Involvement of adenosine receptors in mouse thermoregulation

The effects of the activation of adenosine receptors on core body temperature of mice have been studied in the present investigation. Intraperitoneal (i.p.) injection of non-selective adenosine agonists 5'-N ethyl- carboxamide adenosine (NECA; 0.001, 0.01 and 0.05 mg/kg), R-(N(6)-phenylisopropyl)-adenosine (R-PIA; 0.01, 0.1 and 0.25 mg/kg) and selective A(1) adenosine agonist N(6)-cyclohexyladenosine (CHA; 0.1, 0.25 and 0.4 mg/kg) reduced core body temperature. However, R-PIA and CHA were less potent than NECA in reducing the core body temperature. Theophylline (12.5, 25 and 50 mg/kg) blocked the hypothermia of the adenosine agonists. Pre-treatment of animals with selective A(1) adenosine antagonist 8-phenyltheophylline (8-PT; 0.5, 1 and 2 mg/kg) decreased the hypothermic response of CHA but not of NECA and R-PIA. 8-PT potentiated the hypothermia induced by R-PIA. These results suggest that activation of both A(1) and A(2) adenosine receptors decreases core body temperature in mice.     

Effects of adenosine analogues on apomorphine-induced penile erection in rats

• 1.1. In the present work, the effect of adenosine agonists and antagonists on apomorphine-induced penile erection (PE) has been studied.
• 2.2. Subcutaneous (s.c.) injection of the nonselective D1/D2 dopamine receptor agonist apomorphine (0.05–0.5 mg/kg) induced PE in a biphasic manner. The maximum effect was obtained with 0.1 mg/kg of the drug. The response decreased with increasing doses of apomorphine, from 0.1 to 0.5 mg/kg.
• 3.3. Intraperitoneal (i.p.) injections of adenosine agonists 5′-N-ethylcarboxamidoadenosine (NECA) and N⁶-cyclohexyladenosine (CHA) decreased the response of apomorphine. Apomorphine-induced PE was increased by low doses (25, 50 mg/kg, i.p.) and decreased by high doses (75, 100 mg/kg, i.p.) of the adenosine antagonist theophylline, respectively. Inhibition of PE induced by NECA and CHA was antagonized by 8-PT pretreatment.
• 4.4. Intracerebroventricular (i.c.v.) administration of CHA, NECA, and theophylline produced the same effects as i.p. injections of these agents on PE responses. It is concluded that A-1 and A-2 adenosine receptor activation may inhibit PE induced by dopaminergic mechanism(s), which can be prevented by 8-PT pretreatment.

     

Different influences of adenosine receptor agonists and antagonists on morphine antinociception in mice

• 1.1. Subcutaneous (s.c.) administration of morphine to mice induced a dose-dependent antinociception.
• 2.2. Pretreatment of animals with adenosine receptor antagonists NECA (5′-N-ethylcarboxamidermadenosine) and l-PIA (N⁶-phenylisopropyladenosine) potentiated, while adenosine agonist CHA (N⁶-cyclohexyladenosine) decreased the morphine response.
• 3.3. Adenosine antagonist theophylline decreased, but adenosine receptor antagonist 8-PT (8-phenyltheophylline) increased the antinociception effect of morphine. Inhibitory effect of CHA on morphine antinociception was also reversed by 8-PT pretreatment.
• 4.4. NECA or l-PIA induced a high degree of antinociceptive effect in animals pretreated with 8-PT.
• 5.5. Dipyridamole pretreatment did not alter the effect of morphine.
• 6.6. It is concluded that A-1 and/or A-2 adenosine receptors are involved in morphine antinociception and the adenosine mechanism(s) may exert a modulatory role in this respect.

     

Effects of adenosine agents on apomorphine-induced yawning in rats

In the present work, adenosine agonists and antagonists on apomorphine-induced yawning in rats was investigated. Subcutaneous (SC) injection of apomorphine (0.02, 0.05 and 0.1 mg/kg) induced dose-dependent yawning behaviour in rats. Intracerebroventricular (ICV) administration of different doses of the drug (1, 3, 5 µg/rat) also caused a dose-related yawning. ICV administration of the adenosine receptor agonists 5-N-ethylcarboxami-doadenosine (NECA) and N⁶-cyclohexyladenosine (CHA) decreased apomorphine-induced yawning. The response induced by the adenosine agonists was reduced by 8-phenyladenosine (8-PT) pretreatment. The yawning induced by SC and ICV administration of apomorphine was decreased by ICV or IP injection of theophylline, respectively. It is concluded that at least A₁ adenosine receptors may exert negative influence on the apomorphine-induced yawning. However, the exact mechanism(s) of adenosine receptors in this behaviour remain to be established.     

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

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

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     

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.     

Adenosine receptor modulation of sympathetic neurotransmission in rat isolated kidney

In the present study we set out to define, using discriminatory agonists and antagonists, the adenosine receptors modulating sympathetic neurotransmission in the rat kidney. Isolated kidneys from male Wistar rats were perfused with modified Krebs-Henseleit buffer solution at constant flow. The neuronal noradrenaline stores were labeled with ³H-noradrenaline and the renal nerves stimulated electrically (2 Hz, 3 msec, 9 mA, during 20 sec at intervals of 6 min). ³H overflow was taken as an index of ³H-noradrenaline release. The A₁ receptor selective agonists N⁶-cyclopentyladenosine (CPA), N⁶-cyclohexyladenosine (CHA), and N⁶-[R(−)-1-phenyl-2-propyl]adenosine (R-PIA), and the mixed A₁/A_2A receptor agonists 5′-N-ethylcarboxamidoadenosine (NECA) and 2-chloroadenosine (CADO) inhibited evoked ³H outflow concentration-dependently. The selective A_2A receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS 21680), at concentrations selective for A_2A receptors, failed to modify ³H outflow, whereas at higher concentrations it induced inhibition. The rank order of potency of agonists, CPA > CHA = R-PIA > NECA > CADO >> CGS 21680, is typical for an interaction with the A₁ receptor. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), at concentrations selective for blockade of A₁ receptors, blocked concentration-dependently the inhibitory effects of CPA and NECA; no evidence of an increase in outflow was seen with NECA in the presence of DPCPX. The selective A_2A receptor antagonist 9-chloro-2-(2-furanyl)[1,2,4]triazol[1,5-c] quinazoline-5-amine (CGS 15943) did not influence the agonist effects at concentrations interacting selectively with A_2A receptors but antagonized them concentration-dependently at higher, non-selective concentrations. Taken together, our data establish the presence of inhibitory adenosine A₁ receptors on the terminal sympathetic neurons of rat kidney. No evidence was obtained for the presence of functional A_2A receptors in this preparation. © 1996 Wiley-Liss, Inc.     

Species-dependent effects of adenosine receptor agonists on contractile responses of vas deferens to ATP

1 Experiments were carried out to examine the postjunctional actions of adenosine receptor agonists on the smooth muscle of the vas deferens of the guinea-pig and rabbit. 2 Although they produced neither contraction nor relaxation by themselves, adenosine analogues enhanced contractions of the guinea-pig vas deferens induced by 10 μm ATP. The rank order of potency was N⁶-cyclopentyladenosine (CPA) > 5′-N-ethylcarboxamidoadenosine (NECA) > adenosine > CGS 21680. Dose–response curves for NECA were shifted to the right by the nonselective adenosine receptor antagonist 8(p-sulphophenyl)theophylline (8-SPT; 100 μm ) and by the selective A₁-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 1 m m ). 3 In the rabbit vas deferens, contractions induced by ATP (1 m m ) were inhibited rather than facilitated by NECA. Neither CPA, R(–)-N⁶-(2-phenyl isopropyl)-adenosine (R-PIA) nor CGS 21680 had any effect. 4 The results indicate that the smooth muscle of the guinea-pig vas deferens expresses facilitatory adenosine A₁ receptors but not adenosine A₂ receptors. In contrast, in rabbit there are postjunctional inhibitory adenosine A_2A receptors but not adenosine A₁ receptors.     

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

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

Post-junctional excitatory adenosine A1 receptors in the rat vas deferens

• 1.1. At concentrations between 1 nM and 1 μM, the A₁-selective agonists N⁶-cyclopentyladenosine (CPA) and (R)-N⁶-phenylisopropyladenosine (R-PIA) each enhanced contractions of the rat vas deferens induced by ATP (10 μM), and this enhancement was blocked by an A₁-selective concentration (1 nM) of the antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX).
• 2.2. No such enhancement was observed with the non-selective agonists adenosine and 5′-N-ethylcarboxamidoadenosine (NECA) at concentrations between 1 nM and 100 μM, which instead inhibited the contractions.
• 3.3. These results show that in addition to the previously demonstrated inhibitory A₁ and A₂ adenosine receptors, the rat vas deferens also possesses post-junctional excitatory A₁ adenosine receptors.

     

Functional evidence for the presence of adenosine A2-receptors in cultured coronary endothelial cells

Summary Adenosine and the adenosine receptor agonists, R- and S-N⁶-phenylisopropyladenosine (R- and S-PIA) and 5-N-ethylcarboxamidoadenosine (NECA), enhanced [³H]cAMP accumulation in [³H]adenine-labelled cultured endothelial cells isolated from the microvasculature of guinea pig hearts. As shown by their concentration-response curves, NECA was a more potent agonist than R-PIA or adenosine. Their respective concentrations at half-maximal stimulation of [³H]cAMP accumulation were 0.7 M, 10.5 M and 12.6 M, indicating a 15- to 18-fold potency difference between NECA and the other agonists. The increased [³H]cAMP accumulation elicited by 10^–5 M NECA was inhibited by the xanthine derivative 8-phenyltheophylline, 3-isobutyl-l-methylxanthine, theophylline or caffeine. These findings provide functional evidence for the presence of adenosine receptors of the A₂-type in microvascular coronary endothelial cells in culture. The functional significance of these receptors remains to be established, but they may be involved in the regulation of vascular permeability.Abbreviations NECA 5-N-ethylcarboxamidoadenosine - PIA N⁶-phenylisopropyladenosine - 8-PT 8-phenyltheophylline - IBMX 3-isobutyl-l-methylxanthine - IBMX shirlyDL,-4-(3-butoxy-4methoxybenzyl)-2-imidazolidinone Postdoctoral fellow of the Medical Research Council of Canada Send offprint requests to S. Nees at the above address     

Adenosine receptors involved in the inhibitory control of non-adrenergic non-cholinergic neurotransmission in guinea-pig atria belong to the A1 subtype

Summary We have previously shown that endogenous adenosine inhibits non-adrenergic, non-cholinergic (NANC) neurotransmission in isolated guinea-pig atria. In the present study the effect of adenosine analogues, such as N⁶cyclopentyladenosine (CPA), 5 N-ethylcarboxamide adenosine (NECA), 2 chloroadenosine (2-CADO), R- and S-phenylisopropyladenosine (R- and S-PIA) on the cardiac response to transmural nerve stimulation has been tested in order to characterize the subtype of adenosine receptor involved in the inhibitory control of NANC neurotransmission. The effect of the adenosine antagonist 8-phenyltheophylline (8-PT) was then tested against CPA and NECA.The prototypical A-1 selective agonist CPA was the most active agonist, reducing the response to the stimulation of NANC nerves with an IC₅₀ value of 2.8 nM; RPIA, NECA and 2-CADO showed IC₅₀ values of 9.5, 13.7 and 35 nM respectively. S-PIA was the least active agonist, showing an IC₅₀ value (306 nM) about 30-fold greater than that of R-PIA (9.5 nM). None of the agonists tested was able to modify cardiac response to exogenous CGRR Furthermore, 8-PT competitively antagonized the effect of CPA and NECA with very close pA₂ values (6.77±0.01 and 6.63±0.08 respectively). From these findings we concluded that prejunctional inhibitory adenosine receptors on capsaicin sensitive sensory nerves of cardiac tissue belong to the A-1 subtype. Send offprint requests to A. Rubino at the above address     

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     

Psychomotor stimulant effects of methylxanthines in squirrel monkeys: relation to adenosine antagonism

The behavioral effects of six methylxanthines were studied in squirrel monkeys responding under a fixed-interval (FI) schedule of stimulus-shock termination. Dose-response curves were determined for each drug by administering cumulative doses IV during timeout periods that preceded sequential components of the FI schedule. Low to intermediate doses of caffeine, theophylline, 8-phenyltheophylline (8-PT), 8-cyclopentyltheophylline (CPT), and 3-isobutyl-1-methylxanthine (IBMX) produced dose-related increases in response rate, whereas higher doses increased response rate less or decreased it. Enprofylline did not increase response rate at any dose. Pretreatment with a high dose of the adenosine agonist 5-N-ethylcarboxamide adenosine (NECA) suppressed responding throughout the experimental session. Caffeine, theophylline, 8-PT, CPT, and IBMX, but not enprofylline, antagonized the suppressant effects of NECA in a dose-related manner. The potencies of the methylxanthines for increasing response rate under the FI schedule were positively correlated with their potencies for antagonizing the suppressant effects of NECA, suggesting that the psychomotor stimulant effects of methylxanthines are linked to their antagonistic actions at adenosine recognition sites. CPT, which in vitro has much higher affinity (>10³-fold) than caffeine for adenosine A₁, but not for A₂, recognition sites, was only 3–6 times more potent than caffeine in increasing response rate or in antagonizing the effects of NECA. The psychomotor stimulant effects of methylxanthines therefore appear to be more closely associated with antagonism at adenosine A₂ than adenosine A₁ recognition sites.Animals used in this study were maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School and of the Guide for Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council, Department of Health, Education and Welfare, Publication No. (NIH)85-23, revised 1985     

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

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

The effect of N6-cyclohexyladenosine and 5′-N-ethylcarboxamidoadenosine on body temperature in normothermic rabbits.

• 1.1. Thermal responses to IV administration of N⁶-cyclohexyladenosine (CHA; 0.15 mg/ kg), A₁ adenosine receptor agonist, or 5′-N-ethylcarboxamidoadenosine (NECA; 0.15 mg/kg), A₂ adenosine receptor agonist, were investigated in normothermic rabbits at an ambient temperature (Ta) of 20.0±1.0°C.
• 2.2. Although both compounds inhibited metabolic heat production, only NECA produced hypothermia.
• 3.3. NECA showed strong hypotensive activity.
• 4.4. Both compounds produced vasoconstriction of the ear skin vessels and CHA, in addition, slowed down the respiratory rate.
• 5.5. The role of A₁ or A₂ adenosine receptors in the thermoregulatory activity of these compounds is discussed.

     

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     

Pharmacological characterization of adenosine A1 and A2 receptors in the bladder: evidence for a modulatory adenosine tone regulating non-adrenergic non-cholinergic neurotransmission.

1. The nerve-evoked contractions elicited by transmural electrical stimulation of mouse urinary bladders superfused in modified Krebs Ringer buffer containing 1 microM atropine plus 3.4 microM guanethidine were inhibited by adenosine (ADO) and related nucleoside analogues with the following rank order of potency: R-phenylisopropyladenosine (R-PIA) greater than cyclohexyladenosine (CHA) greater than 5'N-ethylcarboxamido adenosine (NECA) greater than ADO greater than S-phenylisopropyladenosine (S-PIA). Tissue preincubation with 8-phenyltheophylline (8-PT) displaced to the right, in a parallel fashion, the NECA concentration-response curve. 2. The contractions elicited by application of exogenous adenosine 5'-triphosphate (ATP) were also inhibited by ADO and related structural analogues. The rank order of potency to reduce the motor response to ATP was: NECA greater than 2-chloroadenosine (CADO) greater than R-PIA greater than ADO greater than CHA greater than S-PIA. 3. The ADO-induced ATP antagonism was of a non-competitive nature and was not specific. Tissue incubation with 10 microM NECA not only reduced the motor responses elicited by ATP, but also 5-hydroxytryptamine, acetylcholine and prostaglandin F2 alpha. The action of NECA was antagonized following tissue preincubation with 8-PT. The inhibitory action of NECA was not mimicked by 10 microM CHA. 4. The maximal bladder ATP contractile response was significantly increased by tissue preincubation with 5-30 microM 8-PT. 5. The 0.15 Hz evoked muscular twitch was significantly increased by 8-PT while dipyridamole consistently reduced the magnitude of the twitch response. These results are consonant with the hypothesis that an endogenous ADO tone modulates the bladder neurotransmission.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further pharmacological characterization of the adenosine receptor subtype mediating inhibition of oxidative burst in human isolated neutrophils

The aims of this study were to characterize the adenosine receptor subtype mediating inhibition of superoxide anion generation induced by N-formyl-methionyl-leucylphenylalanine (fMLP) in human neutrophils and to test the hypothesis that adenosine 3′:5′-cyclic monophosphate (cAMP) is the second messenger mediating such inhibition. Superoxide anion generation induced by a submaximal concentration of fMLP (1 μM) was inhibited in a concentration-dependent manner by adenosine receptor agonists with a rank order of potency ofN-ethylcarboxamidoadenosine (NECA) > 2(4-[(2- carboxyethyl)phenyl]ethylamino)-5′-N-ethylcarboxamido adenosine (CGS 21680) > (R)-N⁶-phenyl-2-propyladenosine ((R)-PIA) > 2-Chloro-N⁶-(3-iodobenzyl)9-[5-methylcarbamoyl)-β-D-ribofuranosyl] adenine (2-Cl-IB-MECA) > N⁶-cyclopentyladenosine (CPA) > (S)-N⁶-phenyl-2-propyladenosine ((S)-PIA) ≥ N⁶-(4-amino-3-iodobenzyl) adenosine-5′-N-methyl-uronamide (AB-MECA); this order of potency is consistent with the activation of A_2A adenosine receptors. The nonselective A₁, A_2A, and A_2B receptor antagonist 8-p-(sulphophenyl) theophylline (8-SPT; 10 μM) produced blockade of each of the agonists (pK_B values 4.79–5.68). The selective A₁ adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 100 nM) and the selective A₃ adenosine receptor antagonist 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propylxanthine (I-ABOPX; 200 nM) also produced blockade of all the agonists (PK_B values 7.37–7.61 and 6.75–7.52, respectively); however, the concentrations required were higher than those which are considered selective for the A₁ or A₃ receptors. The selective A_2A receptor antagonist, 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; 100 nM), powerfully suppressed the inhibition of the oxidative burst induced by each of the agonists. A Schild analysis of the effects of ZM 241385, 1–100 nM, against NECA and CGS 21680 was carried out. ZM 241385 produced concentration-dependent, parallel shifts of the concentration–effect curves to both NECA and CGS 21680, with pA₂ values of 9.62 and 9.59, respectively. Together, these data establish that inhibition of the oxidative burst in human isolated neutrophils, induced by adenosine receptor agonists, is mediated by the A_2A receptor. NECA (0.01–10 μM) induced a concentration-dependent increase in the intracellular cAMP content of neutrophils. This effect was inhibited in a dose-dependent manner by ZM 241385 (0.001–10 μM), consistent with activation of A_2A adenosine receptors. The results clearly demonstrate that in human neutrophils inhibition of the fMLP—induced oxidative burst by adenosine receptor agonists is mediated via activation of A_2A adenosine receptors linked positively to cAMP. No evidence of A₁, A_2B, or A₃ adenosine receptor-mediated modulation of oxidative burst was found. Drug Dev. Res. 43:214–224, 1998. © 1998 Wiley-Liss, Inc.