabolites. Moreover, CGS-21680 inhibited IL-12 production independently of endogenous IL-10 induction, because anti-IL-10 Abs failed to prevent its effect. The selective A2a antagonist 8-(3-Chlorostyryl) caffeine prevented the inhibitory effect of CGS-21680 on IL-12 production. The phosphodiesterase inhibitor Ro 20-1724 dose-dependently potentiated the inhibitory effect of CGS-21680 and, furthermore, Rp-cAMPS, a protein kinase A inhibitor, reversed the inhibitory effect of CGS-21680, implicating a cAMP/protein kinase A pathway in its action. Thus, ligand activation of A2a receptors simultaneously inhibits IL-12 and stimulates IL-10 production by human monocytes. Through this mechanism, ADO released in excess during inflammatory and ischemic conditions, or tissue injury, may contribute to selective suppression of Th1 responses and cellular immunity.

Role of Adenosine A2 Receptors in Brain Stimulation Reward under Baseline Conditions and during Cocaine Withdrawal in Rats

The present experiments tested the hypothesis that adenosine A2 receptors are involved in central reward function. Adenosine receptor agonists or antagonists were administered to animals that had been trained to self-stimulate in a rate-free brain stimulation reward (BSR) task that provides current thresholds as a measure of reward. The adenosine A(2A) receptor-selective agonists 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido adenosine hydrochloride (CGS 21680) (0.1-1.0 mg/kg) and 2-[(2-aminoethylamino)carbonylethyl phenylethylamino]-5'-N-ethylcarboxamido adenosine (APEC) (0.003-0.03 mg/kg) elevated reward thresholds without increasing response latencies, a measure of performance. Specifically, CGS 21680 had no effect on response latency, whereas APEC shortened latencies. Bilateral infusion of CGS 21680 (3, 10, and 30 ng/side), directly into the nucleus accumbens, elevated thresholds but shortened latencies. The highly selective A(2A) antagonist 8-(3-chlorostyryl)caffeine (0.01-10.0 mg/kg) and the A2-preferring antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) (0.3-10.0 mg/kg) did not alter thresholds or latencies, but DMPX (1.0, 10.0 mg/kg) blocked the threshold-elevating effect of APEC (0.03 mg/kg). In another study, repeated administration of cocaine (eight cocaine injections of 15 mg/kg, i.p., administered over 9 hr) produced elevations in thresholds at 4, 8, and 12 hr after cocaine. DMPX (3 and 10 mg/kg), administered before both the 8 and 12 hr post-cocaine self-stimulation tests, reversed the threshold elevation produced by cocaine withdrawal. These results indicate that stimulating adenosine A(2A) receptors diminishes BSR without producing performance deficits, whereas blocking adenosine receptors reverses the reward impairment produced by cocaine withdrawal or by an A(2A) agonist. These findings indicate that adenosine, via A(2A) receptors, may inhibit central reward processes, particularly during the neuroadaptations associated with chronic drug-induced neuronal activation.

Acute paw oedema induced by local injection of adenosine A 1, A 2 and A 3 receptor agonists

The present study used plethysmometry to examine oedema following local injection of selective adenosine A 1, A 2 and A 3 receptor agonists and inhibitors of adenosine metabolism into the hindpaw of the rat. N 6-Cyclopentyladenosine and l- N 6-phenylisopropyladenosine (A 1), 2-[ p(2-carboxyethyl) phenethylamino]-5′- N-ethylcarboxamidoadenosine hydrochloride (CGS21680) (A 2A) and N 6-benzyl-5′- N-ethylcarboxamido adenosine ( N 6-B-NECA) (A 3) all produced an increase in paw volume ( N 6-B-NECA> N 6-cyclopentyladenosine, l- N 6-phenylisopropyladenosine>CGS21680). At the highest dose, each agent also produced a systemically mediated suppression of oedema. Oedema by N 6-cyclopentyladenosine was blocked by caffeine, 8-cyclopentyl-1,3-dimethylxanthine and enprofylline. Oedema by CGS21680 was blocked by caffeine and 8-cyclopentyl-1,3-dimethylxanthine. Oedema by N 6-B-NECA was blocked by enprofylline, but not by caffeine or 8-cyclopentyl-1,3-dimethylxanthine, or by systemic administration of MRS 1191. Oedema by both N 6-cyclopentyladenosine and N 6-B-NECA was blocked by mepyramine, ketanserin and phentolamine, but that by CGS21680 was not. The adenosine kinase inhibitor 5′-amino-5′-deoxyadenosine and the adenosine deaminase inhibitor 2′-deoxycoformycin produced only a limited increase in paw volume, and this was blocked by caffeine. This study demonstrates an acute paw oedema response following local administration of adenosine A 1, A 2 and A 3 receptor agonists, which likely results from different mechanisms of action in each case.

Afferent arteriolar adenosine A2a receptors are coupled to KATP in in vitro perfused hydronephrotic rat kidney.

Adenosine is known to exert dual actions on the afferent arteriole, eliciting vasoconstriction, by activating A1 receptors, and vasodilation at higher concentrations, by activating lower-affinity A2 receptors. We could demonstrate both of these known adenosine responses in the in vitro perfused hydronephrotic rat kidney. Thus, 1.0 microM adenosine elicited a transient vasoconstriction blocked by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and 10-30 microM adenosine reversed KCl-induced vasoconstriction. However, when we examined the effects of adenosine on pressure-induced afferent arteriolar vasoconstriction, we observed a third action. In this setting, a high-affinity adenosine vasodilatory response was observed at concentrations of 10-300 nM. This response was blocked by both 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3, 5]triazin-5-yl-amino]ethyl)phenol (ZM-241385) and glibenclamide and was mimicked by 2-phenylaminoadenosine (CV-1808) (IC50 of 100 nM), implicating adenosine A2a receptors coupled to ATP-sensitive K channels (KATP). Like adenosine, 5'-N-ethylcarboxamidoadenosine (NECA) elicited both glibenclamide-sensitive and glibenclamide-insensitive vasodilatory responses. The order of potency for the glibenclamide-sensitive component was NECA > adenosine = CV-1808. Our findings suggest that, in addition to the previously described adenosine A1 and low-affinity A2b receptors, the renal microvasculature is also capable of expressing high-affinity adenosine A2a receptors. This renal adenosine receptor elicits afferent arteriolar vasodilation at submicromolar adenosine levels by activating KATP.

Effect of A2A adenosine receptor stimulation and antagonism on synaptic depression induced by in vitro ischaemia in rat hippocampal slices.

1. In the present study we investigated the role of A2A adenosine receptors in hippocampal synaptic transmission under in vitro ischaemia-like conditions. 2. The effects of adenosine, of the selective A2A receptor agonist, CGS 21680 (2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoade nos ine ), and of selective A2A receptor antagonists, ZM 241385 (4-(2-[7-amino-2-(2-furyl)-¿1,2,4¿-triazolo¿2,3-a¿¿1,3, 5¿triazin-5-ylamino]ethyl)phenol) and SCH 58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2, 4-triazolo[1,5-c]pyrimidine), have been evaluated on the depression of field e.p.s.ps induced by an in vitro ischaemic episode. 3. The application of 2 min of in vitro ischaemia brought about a rapid and reversible depression of field e.p.s.ps, which was completely prevented in the presence of the A1 receptor antagonist DPCPX (1, 3-dipropyl-8-cyclopentylxanthine) (100 nM). On the other hand both A2A receptor antagonists, ZM 241385 and SCH 58261, by themselves did not modify the field e.p.s.ps depression induced by in vitro ischaemia. 4. A prolonged application of either adenosine (100 micronM) or CGS 21680 (30, 100 nM) before the in vitro ischaemic episode, significantly reduced the synaptic depression. These effects were antagonized in the presence of ZM 241385 (100 nM). 5. SCH 58261 (1 and 50 nM) did not antagonize the effect of 30 nM CGS 21680 on the ischaemia-induced depression. 6. These results indicate that in the CA1 area of the hippocampus the stimulation of A2A adenosine receptors attenuates the A1-mediated depression of synaptic transmission induced by in vitro ischaemia.

Potent antagonists for the human adenosine A2B receptor. Derivatives of the triazolotriazine adenosine receptor antagonist ZM241385 with high affinity

A series of novel and known 5-substituted 7-amino-2-(2-furyl)[1,2,4]triazolo[1,5-a][1,3,5]triazine derivatives were synthesized and tested for adenosine receptor antagonism in radioligand binding assays at all four adenosine receptor subtypes and for inhibition of the agonist-induced cyclic AMP response at human A2B receptors. The known potent adenosine A2A receptor antagonist, 7-amino-2-(2-furyl)-5-[2-(4-hydroxyphenyl)ethyl]amino[1,2,4]triazolo[1,5-a][1,3,5]triazine (ZM241385, KiA2A = 1.78 nM) had a Ki value of 16.5 nM at A2B receptors in radioligand binding studies on Chinese hamster ovary cells expressing A2B receptors. A pA2 value of 7.9 was measured for the inhibition of the cyclic AMP response by A2B receptors induced by 5′-N-ethylcarboxamidoadenosine (NECA). In a series of 5-phenyl(alkyl)amino analogs the 5-(2-phenylethyl)amino analog LUF5452 and the 5-benzylamino analog LUF5451 were both more potent than ZM241385 in the cyclic AMP assay at A2B receptors. Moreover, Ki values of 9.9 and 7.6 nM were found in binding studies at this receptor subtype, indicating that LUF5451 and LUF5452 are more potent A2B receptor antagonists than ZM241385. The affinity of LUF5451 for the A2A receptor (Ki value = 13 nM) showed that the selectivity for this receptor subtype was lost and that a modest A2B receptor selectivity was achieved. The 5-(2-phenylhydrazino) derivative LUF5475 showed a high A2B receptor affinity (Ki = 7.6 nM), while it was equally active at A2A receptors, being A2B receptor-selective with respect to A1 and A3 receptors. Drug Dev. Res. 48:95–103, 1999. © 1999 Wiley-Liss, Inc.

Adenosine–amino acid interactions in the chick brain: a role in passive avoidance learning

The present work describes interactions between adenosine and the amino acids glutamate and GABA in slices of intermediate medial hyperstriatum ventrale (IMHV), an area of the chick brain known to be involved in learning and memory events associated with a one-trial passive avoidance task. In slices derived from the IMHV of untrained chicks, the A 1 receptor agonist N 6-cyclohexyladenosine (CHA; 10 μM) specifically inhibited glutamate release. Conversely, cyclopentyltheophylline (CPT; 100 μM an A 1 antagonist) increased glutamate release from the slices and blocked the CHA-induced inhibition of glutamate. The A 2 receptor agonist 2- p-(2-carboxylethyl)-phenylamino-5′- N-ethylcarboxamido adenosine hydrochloride (CGS 21680) selectively increased glutamate release when applied at 5 μM while it selectively inhibited GABA release at a lower concentration (10 nM). The addition of NMDA to the medium, resulted in increased adenosine release equivalent to that found following stimulation with 50 mM KCl. Both the NMDA and the KCl-induced increases were eliminated by addition of d-2-amino-5 phosphopentanoic acid ( d-AP5), an NMDA-receptor antagonist. Slices prepared from the IMHV of chicks following successful training on the task showed enhanced adenosine release 30 min, 1, 3 and 6.5 h after training compared to chicks trained to peck a water-coated bead. The results show that changes in adenosine release from the IMHV accompany memory formation in the chick. We suggest that adenosine–amino acid transmitter interactions potentially via the activation of NMDA receptors, a necessary step in long-term memory formation for the task, may modulate the formation of memory for the one-trial passive avoidance task.

Adenosine receptor activation induces vascular endothelial growth factor in human retinal endothelial cells.

Adenosine, released in increased amounts by hypoxic tissues, is thought to be an angiogenic factor that links altered cellular metabolism caused by oxygen deprivation to compensatory angiogenesis. Adenosine interacts with 4 subtypes of G protein-coupled receptors, termed A(1), A(2A), A(2B), and A(3). We investigated whether adenosine causes proliferation of human retinal endothelial cells (HRECs) and synthesis of vascular endothelial growth factor (VEGF) and, if so, which adenosine receptor subtype mediates these effects. The nonselective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), in a concentration-dependent manner, increased both VEGF mRNA and protein expression by HRECs, as well as proliferation. This proliferative effect of NECA was inhibited by the addition of anti-human VEGF antibody. NECA also increased insulin-like growth factor-I and basic fibroblast growth factor mRNA expression in a time-dependent manner and cAMP accumulation in these cells. In contrast, neither the A(1) agonist N(6)-cyclopentyladenosine nor the A(2A) agonist 2-p-(2-carboxyethyl) phenethylamino-NECA caused any of the above effects of NECA. The effects of NECA were not significantly attenuated by either the A(2A) antagonist SCH58261 or the A(1) antagonist 8-cyclopentyl-1, 3-dipropylxanthine. However, the nonselective adenosine receptor antagonist xanthine amine congener completely inhibited the effects of NECA. Addition of antisense oligonucleotide complementary to A(2B) adenosine receptor mRNA inhibited VEGF protein production by HRECs after NECA stimulation. Thus, the A(2B) adenosine receptor subtype appears to mediate the actions of adenosine to increase growth factor production, cAMP content, and cell proliferation of HRECs. Adenosine activates the A(2B) adenosine receptor in HRECs, which may lead to neovascularization by a mechanism involving increased angiogenic growth factor expression.

Autoradiographic comparison of the potency of several structurally unrelated adenosine receptor antagonists at adenosine A 1 and A 2A receptors

We have examined the potency of several adenosine receptor antagonists at adenosine A 1 and A 2A receptors using quantitative autoradiography and have compared the results with those of previous studies using the same radioligands in membrane preparations. The agonists [ 3 H ]cyclohexyladenosine and [ 3 H ]2-[ p-(2-carbonylethyl)-phenylethylamino]-5′- N-ethylcarboxamido adenosine ([ 3 H ]CGS 21680) were used as radioligands for the two receptors. The results show that 1,3-dipropyl-8-cyclopentyl xanthine (DPCPX) is almost 1000-fold and 8-chloro-4-cyclohexyl-amino-1-(trifluoromethyl)[1,2,4]triazolo[4,3- a] quinoxaline (CP-68,247) about 300-fold more potent at adenosine A 1 receptors in cortex and striatum than at striatal adenosine A 2A receptors. Conversely, 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo-[4,3- e]-1,2,4-triazolo [1,5- c]pyrimidine (SCH 58261) is approximately 1000-fold and 4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3- a][1,3,5]triazin-5-yl amino]ethyl)phenol (ZM 241,385) about 400-fold more potent at adenosine A 2A than at A 1 receptors. Caffeine and its metabolites did not show any selectivity. Other studied antagonists were non-selective or showed a modest (20- to 40-fold) adenosine A 2A receptor selectivity. Thus, only a few of the antagonists show such high selectivity that it is not offset by differences in drug distribution and levels of receptor subtype expression.

Upregulation of lysyl oxidase in vascular smooth muscle cells by cAMP: role for adenosine receptor activation.

Lysyl oxidase (LO) is a key participant in the accumulation of insoluble fibers of elastin and collagen by virtue of its role in the initiation of the covalent cross-linkages between and within individual molecules comprising these fibers. In view of the essential role played by LO in the accumulation of the fibrotic components of occlusive arterial lesions in atherosclerosis, identification of the signaling molecules which can affect the expression of the LO gene in vascular smooth muscle is of considerable interest. In the present report, we describe evidence for the role of the second messenger, cAMP, in the modulation of the levels of LO in vascular smooth muscle cells. Elevated intracellular cAMP induces the transcription of the LO gene, as revealed by Northern blot analysis and nuclear run on assays. Transient transfection experiments performed with the wild-type LO promoter and with this promoter mutated at a consensus CREB site, located within the region -100 to -93 base pairs relative to the start of transcription, indicate that cAMP-induced transcriptional activation is partially due to the presence of this CREB site within the promoter. Activation of stimulatory adenosine receptors in vascular smooth muscle cells with 5'-N-ethylcarboxamido adenosine (NECA) increases cAMP, LO mRNA, and enzyme activity. These findings point to the importance of cAMP signaling, potentially initiated by a variety of physiological agents, in the upregulation of LO expression in vascular smooth muscle cells.

Effects of adenosine receptor agonists on induction of contractions to phenylephrine of guinea-pig aorta mediated via intra- or extracellular calcium

The vasorelaxant actions of adenosine and its analogue, 5′-(N-ethylcarboxamido)-adenosine (NECA), were investigated in guinea-pig isolated aortic rings by addition to the tissue prior to induction of a contraction by the α 1-adrenoceptor agonist phenylephrine (PE, 3 × 10 −6 M). The effect was calculated from the ratio (C2/C1) of the contraction to PE before (C1) and in the presence of adenosine or NECA (C2). This was compared with a control ratio obtained at the same time in which no vasorelaxant was present during C2. Experiments were performed in either “normal” or “Ca 2+-free” bathing medium. Both adenosine and NECA caused inhibition of contractions in “normal” and “Ca 2+-free” conditions, the latter indicating that the vasorelaxant action was due in part to inhibition of intracellular Ca 2+ mobilization. To determine whether inhibition of influx of extracellular Ca 2+ is a target for the vasorelaxation, contractions to PE were obtained in “normal” Ca 2+ and in the presence of ryanodine (10 −5 M), which prevents the release of intracellular Ca 2+. These contractions were inhibited by NECA indicating that stimulation of A 2-receptors by NECA interferes with the influx of Ca 2+ via the opening of receptor-operated Ca 2+ channels (ROCs). This study has demonstrated that cell surface A 2-receptor stimulation in the guinea-pig aorta inhibits phenylephrine-induced contractions by interfering with both the release of intracellular Ca 2+ and the influx of extracellular Ca 2+, presumably via ROCs.

2-Substituted N-ethylcarboxamidoadenosine derivatives as high-affinity agonists at human A3 adenosine receptors.

A number of 2-substituted 5'-N-ethylcarboxamidoadenosine (NECA) derivatives was investigated for their affinity and selectivity at human A3 adenosine receptors. The compounds were tested in radioligand competition studies and modulation of adenylyl cyclase activity on membranes from CHO cell lines stably transfected with the four human adenosine receptor subtypes. In binding studies the most potent compound, 2-(3-hydroxy-3-phenyl)propyn-1-yl-NECA (PHPNECA), exhibited a subnanomolar affinity for A3 adenosine receptors with a Ki value of 0.4 nM. As opposed to the limited A3 selectivity of PHPNECA, a 100-fold selectivity compared to both A1 and A2A receptors was found for 2-(2-phenyl)ethynyl-NECA (PENECA; Ki 6 nM). The EC50 values for activation of adenylyl cyclase via A2A adenosine receptors were in good agreement with the respective Ki values from binding experiments. In contrast, IC50 values for A1 and A3 receptor-mediated inhibition of adenylyl cyclase were shifted to higher values compared to the respective affinities determined in radioligand competition studies. Similar discrepancies between binding and functional data have been observed for the inhibitory A1 adenosine receptor in previous studies. Therefore, the same A3 selectivity of PENECA compared to A1 receptors was found in binding and adenylyl cyclase inhibition whereas the selectivity compared to A2A receptors that was detected in ligand binding was obscured in the functional assay. The series of compounds presented in this study identifies 2-substitution of the purine system as a promising target for the development of A3-selective high-affinity ligands.

Spermatogonia of rainbow trout: III. in vitro study of the proliferative response to extracellular ATP and adenosine

Unlike in higher vertebrates, in fish it is not known whether the nerve supply of the testis can influence testicular functions or not. In addition to neurotransmitters, nerve terminals may release ATP and adenosine in the extracellular medium. On the assumption that these molecules might be released by fibers innervating the teleost testis, it is possible that they participate in the control of testicular function and, maybe, in the control of spermatogonia (Go) proliferation. This study addresses this issue. We have investigated the ability for extracellular ATP and adenosine to influence the in vitro incorporation, either basal or GTH-, IGF-I- and suramin-stimulated, of 3H-thymidine (3H-Tdr) by trout Go. Mixed suspensions of somatic and germ cells prepared from testes, which were immature or spermatogenetic, were cultured usually for 4.5 days in the presence or not of the tested molecules; 3H-Tdr was added during the last day in culture. In our cell culture conditions, 25 to 250 microM adenosine, ATP, ADP, and AMP stimulated the 3H-Tdr incorporation by Go from prespermatogenetic testes and from testes starting spermatogenesis, in a dose-dependent way. The effect of these molecules decreased when the testes were more advanced in spermatogenesis and it became inhibiting when the testes were in mid-spermatogenesis. Five'-N-ethylcarboxamidoadenosine (NECA) was as potent as adenosine in stimulating or inhibiting 3H-Tdr incorporation, while R-N6-(2-phenylisopropyl)adenosine (R-PIA) always had a marked inhibiting effect. Adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS; 25-200 microM), a non-hydrolysable analogue of ATP, which had no effect on Go from prespermatogenetic testes (collected October-February) and from testes in advanced spermatogenesis, stimulated 3H-Tdr incorporation by Go from testes at the beginning of spermatogenesis very efficiently. The order of potency of the different ATP analogues was as follows: ATPgammaS > ATP congruent with alpha,beta-methylene-ATP > UTP > 2-methylthio-ATP. These data suggest that A2 adenosine receptors and P2 receptors would be present on unidentified testicular cells. The stimulating effect of adenosine/ATP was additive with that of either GTH-I or IGF-I or suramin when the cells were from testes at the beginning of spermatogenesis, but adenosine suppressed their effect when the cells were from testes in mid-spermatogenesis. In conclusion, our results suggest that in the trout extracellular adenosine and ATP are able to influence the in vitro proliferation of Go, and are potential candidates for mediating the possible influence of the nervous system on the induction, speeding up, then slowing down of spermatogenesis.