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     

Purinoceptor-mediated modulation by endogenous and exogenous agonists of stimulation-evoked [3H]noradrenaline release on rat iris

Summary To investigate whether endogenous purinoceptor agonists affect the sympathetic neurotransmission in the rat isolated iris, and to classify the purinoceptors modulating exocytotic [³H]-noradrenaline release, we have determined the effect of adenosine receptor antagonists on, and the relative potency of selected agonists in modulating, the field stimulation-evoked (3 Hz, 2 min) [³H]-noradrenaline overflow. In addition, the apparent affinity constants of 8-phenyltheophylline (8-PT) and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) in antagonizing the prejunctional effects of purinoceptor agonists were estimated.The relatively A₁-selective DPCPX 10 and 100 nmol/l increased the evoked [³H]-noradrenaline overflow by about 25%–35%a indicating a minor inhibition of evoked release by endogenous purinoceptor agonists probably via an A₁ adenosine receptor. Whereas the A₁/A₂-antagonist 8-PT failed to increase the evoked [³H]-noradrenaline overflow in the absence of exogenous agonists (without or with dipyridamole 1 pmol/l present), the relatively A₂-selective antagonist CP-66,713 (4-amino-8-chloro -1-phenyl(1,2,4)triazolo(4,3-a)quinoxaline) 100 nmol/l decreased it by 20%–30% in the absence and continuous presence of DPCPX. This may be compatible with a minor A₂-mediated facilitation by an endogenous purinoceptor agonist.All exogenous agonists tested (except UTP 100 mol/l) inhibited the evoked [³H]-noradrenaline overflow. The relative order of agonist potency (IC4o, concentration in mol/l for inhibition of evoked release by 40%) was CPA (N⁶-(cyclopentyl)adenosine, 0.004) > R-PIA (R(–)N6-(2phenylisopropyl)adenosine, 0.066) = CHA (N⁶-(cyclohexyl)adenosine, 0.082) > NECA (N⁵-(ethyl-carboxamido)adenosine 0.44) > ADO (adenosine, 4.1). ATP was n early equipotent with ADO. Maximum inhibition was 70%–80% and similar for all agonists. Adenosine deaminase 1 u/ml failed to affect the ATP-induced, but abolished the adenosine-induced prejunctional inhibition. The adenosine uptake inhibitor S-p-nitrobenzyl-6-thioguanosine (NBTG) failed to enhance the potency of ADO and ATP. The A₁-selective antagonist DPCPX 10 nmol/l did not reduce the ATP potency indicating an effect of ATP per se not mediated via an A₁ purinoceptor.Prejunctional affinity constants of 8-PT were 6.07 when tested against adenosine (in the presence of dipyridamole), and 6.60 against CHA. The apparent -log K_B of DPCPX tested against CPA was 9.71. The high DPCPX affinity is compatible with an A₁ adenosine receptor mediating inhibition of sympathetic neurotransmission in rat iris. This receptor may not be the only prejunctional purinoceptor on rat iris sympathetic nerves. The receptor by which ATP acts prejunctionally in this tissue remains to be determined.This study was supported by the Deutsche Forschungsgemeinschaft (Fu 163/2 and 163/3) Send offprint requests to H. Fuder at the above address     

Comparison of A1 adenosine receptors in brain from different species by radioligand binding and photoaffinity labelling

Summary Radioligand binding to A₁ adenosine receptors at brain membranes from seven species was investigated. The antagonist 8-cyclopentyl-1,3-[³H]dipropyl-xanthine ([³H]DPCPX) bound with affinities between 0.17 nM in sheep brain and 2.1 nM in guinea pig brain. Competition of several antagonists for [³H]DPCPX binding showed that the most potent compounds were DPCPX with K _i values of 0.05 nM in bovine brain and 1.1 nM in guinea pig brain and xanthine amine congener (XAC) with K _i values of 0.03 nM in bovine brain and 5.5 nM in guinea pig brain. The differences in affinity of the agonist radioligand 2-chloro-N ⁶-[³H]cyclopentyl-adenosine ([³H]CCPA) were less pronounced, ranging from a K _D value of 0.12 nM (hamster brain) to 0.42 nM (guinea pig brain). Agonist competition for [³H]DPCPX binding of photoaffinity labelling, however, exhibited marked species differences. N-Ethylcarboxamidoa-denosine (NECA) and S-N ⁶-phenylisopropyladenosine (S-PIA) showed 20 to 25-fold different K _D values in different species. NECA had a particularly high affinity in guinea pig brain and was only two-fold less potent than R-PIA. Thus, the difference from the classical A₁ receptor profile (R-PIA > -NECA > S-PIA) is not sufficient to speculate that A₁ receptor subtypes may exist that are coupled to different effector systems. Our data show that these difference can easily be explained by species differences.     

8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) — a selective high affinity antagonist radioligand for A1 adenosine receptors

Summary The properties of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) as an antagonist ligand for A₁ adenosine receptors were examined and compared with other radioligands for this receptor. DPCPX competitively antagonized both the inhibition of adenylate cyclase activity via A₁ adenosine receptors and the stimulation via A₂ adenosine receptors. The K ₁-values of this antagonism were 0.45 nM at the A₁ receptor of rat fat cells, and 330 nM at the A₂ receptor of human platelets, giving a more than 700-fold A₁-selectivity. A similar Al-selectivity was determined in radioligand binding studies. Even at high concentrations, DPCPX did not significantly inhibit the soluble cAMP-phosphodiesterase activity of human platelets. [³H]DPCPX (105 Ci/mmol) bound in a saturable manner with high affinity to A₁ receptors in membranes of bovine brain and heart, and rat brain and fat cells (K _D-values 50–190 pM). Its nonspecific binding was about 1 % of total at KD, except in bovine myocardial membranes (about 10%). Binding studies with bovine myocardial membranes allowed the analysis of both the high and low agonist affinity states of this receptor in a tissue with low receptor density. The binding properties of [³H]DPCPX appear superior to those of other agonist and antagonist radioligands for the A₁ receptor.Abbreviations CHA N⁶-cyclohexyladenosine - DPX 1,3-diethyl-8-phenylxanthine - HPIA N⁶-p-hydroxyphenylisopropyladenosine - NECA, -N-ethylcarboxamidoadenosine - PIA N⁶-phenylisopro-pyladenosine - XAC (xanthine amine congener) 8-{4-[([{(2aminoethyl)amino}carbonyl]methyl)oxy]phenyl-1,3-dipropyl-xanthine. 8-Cyclopentyl-1,3-dipropylxanthine is abbreviated DPCPX (from 1,3-dipropyl-8-cyclopentylxanthine) Send offprint requests to M. J. Lohse at the above address     

Glomeruli and microvessels of the rabbit kidney contain both A1- and A2-adenosine receptors

Summary Rabbit renal cortices were fractionated by collagenase dispersion and glomeruli, microvessels and tubuli purified on a discontinuous sucrose gradient. Binding experiments with (–)[¹²⁵I]N⁶-(4-hydroxyphenylisopropyl)-adenosine ([¹²⁵I]HPIA) provided evidence for the presence of A₁-adenosine receptors in the glomerular and microvascular fraction. With glomeruli, saturation isotherms for specific [¹²⁵I]HPIA binding were mono-phasic with a K _D of 1.3 nmol/l and a B _maxof 7.7 fmol/mg protein. In kinetic experiments, an association rate constant of 4.9 × 105 (mol/ 1)^–1 s^–1 and a dissociation rate constant of 4.3 × 10^–4 s^–1 were obtained, yielding a K _D of 0.9 nmol/l. Adenosine analogs displaced [¹²⁵I]HPIA binding with a rank order of potency typical of A₁-adenosine receptors; furthermore, binding was inhibited by methylxanthines and modulated by GTP. Saturation experiments with the microvessels revealed a K _D of 1.9 nmol/l and a B _max of 13.4 fmol/mg protein. However, no inhibition of glomerular and microvascular adenylate cyclase activity could be demonstrated, but instead both 5-N-ethylcarboxamido-adenosine (NECA) and N⁶-(R-phenylisopropyl)-adenosine (R-PIA) stimulated enzyme activity, with EC₅₀ values of 0.14 mol/l and 1.5 mol/l, respectively. The concentration-response curve for NECA was shifted to the right (factor 9) by 10 mol/l 8-phenyltheophylline. On the other hand, computer simulation of biphasic curves (adenylate cyclase inhibition in the presence of activation via a stimulatory receptor) indicates that the failure to observe an A₁-adenosine receptor-mediated inhibition of adenylate cyclase activity in the presence of stimulatory adenosine receptors may be attributable to methodological constraints. The results demonstrate that both A₁- and A₂-adenosine receptors are present in rabbit glomeruli and microvessels. It is suggested that both receptors are involved in the control of renin secretion.Abbreviations R-PIA (–)N⁶(R-phenylisopropyl)-adenosine - NECA 5-(N-ethyl-carboxamido)-adenosine - S-PIA (+)N⁶-(S-phenylisopropyl)-adenosine - I-HPIA (–)N⁶-(3-iodo-4-hydroxy-phenylisopropyl)-adenosine - HPIA (–)N⁶-(4-hydroxyphenylisopropyl)-adenosine - [¹²⁵I]HPIA (–)N6-(3-[¹²⁵I]iodo-4-hydroxy-phenylisopropyl)-adenosine - ATP adenosine-5-triphosphate - cAMP cyclic 3,5-adenosine-monophosphate - GTP guanosine-5-triphosphate - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - EDTA (ethylenedinitrilo)-tetraacetic acid Send offprint requests to W. Schütz at the above addressThis study was supported by the Fonds zur Förderung der wissenachiftlichen Forschung in Österreich (Project 5712)     

Binding of [H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes — a new, selective antagonist radioligand for A2A 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)>N⁶-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)-7H-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.     

P2-Receptor-mediated inhibition of noradrenaline release in the rat hippocampus

Experiments on hippocampal slices were carried out in order to find out whether the release of noradrenaline in the hippocampus can be modulated through P2-receptors. The slices were preincubated with [³H]-nor-adrenaline, superfused with medium containing desipramine (1 μM), and stimulated electrically, in most experiments by 4 pulses/100 Hz. The adenosine A₁-receptor agonist N⁶-cyclopentyl-adenosine (CPA) and the nucleotides ATP, adenosine-5’-O-(3-thiotriphosphate) (ATPγS) and adenosine-5’-O-(2-thiodiphosphate) (ADPβS) decreased the evoked overflow of tritium by up to 55 %. The adenosine A_2a-agonist 2-p-(2-carboxyethyl)-phenethylamino-5’-N-ethylcarboxamido-adenosine (CGS 21680; 0.003-0.3 μM) caused no change. The concentration-response curve of CPA was shifted to the right by the A₁-antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 3 nM) but not by the P2-receptor antagonists cibacron blue 3GA (30 μM) and reactive blue 2 (30 μM); the apparent pK_B value of DPCPX against CPA was 9.0. In contrast, the concentration-response curve of ATP was shifted to the right by DPCPX (3 nM), apparent pK_B 8.7, as well as by ciba-cron blue 3GA (30 μM), apparent pK_B 5.2, and reactive blue 2 (30 μM), apparent pK_B 5.6; the antagonist effects of DPCPX and cibacron blue 3GA were additive in a manner compatible with the blockade of two separate receptors for ATP. The same pattern was obtained with ATPγS: its concentration-response curve was shifted to the right by DPCPX as well as by cibacron blue 3GA and reactive blue 2. Suramin (300 μM) antagonized neither the effect of ATP nor that of ATPγS. The 5’-nucleotidase inhibitor α,β-methylene-ADP (100 μM) did not change the effect of ATP. Only cibacron blue 3GA (30 μM) but not reactive blue 2 (30 μM), given alone, consistently caused a small increase of the evoked overflow of tritium. Hippocampal slices degraded exogenous ATP, and this degradation was reduced by cibacron blue 3GA (30 μM), reactive blue 2 (30 μM) and suramin (300 μM). The results indicate that the noradrenergic terminal axons of the rat hippocampus possess P2-receptors in addition to the known A₁-adenosine receptors. The presynaptic P2-receptors mediate an inhibition of noradrenaline release, are activated by nucleotides but not nucleosides, and are blocked by cibacron blue 3GA and reactive blue 2. ATP and ATPγS act at both the A₁- and the P2-receptors. An autoreceptor function of cerebral presynaptic P2-receptors remains doubtful. Received: 20 November 1996 / Accepted: 10 February 1997     

腺苷A1受体和NMDA受体在海马齿状回突触传递活动中的关系

目的探讨腺苷A₁受体阻断剂对海马齿状回(DG)突触传递活动的影响及其与NMDA受体的关系。方法 采用在体记录麻醉大鼠LTP的电生理学方法,观察腺苷A₁受体特异性阻断剂8-环戊-1,3-二丙基黄嘌呤(DPCPX)与NMDA受体激动剂、阻断剂在海马DG基础突触传递活动和高频刺激诱导的LTP中作用的相关性。结果DPCPX(6 mg·L^-1,5 μL,icv)或NMDA(0.2 mg·L^-1,5 μL,icv)不影响大鼠海马DG突触传递活动,DPCPX对icv NMDA后高频刺激诱导已形成的LTP维持也无影响;预先给予DPCPX后则可显著增强NMDA的海马DG基础突触传递活动和LTP;AP₅(0.5 mg·L^-1,5 μL)阻断NMDA受体后对LTP的抑制作用不受DPCPX的影响,但预先给予DPCPX则可取消AP₅对LTP的抑制作用。结论DPCPX不影响海马DG突触传递活动,但可影响NMDA受体的效应,增强NMDA受体在海马DG突触传递活动中的作用。     

The role of A3 adenosine receptors in central regulation of arterial blood pressure

1. Pharmacological studies have suggested that A₃ receptors are present on central neurons. Recently this adenosine receptor subtype has been identified in the rat and its presence in the central nervous system has been confirmed.
2. In this study we investigated the effects of acute intracerebroventricular (i.c.v.) injections of N⁶-2-(4-aminophenyl)-ethyladenosine (APNEA), a non-selective A₃ adenosine receptor agonist, on arterial blood pressure (ABP) and heart rate (HR), after treatment with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective antagonist of A₁ adenosine receptors.
3. Anaesthetized rats, after DPCPX (12 μg⁻¹ kg i.c.v.), were treated with APNEA (0.4–4 μg kg⁻¹ i.c.v.) resulting in a transitory and dose-dependent decrease in arterial blood pressure without a change in heart rate. APNEA also induced hypotensive responses after i.c.v. pretreatment with aminophylline, at a dose of 20 μg kg⁻¹. In contrast, pretreatment 48 h before, with 4 μg kg⁻¹ i.c.v. of pertussis toxin reduced the hypotensive effect induced by APNEA. Administration of APNEA at a higher dose (20 μg kg⁻¹ i.c.v.), after DPCPX, induced a decrease in ABP of −66±5.4 mmHg and after 3 min a decrease in heart rate of −62±6.0 beats min⁻¹. Transection of the spinal cord abolished this significant fall in ABP, but not the decrease of HR.
4. These results suggest that a population of A₃-receptors is present in the CNS, whose activation induces a decrease in blood pressure with no change of heart rate.

     

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     

Activation of A1 adenosine receptors decreases the release of serotonin in the rabbit hippocampus,but not in the caudate nucleus

Summary The effects of A1 adenosine receptor ligands on the evoked release of serotonin (5-HT) were studied in slices of the hippocampus and the caudate nucleus of the rabbit, preincubated with ³H-5-HT. In hippocampal tissue electrical stimulation elicited a release which was inhibited by the adenosine receptor agonist N⁶-cyclohexyladenosine (CHA) and enhanced by the selective A₁ receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). The concentration-response curve of CHA was shifted to the right by DPCPX. The shift corresponded to a pA₂ value of 9.4 for DPCPX. CHA, R-N⁶-phenylisopropyladenosine (R-PIA) and DPCPX were ineffective in caudate nucleus tissue. When instead of electrical pulses high K⁺ was used to induce 5-HT release in the presence of the Na⁺ channel blocker tetrodotoxin (TTX), which was present in order to exclude effects mediated by interneurones, CHA was equally effective in the hippocampus but again failed to modify 5-HT release in the caudate nucleus. The disinhibition by DPCPX of the evoked 5-HT release was used to calculate the extracellular concentration of endogenous adenosine at the A₁ receptor. The calculation greatly depended on the dissociation constant of adenosine at the A1 receptor.It is concluded that A1 adenosine receptors, activated by the endogenous agonist at a concentration of about 0.7 mol/l, are located on serotonergic nerve endings in the hippocampus, but not in the caudate nucleus. The estimated extracellular concentration of endogenous adenosine is in reasonable agreement with actually measured concentrations reported in the literature. Send offprint requests to T. J. Feuerstein at the above address     

Pharmacological properties of adenosine receptors and adenosine binding proteins

Two major subtypes of adenosine receptors occur in different tissues which have been distinguished by pharmacological and biochemical criteria. The A₁ adenosine receptor has a high-affinity for adenosine and mediates inhibition of adenylyl cyclase, whereas the A₂ adenosine receptor usually has a lower affinity and mediates stimulation of the enzyme. Furthermore, evidence has been obtained that A₁ receptors increase the conductance of receptor-regulated potassium channels, induce inactivation of calcium channels, and modulate the breakdown of phosphoinositides by phospholipase C. Selective agonists and antagonists have been developed for both receptor subtypes. In addition, both adenosine receptors have extensively been characterized by radioligand binding studies. Suitable radioligands for the A₁ receptor are the agonist [³H]2-chloro-N⁶-cyclopentyladenoisine (CCPA) and the antagonist [³H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX)and for the A_2a receptor [³H]2-[p-(carboxyethyl)phenethylamino]- 5′-N-carboxamidoadenosine (CGS 21860). Furthermore, photoaffinity ligands were developed from adenosine derivatives, which can be covalently incorporated into the binding unit of both receptor subtypes. With this approach, it has been shown that the A₁ receptor has an apparent molecular weight of approximately 36 kDa and the A_2a receptor of 45 kDa. A second approach to elucidate the structure of adenosine receptors involves the purification of receptor protein by affinity chromatography. With this procedure, cerebral A₁ receptors have been purified to apparent homogeneity. More recently, the structure of receptor subtypes has been elucidated by cloning the receptors from a cDNA library. Furthermore, a novel adenosine binding with [³H] 5′ -N-ethylcarboxamidoadenosine ([³H]NECA). The pharmacological profile of this NECA-binding protein has been determined in competition experiments with adenosine receptor ligands. It can be distinguished from that of A_2a adenosine receptors and other adenosine binding proteins. We propose the name A_x for this unique adenosine binding protein. © 1993 Wiley-Liss, Inc.     

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.     

A1 adenosine receptors expressed in CHO-cells couple to adenylyl cyclase and to phospholipase C

A₁ adenosine receptors are in general coupled to inhibition of adenylyl cyclase, but have more recently been reported to be capable of also activating phospholipase C. The present study was done in order to investigate whether these different effects can be elicited by a single A₁ receptor, or whether A₁ receptor subtypes have to be invoked. The cDNA of a rat brain A₁ adenosine receptor was stably expressed in CHO-cells, resulting in clones with varying receptor densities; a clone expressing 1.9 pmol receptors/mg membrane protein was used for further characterization. The ligand binding properties of the expressed receptors were typical for the rat A₁ adenosine receptor. A₁ receptor agonists caused a concentration-dependent inhibition of adenylyl cyclase activity in the membranes, with maximal inhibition by 70%. A₁ receptor stimulation also caused concentration-dependent stimulation of inositol phosphate generation in these cells, with maximal effects of 300%. Both adenylyl cyclase inhibition and enhancement of inositol phosphate generation were essentially abolished after pretreatment of the cells with pertussis toxin. These results indicate that a single A₁ adenosine receptor can couple to two effector pathways, and that both effectors are activated via pertussis toxin sensitive G proteins.Abbreviations CHA N⁶-cyclohexyladenosine - CPA N⁶-cyclopentyladenosine - DPCPX 8-cyclopentyl-1,3-dipropylxanthine - NECA 5-N-ethylcarboxamidoadenosine - R-PIA R-N⁶-phenylisopropyladenosine - IP₁ inositol monophosphates - IP₂ inositol bisphosphates - IP₃ inositol trisphosphates - PCR polymerase chain reaction Correspondence to: M. J. Lohsc at the above address     

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     

Evidence against a regulation of Na+/K+-ATPase by Gi proteins. Failure to detect an influence of G proteins on Na+/Ca2+-exchange in cardiac sarcolemmal membranes

In the myocardium the inhibitory guanine nucleotide-binding regulatory proteins (G_i proteins) mediate negative chronotropic and negative inotropic effects by activation of K⁺ channels and inhibition of adenylyl cyclase. The concept of a uniform inhibitory action of G_i proteins on myocardial cellular activity has been questioned by the recent observations of adenosine-induced activation of the Na⁺/Ca²⁺ exchange and a carbachol-induced inhibition of the Na⁺/K⁺-ATPase activity in cardiac sarcolemmal membranes. The aim of the present study, therefore, was to reinvestigate the putative regulation of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity in purified canine sarcolemmal membranes. These membranes were enriched in adenosine A₁ (Maximum number of receptors, B _max 0.033 pmol/mg) and muscarinic M₂ (B _max 2.9 pmol/mg) receptors and contained G_i2 and G_i3, two G_i protein isoforms, and Go, another pertussis toxin-sensitive G protein, as detected with specific antibodies. The adenosine A₁-selective agonist, (–)-N ⁶-(2-phenylisopropyl)-adenosine, and the muscarinic agonist, carbachol, both inhibited isoprenaline-stimulated adenylyl cyclase activity by 25% and 35% respectively, and the stable GTP analogue 5-guanylylimidodiphosphate inhibited forskolin-stimulated adenylyl cyclase activity by 35% in these membranes. The characteristics of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity as well as those of the ouabain-sensitive, K⁺-activated 4-nitrophenylphosphatase, an ATP-independent, partial reaction of the Na⁺/K⁺-ATPase, were in agreement with published data with regard to specific activity, time course of activity and substrate dependency. However, none of these activities were influenced by adenosine, (–)-N ⁶-(2-phenylisopropyl)-adenosine, carbachol, or stable GTP analogs, suggesting that Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase are not regulated by Gi proteins in canine cardiac sarcolemmal membranes.     

Effects of urea pretreatment on the binding properties of adenosine A1 receptors

The effect of denaturation and/or extraction of nonintegral membrane proteins by 7 M urea on the binding of the antagonist [3H]cyclopentyl-1,3-dipropylxanthine 8 dipropyl-2,3 ([3H]DPCPX), and the agonists adenosine, (-)-N6-(2-phenylisopropyl)-adenosine (R-PIA) and N6-cyclohexyladenosine (CHA), was investigated at human A1 adenosine receptors stably expressed in CHO cells. Pretreatment with urea caused a 56% reduction in membrane proteins. Compared to controls, the use of adenosine deaminase (ADA), 100 microM 5'-guanylylimidodiphosphate (Gpp(NH)p) or urea each caused equivalent increases in specific [3H]DPCPX binding. Neither the binding kinetics nor the affinity of [3H]DPCPX were significantly different in urea-pretreated compared to ADA-pretreated membranes. At 25 degrees C in ADA-pretreated membranes, the competition isotherms for R-PIA and CHA were characterized by two affinity states. Gpp(NH)p (100 microM) reduced, but did not abolish, the value of the high-affinity dissociation constant. Similar results were obtained after treatment with urea for R-PIA, whereas the high-affinity state for CHA was abolished. At 37 degrees C, urea pretreatment, but not 100 microM Gpp(NH)p, abolished high-affinity agonist competition binding. There was no significant effect of any of the treatments on the low-affinity agonist binding state. In urea-pretreated membranes, exogenously added adenosine competed according to a simple mass-action model with a pK(L) of 5.66+/-0.05 (n=3). Compared to the more common approaches of ADA treatment and/or use of guanine nucleotides, our findings suggest that urea pretreatment represents an inexpensive and useful approach for investigating the binding properties of adenosine A1 ligands (including adenosine) to the G protein-uncoupled form of the receptor.     

Guanine nucleotide and cation regulation of radioligand binding to R i adenosine receptors of rat fat cells

Summary The modulation of radioligand binding at R _i adenosine receptors of rat fat cells by guanine nucleotides and cations was investigated. Guanine nucleotides (in the order of potency: GTP=GDP>Gpp(NH)p>5-GMP) decreased the binding of the R _i receptor agonist (–)N⁶-phenylisopropyl[³H]adenosine ([³H]PIA), but did not affect binding of the antagonist 1,3-diethyl-8[³H]phenylxanthine ([³H]DPX). Saturation of [³H]PIA binding revealed that GTP (100 mol/l) converts the high affinity form of the R _i receptor into a low affinity form. This effect was confirmed in kinetic experiments. GTP decreased the potency of agonists in competing for [³H]DPX binding, as shown by a 50-fold shift of the K _i-value for (–)PIA, whereas antagonist-induced inhibition of binding remained unchanged. The divalent cations Mg²⁺ and Ca²⁺ produced a slight increase in [³H]PIA binding but did not affect [³H]DPX binding. Mn²⁺ markedly decreased both agonist and antagonist binding at R _i adenosine receptors. Divalent cations reversed the guanine nucleotide-induced decrease of affinity of the R _i receptor. Na⁺ did not significantly affect agonist or antagonist binding but abolished the stimulatory effect of Mg²⁺ on agonist binding in the presence of GTP. Our data indicate that guanine nucleotides convert the R _i adenosine receptor of rat fat cells from a high to a low agonist affinity state and that the modulation of radioligand binding by mono-and divalent cations differs from that of R _i receptors of other tissues.     

Effects of selective A1 and A2 adenosine receptor agonists on cardiovascular tissues

Summary We investigated the negative chronotropic and vasodilating properties of new selective A₁ and A₂ adenosine agonists such as 2-chloro-N⁶-cyclopentyladenosine (CCPA) and 2-hexynyl-5-N-ethyl-carboxamidoadenosine (2-hexynyl-NECA) as compared with reference adenosine analogues. The potency of these compounds on heart rate was assessed in the rat atrial preparation and their activity on the vascular tone was determined in both rat aorta and bovine coronary artery. CCPA was found to be the most potent At agonist of those currently available in producing negative chronotropic effects (EC₅₀ = 8.2 nM). The A1 antagonist 8-cyclopentyl-1,3-dipropyl-xanthine (DPCPX) blocked CCPA activity in a dose-dependent manner. There was also a significant correlation between its biological effect and the affinity for A₁ receptors as measured in the rat brain by [³H]-N⁶-cyclohexyladenosine (³[H]-CHA) binding. The A₂ selective agonist 2-hexynyl-NECA showed vasodilating properties comparable with those observed with the reference compounds, CGS 21680 and NECA. EC₅₀ values were 596 and 569 nM in rat aorta and bovine coronary artery, respectively. Moreover, the rank order of potency was similar in the two vascular districts examined, suggesting that the rat aorta is a useful model for studying the effects of adenosine derivatives on vascular tone. In addition, the potency of the compounds in inducing vasodilation was found to be correlated with their affinity for A₂ receptors as measured in the rat striatum by ³[H]-CGS 21680 binding.These data further support that A₁ receptors are involved in depressing cardiac activity and A₂ receptors in inducing vasorelaxation.Correspondence to A. Conti at the above address