Characterization of adenosine binding sites in bovine testicular tissue using 8-cyclopentyl-1,3-[3H]dipropylxanthine

The existence of specific adenosine binding sites in bovine testicular tissue was evaluated using the novel antagonist radioligand 8-cyclopentyl-1,3-[3H]dipropylxanthine ([3H]DPCPX). Saturation analysis revealed specific binding that was saturable at approximately 1 nM. Scatchard analysis indicated a single class of binding sites with a KD = 0.26 nM and a Bmax = 0.37 pmol/mg protein. Affinity profiles suggest an A1 subtype recognition site that is different from the classical A1 adenosine receptor. The results presented should prove useful in subsequent studies concerning heterogeneity among adenosine receptors and also aid in discerning the role of adenosine in reproduction.     

Effects of some organophosphorus compounds on the binding of a radioligand (8-cyclopentyl 1, 3-[3H]dipropylxanthine) to adenosine receptors in ovine cardiac membranes.

8-Cyclopentyl-1,3-[3H] dipropylxanthine [( 3H]CPX) is a potent radioligand that specifically binds to the A1 adenosine receptors. Its high specificity makes it a suitable ligand for the characterization of A1 adenosine receptors in tissues with low receptor densities. We have demonstrated that the organophosphorus compounds soman, tabun and sarin, at relatively high concentrations, all bind to the A1 adenosine receptors in ovine cardiac membranes with Ki values of 36.7, 328 and 175 microM, respectively. The binding of soman to the receptor site was found to be totally reversible. We suggested that these organophosphorus compounds affect the mechanical responses of the heart through interaction with a potassium channel that does not seem to be closely linked to the A1 adenosine receptors.     

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.     

Analysis of agonist-antagonist interactions at A1 adenosine receptors

Previous work from our laboratory using sucrose gradient centrifugation and the antagonist radioligand [3H]xanthine amine congener led us to propose that A1 adenosine receptors are coupled to a GTP-binding protein (G protein) in the absence of an agonist and that adenosine receptor antagonists bind to free uncoupled receptors with high affinity and coupled receptors with low affinity and cause a destabilization of receptor-G protein complexes [Mol. Pharmacol. 36:412-419 (1989)]. Because agonists form high affinity ternary complexes composed of the agonist, receptor, and G protein, this hypothesis would imply that interactions between adenosine receptor agonists and antagonists, while competitive, would appear to be "noncompetitive" in nature. Interactions between unlabeled and radiolabeled A1 receptor agonist and antagonist ligands have been investigated using bovine cerebral cortical membranes to further probe this point. The availability of both 3H- and 125I-radioligands allowed us to use both single- and dual-isotope experimental designs. Radioligand antagonist-agonist competition curves along with saturation analyses using filtration and centrifugation to isolate bound radioligand suggested that agonists bind to two sites or receptor states with high affinity and to one site with low affinity. Agonist radioligand saturation curves with or without unlabeled antagonist suggested that antagonists do not bind to all states of the receptor with equal affinity. The computer program EQUIL was used to define models capable of simultaneously fitting all parts of complex experiments in which 125I-N6-aminobenzyladenosine saturation isotherms with or without 8-cyclopentyl-1,3-dipropylxanthine ([3H]CPX) and a saturation isotherm of [3H]CPX were performed. The data were not compatible with two-independent site models or with ternary complex models involving one receptor and one G protein. The data were fit by a model involving one receptor and two G proteins and by a model involving two receptors and one G protein. Both models suggest that 1) a high percentage of the receptor(s) is coupled to a G protein in the absence of an agonist and 2) agonists stabilize whereas antagonists destabilize precoupled receptor-G protein complexes. Because of this, competitive interactions between A1 agonists and antagonists appear noncompetitive in nature.     

Affinity chromatography of A1 adenosine receptors of rat brain membranes

The A1 adenosine receptor of rat brain membranes has been solubilized with digitonin and purified approximately 150-fold by affinity chromatography. The digitonin-solubilized receptor, which can be labeled with 8-cyclopentyl-1,3-[3H]dipropylxanthine([3H]DPCPX), was adsorbed on xanthine amine congener (XAC)-linked agarose. The interaction of the solubilized receptor activity with the affinity gel was biospecific. Adenosine agents blocked adsorption of solubilized receptor activity to the XAC-agarose with the appropriate A1 adenosine selectivity. For agonists, 8-cyclopentyladenosine greater than (R)-phenylisopropyladenosine greater than CV-1808, whereas, for antagonists, 8-cyclopentyltheophylline (CPT) greater than XAC greater than isobutylmethylxanthine = theophylline. The same A1 adenosine receptor specificity was observed for elution of [3H]DPCPX binding activity from the gel. XAC-agarose adsorbed 65-80% of the solubilized [3H]DPCPX binding activity and, after the gel was washed, 30-40% of the adsorbed activity could be eluted with 100 microM CPT, with specific binding activity of approximately 60 pmol/mg of protein. The order of potency of adenosine agonists [8-cyclopentyladenosine greater than (R)-phenylisopropyladenosine greater than 5'-N-ethylcarboxamidoadenosine greater than (S)-phenylisopropyladenosine] and antagonists (DPCPX greater than XAC greater than CPT greater than isobutylmethylxanthine) with the affinity-purified preparation was found to be similar to that of the solubilized adenosine A1 receptor. This affinity chromatography procedure should prove to be valuable in the isolation and molecular characterization of A1 adenosine receptors.     

Effects of benzodiazepine administration on A1 adenosine receptor binding in-vivo and ex-vivo.

The adenosine receptor has been implicated in the central mechanism of action of benzodiazepines. The specific binding of an A1-selective adenosine antagonist radioligand, [3H]8-cyclopentyl-1,3-dipropylxanthine, was measured in-vivo in mice treated with alprazolam (2 mg kg-1, i.p.), lorazepam (2 mg kg-1, i.p.) and vehicle. Binding studies were performed in-vivo and ex-vivo in mice receiving continuous infusion of alprazolam (2 mg kg-1 day-1), lorazepam (2 mg kg-1 day-1) and vehicle by mini-osmotic pumps for 6 days. Continuous infusion of alprazolam and lorazepam significantly decreased specific binding by 34 and 53%, respectively, compared with vehicle treatment (P less than 0.01). Single doses of alprazolam and lorazepam induced a similar trend in specific binding in-vivo (P = 0.07). There were no alterations in A1-receptor density (Bmax) or affinity (Kd) in cortex, hippocampus or brainstem in ex-vivo studies. Benzodiazepine treatment may diminish A1- receptor binding in-vivo by inhibiting adenosine uptake or by direct occupancy of the A1 adenosine receptor recognition site.     

Potentiation of adenosine A1 receptor agonist CPA-induced antinociception by paeoniflorin in mice

The effect of paeoniflorin (PF), a major constituent isolated from Paeony radix, on N6-Cyclopentyladenosine (CPA), a selective adenosine A1 receptor (A1 receptor) agonist, induced antinociception was examined in mice. In the tail-pressure test, CPA (0.05, 0.1, 0.2 mg/kg, s.c.) could induce antinociception in a dose-dependent manner. PF (5, 10, 20 mg/kg, s.c.) alone failed to exhibit any antinociceptive effect in mice; however, pretreatment of PF (20 mg/kg, s.c.) could significantly enhance CPA-induced antinociception. Additionally, pretreatment of 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.25 mg/kg, s.c.), a selective A1 receptor antagonist, could antagonize the antinociceptive effect of combining CPA with PF. Furthermore, in the competitive binding experiments, PF did not displace the binding of [3H]-8-Cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX) but displaced that of [3H]-2-Chloro-N6-cyclopentyladenosine ([3H]-CCPA, a selective A1 receptor agonist) to the membrane preparation of rat cerebral cortex. These results suggested that PF might selectively increase the binding and antinociceptive effect of CPA by binding with A1 receptor.     

3H]MRS 1754, a selective antagonist radioligand for A(2B) adenosine receptors

MRS 1754 [N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-phenoxy]acetamide] is a selective antagonist ligand of A(2B) adenosine receptors. This is the least well-defined adenosine receptor subtype, and A(2B) antagonists have potential as antiasthmatic drugs. For use as a radioligand, MRS 1754, a p-cyanoanilide xanthine derivative, was tritiated on the propyl groups in a two-step reaction using a p-carboxamido precursor, which was dehydrated to the cyano species using trifluoroacetic anhydride. [3H]MRS 1754 (150 Ci/mmol) bound to recombinant human A(2B) adenosine receptors in membranes of stably transfected HEK-293 cells. Specific binding was saturable, competitive, and followed a one-site model, with a K(D) value of 1.13 +/- 0.12 nM and a B(max) value of 10.9 +/- 0.6 pmol/mg protein. Specific binding utilizing 0.7 nM [3H]MRS 1754 was > 70% of total binding. The affinity calculated from association and dissociation binding constants was 1.22 nM (N = 4). Binding to membranes expressing rat and human A(1) and A(3) adenosine receptors was not significant, and binding in membranes of HEK-293 cells expressing human A(2A) receptors was of low affinity (K(D) > 50 nM). The effects of cations and chelators were explored. Specific binding was constant over a pH range of 4.5 to 6.5, with reduced binding at higher pH. The pharmacological profile in competition experiments with [3H]MRS 1754 was consistent with the structure-activity relationship for agonists and antagonists at A(2B) receptors. The K(i) values of XAC (xanthine amine congener) and CPX (8-cyclopentyl-1,3-dipropylxanthine) were 16 and 55 nM, respectively. NECA (5'-N-ethylcarboxamidoadenosine) competed for [3H]MRS 1754 binding with a K(i) of 570 nM, similar to its potency in functional assays. Thus, [3H]MRS 1754 is suitable as a selective, high-affinity radioligand for A(2B) receptors.     

MgCl2和Gpp（NH）p对腺苷A1受体激动剂和拮抗剂结合的作用比较

AIM: To investigate modulation of antagonist and agonist binding to adenosine A1 receptors by MgCI2 and 5'-guanylimidodiphosphate (Gpp(NH)p) using rat brain membranes and the A1 antagonist [^3H]-8-cyclopentyl-1,3-dipropylxanthine ([^3H]DPCPX) and the A1 agonist [^3H]-2-chloro-N^6-cyclopentyladenosine ([^3H]CCPA). METHODS:Parallel saturation and inhibition studies were performed using well-characterised radioligand binding assays and aBrandel Cell Harvester. RESULTS: MgCI2 produced a concentration-dependent decrease (44%), whereasGpp(NH)p increased [^3H]DPCPX binding (19%). In [^3H]DPCPX competition studies, agonist affinity was 1.5-14.6-fold higher and 4.6-10-fold lower in the presence of l0 mmol/L MgCl2 and l0μmol/L Gpp(NH)p respectively;antagonist affinity was unaffected. The decrease in agonist affinity with increasing Gpp(NH)p concentrations was due to a reduction in the proportion of binding to the high affinity receptor state. In contrast to [^3H]DPCPX, MgCl2produced a concentration-dependent increase (72%) and Gpp(NH)p a decrease (85%) in [^3H]CCPA binding.Using [^3H]CCPA, agonist affinities were 5-17-fold higher than those for [^3H]DPCPX, consistent with binding onlyto the high affinity receptor state. Agonist affinity was 1.3-10.5-fold higher and 2.4-4.7-fold lower on addingMgCl2 or Gpp(NH)p respectively; antagonist affinities were as for [^3H]DPCPX. CONCLUSION: The inconsistencies surrounding the effects of MgCl2 and guanine nucleotides on radioligand binding to adenosine A1 receptorswere systematically examined. The effects of MgCl2 and Gpp(NH)p on agonist binding to A1 receptors are consistent with their roles in stimulating GTP-hydrolysis at the G-protein α-subunit and in blocking formation of the highaffinity agonist-receptor-G protein complex.     

A comparison of A2 adenosine receptor-induced cyclic AMP generation in cerebral cortex and relaxation of pre-contracted aorta.

1. A comparative study was carried out between the adenosine receptor mediating a stimulation of cyclic AMP formation in guinea-pig cerebral cortical slices with the adenosine receptor mediating relaxation of phenylephrine precontracted guinea-pig aortic rings. 2. [3H]-cyclic AMP accumulation in [3H]-adenine-prelabelled guinea-pig cerebral cortical slices was stimulated by adenosine and its analogues with the following EC50 values (microM): 5'-N-ethylcarboxamidoadenosine (3.1 +/- 0.3) > 2-chloroadenosine (10 +/- 2) > adenosine (109 +/- 15). 3. 2-Chloroadenosine and adenosine elicited maximal responses for [3H]-cyclic AMP accumulation that were 100 +/- 7 and 71 +/- 6% of the maximal response to 5'-N-ethylcarboxamidoadenosine, respectively. CGS 21680 (100 microM) and DPMA (100 microM) elicited -2 +/- 2 and 12 +/- 3% of the response to 100 microM 5'-N-ethylcarboxamidoadenosine. 4. Estimation of antagonist potencies at the A2 adenosine receptor of cerebral cortex showed a rank order of potency (K1, nM): xanthine amino congener (35 +/- 3) > 8-cyclopentyl-1,3-dipropylxanthine (130 +/- 22) > PD 115,199 (407 +/- 82) > 3,7-dimethyl-1-propargylxanthine (13 +/- 2 microM). 5. Adenosine analogues produced long-lasting relaxation of phenylephrine-precontracted aortic rings with the following rank order of potency (EC50 values, microM): 5'-N-ethylcarboxamidoadenosine (0.68 +/- 0.06) > 2-chloroadenosine (4.3 +/- 0.6) > adenosine (104 +/- 13). Maximal relaxations elicited by these agents were 71 +/- 3, 98 +/- 1, and 100 +/- 1%, respectively. CGS 21680 and DPMA at 100 microM elicited smaller relaxations of the precontracted tissues (12 +/- 2 and 43 +/- 15%, respectively). 6. Antagonism by xanthine derivatives of the 5'-N-ethylcarboxamidoadenosine-induced relaxation of aortic rings showed the following rank order of potency (Ki, nM): xanthine amino congener (17 +/- 4) > 8-cyclopentyl-1,3-dipropylxanthine (171 +/- 36) > PD 115,199 (341 +/- 64) > 3,7-dimethyl-1-propargylxanthine (5520 +/- 820).(ABSTRACT TRUNCATED AT 250 WORDS)     

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     

Interaction of amiloride and its analogues with adenosine A1 receptors in calf brain

Amiloride, a potassium sparing diuretic, is known to interact with a number of ion transport systems, receptors and enzymes. Here, we report on the interaction between this drug and the adenosine A1 receptor as present in calf brain membranes. Adenosine A1 receptors are characterized by a subnanomolar affinity for the antagonists [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) and the agonist [3H]N6-R-1-phenyl-2-propyladenosine ([3H]PIA). Amiloride displaces both agonist and antagonist binding with a Ki value in the low micromolar range. This inhibition is counteracted by NaCl and protons, in contrast to the binding of [3H]PIA and [3H]DPCPX. The results suggest that amiloride interacts with the adenosine A1 receptor at a site distinct from the ligand binding site. In order to elucidate the role of one of the ion transport systems known to be inhibited by amiloride, eight amiloride analogues with different sensitivities for these systems were tested. The potency and order of potency of these compounds towards adenosine A1 receptors excludes the involvement of the epithelial Na+ channel, Na+/H+ exchanger or Na+/Ca2+ exchanger.     

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     

Characterization of the adenosine receptor modulating insulin action in rat skeletal muscle.

The pharmacological profile of adenosine receptors in rat soleus muscle has been investigated by studying the effects of A1-and A2-selective adenosine receptor agonists on glucose utilization and the system A amino acid transporter under conditions where adenosine has been reported to exert a modulatory action on these insulin-sensitive processes. In the presence of adenosine deaminase and a sub-maximally effective concentration of insulin (50 microU/ml), the A1-selective agonists N6-cyclopentyladenosine and R(-)-N6-(2-phenylisopropyl)adenosine (R(-)PIA) caused concentration-dependent inhibitions of 2-deoxy[3H]glucose 6-phosphate and alpha-[14C]methylaminoisobutyric acid accumulations, but had no effect on the rate of [14C]glucose incorporation into glycogen, in incubated soleus muscle strips. These effects on glucose transport/phosphorylation and system A amino acid transport could be antagonized by 8-cyclopentyl-1,3- dipropylxanthine and 8-phenyltheophylline. The A2-selective adenosine receptor agonists CGS 21680 and 2-(phenylamino)adenosine were much less potent in their inhibition of these metabolic processes. These data support the proposal that adenosine exerts a post-receptor insulin-modulatory action in skeletal muscle and strongly suggest that this action is mediated by A1 adenosine receptors: the possible intracellular signalling mechanism(s) for this hormone-modulatory effect of adenosine are discussed.     

Development of pharmacological sensitivity to adenosine analogs in embryonic chick heart: role of A1 adenosine receptors and adenylyl cyclase inhibition

The developing chick heart was employed as a model system to explore temporal correlations between the onset of pharmacological sensitivity to adenosine analogs and the appearance of A1 adenosine receptors coupled to adenylyl cyclase. A characterization of the developmental profile for adenosine analog-induced negative chronotropic response revealed that isolated atria from 5- and 6-day embryos were unresponsive to adenosine analogs. The onset of pharmacological sensitivity occurred on embryonic day 7, as evidenced by a 27% reduction in atrial beating rate in the presence of 2-chloradenosine (2-CIA) (30 microM). The sensitivity of embryonic atria to 2-CIA increased continuously from day 7 to day 12 in ovo, when the atria became fully responsive to the negative chronotropic effect of this adenosine analog. In order to evaluate whether the developmental increase in pharmacological sensitivity to 2-CIA reflected changes in the number of A1 adenosine receptors, the ontogenesis of A1 adenosine receptors was assessed using the antagonist radioligand 8-cyclopentyl-1,3-[3H]dipropylxanthine as a probe. Cardiac membranes from day 5 and day 6 embryos possessed approximately one third of the maximum number of A1 adenosine receptors expressed at later embryonic ages. Additionally, agonist/[3H] DPCPX competition curves revealed that the high affinity state receptors comprised a larger proportion of the total receptor population in membranes from day 6 as compared with day 12 embryos. These results suggest that there are pharmacologically inactive A1 receptors in hearts from day 5 and day 6 embryos. The developmental change in A1 receptor-mediated negative chronotropic response paralleled the increase in [3H]DPCPX binding sites from embryonic day 7 to day 10. Thus, a large fractional occupancy of A1 adenosine receptors is required to express negative chronotropy during this period of embryonic development. Studies of the sensitivity of adenylyl cyclase to inhibition by cyclopentyladenosine as a function of ontogenesis revealed that cyclopentyladenosine inhibited basal adenylyl cyclase activity to a similar maximal extent from embryonic day 5 through day 16. The efficacy of cyclopentyladenosine as an inhibitor of adenylyl cyclase activity was, therefore, stable during a developmental period when A1 receptor density increased approximately 3-fold. Hence, only a fraction of the A1 receptors present during embryogenesis need to be coupled to produce a maximum response with respect to adenylyl cyclase inhibition, which is an indication of the presence of spare receptors. receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

A1 adenosine receptor inhibition of cyclic AMP formation and radioligand binding in the guinea-pig cerebral cortex.

1. A1 adenosine receptors were investigated by radioligand binding and functional studies in slices and particulate preparations from guinea-pig cerebral cortex. 2. Binding of the adenosine receptor antagonist radioligand, 8-cyclopentyl-[3H]-1,3-dipropylxanthine (DPCPX) to guinea-pig cerebral cortical membranes exhibited high density (1410 +/- 241 fmol mg-1 protein) and high affinity (Kd 3.8 +/- 0.3 nM). 3. [3H]-DPCPX binding to guinea-pig cerebral cortical membranes was displaced in a monophasic manner by adenosine receptor antagonists with the rank order of affinity (Ki values, nM): DPCPX (6) < xanthine amine congener (XAC, 153) < PD 115,199 (308). 4. Agonist displacement of [3H]-DPCPX binding was biphasic and exhibited the following rank order at the low affinity site (Ki values): 2-chloro-N6-cyclopentyl-adenosine (CCPA, 513 nM) = N6-R-phenylisopropyladenosine (R-PIA, 526 nM) = N6-cyclopentyladenosine (CPA, 532 nM) < 2-chloroadenosine (2CA, 3.2 microM) = 5'-N-ethylcarboxamidoadenosine (NECA, 4.6 microM) < N6-S-phenylisopropyladenosine (S-PIA, 19.9 microM). 5. In cerebral cortical slices, [3H]-DPCPX binding was displaced by antagonists and agonists in an apparently monophasic manner with the rank order of affinity (Ki values, nM): DPCPX (14) < XAC (45) < R-PIA (266) < PD 115,199 (666) < S-PIA (21000). 6. Cyclic AMP accumulation stimulated by 30 microM forskolin in guinea-pig cerebral cortical slices was inhibited by R-PIA, CCPA and CPA up to 1 microM in a concentration-dependent fashion with IC50 values of 14, 18, and 22 nM, respectively. All three analogues inhibited the forskolin response to a similar extent (82-93% inhibition).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the adenosine receptor mediating contraction in rat colonic muscularis mucosae.

1. The objective of this study was to characterize the adenosine receptor mediating contraction in rat isolated colonic muscularis mucosae (RCMM). 2. Sequential additions of the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA; 0.01-10 microM) elicited reproducible, concentration-related contractions in RCMM. The effects of NECA were mimicked by the adenosine A1 receptor-selective agonists cyclopentyladenosine (CPA), R-phenylisopropyladenosine (R-PIA) and N-[1S, trans)2-hydroxycyclopentyl] adenosine (GR79236) and by S-PIA (the stereoisomer of R-PIA). The adenosine A2 agonists N-[(2-methylphenyl)methyl] adenosine (metrifudil) and 2-[p-(2-carboxyethyl)phenethylamine]-5'-N-ethylcarboxamidoadenosine (CGS21680) also produced contractions in RCMM but were 54 and 165 times less potent respectively than NECA. The rank order of agonist potency for contraction of RCMM was CPA > or = GR79236 = R-PIA > or = NECA > > S-PIA = metrifudil > CGS21680, which is identical to that reported for the inhibition of spontaneous rate in rat isolated right atria and inhibition of lipolysis in rat isolated adipocytes by these same agonists. 3. R-PIA, S-PIA and metrifudil behaved as partial agonists in RCMM. 4. The adenosine A1 receptor-selective antagonist 8-cyclopentyl-1,3- dipropylxanthine (DPCPX) inhibited the contractions produced by all the adenosine agonists tested, with pKB values between 9.2 and 9.5. The non-selective adenosine antagonist 8-phenyltheophylline (8-PT) antagonized the effects of NECA but also markedly potentiated (by 93.0 +/- 10.2% at 3 microM) the maximum contractile response to NECA in RCMM. Neither 8-PT (3 microM) nor DPCPX (0.1 microM) had any effect on the contractions produced by carbachol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential allosteric modulation by amiloride analogues of agonist and antagonist binding at A(1) and A(3) adenosine receptors

The diuretic drug amiloride and its analogues were found previously to be allosteric modulators of antagonist binding to A(2A) adenosine receptors. In this study, the possibility of the allosteric modulation by amiloride analogues of antagonist binding at A(1) and A(3) receptors, as well as agonist binding at A(1), A(2A), and A(3) receptors, was explored. Amiloride analogues increased the dissociation rates of two antagonist radioligands, [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) and [3H]8-ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one ([3H]PSB-11), from A(1) and A(3) receptors, respectively. Amiloride and 5-(N,N-dimethyl)amiloride (DMA) were more potent at A(1) receptors than at A(3) receptors, while 5-(N,N-hexamethylene)amiloride (HMA) was more potent at A(3) receptors. Thus, amiloride analogues are allosteric inhibitors of antagonist binding at A(1), A(2A), and A(3) adenosine receptor subtypes. In contrast to their effects on antagonist-occupied receptors, amiloride analogues did not affect the dissociation rates of the A(1) agonist [3H]N(6)-[(R)-phenylisopropyl]adenosine ([3H]R-PIA) from A(1) receptors or the A(2A) agonist [3H]2-[p-(2-carboxyethyl)phenyl-ethylamino]-5'-N-ethylcarboxamidoadenosine ([3H]CGS21680) from A(2A) receptors. The dissociation rate of the A(3) agonist radioligand [125I]N(6)-(4-amino-3-iodobenzyl)adenosine-5'-N-methyluronamide ([125I]I-AB-MECA) from A(3) receptors was decreased significantly by amiloride analogues. The binding modes of amiloride analogues at agonist-occupied and antagonist-occupied receptors differed markedly, which was demonstrated in all three subtypes of adenosine receptors tested in this study. The effects of the amiloride analogues on the action of the A(3) receptor agonist were explored further using a cyclic AMP functional assay in intact CHO cells expressing the human A(3) receptor. Both binding and functional assays support the allosteric interactions of amiloride analogues with A(3) receptors.     

The binding of 1,3-[3H]-dipropyl-8-cyclopentylxanthine to adenosine A1 receptors in rat smooth muscle preparations.

1. The binding of 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), an antagonist radioligand selective for adenosine A1 receptors, was studied in rat duodenum, colon muscularis mucosae and longitudinal muscle, urinary bladder and vasa deferentia. 2. [3H]-DPCPX bound with high affinity to a single site in all membrane preparations studied with the exception of the rat urinary bladder in which no specific binding was detected. The affinity (Kd) of the binding site for [3H]-DPCPX was similar in all membrane preparations, the colon longitudinal muscle (1.18 +/- 0.47 nM), colon muscularis mucosae (0.84 +/- 0.15 nM), duodenum (1.59 +/- 0.18 nM) and vasa deferentia (0.93 +/- 0.17 nM). The density of [3H]-DPCPX binding sites was similar in the duodenum (38.8 +/- 4 fmol mg-1 protein), muscularis mucosae (43 +/- 3.5 fmol mg-1 protein) and vasa deferentia (43.3 +/- 12.2 fmol mg-1 protein), but in the longitudinal muscle 6-7 fold more binding sites (295 +/- 70 fmol mg-1 protein) were identified. 3. Inhibition studies using DPCPX (0.1-100 nM), N6-cyclopentyladenosine (CPA) (0.1-100 nM), 5'-N-ethylcarboxamidoadenosine (NECA) (2 nM-10 microM) and (R)-N6-phenylisopropyladenosine (R-PIA) (1 nM-1 microM) to displace the binding of [3H]-DPCPX at a concentration around the Kd value (1 nM), demonstrated an order of potency of displacement in all tissues of DPCPX > or = CPA > R-PIA > NECA. This potency order is characteristic of an A1 receptor, indicating that [3H]-DPCPX binds to adenosine A1 receptors in the rat duodenum, colon and vasa deferentia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radioligand binding to adenosine receptors and adenosine uptake sites in different brain regions of normal and narcoleptic dogs

The present study compares the characteristics of radioligand binding to adenosine receptors and adenosine uptake sites in 100- and 50-day-old normal and narcoleptic dogs. Binding to A1 receptors was quantified using a selective A1 agonist ([3H]N6-[(R)-1-methyl-2-phenylethyl] adenosine, [3H]R-PIA) and an antagonist ([3H]dipropyl-8-cyclopentyl-xanthine, [3H]CPX). Differences in the binding of [3H]R-PIA and that of [3H]5'-ethylcarboxamide adenosine ([3H]NECA), which binds to both A1 and A2 receptors with similar affinities, were used to quantify A2 receptors. Nucleoside transport sites were labeled with [3H]nitrobenzylthioinosine ([3H]NBTI), a potent inhibitor of nucleoside transport systems. The present study offered no evidence that either adenosine A1 receptors and adenosine uptake sites in the frontal cortex or adenosine A2 receptors in the putamen were altered in narcoleptic dogs. However, we found that adenosine A1 receptors in the dog exist in different affinity states and that the affinity state in which the receptor is found depends on the brain region examined. A characterization of these low- and high-affinity sites was performed and results indicated that these sites cannot be explained by a single interaction of the A1 receptor with a single G-protein population.