Adenosine A₃ receptors: novel ligands and paradoxical effects

Abstract

The physiological role of the adenosine A₃ receptor is being investigated using newly synthesized, selective ligands. Recently, in addition to agonists, selective antagonists have been developed that belong to three distinct, non-purine chemical classes: flavonoids, 1,4-dihydropyridine derivatives (e.g. MRS1191, which is 1300-fold selective for human adenosine A₃ vs A₁/A_2A receptors, with a K_i value of 31 nM) and the triazoloquinazolines (e.g. MRS1220, which has a K_i value of 0.65 nM). The A₃ receptor has proven enigmatic in terms of antagonist ligand specificity, coupling to second messengers, and biological effects in the CNS, inflammatory system and cardiovascular system. A₃ receptors are also potentially involved in apoptosis. It appears that intense, acute activation of A₃ receptors acts as a lethal input to cells, while low concentrations of A₃ receptor agonists protect against apoptosis. Here, Kenneth Jacobson describes how A₃ receptor agonists might be useful in treating inflammatory conditions, possibly through their inhibition of tumour necrosis factor α (TNF-α) release, which has been shown in macrophages. A₃ receptor antagonists might be useful in treating asthma or acute brain ischaemia. Recently, the versatility of A₃ receptor agonists, administered either before or during ischaemia, in eliciting potent cardioprotection has been shown.

Adenosine has been shown to be a critical modulator of a vast array of physiological functions, through activation of one or more of the four known receptor subtypes: A₁, A_2A, A_2B and A₃ (Refs 1, 2). The adenosine A₁ and A_2A receptors, pharmacologically well characterized through the use of selective ligands, generally have a protective role, i.e. in decreasing energy demand and increasing energy supply, respectively, under conditions of stress. Relatively recently identified through cloning^1,2, the A₃ receptor (Fig. 1) has provided a new challenge to medicinal chemists in search of selective ligands³ and to pharmacologists in defining its role in vivo. With the recent availability of selective agonists and antagonists, both protective and lethal effects of A₃ receptor activation have been discovered.

Fig. 1.

Processes of formation and degradation of adenosine in the cell and actions at A₃ adenosine receptors. The dashed line indicates activity only at micromolar concentrations of IB-MECA. ADA, adenosine deaminase; AK, adenosine kinase; GRKs, G protein-coupled receptor kinases; Ins(1,4,5)P₃, inositol (1,4,5)-trisphosphate; PI, phosphatidylinositol; PKA, cAMP-dependent protein kinase; SAH, S-adenosyl homocysteine.

In humans, A₃ receptors are found in the lungs, liver, heart and kidneys, with a lower density being found in the brain and testes². A₃ receptors are found in both neurones⁴ and astrocytes⁵. Although the density of A₃ receptors in the brain is possibly too low for mapping using either autoradiography with a high-affinity agonist, [¹²⁵I]N⁶-(4-aminobenzyl)-adenosine-5′-N-methyluronamide {[¹²⁵I]I-AB-MECA} (Fig. 2)^6,7, or in situ hybridization⁸, a role for this subtype in the CNS has been proposed^9,10.

Fig. 2.

Structures of highly potent adenosine A₃ receptor agonists. Abbreviations used in the text and receptor binding affinities at rat A₁/A_2A/A₃ receptors (nM) are indicated. Cl-IB-MECA, chloro-N⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide; DBXRM, 1,3-dibutylxanthine-7-riboside-5′-N-methyl-carboxamide; I-AB-MECA, [¹²⁵I]N⁶-(4-aminobenzyl)-adenosine-5′-N-methyluronamide; IB-MECA, N⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide.

The A₃ receptors are under scrutiny in relation to potential therapeutic approaches for treating inflammatory and neurodegenerative diseases, asthma and cardiac ischaemia^10–17. A₃ receptor ligands are protective in cerebral ischaemia models in gerbils¹⁰. In the heart, both A₁ and A₃ receptor agonists appear to protect cardiac myocytes^14–17, but the latter do not cause the hypotension and hypothermia associated with agonists for the other adenosine receptors. Several years ago, a commentary by Beaven et al.¹¹ suggested, on the basis of studies on a rat basophilic cell line, that a then hypothetical A₃ receptor antagonist could be a useful anti-asthmatic drug. Indeed, eosinophils are the only cells in which native human A₃ receptors have been characterized using radioligand binding¹⁸. The occurrence of A₃ receptors on these cells is consistent with the proposed relevance of this subtype to asthma, in which eosinophils may be activated.

Unique pharmacological properties within the adenosine receptor family

The concentration of endogenous adenosine required for half-occupancy of A₁ and A_2A receptors is in the range of 10⁻⁸ to 10⁻⁷ M (Refs 1, 9), concentrations that might be achieved in the basal, resting state of an organ. The K_i value of adenosine in binding to the rat A₃ receptor has not been determined directly, but has been estimated to be 10⁻⁶ M (Ref. 9). Thus, activation of this subtype may require a relatively high concentration of adenosine, such as would occur during hypoxic stress and other cellular damage. Therefore, the pathophysiological role of the A₃ receptor might be very different from the role of the A₁ and A_2A subtypes, in that it would act as an endogenous regulator under conditions of more severe challenge.

The low affinity of xanthines, the classic antagonists of the A₁, A_2A and A_2B subtypes, at rat A₃ receptors is striking^1,2,9. At human, dog and sheep A₃ receptors^19–21, 8-phenyl-substituted xanthines bearing acidic groups are of higher potency as antagonists; however, none have been found to be highly selective. These differences in xanthine affinity among species and the relatively low degree of homology between human and rat receptor sequences (72%) have raised the hypothesis of two potential subtypes of A₃ receptors, although this remains unsubstantiated. The dramatic species differences in antagonist affinity and in pharmacological responses make the extrapolation of studies of A₃ receptors in rodents to the potential treatment of human disease more challenging. In general, one must be be cautious in comparing responses between species in which tissue and cellular expression of A₃ receptors might be different.

In addition to a unique structure–activity profile for agonists, and particularly for antagonists, activation of the A₃ receptor has a characteristic second messenger profile (Fig. 1), in that it has been shown to stimulate directly phospholipases C (Refs 1, 22) and D (Ref. 23) and to inhibit adenylate cyclase¹. In HL-60 cells, activation of A₃ receptors results in the influx of Ca²⁺ and its release from intracellular stores²⁴. In addition, in the RBL-2H3 basophilic cell line, the potency of adenosine receptor agonists in raising [Ca²⁺]_i but not IP₁ levels parallels A₃ receptor affinity²⁵. In the human eye, A₃ receptors also regulate chloride channels of non-pigmented ciliary epithelial cells [M. M. Civan et al., in abstracts from the XIII International Congress of Eye Research (in press)]. There is a strikingly large potency differential among various functional activities of A₃ selective agonists (Table 1), i.e. the same agonists might act functionally in the low nanomolar range (consistent with their affinity in competitive binding assays) for some functional responses, while in other activities, even within the same species, micromolar concentrations of the agonists are needed. Although for many receptors, the measured affinity is typically lower than EC₅₀ values in functional assays, the wide range of these values for A₃ receptors, i.e. spanning ≥4 orders of magnitude, is unusual. The role of spare receptors in this phenomenon has not been explored.

Table 1.

Functional effects of adenosine A₃ receptor agonists

Site	Action	Agonist	Approximate EC50 (nM)	Refs
Primary tissue/culture
Rat hippocampal slicesa	PLC activation	Cl-IB-MECA	50	22
	PLC activation	IB-MECA	180	22
Rat hippocampal slicesa	Inhibition of A1 effects	Cl-IB-MECA	100–1000	4
Rat hippocampal slices	Potentiation of Ca2+ current	APNEA	20	27
Rat cerebellar granule cells	Death
	Increased cAMP	Cl-IB-MECA	15 000	26
Chick ventricular myocytesa	Inhibition of cAMP	Cl-IB-MECA	0.6	15
	Stimulation of PLD	Cl-IB-MECA	2
	Preconditioning	Cl-IB-MECA	8
	Protection during prolonged hypoxia	Cl-IB-MECA	0.2	16
Rat cardiac myocytes	Apoptosis	IB-MECA	≥10 000	28
Human eosinophils	Apoptosis
	Elevation of Ca2+	Cl-IB-MECA	≥10 000	18
	Inhibition of chemotaxis	Cl-IB-MECA	0.1	13
Neutrophils	Degranulation	IB-MECA	1	29
Cell lines
Rat RBL (mast) cellsa	Elevation of Ca2+	Cl-IB-MECA	70	25
		IB-MECA	110	25
Human leukaemia (HL-60)	Apoptosis
	Elevation of Ca2+	Cl-IB-MECA	≥10 000	24
		IB-MECA	≥10 000	24
	Protection against apoptosis	Cl-IB-MECA	10	24
Human U937 macrophagea	Inhibition of TNF-α	IB-MECA	3000	30
		Cl-IB-MECA	3600	31
Human ADF (astroglial) cellsa	Changes in cytoskeleton, Bcl-XL, Rho	Cl-IB-MECA	100	5
Recombinant receptors
Rat A3–CHO cellsa	Inhibition of cAMP	Cl-IB-MECA	70	9
	Inhibition of cAMP	IB-MECA	90	9
Human A3–CHO cellsa	Inhibition of cAMP	IB-MECA	59	31
	Inhibition of growth	Cl-IB-MECA	≥10000	32

^aActivity was antagonized either by a nonselective or selective adenosine A₃ receptor antagonist. APNEA, N⁶-[2-(4-aminophenylethyl)adenosine], a nonselective agonist; IB-MECA, N⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide; PLC, phospholipase C; PLD, phospholipase D; TNF-α, tumour necrosis factor α.

Rat A₃ receptors can interact with G_iα2, G_iα3 and to a lesser extent G_q (Ref. 33). Agonist-induced desensitization of recombinant human A₃ receptors occurs within 20 min, and this is associated with specific down-regulation of G_iα3 and β subunits. The mechanism of desensitization involves phosphorylation of the C-terminal segment of the receptor by G protein receptor-coupled kinases (GRKs), such as GRK 2, 3 and 5 (Ref. 34).

Selective agonists and antagonists

To obtain high potency agonists that selectively activate A₃ receptors, modifications at two sites in the adenosine structure, the N⁶- and 5′-positions, are required⁹. N⁶-(3-iodobenzyl)-adenosine-5′-N-methyl-uronamide (IB-MECA; Fig. 2) was the first highly potent and selective A₃ agonist, both in vitro, in species as diverse as human³¹, dog²¹ and chick¹⁶, and in vivo^9,10. It is approximately 50-fold selective in binding assays for rat A₃ vs either A₁ or A_2A receptors. The radio-ligand [¹²⁵I]I-AB-MECA (Fig. 2) is widely used as a high-affinity radioligand for A₃ receptors⁶, although it is not as selective as IB-MECA (Ref. 7). [¹²⁵I]I-AB-MECA bound to cloned human A₃ receptors expressed in HEK293 cells with a K_d value of 0.59 nM (Ref. 35), but also bound to other subtypes in autoradiographic studies⁷. Substitution at the 2-position of adenosine in combination with modifications at the N⁶- and 5′-positions further enhanced A₃ affinity and selectivity. Thus, 2-chloro-N⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (2Cl-IB-MECA; Fig. 2)⁹ displayed a K_i value of 0.33 nM at A₃ receptors and is 2500- and 1400-fold selective for rat A₃ vs A₁ and A_2A receptors, respectively.

A₃ receptor antagonists, which have been introduced only recently^35–38, were previously hypothesized^10,11 to act as potential anti-asthmatic¹, anti-inflammatory or cerebroprotective agents. Selective antagonists are needed, especially as most of the effects of high concentrations of A₃ agonists (Table 1) have not been ascribed unequivocally to activation of A₃ receptors. Attempts to find leads for selective xanthine-based antagonists were unproductive^9,20. An approach, suggested through molecular modelling, to increase potency of xanthines in A₃ receptor binding by forming the 7-riboside derivative, did indeed enhance subtype selectivity.⁹ Thus, 1,3-dibutylxanthine-7-riboside-5′-N-methylcarboxamide (DBXRM; Fig. 2) is 140-fold selective in binding to rat A₃ vs A₁ receptors. However, as the affinity increased at A₃ receptors, so did the agonist efficacy, and DBXRM proved to be a full agonist at recombinant rat A₃ receptors.

Because xanthines tended to bind only weakly to A₃ receptors, an alternative strategy of screening diverse molecules in chemical libraries for leads was adopted in the design of selective A₃ receptor antagonists. Once the structural principles of A₃ receptor selectivity were discovered in these novel antagonist classes, the leads could be optimized through iterative cycles of chemical synthesis and pharmacological testing. Promising leads for A₃ receptor antagonists appeared among non-xanthine heterocycles (Fig. 3). For example, 1,4-dihydropyridines, known as potent blockers of L-type Ca²⁺ channels and used widely in treating coronary heart disease, were found to bind to human adenosine A₃ receptors. Common dihydropyridine drugs typically bound either non-selectively (for example, nifedipine, with a K_i value of 8.3 μM) or in some cases with selectivity for the A₃ vs other adenosine receptor subtypes (for example, S-niguldipine, with a K_i value of 2.8 μM)³⁶.

Fig. 3.

Structures of selective A₃ adenosine receptor antagonists. Abbreviations used in the text and receptor binding affinities (μM) are indicated.

We have used the 1,4-dihydropyridine core as a template, in which it has been possible to select for affinity at adenosine receptors and completely deselect for affinity at L-type Ca²⁺ channels (K_i <100 μM), principally through introduction of a 6-phenyl group. At human A_2B receptors, such 1,4-dihydropyridines are similarly inactive⁴. For example, a trisubstituted 1,4-dihydro-6-phenylpyridine analogue, MRS1191 (Fig. 3)³⁶, has been found to inhibit radioligand binding at the human A₃ receptor with a K_i value of 31 nM, while the same derivative was nearly inactive in binding at A₁ and A_2A receptor sites (i.e. >1300-fold selective). Even in the rat, MRS1191 was selective pharmacologically in various paradigms; it bound with 28-fold higher affinity for A₃ (K_i value of 1.42 μM) vs A₁ receptors, and at a 10 μM concentration it antagonized only the A₃ subtype in the CA1 region of the hippocampus^4,36. Furthermore, MRS1191 antagonized the effects of the A₃ receptor-selective agonist IB-MECA on inhibition of adenylate cyclase via recombinant human or rat A₃ receptors³¹. In chick ventricular myocyte cultures, MRS1191 antagonized the anti-ischaemic effects of Cl-IB-MECA (Ref. 16). Thus, dihydropyridine derivatives, such as MRS1191, appear to be useful as A₃ receptor antagonists across species, although there is still a need for high-affinity antagonists of rat A₃ receptors.

The structure–activity relationships of analogues of MRS1191, containing both subtle and drastic structural changes at various positions of the dihydropyridine ring (its 3- and 5-acyl substituents, the 4- and 6-aryl/alkyl substituents and the 2-methyl group), have been investigated systematically³⁶. Substitutions of a 5-benzyl ester group provide the greatest versatility for achieving >30000-fold human A₃ receptor selectivity and nanomolar potency. Affinity and selectivity for the human A₃ receptor within this series was optimal in MRS1334 (Fig. 3), which has a K_i value of 2.7 nM (Ref. 36). These racemic dihydropyridines await optical resolution; however, side-by-side comparison of previously known dihydropyridine enantiomers shows that the stereoselectivity at A₃ receptors favours the R-isomer, the opposite of the stereoselectivity at L-type Ca²⁺ channels.

Naturally occurring phenolic derivatives (e.g. the flavones and flavonols) provided another structural lead for development of A₃ receptor antagonists³⁵. The affinity of common phytochemicals at adenosine receptors suggests that a wide range of natural substances in the human diet might potentially antagonize the effects of endogenous adenosine, including those mediated via the A₃ subtype. The flavonoid class has been chemically optimized in the form of MRS1067 (Ref. 35), which is 200-fold selective for human A₃ vs A₁ adenosine receptors. Other high-affinity A₃ receptor-selective antagonists that have been reported recently include a triazolonaphthyridine (L249313; Fig. 3)³⁸, thiazolopyrimidine (L268605; Fig. 3)³⁸ and a derivative of the triazoloquinazoline CGS15943 (MRS1220; Fig. 3)³⁷. L249313 binds to human A₁ and A_2A receptors with K_i values of 6.6 and 1.25 μM, respectively. The K_i value at rat A₃ receptors is 33 μM. Although not as selective as MRS1191, MRS1220 is the antagonist with the highest affinity (K_i 0.65 nM) for human A₃ receptors reported yet. MRS1220 is not A₃-selective in the rat, further emphasizing the species differences in antagonist affinity.

Binding of MRS1067, MRS1191 and MRS1220 at human A₃ receptors was shown to be competitive by Scatchard analysis versus binding of [¹²⁵I]I-AB-MECA (Ref. 31). Antagonism was demonstrated in functional assays consisting of agonist-induced inhibition of adenylate cyclase and the stimulation of binding of [³⁵S]GTP-γ-S to the associated G proteins. MRS1220 and MRS1191, with K_B values of 1.7 and 92 nM, respectively, were highly selective for human A₃ receptor vs human A₁ receptor-mediated effects on adenylate cyclase.

Protective versus lethal effects of A₃ receptor activation

The varied effects of A₃ receptor agonists, in vitro and in vivo, appear to be dual and opposite, i.e. either cytoprotective or cytotoxic, depending on the level of receptor activation and the paradigm studied. The mechanisms involved in these opposite effects are not yet fully understood.

Nanomolar concentrations of selective agonists tend to protect cells, while micromolar concentrations are often toxic (Table 1)²⁴. In certain cultured cell lines, antagonists alone are toxic³⁹. The fact that A₃ receptor antagonists representing three diverse chemical classes evoked the common biological effect of apoptosis (programmed cell death, see below) suggests that a tonic state of activation of the A₃ receptor might exist, and that this possible low level of receptor activation has a protective role. If a tonic A₃ receptor activation does exist, the apoptotic effects of A₃ receptor antagonists might simply be explained on the basis of a block of a protective action induced by endogenous adenosine. To explain how very high doses of agonist alone might induce rather than prevent apoptosis, one could propose differential activation of different second messengers by the same receptor at low and high doses (Fig. 1). Such hypotheses will require further investigation, which would be greatly aided by the development of a high-affinity antagonist radioligand for the A₃ receptor. The low density of A₃ receptors has also made study difficult.

Central nervous system

The first cytoprotective effects of an A₃ receptor agonist were shown following its chronic administration in gerbils in a model of stroke. In an in vivo gerbil model of global ischaemia, the acute administration of IB-MECA during ischaemia exacerbated histological and functional damage, clearly worsening the post-occlusive outcome¹⁰. However, chronic pre-administration of the same agent over several weeks had a highly neuroprotective, post-ischaemic effect, in which the agonist was highly cerebroprotective, preserved microtubule-associated protein 2 (MAP2) immunoreactivity, and depressed NO synthase. In primary astroglial cell cultures, nanomolar concentrations of selective A₃ receptor agonists caused protection against cell death and induced differentiation, while high concentrations increased cell death³². In human ADF cells of astroglial lineage, 100 nM Cl-IB-MECA caused a marked reorganization of the cytoskeleton, with appearance of stress fibres and numerous cell protrusions (which became enriched in the anti-apoptotic protein Bcl-x_L), accompanied by induction of the expression of Rho, a small GTP-binding protein⁵. A high concentration of Cl-IB-MECA (≥10 μM) was lethal to cultured rat cerebellar granule neurons, and the toxic effects of glutamate were also augmented²⁷. In preliminary experiments, acute adminstration of the selective A₃ receptor antagonist MRS1191 proved to be cerebroprotective in the gerbil global ischaemia model⁴⁰.

Several possible explanations for the damaging effects of acute A₃ activation during ischaemia have been offered. These include the detrimental effects seen on cerebral bood flow¹⁰ or the release of a cytotoxic agent. Alternatively, the effects might be via neuronal A₃ receptors. In general, high concentrations of IB-MECA and Cl-IB-MECA directly cause influx of Ca²⁺ (Refs 24, 25); however, this may not be relevant to potent in vivo effects. In addition, examples of cross-talk between the A₃ receptor subtype and other adenosine receptors are being discovered. For example, acute activation of presynaptic hippocampal A₃ receptors antagonizes the action of metabotropic glutamate receptors, thus resulting in enhanced glutamate release⁴¹. Dunwiddie et al.⁴ found that A₃ activation counteracts the protective effects of A₁ receptor activation at the hippocampal synapse, i.e. the depression of excitatory transmission (EPSPs) elicited by A₁ agonists is blunted by selective A₃ agonists. In contrast, Mogul and coworkers²⁷ have shown that A₃ receptor activation increases cellular excitability in these neurones through a pathway independent of A₁ receptors. Activation of A₃ receptors in isolated CA3 pyramidal neurones from guinea-pig hippocampus by a low concentration of a selective agonist was also found to potentiate a Ca²⁺ current through a cAMP-dependent protein kinase (PKA)-dependent/protein kinase C (PKC)-independent mechanism²⁷.

The immune system and inflammation

In a variety of human cell lines of the immune system, A₃ agonists at high concentrations often prove lethal (Table 1). Apoptosis, with characteristic DNA fragmentation, has been shown to occur in human leukaemia HL-60 cells, MCF-7 breast cancer cells and in human peripheral blood eosinophils in response to high concentrations (≥10 μM) of A₃ receptor-selective agonists^24,39. A mediator of apoptosis, bak (pro-apoptotic Bcl-2 homology protein), is upregulated under these conditions³⁹. The protective effects of A₃ receptor activation might involve cytokines. Sajjadi et al.³⁰ have shown that micromolar concentrations of the agonist IB-MECA in U937 cells inhibit the release of tumour necrosis factor α (TNF-α), which in turn might induce apoptosis. Clarification of the need for such high doses of agonists, thousands of times higher than the K_i values at A₃ receptors, has awaited the introduction of selective A₃ receptor antagonists, which are now available for the human A₃ receptor. As discussed above, A₃ receptor antagonists of diverse structures alone cause apoptotic cell death³⁹, and cells may be rescued by subcytotoxic concentrations of selective A₃ receptor agonists.

Walker et al.¹² postulated a role for A₃ receptors in lung inflammation, as adenosine leads to exaggerated airway narrowing in individuals with inflammatory airway disorders. Evidence was found that in humans A₃ receptor gene expression is localized to inflammatory cells (eosinophils, but not mast cells) and that gene expression is upregulated in airway inflammation. Cl-IB-MECA inhibited eosinophil chemotaxic responses to PAF, RANTES or LTB, without affecting adhesion receptors CD18 and selectin, or assembly of F-actin. This effect was blocked by the selective A₃ antagonist L249313 (Ref. 13). Based on this effect, it is not known whether an A₃ agonist or antagonist would be more useful in treating asthma, as, theoretically, eosinophil activation could either augment (via migration to site) or counteract (via migration away from site) inflammation. However, other experiments suggest that an antagonist might be more useful. For example, Meade et al. found that in the BDE-strain rat model of airway disease, A₃ receptor agonists induced bronchospasm via mast-type cells⁴². Although aerosol challenge of antigen-immunized rabbits with the non-selective agonist N⁶-[2-(4-aminophenylethyl)-adenosine] (APNEA) did not elicit dose-dependent changes in either airway resistance or dynamic compliance⁴³, Ali et al. found that the agonists IB-MECA and Cl-IB-MECA caused bronchoconstriction⁴⁴.

Selective activation of A₃ receptors appears to inhibit human neutrophil degranulation, suggesting the anti-inflammatory potential of A₃ receptor agonists in neutrophil-mediated tissue injury²⁹.

There might be an involvement of A₃ receptors in cancer⁴⁵. Activation of A₃ receptors reduced cytotoxic lymphocyte adhesion to tumour cells.

Cardiovascular and renal systems

The acute activation of A₃ receptors in rodents leads to hypotension, exclusively via release of histamine and other mediators from peripheral mast cells⁴⁶. However, canine and human mast cells do not react in this manner²¹. Shepherd et al.⁴⁷ found that in microcirculation of the hamster cheek pouch, activation of A₃ receptors results in vasoconstriction, which also occurs through activation of mast cells.

A₃ receptors occur on ventricular, but not atrial, chick cardiac myocytes¹⁵. There are protective effects of A₃ receptor activation in heart cells, administered both prior to^15,17 and during¹⁶ an ischaemic episode (Fig. 4)¹⁷. IB-MECA also protects against myocardial stunning in conscious rabbits⁴⁸. In cultured chick cardiac myocytes, a brief prior exposure to nanomolar concentrations of the A₃ receptor agonist Cl-IB-MECA protected cells from damage induced by subsequent hypoxia^14,15, thus simulating the protection afforded by a brief hypoxic period, a phenomenon termed ‘preconditioning’. Activation by endogenous adenosine of both A₁ and A₃ receptors is thought to mediate preconditioning. Because the culture consisted almost exclusively of ventricular myocytes, this was not an indirect effect of activation of A₃ receptors on mast cells, as has been reported by Fozard and co-workers⁴⁶ to explain hypotensive effects in vivo. Thus, an A₃ agonist at low concentration is potentially useful therapeutically as a cardioprotective agent, having a more sustained duration of protection and fewer in vivo side-effects than other (e.g. A₁-selective agonists) adenosine agonists^14,16,49. However, high concentrations of the same agonists were shown to be damaging, i.e. they induce apoptosis in rat cardiac myocytes²⁸.

Fig. 4.

Cardioprotection elicited by the selective A₃ adenosine receptor agonists, N⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA) and Cl-IB-MECA, during prolonged ischaemia (modified from Ref. 16). Cardiac ventricular myocytes were cultured from chick embryos 14 days in ovo, and cell injury was induced by a 90 min exposure of the culture to hypoxia with glucose deprivation. Release of creatine kinase into the medium was directly proportional to the degree of cell injury. Curves shown were measured in the presence of 1 μM 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (selective A₁ adenosine receptor antagonist), which had no effect on the cardioprotection observed. The selective A₃ adenosine receptor antagonist MRS1191 antagonized the cardio-protection provided by 10 nM Cl-IB-MECA, with an IC₅₀ of ~10 nM. A₃ adenosine agonists were also cardioprotective following a brief exposure, prior to ischaemia (Ref. 15). Squares, IB-MECA; circles, Cl-IB-MECA.

Concluding remarks

By virtue of regulating programmed cell death, A₃ receptors might play a critical role in human disease states. The relation of the A₃ receptor to apoptosis suggests that both A₃ receptor agonists and antagonists might be useful for treating diseases either in which cytotoxicity is undesirable, such as neurodegeneration, or desirable, such as cancer and inflammation. A₃ receptor antagonists might also be useful in treating asthma. The acute administration of an A₃ receptor antagonist or the chronic adminstration of an A₃ receptor agonist appears to protect brain cells in a global ischaemia model, and thus are potential therapeutic approaches for preventing stroke damage. In the heart, because A₃ receptor activation protects both in a preconditioning model and during prolonged ischaemia, selective agonists might be of great clinical importance.

Chemical names

CGS15943

9-chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c] quinazolin-5-amine

L249313

6-carboxymethyl-5,9-dihydro-9-methyl-2-phenyl-[1,2,4]-triazolo[5,1-a][2,7]naphthyridine

L268605

3-(4-methoxyphenyl)-5-amino-7-oxo-thiazolo[3,2]pyrimidine

MRS1067

3,6-dichloro-2′-(isopropoxy)-4′-methylflavone

MRS1191

3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenyl-ethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate

MRS1220

9-chloro-2-(2-furyl)-5-phenylacetylamino [1,2,4]triazolo[1,5-c]quinazoline

MRS1334

3-ethyl 5-(4-nitrobenzyl) 2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate