The Role of Glial Adenosine Receptors in Neural Resilience and the Neurobiology of Mood Disorders

Adenosine receptors were classified into A₁- and A₂-receptors in the laboratory of Bernd Hamprecht more than 25 years ago. Adenosine receptors are instrumental to the neurotrophic effects of glia cells. Both microglia and astrocytes release after stimulation via adenosine receptors factors that are important for neuronal survival and growth. Neuronal resilience is now considered as of pivotal importance in the neurobiology of mood disorders and their treatment. Both sleep deprivation and electroconvulsive therapy, two effective therapeutic measures in mood disorders, are associated with an increase of adenosine and upregulation of adenosine A₁-receptors in the brain. Parameters closely related to adenosine receptor activation such as cerebral metabolic rate and delta power in the sleep EEG provide indirect evidence that adenosinergic signaling may be associated with the therapeutic response to these measures. Thus, neurotrophic effects evoked by adenosine receptors might be important in the mechanism of action of ECT and perhaps also sleep deprivation.     

Regulation of TrkB receptor translocation to lipid rafts by adenosine A2A receptors and its functional implications for BDNF-induced regulation of synaptic plasticity

Brain-derived neurotrophic factor (BDNF) signalling is critical for neuronal development and transmission. Recruitment of TrkB receptors to lipid rafts has been shown to be necessary for the activation of specific signalling pathways and modulation of neurotransmitter release by BDNF. Since TrkB receptors are known to be modulated by adenosine A_2A receptor activation, we hypothesized that activation of A_2A receptors could influence TrkB receptor localization among different membrane microdomains. We found that adenosine A_2A receptor agonists increased the levels of TrkB receptors in the lipid raft fraction of cortical membranes and potentiated BDNF-induced augmentation of phosphorylated TrkB levels in lipid rafts. Blockade of the clathrin-mediated endocytosis with monodansyl cadaverine (100 μM) did not modify the effects of the A_2A receptor agonists, but significantly impaired BDNF effects on TrkB recruitment to lipid rafts. The effect of A_2A receptor activation in TrkB localization was mimicked by 5 μM forskolin, an adenylyl cyclase activator. Also, it was blocked by the PKA inhibitors Rp-cAMPs and PKI-(14-22) and by the Src-family kinase inhibitor PP2. Moreover, removal of endogenous adenosine or disruption of lipid rafts reduced BDNF stimulatory effects on glutamate release from cortical synaptosomes. Lipid raft integrity was also required for the effects of BDNF upon hippocampal long-term potentiation at CA1 synapses. Our data demonstrate, for the first time, a BDNF-independent recruitment of TrkB receptors to lipid rafts, induced by the activation of adenosine A_2A receptors, with functional consequences for TrkB phosphorylation and BDNF-induced modulation of neurotransmitter release and hippocampal plasticity.     

Adenosine A<Subscript>2A</Subscript> receptors in Parkinson’s disease treatment

Latest results on the action of adenosine A_2A receptor antagonists indicate their potential therapeutic usefulness in the treatment of Parkinson’s disease. Basal ganglia possess high levels of adenosine A_2A receptors, mainly on the external surfaces of neurons located at the indirect tracts between the striatum, globus pallidus, and substantia nigra. Experiments with animal models of Parkinson’s disease indicate that adenosine A_2A receptors are strongly involved in the regulation of the central nervous system. Co-localization of adenosine A_2A and dopaminergic D2 receptors in striatum creates a milieu for antagonistic interaction between adenosine and dopamine. The experimental data prove that the best improvement of mobility in patients with Parkinson’s disease could be achieved with simultaneous activation of dopaminergic D2 receptors and inhibition of adenosine A_2A receptors. In animal models of Parkinson’s disease, the use of selective antagonists of adenosine A_2A receptors, such as istradefylline, led to the reversibility of movement dysfunction. These compounds might improve mobility during both monotherapy and co-administration with L-DOPA and dopamine receptor agonists. The use of adenosine A_2A receptor antagonists in combination therapy enables the reduction of the L-DOPA doses, as well as a reduction of side effects. In combination therapy, the adenosine A_2A receptor antagonists might be used in both moderate and advanced stages of Parkinson’s disease. The long-lasting administration of adenosine A_2A receptor antagonists does not decrease the patient response and does not cause side effects typical of L-DOPA therapy. It was demonstrated in various animal models that inhibition of adenosine A_2A receptors not only decreases the movement disturbance, but also reveals a neuroprotective activity, which might impede or stop the progression of the disease. Recently, clinical trials were completed on the use of istradefylline (KW-6002), an inhibitor of adenosine A_2A receptors, as an anti-Parkinson drug.     

5-Substituted 2-benzylidene-1-tetralone analogues as A1 and/or A2A antagonists for the potential treatment of neurological conditions

Adenosine A₁ and A_2A receptors are attracting great interest as drug targets for their role in cognitive and motor deficits, respectively. Antagonism of both these adenosine receptors may offer therapeutic benefits in complex neurological diseases, such as Alzheimer’s and Parkinson’s disease. The aim of this study was to explore the affinity and selectivity of 2-benzylidene-1-tetralone derivatives as adenosine A₁ and A_2A receptor antagonists. Several 5-hydroxy substituted 2-benzylidene-1-tetralone analogues with substituents on ring B were synthesized and assessed as antagonists of the adenosine A₁ and A_2A receptors via radioligand binding assays. The results indicated that hydroxy substitution in the meta and para position of phenyl ring B, displayed the highest selectivity and affinity for the adenosine A₁ receptor with K_i values in the low micromolar range. Replacement of ring B with a 2-amino-pyrimidine moiety led to compound 12 with an increase of affinity and selectivity for the adenosine A_2A receptor. These substitution patterns led to enhanced adenosine A₁ and A_2A receptor binding affinity. The para-substituted 5-hydroxy analogue 3 behaved as an adenosine A₁ receptor antagonists in a GTP shift assay performed with rat whole brain membranes expressing adenosine A₁ receptors. In conclusion, compounds 3 and 12, showed the best adenosine A₁ and A_2A receptor affinity respectively, and therefore represent novel adenosine receptor antagonists that may have potential with further structural modifications as drug candidates for neurological disorders.     

Anti-inflammatory effects of purine nucleosides, adenosine and inosine, in a mouse model of pleurisy: evidence for the role of adenosine A2 receptors

Adenosine and its metabolite, inosine, have been described as molecules that participate in regulation of inflammatory response. The aim of this study was to investigate the effect of adenosine and inosine in a mouse model of carrageenan-induced pleurisy as well as the participation of adenosine receptors in this response. Injection of carrageenan into the pleural cavity induced an acute inflammatory response characterized by leukocyte migration, pleural exudation, and increased release of interleukin-1β and tumor necrosis factor-α in pleural exudates. The treatment with adenosine (0.3–100 mg/kg, i.p.) and inosine (0.1–300 mg/kg, i.p.) 30 min before carrageenan injection reduced significantly all these parameters analyzed. Our results also demonstrated that A_2A and A_2B receptors seem to mediate the adenosine and inosine effects observed, since pretreatment with selective antagonists of adenosine A_2A (ZM241385) and A_2B (alloxazine) receptors, reverted the inhibitory effects of adenosine and inosine in pleural inflammation. The involvement of A₂ receptors was reinforced with adenosine receptor agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 treatment, since its anti-inflammatory effects were reversed completely and partially with ZM241385 and alloxazine injection, respectively. Moreover, the combined treatment with subeffective dose of adenosine (0.3 mg/kg) and inosine (1.0 mg/kg) induced a synergistic anti-inflammatory effect. Thus, based on these findings, we propose that inosine contributes with adenosine to exert anti-inflammatory effects in pleural inflammation, reinforcing the notion that endogenous nucleosides play an important role in controlling inflammatory diseases. This effect is likely mediated by the activation of adenosine A₂ subtype receptors and inhibition of production or release of pro-inflammatory cytokines.     

Modulation of protein kinase C by adenosine: Involvement of adenosine A1 receptor-pertussis toxin sensitive nucleotide binding protein system

The objective of this study was to determine whether adenosine A₁ or A₂ receptor was responsible for the regulation of protein kinase C (PKC) in porcine coronary artery and its coupling to G-protein. Endothelium denuded arterial rings were incubated with PDBu (200nM) in the presence or absence of adenosine receptor agonists and antagonists for 1 day. Following incubation, the arterial rings were contracted with increasing concentrations of endothelin-1 (ET-1) (10^–10–10^–7M). Arteries incubated with PDBu alone failed to produce contraction in response to ET-1. On the contrary, inclusion of A₁ receptor agonist ENBA at 10^–9M in the incubation media with PDBu protected against the PDBu induced blunting of the ET-1 contractions by 50%. Incubation with ENBA alone increased ET-1 dependent contractions by about 2 fold. Inclusion of A₁ receptor antagonist, N0861 at 10^–6 M along with PDBu and ENBA, completely blocked the protective effect of ENBA against the PDBu induced attenuation of ET-1 contractions. N0861 also completely blocked the increase in ET-1 contractions in the arterial rings incubated with ENBA alone. Another A₁ receptor antagonist DPCPX also produced similar results as N0861. On the contrary, arterial rings incubated with relatively specific A₂ receptor agonist CGS 21680 at 10^–4M did not produce any protection against PDBu induced blunting of the ET-1 contractions. Incubation with CGS 21680 alone also did not significantly alter the ET-1 contractions. Interestingly, inclusion of A₂ receptor antagonist DMPX at 10^–4M in the incubation media along with CGS 21680 mimicked the effects of ENBA alone i.e. produced protection against PDBu and enhanced ET-1 contractions. Incubation of the arteries with ENBA alone caused an accumulation of PKC levels, whereas, incubation with CGS 21680 had no significant effect on PKC levels. To study the coupling of adenosine receptor with G-protein, the tissue was incubated for one day with cholera (CT) or pertussis toxin (PT) in the presence or absence or ENBA and PDBu as described above. Incubation with PT blocked the protective effect of ENBA against PDBu as well as the elevation of ET-1 response when incubated with ENBA alone. On the contrary, incubation with CT did not produce any significant effect on ENBA responses. These results indicate that PKC is modulated by adenosine via A₁ adenosine receptors and through a PT sensitive G-protein.This work was supported by National Heart, Lung and Blood Institute Grant HL-27339.     

Expression of interleukin-1 receptors and their role in interleukin-1 actions in murine microglial cells

Interleukin (IL)-1 is an important mediator of acute brain injury and inflammation, and has been implicated in chronic neurodegeneration. The main source of IL-1 in the CNS is microglial cells, which have also been suggested as targets for its action. However, no data exist demonstrating expression of IL-1 receptors [IL-1 type-I receptor (IL-1RI), IL-1 type-II receptor (IL-1RII) and IL-1 receptor accessory protein (IL-1RAcP)] on microglia. In the present study we investigated whether microglia express IL-1 receptors and whether they present target or modulatory properties for IL-1 actions. RT-PCR analysis demonstrated lower expression of IL-1RI and higher expression of IL-1RII mRNAs in mouse microglial cultures compared with mixed glial or pure astrocyte cultures. Bacterial lipopolysaccharide (LPS) caused increased expression of IL-1RI, IL-1RII and IL-1RAcP mRNAs, induced the release of IL-1beta, IL-6 and prostaglandin-E2 (PGE2), and activated nuclear factor kappaB (NF-kappaB) and the mitogen-activated protein kinases (MAPKs) p38, and extracellular signal-regulated protein kinase (ERK1/2), but not c-Jun N-terminal kinase (JNK) in microglial cultures. In comparison, IL-1beta induced the release of PGE2, IL-6 and activated NF-kappaB, p38, JNK and ERK1/2 in mixed glial cultures, but failed to induce any of these responses in microglial cell cultures. IL-1beta also failed to affect LPS-primed microglial cells. Interestingly, a neutralizing antibody to IL-1RII significantly increased the concentration of IL-1beta in the medium of LPS-treated microglia and exacerbated the IL-1beta-induced IL-6 release in mixed glia, providing the first evidence that microglial IL-1RII regulates IL-1beta actions by binding excess levels of this cytokine during brain inflammation.     

Functional interaction between purinergic receptors: effect of ligands for A2A and P2Y12 receptors on P2Y1 receptor function

A_2A adenosine receptor (A_2AR), P2Y₁ receptor (P2Y₁R) and P2Y₁₂ receptor (P2Y₁₂R) are predominantly expressed on human platelets. The individual role of each of these receptors in platelet aggregation has been actively reported. Previously, hetero-oligomerization between these three receptors has been shown to occur. Here, we show that Ca²⁺ signaling evoked by the P2Y₁R agonist, 2-methylthioladenosine 5’ diphosphate (2MeSADP) was significantly inhibited by the A_2AR antagonist (ZM241385 and SCH442416) and the P2Y₁₂R antagonist (ARC69931MX) using HEK293T cells expressing the three receptors. It was confirmed that inhibition of P2Y₁R signaling by A_2AR and P2Y₁₂R antagonists was indeed mediated through A_2AR and P2Y₁₂R using 1321N1 human astrocytoma cells which do not express P2Y receptors. We expect that intermolecular signal transduction and specific conformational changes occur among components of hetero-oligomers formed by these three receptors.     

Neuroprotection afforded by adenosine A2A receptor blockade is modulated by corticotrophin‐releasing factor (CRF) in glutamate injured cortical neurons

In situations of hypoxia, glutamate excitotoxicity induces neuronal death. The release of extracellular adenosine is also triggered and is accompanied by an increase of the stress mediator, corticotrophin‐releasing factor (CRF). Adenosine A_2A receptors contribute to glutamate excitoxicity and their blockade is effective in stress‐induced neuronal deficits, but the involvement of CRF on this effect was never explored. We now evaluated the interaction between A_2A and CRF receptors (CRFR) function, upon glutamate insult. Primary rat cortical neuronal cultures (9 days in vitro) expressing both CRF₁R and CRF₂R were challenged with glutamate (20–1000 μM, 24 h). CRF₁R was found to co‐localize with neuronal markers and CRF₂R to be present in both neuronal and glial cells. The effects of the CRF and A_2A receptors ligands on cell viability were measured using propidium iodide and Syto‐13 fluorescence staining. Glutamate decreased cell viability in a concentration‐dependent manner. Urocortin (10 pM), an agonist of CRF receptors, increased cell survival in the presence of glutamate. This neuroprotective effect was abolished by blocking either CRF₁R or CRF₂R with antalarmin (10 nM) or anti‐Sauvagine‐30 (10 nM), respectively. The blockade of A_2A receptors with a selective antagonist SCH 58261 (50 nM) improved cell viability against the glutamate insult. This effect was dependent on CRF₂R, but not on CRF₁R activation. Overall, these data show a protective role of CRF in cortical neurons, against glutamate‐induced death. The neuroprotection achieved by A_2A receptors blockade requires CRF₂R activation. This interaction between the adenosine and CRF receptors can explain the beneficial effects of using A_2A receptor antagonists against stress‐induced noxious effects.     

Neuroprotection by adenosine in the brain: From A<Subscript>1</Subscript> receptor activation to A<Subscript>2A</Subscript> receptor blockade

Adenosine is a neuromodulator that operates via the most abundant inhibitory adenosine A₁ receptors (A₁Rs) and the less abundant, but widespread, facilitatory A_2ARs. It is commonly assumed that A₁Rs play a key role in neuroprotection since they decrease glutamate release and hyperpolarize neurons. In fact, A₁R activation at the onset of neuronal injury attenuates brain damage, whereas its blockade exacerbates damage in adult animals. However, there is a down-regulation of central A₁Rs in chronic noxious situations. In contrast, A_2ARs are up-regulated in noxious brain conditions and their blockade confers robust brain neuroprotection in adult animals. The brain neuroprotective effect of A_2AR antagonists is maintained in chronic noxious brain conditions without observable peripheral effects, thus justifying the interest of A_2AR antagonists as novel protective agents in neurodegenerative diseases such as Parkinsons and Alzheimers disease, ischemic brain damage and epilepsy. The greater interest of A_2AR blockade compared to A₁R activation does not mean that A₁R activation is irrelevant for a neuroprotective strategy. In fact, it is proposed that coupling A_2AR antagonists with strategies aimed at bursting the levels of extracellular adenosine (by inhibiting adenosine kinase) to activate A₁Rs might constitute the more robust brain neuroprotective strategy based on the adenosine neuromodulatory system. This strategy should be useful in adult animals and especially in the elderly (where brain pathologies are prevalent) but is not valid for fetus or newborns where the impact of adenosine receptors on brain damage is different.     

Resveratrol inhibits heregulin-beta1-mediated matrix metalloproteinase-9 expression and cell invasion in human breast cancer cells

The growth factor heregulin-β1 (HRG-β1), which is expressed in breast cancer, activates the HER-2 signaling pathway through induction of heterodimeric complexes of HER-2 with HER-3 or HER-4. It has been shown in many studies that HRG-β1 induces the tumorigenicity and metastasis of breast cancer cells. Matrix metalloproteinase (MMP) 9 is a key enzyme in the degradation of extracellular matrices, and its expression may be dysregulated in breast cancer invasion and metastasis. Resveratrol, a major component in grape, exhibited potential anticarcinogenic activities in both in vitro and in vivo studies. However, the inhibitory effect of resveratrol on HER-2-mediated expression of MMP-9 has not been demonstrated yet.

In the present study, we investigated the anti-invasive mechanism of resveratrol in human breast cancer cells. Human breast cancer MCF-7 cells were exposed to resveratrol (2, 5 and 10 μM). The expression activity of MMP-9 was measured by zymogram analysis. Phosphorylated levels of HER-2 and mitogen-activated protein kinase (MAPK)/ERK were measured by Western blot analysis. Total actin was used as internal control for protein expression. HRG-β1 induced the phosphorylation of HER-2/neu receptor and MMP-9 expression in human breast cancer MCF-7 cells. Resveratrol significantly inhibited HRG-β1-mediated MMP-9 expression in human breast cancer cells. MEK inhibitor induced a marked reduction in MMP-9 expression, and it suggested that ERK1/2 cascade could play an important role in HRG-β1-mediated MMP-9 expression. Furthermore, resveratrol significantly suppressed HRG-β1-mediated phosphorylation of ERK1/2 and invasion of breast cancer cells. However, resveratrol had negligible effects on either HRG-β1-mediated phosphorylation of HER-2 receptor or expression of the tissue inhibitor of MMP, tissue inhibitor metalloproteinase protein 1.

Taken together, our results suggest that resveratrol inhibited MMP-9 expression in human breast cancer cells. The inhibitory effects of resveratrol on MMP-9 expression and invasion of breast cancer cells are, in part, associated with the down-regulation of the MAPK/ERK signaling pathway.     

Inflammatory cytokines IL-1β and TNF-α regulate p75NTR expression in CNS neurons and astrocytes by distinct cell-type-specific signalling mechanisms

The p75^NTR (where NTR is neurotrophin receptor) can mediate many distinct cellular functions, including cell survival and apoptosis, axonal growth and cell proliferation, depending on the cellular context. This multifunctional receptor is widely expressed in the CNS (central nervous system) during development, but its expression is restricted in the adult brain. However, p75^NTR is induced by a variety of pathophysiological insults, including seizures, lesions and degenerative disease. We have demonstrated previously that p75^NTR is induced by seizures in neurons, where it induces apoptosis, and in astrocytes, where it may regulate proliferation. In the present study, we have investigated whether the inflammatory cytokines IL (interleukin)-1β and TNF-α (tumour necrosis factor-α), that are commonly elevated in these pathological conditions, mediate the regulation of p75^NTR in neurons and astrocytes. We have further analysed the signal transduction pathways by which these cytokines induce p75^NTR expression in the different cell types, specifically investigating the roles of the NF-κB (nuclear factor κB) and p38 MAPK (mitogen-activated protein kinase) pathways. We have demonstrated that both cytokines regulate p75^NTR expression; however, the mechanisms governing this regulation are cytokine- and cell-type specific. The distinct mechanisms of cytokine-mediated p75^NTR regulation that we demonstrate in the present study may facilitate therapeutic intervention in regulation of this receptor in a cell-selective manner.     

Synthesis and in vitro evaluation of anti-inflammatory activity of ester and amine derivatives of indoline in RAW 264.7 and peritoneal macrophages

A prolonged increase in pro-inflammatory cytokines, TNF-α and IL-6 occurs in inflammatory diseases. Although existing therapies like steroids and TNF-α antagonists are effective they may cause serious adverse effects. We describe the preparation and evaluation for anti-inflammatory activity of 11 novel derivatives of indoline carbamates with a propionic ester, 2-aminoethyl, 3-aminopropyl 2-(dimethylamino)ethyl or 3-(dimethylamino)propyl group in positions 3 or 1. Compounds 25, 26 and 29 were previously shown to inhibit acetylcholinesterase with IC_50s ranging from 0.4 to 55 μM and to prevent cytotoxicity induced by reactive oxygen species in a concentration range of 100 pM–1 μM. Compounds 25, 26, 29, 9, 10, 17 and 18, reduced NO, TNF-α and IL-6 at concentrations of 1–10 pM in LPS-activated RAW-264.7 and mouse peritoneal macrophages. The reduction in cytokines by compound 25 was associated with an increase in IκBα degradation and a decrease in the phosphorylation of p38 but not that of ERK. Conclusion: Indoline derivatives substituted at position 3 with chains carrying ester or amino groups may have potential for the treatment of chronic inflammatory and neurodegenerative diseases.     

P1 receptors and cytokine secretion

Evidence has accumulated in the last three decades to suggest tissue protection and regeneration by adenosine in multiple different cell types. Adenosine produced in hypoxic or inflamed environments reduces tissue injury and promotes repair by receptor-mediated mechanisms. Among other actions, regulation of cytokine production and secretion by immune cells, astrocytes and microglia (the brain immunocytes) has emerged as a main mechanism at the basis of adenosine effects in diseases characterized by a marked inflammatory component. Many recent studies have highlighted that signalling through A₁ and A_2A adenosine receptors can powerfully prevent the release of pro-inflammatory cytokines, thus inhibiting inflammation and reperfusion injury. However, the activation of adenosine receptors is not invariably protective of tissues, as signalling through the A_2B adenosine receptor has been linked to pro-inflammatory actions which are, at least in part, mediated by increased release of pro-inflammatory cytokines from epithelial cells, astrocytes and fibroblasts. Here, we discuss the multiple actions of P1 receptors on cytokine secretion, by analyzing, in particular, the role of the various adenosine receptor subtypes, the complex reciprocal interplay between the adenosine and the cytokine systems, their pathophysiological significance and the potential of adenosine receptor ligands as new anti-inflammatory agents.     

Adenosine A1 receptor: Functional receptor-receptor interactions in the brain

Over the past decade, many lines of investigation have shown that receptor-mediated signaling exhibits greater diversity than previously appreciated. Signal diversity arises from numerous factors, which include the formation of receptor dimers and interplay between different receptors. Using adenosine A₁ receptors as a paradigm of G protein-coupled receptors, this review focuses on how receptor-receptor interactions may contribute to regulation of the synaptic transmission within the central nervous system. The interactions with metabotropic dopamine, adenosine A_2A, A₃, neuropeptide Y, and purinergic P2Y₁ receptors will be described in the first part. The second part deals with interactions between A₁Rs and ionotropic receptors, especially GABA_A, NMDA, and P2X receptors as well as ATP-sensitive K⁺ channels. Finally, the review will discuss new approaches towards treating neurological disorders.     

NF-κB inhibitory action of resveratrol: A probable mechanism of neuroprotection in experimental diabetic neuropathy

Resveratrol has shown array of biological actions, and is under clinical development for various disease conditions. The etiology of diabetic neuropathy revolves around oxidative stress, AGE formation, lipid peroxidation etc. All these stimulate inflammatory processes and NF-κB cascade is considered as one of the major players of inflammatory response. Activation of NF-κB results in elevated levels of inflammatory mediators. COX-2 and TNF-α activity have also been correlated with inflammatory damage in the pathophysiology of diabetic neuropathy (DN). Therefore we investigated the effect of resveratrol on NF-κB inflammatory cascade, COX-2, TNF-α and IL-6 levels in experimental DN.We found that resveratrol protected against various functional and behavioral deficits in diabetic neuropathy in line with our earlier published reports. In this study we found that the resveratrol treatment decreased the expression of p65 and IκB-α in treated rats. Treatment also ameliorated the elevated levels of TNF-α, IL-6 and COX-2. Resveratrol treatment produced significant decrease in nerve MDA levels in treated animals which may also be contributing to reduction in neuro-inflammation. This study confirms the NF-κB inhibitory activity and anti-inflammatory activity of resveratrol which may contribute to neuroprotection in diabetic neuropathy apart from its antioxidant effect.     

New A2A adenosine receptor antagonists: a structure-based upside-down interaction in the receptor cavity

Adenosine receptor antagonists are generally based on heterocyclic core structures presenting substituents of various volumes and chemical-physical profiles. Adenine and purine-based adenosine receptor antagonists have been reported in literature. In this work we combined various substituents in the 2, 6, and 8-positions of 9-ethylpurine to depict a structure-affinity relationship analysis at the human adenosine receptors. Compounds were rationally designed trough molecular modeling analysis and then synthesized and evaluated at radioligand binding studies at human adenosine receptors. The new compounds showed affinity for the human adenosine receptors, with some derivatives endowed with low nanomolar K_i data, in particular at the A_2AAR subtype. The purine core proves to be a versatile core structure for the development of novel adenosine receptor antagonists with nanomolar affinity for these membrane proteins.     

Modulation of adenylyl cyclase by FPP and adenosine involves stimulatory and inhibitory adenosine receptors and g proteins

FPP and adenosine modulate the adenylyl cyclase (AC)/cAMP signal transduction pathway in mammalian spermatozoa to elicit a biphasic response, initially stimulating capacitation and then inhibiting spontaneous acrosome loss. This study addressed the hypothesis that responses to FPP involve interactions between receptors for FPP and adenosine, the biphasic responses involving stimulatory and inhibitory adenosine receptors. Gln‐FPP, a competitive inhibitor of FPP, significantly inhibited binding of an adenosine analogue and responses to adenosine, especially in capacitated suspensions, consistent with interaction between FPP and adenosine receptors. CGS‐21680 (1 μM), a stimulatory A_2a adenosine receptor agonist, significantly stimulated capacitation and cAMP in uncapacitated cells, while cyclopentyl adenosine (1 μM), an inhibitory A₁ adenosine receptor agonist only affected capacitated cells, inhibiting spontaneous acrosome loss. Responses to FPP and adenosine were inhibited in uncapacitated cells by a selective A_2a antagonist and in capacitated cells by a selective A₁ antagonist; subsequent investigations indicated possible involvement of G proteins. Like FPP, cholera toxin stimulated capacitation and cAMP production in uncapacitated cells, suggesting involvement of a G protein with a Gα_s subunit. In contrast, pertussis toxin prevented FPP's inhibition of both spontaneous acrosome loss and cAMP production, suggesting involvement of a Gα_i/_o subunit. Immunoblotting evidence revealed the presence of proteins of the appropriate molecular weights for Gα_s, Gα_i2, Gα _i3, and Gα_o subunits. This study provides the first direct evidence suggesting the involvement of two different types of adenosine receptors and both Gα_s and Gα_i/_o subunits in the regulation of capacitation, resulting in modulation of AC activity and availability of cAMP. Mol. Reprod. Dev. 53:459–471, 1999. © 1999 Wiley‐Liss, Inc.