Beneficial effects of a novel agonist of the adenosine A2A receptor on monocrotaline-induced pulmonary hypertension in rats

Background and Purpose

Pulmonary arterial hypertension (PAH) is characterized by enhanced pulmonary vascular resistance, right ventricular hypertrophy and increased right ventricular systolic pressure. Here, we investigated the effects of a N-acylhydrazone derivative, 3,4-dimethoxyphenyl-N-methyl-benzoylhydrazide (LASSBio-1359), on monocrotaline (MCT)-induced pulmonary hypertension in rats.

Experimental Approach

PAH was induced in male Wistar rats by a single i.p. injection of MCT (60 mg·kg⁻¹) and 2 weeks later, oral LASSBio-1359 (50 mg·kg⁻¹) or vehicle was given once daily for 14 days. Echocardiography was used to measure cardiac function and pulmonary artery dimensions, with histological assay of vascular collagen. Studies of binding to human recombinant adenosine receptors (A₁, A_2A, A₃) and of docking with A_2A receptors were also performed.

Key Results

MCT administration induced changes in vascular and ventricular structure and function, characteristic of PAH. These changes were reversed by treatment with LASSBio-1359. MCT also induced endothelial dysfunction in pulmonary artery, as measured by diminished relaxation of pre-contracted arterial rings, and this dysfunction was reversed by LASSBio-1359. In pulmonary artery rings from normal Wistar rats, LASSBio-1359 induced relaxation, which was decreased by the adenosine A_2A receptor antagonist, ZM 241385. In adenosine receptor binding studies, LASSBio-1359 showed most affinity for the A_2A receptor and in the docking analyses, binding modes of LASSBio-1359 and the A_2A receptor agonist, {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680, were very similar.

Conclusion and Implications

In rats with MCT-induced PAH, structural and functional changes in heart and pulmonary artery were reversed by treatment with oral LASSBio-1359, most probably through the activation of adenosine A_2A receptors.     

Autoradiographic evidence for G-protein coupled A2-receptors in rat neostriatum using [3H]-CGS 21680 as a ligand

Summary Recently [³H]-CGS 21680 (2-[p-(2-carbonylethyl)-phenylethylamino]-5-N-ethylcarboxamidoadeno-sine) has been identified as a selective adenosine A₂-receptor agonist. In this study the binding of [³H]-CGS 21680 to 10 m sections of rat neostriatum was investigated with quantitative autoradiography. Specific, saturable binding was detectable, and Scatchard analysis of saturation experiments gave estimates for K _D and B _max of 1.7 nM and 322 fmol/mg protein, respectively. The rank order of potency for inhibition of [³H]-CGS 21680 binding was 5-N-ethylcarboxamidoadenosine (1.9 nM) > 2-chloroadenosine (18 nM) > R-N⁶-phenylisoprop-yladenosine (59 nM) > S-N⁶-phenylisoprophyladeno sine (460 nM) > 1,3-dipropyl-8-cyclopentylxanthine (700 nM). The binding of [³H]-CGS 21680 was sensitive to GTP, since 1 M GTP reduced binding to 4.7% of control. These data support the identity of CGS 21680 as an agonist at high affinity adenosine A₂-receptors and indicate these receptors in rat striatum are coupled to guanine nucleotide binding proteins. Send offprint requests to F. E. Parkinson at the above address     

Adenosine A2 receptor regulation of apomorphine-induced turning in rats with unilateral striatal dopamine denervation

The ipsilateral intrastriatal administration of the specific adenosine A_2a receptor agonist, 2-[p-(2-carboxyethyl)phenethylamino]-5-N-ethylcarboxamido adenosine (CGS 21680), produced a dose related decrease in apomorphine-induced rotation in the unilaterally 6-hydroxydopamine-lesioned rat. This effect could be reversed by intrastriatal infusions of the A_2a antagonist, 4-amino-l-phenyl[1,2,4]triazolo[4,3-a]quinoxaline (CP 66,713). However, CP 66,713 had no significant effect when infused alone, neither did it influence the response to apomorphine in the absence of CGS 21680. The possible behavioural interactions between A_2a receptors and striatal ACh activity were also investigated using this model. Atropine administered intrastriatally in a dose that had no effect on the response to apomorphine reduced the inhibitory effects of CGS 21680 on apomorphine-induced turning. Naloxone also reduced the effects of apomorphine, an effect which could be prevented by the co-administration of atropine, or CP 66,713. These results indicate that adenosine agonists can modulate apomorphine-induced turning by an interaction with both cholinergic and opioidergic mechanisms in the striatum.     

Functional interaction between pre-synaptic α6β2-containing nicotinic and adenosine A2A receptors in the control of dopamine release in the rat striatum

Background and Purpose

Pre-synaptic nicotinic ACh receptors (nAChRs) and adenosine A_2A receptors (A_2ARs) are involved in the control of dopamine release and are putative therapeutic targets in Parkinson''s disease and addiction. Since A_2ARs have been reported to interact with nAChRs, here we aimed at mapping the possible functional interaction between A_2ARs and nAChRs in rat striatal dopaminergic terminals.

Experimental Approach

We pharmacologically characterized the release of dopamine and defined the localization of nAChR subunits in rat striatal nerve terminals in vitro and carried out locomotor behavioural sensitization in rats in vivo.

Key Results

In striatal nerve terminals, the selective A_2AR agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 inhibited, while the A_2AR antagonist ZM241385 potentiated the nicotine-stimulated [³H]dopamine ([³H]DA) release. Upon blockade of the α6 subunit-containing nAChRs, the remaining nicotine-stimulated [³H]DA release was no longer modulated by A_2AR ligands. In the locomotor sensitization experiments, nicotine enhanced the locomotor activity on day 7 of repeated nicotine injection, an effect that no longer persisted after 1 week of drug withdrawal. Notably, ZM241385-injected rats developed locomotor sensitization to nicotine already on day 2, which remained persistent upon nicotine withdrawal.

Conclusions and Implications

These results provide the first evidence for a functional interaction between nicotinic and adenosine A_2AR in striatal dopaminergic terminals, with likely therapeutic consequences for smoking, Parkinson''s disease and other dopaminergic disorders.     

Effects of adenosine derivatives on human and rabbit platelet aggregation

Summary The inhibitory effects of several adenosine analogues, including the new A2-selective agonists 2-[p-(2-carboxyethyl)phenethylamino]-5-N-ethylcarboxamido-adenosine (CGS 21680) and 2-hexynyl-5-N-ethylcarbox-amidoadenosine (2-hexynyl-NECA), were investigated in vitro on human and rabbit platelet aggregation. The compounds examined inhibited ADP-induced platelet aggregation over a wide range of potency. The rank order of activity was similar between the two species thus showing that the rabbit is a useful animal model for studying the effects of adenosine derivatives on platelet aggregation. 2-Hexynyl-NECA was found to be the most potent adenosine compound of those currently available, having IC₅₀ values of 0.10 and 0.07 M in human and rabbit platelets, respectively. Conversely, the A1 agonists R(–)-N-⁶-(2-phenylisopropyl) adenosine (R-PIA), S(+)-N⁶-(2phenylisopropyl) adenosine (S-PIA) and 2-chloro-N⁶-cyclopentyl-adenosine (CCPA) were the least potent compounds with IC₅₀ values in the micromolar range. The potency of the compounds in inhibiting platelet aggregation was found to be highly correlated with their affinity for A2 receptors as measured using 3H-CGS 21680 binding in rat brain striatum.Correspondence to S. Dionisotti at the above address     

[3H]-SCH 58261 labelling of functional A2A adenosine receptors in human neutrophil membranes

1. The present study describes the direct labelling of A_2A adenosine receptors in human neutrophil membranes with the potent and selective antagonist radioligand, [³H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4 triazolo[1,5-c]pyrimidine, ([³H]-SCH 58261). In addition, both receptor affinity and potency of a number of adenosine receptor agonists and antagonists were determined in binding, adenylyl cyclase and superoxide anion production assays.
2. Saturation experiments revealed a single class of binding sites with K_d and B_max values of 1.34 nM and 75 fmol mg⁻¹ protein, respectively. Adenosine receptor ligands competed for the binding of 1 nM [³H]-SCH 58261 to human neutrophil membranes, with a rank order of potency consistent with that typically found for interactions with the A_2A adenosine receptors. In the adenylyl cyclase and in the superoxide anion production assays the same compounds exhibited a rank order of potency identical to that observed in binding experiments.
3. Thermodynamic data indicated that [³H]-SCH 58261 binding to human neutrophils is entropy and enthalpy-driven. This finding is in agreement with the thermodynamic behaviour of antagonists binding to rat striatal A_2A adenosine receptors.
4. It was concluded that in human neutrophil membranes, [³H]-SCH 58261 directly labels binding sites with pharmacological properties similar to those of A_2A adenosine receptors of other tissues. The receptors labelled by [³H]-SCH 58261 mediated the effects of adenosine and adenosine receptor agonists to stimulate cyclic AMP accumulation and inhibition of superoxide anion production in human neutrophils.

     

Chronic haloperidol treatment leads to an increase in the intramembrane interaction between adenosine A2 and dopamine D2 receptors in the neostriatum

Stimulation of adenosine A₂ receptors (with the selective adenosine A₂ agonist CGS 21680) in rat striatal membrane preparations, produces a decrease in both the affinity of D₂ receptors and the transduction of the signal from the D₂ receptor to the G protein. This intramembrane A₂-D₂ interaction might be responsible for the behavioural depressant effects of adenosine agonists and for the behavioural stimulant effects of adenosine antagonists such as caffeine and theophylline. Dopamine denervation induces an increase in the intramembrane A₂-D₂ interaction, which may underlie the observed higher sensitivity to the behavioural effects induced by adenosine antagonists found in these animals. The present study was designed to examine if chronic treatment with haloperidol, which also produces dopamine receptor supersensitivity, is also associated with an increase in the intramembrane A₂-D₂ interaction in the neostriatum and with a higher sensitivity to the behavioural effects induced by adenosine antagonists. The data showed that: (i) haloperidol pretreatment causes a higher binding of the D₂ antagonist [³H] raclopride in striatal membrane preparations due to an increase in the number of D₂ receptors without changes in their affinity for the antagonist (increase in B_max without changes in k_d); (ii) GCS 21680 decreases the affinity of dopamine for the D₂ receptor, by increasing the equilibrium dissociation constants of high (K_h) and low affinity (K₁) dopamine D₂ binding sites and increases the proportion of high affinity binding sites (R_h); (iii) a low dose of CGS 21680 (3 nM), which is ineffective in membrane preparations from neostriatum of nontreated animals, is effective in membranes from the striatum of haloperidol-pretreated animals; (iv) the nonselective adenosine antagonist theophylline (20 mg/kg SC) causes a higher motor activation in rats pretreated with haloperidol. The possible relevance of these results for the pathophysiology and treatment of tardive dyskinesias is discussed.     

Characterization of the K+-channel-coupled adenosine receptor in guinea pig atria

Summary In the present work we studied the pharmacological profile of adenosine receptors in guinea pig atria by investigating the effect of different adenosine analogues on⁸⁶Rb⁺-efflux from isolated left atria and on binding of the antagonist radioligand 8-cyclopentyl-1,3-[³H]dipropylxanthine ([³H]DPCPX) to atrial membrane preparations. The rate of⁸⁶Rb⁺-efflux was increased twofold by the maximally effective concentrations of adenosine receptor agonists. The EC₅₀-values for 2-chloro-N⁶-cyclopentyladenosine (CCPA), R-N⁶-phenylisopropyladenosine (R-PIA), 5-N-ethylcarboxamidoadenosine (NECA), and S-N⁶-phenylisopropyladenosine (S-PIA) were 0.10, 0.14, 0.24 and 12.9 M, respectively. DPCPX shifted the R-PIA concentration-response curve to the right in a concentration-dependent manner with a K_B-value of 8.1 nM, indicating competitive antagonism. [³H]DPCPX showed a saturable binding to atrial membranes with a B_max-value of 227 fmol/mg protein and a K_D-value of 1.3 nM. Competition experiments showed a similar potency for the three agonists CCPA, R-PIA and NECA. S-PIA is 200 times less potent than R-PIA. Our results suggest that the K⁺ channel-coupled adenosine receptor in guinea pig atria is of an A₁ subtype.Abbreviations CCPA 2-chloro-N⁶-cyclopentyladenosine - DPCPX 8-cyclopentyl-1,3-dipropylxanthine - NECA 5-N-ethylcarboxami-doadenosine - PIA N⁶-phenylisopropyladenosine Send offprint requests to H. Tawfik-Schlieper at the above address     

Adenosine negatively regulates duodenal motility in mice: role of A(1) and A(2A) receptors

BACKGROUND AND PURPOSE

Adenosine is considered to be an important modulator of intestinal motility. This study was undertaken to investigate the role of adenosine in the modulation of contractility in the mouse duodenum and to characterize the adenosine receptor subtypes involved.

EXPERIMENTAL APPROACH

RT-PCR was used to investigate the expression of mRNA encoding for A₁, A_2A, A_2B and A₃ receptors. Contractile activity was examined in vitro as changes in isometric tension.

KEY RESULTS

In mouse duodenum, all four classes of adenosine receptors were expressed, with the A_2B receptor subtype being confined to the mucosal layer. Adenosine caused relaxation of mouse longitudinal duodenal muscle; this was antagonized by the A₁ receptor antagonist and mimicked by N⁶-cyclopentyladenosine (CPA), selective A₁ agonist. The relaxation induced by A₁ receptor activation was insensitive to tetrodotoxin (TTX) or N^ω-nitro-l-arginine methyl ester (l-NAME). Adenosine also inhibited cholinergic contractions evoked by neural stimulation, effect reversed by the A₁ receptor antagonist, but not myogenic contractions induced by carbachol. CPA and 2-p-(2-carboxyethyl) phenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride hydrate (CGS-21680), A_2A receptor agonist, both inhibited the nerve-evoked cholinergic contractions. l-NAME prevented only the CGS-21680-induced effects. S-(4-Nitrobenzyl)-6-thioinosine, a nucleoside uptake inhibitor, reduced the amplitude of nerve-evoked cholinergic contractions, an effect reversed by an A_2A receptor antagonist or l-NAME.

CONCLUSIONS AND IMPLICATIONS

Adenosine can negatively regulate mouse duodenal motility either by activating A₁ inhibitory receptors located post-junctionally or controlling neurotransmitter release via A₁ or A_2A receptors. Both receptors are available for pharmacological recruitment, even if only A_2A receptors appear to be preferentially stimulated by endogenous adenosine.

LINKED ARTICLE

This article is commented on by Antonioli et al., pp. 1577–1579 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01529.x     

Characterization of A2A adenosine receptors in human lymphocyte membranes by [3H]-SCH 58261 binding

1. The present study describes for the first time the characterization of the adenosine A_2A receptor in human lymphocyte membranes with the new potent and selective antagonist radioligand, [³H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4 triazolo [1,5-c] pyrimidine, ([³H]-SCH 58261). In addition, both receptor affinity and potency of reference adenosine receptor agonists and antagonists were determined in binding and adenylyl cyclase studies.
2. Saturation experiments revealed a single class of binding sites with K_d and B_max values of 0.85 nM and 35 fmol mg⁻¹ protein, respectively. A series of adenosine receptor ligands were found to compete for the binding of 0.8 nM [³H]-SCH 58261 to human lymphocyte membranes with a rank order of potency consistent with that typically found for interactions with the A_2A-adenosine receptor. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency similar to that observed in binding experiments.
3. Thermodynamic data indicate that [³H]-SCH 58261 binding to human lymphocytes is entropy and enthalpy-driven, a finding in agreement with the thermodynamic behaviour of antagonists for rat striatal A_2A-adenosine receptors.
4. It is concluded that in human lymphocyte membranes [³H]-SCH 58261 directly labels binding sites showing the characteristic properties of the adenosine A_2A-receptor. The presence of A_2A-receptors in peripheral tissue such as human lymphocytes strongly suggests an important role for adenosine in modulating immune and inflammatory responses.

     

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 α₁-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 μM) the non-selective adenosine receptor agonist, 5′-N-ethylcarboxamido-adenosine (NECA, 1 μM) 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 μM), 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-methyl-carboxamido adenosine (IBMECA) and N⁶-2-(4-aminophenyl) ethyladenosine (APNEA) mimicked this effect (but only at concentrations greater than 10 μM). In the presence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 30 nM) CPA concentration-response curves were shifted, in parallel to the right (apparent pK_B 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 A_2A adenosine receptor selective agonist, CGS 21680 (2-p-(2-carboxyethyl)-phenethylamino-N-ethylcarboxamido adenosine), but not CPA, inhibited phenylephrine (20 μM) stimulated contractions (pIC₅₀ 7.15±0.48). This effect of NECA was blocked by xanthine amine congener (XAC, 1 μM) and the A_2A 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 μM)-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 pK_B 6.88±0.88) and also by the A_2A adenosine receptor antagonist, ZM 241385 (apparent pK_B 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 α₁-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 A_2A subtype, which both inhibit contractility and also stimulate adenylyl cyclase.

     

Characterization of the potency, selectivity, and pharmacokinetic profile for six adenosine A2A receptor antagonists

Antagonists of adenosine A_2A receptors (A_2A-antagonists) with different chemical structures have been developed by several pharmaceutical companies for the potential treatment of Parkinson’s disease. Pharmacological characterization of these antagonists was incomplete, and different assay conditions were used in different labs. Therefore, we characterized the potencies, selectivities, and pharmacokinetic profiles of six prototypical A_2A-antagonists. Displacements of [³H]MSX-2 and of [³H]CGS21680 binding to the human cloned and rat A_2A receptors were performed. The rank order of potency of antagonists to displace [³H]MSX-2 binding to the human A_2A was SCH58261 ≥ Biogen-34 ≥ Ver-6623 ≥ MSX-2 > KW-6002 > > DMPX. For the rat striatal A_2A, the order of potency was Biogen-34 ≥ SCH58261 ≥ Ver-6623 ≥ MSX-2 ≥ KW-6002 > > DMPX. SCH58261 was the most potent antagonist of the human A_2A with a K _i value of 4 nM, whereas Biogen-34 was the most potent antagonist of the rat A_2A with a K _i value of 1.2 nM. Similar results were obtained from cAMP assays. Selectivities of A_2A-antagonists were determined using radioligands [³H]DPCPX, [³H]ZM241385, and [¹²⁵I]-AB-MECA for A₁, A_2B, and A₃ receptors, respectively. KW-6002 and Biogen-34 exhibited the highest selectivity for A_2A vs A₁ (human and rat), respectively. The pharmacokinetic profiles of antagonists were evaluated in vivo in rats. DMPX and KW-6002 had the greatest oral bioavailability. In contrast, SCH58261, MSX-2, and Ver-6623 had low or poor oral bioavailability. In summary, SCH58261, Biogen-34, MSX-2, and Ver-6623 had high affinities for both human and rat A_2A receptors, with reasonable selectivity for A_2A over A₁ and A_2B receptors. They are suitable as A_2A-antagonists for in vitro pharmacological studies. Among the six A_2A-antagonists, KW-6002 is the best for use in in vivo animal studies, particularly for a CNS target, based on its bioavailability, half life, and brain penetration.     

Adenosine receptors mediate both contractile and relaxant effects of adenosine in main pulmonary artery of guinea pigs

In guinea pig main pulmonary artery precontracted with noradrenaline, adenosine exerted an initial phasic contraction followed by a tonic contraction and a slow relaxation. After selective blockade by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX: 10 nM) of A₁ receptors, adenosine only elicited a rapid relaxation. This initial response was characterized by use of adenosine (AR) and its analogues N⁶-cyclopentyl-adenosine (CPA), R-N⁶-phenyllsopropyladeno-sine (R-PIA), 2-chloroadenosine (CADO), 5-N-ethyl-carboxamidoadenosine(NECA), N⁶-2-(4-aminophenyl) ethyl adenosine (APNEA) and 2-p-((carboxyethyl)phenethylamino)-5-carboxamidoadenosine (CGS 21 680). The order of potency of the adenosine analogues for purine-induced phasic contraction was CPA > R-PIA > NECA = APNEA > AR > CGS 21 680 suggesting the involvement of activation of A₁ type adenosine receptors in the contraction phase. DPCPX antagonized the CPA-induced contraction with a pA₂ = 9.27 ± 0.26, but the Schild plot slope parameter was significantly lower than unity (0.58 ± 0.09). In contrast, in electrically driven guinea pig atrial myocardium (a tissue reported to possess A1 receptors), the DPCPX-CPA antagonism was purely competitive (pA₂ = 8.95 ± 0.06; slope = 0.93 ± 0.06). In the presence of 300 nM DPCPX, the rank order of potency for the purine-induced fast relaxation was NECA > CADO = AR > CGS 21 680 = R-PIA > CPA. The NECA- and adenosine-induced relaxation was influenced neither by 300 nM CP 66 713 (an antagonist at A_2a receptors), nor by endothelial removal and inhibition of nitric oxide synthase (100 M N^G-nitro-L-arginine: L-NOARG). The adenosine-induced relaxation was antagonized by 8-phenyltheophylline (8-PT), a potent A₁/A₂ antagonist. However, the rapid relaxation elicited by adenosine in the presence of 8-PT, was reversed and contraction developed. It is concluded that adenosine causes contraction via dual action on A₁ adenosine receptors and on xanthine-resistant sites. Our experiments with APNEA (a prototypic A₃ receptor agonist) did not support the suggestion that A₃ receptors are implicated in the xanthine-resistant component of adenosine-induced contraction, as DPCPX (300 nM) completely abolished and even reversed the APNEA-induced contraction. In addition, cromolyn (a mast cell stabilizing agent) did not influence the xanthine-resistant contraction induced by adenosine in the presence of DPCPX, 8-PT and dipyridamole (an adenosine uptake inhibitor). On the basis of the rank order of agonist potency, the receptors involved in the adenosine-induced rapid relaxation most likely is of the A_2b subtype. The opposing action of the xanthine-resistant contraction, however, did not allow a definitive pharmacological characterization of the receptor mediating relaxation.     

2-Chloro-N6-cyclopentyladenosine: a highly selective agonist at A1 adenosine receptors

Summary 2-Chloro-N⁶-cyclopentyladenosine (CCPA) was synthesized as a potential high affinity ligand for A₁ adenosine receptors. Binding of [³H]PIA to A₁ receptors of rat brain membranes was inhibited by CCPA with a K _i-value of 0.4 nM, compared to a K _i-value of 0.8 nM for the parent compound N⁶-cyclopentyladenosine (CPA). Binding of [³H]NECA to A₂ receptors of rat striatal membranes was inhibited with a K _i-value of 3900 nM, demonstrating an almost 10,000-fold A₁-selectivity of CCPA.CCPA inhibited the activity of rat fat cell membrane adenylate cyclase, a model for the A₁ receptor, with an IC₅₀-value of 33 nM, and it stimulated the adenylate cyclase activity of human platelet membranes with an EC₅₀-value of 3500 nM. The more than 100-fold A₁-selectivity compares favourably with a 38-fold selectivity of CPA. Thus, CCPA is an agonist at A₁ adenosine receptors with a 4-fold higher selectivity and 2-fold higher affinity than CPA, and a considerably higher selectivity than the standard A₁ receptor agonist R-N⁶-phenylisopropyladenosine (R-PIA). CCPA represents the agonist with the highest selectivity for A₁ receptors reported so far.Abbreviations CCPA 2-choro-N⁶-cyclopentyladenosine - CPA N⁶-cyclopentyladenosine - NECA 5-N-ethylcarboxamidoadenosine - PIA N⁶-phenylisopropyladenosine Send offprint requests to M. J. Lohse at the above address     

Postsynaptic Adenosine A2A Receptors Modulate Intrinsic Excitability of Pyramidal Cells in the Rat Basolateral Amygdala

Background:

The basolateral amygdala plays a critical role in the etiology of anxiety disorders and addiction. Pyramidal neurons, the primary output cells of this region, display increased firing following exposure to stressors, and it is thought that this increase in excitability contributes to stress responsivity and the expression of anxiety-like behaviors. However, much remains unknown about the underlying mechanisms that regulate the intrinsic excitability of basolateral amygdala pyramidal neurons.

Methods:

Ex vivo gramicidin perforated patch recordings were conducted in current clamp mode where hyper- and depolarizing current steps were applied to basolateral amygdala pyramidal neurons to assess the effects of adenosine A_2A receptor modulation on intrinsic excitability.

Results:

Activation of adenosine A_2A receptors with the selective A_2A receptor agonist CGS-21680 significantly increased the firing rate of basolateral amygdala pyramidal neurons in rat amygdala brain slices, likely via inhibition of the slow afterhyperpolarization potential. Both of these A_2A receptor-mediated effects were blocked by preapplication of a selective A_2A receptor antagonist (ZM-241385) or by intra-pipette infusion of a protein kinase A inhibitor, suggesting a postsynaptic locus of A_2A receptors on basolateral amygdala pyramidal neurons. Interestingly, bath application of the A_2A receptor antagonist alone significantly attenuated basolateral amygdala pyramidal cell firing, consistent with a role for tonic adenosine in the regulation of the intrinsic excitability of these neurons.

Conclusions:

Collectively, these data suggest that adenosine, via activation of A_2A receptors, may directly facilitate basolateral amygdala pyramidal cell output, providing a possible balance for the recently described inhibitory effects of adenosine A₁ receptor activation on glutamatergic excitation of basolateral amygdala pyramidal cells.     

Binding of the A1-selective adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine to rat brain membranes

Summary 8-Cyclopentyl-1,3-dipropylxanthine (PD 116,948) is a very potent, very A₁-selective adenosine antagonist, with a K _i of 0.46 nM in ³H-CHA binding to A₁ receptors in rat whole brain membranes and 340 nM in ³H-NECA binding to A₂ receptors in rat striatal membranes. Its 740-fold A₁-selectivity is the highest reported for an adenosine antagonist. ³H-PD 116,948 (117 Ci/mmol) was prepared by reduction of the diallyl analog. ³H-PD 116,948 bound to a single site in rat whole brain membranes, with a B _max of 46 pmol/g wet weight and K _d of 0.42 nM. Nonspecific binding was extremely low, amounting to about 3% of total binding under standard conditions and less than 1 % when higher tissue concentrations were used. Affinities of compounds for inhibition of ³H-PD 116,948 binding were highly consistent with an A₁ adenosine receptor. Antagonists were equally potent in ³H-PD 116,948 binding and in ³H-CHA binding, while agonists were consistently about 12-fold more potent in ³H-CHA binding. Hill coefficients were 1.0 for antagonists and about 0.65 for agonists. ³H-PD 116,948 should be a useful antagonist ligand for adenosine A₁ receptors. Send offprint request toR. F. Bruns at above address     

A role for adenosine A(1) receptor blockade in the ability of caffeine to promote MDMA "Ecstasy"-induced striatal dopamine release

Co-administration of caffeine profoundly enhances the acute toxicity of 3,4 methylenedioxymethamphetamine (MDMA) in rats. The aim of this study was to determine the ability of caffeine to impact upon MDMA-induced dopamine release in superfused brain tissue slices as a contributing factor to this drug interaction. MDMA (100 and 300 μM) induced a dose-dependent increase in dopamine release in striatal and hypothalamic tissue slices preloaded with [³H] dopamine (1 μM). Caffeine (100 μM) also induced dopamine release in the striatum and hypothalamus, albeit to a much lesser extent than MDMA. When striatal tissue slices were superfused with MDMA (30 μM) in combination with caffeine (30 μM), caffeine enhanced MDMA-induced dopamine release, provoking a greater response than that obtained following either caffeine or MDMA applications alone. The synergistic effects in the striatum were not observed in hypothalamic slices. As adenosine A₁ receptors are, one of the main pharmacological targets of caffeine, which are known to play an important role in the regulation of dopamine release, their role in the modulation of MDMA-induced dopamine release was investigated. 1 μM 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a specific A₁ antagonist, like caffeine, enhanced MDMA-induced dopamine release from striatal slices while 1 μM 2,chloro-N(6)-cyclopentyladenosine (CCPA), a selective adenosine A₁ receptor agonist, attenuated this. Treatment with either SCH 58261, a selective A_2A receptor antagonist, or rolipram, a selective PDE-4 inhibitor, failed to reproduce a caffeine-like effect on MDMA-induced dopamine release. These results suggest that caffeine regulates MDMA-induced dopamine release in striatal tissue slices, via inhibition of adenosine A₁ receptors.     

Differential regulation of adenosine A1 and A2A 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.     

Facilitatory and inhibitory modulation by endogenous adenosine of noradrenaline release in the epididymal portion of rat vas deferens

Summary The present study aimed at determining the modulation by adenosine of the release of noradrenaline in the epididymal portion of the rat vas deferens. The tissues were treated with pargyline and perifused in the presence of desipramine and yohimbine. Up to four periods of electrical stimulation were applied (5 Hz, 9 min).The A₁-adenosine receptor selective agonist R-N⁶-phenylisopropyladenosine (R-PIA; 100–900 nmol·l^–1) reduced, whereas the A_2A-receptor selective agonist 2-p-(2-carboxyethyl)phenethylamino-5-N-ethylcarboxamidoadenosine (CGS21680; 3–30nmol·l^–1) increased the electrically-evoked noradrenaline overflow in a concentration-dependent manner. The nonselective agonist 5-N-ethy1carboxamidoadenosine (NECA; 30–300 nmol·l^–1) reduced noradrenaline overflow, but the effect did not depend on the concentration. Adenosine deaminase at the concentration of 0.5 ·ml^–1 decreased but at that of 2.0 ·ml^–1 increased noradrenaline overflow. The inhibitors of adenosine uptake, S-(4-nitrobenzyl)-6-thioinosine (NBTI; 50 nmol·l^–1) and dipyridamole (3 mol·l^–1), increased the electrically-evoked noradrenaline overflow. The A₁-adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 20 nmol·l^–1) caused an increase whereas the A₂-adenosine receptor antagonist 3,7-dimethyl-1-(2-propynyl)xanthine (DMPX; 0.1 mol·l^–1) caused a decrease. NBTI (50 nmol·l^–1), partially antagonized the effect of both DPCPX (20 nmol·l^–1) and DMPX (0.1 mol·l^–1).It is concluded that, in the epididymal portion of the rat vas deferens, endogenous adenosine tonically modulates the release of noradrenaline evoked by electrical stimulation, through activation of both inhibitory (A₁) and facilitatory (A_2A) adenosine receptors.Abbreviations CGS 21680 2-p-(2-carboxyethyl)phenethylamino-5-N-ethylcarboxamidoadenosine - DMPX 3,7-dimethyl-l-(2-propynyl)xanthine - DPCPX 1,3-dipropyl-8-cyclopentylxanthine - NBTI S-(4-nitrobenzyl)-6-thioinosine - NECA 5-N-ethylcarboxamidoadenosine - R-PIA R-N⁶-phenylisopropyladenosine Correspondence to J. Gongalves at the above address     

A detailed behavioral analysis of the acute motor effects of caffeine in the rat: involvement of adenosine A<Subscript>1</Subscript> and A<Subscript>2A</Subscript> receptors

Rationale There is no consensus on the contribution of adenosine A₁ and A_2A receptor blockade to motor-activating effects of caffeine.Objective Our aim was to use a detailed and continuous observational method to compare the motor effects induced by caffeine with those induced by selective A₁ and A_2A receptor antagonists.Methods The behavioral repertoire induced by systemic administration of caffeine (3, 10, and 30 mg/kg), A₁ receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 1.2, 4.8 and 7.2 mg/kg), and A_2A receptor antagonist 3-(3-hydroxypropyl)-8-(m-methoxystyryl)-7-methyl-1-propargylxanthine phosphate disodium salt (MSX-3; 1, 3, and 10 mg/kg) was analyzed. The effects of pretreatment with the selective A₁ receptor agonist N ⁶-cyclopentyladenosine (CPA; 0.1 mg/g) and the selective A_2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxyamidoadenosine (CGS 21680; 0.2 mg/kg) on the pattern of motor activation induced by caffeine, CPT, or MSX-3 were also examined.Results The pattern of behavioral activation induced by caffeine was better mimicked by CPT than by MSX-3. Coadministration of CPT and MSX-3 gave different results depending on the dose and the type of behavioral response. CPA was more effective at decreasing the activating effects of caffeine and CPT than those of CGS 21680. On the other hand, CGS 21680 was more effective at decreasing the activating effects of MSX-3 than those of caffeine or CPT. Factor analysis revealed a complex three-dimensional behavioral profile for caffeine that was similar to the profile for CPT and was different from the profile for MSX-3.Conclusions The results indicate a predominant role for A₁ receptors in the motor-activating effects of acutely administered caffeine.