Giulia Arslan

Structure and function of adenosine receptors and their genes

Four adenosine receptors have been cloned from many mammalian and some non-mammalian species. In each case the translated part of the receptor is encoded by two separate exons. Two separate promoters regulate the A1 receptor expression, and a similar situation may pertain also for the other receptors. The receptors are expressed in a cell and tissue specific manner, even though A1 and A2B receptors are found in many different cell types. Emerging data indicate that the receptor protein is targeted to specific parts of the cell. A1 and A3 receptors activate the Gi family of G proteins, whereas A2A and A2B receptors activate the Gs family. However, other G proteins can also be activated even though the physiological significance of this is unknown. Following the activation of G proteins several cellular effector pathways can be affected. Signaling via adenosine receptors is also known to interact in functionally important ways with signaling initiated via other receptors.

Signaling via A2A adenosine receptor in four PC12 cell clones

PC12 cells are genetically labile and so-called wild-type cells comprise multiple subclones. We have examined the A2A adenosine receptor signal transduction pathways in four such clones (denoted clones 1, 19, 21 and 27) of PC12 cells. Adenosine A2A, A2B and A1 receptor mRNAs were detected in all four clones by RT-PCR, whereas no A3 receptor mRNA was found. A2A receptors were quantitated by radioligand binding using the antagonist radioligand [3H]SCH 58261 ([3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4 triazolo [1,5-c] pyrimidine). The Bmax was highest in clone 1 followed by clones 21, 19 and 27. Whereas the amount of Gi protein appeared similar in all four clones, the amount of Gs protein was higher in clones 21 and 27 than in the other two clones. Maximal responses to the non-selective adenosine analogue NECA (5’-N-ethylcarboxamidoadenosine) were similar to those observed with the selective adenosine A2A receptor agonist CGS 21680 (2-[p-(2-carbonylethyl) phenylethylamino]-5’-N-ethylcarboxamidoadenosine), and were approximately equal in clones 1 and 21, but lower in clone 19 and very low in clone 27. For both compounds EC50 was significantly higher in clone 27 than in clone 1. In both clones the response to NECA could be competitively antagonized by a selective adenosine A2A antagonist, SCH 58261. The present results show that different clones of PC12 cells differ widely in the cAMP increase induced by adenosine analogues and that this is due to differences in the amount of adenosine A2A receptor, G protein and effector. A large difference in receptor number resulted in differences in potency of an agonist.

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

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‐triazolo[1,5‐c]pyrimidine, [3H]‐SCH 58261, to human cloned A2A adenosine receptors. 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). 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‐ethylcarboxamido‐adenosine (2HE‐NECA)>5′‐N‐ethylcarboxamidoadenosine (NECA)=2‐phenylaminoadenosine (CV 1808)>2‐[4‐(2‐carboxyethyl)‐phenethylamino]‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680)>R‐N6‐phenylisopropyladenosine (R‐PIA)N6‐cyclohexyladenosine (CHA)>S‐N6‐phenylisopropyladenosine (S‐PIA). 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. 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. 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.

Stimulatory and inhibitory effects of adenosine A2A receptors on nerve growth factor-induced phosphorylation of extracellular regulated kinases 1/2 in PC12 cells

Effects of nerve growth factor (NGF), adenosine and an adenosine A(2A) receptor agonist (CGS 21680) on the phosphorylation of extracellular-regulated kinases 1/2 (ERK1/2) were examined in PC12 cells. Adenosine and CGS 21680stimulated ERK1/2, but inhibited the phosphorylation of ERK1/2 induced by a 10 min incubation with NGF. Longer treatment with CGS 21680 and NGF (1-2h) resulted in an additive effect on the activation of ERK1/2. Forskolin exerted the same effects, suggesting that they are mediated by cyclic AMP. These results indicate that adenosine A(2A) receptor induced increases in cyclic AMP can stimulate ERK1/2 phosphorylation per se, inhibit the initial and enhance the late NGF-induced activation of ERK1/2. These results may be explained by the fact that NGF action is mediated via different pathways at early and late time points.

Adenosine (P1) receptor signalling

The coupling of the four defined types of adenosine receptors to G proteins and the consequent activation of effector pathways is briefly summarized. It is pointed out that the G proteins are able to influence many types of cellular effector systems, and, in particular, that the α and the β,γ‐subunits may activate different signalling pathways that may either act synergistically or antagonistically in the cell. Because adenosine physiologically plays the role of a modulator, particular emphasis is placed on the interactions with parallel signalling pathways. Drug Dev. Res. 39:262–268, 1996.

Locating the neuronal targets for caffeine

Caffeine is the most widely consumed of all psychoactive drugs. In doses that induce behavioral stimulation it acts primarily on adenosine A2A and possibly A1 receptors. Actions on the latter appear to be very much involved with regulation of the stimulatory glutamatergic input to several neuronal structures, including the striatum. Adenosine A2A receptors are found enriched on a subpopulation of GABAergic output neurons from the striatum, where they coexist with dopamine D2 receptors. The two receptors interact, both at the membrane level and by having opposing effects at the level of second messengers. Inhibition of adenosine A2A receptors by the selective antagonist SCH 58261 causes motor stimulation and a decrease in the expression of immediate early genes in these striatopallidal neurons. This effect is also clearly seen when dopamine D2 activation is blocked, either by preventing dopaminergic impulse flow or by receptor blockade. Thus, a tonic effect of endogenous adenosine is specifically antagonized by adenosine receptor antagonists such as caffeine. Despite a strictly delimited primary effect, secondary interactions between neurons lead to changes in the activity of several groups of neurons in interconnected networks. These changes likely underlie the behavioral effects of caffeine. Drug Dev. Res. 45:324–328, 1998.

Chapter 24 Adenosine and P2 receptors in PC12 cells. Genotypic, phenotypic and individual differences

Publisher Summary This chapter investigates the effects of ATP and related nucleotides on PC12 cells. In PC12 cells, ATP is a powerful stimulator of catecholamine release probably through an increase in the intracellular levels of Ca 2+ . The ATP stimulated increase in Ca 2+ appears to be because of several actions possibly mediated through different receptors. The culture conditions of PC12 cells differ among laboratories and PC12 cells have been shown to be able to change their phenotype, not only when cultured with different differentiating factors such as neurotrophins, but also spontaneously. The chapter investigates the possible sources of variability, which can be schematically subdivided into three levels: first, genetic differences within the same cell clone because of spontaneous mutations of some cells leading to the coexistence of different cell types; second, phenotypic differences because of differentiation induced by specific substances such as NGF; third, differences that can be revealed even in the same clone in the same phenotypic phase. The chapter presents the PC12 clones, isolated based on neomycin resistance, which show a relatively stable phenotype and have been employed for studies of calcium movements.