R-N6-phenylisopropyladenosine Research Articles

Study of the novel non-xanthine heterocyclic compound GU285 as a potent non-selective adenosine receptor antagonist in the rat.

The novel pyrazolo[3,4-d]pyrimidine compound GU285 (4-amino-6-alpha-carbamoylethylthio-1- phenylpyrazolo[3,4-d]pyrimidine, CAS 134896-40-5) was examined for its ability (1) to inhibit binding of adenosine (ADO) receptor ligands in rat brain membranes, (2) to antagonise functional responses to ADO agonists in rat right and left atria and coronary resistance vessels, and (3) to reduce the fall in heart rate and arterial blood pressure produced by the ADO A1 agonist N6-cyclopentyladenosine (CPA) in the intact, anaesthetized rat. GU285 competitively inhibited binding of the ADO A1 agonist [3H]-R-N6-phenylisopropyladenosine (R-PIA) yielding a Ki value of 11 (7-18) nmol.l-1 (geometric mean +/- 95% Cl). When assayed against the ADO A2A selective agonist [3H]-2-[p-(2-carboxyethyl)- phenethylamino]-5'-N-ethylcarboxamidoadenosine, (CGS21680), a Ki of 15 (10-24) nmol.l-1 was obtained. In spontaneously beating right atria, GU285 competitively antagonized negative chronotropic effects of R-PIA with a pA2 of 8.7 +/- 0.3 and in electrically paced left atria, GU285 competitively antagonized negative inotropic effects of R-PIA with a pA2 of 9.0 +/- 0.1. In the potassium-arrested, perfused rat heart GU285 (1 mumol.l-1) antagonized only the high sensitivity, ADO A2B mediated component of the biphasic relaxation of the coronary vasculature produced by NECA. The low sensitivity component was unchanged. GU285 (1 mumol.kg-1) antagonized the negative chronotropic and hypotensive effects of the adenosine A1 agonist CPA in anaesthetized rats, producing a 10-fold rightward shift in the dose-response relationship. These data demonstrate that in the rat, GU285 is a potent, non-selective adenosine receptor antagonist that maintains its activity in vivo.

Adenosine A3 receptors regulate heart rate, motor activity and body temperature

To examine the phenotype of mice that lack the adenosine A(3) receptor (A(3)R). We examined the heart rate, body temperature and locomotion continuously by telemetry over several days. In addition, the effect of the adenosine analogue R-N(6)-phenylisopropyl-adenosine (R-PIA) was examined. We also examined heat production and food intake. We found that the marked diurnal variation in activity, heart rate and body temperature, with markedly higher values at night than during day time, was reduced in the A(3)R knock-out mice. Surprisingly, the reduction in heart rate, activity and body temperature seen after injection of R-PIA in wild type mice was virtually eliminated in the A(3)R knock-out mice. The marked reduction in activity was associated with a decreased heat production, as expected. However, the A(3)R knock-out mice, surprisingly, had a higher food intake but no difference in body weight compared to wild type mice. The mice lacking adenosine A(3) receptors exhibit a surprisingly clear phenotype with changes in diurnal rhythm and temperature regulation. Whether these effects are due to a physiological role of A(3) receptors in these processes or whether they represent a role in development remains to be elucidated.

Prevention or reversal of sensorineural hearing loss (SNHL) through biologic mechanisms

The invention is accomplished by preventing and/or reversing inner ear damage due to noise or toxins. In part, this is accomplished by upregulating antioxidant enzyme activity by applying agents such as R-N6-Phenylisopropyl adenosine (R-PIA) to the round window membrane of the inner ear or systemically, and/or by also applying agents such as 1-2-oxothiazolidine-4-carboxylic acid (Procysteine) to the round window membrane. Also, the invention is accomplished by giving the compounds systemically. Selective auditory hair cell protection in the face of gentamicin exposure by concomitant delivery of an NMDA antagonist or glial derived neurotrophic factor (GDNF) with the gentamicin. These and additional agents are also accomplished by curtailing activated programmed cell death pathways and/or inducing/enhancing cell repair mechanisms in the inner ear. The agent (s) may be applied before, during or after the noise trauma or toxin exposure.

Noise-induced hearing loss in chinchillas pre-treated with glutathione monoethylester and R-PIA

The protective effects of glutathione monoethylester (GEE) and GEE in combination with R- N6-phenylisopropyladenosine (R-PIA) were evaluated in the chinchilla when exposed to impulse (145 dB pSPL) or continuous (105 dB SPL, 4 kHz OB) noise. Six groups of 10 chinchillas were used as subjects. Before exposure to noise, the subjects were anesthetized, a 30 μl drop of drug was placed on the round window (GEE [50, 100, 150 mM], GEE 50 mM and R-PIA). Forty minutes later the subject was exposed to either impulse or continuous noise. The 50 mM treatment provided significant protection from impulse noise, but not from continuous noise exposure. The combination provided significant protection from both the continuous and impulse noise. In a separate set of experiments, glutathione (GSH) levels were measured in the perilymph. All the drug treatments elevated GSH levels. The results are discussed in terms of antioxidant treatments as a prophylactic measure against noise-induced hearing loss.

Adenosine receptor–adenylate cyclase system in the trout testis: Involvement in the regulation of germ cell proliferation

To ascertain the presence of adenosine receptors in the trout testis, cells isolated from testes at different spermatogenetic stages were cultured in the presence or absence of adenosine, adenosine receptor agonists, or antagonists and of cAMP analogs, for up to 20 min, or 20 hr, or 4.5 days. Cyclic AMP production was then assayed or 3H-thymidine incorporation was measured. Cellular content of cAMP was enhanced by adenosine, by the adenosine receptor agonist 5′-N-ethylcarboxamidoadenosine (NECA), and by 2-p(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS-21680), an adenosine A2A receptor-selective agonist. The increase in cAMP induced by the adenylate cyclase activator L-858051 was inhibited by the adenosine A1 receptor-selective agonists R-N6-(2-phenylisopropyl)adenosine (R-PIA) and N6-cyclopentyladenosine (CPA). These effects were antagonized by the two adenosine A2 receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX) and 8-(3-chlorostyryl)caffeine (CSC), and by the adenosine A1 receptor-selective antagonist 8-cyclopentyl-1,3dipropylxanthine (CPX), respectively. Increase in the cAMP content induced by adenosine was inhibited by the cell permeable adenylate cyclase inhibitor 2′,5′-dideoxyadenosine. These data suggest that A1 and A2 adenosine receptors which respectively inhibit and stimulate adenylate cyclase activity are present on trout testicular cells (unidentified), while the presence of A3 adenosine receptor subtype was not apparent. 3H-thymidine incorporation decreased in the presence of the adenylate cyclase activator L-858051 and of the cAMP analogs 8-CPT cAMP and Sp-5,6-DCI-cBiMPS, regardless of the presence or absence of the phosphodiesterase inhibitor RO 20-1724. This suggests that an increase in testicular cAMP may act as a negative growth regulator for the mitotic germ cells. In agreement with these data, the activation of A2 stimulatory receptors inhibited short-term (20 hr) DNA synthesis. However, the activation of A1 inhibitory receptors had the same effect. This suggests that events, cAMP-dependent or independent, induced by the activation of testicular adenosine receptors, may participate in the regulation of trout male germ cell proliferation. Mol. Reprod. Dev. 58:307–317, 2001. © 2001 Wiley-Liss, Inc.

Adenosine receptor-adenylate cyclase system in the trout testis: Involvement in the regulation of germ cell proliferation

To ascertain the presence of adenosine receptors in the trout testis, cells isolated from testes at different spermatogenetic stages were cultured in the presence or absence of adenosine, adenosine receptor agonists, or antagonists and of cAMP analogs, for up to 20 min, or 20 hr, or 4.5 days. Cyclic AMP production was then assayed or 3H-thymidine incorporation was measured. Cellular content of cAMP was enhanced by adenosine, by the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), and by 2-p(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680), an adenosine A2A receptor-selective agonist. The increase in cAMP induced by the adenylate cyclase activator L-858051 was inhibited by the adenosine A1)receptor-selective agonists R-N6-(2-phenylisopropyl)adenosine (R-PIA) and N6-cyclopentyladenosine (CPA). These effects were antagonized by the two adenosine A2)receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX) and 8-(3-chlorostyryl)caffeine (CSC), and by the adenosine A1)receptor-selective antagonist 8-cyclopentyl-1,3dipropylxanthine (CPX), respectively. Increase in the cAMP content induced by adenosine was inhibited by the cell permeable adenylate cyclase inhibitor 2',5'-dideoxyadenosine. These data suggest that A(1) and A(2) adenosine receptors which respectively inhibit and stimulate adenylate cyclase activity are present on trout testicular cells (unidentified), while the presence of A3 adenosine receptor subtype was not apparent. 3H-thymidine incorporation decreased in the presence of the adenylate cyclase activator L-858051 and of the cAMP analogs 8-CPT cAMP and Sp-5,6-DCI-cBiMPS, regardless of the presence or absence of the phosphodiesterase inhibitor RO 20-1724. This suggests that an increase in testicular cAMP may act as a negative growth regulator for the mitotic germ cells. In agreement with these data, the activation of A2 stimulatory receptors inhibited short-term (20 hr) DNA synthesis. However, the activation of A1 inhibitory receptors had the same effect. This suggests that events, cAMP-dependent or independent, induced by the activation of testicular adenosine receptors, may participate in the regulation of trout male germ cell proliferation.

Mechanism of adenosine-induced vasodilation in rat diaphragm microcirculation.

The mechanism of adenosine-induced vasodilation in rat diaphragm microcirculation was investigated using laser Doppler flowmetry. Adenosine (10(-5), 3.2 x 10(-5), and 10(-4) M), the nonselective adenosine agonist 5'-N-ethylcarboxamido-adenosine (NECA) (10(-8)-10(-7) M), the specific A(2A) agonist 2-p-(2-carboxyethyl)phenyl-amino-5'-N-ethyl carboxamidoadenosine (CGS-21680) (10(-8)-10(-7) M), and the adenosine agonist with higher A(1)-receptor affinity, R-N(6)-phenylisopropyladenosine (R-PIA) (10(-7), 3.2 x 10(-7), and 10(-6) M) elicited a similar degree of incremental increase of microcirculatory flow in a dose-dependent manner. The ATP-dependent potassium (K(ATP)) channel blocker glibenclamide (3.2 x 10(-6) M) significantly attenuated the vasodilation effects of these agonists. Adenosine-induced vasodilation could be significantly attenuated by the nonselective adenosine antagonist 8-(p-sulfophenyl)-theophylline (3 x 10(-5) M) or the selective A(2A) 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, 10(-6) M), but not by the selective A(1) antagonist 8-cyclopentyl-1, 3-dipropylxanthine (5 x 10(-8) M). Adenylate cyclase inhibitor N-(cis-2-phenyl-cyclopentyl) azacyclotridecan-2-imine-hydrochloride (MDL-12330A, 10(-5)M) effectively suppressed the vasodilator response of adenosine and forskolin. These results suggest that adenosine-induced vasodilation in rat diaphragm microcirculation is mediated through the stimulation of A(2A) receptors, which are coupled to adenylate cyclase activation and opening of the K(ATP) channel.

THE MECHANISM OF CONTRACTION BY 2-CHLOROADENOSINE IN CAT DETRUSOR MUSCLE CELLS

Four adenosine receptors (ARs), designated A1AR (A1 adenosine receptor), A2aAR (A2a adenosine receptor), A2bAR (A2b adenosine receptor), and A3AR (A3 adenosine receptor), have been cloned from various species, but the contraction mechanism via A1ARs in cat detrusor muscle cell is not well known. We examined the cellular mechanism using an A1AR agonist 2-chloroadenosine (2-CA) in cat detrusor cell isolated by enzymatic digestion. To examine which phospholipase mediates the contraction, we used phospholipase inhibitors. The adenosine analog potency order is R-N6-phenylisopropyladenosine (R-PIA) > 5'-N-ethylcarbosamine adenosine (NECA) > 2-chloroadenosine (2-CA) > S-N6-phenylisopropyladenosine (S-PIA). The ratio of equi-effective concentrations of R-PIA/S-PIA was 58.2. 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 300 nM) shifted to the right the concentration-response curves of 2-CA. These results indicate A1ARs mediate 2-CA induced contraction in cat detrusor muscle. G proteins (Gi1, Gi2, Gi3, Go, Gs, and Gq) in cat detrusor muscle were detected by immunoblot analysis. Pertussis toxin (PTX) inhibited 2-CA induced contraction. In permeabilized cells, antibodies against Galphai3 antagonized 2-CA induced contraction, suggesting that the contraction is mediated by Gi3 protein. A phosphatidylinositol-specific phospholipase C (PLC) inhibitor, neomycin, reduced 2-CA induced contraction, but a phospholipase D (PLD) inhibitor, p-chloromercuribenzoic acid, and a phospholipase A2 (PLA2) inhibitor, dimethyl-eicosa-dienoic acid (DEDA), had no effect. We found the presence of the main PLC isozymes, PLC-beta1, PLC-beta3, and PLC-gamma1. 2-CA induced contraction in permeabilized cells was inhibited by PLC-beta3 but not by PLC-beta1 or PLC-gamma1 antibody. These results imply that A1ARs are coupled to PLC-beta3 via PTX-sensitive Gi3 protein. Sr2+ medium and thapsigargin, which replaces intracellular Ca2+ and deplete intracellular calcium stores respectively, inhibited 2-CA induced contraction. These results suggest that A1ARs mediating 2-CA induced contraction exist in cat detrusor muscle and the contraction depends on a PTX-sensitive Gi3 protein, PLC-beta3 and the release of intracellular Ca2+.

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

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

Potentiation by adenosine of histamine-induced bronchospasm in anaesthetized guinea-pig: Receptor subtype/s involved

A potentiating effect by adenosine on the bronchocontractile response to histamine has been previously described in a guinea-pig model (Breschi et al. [1994] Pharmacology 49:42-51). In the present study, the subtype(s) of purinergic receptors involved in this modulatory effect of the nucleoside have been investigated. A possible role for P2 receptors or for the putative P3 receptor subtype was excluded because of the very poor modulatory effect of the P2 agonist α,β-methylene ATP when compared with the effects of all the P1 agonists tested, with the exception of 5′-N-Methyl-N6-(3-iodobenzyl)adenosine. The order of potency revealed for the P1 agonists was R-N6-[2-phenylisopropyl]adenosine (R-PIA) = 5′-N-methylcarboxamidoadenosine > N6-2-(4-aminophenyl)ethyladenosine ≥ adenosine and in the presence of R-PIA, a significantly greater maximal effect was observed. Among the adenosine receptor antagonists evaluated, the A2 antagonist 3,7-dimethyl-1-propargylxanthine even increased the effect of adenosine and no influence was evident with the reported A2B antagonist enprofylline. A significant reduction of the potentiating activity of adenosine was, on the contrary, obtained with the nonselective blocker theophylline or the A1 antagonist DPCPX. In conclusion, the above data suggest that the potentiation of histamine-induced bronchoconstriction by adenosine is mediated by the stimulation of A1 receptors. Drug Dev. Res. 45:17–22, 1998. © 1998 Wiley-Liss, Inc.

Protection by an Adenosine Analogue against Kainate-Induced Extrahippocampal Neuropathology

1. The glutamate analogue kainic acid produces neuronal damage in the central nervous system. We have reported that analogues of adenosine, such as R-N6-phenylisopropyladenosine (R-PIA) can, at doses as low as 10 μg/kg IP, prevent the hippocampal damage that follows the systemic administration of kainate. The present work was designed to examine purine protection against kainate in extrahippocampal regions by using histological methods. 2. The results show that R-PIA, at a dose of 25 μg/kg IP in rats, can protect against the neuronal damage caused by kainate in the basolateral amygdaloid nuclei, the pyriform cortex and around the rhinal fissure. This protection could be prevented by the simultaneous administration of the A1 adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine, confirming that the protection involved adenosine A1 receptors. No protection was observed in the posterior amygdaloid nuclei or the entorhinal cortex, suggesting the absence of relevant adenosine receptors or a different mechanism of excitotoxicity.

Characterization of human A2A adenosine receptors with the antagonist radioligand [3H]-SCH 58261.

1. We have characterized the binding of the new potent and selective antagonist radioligand [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazol o[1,5- c]pyrimidine, [3H]-SCH 58261, to human cloned A2A adenosine receptors. 2. In Chinese hamster ovary (CHO) cells transfected with the human cloned A2A receptor, [3H]-SCH 58261 specific binding (about 70%) was rapid, saturable, reversible and proportional to protein concentration. The kinetic KD value was 0.75 nM. Saturation experiments showed that [3H]-SCH 58261 labelled a single class of recognition sites with high affinity (KD = 2.3 nM) and limited capacity (apparent Bmax = 526 fmol mg-1 protein). 3. Competition experiments revealed that binding of 0.5 nM [3H]-SCH 58261 was displaced by adenosine receptor agonists with the following order of potency: 2-hexynyl-5'-N-ethylcarboxamidoadenosine (2HE-NECA) > 5'-N-ethylcarboxamidoadenosine (NECA) = 2-phenylaminoadenosine (CV 1808) > 2-[4-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosi ne (CGS 21680) > R-N6-phenylisopropyladenosine (R-PIA) > or = N6-cyclohexyladenosine (CHA) > S-N6-phenylisopropyladenosine (S-PIA). 4. Adenosine receptor antagonists inhibited [3H]-SCH 58261 binding with the following order: 5-amino-9-chloro-2-(2-furyl)-[1,2,4]-triazolo[1,5-c] quinazoline (CGS 15943) > SCH 58261 > xanthine amine congener (XAC) > (E,18%-Z,82%)7-methyl-8-(3,4-dimethoxystyryl)-1,3- dipropylxanthine (KF 17837S) > 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) > theophylline. 5. Affinity values and rank order of potency of both receptor agonists and antagonists were similar to those previously obtained in human platelet and rat striatal membranes, except for CV 1808 which was more potent than CGS 21680. SCH 58261 was a competitive antagonist at inhibiting NECA-induced adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in CHO cells transfected with human A2A receptors. Good agreement was found between binding and functional data. 6. Thus, the new antagonist radioligand is preferable to the receptor agonist radioligand [3H]-CGS 21680 hitherto used to examine the pharmacology of human cloned A2A adenosine receptors.

Analysis of the bronchoconstrictor responses to adenosine receptor agonists in sensitized guinea-pig lungs and trachea

Airway perfused lungs and half-lungs and superfused tracheal spirals from ovalbumin-sensitized guinea pigs were set up. Adenosine and the analogues, 5′-( N-ethylcarboxamido)adenosine (NECA), R-N 6-phenylisopropyladenosine ( R-PIA), 2-chloroadenosine, N 6-2-(4-aminophenyl)ethyladenosine (APNEA) and 5′-AMP yielded bronchoconstrictor responses as increases in perfusion pressure or of tension, respectively, of these two preparations. These responses were greater in tissues from sensitized compared with un-sensitized guinea pigs. Cross-tachyphylaxis occurred between the constrictor responses to adenosine and the other constrictor adenosine agonists which indicated a common site of action. The adenosine transport inhibitors, dipyridamole and S-(p-nitrobenzyl)-6-thioinosine (NBTI), inhibited the constrictor responses to adenosine and the analogues, except 2-chloroadenosine. This was attributed to a potentiation of the opposing relaxant effects which generally occurred at higher concentrations of the agonists. The P 1 purinoceptor antagonists 8-phenyltheophylline and 8-cyclopentyltheophylline (A 1 receptor selective) failed to remove the constrictor responses to adenosine either alone or in the presence of dipyridamole. This suggests that the bronchoconstrictor response of sensitized airways tissues is mediated via the novel xanthine-resistant A 3 receptor.

Prevention by a purine analogue of kainate-induced neuropathology in rat hippocampus

Systemic injection of kainic acid produces a characteristic regional and cellular pattern of neuronal loss in the central nervous system by mechanisms which may be relevant to an understanding of neurodegenerative disorders. It has previously been found, by measuring the binding of a glial marker ligand, that analogues of adenosine, such as R- N6-phenylisopropyladenosine (R-PIA), can prevent kainate-induced damage of the hippocampus at doses as low as 10 μg/kg, i.p. The use of gliotic markers, however, is open to misinterpretation, and the present work was designed to re-examine purine protection against kainate using histological methods. The results show that R-PIA, at a dose of 25 μg/kg i.p. in rats, can protect against the neuronal damage caused by kainate and that this protection could be completely prevented by the simultaneous administration of 1,3-dipropyl-8-cyclopentylxanthine, indicating the involvement of adenosine A 1 receptors in the protection.

Binding of the radioligand [3H]-SCH 58261, a new non-xanthine A2A adenosine receptor antagonist, to rat striatal membranes.

1. The present study describes the binding to rat striatal A2A adenosine receptors of the new potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazol o [1,5-c] pyrimidine, [3H]-SCH 58261. 2. [3H]-SCH 58261 specific binding to rat striatal membranes ( > 90%) was saturable, reversible and dependent upon protein concentration. Saturation experiments revealed that [3H]-SCH 58261 labelled a single class of recognition sites with high affinity (Kd = 0.70 nM) and limited capacity (apparent Bmax = 971 fmol mg-1 of protein). The presence of 100 microM GTP in the incubation mixture did not modify [3H]-SCH 58261 binding parameters. 3. Competition experiments showed that [3H]-SCH 58261 binding is consistent with the labelling of A2A striatal receptors. Adenosine receptor agonists competed with the binding of 0.2 nM [3H]-SCH 58261 with the following order of potency: 2-hexynyl-5'-N-ethyl carboxamidoadenosine (2HE-NECA) > 5'-N-ethylcarboxamidoadenosine (NECA) > 2-[4-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosi ne (CGS 21680) > 2-phenylaminoadenosine (CV 1808) > R-N6-phenylisopropyladenosine (R-PIA) > N6-cyclohexyladenosine (CHA) = 2-chloro-N6-cyclopentyladenosine (CCPA) > S-N6-phenylisopropyladenosine (S-PIA). 4. Adenosine antagonists inhibited [3H]-SCH 58261 binding with the following order: 5-amino-9-chloro-2-(2-furyl)-[1,2,4]-triazolo[1,5-c] quinazoline (CGS 15943) > 5-amino-8-(4-fluorobenzyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4-triazolo [1,5-c] pyrimidine (8FB-PTP) = SCH 58261 > xanthine amine congener (XAC) = (E,18%-Z,82%)7-methyl-8-(3,4-dimethoxystyryl)-1,3-dipropylxanthine (KF 17837S) > 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) > or = 8-phenyltheophylline (8-PT). 5. The Ki values for adenosine antagonists were similar to those labelled with the A2A agonist [3H]-CGS 21680. Affinities of agonists were generally lower. The A1-selective agonist, R-PIA, was found to be about 9 fold more potent than its stereoisomer, S-PIA, thus showing the stereoselectivity of [3H]-SCH 58261 binding. Except for 8-PT, the adenosine agonists and antagonists examined inhibited [3H]-SCH 58261 binding with Hill coefficients not significantly different from unity. 6. The present results indicate that [3H]-SCH 58261 is the first non-xanthine adenosine antagonist radioligand which directly labels A2A striatal receptors. High receptor affinity, good selectivity and very low non-specific binding make [3H]-SCH 58261 an excellent probe for studying the A2A adenosine receptor subtype in mammalian brain.

A-type potassium current modulated by A1 adenosine receptor in frog melanotrophs.

1. Transient outward current was recorded in cultured frog melanotrophs with the whole-cell configuration of the patch-clamp technique. The ionic dependence, kinetics and pharmacological properties of the current were studied. The effects of the A1 adenosine receptor agonist R-N6-phenylisopropyl-adenosine (R-PIA) on this current were also investigated. 2. In tetrodotoxin- and cobalt-containing solution, depolarization from -120 mV elicited both transient and delayed outward currents. Pulses from -60 mV activated only a sustained late current. 3. 4-Aminopyridine (4 mM) reduced the transient outward current much more than the delayed outward current. In contrast, tetraethylammonium (10-20 mM) selectively reduced the delayed current. 4. Tail current measurements showed a positive shift in the reversal potential when external K+ concentration was increased, indicating that K+ was the predominant charge carrier. 5. Steady-state inactivation was complete at potentials positive to -10 mV and removed by hyperpolarization. 6. Inactivation of the transient current was slowed and accelerated in oxidizing and reducing conditions, respectively, confirming the involvement of an inactivating 'ball and chain' peptide. 7. R-PIA increased the transient current. The steady-state inactivation curve was shifted towards more positive potentials without changing the activation kinetics. Pretreatment with pertussis toxin (1 microgram ml-1) blocked the response to R-PIA. 8. It is concluded that frog melanotrophs possess an A-type current that is likely to play an important role in excitability. This current, which is directly modulated by A1 adenosine receptors through a Gi/G(o) protein, appears to be responsible for the inhibitory effects of adenosine on electrical activity.

Functional coupling between A1 adenosine receptors and G-proteins in rat hippocampal membranes assessed by high-affinity GTPase activity.

1. In order to investigate the functional interaction between adenosine receptors and G-proteins in native brain membranes, stimulation of high-affinity GTPase activity by adenosine receptor agonists was characterized in rat hippocampal membranes. 2. Addition of 1 microM R-N6-phenylisopropyladenosine (R-PIA), a selective A1 adenosine receptor agonist, augmented the Vmax of the low-KM GTPase by 51%, with a slight increase in the KM value. 3. Several adenosine receptor agonists stimulated the high-affinity GTPase activity in a concentration-dependent manner, with a rank order of potency indicative of the involvement of A1 adenosine receptor subtype as follows: R-PIA > N6-cyclohexyladenosine > 5'-N-ethylcarboxamidoadenosine > or = 2-chloroadenosine > S-PIA > CGS 21680, 2-phenylaminoadenosine. 4. The selective A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine, inhibited R-PIA-stimulated high-affinity GTPase activity in a competitive manner, with a KB value of 2.5 nM. 5. The activating effects on high-affinity GTPase of R-PIA (via A1 adenosine receptors) and of 5-HT (via 5-HT1A receptors) were completely additive, indicating that A1 adenosine and 5-HT1A receptors were coupled to distinct pools of G-proteins in hippocampus. 6. Stimulation of high-affinity GTPase activity by adenosine receptor agonists can be used as a valuable measure for the investigation of the functional coupling between A1 adenosine receptors and G-proteins associated with adenylyl cyclase inhibition.

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-phenylisopropyladenosine (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 66713 (an antagonist at A2a receptors), nor by endothelial removal and inhibition of nitric oxide synthase (100 microM NG-nitro-L-arginine: L-NOARG).(ABSTRACT TRUNCATED AT 250 WORDS)

Role of 5-hydroxytryptamine in mediating adenosine-induced airway contraction

We have investigated the role of 5-hydroxytryptamine (5-HT) on adenosine-induced guinea-pig trachea contraction. R-N6-phenylisopropyladenosine (R-PIA), an A1 receptor subtype agonist, induced a concentration-dependent contraction of tracheal rings. The pD2 values were 7.43 ± 0.26. A 30-min pretreatment with 1,3-dipropyl-8-amino-4-clorophenyl-xantine (PACPX), a selective A1 receptor antagonist, shifted to the right the R-PIA concentration effect curves. Ketanserin (1 μM), a 5-HT2 receptor antagonist, also caused a rightward shift of the R-PIA concentration-effect curves. The changes for the pD2 values comparing the controls and the tissues incubated with ketanserin were statistically significant (P < 0.05). In the same experimental conditions, neither atropine (1 μM), nor diphenydramine (1 μM), nor indomethacin (5 μM) showed any effects. The challenge of R-PIA (1 μM) with said substances induced a release of 5-HT (4.8 ± 0.20 fmol/ml) from guinea-pig trachea in presence or in absence of epithelium; in the same experimental conditions, this effect did not occur in the controls. Our data support the hypothesis that 5-HT plays a role in adenosine-induced airway contraction.

The allosteric enhancer, PD 81,723, stabilizes human A 1 adenosine receptor coupling to G proteins

2-Amino-3-benzoylthiophenes such as PD 81,273 (PD) have been shown to increase agonist, but not antagonist, radioligand binding and to enhance functional effects of A 1 adenosine receptor (A 1AR) activation in tissues derived from rats and guinea-pigs. The mechanism by which PD produces this allosteric enhancement, and its effect on human A 1ARs was not known. In this study, we demonstrate that PD modifies recombinant human A 1AR binding and function in stably transfected CHO cells. In membranes, PD (20 μM) causes: (i) a 3-fold increase in the fraction of receptors found in a high affinity G-protein coupled conformation as assessed by the binding of [ 125 I]N 6-(3- iodo-4- aminobenzyladenosine) ( 125I-ABA), an A 1AR agonist; (ii) a 2.44-fold increase in the potency of the agonist R- N 6-phenylisopropyladenosine ( R-PIA) to compete for binding with the antagonist radioligand, [ 3H]8-cyclopentyl-1,3-dipro-pylxanthine ([ 3H]CPX); (iii) a 1.5-fold increase in the t 1 2 of 125I-ABA to dissociate from A 1AR; and (iv) a 2.2-fold increase in the concentration of guanosine-5′-3-0-(thio)triphosphate (GTPγS) required to half-maximally uncouple receptor-G-protein complexes. In intact CHO cells expressing A 1AR, PD increases the potency of R-PIA to decrease forskolin-stimulated cAMP accumulation by 3.3-fold. We speculate that PD binds to A 1AR at a site distinct from the agonist binding site and stabilizes agonist-R-G complexes.