The C6-2B glioma cell P2Y(AC) receptor is pharmacologically and molecularly identical to the platelet P2Y(12) receptor.

P2Y receptor activation in many cell types leads to phospholipase C activation and accumulation of inositol phosphates, while in blood platelets, C6-2B glioma cells, and in B10 microvascular endothelial cells a P2Y receptor subtype, which couples to inhibition of adenylyl cyclase, historically termed P2Y(AC), (P2T(AC) or P(2T) in platelets) has been identified. Recently, this receptor has been cloned and designated P2Y(12) in keeping with current P2 receptor nomenclature. Three selective P(2T) receptor antagonists, with a range of affinities, inhibited ADP-induced aggregation of washed human or rat platelets, in a concentration-dependent manner, with a rank order of antagonist potency (pIC(50), human: rat) of AR-C78511 (8.5 : 9.1)>AR-C69581 (6.2 : 6.0)>AR-C70300 (5.4 : 5.1). However, these compounds had no effect on ADP-induced platelet shape change. All three antagonists had no significant effect on the ADP-induced inositol phosphate formation in 1321N1 astrocytoma cells stably expressing the P2Y(1) receptor, when used at concentrations that inhibit platelet aggregation. These antagonists also blocked ADP-induced inhibition of adenylyl cyclase in rat platelets and C6-2B cells with identical rank orders of potency and overlapping concentration - response curves. RT - PCR and nucleotide sequence analyses revealed that the C6-2B cells express the P2Y(12) mRNA. These data demonstrate that the P2Y(AC) receptor in C6-2B cells is pharmacologically identical to the P2T(AC) receptor in rat platelets.

P2Y1-receptors in human platelets which are pharmacologically distinct from P2Y(ADP)-receptors.

1. In the present study we have classified the receptor(s) mediating increases in intracellular calcium concentration ([Ca2+]i) in human washed platelets and compared the pharmacological profile obtained with that observed in Jurkat cells, stably transfected with a bovine P2Y1-receptor. 2. The P2Y1-receptor antagonist, adenosine-3'-phosphate-5'-phosphate (A3P5P), competitively antagonized agonist responses in both Jurkat cells, and in platelets with similar affinities (pK(B) of 5.8 and 6.0, respectively). 3. The selective P2Y(ADP) antagonist, AR-C66096, exhibited partial agonism in the Jurkat cells with an affinity (pK(A)) of 4.9. This value is consistent with its known P2Y1-receptor activity. In platelets, AR-C66096 at a concentration (0.1 microM) approximately 100 fold greater than its known P2Y(ADP) receptor affinity, had no effect on ADP-induced increases in [Ca2+]i. 4. The ability of adenine nucleotide analogues to elevate [Ca2+]i in the Jurkat cells was also determined. The rank order of agonist potency (p[A]50) was: 2-MeSADP (8.3)>2-ClATP (7.8)>ADP (7.5)=2-MeSATP (7.4)>ATPgammaS (6.5)>ATP (6.2), with ATP appearing to be a partial agonist. 5. The same rank order of potency was observed when similar experiments were performed in platelets. However, the absolute potencies of all the agonists and the intrinsic activities of both ATPgammaS and ATP were lower in platelets. 6. The operational model of agonism was used to test whether the agonist concentration-effect profiles obtained in these two cell types could be explained on the basis of differences in receptor reserve. The analysis indicated that the data obtained in platelets closely resembled that predicted for a low density or poorly coupled P2Y1-receptor system. 7. The hypothesis that the observed partial agonist behaviour of ATP was the result of receptor activation by contaminating ADP with concomitant receptor blockade by ATP, was tested in the platelet system. This hypothesis was supported by a theoretical analysis, which yielded an affinity value for ATP similar to that obtained previously at P2Y1-receptors. 8. In summary, the results of this study indicate that human washed platelets contain P2Y1-receptors which mediate increases in [Ca2+]i and that this receptor population is pharmacologically distinct from P2Y(ADP)-receptors.

Inhibition of striatal GABA release by the adenosine A2a receptor is not mediated by increases in cyclic AMP.

The adenosine A2a receptor inhibition of potassium (15 mM)-evoked GABA release from striatal nerve terminals has been examined. High extracellular calcium concentrations (4 mM) reduced the effect of the A2a receptor agonist CGS-21680 (1 nM). CGS-21680 inhibited GABA release in the presence of the L-type calcium channel blocker nifedipine, which itself inhibited evoked GABA release (by 16 +/- 4%). omega-Conotoxin inhibited the evoked release by 45 +/- 4% and prevented the action of CGS-21680. Forskolin and 8-bromo cyclic AMP both stimulated evoked GABA release at low concentrations, but at higher concentrations they abolished the inhibition by CGS-21680 without affecting the evoked release. The nonselective protein kinase inhibitor H-7 inhibited both the evoked release and the inhibition by CGS-21680, whereas the selective protein kinase A and G inhibitor HA-1004 had no effect on either evoked release or the action of CGS-21680. Pretreatment with pertussis toxin did not affect the A2a receptor-mediated inhibition. Therefore, the effect of A2a receptor stimulation was not mediated by protein kinases A or G but was inhibited by elevated cyclic AMP levels and mimicked by inhibitors of the N-type calcium channel and protein kinase C.

Further characterization of [3H]-CGS 21680 binding sites in the rat striatum and cortex.

1. The putative high affinity binding site for the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)phenethyl-amino-5'-N- ethylcarboxamidoadenosine (CGS 21680) in the rat cerebral cortex was characterized by use of a number of selective A1 and A2 adenosine receptor ligands, and compared to the characteristics of the more abundant striatal A2A receptor. 2. The binding of [3H]-CGS 21680 to cortical membranes was performed at pH 5.5, in order to increase the amount of specific binding. 3. Reduction of the pH from 7.4 to 5.5 increased the apparent affinity of the striatal binding side for both agonists and antagonists. The relative order of potencies of both groups of ligands were the same at both pH values, and were consistent with binding to the A2A receptor. There was no observable change in the Bmax, the values being 415 and 446 fmol mg-1 protein at pH 5.5 and 7.4 respectively. 4. The cortical binding site yielded a Bmax value of 117 fmol mg-1 protein. The relative order of potencies of the adenosine receptor ligands observed at this binding site were not the same as those observed in the striatum, exhibiting a profile with both A1 and A2 characteristics. 5. Further characterization of this cortical binding site in the presence of the A1 selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) revealed a more typical A2A profile. This indicated that under the conditions used there were two components of [3H]-CGS 21680 binding, approximately 20% of the A1 receptor and 80% to the A2A receptor. 6. It is concluded that in the cerebral cortex there is a CGS 21680 binding site showing the characteristic properties of the striatal A2A receptor, and no evidence was obtained for the existence of a novelA2A-like binding site.

Inhibition by KF17837 of adenosine A2A receptor-mediated modulation of striatal GABA and ACh release.

1. The effect of the A2A adenosine receptor agonist, 2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine (CGS 21680) on the potassium evoked release of [3H]-gamma-aminobutyric acid ([3H]-GABA) from nerve terminals derived from the caudate-putamen and the globus pallidus of the rat was compared. In both preparations CGS 21680 (1 nM) inhibited the [3H]-GABA release evoked by 15 mM KCl but had no effect on that evoked by 30 mM KCl. 2. The ability of CGS 21680 (1 nM) to inhibit the release of [3H]-GABA from striatal nerve terminals was unaffected by the presence of the GABA receptor antagonists, bicuculline (10 microM), phaclofen (100 microM) and 2-hydroxysaclofen (100 microM). Similarly the opioid receptor antagonist, naloxone (10 microM), the adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 40 nM), and the cholinoceptor antagonists, mecamylamine (10 microM) and atropine (100 nM) had no effect on this inhibition. 3. The ability of CGS 21680 (0.1 nM) to stimulate the release of [3H]-acetylcholine ([3H]-ACh) from striatal nerve terminals was unaffected by the presence of bicuculline (10 microM), 2-hydroxysaclofen (100 microM), phaclofen (100 microM), naloxone (10 microM) and DPCPX (4 nM). 4. The novel A2A receptor antagonist, (E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (KF 17837), blocked the CGS 21680 (1 nM)-induced inhibition of [3H]-GABA efflux with an EC50 of approximately 30 nM and also antagonized the CGS 21680 (0.1 nM)-induced stimulation of [3H]-ACh release with an EC50 of approximately 0.3 nM. 5. It is concluded that the A2A adenosine receptor is present on both GABAergic and cholinergic nerve terminals of the rat striatum and that in both the caudate-putamen and the globus pallidus this receptor inhibits [3H]-GABA release. No evidence was seen for a difference in the ligand binding sites of this receptor in the two groups of nerve terminals.

Adenosine A2a receptor-mediated modulation of striatal [3H]GABA and [3H]acetylcholine release.

The ability of adenosine agonists to modulate K(+)-evoked gamma-[3H]aminobutyric acid ([3H]GABA) and acetylcholine (ACh) release from rat striatal synaptosomes was investigated. The A2a receptor-selective agonist CGS 21680 inhibited Ca(2+)-dependent [3H]GABA release evoked by 15 mM KCl with a maximal inhibition of 29 +/- 4% (IC50 of approximately 4 x 10(-12) M). The relative order of potency of three agonists was CGS 21680 > or = 5'-N-ethylcarboxamidoadenosine > R-phenylisopropyladenosine (R-PIA), with the inhibition being blocked by A2a receptor-selective antagonists (CP 66,713 and CGS 15943A) but not by the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). When release of [3H]GABA was evoked by 30 mM KCl, no significant inhibition was observed. In contrast, CGS 21680 stimulated the release of [3H]ACh evoked by 30 mM KCl, with a maximal stimulation of 26 +/- 5% (IC50 of approximately 10(-11) M). This effect was blocked by CP 66,713 but not by DPCPX. The A1 agonist R-PIA inhibited [3H]ACh release, an effect blocked by DPCPX. It is concluded that adenosine A2a receptors are present on both GABAergic and cholinergic striatal nerve terminals where they inhibit and stimulate transmitter release, respectively.

Advantages of achiral h.p.l.c. as a preparative step for chiral analysis in biological samples and its use in toxicokinetic studies.

1. Achiral reverse-phase h.p.l.c. with semi-automated post-column fraction collection and solid-phase sample reconcentration, has been applied as the purification procedure during the enantiomeric quantification of two widely differing experimental drugs; an HMG-CoA reductase inhibitor (I) and an alpha 2-adrenoceptor antagonist (II). 2. The robust and specific achiral methodologies were available prior to the need for chiral analyses and recovery of drug from the fractions provided clean samples from a variety of biological matrices, without the need to develop compatible achiral/chiral mobile phases. 3. Compared with direct chiral chromatography of plasma extracts, this approach decreased the potential for metabolites and endogenous components to interfere or impair the performance of the chiral stationary phase. 4. The availability of quantitative data from achiral analysis of samples negated the need for internal standardization of the chiral analyses, helped confirm assay specificity and provided potential to determine enantiomeric ratios where only one isomer could be accurately measured. 5. Routine enantiomeric analyses were successfully carried out on samples taken from animals dosed orally with the racemic drugs, providing important data on the possible levels of exposure to individual enantiomers during toxicity testing.