Transgenic overexpression of cardiac A(1) adenosine receptors mimics ischemic preconditioning

The role of A(1) adenosine receptors (A(1)AR) in ischemic preconditioning was investigated in isolated crystalloid-perfused wild-type and transgenic mouse hearts with increased A(1)AR. The effect of preconditioning on postischemic myocardial function, lactate dehydrogenase (LDH) release, and infarct size was examined. Functional recovery was greater in transgenic versus wild-type hearts (44.8 +/- 3.4% baseline vs. 25.6 +/- 1.7%). Preconditioning improved functional recovery in wild-type hearts from 25.6 +/- 1.7% to 37.4 +/- 2.2% but did not change recovery in transgenic hearts (44.8 +/- 3.4% vs. 44.5 +/- 3.9%). In isovolumically contracting hearts, pretreatment with selective A(1) receptor antagonist 1, 3-dipropyl-8-cyclopentylxanthine attenuated the improved functional recovery in both wild-type preconditioned (74.2 +/- 7.3% baseline rate of pressure development over time untreated vs. 29.7 +/- 7.3% treated) and transgenic hearts (84.1 +/- 12.8% untreated vs. 42.1 +/- 6.8% treated). Preconditioning wild-type hearts reduced LDH release (from 7,012 +/- 1,451 to 1,691 +/- 1,256 U. l(-1). g(-1). min(-1)) and infarct size (from 62.6 +/- 5.1% to 32.3 +/- 11.5%). Preconditioning did not affect LDH release or infarct size in hearts overexpressing A(1)AR. Compared with wild-type hearts, A(1)AR overexpression markedly reduced LDH release (from 7,012 +/- 1,451 to 917 +/- 1,123 U. l(-1). g(-1). min(-1)) and infarct size (from 62.6 +/- 5.1% to 6.5 +/- 2.1%). These data demonstrate that murine preconditioning involves endogenous activation of A(1)AR. The beneficial effects of preconditioning and A(1)AR overexpression are not additive. Taken with the observation that A(1)AR blockade equally eliminates the functional protection resulting from both preconditioning and transgenic A(1)AR overexpression, we conclude that the two interventions affect cardioprotection via common mechanisms or pathways.     

Involvement of adenosine A1 and A2A receptors in (-)-linalool-induced antinociception

In recent studies performed in our laboratory we have shown that acute administration of (-)-linalool, the natural occurring enantiomer in essential oils, possesses anti-inflammatory, antihyperalgesic and antinociceptive effects in different animal models. The antihyperalgesic and antinociceptive effects of (-)-linalool have been ascribed to its capacity in stimulating the opioidergic, cholinergic and dopaminergic systems, as well as to its interaction with K+ channels, or to its local anaesthetic activity and/or to the negative modulation of glutamate transmission. Activation of A1 or A2A receptors has been shown to induce antinociceptive effects, and the possible involvement of adenosine in (-)-linalool antinociceptive effect, has not been elucidated yet. Therefore, in the present study, we have investigated the effects of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a selective adenosine A1 receptor antagonist and the effects of 3,7-dimethyl-1-propargilxanthine (DMPX), a selective adenosine A2A receptor antagonist on the antinociception of (-)-linalool in mice, measured in the hot-plate test. Both DPCPX (0.1 mg/kg; i.p.) and DMPX (0.1 mg/kg; i.p.) pre-treatment significantly depressed the antinociceptive effect of (-)-linalool at the highest doses tested. These findings demonstrated that the effect of (-)-linalool on pain responses is, at least partially, mediated by the activity of adenosine A1 and A2A receptors.     

Distribution and expression of A1 adenosine receptors,adenosine deaminase and adenosine deaminase-binding protein (CD26) in goldfish brain

The expression patterns of adenosine A(1) receptors (A(1)Rs), adenosine deaminase (ADA) and ADA binding protein (CD26) were studied in goldfish brain using mammalian monoclonal antibody against A(1)R and polyclonal antibodies against ADA and CD26. Western blot analysis revealed the presence of a band of 35 kDa for A(1)R in membrane preparations and a band of 43 kDa for ADA in both cytosol and membranes. Immunohistochemistry on goldfish brain slices showed that A(1) receptors were present in several neuronal cell bodies diffused in the telencephalon, cerebellum, optic tectum. In the rhombencephalon, large and medium sized neurons of the raphe nucleus showed a strong immunopositivity. A(1)R immunoreactivity was also present in the glial cells of the rhombencephalon and optic tectum. An analogous distribution was observed for ADA immunoreactivity. Tests for the presence of CD26 gave positive labelling in several populations of neurons in the rhombencephalon as well as in the radial glia of optic tectum, where immunostaining for ADA and A(1)R was observed. In goldfish astrocyte cultures the immunohistochemical staining of A(1)R, ADA and CD26, performed on the same cell population, displayed a complete overlapping distribution of the three antibodies. The parallel immunopositivity, at least in some discrete neuronal areas, for A(1)Rs, ADA and CD26 led us to hypothesize that a co-localization among A(1)R, ecto-ADA and CD26 also exists in the neurons of goldfish since it has been established to exist in the neurons of mammals. Moreover, we have demonstrated for the first time, that A(1)R, ecto-ADA and CD26 co-localization is present on the astroglial component of the goldfish brain. This raises the possibility that a similar situation is also shown in the glia of the mammalian brain.     

Mechanisms mediating regional sympathoactivatory responses to stimulation of NTS A(1) adenosine receptors

Selective activation of adenosine A(1) and A(2a) receptors in the subpostremal nucleus tractus solitarius (NTS) increases and decreases mean arterial pressure (MAP), respectively, and decreases heart rate (HR). We have previously shown that the decreases in MAP evoked by NTS A(2a) receptor stimulation were accompanied with differential sympathetic responses in renal (RSNA), lumbar (LSNA), and preganglionic adrenal sympathetic nerve activity (pre-ASNA). Therefore, now we investigated whether stimulation of NTS A(1) receptors via unilateral microinjection of N(6)-cyclopentyladenosine (CPA) elicits differential activation of the same sympathetic outputs in alpha-chloralose-urethane-anesthetized male Sprague-Dawley rats. CPA (0.33-330.0 pmol in 50 nl) evoked dose-dependent increases in MAP, variable decreases in HR, and differential increases in all recorded sympathetic outputs: upward arrow pre-ASNA > upward arrow RSNA > or = upward arrow LSNA. Sinoaortic denervation + vagotomy abolished the MAP and LSNA responses, reversed the normal increases in RSNA into decreases, and significantly attenuated increases in pre-ASNA. NTS ionotropic glutamatergic receptor blockade with kynurenate sodium (4.4 nmol/100 nl) reversed the responses in MAP, LSNA, and RSNA and attenuated the responses in pre-ASNA. We conclude that afferent inputs and intact glutamatergic transmission in the NTS are necessary to mediate the pressor and differential sympathoactivatory responses to stimulation of NTS A(1) receptors.     

Agonist-promoted heteromeric oligomerization between adenosine A(1) and P2Y(1) receptors in living cells

We have explored the process of oligomerization of G protein-coupled purinergic receptors, adenosine A(1) receptor (A(1)R) and P2Y(1) receptor (P2Y(1)R), in intact HEK293T cells by means of modified bioluminescence resonance energy transfer technology (BRET(2)) that offers greatly improved separation of the emission spectra of the donor and acceptor moieties compared to traditional BRET. This approach identified both constitutive and agonist-promoted heteromeric oligomerization between Myc-tagged P2Y(1)R fused to a donor, Renilla luciferase (Myc-P2Y(1)R-Rluc) and HA-tagged A(1)R fused to an acceptor, a different form of green fluorescent protein (HA-A(1)R-GFP(2)). The BRET(2) signal increased in a time-dependent manner in the cells expressing HA-A(1)R-GFP(2)/Myc-P2Y(1)R-Rluc upon addition of agonists for both receptors, which could be inhibited by pretreatment with the P2Y(1)R antagonist MRS2179. A high degree of HA-A(1)R-GFP(2) and Myc-P2Y(1)R-Rluc co-localization in the co-transfected HEK293T cells was also observed by confocal laser microscopy. These results indicate that A(1)R and P2Y(1)R can form constitutive hetero-oligomers in living cells and this process is promoted by the simultaneous activation of both receptors.     

The glycoprotein nature of A1 adenosine receptors

A1 adenosine receptors from different tissues and species were photoaffinity labelled and then the carbohydrate content was examined by both enzymatic and chemical treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the labelled membrane receptors shows that neuraminidase treatment alters the electrophoretic mobility of the receptor band indicating the presence of terminal neuraminic acids. Neuraminidase digestion does not influence the binding characteristics of the receptor. The totally deglycosylated receptor protein obtained by chemical treatment has an apparent molecular weight of 32,000.     

Structure and function of A1 adenosine receptors

The A1 adenosine receptor is the best characterized of the widely distributed purinergic receptor family. The purified brain A1 receptor is a monomeric 35- to 36-kDa glycoprotein. A1 receptors can be clearly distinguished from A2 adenosine receptors on the basis of structure activity relationships with selective ligands. Recent structure activity data suggest that subtypes of A1 (A1a, A1b, and A3) and A2 (A2a and A2b) receptors may exist. A1 receptor-mediated responses are coupled via multiple pertussis toxin-sensitive GTP binding proteins (G proteins) to many different effectors in various tissues: adenylate cyclase, phospholipase C, Na+- Ca2+ exchange, Ca2+ channels, Cl- channels, and K+ channels. The formation of calcium-mobilizing inositol phosphates can either be enhanced or inhibited. In general, adenosine has been found to act in concert with other hormones or neurotransmitters in either an inhibitory or a stimulatory way. The myriad modulatory actions of adenosine suggest that: 1) adenosine may simultaneously produce multiple effects within the same cell; and 2) activation of A1 receptors may lead to either a decrease or an increase in the coupling of other receptors to their G proteins.     

ATP-sensitive K(+) channels regulate the concentrative adenosine transporter CNT2 following activation by A(1) adenosine receptors

This study describes a novel mechanism of regulation of the high-affinity Na(+)-dependent adenosine transporter (CNT2) via the activation of A(1) adenosine receptors (A(1)R). This regulation is mediated by the activation of ATP-sensitive K(+) (K(ATP)) channels. The high-affinity Na(+)-dependent adenosine transporter CNT2 and A(1)R are coexpressed in the basolateral domain of the rat hepatocyte plasma membrane and are colocalized in the rat hepatoma cell line FAO. The transient increase in CNT2-mediated transport activity triggered by (-)-N(6)-(2-phenylisopropyl)adenosine is fully inhibited by K(ATP) channel blockers and mimicked by a K(ATP) channel opener. A(1)R agonist activation of CNT2 occurs in both hepatocytes and FAO cells, which express Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B mRNA channel subunits. With the available antibodies against Kir6.X, SUR2A, and SUR2B, it is shown that all of these proteins colocalize with CNT2 and A(1)R in defined plasma membrane domains of FAO cells. The extent of the purinergic modulation of CNT2 is affected by the glucose concentration, a finding which indicates that glycemia and glucose metabolism may affect this cross-regulation among A(1)R, CNT2, and K(ATP) channels. These results also suggest that the activation of K(ATP) channels under metabolic stress can be mediated by the activation of A(1)R. Cell protection under these circumstances may be achieved by potentiation of the uptake of adenosine and its further metabolization to ATP. Mediation of purinergic responses and a connection between the intracellular energy status and the need for an exogenous adenosine supply are novel roles for K(ATP) channels.     

Receptor-G protein gamma specificity: gamma11 shows unique potency for A(1) adenosine and 5-HT(1A) receptors

G protein coupled receptors activate signal transducing guanine nucleotide-binding proteins (G proteins), which consist of an alpha subunit and a betagamma dimer. Whole cell studies have reported that receptors signal through specific betagamma subtypes. Membrane reconstitution studies with the adenosine A(1) and alpha(2A) adrenergic receptors have reached a similar conclusion. We aimed to test the generality of this finding by comparing the gamma subtype specificity for four G(i)-coupled receptors: alpha(2A) adrenergic; A1 adenosine (A(1)-R); 5-hydroxytryptamine(1A) (5-HT(1A)-R); mu opioid. Membranes were reconstituted with Galpha(i)(1) and five gamma subtypes (dimerized to beta1). Using a sensitive alpha-betagamma binding assay, we show that all recombinant betagamma (except beta1gamma1) had comparable affinity for alpha(i)(1). Using high affinity agonist binding as a measure of receptor-G protein coupling, betagamma-containing gamma11 was the most potent for A(1)-R and 5-HT(1A)-R (p < 0.05, one way ANOVA) while gamma7 was most potent for the other two receptors. gamma11 was 3-8-fold more potent for the A(1)-R than were the other gamma subtypes. Also, gamma11 was 2-8-fold more potent for A(1)-R than at the other receptors, suggesting a unique coupling specificity of the A(1)-R for gamma11. In contrast, the discrimination by receptors for the other betagamma subtypes (beta1 and gamma1, gamma2, gamma7, and gamma10) was limited (2-3-fold). Thus the exquisite betagamma specificity of individual receptors reported in whole cell studies may depend on in vivo mechanisms beyond direct receptor recognition of betagamma subtypes.     

2-Aminothiazoles: a new class of agonist allosteric enhancers of A(1) adenosine receptors

This report describes the synthesis and structure-activity relationships of a new class of A(1) adenosine receptor agonist allosteric enhancers, 2-aminothiazolium salts. The EC(50) of compounds 6a, 6b, 7, and 8 were 0.3, 4.5, 3.8, and 1.2 microM, substantially lower than that of the 'Gold Standard' 2-amino-3-benzoyl thiophene (PD 81,723), which has an EC(50) of 38 microM.     

Constitutive activation of A(3) adenosine receptors by site-directed mutagenesis

The objective of this study was to create constitutively active mutant human A(3) adenosine receptors (ARs) using single amino acid replacements, based on findings from other G protein-coupled receptors. A(3) ARs mutated in transmembrane helical domains (TMs) 1, 3, 6, and 7 were expressed in COS-7 cells and subjected to agonist radioligand binding and phospholipase C (PLC) and adenylyl cyclase (AC) assays. Three mutant receptors, A229E in TM6 and R108A and R108K in the DRY motif of TM3, were found to be constitutively active in both functional assays. The potency of the A(3) agonist Cl-IB-MECA (1-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide) in PLC activation was enhanced by at least an order of magnitude over wild type (EC(50) 951 nM) in R108A and A229E mutant receptors. Cl-IB-MECA was much less potent (>10-fold) in C88F, Y109F, and Y282F and mutants or inactive following double mutation of the DRY motif. The degree of constitutive activation was more pronounced for the AC signaling pathway than for the PLC signaling pathway. The results indicated that specific locations within the TMs proximal to the cytosolic region were responsible for constraining the receptor in a G protein-uncoupled conformation.     

Phosphatase inhibitor cantharidin blocks adenosine A(1) receptor anti-adrenergic effect in rat cardiac myocytes

Experiments were performed to examine whether the protein phosphatase inhibitor cantharidin blocks the anti-adrenergic effect of adenosine A(1) receptor stimulation. In electrically stimulated adult rat ventricular myocytes loaded with the intracellular calcium concentration ([Ca(2+)](i)) indicator fluo-3, isoproterenol (10 nM) increased systolic [Ca(2+)](i) by 46%, increased twitch amplitude by 56%, and increased total cellular cAMP content by 140%. The adenosine A(1) receptor agonist 2-chloro-N(6)-cyclopentlyadenosine (CCPA) reduced isoproterenol-stimulated [Ca(2+)](i) and contractility by 87 and 80%, respectively, but reduced cAMP content by only 18%. Cantharidin had no effects on myocyte [Ca(2+)](i), contractility, or cAMP in the absence or presence of isoproterenol but blocked the effects of CCPA on [Ca(2+)](i) and contractility by approximately 44%. Cantharidin had no effect on CCPA attenuation of isoproterenol-induced increases in cAMP. Pretreatment with CCPA also reduced the increase in contractile parameters produced by the direct cAMP-dependent protein kinase A (PKA) activator 8-bromocAMP. These results suggest that activation of protein phosphatases mediate, in part, the anti-adrenergic effect of adenosine A(1) receptor activation in ventricular myocardium.     

Neutrophil adherence to endothelium is enhanced via adenosine A1 receptors and inhibited via adenosine A2 receptors.

We have recently demonstrated that human neutrophils (PMN) possess two different classes of adenosine receptors (A1 and A2) that, when occupied, promote chemotaxis and inhibit the generation of reactive oxygen species (e.g., O2- and H2O2), respectively. We have previously demonstrated that adenosine protects endothelial cells (EC) from injury by stimulated neutrophils (PMN) both by diminishing generation of H2O2 and inhibiting adherence of PMN to EC. We therefore determined whether occupancy of A1 or A2 adenosine receptors regulated adherence of PMN to EC. At concentrations similar to those required to inhibit release of O2- by ligation of A2 receptors, both adenosine (IC50 = 56 nM) and 5'N-ethylcarboxamidoadenosine (NECA, IC50 = 8 nM), the most potent A2 agonist, inhibited adherence to EC by stimulated PMN (FMLP, 0.1 microM). In direct contrast, the specific A1 agonists N6-phenylisopropyladenosine and N6-cyclopentyladenosine (CPA) promoted PMN adherence to EC at concentrations of 1-100 nM. To further investigate the mechanisms by which adenosine receptor agonists affected the adherence of stimulated PMN we examined the effect of NECA (A2) and CPA (A1) on the adherence of PMN to fibrinogen (a ligand for the beta 2 integrin CD11b/CD18) and to gelatin. In a dose-dependent manner (IC50 = 2 nM), NECA inhibited the adherence of FMLP-treated PMN to fibrinogen- but not gelatin-coated plates. In contrast, CPA (A1) promoted adherence of stimulated PMN to gelatin-(EC50 = 13 pM) but not fibrinogen-coated plates. Theophylline (10 microM), an adenosine receptor antagonist, reversed the inhibition by NECA (0.3 microM) of stimulated neutrophil adherence to fibrinogen. These observations not only confirm the presence of A1 and A2 receptors on PMN but also suggest two opposing roles for adenosine in inflammation. Occupancy of A1 receptors promotes neutrophil adherence to endothelium and chemotaxis (a proinflammatory role) whereas occupancy of A2 receptors inhibits adherence and generation of toxic oxygen metabolites (an antiinflammatory role).     

A(1) adenosine receptors in human neutrophils: direct binding and electron microscope visualization.

By occupying specific surface receptors, adenosine and adenosine analogues modulate neutrophil functions; in particular, functional and biochemical studies have shown that A(1) adenosine receptors modulate chemotaxis in response to chemotactic peptides. Until now, the characteristics of the specific agonist binding and the visualization of A(1) receptors in human neutrophils have not been investigated. In the present study, we used the agonist [(3)H] CHA for radioligand binding studies and a CHA-biotin XX probe in order to visualize the A(1) binding sites in human neutrophils, ultrastructurally, by conjugation with colloidal gold-streptavidin. [(3)H] CHA bound A(1) adenosine receptors with selectivity and specificity, although with a low binding capacity. Scatchard analysis showed a Kd value of 1.4 +/- 0.08 nM and a maximum density of binding sites of 7.1 +/- 0.37 fmol/mg of proteins. The good affinity and selectivity of the CHA-biotin XX probe for A(1) adenosine receptors allowed us to visualize them, after conjugation with colloidal gold-streptavidin, as electron-dense gold particles on the neutrophil surface and inside the cell. The internalization of the ligand-receptor complex was followed in a controlled temperature system, and occurred through a receptor-mediated pathway. The kinetics of the intracellular trafficking was fast, taking less than 5 min. These data suggest that the CHA-biotin XX-streptavidin-gold complex is a useful marker for the specific labelling of A(1) binding sites and to follow the intracellular trafficking of these receptors.     

Hetero-oligomerization of adenosine A1 receptors with P2Y1 receptors in rat brains

Adenosine and ATP modulate cellular and tissue functions via specific P1 and P2 receptors, respectively. Although, in general, adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, little is known about the direct interaction between P1 and P2 receptors. We recently demonstrated that the G(i/o)-coupled adenosine A1 receptor (A1R) and G(q/11)-coupled P2Y1 receptor (P2Y1R) form a heteromeric complex with a unique pharmacology in cotransfected HEK293T cells using the coimmunoprecipitation of differentially epitope-tagged forms of the receptor [Yoshioka et al. (2001) Proc. Natl. Acad. Sci. USA 98, 7617-7622], although it remained to be determined whether this hetero-oligomerization occurs in vivo. In the present study, we first demonstrated a high degree of colocalization of A1R and P2Y1R by double immunofluorescence experiments with confocal laser microscopy in rat cortex, hippocampus and cerebellum in addition to primary cultures of cortical neurons. Then, a direct association of A1R with P2Y1R was shown in coimmunoprecipitation studies using membrane extracts from these regions of rat brain. Together, these results suggest the widespread colocalization of A1R and P2Y1R in rat brain, and both receptors can exist in the same neuron, and therefore associate as hetero-oligomeric complexes in the rat brain.     

Downregulation of A(1) and A(2B) adenosine receptors in human trisomy 21 mesenchymal cells from first-trimester chorionic villi

Human reproduction is complex and prone to failure. Though causes of miscarriage remain unclear, adenosine, a proangiogenic nucleoside, may help determine pregnancy outcome. Although adenosine receptor (AR) expression has been characterized in euploid pregnancies, no information is available for aneuploidies, which, as prone to spontaneous abortion (SA), are a potential model for shedding light on the mechanism regulating this event. AR expression was investigated in 71 first-trimester chorionic villi (CV) samples and cultured mesenchymal cells (MC) from euploid and TR21 pregnancies, one of the most frequent autosomal aneuploidy, with a view to elucidating their potential role in the modulation of vascular endothelial growth factor (VEGF) and nitric oxide (NO). Compared to euploid cells, reduced A(1) and A(2B) expression was revealed in TR21 CV and MCs. The non-selective adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased NO, by activating, predominantly, A(1)AR and A(2A)AR through a molecular pathway involving hypoxia-inducible-factor-1 (HIF-1α), and increased VEGF, mainly through A(2B). In conclusion the adenosine transduction cascade appears to be disturbed in TR21 through reduced expression of A(2B) and A(1)ARs. These anomalies may be implicated in complications such as fetal growth restriction, malformation and/or SA, well known features of aneuploid pregnancies. Therefore A(1) and A(2B)ARs could be potential biomarkers able to provide an early indication of SA risk and their stimulation may turn out to improve fetoplacental perfusion by increasing NO and VEGF.