### A. Overview

Extracellular purines (adenosine, ADP, and ATP) and pyrimidines (UDP and UTP) are important signaling molecules that mediate diverse biological effects via cell-surface receptors termed purine receptors. In this review particular emphasis is placed on the discrepancy between the

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Receptors for Purines and Pyrimidines

Four adenosine receptors have been cloned and characterized from several mammalian species. The receptors are named adenosine A(1), A(2A), A(2B), and A(3). The A(2A) and A(2B) receptors preferably interact with members of the G(s) family of G proteins and the A(1) and A(3) receptors with G(i/o) proteins. However, other G protein interactions have also been described. Adenosine is the preferred endogenous agonist at all these receptors, but inosine can also activate the A(3) receptor. The levels of adenosine seen under basal conditions are sufficient to cause some activation of all the receptors, at least where they are abundantly expressed. Adenosine levels during, e.g., ischemia can activate all receptors even when expressed in low abundance. Accordingly, experiments with receptor antagonists and mice with targeted disruption of adenosine A(1), A(2A), and A(3) expression reveal roles for these receptors under physiological and particularly pathophysiological conditions. There are pharmacological tools that can be used to classify A(1), A(2A), and A(3) receptors but few drugs that interact selectively with A(2B) receptors. Testable models of the interaction of these drugs with their receptors have been generated by site-directed mutagenesis and homology-based modelling. Both agonists and antagonists are being developed as potential drugs.

https://pharmrev.aspetjournals.org/content/pharmrev/53/4/527.full.pdf

International Union of Pharmacology. XXV. Nomenclature and Classification of Adenosine Receptors

The role of adenosine and its nucleotides in central synaptic transmission.

Publisher Summary This chapter discusses the physiological role of adenosine in the central nervous system (CNS). In particular, the chapter's attention is directed to the actions of adenosine and related purines in the CNS. One of the major advances in the understanding of purine actions in the central nervous system is the characterization of high affinity specific receptors for purines in brain membranes. The physiological actions of purines, the receptors and biochemical actions that underlie these functional responses and behavioral responses to purinergic drugs are considered. There are three different adenosine receptor sites in brain, which can be distinguished primarily by their effects upon adenylate cyclase, their pharmacological properties, and by their ability to bind labeled analogs of adenosine. One of the best-characterized physiological actions of adenosine is the inhibition of the release of neurotransmitters, an action that has been unequivocally established at many peripheral synapses. If one thing remains clear, it is that purines have a multitude of complex actions at every level, including the biochemical, physiological, and behavioral. There remains little question, but that adenosine or other purines constitute important and rather ubiquitous regulators of neuronalactivity in brain. Despite the evidence that purines play a significant role in neural function, it has remained difficult to define the functional role(s) of purines in brain.

The physiological role of adenosine in the central nervous system.

https://www.cell.com/imto/pdf/0167-5699(88)91220-0.pdf

The adenylate cyclase-cAMP-protein kinase A pathway and regulation of the immune response

(−)N6-Phenylisopropyladenosine, a potent agonist in adenosine-responsive cellular systems, has been labeled with tritium to high specific activity (26 Ci/mmol) and used to identify adenosine binding sites in rat brain membranes. (−)[H3]N6-Phenylisopropyladenosine binding was studied by a vacuum filtration technique. The binding was rapid, rapidly reversible, dependent on pH and temperature and stereospecific since the (−)isomer of N6-phenylisopropyladenosine was 40-fold more potent than the (+)isomer in competition experiments. The stereospecific binding sites were saturable and bound 0.8 pmol of (−)N6-phenylisopropyladenosine per mg of membrane protein. The dissociation constant (KD) of (−)N6-phenylisopropyladenosine for these sites was 5–12 nM as determined independently by saturation and kinetic binding studies. Endogeneous ligands seem to occupy the binding sites since pretreatment with adenosine deaminase increased the specific binding. Adenosine and several adenosine derivatives were studied for their ability to compete with (−)[3H]N6-phenylisopropyladenosine binding. (−)N6-Phenylisopropyladenosine-5′-monophosphate, N6-phenyladenosine, N6-benzyladenosine, 2-chloroadenosine and adenosine were most potent in displacing the radioligand from its binding sites and the IC50-values ranged from 0.3–7 μM. Physiologically inactive compounds such as inosine, hypoxanthine, adenine and the ribose-modified analogues 2′-deoxyadenosine and 2′,5′-dideoxyadenosine did not substantially inhibit binding at concentrations up to 100 μM. The adenosine antagonists isobutylmethylxanthine (IC50 3.2 μM), theophylline (IC50 7.6 μM) and caffeine (IC50 99 μM) competed for the binding sites of (−)[3H]N6-phenylisopropyladenosine in a manner which parallels their known pharmacological activity whereas other phosphodiesterase inhibitors were ineffective. The (−)[3H]N6-phenylisopropyladenosine binding sites in rat brain membranes appear to be equivalent to adenosine receptor sites on the cell surface which have recently been classified as R-site adenosine receptors.

Characterization of adenosine receptors in rat brain by (-)[3H]N6-phenylisopropyladenosine.

Adenosine receptors in human platelet membranes have been characterized by radioligand binding and measurement of adenylate cyclase activity. Binding of 5′-N-ethylcarboxamido[3H]adenosine ([3H]NECA) was rapid, reversible and dependent on protein concentration, pH and temperature. Due to a rapid rate of dissociation (t1/2 approximately 20 s) binding was highest at 0° C. Adenosine deaminase and GTP alone did not influence [3H]NECA binding, whereas several divalent cations decreased binding. Saturation experiments revealed two different binding sites for [3H]NECA, with Kd values of 0.16 and 2.9 μmol/l and Bmax values of 8.4 and 33.4 pmol/mg of protein. In competition experiments NECA was the most potent adenosine agonist (IC50 0.5 μmol/l), followed by 2-chloroadenosine (IC50 6.3 μmol/l) and adenosine (IC50 12μmol/l). A similar rank order of potencies was observed for the stimulatory effect of adenosine analogues on platelet adenylate cyclase. NECA stimulated adenylate cyclase activity with an EC50 value of 0.5 μmol/l and was approximately 4-fold more potent than (−)N6-phenylisopropyladenosine [(−)PIA]. However, (−)PIA and N6-cyclohexyladenosine did not significantly affect [3H]NECA binding, an observation not consistent with the stimulatory effect on adenylate cyclase. The adenosine antagonists 3-isobutyl-1-methylxanthine, theophylline and caffeine showed IC50 values between 98 and 5,600 μmol/l. [3H]PIA bound to platelet membranes with very low affinity and was not displaced by NECA. The [3H]NECA binding to human platelet membranes satisfies essential criteria for Ra adenosine receptors and, with some limitations, should be of value for the characterization of adenosine receptors in Ra subtype selective cells.

Ra adenosine receptors in human platelets. Characterization by 5'-N-ethylcarboxamido[3H]adenosine binding in relation to adenylate cyclase activity.

Direct binding studies of adenosine receptors

The modulation of radioligand binding at Ri adenosine receptors of rat fat cells by guanine nucleotides and cations was investigated. Guanine nucleotides (in the order of potency: GTP=GDP>Gpp(NH)p>5′-GMP) decreased the binding of the Ri receptor agonist (−)N6-phenylisopropyl[3H]adenosine ([3H]PIA), but did not affect binding of the antagonist 1,3-diethyl-8[3H]phenylxanthine ([3H]DPX). Saturation of [3H]PIA binding revealed that GTP (100 μmol/l) converts the high affinity form of the Ri receptor into a low affinity form. This effect was confirmed in kinetic experiments. GTP decreased the potency of agonists in competing for [3H]DPX binding, as shown by a 50-fold shift of the Ki-value for (−)PIA, whereas antagonist-induced inhibition of binding remained unchanged. The divalent cations Mg2+ and Ca2+ produced a slight increase in [3H]PIA binding but did not affect [3H]DPX binding. Mn2+ markedly decreased both agonist and antagonist binding at Ri adenosine receptors. Divalent cations reversed the guanine nucleotide-induced decrease of affinity of the Ri receptor. Na+ did not significantly affect agonist or antagonist binding but abolished the stimulatory effect of Mg2+ on agonist binding in the presence of GTP. Our data indicate that guanine nucleotides convert the Ri adenosine receptor of rat fat cells from a high to a low agonist affinity state and that the modulation of radioligand binding by mono-and divalent cations differs from that of Ri receptors of other tissues.

Guanine nucleotide and cation regulation of radioligand binding to Ri adenosine receptors of rat fat cells.

N6-p-Hydroxyphenylisopropyladenosine (HPIA) has been labelled with carrier-free Na[125I] to very high specific activity (2,175 Ci/mmol) and used as an agonist ligand to characterize Ri adenosine receptors in rat cerebral cortex membranes. The binding is saturable, reversible, stereospecific and dependent on protein concentration. The specific binding at 37°C was of high affinity with an equilibrium dissociation constant KD of 0.48 nmol/l and was saturable with 0.23 pmol of [125I]HPIA per mg of protein. The rate constant of association, k1, was 3.25×108 l mol−1 min−1 and that of dissociation, k2, 0.0110 min−1 yielding a t1/2 of 63 min. In competition experiments the (−)isomer of N6-phenylisopropyladenosine (PIA) was 16-fold more potent than the (+)isomer in competing for the binding sites. Specific binding was most effectively displaced by N6-cyclohexyladenosine (CHA, ki=0.26 nmol/l), (−)PIA (ki=0.33 nmol/l) and HPIA (ki=0.52 nmol/l), whereas 5′-N-ethylcarboxamidoadenosine (NECA, ki-1.42 nmol/l) was less effective. The methylxanthines 3-isobutyl-1-methylxanthine (IBMX), theophylline and caffeine which have been classified as adenosine antagonists had ki values between 5–34 μmol/l. Binding of [125I]HPIA was regulated by guanine nucleotides and divalent cations. The results indicate that [125I]HPIA labels Ri adenosine receptors in rat brain membranes.

U. Schwabe

Papers

Characterization of adenosine receptors in rat brain by (-)[3H]N6-phenylisopropyladenosine.

Ra adenosine receptors in human platelets. Characterization by 5'-N-ethylcarboxamido[3H]adenosine binding in relation to adenylate cyclase activity.

Direct binding studies of adenosine receptors

Guanine nucleotide and cation regulation of radioligand binding to Ri adenosine receptors of rat fat cells.

[125I] N6-p-Hydroxyphenylisopropyladenosine, a new ligand for Ri adenosine receptors.