Fiona E. Parkinson

Autoradiographic evidence for G-protein coupled A2-receptors in rat neostriatum using [3H]-CGS 21680 as a ligand

SummaryRecently [3H]-CGS 21680 (2-[p-(2-carbonylethyl)-phenylethylamino]-5′-N-ethylcarboxamidoadeno-sine) has been identified as a selective adenosine A2-receptor agonist. In this study the binding of [3H]-CGS 21680 to 10 μm sections of rat neostriatum was investigated with quantitative autoradiography. Specific, saturable binding was detectable, and Scatchard analysis of saturation experiments gave estimates for KD and Bmax of 1.7 nM and 322 fmol/mg protein, respectively. The rank order of potency for inhibition of [3H]-CGS 21680 binding was 5′-N-ethylcarboxamidoadenosine (1.9 nM) > 2-chloroadenosine (18 nM) > R-N6-phenylisoprop-yladenosine (59 nM) > S-N6-phenylisoprophyladeno sine (460 nM) > 1,3-dipropyl-8-cyclopentylxanthine (700 nM). The binding of [3H]-CGS 21680 was sensitive to GTP, since 1 μM GTP reduced binding to 4.7% of control. These data support the identity of CGS 21680 as an agonist at high affinity adenosine A2-receptors and indicate these receptors in rat striatum are coupled to guanine nucleotide binding proteins.

FUNCTIONAL CHARACTERIZATION OF ADENOSINE A2 RECEPTORS IN JURKAT CELLS AND PC12 CELLS USING ADENOSINE RECEPTOR AGONISTS

The effect of several adenosine analogues on cyclic AMP accumulation was examined in the rat phaeochromocytoma cell PC12 and in the human T-cell leukaemia cell Jurkat, selected as prototypes of cells predominantly expressing adenosine A2A or A2B receptors. Using the reverse transcription-polymerase chain reaction it was, however, demonstrated that the Jurkat cell and the PC12 cell express both A2A and A2B receptor mRNA, albeit in different relative proportions. In PC12 cells the concentration required for half-maximal response (EC50) for the full agonist 5′-N-ethyl-car-boxamidoadenosine (NECA) was 30 times lower than in Jurkat cells. There was no significant difference in the pA2 for the antagonist 5-amino-9-chloro-2-(2-furanyl)1,2,4-triazolo(1,5-C)quinazolinemonomethanesulphon-ate (CGS 15943) between the two cell types. In the presence of forskolin (1 μM in PC12 cells; 10 μM in Jurkat cells) the EC50 value for NECA was reduced two-to sixfold. Forskolin also increased the maximal cAMP accumulation twofold in PC12 cells and sevenfold in Jurkat cells. A series of 2-substituted adenosine analogues CV 1808 (2-phenylamino adenosine), CV 1674 [2-(4-methoxyphenyl)adenosine], CGS 21680 {2-[p-(2-carbonylethyl)phenylethylamino]-5′-N-ethyl-carboxamido adenosine}, and four 2-substituted isoguanosines, SHA 40 [2-(2-phenylethoxy)adenosine; PEA], SHA 91 [2-(2-cyclohexylethoxy)adenosine; CEA], SHA 118 {2-[2-(p-methylphenyl)ethoxy]adenosine; MPEA}, and SHA 125 (2-hexyloxyadenosine; HOA), all raised CAMP accumulation in PC12 cells, but had minimal or no effect in Jurkat cells. In the PC12 cells the addition of forskolin (1 μM) reduced the EC50 by a factor of 2 (CV 1808) to 12 (SHA 125). In Jurkat cells all the analogues gave a significant, but submaximal, cAMP response in the presence of forskolin (10 μM), but they were essentially inactive in its absence. The results show that a series of 2-substituted adenosine analogues can be used to discriminate between A2A and A2B receptors. The two receptor subtypes appear to coexist, even in clonal cells selected for typical pharmacology. A2 receptor pharmacology can therefore be complex.

Magnesium‐Dependent Enhancement of Endogenous Agonist Binding to A1 Adenosine Receptors: A Complicating Factor in Quantitative Autoradiography

Abstract: Quantitative autoradiography was used to investigate the effects of Mg2+ on agonist and antagonist binding to A1 receptors in rat striatum. A1 receptors were labelled with the selective agonist N6‐[3H]cyclohexyladenosine ([3H]CHA) or the selective antagonist 1,3‐[3H]dipropyl‐8‐cy‐clopentylxanthine ([3HJDPCPX). Mg2+ had no significant effect on equilibrium binding constants for [3H]CHA [control: KD (95% confidence interval) of 0.34 (0.15‐0.80) nM and Bmax of 267 ± 8 fmol/mg of gray matter; with 10 mA/Mg2+: KD of 0.8 (0.13‐4.9) nM and Bmax of 313 ± 8.9 fmol/mg of gray matter] or [3H]DPCPX [control: KD of 0.54 (0.30‐0.99) nM and Bmax of 256 ± 2.3 fmol/mg of gray matter; with 10 mMMg2+: KD of 1.54 (0.2‐11.0) nM and Bmax of 269 ± 35.7 fmol/mg of gray matter]. In contrast, Mg2+ slowed the apparent association rate for both ligands; this was observed as a shift from a one‐component to a two‐component model for [3H]DPCPX. Mg2+ also affected the dissociation rates of both ligands; for [3H]CHA, dissociation in the presence of Mg2+ was not detected. Mg2+ produced a concentration‐dependent inhibition of [3H]CHA binding only prior to equilibrium. HPLC was performed on untreated sections, sections preincubated with adenosine deaminase (ADA), and sections preincubated with ADA and incubated with ADA in the absence or presence of Mg2+ Adenosine was found in measurable quantities under all conditions, and the concentration was not influenced by Mg2+ or by the inclusion of GTP in the preincubation medium. From these data, we conclude the following: (a) adenosine is present and may be produced continuously in brain sections; (b) ADA is not capable of completely eliminating the produced adenosine; (c) Mg2+ apparently does not influence adenosine production or elimination; (d) A1 receptor‐guanine nucleotide binding protein coupling is maximal in this preparation; and (e) Mg2+ decreases the dissociation rate of bound endogenous adenosine from A1 receptors, thus limiting the access of [3H]CHA and [3H]DPCPX to the receptors. Thus, enhancement of endogenous adenosine binding to A1 receptors by Mg2+ is a complicating factor in receptor autoradiography and may be so in other preparations as well.

Identification of a B2-bradykinin receptor linked to phospholipase C and inhibition of dopamine stimulated cyclic AMP accumulation in the human astrocytoma cell line D384.

SummaryWe have examined the activation of a phospholipase C signal transduction pathway by a B2-bradykinin receptor in the human astrocytoma cell line D384 and how this influences D1-dopamine receptor stimulated cyclic AMP accumulation. Addition of bradykinin to D384 cells resulted in a concentration-dependent (10−11 − 10−6 M) increase in the accumulation of [3H]inositol phosphates and a similar concentration-dependent transient increase in specific [3H]β-phorbol-12,13-dibutyrate binding which is indicative of translocation of protein kinase C from the cytosol to the membrane. Changes in intracellular Ca2+ of single cells, measured using the fluorescent indicator dye fura-2, indicated that bradykinin produced a rapid, but transient, increase in intracellular calcium. The Ca2+ response was largely independent of extracellular Ca2+ supporting the idea that receptor activation leads to mobilization of Ca2+ from intracellular stores. However, extracellular Ca2+ was required for a response to a rechallenge with bradykinin. The bradykinin B2-receptor agonist kallidin increased cytosolic Ca 2+ in a similar manner to bradykinin. The Ca2+ response to bradykinin could be partially reduced in the presence of the B2-receptor antagonist [d-Arg10-Hyp,d-Phe7,β-(2-Thienyl)-Ala5,8]-bradykinin, whereas the B1-receptor agonists (Des-Arg9]-bradykinin and [Des-Arg10]-kallidin were ineffective. Bradykinin was also found to attenuate dopamine stimulated cyclic AMP accumulation in D384 cells, at similar concentrations previously observed to stimulate the phospholipase C signal transduction pathway, in the presence of the phosphodiesterase inhibitor, rolipram. In contrast, no attenuation was observed in the presence of the phosphodiesterase inhibitor 1-isobutyl 3-methylxanthine, although the level of dopamine stimulated cyclic AMP observed was lower than in the presence of rolipram. Furthermore, the effect of bradykinin could be mimicked by a calcium ionophore, but not a phorbol ester. These data suggest that D384 cells express a B2 bradykinin receptor coupled to polyphosphoinositide-specific phospholipase C. Activation of this receptor results in elevated levels of cytosolic Ca2+ and to a reduction of D1-dopamine receptor stimulated cyclic AMP accumulation.

Effect of Pertussis Toxin on Radioligand Binding to Rat Brain Adenosine A1 Receptors

Abstract: In a previous study we showed that in vivo treatment with pertussis toxin could inhibit some, but not all, effects of adenosine in the rat hippocampus. In this study we investigated the effect of pertussis toxin on the binding of adenosine analogues to A1 receptors in rat brain. Intraventricular injection of pertussis toxin (10 μg into the lateral ventricle) did not affect A1 receptor binding in any brain region studied, as evaluated by autoradiography. In vitro treatment of brain sections (10 μm) with pertussis toxin for 5 h, under conditions when >80% of the G proteins were ADP ribosylated, did not alter radioligand binding to adenosine A1 receptors. GTP (10 μM) virtually abolished the high‐affinity agonist binding to the A1 receptor. On the other hand, in solubilized cortical membrane preparations, pertussis toxin pretreatment induced a complete shift of the A1 receptors to the low‐affinity state. This suggests that the ability of pertussis toxin to affect G proteins coupled to A1 receptors in brain depends not only on the distribution of the toxin but also on the configuration of receptors and G proteins.

Pharmacological Implications of A Multiplicity of Adenosine Actions in the CNS

Abstract Adenosine (50 nM - 50 μM) in brain extracellular space acts on two major classes of receptors present on virtually every cell. Specificity of action may be achieved by altering brain adenosine levels and by using partial agonists and/or drugs that affect more than one biochemical target.

Autoradiographical Studies of in Vitro and Chronic in Vivo Effects of Propentofylline on Adenosine A1 and A2 Receptors and NBMPR-Sensitive Nucleoside Transporters

Abstract Propentofylline is a novel xanthine that has been shown to limit the extent neuronal damage induced by cerebral ischemia in gerbils (DeLeo et al., 1987). This is in contrast to other xanthines, including, caffeine and theophylline, that increase the extent of damage (Rudolphi et al., 1987; Dux et al., 1987). Propentofylline has been demonstrated to decrease adenosine uptake into human erythrocytes (Fredholm and Lindstrom, 1986), and to increase extracellular concentration of adenosine in ischemic barain (Andine et al., 1990). Therefore, it was proposed that this compound provides protection in cerebral ischemia, in part, by adenosine receptor stimulation due to increased endogenous adenosine levels.